EPA-600/1-77-046
.October 1977
Environmental Health Effects Research Series
THE MECHANISM OF SULFUR DIOXIDE
INITIATED BRONCHOCONSTRICTION
PRO!®-
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711
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EPA-600/1-77-046
October 1977
THE MECHANISM OF SULFUR DIOXIDE
INITIATED BRONCHOCONSTRICTION
Jeffrey M. Charles, Ph.D.
and
Daniel B. Menzel, Ph.D.
Laboratory of Environmental Pharmacology
and Toxicology
Duke University Medical Center
Durham, North Carolina 27710
U.S. Environmental Protection Agency
Region III Information Resource
Contract No. M-tt-1794 ^'chSsL,
Philadelphia, PA 19107
Project Officer
Donald E. Gardner, Ph.D.
Biomedical Research Branch
Clinical Studies Division
Health Effects Research Laboratory
Research Triangle Park, N.C. 27711
U.S. EPA Region III
Regional Center for Environmental
Information
1G50 Arch Street (3PM52)
Philadelphia, PA 19103
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
HEALTH EFFECTS RESEARCH LABORATORY
RESEARCH TRIANGLE PARK, N.C. 27711
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DISCLAIMER
This report has been reviewed by the Health Effects Research
Laboratory, U.S. Environmental Protection Agency, and approved for
publication. Approval does not signify that the contents necessarily
reflect the views and policies of the U.S. Environmental Protection
Agency, nor does mention of trade names or commercial products
constitute endorsement or recommendation for use.
1.1
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FORWORD
The many benefits of our modern, developing, industrial society are
accompanied by certain hazards. Careful assessment of the relative risk
of existing and new man-made environmental hazards is necessary for the
establishment of sound regulatory policy. These regulations serve to
enhance the quality of our environment in order to promote the public
health and welfare and the productive capacity of our Nation's population.
The Health Effects Research Laboratory, Research Triangle Park
conducts a coordinated environmental health research program in toxicology,
epidemiology, and clinical studies using human volunteer subjects. These
studies address problems in air pollution, non-ionizing radiation,
environmental carcinogenesis and the toxicology of pesticides as well as
other chemical pollutants. The Laboratory develops and revises air quality
criteria documents on pollutants for which national ambient air quality
standards exist or are proposed, provides the data for registration of new
pesticides or proposed suspension of those already in use, conducts research
on hazardous and toxic materials, and is preparing the health basis for
non-ionizing radiation standards. Direct support to the regulatory func-
tion of the Agency is provided in the form of expert testimony and prepara-
tion of affidavits as well as expert advice to the Administrator to
assure the adequacy of health care and surveillance of persons having
suffered imminent and substantial endangerment of their health.
Atmospheric sulfur oxides exist in chemically complex particulates
of the respirable size range. Inhalation of these particulates represents
a potential health hazard. Before one can estimate the extent of the
long-term health hazard to man or espouse a particular strategy for abate-
ment, a clear understanding of the mechanism(s) by which sulfates interact
with the mammalian lung is needed. This report provides information
concerning the uptake of sulfate salts by the lung, the interaction of
sulfate salts with specific hormonal systems in the lung and the potential
interrelations between sulfate and heavy metal aerosols as they might
exist in the environment. The data reported here describe the uptake and
elimination kinetics of sulfate ion in mammalian lungs. The release of
histamine by sulfate salts is demonstrated as a potential mechanism of
action and as a means by which the varying potency of different chemical
salts of sulfuric acid may be explained. These studies illustrate that
sulfate aerosols cannot be considered independent of the other inorganic
compounds found in respirable particles. The demonstrated removal of
sulfate ions from the lung clearly shows that such mechanisms are extant
and adds to our knowledge of the pathophysiology of the lung, as it is
related to disease processes.
H. Knelson, M.D.
.Director,
Health Effects Research Laboratory
•m
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PREFACE.
The release of sulfur oxides into the atmosphere is a most
pressing problem. The use of alternative fossil fuel sources
other than petroleum will undoubtedly lead to the emission of
larger amounts of sulfur into the atmosphere. Atmospheric sulfur
oxides exist in chemically complex particulates of the respirable
size range. Inhalation of these particulates represents a poten^
tial health hazard. Before one can estimate the extent of the long-
term health hazard to man or espouse a particular strategy for
abatement, a clear understanding of the mechanism(s) by which
sulfates interact with the mammalian lung is needed. This report
provides the results of a series of experiments into the uptake
of sulfate salts by the lung, the interaction of sulfate salts with
specific hormonal systems in the lung and the potential interre-
lations between sulfate and heavy metal aerosols as they might
exist in the environment. The data reported here describe for the
first time the uptake and elimination kinetics of sulfate ion in
mammalian lungs. The release of histamine by sulfate salts is
demonstrated as a potential mechanism of action and as a means by
which the varying potency of different chemical salts of sulfuric
acid may be explained. In sum, these studies illustrate that
sulfate aerosols can not be considered independent of the other
inorganic compounds found in respirable particles. Hopefully,
IV
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these data will also provide impetus for detailed studies of
electrolyte transport in the lung and its potential relation to
disease in man. The demonstrated removal of sulfate ions from
the lung clearly shows that such mechanisms are extant and adds
to our knowledge of the pathophysiolpgy of the lung, as it is
related to disease processes.
Daniel B. Menzel
Director
Laboratory of Environmental
Pharmacology and Toxicology
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ABSTRACT
In. vitro studies with unsensitized guinea pig lung frag-
ments (ULF) incubated with 10 to 200 mM concentrations of am-
monium ion demonstrated the release of substantial quantities
of histamine. Of the anions tested, sulfate was the most potent,
while nitrate and acetate ions were of intermediate potency and
chloride less potent. An osmotic effect is unlikely since equal
concentrations of NaCl failed to release histamine or LDH, a
cytop'lasmic enzyme into the incubation medium. Drugs known to
modulate the anaphylactic release of histamine through the
cAMP and cGMP systems had no effect on the ammonium ion mediated
release of histamine.
Studies in sensitized guinea pig lung fragments (SLF)
demonstrated the known phenomenon of the ability of the cAMP
and cGMP systems to modulate antigen-antibody release of hista-
mine. Acetylcholine stimulated the release of histamine while
epinephrine and isoproterenol depressed histamine release in
accordance with previous reports. Dibutyryl cAMP and phenyl-
ephrine failed to have significant effects.
The mechanism of absorption of sulfate ions was investigated.
The intracellular sulfate ion space in ULF decreased in the
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presence of 50 mM and 100 mM (NH.^SO, as compared to the presence
of Na^SO,. Since histamine release occurred only in the presence
of (NH,)2SO,, the decrease in the intracellular sulfate ion space
is probably associated with the degranulation process. The intra-
cellular sulfate space in SLF was significantly decreased also
in the presence of 100 mM (NH^SO^.
In both ULF and SLF, drugs capable of modulating the cAMP
and cGMP systems failed to alter the sulfate ion uptake. Sulfate
ion absorption does not appear to be highly dependent on metabolic
energy. At high concentrations of potent metabolic inhibitors
only partial inhibition of sulfate ion uptake was observed.
Phloretin has been reported to inhibit chloride and sulfate up-
take by human red blood cells, however, phloretin had no effect
on the sulfate ion uptake by the lung fragments. The apparent
energy of activation for the initial rate of sulfate absorption
was found to be 3.1 Kcal for ULF and 3.2 Kcal for SLF.
Using the binding of Acridine Orange (AO) to heparin as a
model of histamine binding, the association of AO with heparin
was found dependent on the ionic strength of the incubation
medium. The total number of binding sites for AO per disaccharide
unit was unchanged with increasing ionic strength of medium.
Histamine release by inorganic salts may be a simple ionic ex-
change phenomenon.
The kinetics of sulfate absorption from the airways of the
isolated ventilated and perfused rat lung (IVPL) are presented.
Absorption of sulfate ion appears to be by simple diffusion
vn
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and to be enhanced in the presence of ammonium ions at 0.01 pinole/
lung. Manganous ion was an exception and showed no enhancement.
The t, for the initial rate of sulfate absorption was 8.4 ± 1.8
*
minutes. Sulfate ions introduced into the vasculature have the
same volume of distribution and mean transit time within the lung
as blue dextran, a compound unlikely to leave the intravascular
space. Thus, sulfate ion absorption in the rat IVPL is unidirec-
tional. The administration of 1 pmole (NH,)2SO, intratracheally
led to a rapid decrease in the respiratory volume of the lung,
an effect which could be blocked by prior perfusion with mepyra-
mine maleate (10" M). Ammonium sulfate caused a rapid release
of a large portion of the histamine stores into the lung perfusate,
Neither LDH nor prostaglandins were released by any ions tested.
Experiments in vivo demonstrate that sulfate ion removal
from the rat lung airways appears to be simple diffusion with t,
of 34.5 minutes. Deviations from physiological pH of the sulfate
containing medium and the addition of certain cations (0.1 nano-
mole/lung) enhance sulfate absorption. As in the case of the
IVPL, manganous ion failed to stimulate absorption.
In all systems tested, there is a positive correlation be-
tween the irritant potential associated with a specific sulfate
salt aerosol and the rate at which sulfate ions present in such
solutions are removed from the lung. From these data a model
is proposed for the absorption of sulfate ions and the release
of histamine by ammonium sulfate.
vm
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CONTENTS
Foreword . . . . . . . . . . ... ... . .
Preface . . . ; . . . . . . ... . . . . . iv
•'•.•'. • . . • • • i - . •
Abstract . . . . -.. . ... . . . . . . . .. ..'i vi
List of Figures . . . . . . . . . . . . . . : . x
List of Tables . . '.'•..: ... . . . .... . .J x±i
Acknowledgements . . . .... ..... . . . xiv:
1. Introduction . . . . . . . . . , "..''.; , . 1
2. Conclusions . . ... . . . . ... . . 3
.'••.'• • ' . • ' •
3. Recommendations . . . . . . . . . .... 5
4. Sulfate Ion Uptake and Histamine Release in
Guinea Pig Lung Fragments . . . ... . . . . 7
5. Absorption of Sulfate Ion in the Isolated,
Ventilated and Perfused Rat Lung . . . . ... 46
6. Sulfate Ion Absorption in the Rat Lung In Vivo . . . 72
7. Discussion . . . . .'.'.. . 90
References . .... . . .... . ... . . 99
Bibliography . . , , , . » « » . ,.. t . ,. . .». • ^ .. -,. \ 1Q7
IX
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LIST OF FIGURES
2.1 Standard Histamine Curve . . . . . 14
2.2 Standard DNA :Curve . . 18
2.3 Release of Histamine from Lung Fragments by Salts . • • 21
2.4 Dose Response Release of Histamine by Ovalbumin in
Sensitized Lung Fragments . . 27
35
2.5 Time Course of S-Sulfate Ion Uptake by Unsensitized
Guinea Pig Lung Fragments . . . .28
35 '
2.6 Time Course of S-Sulfate Ion Uptake by Sensitized
Guinea Pig Lung Fragments . . . ... . . ... 33
2.7 Metachromatic Titration of Acridine Orange with
Heparin .... 37
2.8 Absorption Spectra of Acridine Orange-Heparin Solution
in the Presence and Absence of Various Salts . . . . 38
2.9 Metachromatic Titration of Acridine Orange by Heparin
and Varying Ionic Strengths . .39
3.1 Schematic Diagram of the Isolated, Ventilated and
Perfused Lung Preparation . . . .51
3.2 Absence .of Significant LDH Release into the Effluent
of the Perfused Lung . . . . . . . . . . . . 58
3.3 Removal of 35S-Sulfate Ions from the Rat IVPL . . . .59
35
3.4 Absence of Pulmonary Vascular Uptake of S-Sulfate
Ions ... . . . . . . . . . . . ... .62
3.5 Release of Histamine into the Lung Effluent by
Ammonium Sulfate 65
3.6 Effects of Intratracheal Injection of Salts on
Respiratory Volume ... 67
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3.7 Absorption of 3H20 in the Rat IVPL . . . . . . . . 69
4.1 Percent Sulfate Ion Unabsorbed by the Rat Lung
In Vivo . . . . . . ...... . .;.: . . . 80
4.2 Percent Sulfate Ion Unabsorbed by the Rat Lung
Under Varying pH Conditions . , . . . . . . . -85
5.1 Proposed Mechanism for Sulfate Ion Absorption and
Release of Histamine in the Presence of Ammonium .
Sulfate . . . . ...... . . ... . . 97
XI
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LIST OF TABLES
2.1 Release of Histamine from Lung Fragments in the
Presence of 100 mM Salt Solutions . . . . ... .22
2.2 The Absence of Isoproterenol Mediation on Histamine
Release by Sulfate Salts . . ... 24
2.3 The Absence of Acetylcholine Mediation on Histamine
Release by Sulfate Salts . . . . . . ... . . 25
2.4 The Absence of Dibutyryl cAMP Mediation on Histamine
Release by Sulfate Salts . . . 26
2.5 Sulfate Ion Uptake in Unsensitized Guinea Pig Lung
Fragments . . . . . ... ... . .'.. . . . 30
2.6 Effect of Metabolic Inhibitors on the Uptake of
Sulfate Ion by Guinea Pig Lung Slices ... . . . .31
2.7 Failure of Pharmacological Agents to Modulate the Uptake
of Sulfate Ion by Guinea Pig Lung Slices 32
2.8 Sulfate Ion Uptake in Sensitized Guinea Pig Lung
Fragments . . . . .34
2.9 Effects of Pharmacological Agents on the Anaphylactic
Release of Histamine and Sulfate Ion Uptake in Sensitized
Guinea Pig Lung Fragments . . . ... . . . . . 36
2.10 Absence of an Effect of Ionic Strength on the Number of
Acridine Orange Binding Sites on the Heparin
Macromolecule . . . . . . . .40
2.11 Release of LDH from Unsensitized Lung Fragments . . . -42
3.1 Distribution of Intratracheal Injection of
0.1 ml Isotonic Sucrose Solution . . . . . . . . 57
3.2 Effect of Ammonium Ion on the Kinetics of Sulfate Ion
Absorption from the Rat IVPL . .60
3.3 Effect of Heavy Metal Counter Cation on the Kinetics of
Sulfate Ion Absorption from the Rat IVPL . . . . . .63
XII
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4.1 Absorption of 35S-Sulfate in the Rat Lung In Vivo . . .82
4.2 Effect of Counter Cation on Pulmonary Absorption of
35s-Sulfate Ions . . . . . . . .... . . .83
4.3 Effect of Nickel Chloride Aerosol on Pulmonary
Absorption of Sulfate Ion ... . . ... . . . 87
xiii
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ACKNOWLEDGEMENTS
The kind support, encouragement and stimulation of the
scientific personnel of the Health Effects Research Laboratory
is especially appreciated. Drs. Coffin, Gardner and Miller and
Ms. Judy Graham were most cooperative throughout these studies,
participating in many of the experiments. Access to the HERL
animal exposure facilities for Ni aerosol exposures was particu-
larly useful. The consultation of Professors N. A. Porter, T.
Narahashi, J- V. Salzano, and G. M. Rosen are also acknowledged.
Dr/ Ronald Bradow of EPA provided much preliminary information
on the chemical composition of atmospheric sulfates and stimu-
lated our thinking to make our experiments more realistic. These
studies could not have been completed without the able assistance
of Mr. Manley Fuller and Ms. Karen Tilley. Ms. Elaine Smolko
provided much needed editorial guidance and administrative assis-
• . ' •' • • ' ' ' • • . • o
tance.
xiv
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.: . . SECTION i ..
INTRODUCTION
According to data obtained by the United States National
Air Sampling Network (2) the level of particulate sulfate com-
pounds in urban areas was 10.1 yg/m3 arid in rural areas was
5.3 yg/m3 in 1967. In the mid 1970's the automobile catalytic
converter has become a new source of atmospheric sulfate com-
pounds. It is capable of oxidizing up to 80 percent of the
sulfur in the fuels to sulfuric acid.
Environmental exposure to S02 and associated particulates
has been associated with impairment of pulmonary function (67),
as well as increased prevalence of chronic bronchitis (.16,69)
and increased incidence of acute respiratory disease (30,34).
Severe bronchoconstriction due to inhalation of certain sulfate
aerosols has been demonstrated by Amdur (5) and Amdur and Corn
(6).
The respiratory tract epithelium behaves as a highly porous
membrane absorbing a number of solutes (13,14,22). Absorption
rates of a wide Variety of inorganic and organic solutes have
been studied by intratracheal instillation (23,73). The absorp-
tion of most compounds investigated follows first order kinetics
.and appears to be by simple diffusion.
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In light of the above considerations, the present study was
undertaken to elucidate the mechanism of the observed broncho-
constriction due to the inhalation of certain sulfate salts. The
mechanism whereby sulfate ions are removed from the mammalian lung
was also investigated. :.
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SECTION 2
CONCLUSIONS
We have been able to demonstrate that unsensitized guinea
pig lung fragments (ULF) incubated with a variety of ammonium
salts release significant quantities of histamine. The most
efficacious, ammonium sulfate (100 mM), shows maximal histamine
release after 30 minutes. The ammonium sulfate mediated release
is equal to 97% of the total histamine stores. Equal concen-
trations of sodium sulfate also fail to release histamine,
supporting the concept that only certain sulfate salts have
biological actions.
The removal of sulfate ions from the airway appears to be
predominantly by simple diffusion. Absorption of sulfate ions
in the reverse direction, specifically from the vasculature into
the lung, could not be demonstrated. At very low doses ammonium
ion increases the removal process. The t^ at doses of 0.05 ymole
or greater was 8.4 ± 1.8 minutes. The heavy metal cations tested
significantly enhanced the absorption of sulfate ions from the
airways. An exception to this rule was manganous ion, which had
no effect over control.
In all systems tested there is a positive correlation be-
tween the irritant potential associated with a specific sulfate
salt (5,9), and the rate at which sulfate ions are cleared from
the 'lung.
3
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Data presented here suggest a correlation between the rate
of sulfate ion absorption from the mammalian lung and the reported
bronchoconstriction in the presence of certain sulfate salts.
Clearly, the rate of sulfate ion absorption is influenced by the
cationic species present and the pH of the. surrounding extra-
cellular environment. The role of histamine release as a mechanism
for the bronchoconstriction action of ammonium sulfate aerosols is
strengthened by bur data. More research is needed to investigate
the possible release of other vasoactive substances by heavy metal
sulfate salts.
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SECTION 3
RECOMMENDATIONS
Atmospheric sulfate particulates exist in a complex chemical
form associated with a large number of heavy metal ions. The
exact composition undoubtedly varies with the geographic location
and proximity to specific sources of sulfur emission. Nonethe-
less, the data presented in this report, as well as in the lit-
erature, indicate a strong dependence of sulfate ion removal and
bronchoconstrictive effect of sulfate aerosols depending on the
cation associated with sulfuric acid. A further investigation
of the effects of cations (e.g. Zn, Ni, Cd, Co, Hg, Fe, and Mn)
released through energy production is needed. A reciprocal re-
lationship may exist whereby sulfate salts may stimulate directly
or indirectly the uptake of these ions. Studies of the uptake of
these heavy metal ions themselves as well as sulfate ion from the
pulmonary lumen are recommended.
The morphological complexity of the lung places inherent
limits on the kinetic and mechanistic measurements that can be
made with intact animals or perfused, isolated lungs. The develop-
ment of a model system of isolated lung cells, preferably of sus-
tained and uniform composition in culture, to study the uptake of
sulfate and other environmentally present ions is recommended.
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Such studies should be applied to generalized principles to over-
come the limitations of studies restricted to specific kinds of
pollutants. Generalization will allow the application of such data
to a wide variety of sources of emission.
The use of pharmacological methods to mimic human disease
states, such as bronchitis and asthma, is also recommended. The
health hazard of particulate sulfates to specific populations may
be greater due to the independent release of histamine by sulfate
salts which may not be prevented by natural defense mechanisms.
6
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SECTION 4
SULFATE ION UPTAKE AND HISTAMINE RELEASE
IN GUINEA PIG LUNG FRAGMENTS
INTRODUCTION:
Amdur (5) and Amdur and Corn (6) measured an increase in
pulmonary resistance following the inhalation of zinc ammonium
sulfate, zinc sulfate, and ammonium sulfate aerosols. Although
ammonium sulfate was the least potent salt, it was many times
more irritating than sulfur dioxide (802). These observations
raised the question of whether sulfate aerosols arising from
S02.emitted into the atmosphere were more potent than the par-
ent compound, S0«. The experiments of McJilton et al. (52)
support the concept that certain sulfate salts are broncho-
constrictors. Nadel et al. (53) have shown that the inhalation
of a zinc ammonium sulfate aerosol increases pulmonary resis-
tance in guinea pigs as does inhalation of a histamine aerosol.
The bronchoconstriction, in the presence of certain sulfate
aerosols, suggests the possible release of a vasoactive hormone.
Histamine is present in significant quantities in guinea pig
(19-35 yg/g tissue) lung tissue (66). Drazen and Austen (20)
studied the effect of intravenous administration of slow re-
acting substance of anaphylaxis (SRS-A) which led to a 50%
decrease in pulmonary compliance, but had little effect on
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airway resistance. Bradykinin and histamine had similar effects
on compliance but increased airway resistance by 60-140%. These
observations led to the investigation of the direct effects of
ammonium sulfate and other salts on histamine release from
guinea pig lung in vitro (17).
Two theories have been proposed for the site of the his-
tamine binding in mast cell granules. Lagunoff et.al. (47)
have proposed that the negatively charged heparin macromole-
cule, a major constituent of the mast cell granule, is the bind-
ing site for histamine. Alternatively the mast cell granule
protein has been proposed by Uvnas et.al. (80) as the histamine
binding component. Lagunoff (46) has shown that 2.0-2.5 of
the 3.0-3.5 anionic sites per disaccharide unit of heparin in
rat mast cells are available for binding of histamine In situ.
Utilizing the metachromasia associated with Acridine Orange
binding to heparin to study the ionic interactions between
protein, heparin, and histamine, Lagunoff has proposed that
heparin 0-sulfate groups interact with the amine groups of the
granule protein, leaving the glucosamine N-sulfate groups, some
0-sulfate groups and the uronic carboxyl groups of the heparin
macromolecule to interact with histamine. Stone and Bradley (72)
and Lagunoff (46) suggest that the binding sites for Acridine
Orange and histamine on the heparin macromolecule are identical.
To date, a process for the uptake and elimination of in-
spired sulfate salt particulates by the mammalian lung has not
8
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been reported. Gunn (36) has proposed a protonatable carrier
for both mono- and divalent anion transport in human red blood
cells. In the model, the carriers are confined to the membrane
of "the cell. The carrier (C) can interact with from one to
three protons to form species denoted as C-,, C~ and C~. ^i
can complex with monovalent inorganic anions such as chloride
and bicarbonate. C^ can complex with divalent inorganic anions
such as sulfate. Once complexed, the carrier can transverse
the membrane and exchange its complexed anion for one intra-
cellular bicarbonate ion to maintain intracellular electrical
neutrality. €2 can traverse the membrane with one sulfate ion
and exchange the sulfate ion for two bicarbonate ions to main-
tain electrical neutrality.
Gunn et.al. (38) have shown that chloride and sulfate (37)
transport in human red blood cells fit this model. Recently
Levinson and Villereal (48,49) have demonstrated that sulfate
transport in Ehrlich Ascites tumor cells was also consistent
with the idea of a carrier mediated or facilitated transport
system.
Two animal model systems have been employed extensively
to represent the two distinct populations of humans exposed
to atmospheric pollution. Normal guinea pigs mimic the majority
of the population with unimpaired respiratory functions. Guinea
pigs that have been actively sensitized to an antigen, such
as ovalbumin, represent a model of atopic or hypersensitive
individuals such as asthmatics.
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Actively sensitized guinea pig lung fragments release
histamine and SRS-A on stimulation by the immunoglobulin E (IgE)
mediated antibody-antigen reaction (71). From studies by
Orange et..al. (57) with drugs known to influence the cellular
levels of adenosine 3'5'-cyclic monophosphate.(cAMP), it appears
that the cAMP system may be capable of modulating the immuno-
Ipgical release of histamine and SRS-A from human lung fragments,
The 6-adrenergic agonists, isoproterenol and epinephrine, which
are known to increase intracellular levels of cAMP, inhibited
the antigen induced release of histamine and SRS-A from human
lung (58), as well as guinea pig lung fragments (65). Agonists
have predominantly a-adrenergic action, such as phenylephrine,
probably decrease cellular levels of cAMP and enhance the ana-
phylactic release of histamine and SRS-A (44). Acetylcholine,
working through the guanidine, 3',5'-cyclic monophosphate (cGMP)
system has also been shown by Kaliner et.al. (44) to increase
the release of these vasoactiye substances.
In this chapter, the possible release of histamine by
ammonium sulfate and other salts from guinea pig lung fragments
are discussed, as well as studies designed to elucidate param-
eters of sulfate uptake by lung tissue from the two previously
mentioned animal model systems. . Possible pharmacological modu-
lation of sulfate salt mediated histamine release and the
sulfate ion uptake process were also investigated.
10
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METHODS
Active Sensitization of Guinea Pigs: Sensitization was accom-
plished by the method of Sorenby (70), whereby male Hartley
guinea pigs were given a single intraperitoneal injection of
20 mg ovalbumin suspended in 0.5 mg Freund's complete adjuvant.
From 3-5 weeks later, identification of sensitized animals was
accomplished by placing 2 drops of antigen solution (1 mg/tnl
in 0.9% Nad) into one eye of the treated animals. Ten minutes
later the intensity of antigen-induced swelling was judged.
Swelling of the orbital connective tissue sufficient to lift
the rims of both eyelids from the surface of the eyeball was
required before the animal was employed in the preparation of
the lung fragments. This technique has been used successfully
by Taylor and Roitt (76) as a' measure of active Sensitization
of guinea pigs.
Preparation of Lung Fragment s^: Normal or unsensitized guinea
pigs, weighing between 300-400 g were anesthetized with sodium
pentobarbital (20 mg/kg) and the lungs excised. The lungs were
cut into fragments (75-150 mg) with a razor blade and washed
repeatedly with Tyrode's solution until the fragments were free
of blood. Randomized lung fragments, weighing 150-200 mg were
employed in each incubation flask. All incubations were per-
formed in 3.0 ml of Tyrode's solution. The Tyrode's solution
contained 0.9 g NaCl, 0.02 g KC1, 0.02 g CaCl2, 0.01 g MgCl2,
0.1 g glucose, 0.1 g NaHC03 and 0.005 g Na^PO^ per 100 ml ad-
justed to pH 7.40.
11
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Anaphylactic Release of Histamine by Ovalbumin: Sensitized
lung fragments (SLF) were suspended in 3.0 ml of Tyrode's
solution (pH 7.4) containing 0, 2.5, 5.0, 7.5 and 10.0 mg of
ovalbumin. Incubations were carried out for 30 minutes at 37°C,
the fragments removed and the supernatant assayed for histamine.
The total histamine content in SLF was determined by boiling
fresh tissue for 8 minutes and assaying the supernatant.
Release of Histamine by Ammonium Sulfate and Other Salts: Un-
sensitized lung fragments (ULF) were incubated in the presence
of varying concentrations (10-200 mM) of the salts under study
for 30 minutes at 37°C, the fragments removed, and the super-
natant assayed for histamine. The salts studied in this manner
were sodium chloride, sodium sulfate, ammonium chloride, am-
monium siilfate, ammonium nitrate, and ammonium acetate. Total
histamine (yg/g lung wet weight) was determined by boiling
fresh ULF for 8 minutes and assaying the supernatant.
Histamine Assay; Histamine was measured spectrophotofluoro-
metrically (66). Two-tenths milliliter of 707o perchloric acid
was added to 2.0 ml of sample solution and the mixture incubated
for 30 minutes. One milliliter of the supernatant was added to
0.75 g NaCl and then 1.5 ml n-butanol and 0.3 mg 5 N NaOH were
added and mixed. The lower aqueous phase was removed by suction
and 1.5 ml 1 N NaOH saturated with NaCl was added to the re-
maining phase and mixed. One milliliter of the upper butanol
12
-------
layer was transferred to 1.5 ml hexane and 1.25 ml 0.1 N HC1
and the mixture agitated. The upper organic phase was removed
by suction of 0.45 ml of the acid phase and added to 0.1 N NaOH
and 0.05 ml of 0.5% methanolic solution of o-phthaldialdehyde .
The reaction was stopped after 4 minutes by the addition of
0.05 ml 3 N HC1. After the addition of 1.5 ml distilled water,
the fluorescence was measured at 450 nm by excitation at 360 nm
in a Turner model 110 Fluorometer (G. K. Turner Associates, Palo
Alto, Gal.)'. A typical standard histamine curve is shown in
Fig. 2.1.
35
Lung Fragment Uptake of S-Sulfate Ions: ULF were incubated
in the presence of concentrations of 10-100 mM of either sodium
sulfate or ammonium sulfate. The fragments were preincubated
in these solutions for 10 minutes followed by the addition of
35
S-sodium sulfate (lyCi of a 947 mCi/mmole solution) to each
incubation mixture. The fragments were incubated in a shaking
water bath at 0, 22, or 37°C. Incubations were stopped at times
between 0 and 60 minutes, the fragments removed, washed twice
35
in ice cold medium and used in the determination of S radio-
activity. A plot was made of the natural log of percent uptake
versus time, to calculate the rate constants. These values
were used in an Arrhenius plot to determine the energy of acti-
vation of the uptake process.
The SLF were studied in a similar fashion in the absence
and presence of 5.0 mg ovalbumin in the initial incubation medium.
13
-------
o.4 ' o;s 1.2
Histamine (micrograms)
Fig. 2.1. A typical standard histamine curve using histamine
diphosphate as a standard. Each point is the mean ± s.e. of
4 determinations.
14
-------
Pharmacological Modulation of Sulfate Uptake and Histamine
Release: ULF were preincubated at 37°C in 3.0 ml Tyrode's
-3
solution containing 10 M sodium cyanide, sodium fluoride,
or 2-deoxyglucose, or 10" M phenylephrine, epinephrine, iso-
35
proterenol, dibutyryl cAMP, or phloretin. S-sodium sulfate
(1 yCi of 947 mCi/mmole solution) was added to each incubation
mixture after 10 minutes. Incubations were stopped 30 minutes
later, the fragments removed, washed twice in ice cold medium
35
and used in the determination of S radioactivity.
Possible pharmacological modulation of salt mediated
histamine liberation in ULF was investigated by pre-incubation
_0
for 10 minutes with either isoproterenol (10 M), acetylcholine
(10~4 M), or dibutyryl cAMP (10~6 M) . The release of histamine
was determined 30 minutes later upon the addition of ammonium
sulfate, ammonium chloride or sodium sulfate.
Pharmacological modulation of the sulfate release and IgE-
mediated release of histamine in SLF was sought for simultaneously,
_3
SLF were pre-incubated in the presence of 10 sodium cyanide,
sodium fluoride, or 2-deoxyglucose or 10" M phenylephrine,
epinephrine, isoproterenol, dibutyryl cAMP, or phloretin. Five
milligrams ovalbumin was added to each incubation mixture after
35
10 minutes for an additional 10 minutes pre-incubation. S-
sodium sulfate (1 wCi of a 947 mCi/mmole solution) was added
to each flask at the end of the second pre-incubation. Incu-
bations were stopped 30 minutes later, the fragments removed,
15
-------
washed twice in ice cold medium and used in the determination
35
of S radioactivity. The supernatant of the incubation mixture
was used to assay for histamine release.
35
Determination of S Radioactivity: The lung fragments were
prepared for liquid scintillation counting by flask oxygen com-
bustion as described by Buyske et.al. (15) and Abou-Donia
et.al. (1). The tissue was placed on ashless black filter
paper and 0.2 ml of a 1070 sucrose solution was added to aid
combustion. The samples were dried overnight. The paper con-
taining the dried sample was placed in a platinum basket sus-
pended from a glass hook in a stoppered one liter Erlenmeyer
flask containing 5.0 ml deionized water. The flasks were flushed
with oxygen for 20 seconds and ignited with a Thomas-Ogg IR
igniter (A. H. Thomas Company, Philadelphia, Pennsylvania).
A 1 ml aliquot of the resulting solution was added to 10 ml
scintillation solution and counted in a Beckman LS-100C liquid
scintillation counter. The scintillation medium was a mixture
of toluene-Triton X-100 (2:1 v/v) containing 2.79 g/1 2,5-diphenyl-
oxazole and 0.07 g/1 l,4-bis-[2-(5 phenyloxazolyl)]-benzene.
Quench corrections were made from a quench curve prepared util-
35
izing standard S-sodium sulfate (New England Nuclear). The
35
recovery of added S-sulfate was 75.0 ± 1.270.
DNA Determinations: Total DNA in the lung fragments and that
released into the incubation medium was determined by the
16
-------
method of Seibert (68). Lung fragments prepared as above were
suspended in 3.0 ml Tyrode's medium with and without 100 mM
ammonium sulfate and incubated for 30 minutes at 37°C. Aliquots
of the supernatant and the whole aqueous homogenates of the
fragments were taken for DNA analysis. The difference between
the absorbance at 595 and 650 nm of the reaction mixture was
determined with a Varian Techtron Model 635 Spectrophotometer
(Varian Instrument Division, Palo Alto, Cal.) and compared to
a standard curve prepared with calf thymus DNA to determine the
amount of DNA present in the aliquot. A typical standard curve
is shown in Fig. 2.2.
Lactic Dehydrogenase (LDH) Determinations: The LDH determination
was based on the spectrophotometric method of Wroblewski and
LaDue (85). Determinations were made with a LDH Diagnostic kit
obtained from Sigma Chemical Company (St. Louis, Missouri).
ULF were incubated in the presence of 100 mM sodium sulfate
and ammonium sulfate for 30 minutes at 37°C. A 0.05 ml aliquot
from each flask was pipetted directly into different vials con-
taining 0.2 mg NADH and 2.85 ml 0.1 M potassium phosphate buffer,
pH 7.5, mixed and incubated at 25°C. After 20 minutes 0.1 ml
0.02 M sodium pyruvate solution was added to each vial, mixed
thoroughly, and the absorbance measured at 340 nm at 30 second
intervals for 3 minutes. Measurements were made with, a Varian
Model 635 spectrophotometer. Total tissue LDH activity was
determined by homogenizing the tissue in 5 ml phosphate buffer
17
-------
0.5-•
0.4- •
0.3- •
<0
A
0,2- -
0,1- •
260 300 460 500
DMA (mlcrograms)
Fig. 2.2. Standard DNA curve determined by the method of Seibert
(68)using a stock solution of calf thymus DNA (500 yg/ml).
18
-------
and essaying as before. LDH activity was calculated from the
following formula:
LDH activity (units/ml) = AA/min x 20,000
Metachromatic Titrations of Acridine Orange by Heparin: The
absorption spectrum of a 18 yM solution of Acridine Orange dis-
solved in 1 mM sodium phosphate buffer (pH 6.70, ionic strength
0.003 y) , was recorded with a Varian Techtron Model 635 spectro
photometer between 560 and 360 nm. Repeated 10 yl additions of
a 50 yg/ml solution of sodium heparinate were added and the
absorption spectrum recorded. The absorbance at 492 nm of
Acridine Orange-heparin solutions was measured in the presence
of added concentrations (20-100 mM) NaCl, Na2SO,; and
Dye concentrations were calculated using the value of
5.6 x 10 . The number of hypochromatic binding sites per
disaccharide repeating unit in each case was calculated from
a plot of the molar absorptivity versus volume of heparin
solution added (46,72).
Materials : Acetylcholine HC1, Acridine Orange, deoxyribonucleic
6 2
acid (Calf Thymus , Type V), N , 0 -dibutyryl adenosine 3' ,5' -
cyclic monophosphoric acid (monosodium salt), 1-epinephrine,
sodium heparinate (Grade I), histamine diphosphate, d,l-isbpro-
terenol HCL, 1-phenylephrine HC1, and o-phthaldialdehyde were
purchased from Sigma Chemical Company (St. Louis, Mo.).
19
-------
Phloretin was obtained from ICN Pharmaceuticals, Inc.
(Cleveland, Ohio). Ovalbumin (2x crystallized) was purchased
from Worthington Biochemical Corporation (Freehold, N.J.).
Freund's Complete Adjuvant was obtained from Calbiochem (La
35
Jolla, Cal.). S-sodium sulfate (947 mCi/mmole) was purchased
from New England Nuclear (Boston, Mass.).
i
RESULTS
Release of Histamine from Unsensitized Lung Fragments by
Ammonium Ions and Sulfate Ions; ULF incubated with ammonium
sulfate, ammonium nitrate, ammonium acetate, and ammonium
chloride, in concentrations of 10-200 mM, released histatnine
in proportion to the concentration of the salts present (Fig. 2.3)
Sodium sulfate and sodium chloride, however, did not release
any detectable histamine. Those salts that did release hista-
mine, had varying efficacies. The most efficacious, ammonium
sulfate, showed maximal histamine release at concentrations
of 100 mM after 30 minutes. The ammonium sulfate mediated
release was equal to 977o of the histamine content of unsensi-
tized guinea pig lung. The total histamine present in the
unsensitized guinea pig lung was found to be 27.0 ± 3.0 yg per
gram of tissue. This value for the histamine content compares
favorably with that reported by Shore et.al. (66)• The rela-
tive potencies of the salts tested are listed in Table 2.1,
with ammonium sulfate arbitrarily assigned a value of 1007o.
20
-------
30
29-
o
= 20
E '
9
I 15
UJ
U
flE
111
5
(0
10
10 20 90 00 100
SALT CONCENTRATION (mM)
200
Fig. 2.3. Release of histamine from lung fragments by salts.
Each point represents the mean ± s.e. Solid triangles, ammonium
sulfate; open circles, ammonium nitrate; solid circles, ammonium
acetate, open triangles, ammonium chloride; and open boxes,
sodium chloride and sodium sulfate.
21
-------
TABLE 2.1
Release of Histamine from Lung Fragments in the
Presence of 100 mM Salt Solutions
Compound
Sodium Chloride
Sodium Sulfate
Ammonium Chloride
Ammonium Acetate
Ammonium Nitrate
Ammonium Sulfate
No. of
Experiments
6
6
9
4
5
6
Histamine
Release
(vg/gm lung)
0
0
9.4 ±0.8
15.5+0.3
15.7 ± 1.3
26.3 ± 1.2
Percent Total
Histamine
Released
0
0
34.8
57.4
58.1
97.4
Relativea
Potency
(%)
0
0
35.7
58.9
59.7
100.0
Ammonium sulfate was arbitrarily assigned a value of 100% since it released
approximately 10070 of the histamine stores.
-------
Failure of cAMP or cGMP Mediated Systems to Modify Salt Mediated
Release of Histamine from Unsensitized Lung Fragments; The
possible modulation of salt mediated histamine release in the
-3
presence of 10 isoproterenol is shown in Table 2.2. Isopro-
terenol had no effect on histamine release.
Similarly, the presence of 10" M acetylcholine did not
significantly change the histamine release by sulfate salts
(Table 2.3). Dibutyryl cAMP was preincubated with ULF at a
concentration of 10 M for 5 minutes before the addition of
the salts and again no significant change in total histamine
release was noted (Table 2;4).
Sensitized guinea pig lung was found to contain 15.8 ±
1.3 yg per gram of tissue. This value is lower than the range
of 19-35 yg/g tissue reported by Shore et.al. (66). SLF
released histamine in the presence of the antigen, ovalbumin,
in a dose response fashion (Fig. 2.4).
Sulfate Ion Uptake by Unsensitized Lung Fragments: The uptake
of sulfate ions by ULF in the presence of 10, 50 and 100 mM
NaoSO, and (NH/^SCK is shown in Fig. 2.5. A maximum uptake
of 14.5 nanomoles of sulfate ions per mg tissue was observed.
The time course for the uptake was similar with both salts,
reaching maximal uptake in 30 minutes. A smaller sulfate ion
uptake occurred'with (NIL^SO/ at 50 and 100 mM concentrations
having decreased by 8% and 257» respectively compared to the
equivalent concentrations of NaSO,. Sulfate ion uptake was
23
-------
TABLE 2.2
The Absence of Isoproterenol Mediation on
Histamine Release by Sulfate Salts
Compound
•No. of
Experiments
Concentrations
(mM)
Histamine Release
in absence of
Isoproterenol
(10-3 M)
(vg/gm lung)
Histamine Release
in presence of
Isoproterenol
(10-3 M)
(yg/gm lung)
Ammonium Chloride
Ammonium Sulfate
Sodium Sulfate
6
6
6
100
50
100
11.
20.
6
0
±
±
0
2
3
.5
.8
11.
20.
1
7
+
+
0
1.2
4.9
-------
TABLE 2.3
The Absence of Acetylcholine Mediation on
Histamine Release by Sulfate Salts
N>
Ln
Compound
No. of
Experiments
Concentrations
(mM)
Histamine Release
in absence of
Acetylcholine
(10-4 M)
(yg/gm lung)
Histamine Release
in presence of
Acetylcholine
(10-4 M)
(ug/gm lung)
Ammonium
Ammonium
Chloride
Sulfate
Sodium Sulfate
6
6
6
100
50
100
10.
20.
2
2
± I.
± 4.
0
6
0
8.
17.
9
9
± 1
± 4
o
.3
.4
-------
TABLE 2.4
The Absence of Dibutyryl cAMP Mediation on
Histamine Release by Sulfate Salts
K5
Compound
No. of
Experiments
Concentrations
(mM)
Hi si-amine Release
in absence of
Dibutyryl cAMP
(10-o M)
(yg/gm lung)
Histamine Release
in presence of
Dibutyryl cAMP
(10-6 M)
(ug/gm lung)
Ammonium Chloride
Ammonium Sulfate
Sodium Sulfate
6
6
6
100
50
100
8.89 ± 0.96
21.31 ± 0.54
0
8.87 ± 0.43
18.46 ± 4.60
0
-------
50--
•o
o
S 40+
9
"5
oc
S 30+
I
CO
I 20+
**
s
o
o 10+
ji
2.5 5.0 7.'5
Ovalbumln (mg)
Fig•. 2.4 Dooe Response release of histamine by antigen (oval-
bumin) in sensitized lung fragments. Incubations in the
presence of antigen were for 3D minutes in Tyrode's solution
at 37°C.
27
-------
I
tO iO 40
TIM* (MM.)
•0
OC
Fig. 2.5 Time course of S-sulfate ion uptake by guinea pig
lung fragments in the presence of various concentrations of
ammonium sulfate and sodium sulfate. Solid triangles, 10 mM
Na2S04 and (NH^SO^. Open triangles, 50 mM (NH2)S04< Half solid
triangles, 50 mM Na2SO^, closed circles, 100 mM (NH^)2SO^ and
open circles, 100 mM Na2SO,.
28
-------
dependent on the temperature (Table 2.5). Two processes appear
to account for the uptake as shown by a plot of the natural
logarithm of the uptake versus time. An Arrhenius plot of these
data indicated an apparent energy of activation of 3.1 Kcal
for the initial kinetic process and 6.1 Kcal for the second.
Sulfate ion uptake was uneffected by NaCN but inhibited
by NaF and 2-deoxyglucose (Table 2.6). Even though high con-
centrations of HaF and 2-deoxyglucose in a glucose-free medium
were used complete inhibition could not be achieved.
Isoproterenol, epinephrine, and phenylephrine failed to
alter the sulfate ion uptake significantly (Table 2.7). Addi-
tion of dibutyryl cAMP also had no effect. Phloretin did not
alter sulfate ion uptake. ;
Sulfate Ion Uptake by Sensitized Lung Fragments: The uptake of
sulfate ions by SLF in the presence of antigen, and 50 and 100 mM
concentrations of either Na^SO^ or (NH.^SO, is shown in Fig. 2.6.
A maximum uptake of 14.6 ± 2.1 nanomoles/mg tissue was observed.
Differing from ULF, there was no significant decrease in sulfate
ion uptake with 50 mM (NH, ^SO versus Na^SO, but there was a
15.1% decrease in the presence of 100 mM (NH,)2SO,. The uptake
was again dependent on temperature (Table 2.8) either in pres-
ence or absence of antigen. There was a tendency for less up-
take to occur in the presence of the antigen. An Arrhenius
plot of the data indicated an apparent energy of activation of
3.3 Kcal/mole for the initial kinetic process and 3.2 Kcal/mole
for the second.
29
-------
TABLE 2.5
Sulfate Ion Uptake in Unsensitized
Guinea Pig Lung Fragments
f) ' ' ' '
Temperature ( C) Time (Min) Sulfate Ion Uptake (picomoles)
0°C 1
• ' - . 5
, '. '. •".. '-• '' / ' 15 '
• . . : ' . ; .. '. 30- • "• .
60
22°C 1
••'••'• .-'.'. " 5
15
30
60 :
37°C ' 1
•5
15
30
60
6.
16.
45.
57.
67.
11.
28.
51.
67.
86 .
13.
30.
67.
84.
122.
6
2
1
2
6
6
0
7
1
9
2
8
1
2
1
+ .
+
±
±
±
±
±
±
±
+
±
±
±
±
±
1.
1.
3.
6.
5.
1.
3.
1.
6.
4.
1.
3.
1.
8.
3.
1
7
3
6
0
1
3
6
0
4
7
3
0
2
3
(6)
(6)
(7)
(5)
(4)
(7)
(4)
(6)
(5)
(8)
(5)
(4)
(7)
(6)
(5)
aThe values are expressed as the mean -± s.e.; the number in
parentheses is the number of determinations.
30
-------
TABLE 2.6
Effect of Metabolic Inhibitors on the Uptake
3.
of Sulfate Ion by Guinea Pig Lung Slices
Additions Per Cent Uptake of 35-S-Sulfate
None 100
Sodium Fluoride (10"3 M) 85 ± 4
Sodium Cyanide (10~3 M) 111 ± 3
2-Deoxyglucose (10 M) 85 ± 4
aLung slices were preincubated for 10 min with the inhibitors
35
prior to the addition of 1 yCi S-Na^SO/. Incubations were
carried out in Tyrode's solution for 30 min at 37 C. Each
value is the mean ± s.e. of 8 experiments.
31
-------
TABLE 2.7
Failure of Pharmacological Agents to Modulate
the Uptake of Sulfate Ion by Guinea Pig Lung Slices3
35
Additions Per Cent Uptake of S-Sulfate
Phenylephrine .101 ±6
Epinephrine 95 ± 16
Isoproterenol 104 ± 10
Dibutyryl Cyclic AMP 96 ± 9
Phloretin 102 ± 17
St . ' ' '
Lung slices were preincubated for 10 rain with the pharmacolo-
logical agents (10 M) prior to the addition of 1 pCi
S-Na^SO,. Incubations were carried out in Tyrode's solution
for 30 min at 37°C. Each value is the mean ± e.e. .of 8
experiments.
32
-------
20 30 40
TIME (MIN.)
60
eo
35
Fig. 2.6. Time Course of S-Sulfate ion uptake by sensitized
guinea pig lung fragments in \the presence of various concentra-
tions of sodium sulfate andxammonium sulfate. Open triangles,
50 mM sodium sulfate; solid triangles, 50 mM ammonium sulfate;
open circles 100 mM sodium sulfate; and solid circles, 100 mM
ammonium sulfate.
33
-------
TABLE 2.8
Sulfate Ion Uptake in Sensitized
Guinea Pig Lung Fragments
Temperature Time
(OC) (min)
0° 1
. 5
15
3°
60
22° 1
: . V '5 '
15
30
60
37° 1
5
15
30
60
Sulfate
Absence of
15.
23.
42.
53.
60.
14.
39 .
48.
6.0.
99.
17.
20.
46.
64.
96.
7
2
2
1
7
5
9
4
1
7
4
6
8
0
4
•±
±
1.
5.
±10.
±-
±
±
±
±
•±
±
f
±
±
±
8.
8-
0.
1.
1.
5.
6.
2.
1.
4.
5.
±11.
Ion Uptake (Picomoles)c
Ovalbumin
5
2
8
2
5
9
2
6
1
4
1
2
4
2
2
(6)
(6)
(7)
(6)
(5)
(6)
(7)
(8)
(6)
(6)
(5)
(7)
(5)
(6)
(6)
Presence of
12
20
35
45
55
13
27
48
57
78
8
39
55
71
89
.4
.5
.1
.5
.6
.7
. 0
.2
.5
.0
.6
.3
.8
.5
.0
± 1.
± 5.
± 4.
± 2.
± 3.
± 0.
± 1.
± 2.
± 4.
± 4.
•± 1.
± 1.
± 1.
•± 4.
±11.
0\
1
9
3
6
1
5
9
2
5
6
1
9
7
8
0
i
ralbumin
(7)
(7)
(7)
(8)
(6)
(7)
(6)
(7)
(7)
(7)
(7)
(6)
(7)
(7)
(9)
aThe values are expressed as the mean ± s.e.; the number in
parentheses is the number of determinations.
34
-------
Sulfate ion uptake was uneffected by 2-deoxyglucose in a
glucose-free medium, but inhibited by NaF and NaCN. Even though
high concentrations were used, complete inhibition could not be
achieved (Table 2.9).
Isoproterenol, epinephrine, phenylephrine, acetylcholine
and dibutyryl cAMP modulated the anaphylactic release of hista-
mine as reported previously (44,50.57). but failed to alter
sulfate ion uptake significantly. Phloretin also failed to
alter the sulfate uptake but decreased histatnine release. The
results are shown in Table 2.9.
Dependence of Metachromasia of Acridine Orange on Ionic Strength:
Acridine Orange solutions exhibited metachromasia in the pres-
ence of heparin (Fig. 2.7), which was dependent on the ionic
strength of the solution. Addition of (NH^SO^, NH^Cl, Na2SO^,
and NaCl decreased the metachromasia of the Acridine Orange-
heparin solutions an equivalent amount at the same ionic strength
(Fig. 2.8). The metachromatic titration of Acridine Orange with
heparin in the presence of these salts is shown in Fig. 2.9.
Equivalent end points were found under all conditions. While
the affinity of heparin for Acridine Orange decreases with in-
creasing ionic strength, as shown by the decrease in metachro-
masia with increasing ionic strength, the number of binding
sites per disaccharide unit remains constant (Table 2.10).
35
-------
TABLE 2.9
Effects of Pharmacological Agents on the Anaphylactic Release of Histamine
and Sulfate Ion Uptake in Sensitized Guinea Pig Lung Fragments3
CO
Additions
, '
2-Deoxyglucose
Sodium Cyanide
Sodium Fluoride
Acetylcholine
Dibutyryl cAMP
Epinephrine
Isoproterenol
Phenylephrine
Phloretin
Con c en t r a t ion
IO"3 M
o
10" J M
IO"3 M
c
10° M
IO"5 M
10~5 M
IO"5 M
10~5 M
C
10" J M
No. of Percent Release
Determinations of Histamine
20
*- v*
17
17
16
17
18
19
18
16
15
100
• JL\J \J
100. b
60.1
97.2
110.2
95.3
79.0
64.5
92.4
84.6
±5.1
**
± 8.1
±7.8
**
± 6.4
±3.1
**
± 5.5
. „**
± 4.8
± 5.1
**
± 4.3
Percent Uptake of
35s-Sulfate Ions
100
101.8
89.6
88.5
93.6
96.6
97.4
99.7
97.6
98.8
± 4.5
**
±5.7
/ «**
±4.0
± 5.1
± 2.5
±4.6
±5.6
± 3.2
± 5.2
All preincubations with the agents were for 10 minutes followed by the addition of 5 mg of
Ovalbumin. Ten minutes later radioactivity was added and 30 minutes later the supernatant
was assayed for histamine and the fragments oxygen combusted to determine 35s-radioactivity.
The values are expressed as the mean ±s.e.
t* -. "
Values differ significantly from control at p < 0.05
-------
560
520
480 440
WAVELENGTH
400
360
Fig. 2.1. Metachromatic titration of a solution of Acridine
Orange (0.003n) with 10 microliter additions of heparin solution
(50 ng/microliter). Top curve is absorption spectrum of Acridine
Orange alone. Each subsequent curve represents the metachromatic
shift of the spectrum upon the addition of 10 microliters of the
heparin solution.
37
-------
6fO
020
490 440
WAVKLENOTH
400
BfO
Fig. 2.8, Absorption spectra of (A) Acridine Orange (0.003v),
(B) 3.0 yg sodium heparinate added to Acridine Orange (0.003y)
in presence of. 0.063y of the various salts tested, (C) 3.0 yg
sodium heparinate added to Acridine Orange at 0.003u,
38
-------
1.0-
tO 40 60 tO ^00 ItO 140 WO
MICftOLITIM HIMftlN ADDED
Fig. 2.9. Metachromatic Titration of Acridine Orange by Heparin
Varying Ionic Strengths and Salt Compositions. Heparin denotes
the'tit rat ion at 0.003 y; NajSO^ 0.060 y; (NH,)2SO, 0.060 y';
NH^Cl 0.020 y; NaCl 0.020 y. The point of inflection is noted
in yl of added heparin solution (50 ng/yl). See text for
experimental details.
39
-------
TABLE 2.10
Absence of an Effect of Ionic Strength on the Number of Acridine
Orange Binding Sites on the Heparin Macromolecule
«a
Additions to Acridine Orange Binding Sites
Acridine Orange Solution Ionic Strength (y) per Dissacharide Unit
Heparin 0 3,53 ± 0.10 (4)
Heparin + NaCl 0.020 3.69 ± 0.03 (3)
Heparin + NH4C1 0.020 3.37 ± 0.11 (4)
Heparin + (NH4>2S04 0.060 3.58 ± 0.05 (4)
Heparin + Na2S04 0.060 3.58 ± 0.05 (4)
aAcridine Orange binding sites were obtained by metachromatic titration and are
recorded as the mean ± s.e.; the number in parentheses is the number of
determinations.
-------
Absence of Cell Lysis in the Presence of Salts: Cell lysis
does not appear to contribute significantly to either salt
mediated histamine release or the decrease in sulfate ion up-
take at high concentrations of (NH,)2SO,. All of the DNA
originally present in the tissue slice could be found in the
fragments (4.7 ± 0.2 mg DNA/gm vs. 4.8 ± 0.2 mg/gm). LDH re-
lease into the incubation medium in the presence of 100 mM
Na2S04 or (NH^) SO^ did not significantly differ from the
control incubated in Tyrode's alone (Table 2.11).
SUMMARY AND CONCLUDING REMARKS
Because sulfate residues exist mainly as particles in the
atmosphere, the local alveolar concentration resulting from the
dissolution of a respirable particulate could be high. It ap-
pears that ammonium sulfate, in the concentration of 100 mM,
releases approximately 10078 of the stored histamine in the
guinea pig lung. An equal ammonium ion concentration (200 mM
ammonium chloride) released only half of the histamine stores.
Since lung fragments in the presence of 200 mM sodium chloride
exhibited no detectable release of histamine, the histamine
released by ammonium chloride is ascribed to the ammonium
ion. Similarly, the difference observed in histamine released
between 100 mM ammonium sulfate and 200 mM ammonium chloride
must be some function of the sulfate anion. The sulfate ion,
per se, has no inherent effect alone, since sodium sulfate
41
-------
TABLE 2.11
Release of LDH from Unsensitized Lung Fragments
No. of LDH Activity Released3
Treatment Determinations (units/mg tissue)
Homogenate 5 98.9 ± 9.40
Tyrode's solution 4 3.02 ± 0.36
alone
Tyrode's solution 8 3.23 ± 0.17
containing 100 mM
Na2S04
Tyrode's solution 8 3.28 ± 0.20
containing 100 mM
(NH4)2S04
aThe values are expressed as the mean ± s.e.
42
-------
at concentrations ranging from 10 to 200 mM caused no hista-
mine release. The presence of ammonium ion seemed to be a
necessary factor for histamine release. Ammonium nitrate
and acetate were of intermediary potency in histamine release.
These experiments demonstrate that the histamine released
by ammonium and sulfate ions in ULF cannot be modulated by
either cAMP or cGMP, since isoproterenol, acetylcholine and
dibutyryl cAMP failed to influence histamine release.
Studies in SLF demonstrated the known phenomenon of the
ability of the cAMP and cGMP systems to modulate antigen-
antibody release of histamine. Acetylcholine, epinephrine
and isoproterenol all had significant effects (p < .05) on
histamine release in accordance with previous reports (44,57,58)
Dibutyryl cAMP and phenylephrine failed to have significant
effects, although reported to be capable of modulation (57,58).
An unexpected observation was the ability of phloretin to
decrease the release of histamine.
Release of histamine through lysis of the mast cells is
not likely since neither DNA nor LDH, a cytoplasmic enzyme,
were released into the supernatant in the presence of ammonium
sulfate as compared to control. Total DNA in the fragments
also remained constant in the presence of concentrations of
ammonium sulfate known to release histamine. Thus, the ob-
served histamine release is likely to be a degranulation
phenomenon.
43
-------
The intraceilular sulfate ion space in ULF and SLF de-
creased in the presence of (NH.^SO, , the decrease in the
intraceilular sulfate ion space is probably associated with
the degranulation process. The intraceilular sulfate.. space .
in SLF was significantly altered in the presence of 100 tnM
(NH4)2S04.
In both experimental systems, drugs capable of modulating
the cAMP and cGMP systems failed to alter the sulfate ion up-
take. The sulfate ion transport system does not appear to be
highly dependent on the availability of metabolic sources of
energy. At high concentrations of potent metabolic inhibitors
only partial inhibition of sulfate ion uptake was observed.
In the case of ULF, both sodium fluoride and 2-deoxyglucose
showed inhibition and in SLF sodium fluoride and sodium cyanide
had effects. Phloretin has been reported to inhibit chloride
and sulfate uptake by human red blood cells (83), however,
phloretin had no effect on the sulfate ion uptake by the
lung fragments.
Data presented here, concurs with the observations of
others (72), that the metachromasia associated with Acridine
Orange binding to heparin is a function of ionic strength.
The number of Acridine Orange binding sites found in our
experiments (3.53±0.10 binding sites per disaccharide unit)
corresponds favorably with that reported by Lagunoff (46) of
3.31 ± 0.09 binding sites per disaccharide unit. The number
of binding sites remained constant with increasing ionic
44
-------
strength. Stone and Bradley (72) and Lagunoff (46) sug-
gest that the binding sites for Acridine Orange and hista-
mine on the heparin tnacromolecule are identical. Since we
observed a decrease in the extent of Acridine Orange binding
to the heparin macromolecule with increasing ionic strength,
a local increase in the ionic strength within the granule
is likely to cause displacement of histamine bound to hepa-
rin. Since the mast cell granule is freely permeable to
the external ionic environment, intracellular uptake of
ammonium or sulfate ions could result in the displacement
of bound histamine.
45
-------
SECTION 5
ABSORPTION OF SULFATE ION IN THE ISOLATED,
VENTILATED, AND PERFUSED RAT LUNG
INTRODUCTION
The isolated, ventilated and perfused lung (IVPL) would
seem to be most similar to the lung in vivo but it is technically
difficult to maintain free of edema with normal perfusion rates.
However, when compared with other in vitro preparations it
has several merits.
The tissue slice method utilized in the previous chapter
has certain intrinsic shortcomings. The lung is damaged during
the slicing procedure, substrates are delivered to the cut edge
of the tissue or alveolar epithelium rather than through the
capillaries, and oxygen must be supplied through a liquid medium
to the edge of the slice instead of through gas in the air
spaces and physiological medium in the capillaries.
The IVPL has been used extensively to study metabolism
and uptake by the lung vasculature of numerous compounds. The
techniques employed for the IVPL are modifications of those de-
veloped by Niemeier and Bingham (55) and Rosenbloom and Bass (62).
Gassenheimer and Rhodes (31) studying the influence of
ventilation frequency on glucose and palmitate uptake and
46
-------
§?^ <
:'^.•;• :".?.:•$>*3-•^:-:v".>• ••"'.• - '
metabolism in the rat IVPL, found that oxidation of glucose
reached a maximum at 70 cycles per minute and decreased there-
after to 215 cycles per minute. Palmitate incorporation into
the lung lipids increased non-linearly with frequency. Angio-
tensin I in the perfusate is converted to Angiotensin II in
the dog and rat IVPL (25,64). Radioactivity is not retained
by the lungs and ,has the same volume distribution and mean
transit time as blue dextran, a compound unlikely to leave the
intravascular space (64), suggesting that angiotensin is hydrp-
lyzed by enzymes located on the luminalsurface of pulmonary
-.-'•• • .• . .. .• .' .' . ,.-'.>.•.',••
endothelial cells. Prostaglandin F-. is largely eliminated
from the circulation during a single passage through the pul-
monary vascular bed and the volume of distribution and the mean
transit time are greater than blue dextran (63). Metabolism
therefore requires cellular uptake." •••••'-"••*• •
The effect of CO^ concentration oh phosphblipld metabolism
has been investigated by Longmore et'.al. (51) . Glucose'was fouhd
to be a precursor of the palmitate of phosphatidylchbline. The
CO^ concentration of the perfusioh was found to effect the in-
corporation of glucose in palmitate.
Both serotonin and norepinephrine are taken up by the lung
vasculature in the rabbit (10,40,41) and rat (43,54) IVPL.
Willis and Kratzing (84) utilized the rat IVPL to determine the
location of ascorbic acid in the lung. Ascorbic acid was riot
present in the effluent after perfusion of the pulmonary
47
-------
ni
l abiqil
^
s
suid nsrl^ isissig s^s smiif
Only a few studies of 9^e^^fn^^^f
ion
^
bladder (27) and frog
itt;IW
the
4^
-------
in the absence and presence of various ions is described. The
release of histamine and lactic dehydrogenase (LDH), a cyto-
plasmic enzyme, by the ions is also presented.
METHODS
Preparation of the Isolated, Ventilated, and Perfused Lung:
Female Sprague Dawley rats, weighing 250-300 g, were anesthe-
tized with pentobarbital (35 mg/kg). The isolated and perfused
rat lung was prepared by a modification of the technique of
Niemeier and Bingham (55). The trachea was isolated through
a midline incision and a cannula (PE 240) was inserted. The
aorta and inferior vena cava are severed to allow exsanguination
of the animal. The lungs and heart are exposed through a mid-
line sternotomy and the rib cage retracted. The pulmonary
artery was cannulated through a small incision made in the
right ventricle with a cannula (PE 200) filled with modified
Tyrode's solution. The entire right ventricle and right atrium,
together with most of the left ventricle were removed. The
modified Tyrode's solution (pH 7.35, 37°C) contained 35.0 g/1
polyvinylpyrrolidone (average molecular weight 40,000) to main-
tain a physiological oncotic pressure. Perfusion of the lungs
was started immediately to remove any remaining blood in the
pulmonary circulation. The perfusion rate was maintained at
a constant 2.0 ± 0.1 ml per minute.
The lungs were removed from the animal and suspended by
the tracheal cannula in an artificial thorax (25°C) where
49
-------
respiration was maintained mechanically by an alternating nega-
tive pressure (-3 to -15 cm of water). Positive pressure ven-
tilation may lead to edema, destruction of alveolarseptae by
over-inflation, and is reported to contribute to alveolar col-
lapse and progressive atelectasis (21,42,82). The respiratory
rate was kept at 90 inspirations per minute. A Harvard small
animal respirator (Harvard Apparatus, Millis, Mass.), was
used to provide an alternating +6 and -6 cm H?0 pressure, super-
imposed over a background -9 cm of HjO within the chamber,
generated by a vacuum pump. Pressure was monitored by a Magna-
helic gauge. Respiratory volume was monitored by a Grass volu-
metric pressure transducer connected to the tracheal cannula.
The system for ventilating and perfusing the lung is shown in
Fig. 3.1.
Determinationcof the Relative Distribution of an Intratracheal
Injection: A study was undertaken to determine the relative
distribution within the airways of a single intratracheal in-
jection of 100 yl of isotonic (0.3 M) sucrose. Following in-
35
jections of 4 yCi of S-NajSO,, perfusion was continued for
35
1 minute and the S-radioactivity within each lobe was de-.
tennined by flask oxygen combustion as described previously.
Results are expressed as dpm per mg of wet lung tissue.
All subsequent experiments were carried out with a single in-
jection of 100 yl of 0.3 M sucrose.
50
-------
.FLOW REGULATOR
AND METER
3-WAY VALVE
RESPIRATOR
FRACTION COLLECTOR
Fig. 3.1. Schematic Diagram of the Isolated, Ventilated and
Perfused Lung Preparation.
51
-------
Possible Release of LDH by Lung Perfusion: The possible re-
lease of LDH during perfusion of the pulmonary circulation with
modified Tyrode's solution was investigated. The LDH deter-
mination was based on the spectrophotometric method of Wroblewski
and LaDue (85). Lungs were perfused until free of blood. Per-
fusion was continued for 30 minutes while one minute aliquots
(2 ml) were collected. A 0.05 ml aliquot from each minute sample
was taken for the determination of LDH activity as described in
Chapter II. The limit of the assay was such that 29 lU/min
released from the lung could be detected. Total tissue LDH
activity was determined by homogenizing the lung tissue in 5 ml
ice cold 0.10 M potassium phosphate buffer (pH 7.5) and assaying
as before. All determinations were made with a LDH Diagnostic
Kit obtained from Sigma Chemical Company (St. Louis, Mo.).
Kinetic Characterization of Airway Sulfate Ion Uptake: Kinetic
characterization of airway sulfate uptake by the pulmonary cir-
culation was accomplished by intratracheal injection of total
sulfate salt doses of 0.01, 0.05, 0.10 and 1.00 pinole of sulfate
35
containing 0.65 yCi/mmole of S-Na2SO,. The pH of the solutions
was adjusted to 7.40. Aliquots were collected from the lung
35
effluent each minute (2 ml) and S-radioactivity determined
by liquid scintillation counting. Results are expressed as the
logarithm of the percent sulfate ions unabsorbed by the lung
versus time.
52
-------
Investigation of Possible Sulfate Ion Uptake from the Vasculature
into the Lung: To investigate the possibility of sulfate ion
35
uptake from the vasculature into the lung, S-sodium sulfate
dissolved in modified Tyrode's solution was perfused into the
lung at 1.6 pCi/min (256 ng/min) at a flow rate of 2 ml/min for
10 min using a syringe pump (Fig. 3.1). The perfusion solution
also contained blue dextran, 5 mg/ml. Blue dextran (ave. mol.
wt. 2,000,000) was used as a compound unlikely to leave the
vascular space during a single circulation and has been used
as a basis for calculating apparent mean transit times and
volume of distribution within the lungs (18). The perfusion
was continued for another 20 minutes with the modified Tyrode's
solution alone. The venous effluent was collected every tenth
of a minute and 3 ml distilled H^Owere added to each sample.
The blue dextran content was estimated from the absorbance at
600 nm. Aliquots of each sample were taken for the determina-
tion of S-radioactivity.
Modulation of Sulfate Ion Absorption from the Airways by the
Counter Cation: To investigate the absorption of sulfate ioris
in the presence of other cations, an unlabeled chloride salt
of the cation in question was dissolved in the isotonic sucrpse
35
solution together with the S-sodium sulfate (specific activity -
0.64 pCi/mmole) prior to injection. Injections of 0.1 ml iso-
35
tonic sucrose solution were used containing 0.1 pmole S-sodium
sulfate and either 0.1, 1.0 or 10.0'pinole of chloride salt.
53
-------
Absorption was allowed to proceed for 30 minutes. Aliquots
(2 ml) were collected from the lung effluent each minute and
35
the S-radioactivity determined. Chloride salts studied in
this manner were ammonium, cadmium, cobaltous, ferric, man-
ganous. mercuric, nickelous, and zinc.
Possible Release of LDH, Prostaglandins and Histamine by
Sulfate Salts: The possible release of LDH into the lung
effluent was determined, after the injection of 0.1 ml isotonic
sucrose solution containing 1.0 ymole sodium sulfate alone and
in combination with 1.0 ymole of the chloride salts tested
separately.
The possibility of release of prostaglandins by the salts
was also investigated. The lung effluent was mixed with a
stream of modified Tyrode's medium containing mepyramine maleate
(4.2 x 10~7 g/ml), atropine sulfate (9.6 x 10"7 g/ml), methyser-
gide (8.3 x 10~7 g/ml), propranolol (1.3 x 10 g/ml), and as-
pirin (6.4 x 10 g/ml). This mixture was then superfused onto
a rat stomach strip prepared according to the method of Vane
(81). With the inhibitor mixture, the rat stomach strip response
is specific for prostaglandins. The release of histamine by
the salts was also determined spectrophotoflurometrically by
the method of Shore (66).
Bronchoconstriction Mediated by Certain Sulfate Salts: Broncho-
constriction by sulfate salts was investigated by monitoring the
54
-------
respiratory volume prior to and after intratracheal administra-
tion of 0.1 ml of 1 p mole of various salts in isotonic sucrose
solution. The salts tested in this fashion were Na2SO, , NH,C1,
and (NH/^SO,. The effect on intratracheal injection of 14 yg
histamine in 0.1 ml isotonic sucrose solution on respiratory
volume was examined. With those salts decreasing the respira-
tory volume, another experiment was performed perfusing the lung
with Tyrode's medium containing 1.5 x 10 mepyramine maleate
(H-l antihistamine) to determine if the bronchoconstriction
could be prevented.
3 ' ' ; '•' '
Absorption of HpO by the Rat IVPL: Removal of water from the
airways by the pulmonary circulation was determined by intra-
•3
tracheal injection of 100 yl of 1^0 (1 yCi/mmble) containing
0.3 M sucrose. Samples were collected each minute, as before,
3
from the lung effluent and H-radioactivity determined by liquid
scintillation counting. The effect of 0.10 ymole ^280^ and
0.10 ymole (NH/^SO, on removal of water from the airways was
3
also examined by addition of these salts to the H20. Each
curve is the mean of three experiments .
Materials : Blue dextran 2000 was obtained from Pharmacia Fine
335
Chemicals. H^O and S- sodium sulfate were purchased from New
England Nuclear.
RESULTS
Distribution of an Intratracheal Injection in the Rat IVPL: In
order to determine the reproducibility of intratracheal
55
-------
instillation as a route of exposure of the lung to sulfate salts,
a single injection of 100 yl of isotonic sucrose containing 4 yCi
35
of S-Na^SO, was given and the perfusion and ventilation were
continued for one minute. The lung was then removed from the
35
apparatus, divided into five portions and the S-radioactivity
was determined in each lobe (Table 3.1). The right lower and
left upper lobes of the lung contained the highest radioactivity
while the right middle lobe contained the lowest. While the
distribution of radioactivity was not uniform, intratracheal
instillation did provide a reproducible method of exposure.
Absence of Release of Significant Quantities of LDH by Perfusion;
Figure 3.2 depicts the LDH activity released by perfusion of
the lung vasculature with modified Tyrode's solution. The amount
released never exceeded 40 lU/min. This release is insignificant
when compared to the total LDH activity of 63.8 ±9.9 lU/mg
tissue wet weight. An average rat lung would therefore contain
approximately 84,000 IU.
Kinetics of Sulfate Ion Removal from the Airways: Figure 3.3
shows a semilogarithmic plot of the removal of doses of 0.01
35
and 0.10 pinole S-sulfate ions from the rat IVPL following
intratracheal administration. From such curves the initial first
order rate constants and t, were calculated. A summary of these
data are presented in Table 3.2. The rate of removal of 0.05 pinole
^280^ or greater was constant. The average t, for sulfate ion
56
-------
TABLE 3.1
Distribution of Intratracheal Injection of 0.1 ml Isotonic
35
Sucrose Containing 4 pCi of S-Sddium Sulfate
Lobe of Lung Weight (gm) dpm/mg wet lung tissue
Right upper 0.111 ± 0.010 2456 ± 758
Right middle 0.151 ± 0.012 1344 ± 303
Right lower 0.368 ± 0.022 5319 ± 1407
Left upper 0.155 ± 0.012 6462 ±1024
Left lower 0.438 ± 0.025 3601 ± 1029
Total lung wg. - 1.210 ± 0.060
Expressed as mean ± s.e. of 10 experiments.
-------
10.0--
-------
100
O 80
60
ui
oc
* 40
ii i i i i ii
5 10
MINUTES
35
Fig. 3.3. Removal of S-sulfate ions from the isolated, ven-
tilated and perfused rat lung. Solid circles represent 0.01 vimole
sodium sulfate; open circles 0.1 nmole.
59
-------
TABLE 3.2
Effect of Ammonium Ion on the Kinetics of
Sulfate Ion Absorption from the Rat IVPL
. Dose per Lung
Compound (nmole)
Sodium Sulfate 0
0
0
1
Ammonium Sulfate 0
0
1
.01
.05
.10
.00
.01
.10
.00
No. of
Experiments
3.
4
11
5
4
3
4
Initial
K (minutes- l)b
0.
0.
0.
0.
0.
0.
0.
021
082
085
080
044
086
093
± 0.
± 0.
± 0.
± 0.
± o.
± 0.
± 0.
003
025
016
018
007
047
031
(minutes )b
33.
8.
8.
8.
15.
8.
7.
0
4
1
7
7
1
5
± 4.
±2,
± 1.
± 2.
± 2.
± 3.
± 2.
7
6
3
0
5
5
5
**
c
d
**
c
d
All solutions were administered intratracheally in 100 yliters isotonic sucrose, pH 7.4.
Values are expressed Mean ± s.e.
c an Paired "t" test between the indicated means show no significant difference at 0.05
level of confidence.
Statistical significant difference between sodium sulfate and ammonium sulfate, p < 0.01.
-------
removal by the rat IVPL for doses of 0.05 pinole and larger is
8.4 ± 1.8 minutes (20 determinations).
35
Absence of Pulmonary Vascular Uptake of S-Sulfate Ions:
Figure 3.4 shows the appearance and then disappearance of blue
dextran in the venous effluent, following a pulse of blue dex-
tran added to the perfusate. Similarly, the appearance and then
35
disappearance of S-sulfate ions follows the same pattern.
Thus sulfate ions have the same volume of distribution and mean
transit time within the lung as blue dextran, a compound unlikely
to leave the intravascular space.
Modulation of Airway Sulfate Ion Absorption by Certain Counter
Cations: Ammonium ions accelerated the removal of sulfate ions
at doses of 0.01 ymole but not at greater doses (Table 3.2).
The t, of 0.01 ymole sodium sulfate was 33.0 ± 4.7 min compared
%
to 15.7 ±2.5 min for 0.01 ymole ammonium sulfate. All heavy
metal cations, except manganous ion, enhanced the absorption
(Table 3.3).
Lack of Release of LDH and Prostaglandins but Release of Histamine
by Certain Sulfate Salts: Histamine was released into lung per-
fusate following an intratracheal injection of 1 ymole (NIL^SO/
(Fig. 3.5). A total of 11.4 yg of histamine was collected in the
perfusate from a single injection. The release of prostaglandins
from the lung into the perfusate following the intratracheal
injection of 1 ymole of either Na2SO^, (NH,)2SO, or NH^Cl was not
observed. The bioassay system has been used in other experiments
61
-------
0.6-f
£
§0.4
(O
UJ
O
.-z . '
<
CO
tr
O
I
-3888888
-•20
-•15
- -10
-•5
O
x
2 4 6 8 10 12 14 16
MINUTES
Fig. 3.4. Absence of pulmonary vascular uptake of S-sulfate
ions. Lungs were perfused continuously with modified Tyrode's
medium. A syringe pump provided a pulse of 35s_Sodium sulfate
and blue dextran in modified Tyrode's solution into the main
perfusion line (2 ml/min). The arrows indicate the times at
which the syringe pump was turned on (first arrow) and off
(second arrow). Blue dextran (o) and 35g-sulfate ions ( • )
were measured in the pulmonary venous effluent.
62
-------
TABLE 3.3
Effect of Heavy Metal Counter Cation on the Kinetics
of Sulfate Ion Absorption from the Rat IVPL
OJ
Compound3
Na2S04
MnCl2
CoCl2
NiCl2
ZnClo
Dose per Lung
(umole)
0.1
0.1
0.1
0.1
0 . 1
No. of
Experiments
11
5
4
4
4
Initial k
(Minutes ~l)b
0.085 ± 0.016
0.098 ± 0.013
0.169 ± 0.034
0.171 ± 0.028
0.172 ± 0.018
(Minutes )b
8.12 ± 1.32
7.10 ± 1.29C
4.11 ± 1.00**
4.05 ± 0.70**
**
4.03 ± 0.37
-------
TABLE 3.3 (Continued)
Compound3
CdCl2
FeCl3
HgCl2
Dose per Lung
(pmole)
0.1
0.1
0.1
No. of
Experiments
4
4
4
Initial k
(Minutes- l)t>
0.195 ± 0.047
0.194 ± 0.033
0.224 ± 0.047
fc%
(Minutes)0
**
3.56 ± 0.82
**
3.56 ± 0.57
•fck
3.10 ± 0.62
a
All chloride salts were administered intratracheally in 100 pliters of isotonic
35
sucrose, pH 7.4, in the presence of 0.1 pinole S-NaASO, (specific activity - 0.65
35
vjCi/mmole). Control was 0.1 ymole S-NaxSO, alone.
Values are expressed as mean ± s.e.
°Paired "t" test between indicated means and mean of control shows no significant
difference.
vtvt
Paired "t" test between indicated means and mean of control show significant
difference p < 0.05.
-------
•«.
Ill
III
' -I
III
III
z
i
H
co
10 14
Fig. 3.5. Release of histamine into the lung effluent after
intratracheal injection of 1 umble ammonium sulfate. Injection
is indicated by arrow.
65
-------
to detect the release of about 0.5 ng/ml prostaglandin E2 and
5 ng/ml prostaglandin F^ from the rat lung. No detectable
amounts of LDH were released by intratracheal administration by
any of the salts tested. Total LDK level in the rat lung was
found to be 63.8 ± 9.9 IU/mg tissue wet weight. An average lung
would therefore contain 84,000 IU. The limit of the assay was
such that 29 lU/min released from the lung could be detected.
Bronchoconstriction Mediated by Certain Sulfate Salts: A dose
of either 1 ymole (NH/^SO, or 2 ymoles of NH.C1 caused a re-
duction in the respiratory volume of lung. Typical tracings of
the respiratory volume following injection of these salts are
shown in Fig. 3.6. Instillation of the control solution, iso-
tonic sucrose, o.r 1 ymole Na2SO/ produced a minimal reduction
in respiratory volume. The reduction in respiratory volume
could also be accomplished by the injection of 14 yg of hista-
mine. This dose of histamine was equivalent to that released
by 1 ymole (NH/^SO, in prior experiments. Ammonium chloride
was less effective, producing a 2670 decrease compared to a 5670
for both (NH.KSO, and histamine. Prior perfusion of the lung
with 1.5 x 10" M mepyramine maleate added to the Tyrode's me-
dium blocked the reduction of the respiratory volume by both
histamine and (NH,)2SO,.
3
Absorption of HpO by the Rat IVPL: Since histamine was re-
leased by ammonium salts, the enhanced sulfate absorption from
66
-------
ui
8
K
O
£
CO
g
O4 -T CONTMOl INJECTION
ao- - —•
0.1-*.
ao- •
-aa-L
• o.t- •
ao-|-
-04
• at
ao
B
Fig. 3.6. Effects of intratracheal injection of 1 pinole of salts
in 0.1 ml isotonic sucrose solution on respiratory volume in the
isolated rat lung. A. Isotonic sucrose alone or with sodium
sulfate; B. Ammonium sulfate or 14 yg histamine; C. 2 ymole
ammonium chloride; and D. Perfusion of lung with 10-5 M mepyr-
amine maleate prior to injection of ammonium sulfate, ammonium
chloride or 14 yg histamine.
67
-------
Che airways in the presence of ammonium ions could be due to
3
increased vascular permeability. The removal of HjO from the
airway was determined (Fig. 3.7). Water was removed from the
airway at the same rate regardless of the presence of 0.1 pinole
Na2SO, or (NH.^SO,. The calculated t^ in sucrose alone was
0.94 ±0.18 min compared to 1.1.3 ± 0.42 min and 0.92 ± 0.15 min
for solutions containing Na2SO, and (NH,)pSO, respectively.
SUMMARY AND CONCLUDING REMARKS
The release of LDH by the IVPL was monitored as an indi-
cation of the viability of the preparation. Cellular integrity
of the lung was maintained as evidenced by the lack of signifi-
cant LDH release over the time course of an experiment. Gross
edema failed to appear over the same time period. It was ob-
served that perfusion with Tyrode's solution (25°C, pH 7.4) in
the absence of the PVP led to the rapid development of edema
within 5 minutes from the start of perfusion. This is under-
standable when one considers the lack of an oncotie pressure
in the perfusion solution, in the latter case.
In the present studies, the removal of sulfate ions from
the airway appears to be predominantly by simple diffusion.,
At very low doses ammonium ion increases the removal process.
The t, at doses of 0.05 pinole or greater of Na2SO, was
8.4 ± 1.8 minutes.
The heavy metal cations tested, increased sulfate ion
absorption from the rat IVPL 199-264% as compared to absorption
68
-------
100
MINUTES
Fig. 3.7. Absorption of H«0 in the isolated, ventilated and
3
perfused rat lung. Open circles, H«0 alone; solid circles,
3 3
H20 and 0.10 ymole sodium sulfate; and open triangles H«0
and 0.10 ymole ammonium sulfate.
69
-------
in the presence of sodium ions. An exception to the above was
the case of manganese ion in which sulfate ion absorption did
not differ significantly from control.
Amdur (5) and Amdur and Underhill (9) have previously re-
ported an irritant potential associated with certain sulfate
salts. Sodium and manganese sulfate salts, however, failed to
alter flow resistance at similar concentrations. Thus not all
sulfate compounds are irritants. This suggests a possible cor-
relation between irritant potential and the rate of sulfate ion
absorption into the lung vasculature as modified by the presence
of the counter cation. Because of the cellular complexity of
the mammalian lung, it is difficult to be certain of the mechanism
of sulfate ion absorption and how the counter cation enhances
absorption.
Sulfate ions introduced into the vaseulature have the
same volume of distribution and mean transit time within the
lung as blue dextran, a compound unlikely to leave the intra-
cellular space. Therefore, it can be stated with some certainty
that sulfate ion absorption in the rat IVPL is unidirectional.
As in the studies with guinea pig lung fragments, ammonium
ions play an important role in the release of histamine in per-
fused lung. The release of 11.4 yg of histamine by a single
dose of 1 ymole of (NH/J^SO, represented an almost complete
degranulation (see Shore et.al. (66) for histamine content of
tissues). All other cations studied, failed to release detectable
70
-------
amounts of histamine. No measurable amounts of LDH were re-
leased by any salt tested. Although prostaglandins were spe-
cifically sought for, none were released. Using the same
preparation for other studies, prostaglandins are detected in
the perfusate.
Intratracheal instillation of ammonium sulfate solutions
decreased the respiratory volume of the preparation by 56 per-
cent while ammonium chloride caused only a 26 percent decrease.
Histamine (14 yg) applied intratracheally also decreased the
respiratory volume by the same percentage. The release of
histamine was shown to be rapid and followed the same time
course as the decrease in respiratory volume. Histamine appeared
to be the only vasoactive hormone elaborated. Since prior per-
fusion with an H-l antihistamine prevented a decrease in the
respiratory volume by ammonium sulfate, histamine is most likely
the only mediator.
Little or no changes in the vascular permeability could be
observed with doses of (NIL^SO, causing histamine liberation.
The t, for the removal of 3H2
-------
SECTION 6
SULFATE ION ABSORPTION IN THE RAT LUNG IN VIVO
INTRODUCTION
The respiratory tract epithelium behaves as a highly
porous membrane permeable to a number of solutes. Removal
of organic compounds from the airway has been studied by
Schanker and his colleagues. Schanker and coworkers admin-
istered intratracheally a small volume (0.1 ml) of Krebs-
Ringer phosphate solution containing C-labelled compounds
to anesthetized rats. Removal was determined by assay of the
C-radioactivity remaining in the liing. A number of lipid
insoluble neutral compounds, including urea, erythritol, man-
nitol and sucrose, administered over a 100-1,000 fold range of
concentration, disappeared from the lungs at rates directly pro-
portional to the concentration (22). The relative rates of ab-
sorption ranked in the same order as the diffusion coefficients
of the compounds. Simple diffusion appears to account for re-
moval. Enna and Schanker suggested that, due to the extremely
low lipid solubility of these compounds, absorption was pre-
dominantly by passage through aqueous channels or pores in the
membrane rather than through lipid regions. Diffusion through
at least three different populations of pore size could explain
72
-------
the absorption of these compounds. The smallest diameter pore
prevents the diffusion of all the saccharides relative to that
of urea. A larger diameter pore allows the diffusion of only
erythritol, while the largest pore size permits the passage of
all compounds except dextran (MW - 70,000).
In another study involving organic anions and cations,
sulfanilic acid, tetraethyl ammonium ion, p-aminohippuric acid,
and p_-acetylaminohippuric acid, and procaineamide ethobromide
appeared to be absorbed by diffusion through aqueous pores
since their absorption was non-saturable and roughly related
to molecular size rather than to partition coefficient (23).
Phenol red, a lipid insoluble organic anion is an exception
to the above cases. This compound is absorbed from the rat lung
not only by diffusion but also in part by a carrier-type trans-
port process (t, = 20 minutes). This process, which becomes
*z • .
saturated at high concentrations of the dye, is inhibited by
certain organic anions including benzylpenicillin and cephalo-
thin (24). The main barrier to the diffusion of water soluble
compounds is the alveolar membrane. In studies with the iso-
lated perfused dog lung, Taylor and Gaar (73) calculated an
o
equivalent pore radius of 8-10 A for the alveolar membrane and
a much larger radius for the capillary endothelium.
Lipid soluble compounds are thought to be absorbed mainly
by diffusing through lipoid regions of the membrane. Burton
and Schanker (13) administered five antibiotics intratracheally
73
-------
to anesthetized rats. The t, ranged from 1.9 to 33 minutes.
^
Chloramphenicol was absorbed most rapidly followed by doxycy-
cline, erythromycin and tetracycline, with benzyl penicillin
showing the slowest rate. A comparison of pulmonary absorption
rate, molecular weight and chloroform/water partition coeffi-
cient of the drugs indicated that lipid solubility was more
closely associated with the relative rate of absorption than
molecular size. Corticosteroids are rapidly absorbed from the
respiratory tract with t, of absorption ranging from 1.0-1.7
• • *
minutes (14).
In other studies on lipid soluble compounds Normand et.al.
(56) investigated the permeability of alveoli and capillaries
in the fluid filled lungs of the fetal lamb. They reported
that urea, thiourea and N-ethylthiourea penetrated the alveolar
wall at rates which increased with the lipid solubility of the
compounds. Earlier, Taylor et.al. (74) in a study of the al-
veolar membrane of the isolated perfused dog lung, reported
that the permeability coefficient of a lipid soluble compound,
dinitrophenol, was much greater than that of lipid insoluble
compounds, such as glucose and urea.
Only a few studies of the movement of simple anions across
the lung have appeared. Gatzy (32) proposed that chloride ion
transport accounts for the potential difference found across
the alveolar membranes of the amphibian lung. Chloride ion
transport also is found in other epithelia such as the toad
74
-------
bladder (27) and frog epithelium (39). Using the permeability
coefficient of sodium ion, the t, for sulfate ion transport in
*
the turtle lung can be calculated as 27.8 minutes (19). Sul-
fate ion transport in mammalian lungs has not been reported
previously.
It must be pointed out that in all studies cited, the
actual site of solute absorption is unknown. Absorption could
be occurring across the alveolar epithelium, across the bronche-
olar epithelium of the airways or at a combination of these
sites. Burton and Schanker (12) have reported that lung ab-
sorption rates are increased by at least two-fold when solutes
are administered as aerosols.
The studies presented in this chapter were designed to
determine the manner by which the mammalian lung disposes of
inspired sulfate ions. Possible modulation of the absorption
process by the associated cation was also investigated.
METHODS
In Vivo Rat Lung Preparation; The technique is a modification
of the method of Enna and Schanker (23). Female Sprague Dawley
Rats weighing 150-200 g were anesthetized with sodium pentobar-
bital (35 mg/kg). The animal was placed on its back on an
animal board and the limbs were secured with masking tape.
After exposing the trachea through a longitudinal incision in
the neck, the trachea was cut transversely halfway through be-
tween two tracheal rings. Two centimeters of a 4 cm long
75
-------
polyethylene cannula (PE 240) was inserted into the trachea
to a point approximately 0.5 cm above the hylum and sutured.
The incision was then covered with sterile gauze dipped in
Tyrode's medium (pH 7.4). The mounting board was placed-in
an upright position. One-tenth milliliter of an isotonic
sucrose solution (300 mM, 25°C, pH 7.4) containing S-sodium
sulfate alone and with variable chloride salts, was injected
into the lungs via the tracheal cannula through a 1.5 inch
22 gauge needle attached to a 100 yl syringe (Hamilton). The
solution was injected over a 1 to 2 second interval. The needle
and syringe were withdrawn completely, the animal removed from
the mounting board, laid on its stomach, and maintained under
light anesthesia.during the specified experimental time period.
35
Absorption of S-Sulfate Ions from the Rat Lung: One-tenth
milliliter of the isotonic sucrose solution containing either
1.0, 10.0 or 100.0 nanomole 35S-sodium sulfate (specific activity
0.014 yCi/nanomole) was injected into the lungs via the tracheal
cannula. Absorption of sulfate ions was allowed to occur for
various times between 0 and 120 minutes. One minute before
the end of the absorption period, removal of the lungs was
begun. The pleural cavity was carefully opened and the trachea
gently separated from surrounding tissue. At the end of the ab-
sorption period, the blood supply to the lungs was quickly
stopped by cutting around both sides of the lung. The lungs
with the heart, a portion of the trachea and the trachea cannula
76
-------
attached were removed from the body. The heart, thymus gland,
and the esophagus were trimmed away and the cannula removed.
The lungs and trachea were immediately prepared for the de-
35
termination of S-radioactivity remaining in the lungs by
oxygen combustion described previously. These preliminary ex-
periments were used to characterize the mechanism of sulfate
ion removal and to establish the approximate half-life.
Modulation of Sulfate Ion Absorption by the Counter Gation: To
investigate the absorption of sulfate ions in the presence of
other cations, an unlabelled chloride salt of the cation in
question was dissolved in the isotonic sucrose solution together
35
with S-sodium sulfate (specific activity - 0.014 yCi/nanomole) .
Injections of 0.1 ml isotonic sucrose solution were used con-
35
taining 0.1 nanomole S-sodium sulfate and either 0.1, 1.0 or
10.0 nanomole of the chloride salt. Absorption was allowed to
proceed for 30 minutes, approximately equal to the t, of sulfate
ion absorption from the rat lung. As before, one minute prior
to the end of the absorption period, removal of the lungs was
35
begun and the S-radioactivity remaining in the lung de-
termined. Chloride salts studied in this manner were ammonium,
cadmium, cobaltous, ferric, manganous , mercuric, nickelous,
and zinc.
Modulation of Sulfate Ion Absorption by pH: To investigate the
absorption of sulfate ions under varying pH conditions, the
77
-------
35
isotonic sucrose solution containing S-Na^SO, was adjusted
to a known pH (pH 4.4-9.4) with either HC1 or NaOH prior to
injection. Injections of 0.1 ml of these solutions were used
35
containing 0.1 namomole S-sodium sulfate (specific activity -
0.014 iiCi/nanomole) . Absorption was allowed to proceed for
30 minutes. As before, one minute prior to the end of the ab-
35
sorption period removal of the lungs was begun and S-radio-
activity remaining in the lung determined.
Exposure of Rats to Nickel Chloride Aerosol Prior to the Deter-
mination of Sulfate Ion Absorption: The inhalation exposures
were conducted in a plexiglass exposure chamber expecially
designed for exposure to particulate aerosols. The animals
were isolated in individual cells which allowed only their heads
to be exposed to the aerosol. All exposures were for two hours.
A fluid atomizer generator (Environmental Research Corporation,
Model 7330) was used to generate the nickel chloride aerosol
from solutions ranging in concentration from 2.61 to 13.05 gm/1
of deionized water. Samples were collected on membrane filters
having 0.22 ytn porosity for determining the concentration within
the chamber. The deposited salt was eluted from the filter by
placing it in a flask containing 19.8 ml of deionized water
and 0.2 ml of HNO-. After shaking for several hours the result-
ing solution was analyzed using a Perkin-Elmer Model 306 atomic
absorption spectrophotometer with a Model 2100 heated graphite
78
-------
accessory. For monitoring the aerosol exposure by particle
size distribution, a Royco Instrument Model 225 Particle Counter
with a Model 507 module was used. The mass median diameter
of the NiCl2 aerosol was 1 ym or below in these experiments.
The mass median aerodynamics diameter was determined to be less
than or equal to 2pm. Control animals were exposed for 2 hours
to a deionized water aerosol.
After the exposure period, the animals were removed from
their cells and 4 animals were used in the determination of
I I
the amount of Ni deposited after a 2 hour exposure. Their
I |
lungs were excised, ashed, and analyzed as before for Ni
content.
The exposed animals were used to determine sulfate ion
absorption. A tracheal cannula was inserted as before. In-
jections of 0.1 ml isotonic sucrose containing 0.1 nanomole
35
S-sodium sulfate (specific activity - 0.014 yCi/nanomole)
pH 7.40. Absorption was allowed to proceed for 30 minutes.
35
The lungs were removed at the end of this time and S-radio-
activity determined.
35
Materials: S-sodium sulfate was purchased from New England
Nuclear.
RESULTS
Kinetics of Sulfate Ion Removal from the Airways: Fig. 4.1
35
shows a semilogarithmic plot of the removal of sodium S-sulfate
79
-------
100
80
60
40-
20
IS
60
MINUTES
120
Fig. 4.1 Percent sulfate ion unabsorbed by the rat lung versus
time in the?presence of various concentrations of sodium sulfate
Each poirit_is the mean s.e. of three animals. Closed circles
1.0 nanomole; open circles, 10.0 nanomole; open triangles 100 '
nanomole. ° '
80
-------
from the lung following intratracheal administration. Three
concentrations, 1.0, 10.0 and 100.0 namomole Na2SO, were used.
Although the concentration of Na^SO, varied over a 100-fold
range, the percentage absorbed at a given time was constant.
The total amount of Na2SO, absorbed was proportional to the
initial concentration administered (Table 4.1). These results
suggested that absorption occurred by a nonsaturable process,
such as simple diffusion. The half-life for sulfate absorption
in the presence of sodium ions, calculated from the slopes of
these lines was found to be 34.5 minutes.
Modulation of Airway Sulfate Ion Absorption by Certain Counter
Cations: The removal of sulfate ions was accelerated in the
presence of certain cations (Table 4.2). The dose of cation
at which maximum augmentation of sulfate absorption occurred
I I I I
was variable. Co and Hg ions produced maximal effect at
I I | [
0.1 nanomole. Cd and Ni were of intermediary potency.
I I I I I _L
Fe , Zn and NH, were the least effective reaching maximal
effect at 10.0 nanomoles. As was observed in the case of the
I [
isolated perfused rat lung, Mn failed to alter sulfate ion
absorption. Sodium chloride depressed slightly the sulfate
ion absorption when present at 10 nanomole.
Modulation of Airway Sulfate Ion Absorption by pH: The ab-
sorption of sulfate ions from the rat lung in_ vivo was enhanced
at pH values departing from physiological values (Fig. 4.2).
81
-------
TABLE 4.1
35
Absorption of S-Sodium Sulfate from the Rat Lung
Concentration of
Na^SO, (nanomole)
1.
10.
100,
0
0
o
No. of
Animals
11
3
3
30
Min,
% Dose
43
42
47
.2 ±
.0 ±
.4 ±
, Absorption
±
2
5
6
s. e.
.5a
.Oa
.8a
0.
0.
4.
Sulfate
Amount (
041 ±
403 ±
550 ±
0.
0.
0.
Ion
ug)
002
048
653
aNo statistical difference at p < 0.01.
82
-------
TABLE 4.2
35,
Effect of Counter Cation on Pulmonary Absorption of S-Sulfate Ions
Concentration
Salta (nanomole)
Na~SO, alone 1.
MnCl2 1.
NH^Cl 10.
oo ZnCl0 0.
LO i.
1.
10.
FeCl3 0.
1.
10.
CdCl2 0.
1.
10.
0
0
0
1
0
0
1
0
0
1
0
o
No. of
Animals
11
8
7
5
5
5
6
6
5
6
8
7
30 Minute Absorption Sulf ate Ion
"» Dose ± s.e.
43.
44.
54.
49.
55.
64.
50.
53.
72.
52.
57.
56.
2 ±
8 ±
1 ±
3 ±
2 ±
2 ±
3 ±
8 ±
4 ±
4 ±
2 ±
6 ±
2.4
1.7
2.3 .
5.2
3.1
2.8
4.9
1.7
2.9
3.0
4.3
2.6
Amount
41.
43.
51.
47.
53.
61.
48.
51.
69.
50.
54.
54.
5
0
9
0
0
6
0
7
5
3
9
3
+
+
+
+
+
+
±
±
~±
+
±
-t-
(ng)
2.4
1.6
2.2
5.0
3.0
2.7
5.0
1.6
2.7
3.1
4.1
2.4
% Enhancement
over Control
0.0
3.7 ±
25.2 ±
14.2 ±
27.8 ±
48.6 ±
16.4 ±
24.5 ±
46.2 ±
21.3 ±
32.4 ±
31.0 ±
3.9b
5.3
12.0
7.2
6.5
11.3
3.9
6-7
6v9
9.9
6.0
-------
TABLE 4.2 (Continued)
00
Salta
NiCl2
HgCl3
CoCl2
NaCl
Concent rat ion
(nanomole)
0.
1.
10.
0.
1.
10.
0.
1.
10.
10.
1
0
0
1
0
0
1
0
0
0
No. of
Animals
4
10
5
5
6
5
8
10
8
11
30 Minute
7o Dose ± s
54.8
56.8
56.5
55.0
57.2
55.3
62.2
56.8
56.9
36.6
± 2.
± I-
± 1.
± 2.
± 4.
± 3.
± 2.
± 2.
± 2.
± 3.
Absorption Sulfate Ion
.e.
2
7
3
1
6
5
6
8
8
0
Amount (ng)
53 . 2
54.5
54.2
53.3
54.9
53.1
60.0
54.5
54.6
35.1
± 2.1
±1.6
± 1.2
± 2.0
± 4.6
± 3.4
±2.2
± 2.5
± 2.6
± 2.8
70 Enhancement
over Control
26.7
31.5
31.0
27.3
32.4
28.0
44.0
31.5
31.7
-15.3
± 5.
± 3.
± 3.
± 4.
±11.
± 7.
± 6.
± 6.
± 6.
± 6.
0
9
0
9
1
8
0
o
0
9
o c
All chloride salts were studied in the presence of 1.0 nanomole S-Na^SO, (specific
activity - 0.014 yCi/mmole).
No statistical difference over control at D < .001.
-------
60--
Q
at
oo
O 50
CO
D
. o
40--
4.4
5.4
64 7.4
PH
QA
Fig. 4.2. Percent sulfate ion unabsorbed by the rat lung after
30 minutes under varying pH conditions in the presence of 1.0
nanomole 35s-sodium sulfate (specific activity — 0.014 yCi/mmole)
Each point represents the mean ± s.e. The number of animals used
at each pH is given.
85
-------
In basic solutions the maximum enhancement over control
values (pH 7.4) of 26.9 ± 4.470 were observed at pH 9.4.
In acid solutions, a maximum enhancement of 32.4 ± 5.670
was observed at pH 4.4. Sulfate ion absorption was rela-
tively constant between pH 6.4 and 7.4.
Effect of Exposure to Nickel Chloride Aerosol on Sulfate Ion
3
Absorption : Exposure of rats to an aerosol of 480 yg/m
for 2 hours prior to the determination of sulfate ion absorp-
tion led to a 12. 0 ± 2.7% enhancement of absorption (Table
4.3). During this time period 0.856 ± 0.060 yg Ni** was de-
posited in the lungs of the test rats. Exposure to aerosol
3
concentrations of 113 and 279 yg/m had no significant
effect on the absorption.
SUMMARY AND CONCLUDING REMARKS
These experiments demonstrate that sulfate ion removal
by the rat lung is a nonsaturable process and appears to occur
by simple diffusion, with a t, of 34.5 minutes. This tv com-
pares favorably with the value of 27.8 minutes for sulfate
ion removal in the turtle lung reported by Deitchman and
Paganelli (19). We have also observed that ammonium ions and
certain heavy metals enhance the absorption whereas sodium
chloride depresses slightly the absorption of sulfate.
I [
As was shown in the isolated perfused rat lung Mn
failed to effect sulfate ion absorption. The ability of
86
-------
TABLE 4.3
Effect of 2 Hour Exposure to Nickel Chloride Aerosol Prior to
35
Determination of Pulmonary Absorption of S-Sulfate Ions
00
Concentration
of NiCl2
Aerosol
(pg Ni/m3)
Control
113
279
480
No. of
Animals
5
6
5
6
I i
Amount of Ni' '
Deposited
(yg/lung)
0
0.141 ± 0.046
0.189 ± 0.039
0.856 ± 0.060
30 Min.
% Dose
57.9 ±
60.0 ±
60.8 ±
64.9 ±
Absorption
± s.e.
2.0
1.7
2.4
1.6**
Sulfate Ionsa
Amount (yg)
0.056 ± 0.002
0.058 ± 0.002
0.058 ±0.002
0.062 ± 0.022**
% Enhancement
over Control
0
4.6 ± 2.9
5.0 ± 4.2
12.0 ±2.7**
a 35
In the presence of 1 nanomole S-sodium sulfate (specific activity - 0.014 uCi/nariomole).
Significant difference from control animals p <.05.
-------
heavy metals, given as an aerosol to test animals, was also
demonstrated to enhance the absorption of sulfate ions.
Sulfate ion absorption was enhanced under basic and acidic
conditions, but remained relatively constant between pH 6.4
to 7.4.
Assuming the sulfate aerosol levels reported in the
CHESS study (26) a total burden of 0.12 yg per hour would
be inhaled by the rat exposed to ambient levels of particulate
sulfates of 20 yg per cubic meter. This calculation assumes
that all of the suspended sulfate would be deposited in lung
and that the rat would have a tidal volume of 1 cc and a
respiratory rate of 100 breaths per minute. The rate of ab-
sorption was the same over a 100 fold range of 0.096 yg of
sulfate to 9.6 yg of sulfate ion given in this study. From
the data presented, 31 percent of 0.04 yg of the inhaled
sulfate from the one hour burden would remain after one hour
post exposure. If one were to assume the same rate of removal
from the human lung as for the rat lung, then 7.20 yg of,sul-
fate would be inhaled and 2.23 yg would remain after 1 hour
of exposure.
It must be pointed out that in all studies cited, in-
cluding the present one, the actual site of solute removal
by the lung is unknown. Burton and Schanker (12) have re-
ported that lung absorption rates are increased by at least
88
-------
two fold when solutes are administered as aerosols. While
sulfate ions are readily transported from the lumen of the
lung, residual sulfate concentrations are likely to remain.
Histamine release and bronchoconstriction may as a conse-
quence occur.
89
-------
SECTION 7
DISCUSSION
Until recently the prime source of sulfate air pollution
was the atmospheric oxidation of sulfur dioxide (802)• Measure-
ments of ambient sulfate levels in the Hudson River Valley dur-
ing 1970-1971 indicate that ammonium sulfate is a principal
component of sulfate residues in the atmosphere (Dr. R. Bradow,
personal communication). The automobile catalytic converter
has introduced a new source of sulfate in the form of sulfuric
acid mist. Ammonia and other cations present in the environ-
ment probably convert this mist to a mixture of sulfate salts
and sulfuric acid.
Amdur (5) and Amdur and Corn (6) measured the increase in
pulmonary resistance following the inhalation of zinc ammonium
sulfate, zinc sulfate and ammonium sulfate aerosols. Although
ammonium sulfate was the least potent salt, it was many times
more irritating than its parent compound, S02- In further
studies, Amdur .and Underbill (9) demonstrated that an equivalent
amount of sulfur present as S02 gas produced a lesser irritant
response than sulfuric acid and most sulfate salts. Exceptions
to this were ferrous sulfate and manganous sulfate. Thus, not
all sulfate salts are irritant in nature.
90
-------
The observations of McJilton et.al. (52) support the con-
cept that-certain sulfate salts are bronchoconstrictors.
Nadel et.al. (53) have shown that the inhalation of a zinc
ammonium sulfate aerosol increases pulmonary resistance in
guinea pigs as does inhalation of a histamine aerosol.
We have been able to demonstrate that unsensitized guinea
pig lung fragments (ULF) incubated with a variety of ammonium
salts release significant quantities of histamine. The most
efficacious, ammonium sulfate (100 mM), shows maximal histamine
release after 30 minutes. The ammonium sulfate mediated release
is equal to 97% of the total histamine stores. Cell lysis
through osmotic shock is unlikely since equal concentrations
of sodium chloride fails to release histamine. Lysis of the
mast cells is not likely since neither LDH nor DNA are released
into the supernatant in the presence of ammonium sulfate. Total
DNA in the fragments remains .constant in the presence of ammonium
sulfate. Total DNA in the fragments remains constant in the
presence of concentrations of ammonium sulfate known to release
histamine. Equal concentrations of sodium sulfate also fail
to release histamine, supporting the concept that only certain
sulfate salts have biological actions. These studies suggest
that the inhalation irritation associated with certain sulfate
salts may be a function of their ability to release histamine
in the presence of ammonium ion.
91
-------
The intracellular sulfate ion space in both ULF and SLF
decreases in the presence of (NH/)~SO, when compared to that
measured in the presence of Na2SO,. Since histatnine release
only occurs in the presence of (NH/^SO, , the decrease in the
intraeellular sulfate ion space is probably associated with
the histamine release process.
Sensitized guinea pig lung fragments (SLF) have been shown
to release histamine and slow reacting substance of anaphylaxis
(SRS-A) on stimulation by the immunoglobulin E (IgE) mediated
antibody-antigen reaction (71). This process has been shown
to be modulated by the cAMP and cGMP systems (44,57,58,65).
Ammonium sulfate mediated histamine release from ULF cannot be
modulated by drugs acting on these systems.
Sulfate ion uptake by ULF and SLF is uneffected by phar-
macological agents known to modulate cellular cAMP and cGMP
levels. The absorption of sulfate ions does not appear to
be highly dependent on the availability of metabolic sources
of energy. At high concentrations of potent metabolic inhibi-
tors only partial inhibition of sulfate ion uptake is observed.
Phloretin has been reported to inhibit chloride and sulfate
uptake by human red blood cells (83), however, phloretin has
no effect on the sulfate ion uptake by ULF and SLF.
Data presented here, concurs with the observations of
others (46), that the metachromasia associated with Acridine
Orange binding to heparin is a. function of ionic strength.
92
-------
The number of Acridine Orange binding sites found in our experi-
ments of 3.53 ±0.10 binding sites per disaccharide unit cor-
responds favorably with that reported by Lagunoff (46) of
3.31 ± 0.09 binding sites per dissacharide unit. The total
number remains constant with increasing ionic strength. Stone
and Bradley (72) and Lagunoff (47) suggest that the binding
sites for Acridine Orange and histamine on the heparin macro-
molecule are identical. Since we observe a decrease in the
extent of Acridine Orange binding to the heparin macromolecule
with increasing ionic strength, a local increase in the ionic
strength within the granule is likely to cause displacement
of histamine bound to heparin. Since the mast cell granule is
freely permeable to the external ionic environment, intracel-
lular uptake of ammonium or sulfate ions could result in the
displacement of bound histamine.
In experiments with the isolated, perfused and ventilated
lung (IVPL), the removal of sulfate ions from the airway
appears to be predominantly by simple diffusion. Absorption
of sulfate ions in the reverse direction, specifically from
the vasculature into the lung, could not be demonstrated. At
very low doses ammonium ion increases the removal process. The
t, at doses of 0.05 ymole or greater was 8.4 ± 1.8 minutes.
The heavy metal cations tested significantly enhanced the ab-
sorption of sulfate ions from the airways. An exception to
this rule was manganous ion, which had no effect over control.
93
-------
Intratracheal instillation of ammonium sulfate solutions
decreases the tidal volume of the preparation by 56% while am-
monium chloride causes only a 2670 decrease. Histamine (14 yg)
applied intratracheally also decreases the tidal volume by the
same percentage. As in the studies with guinea pig slices,
ammonium ions played an important role in the release of hista-
mine in perfused lungs. Histamine appears rapidly in the per-
fusate in about the same amount, 11 jag, and produces an equiva-
lent decrease in tidal volume. The release of histamine is
rapid and follows the same time course as the decrease in tidal
volume and represents an almost complete degranulation. Al-
though prostaglandins were specifically sought for, none were
released. Histamine appears to be the only vasoactive hormone
elaborated. Since prior perfusion with an H-l antihistamine
» . •
prevents a decrease in the tidal volume by ammonium sulfate,
histamine is most likely the principle mediator. All the other
cations tested failed to release histamine or LDH into the
lung effluent.
In the final set of studies it is shown that sulfate ion
removal by the rat lung in vivo is a non-saturable process
and appears to occur by simple diffusion. A t, of 34.5 minutes
"a
is observed. Deviations from the physiological pH and the
presence of certain cations lead to an enhanced absorption of
sulfate ions from the airways. As in the case of the rat IVPL,
manganous ions failed to modulate absorption.
94
-------
The more rapid diffusional process found in IVPL was not
detected. Ammonium ions augment the release of histamine in
lung fragments and the removal of sulfate ions in both perfused
and living lungs. In the intact animal ammonia is always present
in the blood (0.1 mM) and the level of intracellular ammonium
ions within the lung may be great enough to obscure the more
rapid process seen in IVPL. Other factors such as limitations
on the redistribution and elimination of sulfate ions from the
blood or feed-back effects from the release of histamine or
other vasoactive substances could also alter the removal of
sulfate ions from the lung in vivo. As a consequence, one
should consider the perfused lung model as the simplest case.
Until the intraluminal concentration of sulfate compounds is
determined from the inhalation of ambient levels of particu-
late sulfate compounds, it is difficult to predict which process
will predominate.
In all systems tested there is a positive correlation be-
tween the irritant potential associated with a specific sulfate
salt (5,9), and the rate at which sulfate ions are cleared from
the lung.
Althouth most heavy metals are known to exert biological
effects through combination with sulfhydryl groups, they also
combine with hydroxyl, carboxyl, imidazole and amine groups
(59,61). These interactions can lead to membrane changes.
The enhanced sulfate ion absorption observed in the experiments
95
-------
presented is probably related to membrane changes resulting
from these interactions. The transport of ions across absorbing
or excreting membranes has been shown to be sensitive to the
action, of heavy metal ions (11,28,35,45,75).
Based on these observations, we wish to suggest a possible
mechanism for the absorption of sulfate ions and observed hista-
mine release due to ammonium and sulfate ions. The proposed
model is depicted in Figure 5.1. Gatzy (33) has demonstrated
that chloride ions are actively transported from the vasculature
into the lungs. This results in an electrochemical gradient,
with the airways being negative with respect to the capillary
lumen (32). This electrochemical gradient favors the diffusion
of sulfate ions from the airways to the vascular space. Ab-
sorption of sulfate ,ions from the vasculature into the lungs
fails to occur, since it is against the electrochemical gradient
Sulfate ions can also diffuse into mast cells. Ammonium ions
can dissociate in solution (pK = 9.24) to hydrogen ion and
£1 .
ammonia. Ammonia is lipid soluble and can diffuse freely across
the cell membrane because it is uncharged. Once in the cell
ammonium ion can be formed.
The mast cell granule is freely permeable to the cellular
ionic environment. Ammonium and sulfate ions could diffuse
into the granule and result in the ion exchange reaction de-
picted in Figure 5.1, leading to the release of (histamine^SO, .
96
-------
Vascular
Space
Cl
Histamine
Alveolar
Space
>cr
Vascular
Endothelial
Cells
Extra-
Cellular
Space
Within the granule:
+-
2(HistNH3S04-Hep-P)
+~
2(NH4~S04-Hep-P)
2NH4 + S04
Figure 5.1. Proposed mechanism for sulfate ion absorption and
release of histamine in the presence of ammonium sulfate.
97
-------
Such a model would be consistent with the concomitant decrease
in intracellular sulfate ion space with histamine release.
Once released, histamine would lead to either a decrease in
compliance or as previously reported, a bronchoconstriction.
Date presented here suggest a correlation between the
rate of sulfate ion absorption from the mammalian lung and
the reported bronchoconstriction in the presence of certain
sulfate salts. Clearly, the rate of sulfate ion absorption
is influenced by the cationic species present and the pH of
the surrounding extracellular environment. The role of hista-
mine release as a mechanism for the bronchoconstriction action
of ammonium sulfate aerosols is strengthened by our data. More
research is needed to investigate the possible release of other
vasoactive substances by heavy metal sulfate salts.
98
-------
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BIBLIOGRAPHY
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Abstracts - Continued
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108
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1..REPORT NO.
EPA-600/1-77-046
3. RECIPIENT'S ACCESSION-NO.
FITLE AND SUBTITLE
THE MECHANISM OF SULFUR DIOXIDE INITIATED
BRONCHOCONSTRICTION
5. REPORT DATE
October 1977
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Jeffrey M. Charles, Ph.D.
Daniel B. Menzel, Ph.D.
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Laboratory of Environmental Pharmacology and Toxicology
Duke University Medical Center
Durham, North Carolina 27710
10. PROGRAM ELEMENT NO.
1AA601
11. CONTRACT/GRANT NO.
68-02-1794
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Triangle Park, N.C. ?7711
RTP,NC
13: TYPE OF REPORT AND PERIOD COVERED
Final Report 9/1/75-8/31/76
14. SPONSORING AGENCY CODE
EPA-600/11
15. SUPPLEMENTARY NOTES
16. ABSTRACT
In vitro studies with unsensitized guinea pig lung fragments (ULF) incubated
with 10 to 200 mM concentrations of ammonium ion demonstrated the release of sub-
stantial quantities of histamine. Of the anions tested, sulfate was the most potent,
while nitrate and acetate ions were of intermediate potency and chloride less potent.
Absorption of sulfate ion from the airways of the isolated ventilated and perfused
rat lung (IVPL) appears to be by simple diffusion and to be enhanced in the presence
of arrmonium ions at 0.01 ymole/lung. Manganous ion was an exception and showed no
enhancement. The t^ for the initial rate of sulfate absorption was 8.4 ±1.8 minutes.
The administration of 1 ymole (NHtt)2SOit intratracheally led to a rapid decrease in
the respiratory volume of the lung, an effect which could be blocked by prior per-
fusion with mepyramine maleate (10~5 M). Experiments in vivo demonstrate that sulfate
ion removal from the rat lung airways appears to be simple diffusion with t^ of 34.5
minutes. Deviations from physiological pH of the sulfate containing medium2and the
addition of certain cations (0.1 nanomole/lung) enhance sulfate absorption. In all
systems tested, there is a positive correlation between the irritant potential
associated with a specific sulfate salt aerosol and the rate at which sulfate ions
present in such solutions are removed from the lung.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
sulfur dioxide
bronchi
respiratory system
in vitro
in vivo
biochemistry
bronchoconstriction
06 A, F, T
12. - 3TRISUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (This Report)
UNCLASSIFIED
21. NO. OF PAGES
123
20. SECURITY CLASS (This page)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
109
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