United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
EPA-600 1-79-031
August 1979
Research and Development
Effects of
Chlordimeform on
Vascular Tissue
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
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The nine series are:
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This document is available to the public through the National Technical Informa-
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EPA-600/1-79-031
August 1979
EFFECTS OF CHLORDIMEFORM ON VASCULAR TISSUE
Casey P. Robinson
University of Oklahoma
Health Sciences Center
P.O. Box 26901
Oklahoma City, Oklahoma 73190
R804975
Jeffrey Charles
Health Effects Research Laboratory
Environmental Protection Agency
Research Triangle Park, North Carolina 27711
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 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. Environ-
mental Protection Agency, nor does mention of trade names or commercial products
constitute endorsement or recommendation for use.
11
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FOREWORD
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 participates in the development
and revision of 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 primarily responsible for providing the health basis for non-
ionizing radiation standards. Direct support to the regulatory function
of the Agency is provided in the form of expert testimony and preparation
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.
The use of pesticides over the last several years has reduced human
and animal disease and increased crop yields. Although alternate pest
control measures are being used to an increasing extent, it appears that
the need for chemical measures for controlling pests will continue
strong for the forseeable future. Many of the mainstays of chemical
pest control of previous years have proved to pose to great a risk to
human health and have been removed from distribution. This has of
course increased useage of other pesticides and increased the urgency of
finding more specific pesticides which are less toxic to humans and
other non-target organisms.
One group of pesticides with a good deal of specificity (as acaricides)
and seemingly low acute toxicity to higher animals is available. This
type information is necessary in assuring safety of these pesticides.
F. G. Hueter, Ph.D.
Director
Health Effects Research Laboratory
iii
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PREFACE
In order to fully understand potential risk to human beings of exposure to a chemi-
cal, one needs to understand the effects of that chemical upon each organ system and to
determine its mode of action. For some classes of pesticides their primary mode of
toxicity has been established. An example of this is the organophosphorus cholinesterase
inhibitors. There is general agreement that their acute lethality and most of their
toxicities result from cholinesterase inhibition and subsequent acetylcholine accum-
ulation. Even among this group of compounds, however, there are toxic effects which
seem to be due to other actions.
For other classes of compounds much less is known of their mechanisms of lethality
and effects on organ systems. An example of this is the formamidines, the first useful
one of which was chlordimeform. Little information on the mode of action of
chlordimeform was available, and on its other effects. This study was initally proposed
to determine vascular actions of chlordimeform. The project was expanded to examine
the cardiovascular actions of other available formamidines including formamidine
metabolites which also retained the formamidine structure.
IV
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ABSTRACT
In this study the effects of formamidine pesticides on isolated arteries and on
cardiovascular responses of the dog were determined. The mode of these actions was
also investigated.
Effects of formamidine pesticides were determined in vitro on the rabbit central
ear artery and aorta. Demethylchlordimeform (DCDF), formetanate and U-40481, but
not chlordimef orm (CDF) contracted strips of rabbit central ear artery.
_q DCJDF caused contractions of the rabbit central ear artery in concentrations from
10 -10 M. The maximal contraction was approximately 40% of the maximal contrac-
tion to norepinephrine, and was obtained with 10 M. Neither .1 mg/ml of atropine,
pyribenzamine nor methysergide affected these contractions, but .001 mg/ml of phen-
tolamine reduced, and .1 mg/ml of phentoiamine almost abolished them. These contrac-
tions were not decreased in reserpinized rabbits. Formetanate and U-40481 contracted
the rabbit central ear artery with maximal contractions 22 + 8% and 49 ±36% of NE
contractions, respectively. Maximal contractions were obtained with 10 M forme-
tanate and 10 IM U-40481 and cumulatively-added higher concentrations caused a
decrease irutensiofTfrom that maximum. Their contractions were antagonized by 10 M
and 3 x 10 M phentoiamine.
Three of the four formamidines antagonized contractions to vasoactive agents.
CDF antagonized contractions of the rabbit aorta induced by several vasoactive agents,
with the order of antagonism being potassium histamine serotonin norepinephrine.
DCDF antagonized contractions of the vascular strip caused by two vasoactive agents. A
concentration of 10 M antagonized contractions caused by norepinephrine and
potassium but not by serotonin and histamine. U-40481 reversibly antagonized
contractions induced by serotonin, norepinephrine and histamine, and to some extent
potassium. Formetanate had little antagonist activity.
45
CDF increased the rate of Ca washout from the medial-intLmal layer of rabbit
aorta, but did not affect the norepinephrine-induced decrease in Ca washout rate.
CDF also did not affect Ca uptake by the medial-intimal strips from rabbit aorta.
Neither DCDF, U-40481 nor formetanate altered the resting rate of norepinephrine
efflux from the pre-loaded rabbit central ear artery. Both U-40481 and formetanate
reduced electrically-induced norepinephrine release. DCDF, applied in a lower con-
centration, did not alter norepinephrine release induced by either electrical stimulation
or by nicotine addition.
In the anesthetized dog, CDF caused marked cardiovascular effects. These
included a depressor effect on blood pressure, an initial decrease followed by an increase
in musculocutaneous blood flow, and in high doses an increase in heart rate. The
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mechanism of these effects does not involve stimulation of B-adrenergic nor muscarinic
receptors. It enhances the vasodilation caused by isoproterenol, acetylcholine and
histamine. It does not involve blockade of °^ or s8 -adrenergic, histaminergic nor
muscarinic receptors, nor does it involve an inhibition of monoamineoxidase.
This report was submitted in fulfillment of Grant No. R804975 by the University of
Oklahoma, Health Sciences Center under the sponsorship of the Environmental Pro-
tection Agency. This report covers the period June 20, 1977 to May 20, 1979, and work
was completed as of June 20, 1979.
VI
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CONTENTS
Foreword i i i
Preface i v
Abstract v
Figures viii
Tables xi
Abbreviations xi i
Acknowledgements xiii
1. Introduction 1
2. Conclusions 2
3. Recommendations 3
4. Methods 4
5. Chemicals Used 11
6. Results 12
7. Discussion 42
References 47
vn
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FIGURES
Number
1 Effect of norepinephrine or demethylchlordimeform on the
contractile tension of strips of rabbit central ear artery. ... 12
2 Contractile response of the rabbit central ear artery in response
to norepinephrine, formetanate 13
3 Effect of NE, U-40481, or formetanate on the contractile
tension of strips of rabbit central ear artery 14
4 Effect on the contractile tension of strips of rabbit central ear
artery of demethylchlordimeform alone and in strips pre-
treated with phentolamine 15
5 Contraction of a strip of rabbit central ear artery caused by
addition of demethylchlordimeform and phentolamine.
B. Effect of phentolamine on tension of rabbit central ear
artery strip contracted with demethylchlordimeform 17
6 Contractile tension of rabbit central ear artery strips to U-40481
and formetanate before and during contact with phen-
tolamine 18
7 Responses to demethylchlordimeform of rabbit central ear
artery strips from control and reserpinized rabbits 19
8 Effect of CDF on contractions of rabbit aortic strips induced
by serotonin, norepinephrine, potassium, or histamine .... 20
9 Isometric recordings of the effect of CDF on responses of
aortic strips to agonists 21
10 Isometric recordings of the effect of CDF on rabbit aortic
strips contracted by exposure to cumulative" j increasing
concentrations of agonist 22
11 Responses of strips of rabbit central ear artery to norepinephrine,
potassium, serotonin, and histamine with and without
DCDF 23
12 Contractile tension of rabbit central ear artery strips induced
by norepinephrine, histamine, serotonin, or potassium in
the absence or presence of formetanate 24
viii
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13 Contractile tension of rabbit central ear artery strips induced
by norepinephrine, histamine, serotonin, or potassium in
the absence or presence of U-40481 25
14 Contractile tension of rabbit central ear artery strips to
norepinephrine, histamine, serotonin, or potassium and
the effect of U-40481 on the contractile tension 26
45
15 Effects of CDF on Ca efflux from media-intima of
rabbit aortic strips 27
45
16 Uptake of Ca by media-intima strips of rabbit aorta
in the presence or absence of CDF 28
17 Uptake of Ca in the absence and presence of forme-
tanate, U-40481 and DCDF 29
18 Radioactivity in four-minute fractions of superfusate from
H-norepinephrine loaded rabbit central ear artery, as
a percent of total radioactive efflux after initial
eight minute washout 30
19 Radioactivity in four-minute fractions of superfusate from
H-NE loaded rabbit central ear artery as a percent of
total radioactive efflux after initial eight minute
washout 31
20 Effect of chlordimeform administered at the arrow on blood
flow and blood pressure recorded from femoral arteries
of the anesthetized dog 33
21 Reduction in systolic and diastolic blood pressure following i.v.
chlordimeform in dogs with no pretreatment, pretreatment
with atropine, pretreatment with physostigmine, or pre-
treatment with propranalol 34
22 Alteration in blood flow in the dog femoral artery following
chlordimeform administration 35
23 Effect of chlordimeform on heart rate of dogs 36
24 Effect of isoproterenol on dog heart rate, systolic blood pressure,
diastolic blood pressure, and musculocutaneous blood flow
before and after administration of chlordimeform 37
25 Effect of acetylchloline on cardiovascular responses before and
after chlordimeform 38
26 Effect of epinephrine on cardiovascular responses before and
after chlordimeform 39
IX
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27 Effect of histamine on cardiovascular responses before and
after chlordimeform 40
28 Effect of tyramine on cardiovascular responses before and
after chlordimeform 41
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TABLES
Number Page
1 Effect of Preincubation of Artery Strips with Atropine,
Methysergide, Pyribenzamine and Phentolamine
on Demethylchlordimeform-Induced Contractions
of Strips of Rabbit Central Ear Artery 16
2 Effect of the Addition of Atropine, Methysergide,
Pyribenzamine and Phentolamine on Strips of
Rabbit Central Ear Artery Contracted with Deme-
thylchlordimeform 17
3 Effects of Demethylchlordimeform on Nicotine-Induced
Efflux of Radioactivity from and Contractions
of, the Rabbit Ear Artery 32
4 Radioactivity of Rabbit Ear Artery Strips Incubated
with Tritiated Norepinephrine in the Presence
of Formamidines or Cocaine 32
XI
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LIST OF ABBREVIATIONS
ACh - - acetylcholine
CDF - - chlordimeform
DCDF - - demethylchlordimeform
MAO - - monoamine oxidase
NE - - norepinephrine
Xll
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ACKNOWLEDGMENTS
Generous supplies of several chemicals were gifts of their manufacturers. These
were greatly appreciated, and are listed below.
Ayerst Laboratories: Propranolol
Ciba Pharmaceutical Co.: Phentolamine, pyribenzamine and reserpine
Ciba-Geigy Agricultural Chemicals: Chlordimeform
Nor-Am Agricultural Products, Inc.: Demethylchlordimeform and formetanate
Sandoz Pharmaceuticals: Methysergide
The Upjohn Company: Amitraz and U-40481
Winthrop Laboratories: 1 -Norepinephrine
xni
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SECTION I
INTRODUCTION
Some chemicals containing the formamidine moiety in their structure are active as
broad spectrum acaricides. The first commercially important one was chlordimeform
(CDF). CDF inhibits monoamine oxidase, an enzyme involved in inactivation of nore-
pinephrine in blood vessels as well as many other places in the body (Aziz and Knowles,
1973; Beeman and Matsumura, 1973). Although this effect does not contribute to acute
lethality of chlordimeform ( Robinson et al., 1975; Robinson and Smith, 1977), it was
thought that it might affect cardiovascular "Function.
In this study the effects of several formamides on isolated rabbit blood vessels and
anesthetized dogs have been investigated. The structures of the compounds investigated
are below:
CH-
N=CH-N
CH3
H
N=CH-N
CH3
Chlordimeform
Dem ethylchlordi m ef orm
CH3NHCOO
N=CH-N.
.CH.
'H
HCI
=CH-N*
.CH
HCI
U-40481A
Formetanate
CDF was the original formamidine investigated, and DCDF has been shown to be a meta-
bolite of CDF in vivo (Knowles and Sen Gupta, 1970). U-40481 is a metabolite of amitraz
(Knowles and Kbulston, 1973), an acaricide which currently has Section 18 approval for
use on pear trees. Formetanate is another formamidine with acaricidal activity (Knowles
and Ahmad, 1971). These compounds are all available in forms which are sufficiently
water soluble to allow for their in vitro study.
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SECTION 2
CONCLUSIONS
This study has demonstrated that formamidine acaricides and the formamidine
metabolites investigated have marked effects on isolated vascular muscle and on cardio-
vascular function. These actions have been quantified. The direct contractile activity of
some of them in isolated arteries is due to direct stimulation of alpha-adrenergic
receptors. They also interfere with contractions induced by norepinephrine, serotonin,
his tarn ine and potassium. Some of them affect calcium flux, and some interfere with
electrically-induced release of norepinephrine.
In anesthetized dogs CDF affects both blood pressure and blood flow, but does not
accomplish this through stimulation of the commonly known receptors. It increases the
vasodilation caused by some vasoactive agents. From the in vitro studies it is thought
that alteration in calcium availability may be involved in its actions.
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SECTION 3
RECOMMENDATIONS
Further study should be made of the direct cardiovascular effects of form ami dine
pesticides in intact animals. Marked CDF-induced cardiovascular effects have been
demonstrated, and other pesticides should be investigated in a similar fashion.
The marked potentiation of vasodilation in the presence of CDF should be further
investigated. This probably does not involve any of the receptors usually involved in
vasodilation, and may well involve calcium availability.
Effects of CDF on calcium mobilization on the cellular level should be
investigated. This could be done on isolated mitochondria and sarcoplasmic reticulum
preparations from vascular smooth muscle.
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SECTION 4
METHODS
The effects of fomamidines on the cardiovascular system were investigated in two
types of experiments. In one type, their effects on isolated arteries were investigated.
These studies included evaluating their contractile activities on isolated arteries as well
as their activities and selectivities at blocking contractions of other vasoactive agents.
They also included a determination of their effects on calcium flux, and on
norepinephrine (NE) release and reuptake.
In the other type of experiment the effects of these compounds injected into the
whole anesthetized dog were investigated. In these studies the effects on blood pressure,
heart rate, and musculo-cutaneous blood flow were observed.
In these studies chlordimeform (CDF) was used as a prototype. As time has per-
mitted the effects of other foramidines have also be investigated. These include U-
40481, formetanate and demethylchlordimeform (DCDF).
PROCEDURES FOR IN VITRO STUDIES IN RABBIT ARTERIES
Superfusion of Strips of Aorta Used in Chlordimeform Study
In this study strips of whole aorta (not deadventitiated) were prepared and treated
as in Zelenski, et. al, 1978. Male New Zealand white rabbits were sacrificed by cervical
dislocation, an
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Responses to cumulatively added histamine were obtained on all six muscles using
concentrations of 1 to 3000 /rM. All concentrations were superfused for 2 min except for
the 1/i M solution which was applied for 5 min. After obtaining responses to all of the
concentrations of histamine the first time, tissues were washed for 2 hr with normal
Ringers solution. Four muscles were superfused for 7 min with normal Ringers solution
containing 2 x 10 g/ml of CDF. Responses were then obtained to the same series of
histamine solutions containing 2 x 10~ g/ml of CDF. Tissues were washed withgRingers
solution for 2 hr, and the procedure described above was repeated using 3 x 10 g/ml of
CDF in the four treated muscles. Dose response curves were obtained in two control
muscles each time to verify the stability of the responses over the experimental time
period.
This basic procedure was also used for NE, potassium, and serotonin with minor
modifications: The concentration of NE used was 10 to 10 g/ml with the muscles
being exposed to each concentration for 4 min. CDF concentrations used were 2 x 10
and 3 x 10~ g/ml. The concentrations of potassium were 10 to 140 mM. The osmolarity
of the various potassium solutions was maintained constant by altering sodium concen-
tration of the solutions. Muscles were exposedJto each concentration of potassium for 6
min; the CDF concentrations used were 5 x 10 and 10~dg/ml. Serotonin concentrations
were 10 to 10 g/ml. Muscles were exposed to each concentration for 4 min.
Concentrations used were 2 x 10 and 3 x 10 g/ml.
The effects of CDF on the contractions of aortic strips elicited by a single con-
centration of each of the agonists was observed using approximately equipotent
submaximal concentrations of each agonist. The concentrations used were: serotonin,
10 _g/ml; NE, 10 g/ml; potassium, 30 m ]VI; and histamine, 10^ M. Superfusion with
(10~ g/ml) was begun 5 min before addition of each agonist and was continued throughout
exposure to the agonist. Control responses to each agonist were obtained both before
and after exposure to CDF.
The CDF used in these studies was 95% pure. Most of the remaining 5% would be
4-chloro-N-formyl-O-toluidine. To determine whether the antagonism observed could be
due to this impurity, studies were done using the maximal concentration of that
compound which could be present in the highest concentration of CDF used with each of
the four agonists. These concentrations of the impurity were without effect on con-
tractions induced by any of the agonists.
Method of Determining Contractile Activity of Rabbit Ear Artery
Strips Used in Studies with DCDF, Formetanate, U40481A and BAAM
In these studies, central ear artery strips were used to determine agonist activity.
The method is as reported in Robinson and Bittle, 1979. New Zealand White rabbits
weighing 1.5 to 2.5 kg were sacrificed by cervical dislocation. The proximal half of each
central ear artery was removed, cleaned, spirally-cut and divided into four sections
approximately equal in length. Each strip was tied at both ends and suspended in Ringers
solution (pH 7.4) in a temperature controlled muscle chamber at 38 + 0.5° C. The
incubation solution was as previously described.
The Ringers solution was aerated with 95% 02-5% COg for at least 30 min prior to
use, and continuously while in the muscle chamber. Tension was measured using
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isometric transducers and recorded by DMP-4A Physiographs (Narco Biosystems). Before
all experiments, strips of artery were allowed to equilibrate under 1 g tension for 60 min
with changes of bathing fluid every 15 min. Repeated additions of a sub-maximal
concentration of NE were made until contractions obtained were consistent. The muscle
strips were then considered to be equilibrated.
Determination of agonist activity. Contractile effects,,on the central ear artery were
determined by the cumulative addition of 10 to 10 R9 formamadine_£o the muscle
strips. Tension changes were stated as percent of tension generated to 10 M NE.
Investigation of the Mode of Agonist Action of DCDF in the Central Ear Artery
The mode of contractile activity of DCDF was investigated in the rabbit central
ear artery from both normal and reserpinized rabbits as reported in Robinson and Bittle,
1979. It was observed that DCDF had considerable agonist activity in the rabbit ear
artery. Experiments were designed to observe the effects of an anthihistamine (pryi-
benzamine hydrochloride, J mg/ml) an antiserotonergic (methysergide, J mg/ml), an
anticholinergic (atropine sulfate, J mg/ml), and an alpha-adrenergic blocking agent
(phentolamine, .001 & J mg/ml) on DCDF - induced contractions. In one type of experi-
j&cnt, responses to cumulatively increasing concentrations of DCDF were obtained, then
DCDF was removed by several washings. Fifty min later one of the antagonists was
added to the bath and the responses to cumulatively added DCDF were again observed.
The effects of depleting intraneuronal stores of NE on DCDF-induced contractions
were also determined. This was accomplished by the intramuscular injection of 2.5
mg/kg of reserpine 72 and 24 hours prior to removing the arteries. Responses of cumu-
latively added DCDF were observed in muscle strips form reserpinized rabbits as pre-
Triously described. Amine depletion within the strips was verified by obtaining reduced
responses to nicotine.
Determination of Activity as Antagonists of Vasoactive Agents
Three kinds of experiments were designed to determine antagonist activity of the
formamadines. In one type, effects- on the concentration-response curve to NE,
histamine, serotonin and potassium were examined. Responses to a cumulatively added
vasoactive agent were obtained, and then the agonist was removed by washing. Fifty min
later a formamidine was added, and 10 min after that responses to the same agonist were
again determined.
In a second type of experiment, formamidine-induced effects on the response to a
single concentration of the same four agonists were observed. In these studies, 10 M
NE, 10 M histamine, 10 M serotonin and 40m M potassium were used as agonists. The"
response To one of these agonists was obtained", the _drv* was removed by multiple
washings and 80 min later one of the formamidines (10 M) was added. After ten min
contact time, the original agonist was re-added to the strip and the effects on the shape
of the contraction and maximal contractile tension were observed. In a third type of
experiment formamadine effects on an agonist-contracted muscle strip were examined.
Concentrations of each compound were the same as in the previous study. The response
to a single agonist was obtained, washed out and elicited again 90 min later. When the
contractile tension was maximal, the formamidine was added and the response observed.
Reversibility of the antagonism was determined by recovery of control activity
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after washing out the formamidine and re-exposing the strip to the same agonist given
before the formamidine.
Determination of the Effects of Chlordimeform on Calcium Flux
Calcium efflux—-
In these studies calcium flux was studied in deadventitiated aorta. Methods for
CDF effects were reported in Zelenski, et al., 1978.
In this investigation, strips were prepared from the aorta as described previously,
and the adventitia was removed according to the method of Maxwell et al. (1968). The
deadventiated strip was then cut into two JL5-cm strips and hung at 2 g tension in 20 ml
of aerated Ringers solution. Sufficient Ca was added to the incubation medium to
make a Ca concentration of l.S/fcCi/ml. During a 3-hr, incubation period, the Ringers
solution was continuously aerated with 95% 02-5% COo and maintained at 38 + 0.5 C.
At the end of 3 hr, the bath solution was drained, and superfusion of both muscles
was started immediately with calcium-free (0-Ca) Ringers solution (CaCl2,2.4 mM omit-
ted). The superfusate was collected using a Buchler continuous automatic fraction
collector Model 3T-4002 and fraction collector activator Model 3-400 8T. The rate of
superfusion was 2ml/min. Samples were collected at 5-min intervals. Muscles were
maintained under 2 g tension throughout the experiment.
At the end of 30 min of washing with 0-Ca Ringers solution, superfusion of one
muscle with 10 g/ml of CDF was begun. The control muscle continued to receive 0-Ca
Ringers solution.
After 45 min of CDF superfusion, solutions were changed for both muscles. The
muscle previously exposed to 0-Ca Ringer^solution containing CDF was superfused with
0-Ca Ringers solution containing CDF (10 g/ml) and NE (10/g/ml). The control muscle
was superfused with 0-Ca Ringers containing lOylg/ml of NE. Samples were collected for
an additional 50 min.
45
To determine the Ca concentration of the superfusate samples, 0.4-ml aliquots
were removed and placed in liquid scintillation mini vials containing 5 ml of Aquasol-2.
At the end of the washout period, muscle strips were removed, blotted, weighed, and
dissolved in 0.2.ml of Soluene-100. Five milliliters of Aquasol-2 was then added to each
sample. The Ca concentration in each sample was determined using a Beckman liquid
scintillation counter (Model LS-100). The counting time was automatically adjusted to
obtain a counting error of less than 1%, and the counting rate of each sample was
corrected for quenching.
The mean of three calcuim efflux experiments was plotted as desaturation curves
showing the percentage of Ca remaining in the tissue during the continuous super-
fusion. (Bianchi, 1961; Weiss, 1966). Methods for studying effects on Ca efflux of
DCDF, formetanate and U-40481 were similar.
Calcium uptake - -
45
Methods for studying the effects of CDF on 4cCa uptake were reported in Zelenski,
et al., 1978. The effect of CDF on the uptake of Ca was also determined. A deadven-
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titiated aortic strip was cut into 16 approximately equal pieces weighing 5 to 10 mg each
and incubated in aerated Ringers solution for 1 hr, then transferred to 0.1 mM calcium
Ringers solution for an additional 90 min. Each piece was gently blotted on filter paper
and placed.Jnto a minivLal containing 4.2 ml of 0.1 mM calcium _Ringers solution
containing Ca, 3.33 x 10 cpm. Eight of the vials contained CDF (10 g/ml). Control
and exposed strips were removed at 1, 2, 5, 10, 20, 40, 60, and 90 min. Upon removal,
strips were blotted on filter paper and rinsed for 5 sec in each of four successive 20-ml
vol of a Tris buffer containing 10 mM lanthanum, then blotted again. (Lanthanum was
used to remove extracellular calcium and to prevent the efflux of intracellular calcium.)
The strips were weighed and solubilized in 0.1 ml of soluene, and their radioactivities
were determined as previously described.
The significance of differences between groups was determined using Student's t
test for nonpaired data, with P <0.05 considered significantly different.
Methods for studying the effect of DCDF, U-40481 and formetanate on Calcium
uptake were similar.
Initial calcium uptake on the addition of NE —
Ten small approximately equal sections of deadventitiated aorta were prepared
from the thoracic aorta of a single rabbit as previously described in this report. A single
piece was placed into a tube in Ringers solution in a shaker-heater bath for 10 minutes at
38 , then placed into another tube containing Ringers solution, Ca, C-mannitol, and
DCDF. It was removed in 15 seconds, lightly blotted, weighed and prepared for
determination of the radioactive isotopes. In a single experiment, the 9 other strips were
treated in a similar manner, with a total of 5 being treated as described and 5 being
exposed to the same solutions without DCDF (i.e., control). One control and one treated
strip were exposed to 15, 30, 60, 120 and 390 seconds each. Mannitol was used to enable
a differentiation of intracellular Ca from true intracellular uptake. This experiment
was repeated 5 times.
Similar experiments were done with U-40481 and formetanate using 5 or 6 rabbits
for each drug.
Determination of the Effects on Norepinephrine Release and Uptake
Electrically-induced NE release —
The method of determining the effects of formamidines on the electrically-induced
release of NE was that described for DCDF in Robinson and Bittle, 1979. A helically cut
strip was prepared from the proximal 5 cm of each central ear artery as previously
described. Strips from each ear were mounted between tw" parallel platinum electrodes
for superfusion and transmural stimulation as previously described (Su and Bevan, 1970;
Bevan et al., 1972), in temperature controlled organ baths (38 ) at 1 g tension. The strips
were incubated for 60 min in aerated Ringers solution containing .5 Ci/ml of 1 -7- H-
NE (specific activity 15 Ci/mmol). The baths were then drained and the strips superfused
with Ringers solution containing 10 M NE for 2.5 min. Superfusion was continued with
Ringers solution, or Ringers solution containing DCDF throughout the experiment.
Stimulation periods were four min in length with 20 min between them. When the effects
of 10 M DCDF were to be observed, it was added to the superfusate 8 min before
electricaT~stimulation and maintained in the superfusate for the duration of electrical
-------
stimulation. The superfusate was collected at 4 min intervals and an aliquot was
removed for radioactivity determination by scintillation spectrometry. Radioactivity at
each time period was expressed as percent of the radioactive efflux during the experi-
ment starting with the third collection period through the end of the experiment
(lOOmin). Eight strips from 4 rabbits were used in the study.
Similar studies were done with U-40481 and formetanate using 5 and 6 rabbits.
Nicotine induced release of NE —
Effects of DCDF on nicotine-induced NE release were described in Robinson and
Bittle, 1979. Effects of DCDF on nicotine-induced contractions were studied in an
experiment similar to the above except that nicotine was added to the superfusate for
2.5 min every 30 min, timed so that nicotine addition and collection of a 4 min sample
would begin simultaneously. Maximal tension generated by the NE released by nicotine
was calculated as a percent of the contraction to 10 M NE.
Effects on Uptake of NE —
3
Effect of the formamidines on the uptake of -H-NE were determined in the rabbit
central ear artery. Both ear arteries were removed and cut into 4 segments each as
previously described in this report. They were allowed to equilibrate for 1 hour at room
temperature and transferred to a shaker-heater bath for an additional 30 min. At the
time of transferring the strips to the shaker-heater bath, either CDF, DCDF,
formetanate, cocaine, or U-40481 was added to the strip, or an equal volumegof water
(10 1) as a control. After 30 min of incubation with the various chemicals, 10 M 1- H-
NE was added to the bath and 60 min later the strips were removed, rinsed in 3
consecutive baths and placed into freshly aerated Ringers solution and allowed to remain
there 30 min. The strips were then removed, blotted, weighed and prepared for
redioactivity determination as previously described.
PROCEDURES FOR IN VIVO STUDIES IN THE ANESTHETIZED DOG
Mongrel dogs of either sex weighing between 14 and 23 kg were used. All dogs
were vaccinated against canine distemper and infectious canine hepatitis, were free from
internal and external parasites, and had normal hematocrit values. Dogs were
anesthetized with 30 mg/kg pentobarbital i.v. and were given supplements as needed
throughout the experiment. Heparin sodium was administered i.v. to prevent blood
clotting. Animals that were to receive physostigmine were intubated with an endo-
tracheal tube attached to an animal pump respirator (Harvard Apparatus).
The right cephalic vein and left femoral artery were cannulated for drug injections
and for blood pressure recording, respectively, and the right femoral artery was exposed
and a blood flow transducer placed around it. Musculo-cutaneous blood flow to the right
hind limb, blood pressure and heart rate were constantly recorded throughout the
experiment.
Experiments were designed to study the effects of CDF on the cardiovascular
system of the dog, and to determine whether certain compounds would modify the re-
sponses to CDF. These compounds were the beta adrenergic receptor blocking drug
propranolol, .5 mg/kg, the muscarinic receptor blocking drug atropine, 2 mg/kg, and the
cholinesterase inhibitor physostigmine, 0.5 mg/kg. Appropriate test drugs were given
-------
SECTION 6
RESULTS
EFFECTS OF FORMAMIDINES ON RABBIT ARTERIES IN VITRO
Direct Agonist (Contractile) Activity
DCDF, Formetanate and U-40481 (but not CDF) all caused contractions of the
rabbit ear artery.
O
CO
X
<
100
90
80
70
60
50
40
30
20
10
0
lO'10 10'9
10"8 10'7 10" 10
CONCENTRATION (M)
10
-3
Fig. 1. Effect of norepinephrine ( O ) or demethylchlordimeform ( 0 ) on the contrac-
tile tension of strips of rabbit central ear artery. Each value represents the Mean + SEM
of 26 or 28 observations. (From Robinson and Bittle, 1979).
12
-------
DCDF- -
Effects of DCDF as an agonist were reported in Robinson and Bittle, 1979.
Cumulatively added DCDF increased contractile tension of strips of rabbit central ear
artery in concentrations from 10 to 10 M (Fig 1). Maximal tension was usually
observed at 10 IM with the addition of higher concentrations often resulting in a
decrease in contractile tension. Concentrations of 10 M or higher almost always
relaxed the muscle strips to below resting tension. Maximal tension generated in
response to DCDF was 39% of the maximal NE corjlraction. The EC^ for contracting
the strips was 4 x 10 M for DCDF compared to 10 _M for NE.
Formetanate and U-40481- -
Effects of Formetanate and U-40481 as an agonist are reported together. (Robin-
son, 1979).
Low concentrations of both formamidines increased contractile tension of ear
artery strips and high concentrations decreased their tension (Fig. 2, 3). U-40481
U-40481 FORMETANTATE
5 min
~5
Fig. 2. Contractile response of the rabbit central ear artery in response to 10~ M
norepinephrine (NE), 10~ M_ formetanate (Form.), and 10 M U40481. ~~
(From Robinson 1979).
13
-------
achieved a maximal contractile tension of 49 + 6% of the maximal tension to 10 M NE.
Formetanate was less potent, achieving a maximal tension of 22 + 8% that of NE. U-
40481 elicited itsinaximal contraction at 10~ M, while that of formetanate was
obtained with 10 M. In concentrations higner than those eliciting maximal
contractions, both compounds partially relaxed the strips from the maximal contraction
achieved.
o
I—
o
cr
O
o
x
<
-4 -3 -2
M
CONCENTRATION
Fig. 3. Effect of NE (A ) U-40481 (O ) or formetanate (0 ) on the contractile tension
of strips of rabbit central ear artery. Each value represents the Mean + SEM of 6 obser-
vations.
(From Robinson, 1979).
14
-------
LU
z
z
o
X
<
100
90
80
70
60
50
40
30
20
10
10'
.-o
-7
10^ 10" 10' 10" 10
CONCENTRATION OF DCDF (M)
-5
10'
Fig. 4. Effect on the contractile tension of strips of rabbit central ear artery of deme-
thylchlordimeform alone ( O ) and in strips pretreated with phentolamine. .001 mg/ml
( 0 ) or .1 mg/ml ( A ). Each value is the Mean + SEM from 5 or 7 observations.
(From Robinson and Bittle, 1979).
15
-------
Mechanism of the Direct Agonist Activity
Use of Selective Blocking Drugs- -
DCDF--
Specific receptor blocking drugs were used to investigate the nature of the DCDF-
induced contractions as reported in Robinson and Bittle (1979). The prior addition of
either atropine, methysergide or pyribenzamine did not affect the contractions, but
phentolamine greatly reduced them (Table 1). To observe the effects of phentolamine
TABLE 1
EFFECT OF PREINCUBATION OF ARTERY STRIPS WITH ATROPINE, METHYSER-
GIDE,PYRIBENZAMINE AND PHENTOLAMINE ONT DjEMETHYLCHLORDIMEFpRM-
INDUCED CONTRACTIONS OF STRIPS OF RABBIT CENTRAL EAR ARTERY
CONTRACTILE TENSION (g) TO
10~& M DEMETHYLCHLORDIMEFORM
ANTAGONIST CONTROL WITH ANTAGONIST (n)
MEAN + SEM MEAN + SEM
ATROPINE, .Img/ml
METHYSERGIDE, .Img/ml
PYRIBENZAMINE, .Img/ml
PHENTOLAMINE, .Img/ml
.27 + .08
.21 + .09
.31 + .07
.27 + .03
.27 + .08
.22 +.07
.28 + .09
.02 + .01
(6)
(8)
(5)
(5)
on DCDF-induced contractions more fully, the effects of two concentrations of
phentolamine on concentration response curves to DCDF were determined. In these
experiments, .001 mg/ml phentolamine shifted the concentration response curve to the
right, while .1 mg/ml phentolamine almost abolished DCDF-induced contractions (Fig. 2).
The effect of phentolamine on the contraction induced by a single concentration of
DCDF (10 M) was observed (Fig. 5a) Incubation with .001 mg/ml phentolamine reduced
the contraction toadded DCDF (Fig. 5b) and with .1 mg/ml phentolamine abolished the
contraction to added DCDF (Fig. 5c). In strips' contracted with 10 M DCDF, the
addition of .001 mg/ml phentolamine partially relaxed the strips and .1 mg/ml almost
abolished induced tension within the strip (Fig. 5B). Neither atropine, methysergide, nor
pyribenzamine reduced contractile tension of strips contracted with DCDF (Table 2).
16
-------
.8
^ 4
to
DCDF
.61-
.2° .4
z
o
C/5
z ,
Ph DCDF
.0001 mg/ml
Ph DCDF
.01 mg/ml
Ph (.0001 mg/ml)
Ph (.01 mg/mi;
DCDF
Tig. 5.A. Contraction of a strip of rabbit central ear artery caused by addition of 10 M_
demethylchlordimeform before (a) and after phentolamine, .001 mg/ml (b) and phentola-
mine, .1 mg/ml (c). B. Effect of phentolamine, .0_Q1 mg/ml and .1 mg/ml on tension of
rabbit central ear artery strip contracted with 10 M demethylchlordimeform. (From
Robinson and Bittle, 1979). ~~
TABLE 2
EFFECT 'OF THE ADDITION "OF
MENZAMINE AND
PHENTOLAMINE ON STRIPS OF RABBIT fiNTRAL EAR ARTERY CONTRAC
WITH DEMETHYLCHLORDIMEFORM
, CONTRACTILE TENSION (g) TO
10 ° M DEMETHYLCHLORDIMEFORM
ANTAGONIST
CONTROL
MEAN +SEM
AFTER ANTAGONIST
MEAN + SEM
(n)
ATROPINE, .lmg/ml
METHYSERGIDE, .lmg/ml
PYRIBENZAMINE, .lmg/ml
PHENTOLAMINE, .OOlmg/ml
PHENTOLAMINE, .lmg/ml
.40 + .04
.29 + .03
.26 + .05
.39 + .02
.39 + .02
.40 + .04
.29 + .03
.25 + .05
.30 + .10
.00 + .00
(7)
(5)
(5)
(12)
(12)
17
-------
Formetanate and U-40481- -
Antagonism by phentolamine of contractions caused by formetanate and U-40481
are in Robinson, 1979. Phentolamine reduced contractions to both U-40481 (Fig 6A) and
formetanate (Fig 6B). On the sam£ strips 3 x 10 M[ phentolamine reduced contractions
to 10 M NE to 1 + .8% and to 10~5 M NE to 42 + 8% of their control values. Thus the
contractile effects seem to be cause? by alpha-adrenergic-receptor stimulation.
B
H-
O
O
o
X
20 -
15 -
10 -
5 -
0
ITIII
10-9 -8 -7 -6 -5 -4 -3 -2 M
10-9 -8 -7 -6 -5 -4 -3 -2!M
U-40481
FORMETANATE
Fig. 6. Contractile tension of rabbit central ear artery strips to U-40481 (A) and former
tanate (B) before ( £ ) and during contact with phentolamine 10 RI ( • ) and 3 x 10
M ( D ). Each value represents the Mean + SEM of 8 observations. (From Robinson,
T979).
Use of Reserpinized Rabbits - -
Reserpinized rabbits were used to investigate the mechanism of the agonist action
of DCDF as reported in Robinson and Bittle, 1979.
Rabbits that had received reserpine injections were less active than normal. De-
pletion of intraneuronal NE stores within the muscle strips was demonstrated by a
marked reduction in responsiveness of the strips to 2 mg/ml of tyramine. The sensitivity
of the central ear artery to DCDF was not reduced in arteries from reserpinized rabbits
(Fig. 7). Thus contractions caused by DCDF are not dependent on NE stores.
18
-------
e
4
I
K
I
M
COHC. (M)
Fig. 7. Responses to demethylchlordimeform of rabbit central ear artery strips from
control ( O ) and reserpinized rabbits ( Q ). Each value is the Mean + SEM from 6 or 7
observations. (From Robinson and Bittle, 1979).
Activity at Antagonizing Contractions Elicited by Vasoactive Agents
CDF--
Effects of CDF on contractions elicited by vasoactive agents were reported in
Zelenski et aL (1978). Figure 8 shows the effects of two concentrations of CDF on log
concentraHon-response curves obtained with serotonin (Fig. 8A), NE (Fig. 8B), potassium
(Fig. 8C), and histamine (Fig. 8D). CDF caused a parallel shift to the right in the NE and
serotonin concentration-response_
-------
curves were not achieved in the presence of CDF. However, there was no indication
that the tension in these muscles had reached a plateau. Potassium and histamine
concentration-response curves were not shifted to the right in a parallel fashion. After
treatment with CDF, maximal responses compared to control muscles were unattainable,
although maximal muscle tension in the presence of CDF had been achieved.
IOO
UJ
§ 80
| 60
| 40
1 20
100
80
UJ
K 6O
i 4O
2 20
3?
B
10 '
-8 -6
CONCENTRATION OF
SEROTONIN (g/ml)
10 2O 40 80 I4O
CONCENTRATION OF
-4
CONCENTRATION OF
NE (g/ml)
3 10 3O I02
CONCENTRATION OF
HISTAMINE
Fig. 8. Effect of CDF on contractions of rabbit aortic strips induced by serotonin (A),
norepinephrine (B), potassium (C), on histamine (D). .Control response (_n£ ) and in the
presence of chlordimeform: 5 x 10 g/ml ( A ), 10 g/ml ( Q ), 2 x 10 g/ml ( A ), or
g/ml ( • ). (From Zelenski, et al., 1978)
3 x 10 -
20
-------
The effects of CDF on contractions elicited by each of the agonists may be seen in
Fig. 9. In each case, CDF caused a decrease in both the maximal tension generated and
in the rate of tension development by the muscle strips. The slowed rate of tension
development in the presence of CDF resulted in a change in the single tension peak in the
control serotonin contraction into a double-peaked response in each of six muscles (Fig.
9A). This same effect of CDF on the shape of the response was also seen in some but
not all of the responses to NE (Fig. 9B). A single-peaked response after exposure to CDF
was observed in both potassium-and histamine-induced contractions (Figs. 9C and 9D).
9
CM
SEROTONIN 10"* g/ml
CDF I x 10'g/ml
SEROTONIN K)~6g/ml
NE KTg/ml
CDF Ixl0~3g/ml
NE I0"7g/ml
K* 30 mM
CDF Ixl0*g/ml
K* 30 mM
5min
HISTAMINE
CDF I x 10"* g/ml
HISTAMINE 10 mM
Fig. 9. Isometric recordings of the effect of CDF on responses of aortic strips to
agonists. Drugs perfused during periods indicated ( i—I ); at other times perfused with
physiological solution only.
21
-------
The addition of chlordimeform to muscle strips contracted by cumulatively added
agonists resulted in a decrease in contractile tension in each case (Fig. 10). The initial
decrease in tension was more rapid than that observed when the agonist was removed in
the absence of CDF. Tension continued to decrease to almost baseline in muscles
contracted by serotonin (Fig. 10A), potassium (Fig. IOC), and histamine (Fig. 10D).
However, in the presence of NE (Fig. 10B), muscles relaxed an average of only 48%
(range, 41-61%) within the 2-hr observation period.
'3xlO~3 g/ml CDF. SEROTONIN
MAINTAINED
'3xlO~3 g/ml CDF,
NOREPINEPHRINE MAINTAINED
(b)
POTASSIUM REMOVED
7n*i
f ^
IO"3 g/ml CDF,
POTASSIUM MAINTAINED
7min
3xK>~3 g/ml CDF,
HISTAMINE MAINTAINED
Fig. 10. Isometric recordings of the effect of CDF on rabbit aortic strips contracted by
exposure to cumulatively increasing concentrations of agonist. Drugs and final con-
centration of agonist are as follows: (A) serotonin, 10 g/ml; (B) norepinephrine, 10
g/ml; (C) potassium, 140 mM; and (D) histamine, 3 AM. Upper tracings show relaxation
on removal of agonist (arrow); lower, relaxation on addition of CDF, 3 x 10 g/ml,
(arrow) without removing agonist. (From Zelenski et al, 1978).
22
-------
DCDF--
Effects of DCDF on contractions elicited by vasoactive agents were reported in
Robinson and Bittle (1979).
In experiments in which muscle strips were pre-incubated with 10 M DCDF
before the cumulative addition of agonist, there was a reduction in contractions induced
by NE (Fig. 11 A) and to a lesser extent to those induced by potassium (Fig. 11B). Sero-
tonin-induced contractions were not altered by this low concentration of DCDF (Fig.
11C), and contractions to low concentrations of histamine were potentiated (Fig. 11D).
100
I
-7
&M 10 20 4O 60 1OO ZOOmM
s
a
o
o
i
M
40
20
100
-7
-B -4 -S -2M io-«
20
CONCENTIATION OF AOOHIST
Fig. 11. Responses of strips of rabbit central ear artery to norepinephnne (a), potassium
(b), serotonin (c) and histamine (d) with ( Q ) and without ( 9 ) 10 M DCDF. Each
value is the Mean + SEM from 6-14 observations. (From Robinson and Bitfle, 1979).
23
-------
Formetanate and U-40481- -
Effects of formetanate and U-40481 on agonist-induced contractions are from
Robinson (1979). 10 M_ formetanate had little effect oncontractions elicited by any of
the vasoactive agents (Fig. 12). With lower concentrations of NE there was some
depression of contractile responses (Fig. 12A). With lower concentrations of histamine
(Fig. 12B) and through much of the curve to serotonin (Fig. 12C), contractile tension was
higher than control levels. The concentration response curve to potassium was little
affected by formetanate (Fig. 12D). 10~ Rl U-40481 had much more marked effects on
responses to three of the agonists.
o
Q-
C/3
2 M
NOREPINEPHRINE
HISTAMINE
ts>
z
o
Q-
tr
i
<
5
x
S
10 6
10
I 1 I I IT
20 3040 6080100mM
SEROTONIN
POTASSIUM
Fig. 12. Contractile tension of rabbit central ear artery strips induced by norepiner
phrine, histamine, serotonin, or potassium in the absence ( Q) or presence ( £ ) of 10
M formetanate. Each value represents the Mean + SEM from 5-8 observations. (From
Robinson, 1979).
24
-------
Responses to NE (Fig. 13A), histamine (Fig. 13B), and serotonin (Fig. 13C) were all
markedly depressed by U-40481. Effects on potassium contractions were much less, with
lower concentrations eliciting somewhat higher contractions in the presence of U-40481,
and higher concentrations eliciting slightly depressed responses (Fig. 13D).
10-9 -8 -7 -6 5 M
NOREPINEPHRINE
2 M
HISTAMINE
o
a.
CO
SEROTONIN
\ I I I I T
10 20 3040 eOSOlOOmU
POTASSIUM
Fig. 13. Contractile tension of rabbit central ear artery strips induced by norepine?
phrine, histamine, serotonin, or potassium in the absence ( Q ) or presence ( % ) of 10
M U-40481. Each value represents the Mean + SEM from 6-10 observations. (From
Robinson, 1979).
The addition of U-40481 to strips contracted by 10 M NE caused a rapid reduction
(Fig. 14A, center) of 68 + 20% of muscle tension. On serotonin-contracted strips there
was a rapid reduction (Fig.ISC, center) in tension almost to baseline (97 + 37%). Hista-
mine-contracted strips were also rapidly affected, but were reduced only~31 + 6% (Fig.l4B
center). Potassium contractures were sometimes slightly but slowly reducedT The mean
effect was a reduction of 19 + 10% in tension.
25
-------
A.
T r~
\ QD I
r J
NE
B
r—.
TM
. r J -
4. *
H
00
Lf>
c. 1
0.1
Adj. S
U
DO
4 4
K+ K+
U Adj. K+
5 min
Fig. 14. Contractile tension of rabbit central ear artery strips to 10 M norepinephrine
(A), 10" M histamine (B), 10 M_ serotonin (C), or 40 mM potassium (SJ and the effect
of 10 MTr-40481 on the contractile tension. Left, control contraction. Center, effect
of U-40481 added to contracted strip. Right, effect of presence of U-40481 on
contractions to added agonist. Tension adjusted to baseline at Adj. during contact to U-
40481 before exposure to agonist.(From Robinson, 1979).
26
-------
Preincubation of U-40481 with muscle strips greatly diminished responses obtained
to added NE (Fig. 14A, right), serotonin (Fig. 14C, right), and to a lesser extent histamine
(Fig. 14B, right). Total tension induced in strips by potassium in the presence of U-40481
was not always depressed but the normally biphasic responses were separated more
clearly into two components (Fig. 14D, right) by a slowing of the second phase.
Effects of Formamidines on Calcium Flux
Calcium Efflux
Effects of CDF on the efflux of 45Ca was reported in Zelenski, et aL, 1978.
Figure 15
containing NE,
control muscles
70-min washout
NE (10^ g/ml)
The projected
extrapolated.
illustrates the " Ca efflux rates during superfusion with 0-Ca media
CDF, or a combination of both. The upper desaturation curve is from
. The first part depicts a normal rate of calcium efflux during an initial
with the 0-Ca medium. The latter part of the control curv.e shows that
when added to the superfusate caused a slight decline in Ca release.
slope of a normal washout curve with 0-Ca Ringers solution was
100
lil ^^
35
co 70
i—
\ *°
z 4O
| 30
*
o
0 on
D 2O
fr
IO
r 100
- 90
O 70
°0 60
°0 NE,IOug/ml 50
O i **vx
O I
°0 1 40
O A W
o •
x O
^x. O
x o
xx O
XX0 o 20
xv
.
• O Chlordimeform, K)"3 g/ml
- °°o|
°^ ChJordimeform,IO"3g/ml
^x NE, lOug/ml
°oX 1
O J.
O If
°°0
V " O ft A
x ^^ O
x"»
x
.
i i . t i i • i . i • i 1 1 • 1 1 i . i . i
20 4O 60 80
TIME (min)
45.
100
20 40 GO 80
TIME (min )
IOO
Fig. 15. Effects of CDF on 45Ca efflux from media-intima rabbit aortic strips. Strips
were incubated for 3 hr with Ca prior to perfusion with calcium-free Ringers solution.
Arrows indicate the addition to the perfusate of (A) norepinephrine, ID ^ g/ml; (B)
Chlordimeform, 10~ g/ml; or (C) norepinephrine, 10>^g/ml plus CDF, 10~3g/ml. (From
Zelenski et aL, 1978)
45
CDF produced an initial increase in Ca efflux which was maintained for approxi-
mately 25 min before a gradual decrease in efflux was observed. The rate of Ca
washout at this time was approximately that of the control washout curve. Addition of
NE (10/rg/ml) to the Ringers-chlordimeform solution resulted in an immediate decrease
in calcium efflux from the tissue. This change was even more pronounced than that
observed when NE was added to the muscle strips in the absence of CDF.
27
-------
Calcium Uptake
45
Effects of CDF on the uptake of ™Ca by deadventitiated aorta strips was reported
in Zelenski et al., 1978. CDF did not affect either the rate of Ca uptake or the total
uptake at 9(TrnTn in deadventitiated muscle strips (Fig. 16).
0 5 10 20
40
TIME (min)
Fig. 16. Uptake of Ca bv media-intimal strips of rabbit aorta in the presence ( • ) or
absence ( • ) of CDF, 10 g/ml. Each value represents the mean + SEM of observations
of strips from five or six rabbits. Values compared at each time interval were not
significantly different (P 0.05). (From Zelenski et al., 1978).
Initial Calcium Uptake on the Addition of Norepinephrine
45,
Results of the effects of DCDF, formetanate and U-40481 on the initial ""Ca
uptake upon addition of NE are presented in Robinson and Pento, 1979. Neither
compound altered Ca uptake significantly (Fig. 17).
28
-------
TIME (MIN.)
45
Fig. 17. Uptake of Ca in the absence ( Q ) and presence ( £ ) of U-40481 (A), DCDF
(B), and f ormetanate (C). (From Robinson and Pento, 1979).
29
-------
Activity at Altering Norepinephrine Release From Sympathetic Nerves by Nerve Stimu-
lation
DCDF- -
3
The effects of DCDF on electrically-induced release of radioactivity from H-NE
loaded central ear arteries is described in Robinson and Bittle, 1979. In the experiments
in which helically cut strips from the central ear artery were mounted between parallel
platinum electrodes and superfused with Ringers solution, the addition of DCDF did not
alter (P>.05) the efflux of tritiated NE fromthe strips (Fig. 18). Transmural stimulation
causes an increased release of radioactivity which decreases with each succeeding
stimulation period. Prior and concurrent exposure to DCDF did not block the increased
tritiated outflow from the muscle strips caused by transmural stimulation (Fig. 18).
40
35
30
• 25
o 20
o
o
Of
15
10
I
tmd
TT-r
"hhr-n-Tl-n
TS
TS
TS
TS
DCDF
DCDF
Fig. 18. Radioactivity in four-minute fractions of superfusate from H-norepinephrine
loaded rabbit central ear artery, as a percent of total radioactive efflux after initial
eight minute washout. Transmural stimulation (TS) and demethylchlordimeform (DCDF)
where indicated (— ). From Robinson and Bittle, 1979.
30
-------
Formetanate and U-40481- -
The effects of formetanate and U-40481 on electrically-induced release of radio-
activity from H-NE loaded central ear arteries is reported in Robinson, 1979. Control
muscle strips pre-loaded with H-NE, and transmurally stimulated had a progressive
decrease in the radioactivity released during each of the four stimulation periods (Fig.
19A). The control strips and those treated with U-40481 were from the same rabbits, and
both data were obtained simultaneously.
Neither formamidine altered the resting rate of radioactivity washout (Fig. 19B, C), but
both depressed the stimulation-induced radioactive efflux. This depression of elec-
trically-induced release was still present in the formetanate exposed strip on the second
control response, despite three or more washes and a 20 min resting period (Fig. 19).
2 10 -
I
I
10-
Vrr
S
TrrlriTrr
rnrnn
1
TNS TNS
TNS TNS TNS TNS
U 40481 U 40481
TNS TNS
TNS
Fig. 19. Radioactivity in four-minute fractions of superfusate from 3H-NE loaded rabbit
central ear artery as a percent of total radioactive efflux after initial eight minute
washout. Transmural stimulation (TNS) and U-40481 (in B) or formetanate (Form.) (in C)
where indicated ( — ). Each value represents the Mean + SEM of either 5 or 6 observa-
tions. From Robinson, 1979.
31
-------
Activity at Altering Nicotine-Induced Release of Norepinephrine
3
Effects of DCDF on nicotine induced release of H-NE was reported in Robinson
and Bit tie, 1979. In the experiments in which nicotine was added to the superfusate, the
increase in tritiated efflux was not reduced, (Table 3), while the nicotine-induced
contractions were abolished. Thus, from the H-NE experiments it was learned that
DCDF does not alter the rate of release of NE either in the absence of releasing stimuli,
or during electrical or nicotine-stimulation.
TABLE 3
EFFECTS OF DEMETHYLCHLORDIMEFpRM ON NICOTINE INDUCED
EFFLUX OF RADIOACTIVITY FROM AND CONTRACTIONS
OF, THE RABBIT EAR ARTERT
%Increase in Contraction to
Radioactive Efflux Nicotine
Mean + S.E. (n) Mean + S.E. (n)
Nicotine, 10"5 M 50.5 + 19.1 (9) 16.1 + 3.9 (10)
Nicotine + DCD~F, 49.8 + 34.9 (7) .0 f .0 (7)
Q
a. Incubated for 60 min, in H-NE before,-beginmng superfusion.
b. As a percent of the contraction to 10 ]VI NE.
Activity at Altering the Uptake of Norepinephrine
None of the formamidines tested significantly altered uptake of radioactivity by
strips of rabbit central ear artery (Table 4).
TABLE 4
RADIOACTIVITY OF RABBIT EAR ARTERY STRIPS INCUBATED WITH TRITIATED
NOREPINEPHRINE IN THE PRESENCE OF
FORMAMIDINES OR COCAINE
Chemical Uptake as Percent of
Control Uptake
Mean + SE (n)
Chlordimeform, 10 M _5
Demethylchlordmeform, 10 M
FormetanateJ.0 M
U-40481, 10^4 M
Cocaine, 10 M
93 + 19
83 + 28
98 + 24
103 + 31
32 + 11
(6)
(5)
(6)
(6)
(6)
-8 3
9. Incubated for 30 minutes with chemicals, then 60 min with 10 M 1- H-norepine-
phrine.
32
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EFFECTS OF CHLORDIMEFORM ON CARDIOVASCULAR RESPONSES OF THE DOG
Chlordimeform caused several dose related effects on the cardiovascular system.
These include a fall in diastolic pressure (Fig. 20, 21A), a fall in systolic pressure (Fig.
20, 21B), an initial decrease (Fig. 20, 22A) followed by an increase in musculo^cutaneous
blood flow (Fig. 20, 22B), and, in the highest dose, an increased heart rate (Fig. 23). The
mean time to the maximal decrease in blood pressure following CDF injection for all
doses of CDF taken together was 26 + 2.1 sec. The mean time from the injection to the
maximal initial decrease in blood pressure was 27 + 2.0 sec. Thus the initial decrease in
blood flow seems to be related to the initial fall in blood pressure.
35
m
1/min.
10 sec.
8.5 min. later
180
140
100
60
20'
mmHg
Fig. 20. Effect of 10 mg/kg chlordimeform administered 10 sec. before start of tracing
on blood flow (upper tracing) and blood pressure (lower tracing) recorded from femoral
arteries of the anesthetized dog. (From Robinson et al., 1979).
33
-------
Chlordimeform Doselmg/kg)
Fig. 21. Reduction in systolic (A) and diastolic (B) blood pressure following i.v. chlor-
dimeform in dogs with no pretreatment ( D ), pretreatment with 2 mg/kg atropine
( A ),.5 mg/kg physostigmine ( Q ) or with 15 mg/kg propranolol ( • ). Each value is
the mean + SEM of observations from 5 dogs. (From Robinson et al., 1979).
34
-------
Chlordimeform (mg/Kg)
Fig. 22. Initial decrease (A) and subsequent increase (B) in blood flow in the dog femoral
artery following chlordimeform. Symbols same as in Fig. 21. Each value is the mean +
SEM of observations from 5 dogs. (From Robinson et al., 1979).
35
-------
o
z
<
X
u
Z
III
u
oc
oT
Chlordimcform Dose(mg/kg)
Fig. 23. Effect of chlordimeform on dog heart rate. Symbols same as in Fig. 21. Each
Value is the mean + SEM of observations from 5 dogs. (From Robinson et al., 1979).
The maximal increase in blood flow occurred 42 + 1.9 sec. after injection of CDF
and was after blood pressure had only recovered slightly (Fig.20).
Propranolol, the beta-adrenergic receptor blocking agent, was one of two receptor-
blocking drugs used to investigate the mechanism of the CDF-induced cardiovascular
responses. Propranolol did not significantly reduce the CDF-induced fall in blood
pressure at any point, and at one point the decrease in diastolic blood pressure was even
36
-------
greater in propranolol-treated dogs (Fig. 21). Propranolol reduced the initial decrease in
blood flow at only the two highest CDF-doses, and did not reduce the secondary increase
in blood flow (Fig. 22) nor affect CDF-induced alterations of heart rate (Fig. 23).
The muscarinic blocking drug atropine did not significantly affect CDF alterations
of blood pressure (Fig. 21). At only one CDF-dose did it reduce the initial decrease in
blood flow, and at only one dose did it reduce the secondary increase in blood flow (Fig.
22). It did not affect the small changes in heart rate which were caused only by higher
doses of CDF (Fig.23).
200T
-80J-
H.R.
tnitid
SYS. DIA. Bkxxi Flow
Fig. 24. Effect of 1/fg/kg isoproterenol on dog heart rate (H.R.), systolic blood pressure
(Sys.), diastolic blood pressure (Dias.) and musculocutaneous blood flow before ( D ) and
after ( • ) administration of 20.8 mg/kg of chlordimeform. Bar represents mean
percent change + SEM from 3 dogs. (From Robinson et aL, 1979)
The cholinesterase inhibitor physostigmine-treated dogs showed greater CDF-
induced effects on both blood pressure and blood flow. At several CDF doses, falls in
systolic blood pressure (Fig.21A), diastolic blood pressure (Fig. 21B), and initial blood
flow (Fig. 22A) and the increase in secondary blood flow (Fig. 22B) were all larger in
these dogs that had received physostigmine. Physostigmine treatment caused a signifi-
cantly faster heart rate following CDF treatment than was observed in control dogs.
37
-------
Isoproterenol increased heart rate, decreased both systolic and diastolic blood
pressure, slightly decreased initial blood flow in the femoral artery and markedly
enhanced the subsequent increase in blood flow (Fig. 24). Chlordimeform altered only
the increase in blood flow, increasing it from a 68% increase in control dogs, to a 157%
increase in CDF-treated dogs.
The effects of ACh on blood flow were qualitatively similar to those of isoprote-
renol (Fig. 25). CDF effects on ACh responses also resembled those isoproterenol re-
sponses, i.e. with a significant effect only on the secondary increase in blood flow.
Acetylcholine increased blood flow by 23% in control dogs and by 43% in CDF-treated
dogs.
100-r
UJ
-60 J-
Initial Subsequent
H.R. SYS. DIA. Blood Flow
Fig. 25. Effect of .4,//g/kg acetylcholine on cardiovascular responses before and after
20.8 mg/kg chlordimeform. Symbols, etc. as in Fig. 25. (From Robinson et al., 1979).
38
-------
Administered alone in a dose of 0.4 Xf g/kg, ACh caused a slight slowing of the
heart, a fall in blood pressure, and a decrease in blood flow in the femoral artery (Fig.
26). After CDF the decreases in blood flow and systolic blood pressure were
significantly different whereas heart rate and diastolic pressure were not.
Responses to epinephrine included increased blood pressure, a brief decrease in
blood flow, and a secondary increase in blood flow (Fig. 26), all of which were unaffected
by CDF. The compensatory slowing of the heart rate following epinephrine was blocked
by CDF, with a resulting increase in heart rate.
300-r
-100-1-
H.R. SYS. DIA. Blood Flow
Fig. 26. Effect of 2j4 g/kg epinephrine on cardiovascular responses before and after 20.8
mg/kg chlordimeform. Symbols, etc., as in Fig. 25. (From Robinson et al., 1979).
39
-------
Histamine also increased heart rate, decreased blood pressure, caused an initial fall
in blood flow and a subsequent increase in blood flow (Fig. 27), all quantitatively similar
to the effects of both isoproterenol and ACh. CDF altered only the secondary blood flow
increase, causing a large increase in blood flow from 16% in control dogs to 67% in CDF-
treated dogs.
100-r
Intkil Subsequent
H.R. SYS. DIA. Blood Flow
Fig. 27. Effect of 10/( g/kg histamine on cardiovascular responses before and after 20.8
mg/kg chlordimeform. Symbols, etc., as in Fig. 25. (From Robinson et al., 1979).
40
-------
Responses to tyramine before CDF were as expected for a compound that releases
catechlolamines, and to a lesser extent directly stimulates adrenergic receptors (Fig. 28).
These effects were not altered by CDF treatment.
240 -r
Initial Subsequent
H.R. SYS. DIA. Blood Flow
Fig. 28. Effect of 40/f g/kg tyramine on cardiovascular responses before and after 20.8
mg/kg chlordimeform. Symbols, etc., as in Fig. 2. (From Robinson et al., 1979).
41
-------
SECTION 7
DISCUSSION
STUDIES IN ISOLATED BLOOD VESSELS
Actions of Chlordimeform
This is as reported in Zelenski et al., (1978), Pharmacornechanical coupling has been
suggested as a name for the processes through which drugs can affect smooth muscle
contraction without a necessary change in membrane potential (Somlyo and
Somlyo, 1968). Contractile responses are elicited by a rise in free intracellular calcium
levels, while relaxation is brought about by a fall in the cytoplasmic concentration of
calcium (Bianchi, 1968). Drug-induced pharm acorn echanical coupling may result, at least
in part, from a rise in free intracellular calcium levels caused by an increased influx of
extracellular calcium (Somlyo and Somlyo, 1968a; Durbin and Jenkinson, 1961).
Activator calcium may also arise from displacement of membrane-bound calcium (Briggs,
1962). Thus druginduced contractions of smooth muscle may involve mobilization of
activator calcium from two sources: one, an extracellular or perhaps loosely bound
calcium; the other intracellular, membrane-bound or "sequestered" calcium (Hudgins and
Weiss, 1969; Hinke, 1965). Differences in maximal responses to vasoactive agents may
be due in part to either their unequal abilities to translocate calcium to contractile
proteins from an intracellular store or to an unequal maximal increase in calcium
permeability. In eliciting their responses, vasoactive agents seem to have considerable
selectivity in the sources from which they mobilize calcium (Hudgins and Weiss, 1969,
Hudgins and Weiss, 1969a; Goodman and Weiss, 1961; Adams et al., 1974; Goodman and
Weiss, 1974).
It appears that CDF affects both stores of calcium but has a proportionally greater
effect on extracellular or loosely bound calcium stores, if NE primarily releases bound,
intracellular calcium and potassium primarily affects extracellular or loosely bound cal-
cium as has been reported (Briggs, 1962; Hudgins and Weiss, 1968). Results of the effects
of vascular strips to various agonists, on their rates of contraction after a singlejconcen-
tration of agonist, on their rates of relaxation after addition of CDF, and on Ca flux
are consistent with this interpretation and will be discussed individually.
CDF antagonized contractions to all four agonists examined, with the order of
antagonism being potassium, histamine, serotonin, NE. The order of antagonism for
potassium, histamine, and NE is that reported by Hudgins and Weiss (1969a) in decreasing
order of mobilization of extracellular calcium and by Kalsner et al. (1970) in decreasing
order of effect of /^-diethyl-aminoethyl-diphenylpropylacetate which they judged to
selectively antagonize utilization of calcium from extracellular or loosely bound sites.
However, the antagonism of CDF for serotonin-induced contractions was not as great as
would have been anticipated from the results of the studies of Kalsner et al. (1970) with
/? -diethyl-aminoethyl-diphenylpropylacetate, which they found to resemble potassium
as to the pools of calcium which it affects. We are unable to explain this difference,
42
-------
except that the effects of serotonin in the present study may have been somewhat po-
tentiated by CDF inhibition of the enzyme MAO which inactivates serotonin, thus par-
tially overcoming the antagonism of CDF. Thus the relative antagonism of CDF for NE,
potassium, and histamine (but not serotonin) are in the order anticipated for an agent
that interferes more with intracellular or loosely bound calcium stores.
CDF slowed contractions induced by all of the agonists. Serotonin and sometimes NE
responses were thereby resolved into two peaks. The response to NE is biphasic, with the
initial response probably being mediated by release of intracellular calcium and the slow
phase by the release of loosely bound calcium or an influx of extracellular calcium
(Brodie et al., 1959; Bohr, 1963; Sitrin and Bohr, 1971). Thus the resolution by CDF of
the biphasic response to NE into two partially separated peaks must result from a slowing
of the second phase of contraction.
CDF caused a rapid decrease in tension in muscles contracted by all of the agonists.
Even if CDF affected only extracellular of loosely bound calcium, it would be expected
to partially relax NE-induced contraction as the slow phase of contraction seems to be
mediated through this calcium source (Goodman and Weiss, 1971; Kalsner et al., 1970).
That NE contractures could only be partially antagonized may indicate that ITE mobilized
calcium from some sources unaffected by CDF.
Actions of Demethylchlordimeform
This is as reported in Robinson and Bittle, 1979. DCDF causes contractions of the
rabbit central ear artery which are not reduced by blocking the muscarinic, histaminergic
or serotonergic receptors, but are reduced by blocking alpha-adrenergic receptors. There
are two methods by which alpha-adrenergic stimulation can occur within the rabbit
central ear artery. One is by increasing the concentration at the receptor of NE from
sympathetic nerve stores; the other is by a direct action on the alpha-adrenergic
receptor. Among the several mechanisms whereby DCDF could increase NE con-
centrations at the receptor is inhibition of MAO. DCDF does inhibit MAO and this
could, theoretically at least, cause an increase in NE accumulation at alpha-adrenergic
receptors. However, because reduction of NE stores by prior reserpimzation of the
rabbits did not reduce DCDF-induced contractions and because the addition of DCDF to
vascular strips preloaded with tritiated NE did not affect tissue stores of NE, it thus
seems to be acting directly at the alpha-adrenergic receptor as a partial agonist.
The effects of DCDF on vascular tissue have not been previously reported, but in the
rabbit aorta (Zelenski, et al., 1978) as well as in the central ear artery (unpublished
observations) CDF, unlike DCDF in the central ear artery, had almost no agonist
activity. CDF does have agonist activity_jn the rectus abdominis muscle from some
species of frogs (Rana nigromaculata in 10 M, and Rana Catesbianabut only in 10 _M
concentrations, Watanabe et al., \L975). In another speciesTTlana pipiens, CDF does not
cause contractions of the esennized rectus muscle in 10 IM (Beeman and Matsumura,
1974). In the one species in which 10 M DCDF has agonist activity, it caused a slow
contraction unaffected by d-tubocurarine~lWatanabe et al., 1975). The authors specu-
lated that the contraction may result from depolarization of the end-plate membrane,
but it is apparent that it did not result from a direct stimulation of the cholinergic
receptor. Thus, even in the tissue where agonist activity has been shown for the parent
compound, it does not seem to result from receptor stimulation. That DCDF has agonist
activity at the alpha-adrenergic receptor and the parent compound CDF does not, may
seem unusual, as DUDF differs from CDF only in that a methyl group has been replaced
43
-------
by a hydrogen on the terminal nitrogen. However, if this nitrogen is considered anala-
gous to the one in epinephrine, then both DCDF and epinephrine have the same substi-
tuent groups on this nitrogen, and CDF has the same groups as does N-methylepinephrine.
Epinephrine is an excellent alpha-receptor stimulant, while N-methylepinephrine, like
other teniary amines of similar structure, does not stimulate alpha-adrenergic receptors.
DCDF has antagonist activity as well as agonist activity. That a partial agonist such
as DCDF antagonized contractions to the full agonist NE is expected, as partial agonists
are also partial antagonists. DCDF also reduced potassium contractures. The
contractions of rabbit aorta to these two agonists as well as to serotonin and histamine
were also antagonized by CDF in the rabbit aorta (Zelenski; et al., 1978). CDF antago-
nized contractions of non-vascular muscle. Watanabe et al. reported that CDF non-com-
petitively antagonized contractions of the frog rectus abdominis muscle caused by potas-
sium and acetylcholine (Watanabe et al., 1978), and Yamamoto and Fukami reported that
it blocks at the neuromuscular juncTTon of the larvae of the waxmoth, Galleria mellonella
(Yamamoto and Fukami, 1976). In the studies on NE flux using labeled NE, there was
little effect on either resting NE flux or on the amount of NE released by electrical
stimulation or by nicotine. No comparable studies with either DCDF or the parent
compound on NE flux have been previously reported. However, CDF did not alter the
release of another transmitter, acetylcholine, in the frog sciatic nerve sartorius Muscle
preparation(Wang et al., 1975).
Thus DCDF, an active metabolite of CDF, stimulates alpha-adrenergic receptors, and
in high concentrations can block alpha-adrenergic and other receptors. If these actions
observed at the alpha-adrenergic receptor in the central ear artery also occur at alpha-
receptors within the brain, it is possible that this direct effect of DCDF may account for
part of the central toxic signs (Beeman and Matsumura, 1973; Aziz and Knowles, 1973)
observed after the injection of the parent compound CDF.
Actions of U-40481 and Formetanate
This is as reported in Robinson, 1979. These two formamidines, one a cholinesterase
inhibiting acaricide and insecticide, the other a metabolite of an acaricide and insec-
ticide, have several common effects on isolated arteries. One of these effects is agonist
activity. Lower concentrations of both compounds cause dose-related ear artery con-
tractures which are markedly reduced by phentolamine. Thus they both seemingly cause
activation of the alpha receptor. U-40481, a secondary amine, is much more potent at
this effect than the tertiary amine formetanate. DCDF also had alpha-adrenergic
activity, whereas the N-methylated tertiary amine CDF did not.
Activation of alpha-adrenergic receptors does not seem to be due to release of NE
because neither compound altered the resting rate of radioactive efflux from HNE
loaded strips and in fact both decreased the electrically-induced radioactive efflux.
MAO-inhibiting activity has been demonstrated for several formamidines including U-
40481 (Aziz and Knowles, 1973; Beeman and Matsumura, 1973). However, it is not likely
that MAO-inhibition and a resulting decreased amine metabolism contributes to the
agonist activity of these compounds since acute exposure even to potent MAO inhibitors
does not usually cause contractions of arterial strips.
The antagonist activities of the two formamidines also varied considerable. For-
metanate had little, while an equimolar concentration of U-40481 markedly reduced
44
-------
contractions ot NE, serotonin and histamine, but not to potassium. That antagonism by
U-40481 to NE and serotonin contractures is similar is consistent with an earlier report
that both of those amines contract the rabbit ear artery through stimulation of the same
phentolamine sensitive receptor (Apperley et al., 1974).
The antagonism for histamine-induced contractions was somewhat less than for NE
and serotonin contractures, and must be at a different receptor since histamine
contractures do not result from alpha-adrenergic receptor stimulation (Carrol et al.,
1977). Since all antagonist activities were reversible, they did nqt result from irrever-
sible binding or from damaging components of the contractile mechanism.
U-40481 and formetanate-inhibition of electrically-induced release of radioactivity,
may be related to local anesthetic-like activity similar to that reported for another for-
mamidine (Chinn et al., 1976). These compounds have not been examined for that ac-
tivity. On the other "Rand, DCDF did not inhibit electrical release but the concentration
used was one-one hundreth of the one used in this study.
Structurally, U-40481 and formetanate differ in two respects. One is that U-40481
has a hydrogen and a methyl group on the terminal nitrogen, while formetanate has two
methyl groups. The other is on the ring where U-40481 has two methyl groups while
formetanate has a carbamyl group. It is the carbamyl moiety that confers upon forme-
tanate cholinesterase-inhibiting activity, and it may, in addition to the extra methyl
group on the nitrogen, contribute to the reduced alpha-adrenergic agonist activity com-
pared to U-40481. This bulky group may also contribute to the relative inactivity as an
antagonist that was observed with formetanate.
STUDIES IN THE ANESTHETIZED DOG
Effects of Chlordimeform
CDF markedly affected cardiovascular function in the anesthetized dog. It caused an
initial decrease followed by an increase in musculocutaneous blood flow. A decrease in
blood pressure was also observed at the same time the maximal increase in blood flow
occurred. Thus, these were both probably caused by vasodilation. The initial decrease in
blood flow may have been caused by constriction of resistance vessels of the hindlimb.
This could result form central stimulation, or from peripheral actions. In the
experiments on isolated rabbit arteries, although other formamidines did contract ar-
teries directly, this was not consistantly observed with CDF. It is possible that CDF may
directly constrict smaller resistance vessels initially and then cause vasodilation due to
some direct effects on the blood vessels later.
Effects of Receptor Antagonists on Chlordimeform Induced Cardiovascular Changes
CDF could relax blood vessels by stimulating either y^-adrenergic or muscarinic
receptors found in the medial layer of blood vessels. However, because neither blockade
of /fi'-adrenergic receptors by propranolol, nor blockade of muscarinic receptors by atro-
pine blocked the effects of CDF, it seems that CDF-induced cardiovascular effects do
not involve stimulation of those two receptors. With physostigmine, however, there were
some increased effects. This may indicate some involvement of muscarinic sites, or
perhaps the effect is due to hemodynamic or other changes caused by physostigmine.
45
-------
Effects of Cholrdimeform on Cardiovascular Actions of Vasoactive Compounds
Tyramine exerts its pressor effects through stimulation of adrenergic receptors to an
extent, and to a much greater extent through release of catecholamines stored in
sympathetic nerves. It is inactivated by MAO. If CDF inhibits MAO sufficiently to
reduce the rate of inactivation of tyramine, then tyramine's effects should be intensified.
Since tyramine's cardiovascular effects were not significantly altered by CDF, several
inferences as to CDF actions can be made. One is, that CDF inhibition of MAO, even at
this high dosage is not physiologically important, at least not as far as the vascular
system is concerned. Another is, that CDF does not reduce the tyramine-induced release
of catecholamines from sympathetic nerves. A third is, that CDF blocks neither alpha
r the
nor beta adrenergic receptors in the dog cardiovascular system. Further evidence for
lack of alpha- or beta-blocking ac
blocking activity with isoproterenol.
lack of alpha- or beta-blocking activity was obtained with epinephrine, and for beta-
activi
«
Histamine exerts its effects by stimulating two kinds of receptors: H, receptors and
receptors. Both are found in the cardiovascular system, with both receptors being
involved in vasodilation, (Turker, 1973; Roberts et al., 1979), and probably primarily H2
receptors involved in the stimulant action on theTeart (Shimizu et al., 1970). From the
data on blood flow there is no evidence of blocking the vasodilatioTTinduced by histamine,
nor is there any decrease in heart rate. Heart rate in vivo following a fall in blood
pressure is increased primarily by non-histaminergic me"chanisms, and therefore drawing
firm conclusions about histamine effects form an innervated heart in vivo is not possible.
CDF enhancement of vasodilation as demonstrated by increased blood flow following
several different vasodilator drugs may be related to calcium mobilization, but no studies
were done to directly study this possibility in vivo.
Thus, in summary of the study of the mode of action of the profound cardiovascular
effects of CDF, we have ruled out several possible modes of action, because the effects
appear not to be caused primarily by stimulation or blockade of the common vascular
receptors. CDF may affect calcium availability, as CDF effects on calcium flux have
been noted in the studies on isolated arteries.
46
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49
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
. REPORT NO.
EPA-600/1-79-031
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
Effects of Chlordimeform on Vascular Tissue
5. REPORT DATE
August 1979
6. PERFORMING ORGANIZATION CODE
. AUTHOR(S)
Casey P. Robinson
8. PERFORMING ORGANIZATION REPORT NO.
9. °ERFORMING ORGANIZATION NAME AND ADDRESS
University of Oklahoma
Health Sciences Center
P.O. Box 26901
Oklahoma City, OK 73190
10. PROGRAM ELEMENT NO.
1EA615
ill. CONTRACT/GRANT NO.
R804975
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
13. TYPE OF REPORT AND PERIOD COVERED
RTP, NC
14. SPONSORING AGENCY CODE
EPA 600/11
15. SUPPLEMENTARY NOTES
16. ABSTRACT
In order to fully understand potential risk to human beings of exposure to a chemical
one needs to understand the effects of that chemical upon each organ system and to deter-
mine its mode of action. For some classes of pesticides their primary mode of toxicity
has been established. An example of this is the organophosphorus cholinesterase inhibi-
tors. There is general agreement that their acute lethality and most of their toxicities
result from cholinesterase inhibition and subsequent acetylcholine accumulation. Even
among this group of compounds, however, there are toxic effects which seem to be due to
other actions.
For other classes of compounds much less is known of their mechanisms of lethality
and effects on organ systems. An example of this is the formamidines, the first useful
one of thich was chlordimeform. Little information on the mode of action of chlordime-
form was available, and on its other effects. This study was initially proposed to deter-
mine vascular actions of chlordimeform. The project was expanded to examine the
cardiovascular actions of other available formamidines including formamidine metabolites
which also retained the formamidine structure.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b-IOENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Demethylchlordimeform
Pesticides
Serotonin
In Vitro
Phentolamine
06B,T
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)
UNCLASSIFIED
21. NO. OF PAGES
63
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (Rev. 4-77) PREVIOUS EDITION is OBSOLETE
50
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