EPA-600/1-76-035
November 1976
Environmental Health Effects Research Series
FOR
EFFECTS OF
>
EP 600/1
76-035
f
55
\
5SSZ
\
LU
of Research and Development
** '*' '- ''
-------
RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environ-
mental Protection Agency, have been grouped into five series. These five broad
categories were established to facilitate further development and application
of environmental technology. Elimination of traditional grouping was con-
sciously planned to foster technology transfer and a maximum interface in
related fields. The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS
RESEARCH series. This series describes projects and studies relating to the
tolerances of man for unhealthful substances or conditions. This work is gener-
ally assessed from a medical viewpoint, including physiological or psycho-
logical studies. In addition to toxicology and other medical specialities, study
areas include biomedical instrumentation and health research techniques uti-
lizing animals—but always with intended application to human health measures.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
-------
EPA-600/1-76-035
November 1976
IN-VITRO METHODS FOR EVALUATING SIDE EFFECTS
OF PESTICIDES AND TOXIC SUBSTANCES
By
Toshio Narahashi, Ph.D.
Department of Physiology and Pharmacology
Duke University Medical Center
Durham, North Carolina 27710
Contract No. 68-02-1289
Project Officer
Lawrence Rosenstein, Ph.D.
Environmental Toxicology Division
Health Effects Research Laboratory
Research Triangle Park, N.C. 27711
..jlcCTION AGENCY
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
HEALTH EFFECTS RESEARCH LABORATORY
RESEARCH TRIANGLE PARK, N.C. 27711
-------
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.
-------
FOREWORD
The many benefits of our modern, developing, industrial society are
accompanied by certain hazards. Careful assessment of the relative risk
of existino 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 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 project described herein was initiated to meet program needs relevant
to the development of in vitro test systems for pesticide toxicity. From a
research point of view, the type of methodology developed can be used not only
to predict the relative degree of toxicity but also to corroborate the results
obteined using animal dose response models.
John H. Knelson, M.D.
Director,
Health Effects Research Laboratory
m
-------
CONTENTS
Page
I. INTRODUCTION 1
II. METHODS 2
A. Materials 2
B. Methods of Recording of Muscle Contractions 5
III. RESULTS 9
A. Guinea Pig Ileum 9
B. Guinea Pig Heart 18
C. Guinea Pig Vas Deferens 20
D. Rat Diaphragm 21
E. Frog Rectus Abdominis 22
IV. SUMMARY AND CONCLUSIONS 23
V. REFERENCES 28
VI. TABLES 29
VII. FIGURES 55
-------
ABSTRACT
Several skeletal muscle and smooth muscle preparations have been examined
for their usefulness in evaluating the toxic effects of a variety of insecticides.
The following preparations were found satisfactory for such test: guinea pig
ileum for muscarinic receptors, guinea pig heart for B-adrenergic receptors,
guinea pig vas deferens for a-adrenergic receptors, frog rectus abdominis for
nicotinic receptors of tonic muscle, and rat diaphragm for nicotinic receptors
of phase muscle. Five carbamate insecticides, four organophosphate insecticides
and chlordimeform were studied. None of the insecticides tested had any direct
and potent effect on these receptors except the effect on cholinergic receptors
via cholinesterase inhibition. Carbofuran, propoxur and formetanate had potent
stimulating actions on the guinea pig ileum, but these effects could entirely
be attributed to the accumulation of acetylcholine in the synaptic cleft as a
result of cholinesterase inhibition. Thus it can be concluded that these
insecticides exert no direct action on cholinergic and adrenergic receptors.
-------
I. INTRODUCTION
A variety of chemicals which are in use in agriculture, industry, and homes
are potentially hazardous to humans. In addition, a large number of new chemicals
are being developed for use, many of which are also potential hazardous compounds.
Although the mechanism of major toxic action of some of these compounds is known,
there could be a variety of side effects which might be responsible for acute and/or
chronic toxicity caused by these substances.
In our previous study under the EPA Contract No. 68-02-1289, efficient,
accurate and inexpensive methods have been developed to evaluate the neural toxicity
of various pesticides and toxic compounds (Narahashi, 1976). The abdominal nerve
cord preparation of the crayfish has been found to be most satisfactory among the
various preparations examined. A variety of toxic compounds including organophosphate,
carbamate, and pyrethroid insecticides exert very potent actions on this preparation.
In our second year study, we have developed methods whereby various side effects
of toxic compounds can be evaluated. One of the most important examples is
organophosphate and carbamate pesticides. It has been shown by a number of inves-
tigators that the inhibition of cholinesterases is the major mechanism whereby both
mammals and insects are intoxicated by these pesticides. However, there is some
notion that certain anticholinesterase pesticides do exhibit effects through the
mechanisms other than the inhibition of cholinesterases. Such actions, sometimes
called "direct actions", could pose serious problems when the aspects of safety,
environment and treatment of intoxication by these compounds are considered. These
possible side effects produced by the mechanisms other than cholinesterase inhibition
are most probably responsible for a variety of systemic and behavioral effects which
cannot be ascribed to excess stimulation of cholinergic receptors as a result of the
-------
inhibition of cholinesterases. This problem has been largely ignored, and only
very limited information is available despite its paramount importance. If there
were serious side effects other than cholinesterase inhibition, routine approaches
to the therapeutics such as those utilizing atropine and pralidoxime would not
guarantee the complete safety. The same consideration applies to any other
chemicals in use which are potentially hazardous and have multiple effects on human
and other mammals.
The purpose of this study is to accomplish the following goals:
1. To compare various skeletal muscle and smooth muscle tissues for their
usefulness in evaluating the toxic effects of a variety of substances
including organophosphate and carbamate insecticides on cholinergic and
adrenergic receptors.
2. To establish techniques whereby the toxic side effects other than those
ascribed to cholinesterase inhibition can be evaluated with the tissues
selected in project (1) above.
3. To study some toxic substances for their side toxic effects using the
method established by project (2) above.
II. METHODS
A. Materials
One of the main purposes of this project was to test some skeletal and smooth
muscle tissues for evaluation of toxic nature of various chemicals. Some muscles
have nicotinic receptors but are devoid of muscarinic receptors and catecholamine
receptors. Some other smooth muscles are innervated by both sympathetic and
parasympathetic nerves, so that both muscarinic and adrenergic receptors exist.
By using an appropriate blocking agent acting on one of the receptors, one should
-------
be able to study the effect of a test compound on the other receptor of the tissue.
In fact, this is a routine technique to study the interaction of a test compound
with any particular type of receptors, and has been used very often. However,
the data on pesticides and other environmental agents with respect to such interaction
are very limited. The selection of the material was based on specificity for each
type of receptor (such as nicotinic, muscarinic, a-adrenergic and 3-adrenergic),
easiness of handling, reproducibility of results, and costs. The following prepara-
tions were examined.
Guinea pig ileum
The isolated preparation of the ileum of the guinea pig contains the
parasympathetic ganglia, the parasympathetic preganglionic and postganglionic
fibers, the sympathetic postganglionic fibers, and the smooth muscle with both
muscarinic and adrenergic receptors. With combination of appropriate blocking
agents, it is possible to study the effect of a toxic substance on one of these
systems. For example, with the preparation treated with an adrenergic blocking
agent and a gangl ionic blocking agent the effect of a test compound on the
muscarinic receptor can be examined. If the test compound had a muscarinomimetic
action, stimulation of the preparation would be manifest in the form of contraction.
If it were a muscarinic blocking agent, the contractile response of the preparation
to acetylcholine would be diminished.
For anticholinesterases such as organophosphate and carbamate insecticides,
the direct effects other than those produced by cholinesterase inhibition can be
studied with prior application of an inhibitor of transmitter release such as
magnesium ions and black widow spider venom. An alternative method is to denervate
the muscle.
-------
Guinea pig heart
The guinea pig heart contains B-adrenergic receptors, and has been used for
the study of sympathomimetic drugs with a specific affinity for B-receptors.
Muscarinic blocking agents such as atropine can be used to block the muscarinic
receptors on this heart preparation.
Guinea pig vas deferens
The guinea pig vas deferens contains a-adrenergic receptors, and has been
used for the study of sympathomimetic drugs with a specific affinity for a-receptors.
Muscarinic blocking agents such as atropine can be used to block the muscarinic
receptors on this preparation.
Frog rectus abdominis
The frog rectus abdominis has been widely used for the study of drugs acting
on nicotinic cholinergic receptors. It is routinely used to examine the potency
of agonists acting on nicotinic receptors. There is no sympathetic innervation, so
that no complication will arise as a result of the possible effect on the adrenergic
receptors. This preparation is composed of tonic muscle fibers, and nicotinic
agonists cause a contracture.
Rat diaphragm
The phrenic nerve-diaphragm preparation isolated from the rat has been used
for the study of drugs acting on nicotinic cholinergic receptors. Unlike the
frog rectus abdominis, the diaphragm is composed of phasic muscle fibers. Therefore,
nicotinic agonists cause a transient depolarization of the end-plate membrane
evoking tetanus. This phase is followed by a desensitization block.
-------
B. Methods of Recording of Muscle Contractions
Guinea-pig 11 euro
The ileum was isolated from the male guinea pig weighing 450-550 g. The
isolated tissue was cut in a length of 2 cm, tied off with silk threads at both
ends, and mounted in a chamber of 15 ml capacity as illustrated in Fig. 1. One
of the threads was connected with a force-displacement transducer (Grass model
FT03C). The other thread was connected with a metal rod mounted on a micromanip-
ulator which permitted adjustment of the muscle tension. Air was introduced from
the bottom of the chamber, and the glass filter was effective in making fine air
bubbles. Tyrode's solution (solution A, Table 1) was led into the chamber and
was sucked up from the surface. The chamber was immersed in a water bath except
for its top portion to maintain the temperature constant at 37°C. The output of
the transducer was fed into a preamplifier (Grass model 5E) and contractions were
recorded on a Grass Polygraph (model 5DWC.1).
Test solution (0.2 ml) was injected into the bath in the chamber (15 ml) while
suspending perfusion. Thus the test solution was diluted by a factor of 75. The
final concentration of test compound was given in this paper.
In some experiments, denervated preparations were used. Two methods were
employed, one being turned the ileum inside out and the other being the one
developed by Paton and Zar. In the former method, a piece of the ileum in about 10
cm long was turned inside out on a glass rod of 6 mm in diameter. The internal
surface of the ileum, which was now faced outside, was stroked tangentially by
means of a wisp of cotton wool so as to remove the circular muscle and the Auerbach's
plexus. Thus only the longitudinal muscle layer remained on the glass rod.
The method of Paton and Zar is summarized as follows:
The ileum was stretched on a glass rod of 6 mm diameter, and was gently
pulled upwards by applying traction at the proximal end to obtain a
a stip of longitudinal muscle. Auerbach's plexus could be visible under
-------
a dissecting binocular microscope, but most regions of the strip
were free of the plexus. A stretch of the strip where no plexus
was found was cut off and used as the nerve-free ileum preparation.
The denervated ileum was mounted in the chamber in the same manner as that
for the intact ileum. The only differences were that a mixture of 95% CL and
5% of CO,, instead of air was used to bubble the bathing medium, and that solution
C was used instead of solution A (Table 1).
Guinea pig vas deferens
The vas deferens was isolated from the male guinea pig weighing 450 to 550 g,
and cut in a length of 1.5 cm. The preparation was tied off at both ends with
silk threads, and mounted in a 15 ml glass filter chamber as described in the
preceding section for the guinea pig ileum. The methods of recording contractions
and application of test compounds, and temperature were the same as those for the
ileum. Solution C (Table 1) was used as the bathing medium.
Guinea pig heart
The heart was isolated from the male guinea pig weighing 450 to 550 g. The
aorta was cannulated by a glass tubing of 2 mm in outer diameter which was in
turn connected with the inlet polyethylene tubing (Fig. 2). The side arm of
the cannula was capped with a rubber membrane through which test solution (0.2 ml)
was injected into the perfusate. The reservoir of Tyrode-Locke solution (solution E,
Table 1) was kept about 1.5 m above the heart, and was bubbled with a gas mixture
of 95% 02 and 5% CO,,. The solution was then passed through a water bath with a
temperature of 45°C, and then perfused through the heart. The temperature of the
heart was maintained at 33-40°C The ventricle of the heart was attached to a
hook which in turn was connected with a force displacement transducer (Grass model
FT03C), and contractions were recorded on a Grass Polygraph (model 5DWC.1) via a
preamplifier (Grass model 5E).
-------
Rat diaphragm
The diaphragm was isolated with a stretch (1.5 cm) of the phrenic nerve
attached from the rat weighing 90 to TOO g (male and female mixed). One end
of the muscle was tied off with a silk thread which in turn was connected with
a force-displacement transducer (Grass model FT03C) (Fig. 3). The other end was
tied off at a few points with silk threads which were connected with a metal
rod. The rod was mounted on a micromanipulator which permitted adjustment of the
tension of the muscle. The preparation was placed in a 60 ml glass filer chamber
containing physiological saline solution. Solution D (Kreb's solution II) or
solution B (Tyrode's solution II with a double amount of glucose) (Table 1) was
used as the bathing medium. The perfusate was introduced to the glass filter
in a water bath at a temperature of 37°C, and was sucked from the surface of the
chamber. A gas mixture of 95% 0? and 5% COp was introduced from the bottom of
the chamber, and was converted into fine bubbles when it passed through the glass
filter. The cut end of the phrenic nerve was sucked into a suction electrode which
consisted of a glass capillary of 0.1 mm inner diameter. The suction electrode
was connected with a stimulator via an Ag-AgCl wire.
Electric pulses of 15-30 V in intensity and 0.3-10 msec in duration were
applied to the nerve every 5 sec as supramaximal stimuli. The nerve evoked
diaphragm contractions were recorded on a Grass Polygraph (model 5DWC.1) via a
preamplifier (Grass model 5E). Test solution (0.3 ml) was injected into the
bath (60 ml) while suspending the perfusion. Thus the test solution was
diluted by a factor of 200. Dose was expressed as the final concentration in
the bath.
Frog rectus abdominis
The rectus abdominis was isolated from the frog Rana pi pi ens about
two inches in length. The methods of mounting in the chamber and recording
contractions were the same as those for the guinea Dig ileum. Air bubbled
7
-------
frog Ringer's solution (solution F, Table 1) was used as the bathing medium which
was kept at a constant temperature of 20°C.
C. Chemicals
The following chemicals acting on the autonomic nervous system were used in
the present study. Unless otherwise stated, pure chemicals were used: Acetylcholine
chloride (Sigma Chemical Co., St. Louis, Mo.), physostigmine salicylate (Sigma
Chemical Co.) and Antilirium ampules from O'Neal, Jones and Feldman, Inc., St. Louis,
Mo.), neostigmine mesylate (Prostigmin from Rocke Lab., Division of Hoffman-La Roche,
Inc., Nutley, N. J.), atropine sulfate (Atropine from Elkins Sinn, Inc., Cherry
Hill, N. J.), phentolamine mesylate (Regitine from CIBA Pharmaceutical Co., Division
of CIBA-GEIGY Co., Summit, N. J.), propranolol hydrochloride (Sigma Chemical Co.),
carbamylcholine chloride (Aldrich Chemical Co., Inc., Milwaukee, Wis.), isoproterenol
hydrochloride (Isuprel from Winthrop Lab., Division of Sterling Drug Inc., New York,
New York), norepinephrine hydrochloride (Sigma Chemical Co.), d-tubocurarine (Lilly
Laboratories, Indianapolis, Ind.), diphenhydramine hydrochloride (Benadryl from
Parke-Davis, Detroit, Michigan), hexamethonium bromide (Sigma Chemical Co.),
nicotine dihydrochloride (J. T. Baker Chemical Co., Phillipsburg, N. J.), and
histamine dihydrochloride (K & K Laboratories, Plainview, New York).
The insecticides examined were provided by the Environmental Protection Agency,
National Environmental Research Center, Research Triangle Park, North Carolina.
They were carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl methylcarbamate),
carbaryl (1-naphthyl N-methylcarbamate), leptophos (0-(4-bromo-2,5-dichlorophenyl)
0-methyl phenylphosphonothioate), monocrotophos (3-hydroxy-N-methylcrotonamide
dimethyl phosphate), dichlofenthion (diethyl 2,4-dichlorophenyl phosphorothionate),
dursban (0,0-diethyl 0-3,5,6-trichloro-2-pyridyl phosphorothioate), propoxur (2-
isopropoxyphenyl N-methylcarbamate), ferbam (ferric dimethyldithiocarbamate),
formetanate (3-(((dimethylamino)methylene)amino)phenyl N-methylcarbamate hydrochloride;
and chlordimeform (N'-(4-chloro-o-tolyl)-N-N-dimethylformamidine). Formetanate was
8
-------
directly dissolved in aqueous solutions. Ferbam was first dissolved in
dimethylsulfoxide (DMSO) to make up a stock solution which in turn was diluted
with perfusate before experiment. The final maximum concentration of DMSO in
test solutions was 0.0133% (v/v), and had no effect on the preparations used.
All other insecticides were dissolved in ethanol to make up stock solutions
which in turn were diluted with perfusate. The final maximum concentration
of ethanol was 0.0133% (v/v), and had no effect on the preparations used.
III. RESULTS
A. Guinea Pig Ileum
Drugs acting on autonomic nervous system
A variety of drugs acting on the sympathetic and parasympathetic nervous
systems were examined for their effects on the guinea pig ileum to establish
the control picture of drug action. A representative drug was selected for
each class of action.
a. Acetylcholine. Acetylcholine (ACh) was tested as a representative of
agonists acting on muscarinic and nicotinic receptors. It caused a contraction
of the ileum at low concentrations. An example of such a record is illustrated
in Fig. 4. The effect of ACh was completely reversed after washing with drug-
free medium. The dose-response relation is shown in Fig. 5. On the assumption
of a one-to-one stoichiometric interaction between ACh and receptor, the apparent
dissociation constant was estimated to be 2,67 x 10 M from the Lineweaver-Burk
plot.
-9
b. Physostigmine. At low concentrations ranging from 1.33 x 10 M to
_n
1.33 x 10 M, physostigmine itself did not cause a sizable contraction of the
ileum. However, the ACh contraction was potentiated by physostigmine at
-9 -8
concentrations of 3.99 x 10 M to 3.99 x 10 M. An example of the potentiated
contraction is illustrated in Fig. 4. In order to assess the optimal time for
-------
o
physostigmine pretreatment, the contraction induced by ACh (1.55 x 10 M to
-7 8
1.55 x 10 M) was measured after pretreatment with 1.33 x 10 M physostigmine
for various periods of time. The results are summarized in Fig. 6. The
physostigmine-induced potentiation appeared after 1 min of pretreatment, and
after 5 min the potentiation was observed invariably. Therefore, the ileum was
pretreated with physostigmine for 5 min in the subsequent experiments. Table 2
gives the results of two experiments in which the relative potency of various
concentrations of physostigmine in potentiating the ACh response was examined.
_Q
Physostigmine had no potentiating effect at 1.33 x 10 M, and the threshold
-9
concentration was estimated to be 4 x 10 M. The physostigmine-induced
potentiation of ACh response is interpreted as being due to inhibition of
acetylcholinesterase (AChE).
At high concentrations, physostigmine itself induced contractions. An
example of such a record is illustrated in Fig. 7. Unlike the contraction
induced by ACh, the physostigmine-induced response was characterized by a
long latent period, a slow onset, and repetitive contractions. A slight sign
-8
of contractions or increasing tone was visible with 1.33 x 10 M physostigmine,
-8
and the response was clear at 3.99 x 10 M or higher concentrations. Data are
given in Table 3. The mechanism underlying the direct stimulation of ileum by
physostigmine will be discussed later.
c. Neostigmine. As is expected from the ability to inhibit AChE, neostigmine
had a potentiating action on ACh response. An example of dose-response curves
of ACh before and after treatment with 1.14 x 10~ M neostigmine is illustrated
in Fig. 8.
d. Atropine. Atropine is a competitive antagonist agains the ACh stimulation
of muscarinic receptors. It suppressed the response of the ileum to ACh (Fig. 9).
The time course of the action of atropine is shown in Fig. 10. At a concentration
Q
of 1.33 x 10" M, the atropine treatment for a period of 3-5 min was sufficient
10
-------
to block the contraction caused by 7.74 x 10~ M ACh. Therefore, atropine was
applied for 5 min in the subsequent experiments. Dose-response curves for ACh
_o
contraction before and after treatment with 1.33 x 10 M atropine are illustrated
in Fig. 11. The curve with atropine is shifted along the abscissa in the direction
of higher ACh concentration indicating a competitive antagonism between these two
drugs.
e. Phentolamine. Phentolamine is an a-blocking agent, and was tested on
tha guinea pig ileum for its possible action. It had no marked effect on the ACh-
induced contraction at low concentrations up to 1.33 x 10" M, but suppressed the
ACh response at higher concentrations (Fig. 12). The time course of action of a
higher concentration of phentolamine (1.33 x 10" M) is illustrated in Fig. 13.
-8
The contraction induced by 7.74 x 10 M ACh was gradually suppressed as the
phentolamine pretreatment was prolonged, and a near maximum suppression was
observed with a 3 min pretreatment. Thus the phentolamine pretreatment was set
at a 5 min period in the subsequent experiments. Dose-response curve of
phentolamine to suppress the ACh contraction is illustrated in Fig. 14. The
concentration of phentolamine to produce a 50% effect was of the order of 10 M.
Fig. 15 shows an example of dose-response curves of ACh contraction before and
after treatment with 1.33 x 10" M phentolamine. The curve is shifted in the
direction of high concentration of ACh after pretreatment with phentolamine,
indicating a competitive antagonism between the two drugs. The last observation
strongly suggests that the phenotlamine-induced suppression of ACh contraction
is not due to its effect on the a-receptor but due to its inhibitory effect on
the muscarinic receptor.
f. Propranolol. Propranolol is a 3-blocking agent, and was tested on
the guinea pig ileum for its possible effect. It had no effect on the ACh
contraction except when it was given at a very high concentration (Fig. 16).
11
-------
Dose-response relation is illustrated in Fig. 17. The inhibitory effect of the
high concentration of propranolol is presumably due to direct action on the
muscarinic receptor.
Insecticides
Ten insecticides were examined for their direct effects on the guinea pig
ileum and their ability to change the contractions induced by ACh and carbachol.
Carbachol was used to distinguish the effect through the inhibition of AChE and
the direct effect on the muscarinic receptor.
a. Carbofuran. Carbofuran caused little or no contraction at a concentration
_o
of 1.33 x 10 M (Fig. 18). However, potent contractions were induced at higher
concentrations ranging from 1.33 x 10 M to 1.33 x 10 M (Fig. 18). Despite
the strong stimulating action, carbofuran did not exhibit marked potentiating or
depressing effect on the ACh- or carbachol-induced contraction (Fig. 19). The
data are summarized in Table 4.
b. Carbaryl. An example of a record of muscle tension in the presence of
1.33 x 10-1.33 x 10 M carbaryl is illustrated in Fig. 20. There was practically
no effect on the ileum. Carbaryl exerted no direct effect on the ileum, and did not
affect the ACh- or carbachol-induced contraction (Fig. 20B, Table 4).
-9 -6
c. Leptophos. In concentrations ranging from 1.33 x 10 M to 1.33 x 10 M,
leptophos did not induce any sizable contraction by itself (Fig. 21), and had no
effect on the ACh- or carbachol-induced contraction (Table 4).
d. Monocrotophos. Monocrotophos did not induce contraction by itself and
had no influence on the ACh- or carbachol-induced contraction at concentrations
ranging from 1.33 x 10"8 M to 1.33 x 10"6 M (Fig. 22, Table 4).
e. Dichlofenthion. Dichlofenthion did not induce contraction by itself
and had no effect on the ACh- or carbachol-induced contraction at concentrations
ranging from 1.33 x 10"8 M to 1.33 x 10"6 M (Fig. 23, Table 4).
12
-------
8 fi
f. Dursban. At concentrations ranging from 1.33 x 10 M to 1.33 x 10 M,
dursban did not induce any contraction by itself (Fig. 24, Table 4). It had a
slight tendency to decrease the ACh- or carbachol-induced contraction after 3 min
pretreatment, but the effect eas small (Table 4).
-9
g. Propoxur. At a concentration of 1.33 x 10 M, propoxur did not induce
any measurable contraction (Table 4). At higher concentrations ranging from
_ 0 _C
1.33 x 10 M to 4 x 10~ M, it induced slow contractions which increased in
intensity with the concentration (Table 4). Examples of records of the propoxur-
induced contractions are illustrated in Fig. 25. However, the effect of propoxur
on the ACh- or carbachol-induced contraction was negligible (Fig. 26, Table 4).
n. Ferbam. Because of difficulty in solving ferbam in ethanol which was
used as the solvent for the other insecticides tested excepting formetanate,
methanol was first employed to dissolve ferbam. The ferbam solution thus prepared
stimulated the ileum in producing contractions at concentrations ranging from
1.33 x 10"8 M to 1.33 x 10~6 M, but did not alter the ACh-induced contraction.
However, when dimethylsulfoxide (DMSO) was used as the solvent, no such stimulating
effect of ferbam was observed (Fig. 27, Table 4). The ACh-induced contraction was not
affected by ferbam pretreatment (Fig. 27, Table 4). Thus the stimulating effect
observed with ferbam solutions containing methanol as a solvent is due to the
action of methanol.
i. Formetanate. Formetanate induced contractions at concentrations ranging
from 1.33 x 10~8 M to 1.33 x 10"6 M, but had little or no effect on the ACh- or
carbachol-induced contraction (Fig. 28, Table 4).
_Q
j. Chlordimeform. At concentrations ranging between 1.33 x 10 M and
1.33 x 10" M, chlordimeform did not induce any measurable contraction (Fig. 29,
Table 4). The contraction induced by ACh or carbachol was not influenced by
13
-------
chlordimeform at a concentration of 1.33 x 10 M, but was depressed somewhat
by 1.33 x 10"6 M (Table 4).
Effects of specific Inhibitors on insecticide-Induced contractions
Of the ten insecticides that were examined, carbofuran, propoxur and
formetanate had a potent stimulating action on the guinea pig ileum causing
contractions. The effect could be exerted as a result of stimulation of the
muscarinic receptor in the muscle, the nicotinic receptor in the muscle, the
nicotinic receptor in the parasympathetic ganglia, or the histaminic receptor
in the muscle. In order to determine the site of action of the three insecticides,
experiments were carried out using specific inhibitors of these receptors. These
inhibitors of these receptors. These inhibitors included atropine for the
muscarinic receptor, hexamethonium for the nicotinic receptor, and diphenhydramine
for the histaminic receptor.
_Q
a. Carbofuran. Pretreatment of the ileum with 1.33 x 10" M atropine
-4
abolished the carbofuran-induced contraction. Pretreatment with 1.33 x 10 M
hexamethonium or with 1.33 x 10 M diphenhydramine was ineffective in preventing
the contraction. Examples of contraction records under these conditions are
illustrated in Fig. 30, and the data are summarized in Table 5. These results
indicate that the carbofuran-induced contraction is the result of direct
stimulation of the muscarinic receptor in the ileum, or stimulation of the
muscarinic receptor in the parasympathetic ganglia which in turn stimulates
the ileum via the activity of the postganglionic parasympathetic nerve fibers,
or both.
b. Propoxur. Similar to the case of carbofuran, the propoxur-induced
_o
contraction was prevented by pretreatment with 1.33 x 10" M atropine, but
not by 1.33 x 10~ M hexamethonium or 1.33 x 10~ M diphenhydramine (Fig. 31,
Table 5). Thus the same conclusion as that for carbofuran applies to propoxur.
14
-------
c. Formetanate. Ine formetanate-induced contraction was almost completely
_Q
abolished by pretreatment with 1.33 x 10 M atropine, was slightly decreased by
pretreatment with 1.33 x 10 M diphenhydramine, and was not affected by
pretreatment with 1.33 x 10" M hexamethonium (Fig. 32, Table 5). Thus the
major stimulating action of formetanate is exerted on the muscarinic receptor
in the ileum or in the parasympathetic ganglia or both, and there is a minor
stimulating action on the histaminic receptor.
Effects of low Ca -high Mg solutions on insecticide-induced contractions
As described in the preceding section, the contractions of the ileum induced
by carbofuran, propoxur and formetanate are due to the stimulation of the
muscarinic receptor in the muscle, the muscarinic receptor in the parasympathetic
ganglia, or both. If the stimulation of the muscarinic receptor in the ganglia
were the major cause of contraction, the effect would be abolished by preventing
release of transmitter from nerve terminals. One way of inhibiting the transmitter
release is to lower calcium concentration and raise magnesium concentration.
Therefore, the effects of the three insecticides on the ileum were studied in
low Ca -high Mg media. Prior to performing such experiments, control experiments
were carried out to see whether low Ca -high Mg media had any effect on the
ACh- and physostigmine-induced contractions.
a. ACh contraction. When the magnesium concentration in Tyrode's solution
was increased from the normal level of 1.03 mM to 1.70-2.40 mM without changing
the calcium concentration (1.80 mM), the contraction induced by ACh tended to
decrease. The inhibitory effect was almost negligible in 1.7 mM Mg , but became
more marked at 2.30-2.40 mM Mg (Table 6). In contrast, decreasing the calcium
concentration from 1.80 mM to 0.18-0.36 mM with the magnesium concentration elevated
to 1.70-2.20 mM had a marked depressant effect on the ACh-induced contraction
(Fig. 33B, Table 6). Therefore, the effects of physostigmine and insecticides in
15
-------
the modified Tyrode's solution were always normalized to the levels observed in the
same Ca and Mg concentrations.
b. Physostigmine. Unlike the contraction induced by ACh, the response caused
by physostigmine was characterized by a long latent period and slowly developing,
repetitive contractions. It was therefore difficult to measure a maximum amplitude
of the physostigmine-induced contraction as a measure of activity. Thus the latent
period for the physostigmine-induced contraction to attain a level equivalent to
the maximum amplitude of the contraction evoked by ACh was measured as an index of
response. An example of a record is illustrated in Fig. 33. Decreasing the
calcium concentration from 1.80 mM to 0.036-0.36 mM with the magnesium concentration
elevated from 1.03 mM to 2.20-2.30 mM tended to prolong the latent period and to
decrease the amplitude of the physostigmine response, but the difference was not
marked (Table 7). Thus it can be said that the contraction induced by physostigmine
is at least in part due to an increase availability of ACh released from the
postganglionic nerve fibers as a result of inhibition of AChE.
c. Insecticides. The records of ileum contractions induced by carbofuran,
propoxur and formetanate in modified Tyrode's solution are illustrated in Figs. 34,
35 and 36, respectively. For the three insecticides, the contraction was delayed
in onset and decreased in amplitude by an increase in magnesium concentration from
1.03 mM to 2.20 mM and a concurrent decrease in calcium concentration from 1.80 mM
to 0.36 mM (Table 8). Therefore it can be concluded that the contractions induced
by the three insecticides are at least in part due to an increased availability
of ACh released from the postganglionic nerve terminals as a result of AChE
inhibition.
Effects of black widow spider venom on insecticide-induced contractions
Venom from the black widow spider is known to inhibit the release of transmitter
from nerve terminals. Therefore, the venom was used in the present study in an
16
-------
attempt to block the transmitter release. If the venom completely inhibited
the transmitter release under the present experimental conditions, any contraction
induced by the released ACh would be abolished.
Experiments were performed according to the following protocol. First the
ileum preparation was soaked in Tyrode's solution containing black widow spider
venom at various concentrations (0.2-4 glands/ml) and for various periods of time
(5-60 min). Then the preparation was mounted in the chamber and washed with
ncrmal Tyrode's solution free of venom until the ileum regained the ability to
respond to ACh. For some unknown reason, the ileum did not respond to ACh immediately
after treatment with venom. Physostigmine was then applied to the preparation and
the response was recorded.
Fig. 37 illustrates the contraction evoked by physostigmine application after
treatment with black widow spider venom at a concentration of 0.4 glands/ml for 15
min at room temperature. The response was smaller than that in normal preparation,
but was not abolished by venom treatment. Data from 13 ileum preparations in 6
series of experiments are summarized in Fig. 38. The ordinate represents the recip-
rocal of the time necessary for the contraction induced by 4 x 10 M physostigmine
-8
to reach the same level as the contraction induced by 6.65 x 10 M ACh. Thus
increasing the ordinate value means greater response to physostigmine. The abscissa
represents the time of venom treatment. It is clearly shown that the physostigmine
i
response decreases with prolonging the time of venom treatment in 5 out of 6 series
of experiments, but that venom fails to abolish the response. Thus black widow
spider venom does not seem to be totally effective in abolishing transmitter release
in the ileum preparation.
Denervated preparations
The most straightforward way to eliminate the effect of the transmitter ACh
released from nerve terminals would be to remove the ganglia from the ileum
17
-------
preparation. Two methods of denervation were attempted as described in the
Methods section.
a. Denervation by inside-out. The ileum preparation in which its inside
was turned over to the outside and the ganglia were removed by gently brushing
them off was still responsive to physostigmine (Table 9). Thus the ganglia
did not seem to be completely eliminated by this method, and this preparation
was not used for the study of insecticides.
b. Denervation by Paton-Zar method. The ileum preparations denervated by
the method developed by Paton and Zar (1968) did not respond to physostigmine
(Fig. 39). This indicates that the ganglia were completely eliminated from the
ileum. The ACh contraction was potentiated by physostigmine pretreatment as a
result of inhibition of AChE.
The effects of carbofuran, propoxur and formetanate on the denervated
preparations are illustrated in Fig. 39. None of them caused any contraction.
The results are summarized in Table 10. Nicotine was also tested to insure the
absence of ganglia. There was no effect of nicotine (Table 10).
Thus it can be concluded that carbofuran, propoxur and formetanate have
no direct stimulating action on the muscarinic receptor in the ileum. The
contractions induced by these insecticides in the intact ileum preparation are
due to an increased availability of ACh released from the postganglionic nerve
fibers as a result of inhibition of AChE.
B. Guinea Pig Heart
Drugs acting on autonomic nervous system
The guinea pig heart was chosen as material to study the effects of insecticides
on B-adrenergic receptors. Before examining the insecticide action, a few drugs
known to act on muscarinic and 3-adrenergic receptors were tested to establish the
18
-------
basic pattern of responses of the heart preparation.
a. Carbachol and atropine. Carbachol is an agonist acting on both nicotinic
and muscarinic receptors. As expected from the muscarinic stimulating action,
carbachol exerted a potent negative inotropic effect on the heart. An example of
a record is illustrated in Fig. 40, and numerical data are given in Table 11. The
amplitude of the contraction became smaller with increasing the dose, and the
contraction was stopped after an 0.2 ml injection of 5 x 10~ M carbachol (Fig. 40).
Atropine itself had no effect on the contraction even at a very high dose
(1 x 10 M, 0.2 ml) (Fig. 41, Table 11). However, atropine effectively eliminated
the negative inotropic action of carbachol (Fig. 41, Table 11). It should be noted
that the effect of atropine lasted for a long period of time, and the muscarinic
blocking action was quite evident even after a 60 min washing with atropine-free
medium.
b. Isoproterenol and propranolol. Isoproterenol is an agonist acting on
8-adrenergic receptors. Since isoproterenol is relatively unstable in aqueous
solutions, its stability was first examined under various experimental conditions.
The results are illustrated in Fig. 42. When isoproterenol was simply dissolved
in Locke's solution, the potency of the test solution declined with time. Keeping
the test solution in dark or adding Na2S~Ot- was not particularly effective in
preventing degradation of isoproterenol. Ascorbic acid was satisfactory in this
-4
regard, and was added to isoproterenol solutions at a concentration of 1 x 10 M
in all the subsequent experiments. The pH of the solution was adjusted to 4.0.
As expected from the 3 stimulating action, isoproterenol exerted a positive
inotropic effect on the heart (Fig. 43, Table 12). In order to exclude the
possibility that isoproterenol acts on the muscarinic receptor of the heart,
the effect of atropine on the isoproterenol stimulation was examined. Atropine
did not modify the positive inotropic action of isoproterenol (Fig. 43, Table 12).
Propranol is a B-adrenergic blocking agent. It had no effect on the
19
-------
contraction when applied alone, but effectively suppressed the propranolol-induced
positive inotropic effect (Fig. 44, Table 13).
Insecticides
Carbofuran, propoxur and formetanate had a potent stimulating action on the
guinea pig ileum as described in Section A, and were chosen to study the possible
action on the heart 3-adrenergic receptor. Examples of records of the heart
contraction in the presence of carbofuran, propoxur and formetanate are illustrated
in Figs. 45, 46 and 47, respectively, and numerical data are given in Table 14.
These experiments were performed according to the following protocol: First 0.2 ml
-4
of 1 x 10 M atropine solution was added to the perfusate to block the muscarinic
receptor. One minute later, 0.2 ml of 154 mM NaCl solution with or without an
insecticide was added to see the direct insecticide action on the 3-adrenergic
receptor. Three minutes later, 0.2 ml of 1 x 10~ M isoproterenol solution was
added to see the suppressive effect of the insecticide on the 6-adrenergic receptor.
None of the three insecticides tested had any direct stimulating action or suppressive
action in the 3-adrenergic receptor.
Thus it can be concluded that carbofuran, propoxur and formetanate have no
effect on the 3-adrenergic receptor of the guinea pig heart.
C. Guinea Pig Vas Deferens
Drugs acting on autonomic nervous system
The guinea pig vas deferens was chosen as a material to study the insecticide
action on a-adrenergic receptor. Stimulation of this receptor causes contractions
in the vas deferens. Norepinephrine was effective in causing contractions at a
-5 -6
concentration of 2.66 x 10 M, and the effect was abolished by 1.33 x 10 M
phentolamine, an a-blocker (Fig. 48, Table 15).
20
-------
Insecticides
Carbofuran, propoxur and formetanate, insecticides capable of stimulating
the guinea pig ileum at low concentrations, were examined for their effects on the
a-adrenergic receptor of the guinea pig vas deferens. Figures 49, 50 and 51
represent examples of records in carbofuran, propoxur and formetanate, repectively,
and numerical data are summarized in Table 16. The protocol of experiments is as
-8
follows: First 1.33 x 10 M atropine was applied for 2 minutes to block the
muscarinic receptor. Second an insecticide solution was applied for 3 minutes to
_5
see direct stimulating effect. Then 2.66 x 10 M norepinephrine was introduced
to examine the blocking action of the insecticide on the a receptor. None of the
three insecticides had any stimulating or blocking effect on the a-adrenergic
receptor of the guinea pig vas deferens.
D. Rat Diaphragm
Drugs acting on nicotinic receptors
The rat diaphragm was used to study the effect of insecticides on nicotinic
receptors. As control experiments, the effects of physostigmine was first
examined. At low concentrations (5 x 10 M, 1.5 x 10 M), physostigmine
potentiated the contractile response evoked by nerve stimulation, whereas at
a higher concentration (5 x 10" M) it suppressed the contraction (Figs. 52
and 53, Table 18). This dual action is interpreted as being due to an increased
availability of the release ACh as a result of inhibition of AChE by physostigmine.
a-Tubocurarine suppressed the nerve evoked contraction of the diaphragm
(Fig. 54, Table 17).
Insecticides
-9 -8
a. Carbofuran. Carbofuran, at concentrations of 5 x 10 M and 5 x 10 M,
had no effect on the diaphragm contraction evoked by nerve stimulation (Table 18).
At higher concentrations (5 x 10 M, 5 x 10 M), however, it potentiated the
contraction (Table 18). Examples of records are illustrated in Fig. 55.
21
-------
-9 -7
b. Propoxur. At concentrations ranging from 5x10 M to 5 x 10 M,
propoxur had no effect on the diaphragm contraction. At a higher concentration
of 5 x 10" M, the contraction was unaffected in most cases (Fig. 56), but
potentiated in others to make the average value slightly higher than the control
(Table 18).
c. Formetanate. Formetanate had no effect on the contraction at concentratior
ranging from 5 x 10" M to 5 x 10"7 M. It potentiated the contraction at 5 x 10"6 H
(Fig. 57), but the effect was much less at a higher concentration of 5 x 10" M
(Table 18).
In summary, all of the three insecticides tested potentiated the nerve evoked
contractile response of the rat diaphragm at concentrations beyond a certain
threshold level. This effect is presumably due to the inhibition of AChE which in
turn would cause an accumulation of ACh in the end-plate region. If this is the ca:
then prior inhibition of AChE by an anticholinesterase would abolish this effect.
This was tested by using physostigmine as an anticholinesterase.
d. Effects on physostigmine-treated diaphragm. As is given in Table 19, the
rat diaphragm pretreated with physostigmine at a concentration of 1 x 10 M did
not respond to carbofuran, propoxur or formetanate in the form of potentiated
contraction (Figs. 58, 59 and 60). Thus it can be concluded that the potentiating
effect of the three insecticides tested on the rat diaphragm is due to the AChE
inhibition which in turns causes an accumulation of ACh.
E. Frog Rectus Abdominis
Physostigmine
The frog rectus abdominis contains nicotinic receptors, and responds to
nicotinic agonists to produce contracture. Therefore, this is another convenient
preparation to examine the effects of insecticides on nicotinic receptors.
22
-------
Examples of ACh contractions with and without physostigmine are illustrated
in Fig. 61. The contraction caused by ACh was potentiated by physostigmine at
-5 -4
concentrations ranging from 1 x 10 M to 1 x 10 M (Figs. 62 and 63). At a
higher concentration of 3.99 x 10 M, physostigmine tended to inhibit the ACh
contraction. Both the potentiation and inhibition by physostigmine can be
interpreted as being due to the accumulation of ACh in the end-plate region.
Insecticides
When applied to the preparation pretreated with physostigmine to inhibit
AChE, carbofuran, propoxur and formetanate did not evoke any contraction by
themselves and did not modify the ACh contraction at any concentrations used
ranging from 1.33 x 10"8 M to 1.33 x 10"5 M or 1.33 x 10"4 M (Figs, 64, 65 and
66, Table 20).
It can be concluded that none of the three insecticides has any direct
effect on the nicotinic receptor of the frog rectus abdominis.
IV. SUMMARY AND CONCLUSIONS
1. A variety of skeletal muscle and smooth muscle tissues have been
examined for their usefulness in evaluating the toxic effects of environmental
agents, organophosphate and carbamate insecticides in particular. Methods
have been established whereby the toxic side effects on various receptors
caused by cholinesterase inhibition and by other mechanisms can be evaluated,
Several insecticides were studied for their effects on various receptors using
these methods.
2. The guinea pig ileum is a convenient preparation for the study of
drug action on muscarinic receptors. Acetylcholine caused contractions at
concentrations in the order of 10 M. Physostigmine potentiated the acetylcholine-
_o
induced contraction at low concentrations (M0~ M), and induced contractions by
23
-------
_Q
itself at higher concentrations (> 4 x 10~ M). The potentiation is interpreted
as being due to the increased availability of acetylcholine as a result of
acetylcholinesterase inhibition. Neostigmine (10 M) also potentiated the
_o
acetylcholine contraction. Atropine (10" M) effectively antagonized the
acetyl chol i ne-i nduced contract!'on.
The a-adrenergic blocking agent phentolamine competitively antagonized
the contraction by acetylcholine only at high concentrations (10" M). Thus
the phentolamine suppression is not due to its effect on the a-adrenergic
receptor but due to its inhibitory effect on the muscarinic receptor. The
3-adrenergic blocking agent propranolol suppressed the acetylcholine contraction
only at a very high concentration (10" M). This effect is presumably due to
direct action on the muscarinic receptor.
3. Among the ten insecticides tested on the guinea pig ileum, the following
seven had no direct effect and did not affect the acetylcholine- and carbachol-
induced contraction at concentrations indicated: carbaryl (10~ - 10" M),
lepthophos (10"9 - 10"6 M), monocrotophos (10~8 - 10"6 M), dichlofenthion (10~8 -
10"6 M), dursban (10~8 - 10"6 M), ferbam (10~8 - 10"6 M), and chlordimeform at
a higher concentration (10~ M) slightly depressed the acetylcholine- and
carbachol-induced contractions.
4. Three insecticides had a potent stimulating action on the guinea pig
ileum. Carbofuran induced potent contractions at concentrations of 10 to
10" M. However, it did not exhibit any potentiating or depressing effect on
the acetylcholine- or carbachol-induced contraction. Propoxur and formetanate
_o _(:
induced contractions at concentrations of 10" - 10~ M, but had no effect on
the acetylcholine- or carbachol-induced contraction, The contraction induced by
_o
carbofuran or propoxur was abolished by pretreatment with atropine (10 M)
but not by hexamethonium (10~ M) or diphenhydramine (10 M). Thus the
24
-------
contraction by either of these two insecticides is the result of stimulation of
the muscarinic receptor. The formetanate-induced contraction was abolished by
_Q
pretreatment with atropine (10 M), was slightly suppressed by diphenhydramine
(10 M), but was not affected by hexamethonium (10~ M). This formetanate
stimulates the muscarinic receptor and slightly stimulates the histaminic
receptor.
5. The chemicals effective on the guinea pig ileum may exert effects through
inhibition of acetylcholinesterase or may directly act on the muscarinic receptors
of the muscle. In an attempt to distinguish these two possibilities, use was made
of low Ca - high Mg media which were known to inhibit transmitter release from
nerve terminals. The onset of the contraction induced by physostigmine, carbofuran,
propoxur and formetanate was delayed by decreasing Ca concentration from 1.80 mM
to 0.036-0.36 mM and simultaneously increasing Mg concentration from 1.03 mM to
2.20-2.30 mM, but was not abolished. Thus the contraction induced by these drugs
is at least in part due to an increased availability of acetylcholine released from
nerve terminals as a result of acetylcholinesterase inhibition.
6. Black widow spider venom has been known to inhibit transmitter release
from nerve terminals. The use of the venom in the guinea pig ileum still failed
to distinguish the two possible mechanisms described in the preceding paragraph (5),
since the venom suppressed the acetylcholine response itself and also since
physostigmine produced contractions after venom treatment.
7. Denervation of the guinea pig ileum was successful in locating the site
of action of the chemicals effective in producing contractions. Turning over the
ileum inside out to brush off the ganglia did not give satisfactory results,
because physostigmine was still effective. The method developed by Paton and Zar
(J. Physio!. 194, 13 [1968]) was satisfactory, and physostigmine became ineffective.
25
-------
Carbofuran, propoxur and formetanate also failed to stimulate the denervated
ileum. Thus it can be concluded that these insecticides have no direct stimulating
action on the muscarinic receptor in the ileum, and that the contractions induced
by them in intact ileum preparations are due to an increased availability of
acetylcholine released from the postganglionic nerve fibers as a result of
inhibition of acetylcholinesterase.
8. The guinea pig heart is a suitable preparation to study drug action on
B-adrenergic receptors. Carbachol had a potent negative inotropic effect through
the stimulation of the muscarinic receptor, and the effect was antagonized by
atropine. Isoproterenol had a positive inotropic effect through the stimulation
of the B-adrenergic receptor, and the effect was antagonized by propranolol, a
B-blocker. Carbofuran, propoxur and formetanate had no effect on the atropinized
heart, and did not alter the positive inotropic effect of isoproterenol. Thus it
can be concluded that these three insecticides have no effect on the B-adrenergic
receptor of the guinea pig heart.
9. The guinea pig vas deferens was chosen as a material to study the drug
action on a-adrenergic receptor. Norepinephrine (10 M) was effective in causing
contractions through the stimulation of a-receptors, and the effect was antagonized
by phentolamine (10" M), an a-blocker. Carbofuran, propoxur and formetanate (up
to 10" M) did not cause contraction in the atropinized vas deferens, and did not
alter the norepinephrine-induced contraction. It can be concluded that none of
the three insecticides has any stimulating or blocking effect on the a-adrenergic
receptor of the guinea pig vas deferens.
10. The rat diaphragm was used as a representative of nicotinic receptors
in mammalian phasic muscles. Physostigmine potentiated the nerve evoked contraction
at low concentrations (%10 M), but suppressed it at higher concentrations (5 x 10
d-Tubocurarine (10~ M) suppressed the contraction. Carbofuran potentiated the nervt
evoked contraction at high concentrations (5 x 10~ M, 5 x 10" M). Propoxur and
-------
formetanate also potentiated the contraction at a high concentration (5 x 10" M).
However, none of the three insecticides had any effect on the contraction in the
muscle pretreated with physostigmine (10~ M). Thus it can be concluded that
the potentiating effect of these insecticides on the rat diaphragm is due to the
inhibition of acetylcholinesterease which in turn causes an accumulation of acetylcho-
line.
11. The frog rectur abdominis was chosen as a representative of nicotinic
receptors in the tonic muscle of cold blooded animals. Physostigmine potentiated
-5 -4
the acetylcholine-induced contraction at 10 - 10 M, but suppressed it at a
higher concentration (4 x 10" M). Carbofuran, propoxur and formetante (up to
10 M) did not evoke any contraction and did not modify the acetylcholine contraction
in the muscle pretreated with physostigmine (10~ M). It can be concluded that
none of the three insecticides has any effect on the nicotinic receptor of the
frog rectus abdominis.
12. For the purpose of studying the side effects of various insecticides and
other environmental agents on postsynaptic receptors, the guinea pig ileum
(muscarinic receptor), the guinea pig heart (S-adrenergic receptor), the guinea pig
vas deferens (a-adrenergic receptor), the rat diaphragm (nicotinic receptor in
phasic muscle), and the frog rectus abdominis (nicotinic receptor in tonic muscle)
have proved quite satisfactory. The potent stimulating action of carbofuran,
propoxur and formetanate on the guinea pig ileum is due to acetylcholinesterase
inhibition, and they have no direct effect on the muscarinic, nicotinic, a-adrenergic
and g-adrenergic receptors.
Acknowledgements
The author wishes to express his sincere thanks to Dr. Keiichiro Nishimura
who performed the experiments, and Ginny Arnold and Arlene McClenny for secretarial
assistance.
27
-------
V. REFERENCES
Narahashi, T. (1976) In vitro screening methods evaluating the neurotoxic
potential of pesticides. Environmental Health Effects Research Series,
EPA-600/1-76-005, Environmental Protection Agency, Office of Research
and Development.
Paton, W. D. M. and M. A. Zar. (1968) The origin of acetylcholine released
from guinea pig intestine and longitudinal muscle strips. J. Physiol.
(London) 194, 13-33.
28
-------
TABLE 1
Compositions of Bathing Media (mM)
E
A B C D Tyrode- F
Tyrode I Tyrode II Krebs I Krebs II Locke Ringer
NaCl
KC1
CaCl2
MnCf)
nyoUfl
NaHC03
3 2 4
2 4
Glucose
136.9 136.9 113
2.68 2.68 4.7
1.80 1.80 2.5
i m i 03 1?
1 . UJ 1 . UO 1 . L-
11.9 11.9 25
Ooc n oc
1 0
5.55 11.1 11.5
94.1
4.69
2.52
0/1£
25
1 IS
11.1
154 111.2
5.63 1.88
2.16 1.08
1.99 2.38
n n?
5.55 11.1
Na pyruvate
Na fumarate
Na glutamate
pH
2.45
8.0-8.1
7.6 7.4
2.66
7.4
8.1
7.9
29
-------
TABLE 2
Effects of 5-Minute Pretreatment with Physostigmine on Acetylcholine-Induced
Contraction in Guinea Pig Ileum
Physostigmine (M) Maximum Contraction
7.74 x TO"3 Ma
0 1
1.33 x 10"9 0.97
3.99 x 10"9 1.12
1.33 x 10~8 1.33
3.99 x 10"8 1.24
by Acetylcholine
1.55 x 10~7 Mb
1
1.00
1.15
1.23
1.77
Preparation 6-16-75.
Preparation 5-30-75.
30
-------
TABLE 3
Contraction Induced by 3.99 x 10 M Physostigmine in Guinea Pig Ileum
Contraction after exposure to physostigmine (min)
Preparation
0.5 1 1.5 2 2.5 3 3.5 4 4.5 5
0 0 + + + ++++++++++
0 0 0 0 + + + ++++++
0 0 + + + ++++++++++
aO, no contraction; +, slight contraction; +, contraction; ++, strong contrac-
tion.
31
-------
TABLE 4
Effects of Insecticides on Guinea Pig Ileum
Insecticides Concentra-
Insecticide- Insecticide pretreatment on
tion » induced
1.33x10 Contraction Acetylchol
^ ' induced
ine-
contraction
Carbofuran 8
7
6
Carbaryl 9
8
7
6
Leptophos 9
8
7
6
3 min 1 min 3
+,+,0 1.12
0.96
+,+,+,+ 1.35
1.03
+,+,+ 1.00C
0 0.84
0.89
0,0,0 1.13
0.95
0,0,0 1.23
1.15
0.89
0,0,+ 1.30
0.81
0.82
0.85
0 1.02
0,0,+ 1.00
0.93
0,0,+ 1.10
1.00
0,0,+ 1.02
1.13
1.02
min
1.06
0.89C
1.03C
1.08
0.81
0.95
1.35
0.93C
0.92
0.93
Carbachol-
induced
contraction
1 min 3 min
0.93 1.
1.00
1.05
0.93
1.71C 1.
1.00
1.05 1.
1.02C 0.
0.95 1.
1.08C
0.46 0.
0.96C 1.
0.89
0.85 0.
0.83 0.
0.89 1.
0.93
0.95C
04
07
09C
80C
04C
99
12
73C
85C
07C
(continued)
32
-------
TABLE 4 (continued)
Insecticides Concentra- Insecticide-
tion induced
1 33 x "10"^ Contraction
(X)
Insecticide Pretreatment
Acetylcholine-
induced
contraction
Monocrotophos
Dichlofenthion
Dursban
Propoxur
3 min
8 0, + ,0
7 0,0,0
6 0,0,0,0
8 0,0,0
7 0,0,0
6 0,0,0,0,0
8 0,0
7 0,0
6 0,0,0,0
9 0
8 +,0,+
1 t>t
6 +,0,+,+,+,+
1 min
1.07
0.98
0.78
1.08
0.99
0.95
1.00
0.94
1.00
1.04
0.91
0.93
0.98
0.95
0.93
3 min
0.93
0.68
0.96
0.79
1.00
0.92
0.90
0.79
0.87C
0.87
0.88
0.85
0.87
0.98
1.03
1.11
on
Carbachol-
induced
contraction
1 min
1.03
0.86
1.04C
0.97C
1.01C
0.97C
1.02C
0.97
0.96C
0.94C
0.88C
0.94C
0.96C
0.74C
0.94
1.07
0.91C
3 min
1.02C
0.96C
0.94C
1 .08C
1.38
1.02C
1.24C
0.79
1.00
0.88C
1.89C
0.71C
0.81C
0.86C
1.14°
0.97C
1.09C
(continued)
-------
TABLE 4 (continued)
Insecticides Concentra- Insecticide-
tion induced
1.33 x 1Q-X c°^action
(X)
3 min
Ferbam 8 0,0,0
7 0,0,0
6 0,0,0
Formetanate 8 + ,+
7 +,+,+
6 +,+,+,+
Chlordimeform 8 0,0
7 0,0,0,0,0
* —
6 0,0,0,0,0
Insecticide Pretreatment on
Acetylchol ine-
induced
contraction
1 min 3 min
0.82
0.95
1.05C
0.96
1.06
0.96
1.12
1.00
0.80
0.82
Carbachol-
induced
contraction
1 min 3 min
0.69C
0.98
0.84C
0.86C
0.89C
0.90C
0.90
1 .34
1.03
1.19
0.92C
0.91
0.94C
0.79C
0.60r
0.36C
alnsecticide-induced contraction was measured 3 min after treatment. Each
observation was made with different preparation. 0, no effect; +, slight
contraction; + , contraction.
Effects of 1-min and 3-min pretreatm-?nt with insecticides on the contractions
7 —^
induced by acetylcholine (1.33 x 10" M or 7.65 x 10 ° M) and by carbachol
(continued)
-------
TABLE 4 (continued)
(1.33 x 10 M). The amplitude of the maximum contraction is given in a
value relative to the control before treatment with insecticides. Each
measurement was made with different preparation
cContraction slowly increased with time.
Dimethylsulfoxide was used as solvent instead of ethanol.
35
-------
TABLE 5
Effects of 4-Minute Pretreatment with Atropine, Hexamethonium
and Diphenhydramine on Insecticide-Induced Contraction of Guinea Pig Ileumc
Pretreatment
Insecticide-induced contraction
Carbofuran Propoxur Formetanate
1.33 x TO"6 M 3.99 x 10~6 M 1.33 x 10~6 M
None
Atropine 1.33 x 10 M
0
0
0
0
0
0
0
0
1
-4
Hexamethonium 1.33 x 10 M
++
Diphenhydramine 1.33 x 10~ M
aEach measurement represents response of individual ileum preparation.
0, no contraction; +, slight contraction; +, contraction; ++, strong contrac-
tion.
36
-------
TABLE 6
Effects of Mg and Ca Concentrations on Contraction Induced
_o
by 6.67 x 10 M Acetylcholine in Guinea Pig Ileum
Mg++ (mM)
1.70
1.80
1.90
2.00
2.03
2.10
2.20
2.30
2.36
2.40
Ca++ (mM)
1.80
1.80
1.80
1.80
1.80
1.80
1.80
1.80
1.80
1.80
Relative contraction3
1.00
0.70
0.90
1.00
0.96
1.29
1.07
0.80
0.94
0.84
0.90
0.56
0.92
0.88
1.35
1.05
1.00
0.73
0.73
0.68
0.61
Mean
0.99
0.80
0.94
0.84
0.73
0.92
1.07
0.73
0.71
0.61
(continued)
37
-------
TABLE 6 (continued)
Mg (mM) Ca (mM) Relative contractiona Mean
1.70 0.36 0.71 0.71
1.70 0.18 0.67 0.65
0.63
2.20 0.36 0.39 0.58
0.35
1.00
2.20 0.18 0.48 0.48
Amplitude of maximum contraction in modified Tyrode's solution relative to that
in normal Tyrode's solution containing 1.03 mM Mg and 1.80 mM Ca
38
-------
TABLE 7
Effects of Mg and Ca Concentrations on Physostigmine-Induced
Contraction in Guinea Pig Ileuma
Latent time
Mg (mM) Ca (mM) (Min., Sec.)
1.03 1.80 2 '40"
2 '30"
2.20 1.80 3'10"
2 '40"
2.20 0.36 2'50"
5'30"
3'30"
2.20 0.18 5'30"
3'00"
2.20 0.09 3'00"
2.20 0.036 4'20"
3 '40"
Mean
2'35"
2'55"
4 '00"
4'15"
3 '00"
4 '00"
aData are given in latent time for the contraction induced by 4.00 x 10~ M
physostigmine to attain the same amplitude as the maximum contraction
— ft
induced by 6.67 x 10" M acetylcholine.
39
-------
TABLE 8
Effects of Mg and Ca Concentrations on Insecticide-Induced
Contraction in Guinea Pig Ileuma
Insecticide Mg Ca ,
(Concentration) (mM) (mM)
Carbofuran 1.03 1.80
(1.33 x 10"6 M)
2.20 0.36
Propoxur 1.03 1.80
(3.99xlO-6M) 2_2Q Q>36
Formetanate 1.03 1.80
(1.33 x 10"6 M)
2.20 0.36
1
+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
In insecticide (min)
2345
+ ++ ++ ++
t t
t + +
0 + + +
0 0
0 +
0 + + +
000 +
00 + +
000 +
0 + + ++
0 + + +
0 + + +
000 +
00 + +
00 + +
Washing
(min)
++ (5)
+ (5)
++ (5)
++ (5)
++ (3)
++ (3)
+ (5)
+ (5)
+ (5)
++ (5)
++ (5)
+ (5)
++ (5)
+ (5)
++ (5)
++ (5)
aO, no contraction; +, slight contraction; +, contraction; ++, strong contrac-
tion.
Washing with insecticide-free normal Tyrode's solution containing 1.03 mM Mg
1.80 mM Ca++.
-------
TABLE 9
Effects of Drugs and Insecticides on the Guinea Pig Ileum Denervated
by Inside-Out Method
Drug
Concentration
(M)
Contraction3
Acetylcholine
Nicotine
Physostigmine
Carbofuran
Propoxur
Formetanate
6.67 x 10
-8
6.67 x 10
-6
3.99 x 10
-7
1.33 x 10
-6
1.33 x 10
-6
1.33 x 10
-6
'individual symbols represent contractile responses of individual preparations.
0, no contraction; +, slight contraction; +, contraction.
41
-------
TABLE 10
Effects of Drugs and Insecticides on the Guinea Pig Ileum Denervated
by Paton-Zar Method
Drug Concentration Contraction3
(M)
Acetylcholine 6.67 x 10~8 + +
Nicotine 6.67 x 10"6 0 0
Physostigmine 3.99 x 10 0 0
Carbofuran 1.33 x 10"5 0 0
Propoxur 1.33 x 10" 0 0
-4
Formetanate 1.33 x 10 0 0
+
0
0
0
0
0
Individual symbols represent contractile responses of individual preparations,
0, no contraction; +, contraction.
42
-------
TABLE 11
Effects of Carbachol and Atropine on the Contraction of Guinea Pig Heart3
Drug Concentration Relative0 contraction
(M)
Carbachol 1 x
2 x
5 x
Atropine 1 x
1 x
1 x
1 x
io-6
io-6
io-6
io-7
io-6
io-5
ID'4
m-4
0.43
0.68
0.59
0.56
0.17
0.20
0.16
0
0.94
0.97
0.97
1.05
+ + 0.98
Carbachol 2x10"
aPreparation 9-22-75.
0.2 ml test solution added to the perfusate.
cAmplitude of contraction relative to the control before application of drugs,
43
-------
TABLE 12
Effects of Isoproterenol and Atropine on the Contraction of Guinea Pig Heart
Preparation
9-23-75
9-24-75
9-25-75A
Drug
Isoproterenol
Atropine
Isoproterenol
Isoproterenol
Atropine
Isoproterenol
Isoproterenol
Atropine
Isoproterenol
Concentration9
(M)
1 x 10"5
1 x 10"4
1 x 10"5
1 x 10"5
1 x 10"4
1 x 10"5
1 x 10'6
1 x 10"4
1 x 10'6
Relative After atropine
Before atropine
4.40
1.29
2.88 0.66
8.00
0.80
9.25 1.16
1.56
0.90
1.44 0.92
0.2 ml test solution added to the perfusate.
''Amplitude of contraction relative to the control before application of drugs.
44
-------
TABLE 13
Effects of Isoproterenol and Propranolol on the Contraction of Guinea Pig Heart
Preparation
Drug
Concentration3
(M)
Relative13
Contraction
After propranolol
Before propranolol
9-23-75
9-24-75
Isoproterenol
Propranolol
Isoproterenol
Isoproterenol
Propranolol
Isoproterenol
1
1
1
1
1
1
x 10"5
x 10"4
x 10"5
x 10'6
x 10"5
x 10"6
4.25
1.00
0.88
5.50
0.89
1.13
0.21
0.21
0.2 ml test solution added to the perfusate.
^Amplitude of contraction relative to the control before application of drugs.
45
-------
TABLE 14
Effects of Insecticides on the Contraction of Guinea Pig Heart0
Insecticide
Carbofuran
Propoxur
Formetanate
Concentration
(M)
6
5
4
6
5
4
6
5
4
3
2
Direct0
Effect
0
0
0
0
0
0
0
0
0
0
0
In the presence
of isoproterenol
0.96 + 0.02
1.12 + 0.17
0.94 + 0.02
0.99 + 0.04
1.02 + 0.04
1.00 + 0.04
0.94 + 0.03
0.96 + 0.03
0.97 + 0.04
0.98 + 0.06
0.87 + 0.07
N
3
4
7
3
3
4
3
3
4
3
5
aProtocol: 0.2 ml of 1 x 10~4 M atropine; 1 min later, 0.2 ml of 154 mM NaCl
without (control) or with (test) insecticide; 3 min later, 0.2 ml of
1 x 10~ M isoproterenol.
Concentration of test solution added to the perfusate.
C0, no effect.
dn ... £ ., . . • /isoproterenol + insecticidew
Ratio of the contractions ( K insecticide )7
(isoproterenol + control solution) . h + $>E_M-
v control solution '
46
-------
TABLE 17
Effects of d-Tubocurarine on the Rat Diaphragm Contraction Evoked
by Nerve Stimulation
Perfusate Concentration
(M)
Solution E 0
5 x 10"8
5 x 10"7
2.5 x 10"6
Solution F 0
3.33 x 10"9
3.33 x 10"8
3.33 x 10"7
1.67 x 10"6
3.33 x 10"6
Relative contraction
Without With
physostigmine physostigmine
4 x 10"7M
1
0.93
0.95
0.35 0.71
0.20 0.68
1
1.00
0.83
0.76
0
0
49
-------
TABLE 18
Effects of Physostlgmine and Insecticides on the Rat Diaphragm Contraction
Evoked by Nerve Stimulation
Insecticide
Physostigmine
Carbofuran
Propoxur
Formetanate
Concentration
(5 x 10"X M)
7
6
9
8
7
6
9
8
7
6
9
8
7
6
5
Relative contraction
1.12 + 0.04
3.01 + 0.48
1.15 + 0.05
0.96 + 0.04
2.25 + 0.78
2.27 + 1.33
0.84 + 0.12
0.96 + 0.06
0.95 + 0.06
1.46 + 0.41
0.89 + 0.07
0.94 + 0.03
0.92 + 0.02
3.32 + 0.52
1.19 + 0.28
N
3
4
4
4
3
5
4
5
8
6
4
7
10
5
50
-------
TABLE 19
Effects of Insecticides on the Nerve Evoked Contraction of Rat Diaphragm
Pretreated with 1 x 10" M Physostigmine for 10 Minutes
Insecticide Final
concentration
(M)
-9
Carbofuran 5 x 10
5.5 x 10"8
5.55 x 10"7
5.56 x 10"6
-9
Propoxur 5 x 10
1 x 10"8
6 x 10"8
1.1 x 10"7
5 x 10"7
6.1 x 10"7
Relative
contraction
1.04
1.02
1.00
1.03
1.04
1.02
1.06
1.08
1.03
0.97
1.00
0.98
0.97
0.97
0.94
0.93
1.17
1.03
1.04
1.12
1.02
1.02
1.04
1.00
1.00
1.02
1.00
Mean
+ S.E.M.
1.02 + 0.01
1.05 + 0.01
1.00 + 0.01
0.95 + 0.01
1.10
1.08
1.02
1.02
1.01
(continued)
51
-------
TABLE 19 (continued)
Insecticide Final
concentration
(M)
Propoxur 1 x 10"
1.11 x 10"6
5.61 x 10"6
6 x 10"6
6.11 x 10"6
1.1 x 10"5
-9
Formetanate 5 x 10
5 x 10~8
5.5 x 10"8
5 x 10"7
5.55 x 10"7
5 x 10"6
5.56 x 10"6
5 x 10"5
5.56 x 10"5
Relative Mean
contraction + S.E.M.
1.00
1.07
0.98
0.94
1 .08
0.89
1.00 1.05+0.03
1.00
1.09
1.09
1.00 1.00
1.00
1.09 1.09
1.09
1.00 1.03
1.06
1.05 1.03
1.00
1.00 1.00
1.00
1.02 0.97
0.92
0.77 0.74
0.70
1.05 1.00
0.94
52
-------
TABLE 20
Effects of Insecticides on the Contraction of Frog Rectus Abdominis Pretreated
with Physostigmine
Experiment Insecticide Concentration
(1.33 x 10~X M)
I Carbofuran 8
7
6
5
II Carbofuran 8
7
6
5
Propoxur 8
7
6
5
Formetanate 8
7
6
5
4
Direct0
Effect
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
In the presence
of acetylcholine
0.96 + 0.06
1.01 + 0.01
1.03 + 0.02
1.07
0.89 + 0.04
1.00 + 0.05
0.83 + 0.06
1.07 + 0.04
0.88 + 0.05
0.90 + 0.06
0.91 + 0.02
1.00 + 0.02
0.97 + 0.02
0.93 + 0.04
0.96 + 0.07
1.02 + 0.06
0.89 + 0.08
N
4
4
4
2
6
4
8
3
8
8
8
9
4
4
8
7
7
(continued)
53
-------
TABLE 20 (continued)
Protocol: Physostigmine for 5 min; insecticide for 3 min; acetylcholine 3-4 min.
b I, 3.99 x 10"6 M acetylcholine and 8.07 x 10~5 M physostigmine.
-6 -4
II, 1 x 10 M acetylcholine and 1.33 x 10 M physostigmine.
C0, no effect.
Ratio of the contraction in acetylcholine + insecticide to that in acetylcholine
in the mean + S.E.M.
54
-------
MICROMANIPULATOR
TRANSDUCER
\
TO POLYGRAPH
SUCTIO
^-PERFUSATE
F=*-AIRorGAS
CHAMBER
(15ml)
GLASS FILTER
CONSTANT TEMPERATURE WATER BATH
Figure 1. Diagram of experimental set-up for guinea pig ileum.
55
-------
VPERFUSATE
RESERVOIR
TO POLYGRAPH
CONSTANT TEMPERATURE
WATER BATH
-PERFUSATE
TRANSDUCER
MICROMANIPULATOR
Figure 2. Diagram of experimental set-up for guinea pig heart.
56
-------
TO POLYGRAPH
MICROMANIPULATOR TRANSDUCER
*
SUCTION
CONSTANT TEMPERATURE WATER
BATH
:*-PERFUSATE
GAS
Figure 3. Diagram of experimental set-up for rat diaphragm.
57
-------
3.66x)0"8M ACh
1.33 xl(T8M PHYSOSTIGMINE-*-
3.66x|(T8MACh
6-16-75D
2 min
Figure 4. Contraction of guinea pig ileum induced by acetylcholine (ACh)
-8
(3.66 x 10 M) and potentiation by a low concentration of physostigmine
1.33 x 10"8 M).
58
-------
t 5
on
< 4
OQ
a:
< 3
z
o
Of.
p 1
0
11
1
0.3 0.4 0.6 0.8 1 2
ACETYLCHOLINE (x]0'7M)
J I
Figure 5. Dose-response relation for contraction of guinea pig ileurn by
acetylcholine. Data are given in the mean + S.E.M. with the nur',r>.-r of exper-
iments beside the symbol.
59
-------
1.5
UJ
2 i
LU
o
i
i
i
i
0123456
TIME FOR PHYSOSTIGMINE PRETREATMENTfmin.)
Q -I
Figure 6. Potentiation of acetylcholine (1.55 x 10~ - 1.55 x 10 Mj contract ion
of guinea pig ileum by pretreatment with physostigmine (1.33 x 10"d Mj for
periods of time. Data are given in the mean + S.E.M. with the number of <-
iments beside the symbol. The ordinate represents the amplitude of d
contraction relative to that without physostigmine.
60
-------
6.65xlO"8M ACh
-3.99 x |Q"7M PHYSOSTIGMINE
0.5 g
•Wft*
12-3-75B
2min
-8
Figure 7. Contraction of guinea pig ileum induced by 6.65 x 10 M acetylcholine
(ACh) and repetitive contractions induced by a high concentration of physostignine
(3.99 x 10"7 M).
61
-------
5r-
3
02
h-
U
<
QC. ,
0
AFTER
- NEOSTIGMINE
I I
BEFORE
NEOSTIGMINE
I
I I
0.6 0.8 1 23
ACETYLCHOLINE (xlO"7M)
Figure 8. Dose-response relations for contraction of guinea pig ileurn by
acetylcholine before and after treatment with neostigmine (1.14 x 10 K;
for 6 min.
62
-C,
-------
7.32x]0"8M ACh
.33*]0'8M ATROPINE-
ACh
0.5 g
7-2-75H
2 min
_o
Figure 9. Contraction of guinea pig ileum by 7.32 x 10" M acetylcholine fACh)
_Q
and suppression by 1.33 x 10~ M atropine.
63
-------
0
12345
ATROPINE PRETREATMENT(min.)
Figure 10. Suppression of acetylcholine (7.74 x ICf M) contraction of guinea
-8
pig ileum by pretreatment with 1.33 x 10 M atropine for various periods r,f
time.
64
-------
>- •»
oe.
<
a:
j—
co 3
O 2
u
<
oe
»—
8
WITHOUT
ATROPINE
ATROPINE
PRETREATMENT
3 10 30
ACETYLCHOLINE (xlO~7 M)
200
Figure 11. Dose-response relations for contraction of guinea pig ileurn by
_p
acetylcholine with and without pretreatment with 1.33 x 10 M atropine for
4 min.
65
-------
A
6.65x10"8M ACh
0.5
7-I8-75C
-1.33*10"8M PHENTOLAMINE-*
6.65 xlO'8M ACh
2 mm
8
6.65xlO"8M ACh
-1.33xl(r6M PHENTOIAMINE—»-
6.65x)(T8M ACh
7-18-75 G
2 min
Figure 12. Ineffectiveness of a low concentration of phentolamine (1.33 x 10"8 M)
in suppressing acetylcholine (ACh)-induced contraction in guinea pig ileum (A),
and suppression by a high concentration of phentolamine (1.33 x 10 M) (B),
66
-------
z
o
o
0.8
k^J
Z 0.6
o
x
u
>-
t—
UJ
U
<
UJ
UJ
CtL
0.4
0.2
0
I
I
I
I
I
1
012345
PHENTOLAMINE PRETREATMENT(min.)
•* H
Figure 13. Suppression of acetylcholine (7,74 x 10 M) contraction of guinea
_5
pig ileum by pretreatment with 1.33 x 10 M phentolamine for various periods
of time.
67
-------
z
o
I—
u
Z
o
0.8
Z 0.6
o
—I
>-
I—
UJ
u
UJ
UJ
0.4
0.2
0
1
1
1
1
1
1.33xlO'9 | 1.33xlO'7 | I33xl0'5
1.33xlO'8 1.33xlO"6
PHENTOLAMINE (M)
Figure 14. Dose-response relation for phentolamine suppression of acetylcholine
-8
(6.65 and 7.74 x 10 M) contraction in guinea pig ileum. Tissue is treated by
phentolamine for 5 min prior to acetylcholine application. Data are given in
the mean + S.E.M. with the number of experiments beside the syrr.bcl.
68
-------
QC
Q 3
t—
u
Q^ /*\
Z
O
u
0
BEFORE
PHENTOLAMINE
AFTER
PHENTOLAMINE
±
J L
0.1 0.2 03 0.6 1 23 6
ACETYLCHOLINE (x!0'7M)
10
12
Figure 15. Dose-response relation for contraction of guinea pig ileum by
-5
acetylcholine before and after treatment with 1.33 x 10 M phentolamine for
4 min.
69
-------
6.65 x 1(T8M ACh
7-15-75G
B
6.65x)0-8MACh
7-17-75 F
1.33 x 10"'°M PROPRANOLOL—*-
6.65 x 1(T8M ACh
2 min
-1.33 xlO~5M PROPRANOLOL—*-
6.65 xlO^M ACh
2 min
Figure 16. Ineffectiveness of a low concentration of propranolol (1.33 x 10" M)
in suppressing acetylcholine (ACh)-induced contraction in guinea pig ileum (A),
and suppression by a high concentration of propranolol (1.33 x 10" M) (B).
70
-------
1.2
Z
o
u 1.0
<
Of.
\—
00.8
LU
Z
00-6
x
u
fc 0.4
^ 0.2
QC
0
•2
I
?
1
1
1.33 x]0'8 1.33 xl
PROPRANOLOL(M)
Figure 17. Effects of various concentrations of propranolol on acetylcholine
_ o
(6.65 x 10 M) contraction of guinea pig ileum. Tissue is pretreated with
propranolol for 5 min prior to acetylcholine application. Data are given in
the mean + S.E.M. with the number of experiments beside the symbol.
71
-------
1.33 x|0'8M CARBOFURAN
1.33 x 10"7M CARBOFURAN
0.5 91
8-1I-75A
B
0.5 g
8-H-75F
2 min
—I.33«IO"6M-
CARBOFURAN
2 mm
Figure 18. Contractions of guinea pig ileum caused by various concentrations
cf carbofuran. The contractions are much slower in onset than the acetylcholine
(ACn)-induced contraction, and are repetitive in nature at a high concentration
— ft 9
(1.33 x 10 M). The contraction induced by acetylcholine (ACh) (6.65 / 10"" Mj
is ret affected by pretreatment with carbofuran.
72
-------
6.65xlO'8M ACh 6.65xlO~8M ACh
.33 x)(r6M CARBOFURAN
•^ ^-
6.65xlO"8M ACh
0.5gj"
7-24-75G
2 min
Figure 19. Absence of the effect of carbofuran (1.33 x 10 M) pretreatment
on the acetylcholine (ACh)-induced contraction in guinea pig ileum. Small
repetitive contractions are caused by the high concentration of carbofuran
itself (see Fig. 18).
73
-------
133x|0"9MCARBARYL
1.33x)0"8MCARBARYL
1.33xlO~7MCARBARYL
0.5 a
7-22-75A
B
6.65x]0"8M ACh 6.65x)0~8M ACh
2 mm
.33x)0"6MCARBARYL
^»
6.65 x)0"8M ACh
8-4-75C
IV.
^g^L^^Jfc
^^^^^^^R
2mm
Figure 20. A, absence of direct stimulating action of carbaryl (1.33 x 10 -
1.33 x 10 M) in guinea pig ileum. B, it also fails to modify acetylcholine
(ACh)-induced contraction.
74
-------
9.10xlO~8MACh
1.33xlO~6MLEPTOPHOS
6-IO-75D
2 min
Figure 21. Acetylcholine (ACh)-induced contraction in guinea pig ileum, and
absence of direct stimulating action of 1,33 x 10~ M leptophos.
75
-------
1.33
6.65xlO"8M ACh
M MONOCROTOPHO5
6.65 x ]0~8M ACh
0.5 g
8-15-75A
2 min
Fsgure 22. Absence of direct stimulating action of 1.33 x 10 M monocrotophos
in guinea pig ileum. Acetylcholine (ACh)-induced contraction is not appreciably
i
affected by pretreatment with monocrotophos.
76
-------
6.65xlCT8M ACh 6.65x10'8M ACh
-).33xlO"6M DICHLOFENTHION—*•
6.65xlO'8M ACh
O.i
8-14-75 C
2 min
Figure 23. Absence of direct stimulating action of 1.33 x 10~ M dichlofenthion
in guinea pig ileum. Acetylcholine (ACh)-induced contraction is not affected
by pretreatment with dichlofenthion.
77
-------
-1.33
DURSBAN-
6.65xlO"8M ACh
6.65xlO~8M ACh
8-18-75F
2 m in
Figure 24. Absence of direct stimulating action of 1,33 x 10" M dursban in
guinea pig ileutn. Acetylcholine (ACh)-induced contraction is only slight
reduced by pretreatment with dursban.
78
-------
1.33
6.6 5 x 10~8M ACh
0.5 g
8-15-/5A
Figure 22. Absence of direct stimulating action of
in guinea pig ileum. Acetylcholine (ACh)-induced Co.,
affected by pretreatment with monocrotophos.
76
-------
9.10x)0"8MACh
].33xlO~6MLEPTOPHOS
0.5o
6-10-75D
2 m in
Figure 21. Acetylcholine (ACh)-induced contraction in guinea pig ileum, and
absence of direct stimulating action of 1,33 x 10" M leptophos.
75
-------
l.33x IO'8M PROPOXUR
05 g
8- 19-75 A
B
6 65 x |Q'8M ACh
05g
9-9-75C
1.3 3 x 10 ~7M PROPOXUR
3.99 x U^K PROPOXUR
2 min
1.33 x I06M PROPOXUR
1 -.»..i .j il
.itii i"irnr^™*™*i
2min
Figure 25. Contractions induced by various concentrations of propoxur in
guinea pig ileum. The contractions are small in amplitude and slow in onset
compared to the acetylcholine (ACh)-induced contraction.
79
-------
6.65x]Q"8M ACh
8-19-75C
1.33x|0"6MPROPOXUR-
6.65 xlQ'8M ACh
******
2 min
Figure 26. Absence of the effect of propoxur (1.33 x 10~ M) pretreatment on
the acetylcholine (ACh)-induced contraction in guinea pig ileurn.
80
-------
6.65x]0"8M ACh 6.65 xlO"8M ACh
0.5g
.33x]0~7M FERBAM *-
6.65xlO'8M ACh
r,fcftrf'V«**V>
8-20-75C
2 min
Figure 27. Absence of direct stimulating action of 1,33 x 10 M ferbam in
guinea pig ileum. Acetylcholine (ACh)-induced contraction is not affected
by pretreatment with ferbam.
81
-------
•—I 33 « ICT8M—i
FORMETANATE
— I 33x|0'7M-"-
FORMETANATE
05B
8-27-75A
-•1.33 " 10'6M FORMETANATE-
2 mm
B
6.65«10"8M ACh 6.65 xl(3"8M ACh
•*-l.33x|0~6M FORMETANATE*
6.65* If^M ACh
0.5 a
8-27-75D
-P fi
Figure 28. A, contractions induced by formetanate (1.33 x 10" - 1.33 x 10 M)
in guinea pig ileum. The contractions become more intense with increasing the
contraction, but are slow in onset. B, absence of the effect of 1.33 x 10 M
formetanate pretreatment on the acetylcholine (ACh)-induced contraction.
Repetitive contractions are caused by formetanate itself.
82
-------
6.65xlO"8M ACh
-).33x)0"6M CHLORDIMEFORM-^
6.65xlO~8M ACh
0.5 g
9-5-75D
2 min
Figure 29. Absence of direct stimulating action of 1.33 x 10~ M chlordimeform
in guinea pig ileum. Acetylcholine (ACh)-induced contraction is slightly
suppressed by pretreatment with chlordimeform.
83
-------
6.65xlO"8MACh
8-1I-75K
B
6.65x|(T8MACh
0.2 g I
\
9-3-75G
8-22-75B
-1.33x1(T8M ATROPINE •
1.33x)0~6M CARBOFURAN
6.65xlCT8M ACh
2 mm
-133xlO~4V HEXAMETHONIUM-
1.33xl(T6M CARBOFURAN
2 mm
-1.33x|0~7M DIPHENHYDRAMINE »-
1.33 x |Q"6M CARBOFURAN
2 mm
o
Figure 30. Effects of pretreatment with atropine (1.33 x 10" M), hexamethonium
(1.33 x 10"4 M), diphenhydramine (1.33 x 10"7 M), and physostigmine (1.33 x 10"8
on the contractions induced by carbofuran (1.33 x 10" M) in guinea pig ileum.
None of these pretreatments themselves causes contraction. The carbofuran
contractions are completely abolished by atropine (A), but not appreciably
affected by hexamethonium (B), or diphenhydramine (C). Acetylcholine (ACh)-
induced contractions are also illustrated for comparison. See Fig. 18 for the
contractions in the presence of carbofuran alone.
M)
84
-------
665«10"°M ACh
058
M ACh
I 33«IO"8M ATROPINE-
-399xIO-'M PROPOXUR-
33«IO~4M HEXAMETHONIUM-
-3.99 x IO"4M PROPOXUR-
-I33«IO"7M OIPHENHYDRAMINE-
-399»10"*V PROPOXUR-
-8
Figure 31. Effects of pretreatment with atropine (1.33 x 10" M), hexamethonium
(1.33 x 10 M), and diphenhydramine (1.33 x 10~ M) on the contractions induced
by propoxur (3.99 x 10" M) in guinea pig ileum. The propoxur contractions are
completely abolished by atropine (A), but not appreciably affected by hexarr>ethor,iur
•B) or diphenhydramine (C). Acetylcholine contractions are also illustrated for
comparison. See Fig. 25 for the contractions in the presence of propo/ur alone.
85
-------
A
-« I.33«10'8M ATROPINE *-
6.65»IO"*M ACh
«-» -. 133 xlCr°W FORMETANATE-»•
05g
9.4.750
B
l33xlr
-------
-TYRODE-
6.65x|0"8M ACh 3.99xlO'7MPHYSOSTIGWIN
0.5 g
[_JV
1I-28-75A
2 mm
B
TYRODE
6.65xlO~8M ACh
05g
I-28-75B
-HIGH Mg-H- LOW Co* TYRODE-
6.65xlO~8M ACh 6.65x|0"8M ACh
r\ (V
-3.99xlO~7M PHYSOSTIGMINE
-TYRODE*
2 min
Figure 33. Effect of high Mg++ - low Ca++ (2.2 mM Mg++-0.36 mM Ca++) Tyrode's
solution on the contractions of guinea pig ileum induced by acetylcholine (ACh)
o 7
(6.65 x 10 M) and by physostigmine (3.99 x 10 M). The ACh contraction is
smaller in amplitude and the physostigmine contraction is smaller in amplitude
and longer in latency in the modified Tyrode than in normal Tyrode.
87
-------
6.65xlO~8M ACh
-TYRODE
L33xlO"6M CARBOFURAN*:
11-28-75F
2 mm
B
-HIGH Mg* LOW Co* TYRODE-
-•-TYRODE-
665xlO~8M ACh
1.33xlO"6MCARBOFURAN
11-28-75G
2 min
Figure 34. Effect of high Mg++ - low Ca++ (2.2 mM Mg++-0.36 mM Ca++) Tyrode's
solution on the contractions of guinea pig ileum induced by carbofuran
(1.33 x 10 M). The carbofuran contractions are smaller in amplitude and
longer in latency in the modified Tyrode than in normal Tyrode.
88
-------
-« TYRODE
6.65x10"8M ACh
3.99x)0'6MPROPOXUR-
12-5-75A 2 mm
B
•* HIGH M8* LOW Co* TYRODE —
6.65 x]Q~8M ACh ,
•**> —'—3.99xlQ~6M PROPOXUR-
0.5 gF
12-5-75B 2 min
Figure 35. Effect of high Mg++ - low Ca++ (2,2 mM Mg++-0.36 mM Ca++) Tyrode's
solution on the contractions of guinea pig ileum induced by propoxur
(3.99 x 10" M). The propoxur-induced contractions are smaller in amplitude
and longer in latency in the modified Tyrode than in normal Tyrode.
89
-------
ACh
TYRODE
].33xlO~6M FORMETANATE*
H-28-75D
2 min
B
6.65x10~°M ACh
11-28-75E
-HIGH Mg* LOW CottTYRODE «~»-TYRODE»
1.33 xlO"6M FORMETANATE »~
2 min
Figure 36. Effect of high Mg++ - low Ca++ (2.2 mM Mg++-0.36 mM Ca++) Tyrode's
solution on the contractions of guinea pig ileum induced by formetanate
(1.33 x 10" M). The formetanate contractions are smaller in amplitude and
longer in latency in the modified Tyrode than in normal Tyrode.
90
-------
6.65*10"6M NICOTINE
6.65x7(f8M ACh
-3.99xlQ'8M PHYSOSTIGMINE-
20 mg
3-4-76A
2 min
Figure 37. Effects of 6,65 x 10" M nicotine, 6,65 x 10"8 M acetylcholine
-8
(ACh) and 3,99 x 10 M physostigmine on the guinea pig ileum treated with
black widow spider venom (0.4 glands/ml) for 15 minutes. Although nicotine
fails to stimulate the preparation, acetylcholine response is small and
physostigmine still induces contractions.
91
-------
0,015
K °-01
0.005
0
0 5
1
1
1
15 30 45 60
VENOM TREATMENT (min.)
75
Figure 38, Effect of pretreatment of guinea pig ileum with black widow spider
venom at various concentrations (0.2-4 glands/ml) at 24°C or 37°C on physostigmine
contraction. Each symbol represents each preparation. The ordinate represents
the reciprocal of the time for the physostigmine (3.99 x 10" M) contraction to
-8
attain the same level as the acetylcholine (6.65 x 10 M) contraction in the
same preparation. The dotted line shows the mean control value without /eno-
treatment.
92
-------
6«5»IO'sMACh 399»10'7MPHYSOS7IGMINE 665«IO"*MACh
B
663»IO"8M ACh 6 65 HIO'AM NICOTINE
—— »— ..—» 665*IO'*M ACh
TV
C
«u65.|CT«MACk
** •. 133"ICT6M CARBOFURAN—M U6« 10"5MCAR6OFUfiAN »
46* \0'*M PBOPOXUR *
••—I 33 « IO'*M FO8METANATE—!««» IO'5M FOKMETANATE-*- Dt * lO^M FOtMtTANATE-*-
Figure 39. Responses of the denervated guinea pig ileum (Paton-Zar method) to
drugs and insecticides. A, physostigmine (3.99 x 10" M) itself causes no
contraction, but potentiates the acetylcholine (ACh)-induced contraction.
B, Nicotine (6.65 x 10~ M) fails to induce contraction. C, carbofurar,
(1.33 x 10~6 M and 1.46 x 10"5 M) fails to stimulate the ileurn. D,
(1.33 x 10"6 M and 1.46 x 10"5 M) fails to stimulate, t, forretanate
(1.33 x 10"6 M, 1.45 x 10"5 M, and 1.59 x 10"4 M) fails to stir.ulate.
93
-------
2x)0'6M
CARBACHOL, 02ml
*
5x](T6M
CARBACHOL, 0.2 ml
I
9-22-75 B
1 mm
Figure 40. Negative inotropic effect of carbachol on the guinea pig heart.
Carbachol solutions are injected to the perfusate at arrows in the amount
and concentrations indicated.
94
-------
I xl(T4M ATROPINE, 0.2mi
I
9-22-75B
1 x]0~4M ATROPINE,0.2ml
2x10-6MCARBACHOL,0.2ml
I
1 mm
Figure 41. Effect of atropine on the contraction of the guinea pig heart. Drug
solutions are injected to the perfusate at arrows in the amount and concentra-
tions indicated. Although atropine itself has no effect on the contraction, it
abolishes the negative inotropic action of carbachol seen in Fig. 40.
95
-------
2.0
2,.s
U
t—
01.0
0.5
0
ISOPROTERENOL IN :
O LOCKE'S SOLUTION
LOCKE'S SOLUTION IN DARK
LOCKE'S SOLUTION + Na2S2O5
Tl AAC f _-: - \
ISOPROTERENOL IN:
O LOCKE'S SOLUTION
LOCKE'S SOLUTION + ASCORBIC ACID
I
I
0 10 20 30 40 50 60
TIME (min.)
70
80
90
100
Figure 42. Lffects of storage of isoproterenol solution under various experimental
conditions on its potency in causing a positive inotropic action on guinea pig
heart. The ordinate represents the amplitude of contraction relative to that
observed in freshly prepared solution, and the abscissa is the storage time.
Concentrations of the test solutions (0.2 ml) injected are 1 x 10" M for
4 -4
isoproterenol, 4.26 x 10 M for Na^S^U,- and 1 x 10 M for ascorbic acid.
96
-------
1«IO"4M
ATROPINE, 0.2ml
I
05g
lxlO~5M
ISOPROTERENOL,02r
I « IO'5M
ISOPROTERENOL,02ml
I
9-23-75
I mm
Figure 43. Positive inotropic effect of isoproterenol on the guinea pig heart
(A). Drug solutions are injected to the perfusate at arrows in the amount and
concentrations indicated. Atropine does not affect the isoproterenol action (B)
97
-------
U10'5M
ISOPROTERENOL
0.2ml
0.5 g
B
PROPRANOLOL, 0.2ml
*
9-23-75
|x)0"5M
ISOPROTERENOL, 0.2 ml
\
iilitiiuuuuliilftUUIllllUllilUUUltllttllllllll
Figure 44. Abolition of the positive inotropic action of isoproterenol on the
guinea pig heart (A) by propranolol (B). Drug solutions are injected to the
perfusate at arrows in the amount and concentrations indicated.
98
-------
lxlO"4M CONTROL SOLUTION
ATROPINE ,0.2ml 0.2ml .] x 10'6M ISOPROTERENOL
\ \ * 0.2ml
B
10-20-75
)x|0"4M lxi(T4M CARBOFURAN 1 xl
-------
lx!0'4M CONTROL SOLUTION
ATROPINE.0.2 ml 0.2 ml ] x 1Q'6M ISOPROTERENOL
| 0.2 ml
10-24-75
B
lxlO"4M lxlO"4M PROPOXUR
ATROPINE,0.2ml 0.2ml lxiO'6M ISOPROTERENOL
| I 10.2 ml
0.5 g
2 min
Figure 46. Effect of propoxur on the contraction of the guinea pig heart, The
positive inotropic action of isoproterenol on the atropinized preparation (A)
is not affected by propoxur (B). Propoxur itself has no direct effect on the
contraction (B). Drug solutions are injected to the perfusate at errors in the
amount and concentrations indicated.
100
-------
lxlO"4M CONTROL
ATROPINE SOLUTION
0.2ml 0.2ml
I I
lxlO'6M
ISOPROTERENOL
0.2ml
B
\ x 10'4M
ATROPINE
0.2ml
1x)0~2M
FORMETANATE
0.2ml
10-24-75
ISOPROTERENOL
0.2ml
I
0.5 9
2 min
Figure 47. Effect of formetanate on the contraction of the guinea pig heart.
The positive inotropic action of isoproterenol on the atropinized preparation
(A) is not affected by formetanate (B). Formetanate itself has no direct action
on the contraction (B). Drug solutions are injected to the perfusate at STOW:
in the amount and concentrations indicated.
101
-------
-l33x10~6M PHENTOLAMINE-
2.66xtO'5MIMOREPINEPHRINE
lg
2.66xlO"5M NOREPINEPRINE
10-31-75A
2 min
Figure 48. Contractions of the guinea pig vas deferens induced by norepinephrine
(2.66 x 10 M), and suppression by phentolamine (1.33 x 10 M).
102
-------
-I33*IO~8M ATROPINE-
-133"10"8M ATROPINE-
-CONTROL SOLUTION »-
2.66*ICT5M NOREPINEPHRINE
-l.33x|0~5M CARBOFURAN—»-
266xlO'5M NOREPINEPHRINE
11-3-75 D
2 mm
Figure 49. Effect of carbofuran on the atropinized guinea pig vas deferens.
Carbofuran (1-33 x 10" M) does not stimulate, and fails to affect the norepinephrine-
induced contraction.
103
-------
le
-l.33xlO'8M ATROPINE-
*-CONTROL SOLUTION—»>
2.66xlOT5M NOREPINEPHRINE
-1.33x]0"8M ATROPINE-
-l.33xlO'5M PROPOXUR-*
2.66xlO'5M NOREPINEPHRINE
11-6-75 D
2 min
Figure 50. Effect of propoxur on the atropinized guinea pig vas deferens,
Propoxur (1.33 x 10~ M) does not stimulate, and fails to affect the norepinephrine-
induced contraction.
104
-------
-1.33xl(T8M ATROPINE-
-CONTROL SOLUTION +
2.66 x)0'5M NOREPINEPHRINE
lg
•*1.33xlO'4M FORMETANATE*
2.66x|0'5M NOREPINEPHRINE
11-4-75 A
2 mm
Figure 51. Effect of formetanate on the atropinized guinea pig vas deferens.
Formetanate (1,33 x 10~ M) does not stimulate, and fails to affect the
norepinephrine-induced contraction.
105
-------
xlO^MPYSOSTIGMINE-
0.2 a
2-28-76C
2 min
Figure 52. Potentiatlon of the contractions of the rat diaphragm evoked by
nerve stimulations by 1 x 10" M physostigmine.
106
-------
2.5
2.0
u
< 15
L0
z
o
u
1.0
LLJ
a:
0.5
0
I I
I
I
I
3 6 10 20 30 60
PHYSOSTIGMINE (x]0'7M)
100 200
Figure 53. Effects of physostigmine pretreatment for 5 min on the contraction
evoked by nerve stimulation in rat diaphragm. The ordinate represents the
height of contraction relative to that without physostigmine.
107
-------
-KREBSH(SOLUTION DJ-
-2.50*10'6M d-TUBOCURARlNE-*-
O.lg
il
2-28-76C
11
2 min
B
-TYRODE 31 (SOLUTION B)-
-1.67xlO"6M d-TUBOCURARINE-
0.1
niti.it,
I-19-76A
2 min
Figure 54. Suppression of the contractions of the rat diaphragm evoked by
nerve stimulations by 2.50 x 10" M d-tubocurarine in solution D (record A)
and by 1.67 x 10" M d-tubocurarine in solution B (record B).
108
-------
3-I-76A
-SXIO^MCARBOFURAN
2 mm
Figure 55. Potentiation of the nerve evoked contraction of rat diaphragm by
5x10 M carbofuran. The sensitivity of recording system is reduced to one-
half at vertical arrow.
109
-------
0.5 g
-5xlQ'6M PROPOXUR-
l!h HI
UuHMlh
1-29-76 A
2 mm
Figure 56. Absence of the effect of 5 x 10 M propoxur on the nerve evoked
contraction of rat diaphragm.
110
-------
-SMO^MFORMETANATE——
Figure 57. Potentiation of the nerve evoked contraction of
5 x 10" M formetanate,
rat diaphragm by
111
-------
-1 x]Q'6M PHYSOSTIGMINE •
0.2 g
•5*10~VM CARBOFURAN-
2 mm
2-27-76 B
-]xlO~*M PHYSOSTIGMINE-
-«-5.5xlO"8MCARBOFURAN-
-5 55 MO"'M CARBOFURAN-
-556xlO°MCARBOFURAN-
-9
,-6
— M r\
Figure 58. Absence of the effects of carbofuran (5 x 10 M to 5.56 x 10" M)
on the nerve evoked contractions of the rat diaphragm pretreated with 1 x 10" M
physostigmine.
112
-------
ICT6M PHYSOSTIGMINE-
02g
Olg
-5 «]0"9W PROPOXUR-
I « IO"6M PHrSOSTIGMINE-
— I » 10"8M PROPOXUR
2-26-76B
-6»IO"<1M PROPOXUR-
-lx|0"6M PHYSOSTIGMINE-
I « IO"7M PROPOXUR -*-• 6 I »10"7M PROPOXUR-
2 .1,1
Figure 59. Effects of propoxur (5 x 10"9 M to 5.61 x TO"6 M) on the nerve
evoked contractions of the rat diaphragm pretreated with 1 x TO"6 M physostigmine.
The contractions are potentiated only slightly.
113
-------
-1 x IO~6M PHY5OSTIGVINE •
55* IO"8M FORMETANATE-
2 mm
2-28-76A
t-555«IO~7M FORMETANATE
-1»IO~6M PHYSOSTIGMINE-
FORMETAN ATE -"5.56 * 1
-------
-399"IO"6M ACh
B
<9
•l.33x|0"4M PHYSOSTIGMINE-
-3.99 x 10'6M ACh-
5 mm
I2-15-75B
r-igure 61. Contraction of the frog rectus abdominis by 3.99 x 10" M acetylcholine
CACh) (A), and potentiation by 1.33 x 10~4 M physostigmine fB).
115
-------
5 i-
at:
K—
cO 3
O 2
u
o
u
I I
1
CONTROL
1 i 1 I
I
3 4 6 10 20 30 40 60 100
ACETYLCHOLINE (xlO"7M)
200
Figure 62. Dose-response relation for acetylene!ine contraction of frog rectus
abdominis with and without pretreatment with 8,07 x 10" M physostigrnine for
5 min.
116
-------
10'
10"5 10'4
PHYSOSTIGM!NE(M)
10
,-3
Figure 63. Effects of pretreatment with various concentrations of :/r/:ostigr.ine
for 5 min on acetylcholine (3.99 x 10" M) contraction of frog rec"^ ^cor-inis.
Each symbol represents each preparation.
117
-------
B
I 33 x 10"4M PHYSOSTIGMINE-
33"10"4M PHYSOSTIGMINE-
1.33xlO"5M CARBOFURAN
1-7-76A
5 mm
igure 64. Effect of 1,33 x 1 0" M carbofuran on the frog rectus 3bdo~:inis
-4
-
pretreated with 1.33 x 10 X physostigmine. Carbofuran does not cejse
contraction itself, and fails to potentiate markedly the contraction e/oked
"6
by 3.99 x TO" M acetylcholine (ACh).
118
-------
-].33x](T4M PHYSOSTIGMINE-
-3.99x1(7^ ACh-
B
-|.33xlO'4M PHYSOST1GMINE-
1 33 x 10~5M PROPOXUR *-
*—3 99x)0~6M ACh-*-
5 mm
12-18-75A
Figure 65. Effect of 1.33 x 10 M propoxur on the frog rectus abdorr-irn's tretreated
with 1.33 x 10 M physostigmine. Dropoxur does not initiate contraction by itself,
-6
and fails to potentiate the contraction evoked by 3.99 x 10 M acetylcnoline 'AChj.
119
-------
-1.33xlO'4M PHYSOSTIGMINE-
-3 99*10~6M ACh-»-
B
I2-I7-75B
-1.33 « ICf4 M PHYSOSTIGMINE-
I33»10'4M FORMETANATE-
•• 3.99>
-------
TECHNICAL REPORT DATA
ase read instructions on the reverse before completing)
1 REPORT .\o
EPA-600/1-76-035
4 TITLE A\2 S^ 3~ TL£
IN-VITRO METHODS FOR EVALUATING SIDE EFFECTS OF
PESTICIDES AND TOXIC SUBSTANCES
6. PERFORMING ORGANIZATION CODE
7 AUTHOR, S'
Toshio Narahashi, Ph.D.
8. PERFORMING ORGANIZATION REPORT NO
3. RECIPIENT'S ACCESSION-NO.
5 REPORT DATE
November 1976
9 PERFORVING ORGANIZATION NAMb AND ADDRESS
Department of Physiology and Pharmacology
Duke University Medical Center
Durham, North Carolina 27710
10. PROGRAM ELEMENT NO.
661526HEAO
11. CONTRACT/GRANT NO.
68-02-1289
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park. N.C. 27711
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA-ORD
15. SUPPLEMENTARY NOTES
16 ABSTRACT
Several skeletal muscle and smooth muscle preparations have been examined for
their usefulness in evaluating the toxic effects of a variety of insecticides. The
following preparations were found satisfactory for such test: guinea pig ileum for
muscarinic receptors, guinea pig heart for 3-adrenergic receptors, guinea pig vas
deferens for a-adrenergic receptors, frog rectus abdominis for nicotinic receptors
of tonic muscle, and rat diaphragm for nicotinic receptors of phase muscle. Five
carbamate insecticides, four organophosphate insecticides and chlordimeform were
studied. None of the insecticides tested had any direct and potent effect on these
receptors except the effect on cholinergic receptors via cholinesterase inhibition.
Carbofuran, propoxur and formetanate had potent stimulating actions on the guinea
pig ileum, but these effects could entirely be attributed to the accumulation of
acetylcholine in the synaptic cleft as a result of cholinesterase inhibition. Thus
it can be concluded that these insecticides exert no direct action on cholinergic
and adrenergic receptors.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
Insecticides
Toxicity
In vitro analysis
Adrenergics
Cholinergics
DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
b.IDENTIFIERS/OPEN ENDED TERMS
19 SECURITY CLASS (This Report/
-II
COSATI f icld/Group
06, F, 0
20 StCTJR'TY CLASS (Thispaqe)
UNCLASSIFIED
21 NO OF PAGEe
J 125
'22 PRICE
EPA Form 2220-1 (9-73)
121
-------
INSTRUCTIONS
1. REPORT NUMBER
Insert the EPA report numner as it appears on the cover of the publication.
2. LEAVE BLANK
3. RECIPIENTS ACCESSION NUMBER
Reserved for use by each report recipient.
4. TITLE AND SUBTITLE
Title should indicate clearh and briefly the subject coverage of the report, and be displayed prominently. Set subtitle, it used, in smaller
t>pe or otherwise subordinate it to mam title. When a report is prepared in more than one volume, repeat the priman title, add volume
number and include subtitle for the specific title.
5. REPORT DATE
Each report shall carr> a date indicating at least month and year. Indicate the basis on which it was selected (e.g., date of issue, date of
approval, date of preparation. etc.).
6. PERFORMING ORGANIZATION CODE
Leave blank.
7. AUTHOR(S)
Give name(s) in conventional order (John R. Doe, J. Robert Doe, etc.). List author's affiliation if it differs from the performing organi-
zation.
8. PERFORMING ORGANIZATION REPORT NUMBER
Insert if performing organization wishes to assign this number.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Give name, street, city, state, and ZIP code. List no more than two levels of an organizational hirearchy.
10. PROGRAM ELEMENT NUMBER
Use the program element number under which the report was prepared. Subordinate numbers may be included in parentheses.
11. CONTRACT/GRANT NUMBER
Insert contract or grant number under which report was prepared.
12. SPONSORING AGENCY NAME AND ADDRESS
Include ZIP code.
13. TYPE OF REPORT AND PERIOD COVERED
Indicate interim final, etc., and if applicable, dates covered.
14. SPONSORING AGENCY CODE
Leave blank.
15. SUPPLEMENTARY NOTES
Enter information not included elsewhere but useful, such as: Prepared in cooperation with, Translation of, Presented at conference of,
To be published in, Supersedes, Supplements, etc.
16. ABSTRACT
Include a brief (200 words or less) factual summary of the most significant information contained in the report. If the report contains a
significant bibliography or literature survey, mention it here.
17. KEY WORDS AND DOCUMENT ANALYSIS
(a) DESCRIPTORS - Select from the Thesaurus of Engineering and Scientific Terms the proper authorized terms that identify the major
concept of the research and are sufficiently specific and precise to be used as index entries for cataloging.
(b) IDENTIFIERS AND OPEN-ENDED TERMS - Use identifiers for project names, code names, equipment designators, etc. Use open-
ended terms written in descriptor form for those subjects for which no descriptor exists.
(c) COSATI FIELD GROUP - Field and group assignments are to be taken from the 1965 COSATI Subject Category List. Since the ma-
jority of documents are multidisciplinary in nature, the Primary Field/Group assignment(s) will be specific discipline, area of human
endeavor, or type of physical object. The application(s) will be cross-referenced with secondary Field'Group assignments that will follow
the primary postmg(s).
18. DISTRIBUTION STATEMENT
Denote releasabihty to the public or limitation for reasons other than security for example "Release Unlimited." Cite any availability to
the public, with address and price.
19. & 20. SECURITY CLASSIFICATION
DO NOT submit classified reports to the National Technical Information service.
21. NUMBER OF PAGES
Insert the total number of pages, including this one and unnumbered pages, but exclude distribution list if any.
22. PRICE
Insert the price set by the National Technical Information Service or the Government Printing Office, if known.
EPA Form 2220-1 (9-73) (Reverse)
------- |