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Smooth muscle stimulating action of venom from the Gila monster, Heloderma suspectum
Toxkox 1967, Vol. 3, pp . 11-15. Pergamon Prep Ltd., Printed In Great Britain
SMOOTH MUSCLE STIMULATING ACTION OF VENOM FROM THE GILA MONSTER, HELODERMA SUSPECTUM* ROBERT A. PATTERSON Department of Zoology, Arizona State University, Tempe, Arizona, U.S.A . (Accepted for publication 17 March 1967)
Abstract-Venom from the Gila monster, Heloderma suspectum Cope, was tested on isolated sections of the guinea pig ileum, rat ascending colon, and rat estronized uterus . A stimulating effect of a delayed nature was found which was neither blocked by atropine nor by the antihistaminic agent Henadryl . While incubation in a boiling water bath for 20 min destroyed this smooth muscle stimulatory effect, shorter incubation periods did not . INTRODUCTION
work [1] has shown that intravenous injections of Gila monster venom caused tachycardia, hypotension, and respiratory distress in anesthetized dogs and rats . Intracardial injections of this venom in non-anesthetized rats produced symptoms indicating similar responses [1]. This study was initiated as part of a survey of effects produced by venom from the Gila monster (Heloderma suspectum Cope) on various smooth muscles. Tissues were selected to test the histaminic and/or cholinergic properties of this venom. These tissues included guinea pig ileum which is sensitive to histamine, rat colon relatively insensitive to histamine, and estronized rat uterine tissue sensitive to various other types of smooth muscle stimulating agents . Atropine and Benadryl were chosen to block the actions of acetylcholine and histamine. In addition, the rat uterus was used to test venom samples which had been heated on a boiling water bath . PREvious
Venom preparation
MATERIALS AND METHODS
Venom was obtained from the Poisonous Animals Research Laboratory of Arizona State University. The venom secreted from the labial glands had been pooled, then lyophilized and weighed. Finally, the venom was diluted in mammalian saline (0-85 per cent NSCI) and subdivided into small quantities in ampoules which were lyophilized, sealed, and stored at freezing temperatures . As needed, the ampoules were opened and the dehydrated venom diluted with distilled water . The final concentration used in the different tests varied but the volume used was always less than 0-5 ml . *This study was supported by the United States Public Health Service Research Grant HE 04923 from the National Heart Institute.
12
ROBERT A . PATTERSON
Heat stability To test the heat stability of the diluted venom, five ampoules of diluted venom were heated on a boiling water bath . At intervals of 3, 5, 10, 20 and 30 min a vial was removed and frozen after replacing any fluid lost by evaporation . Contents of these ampoules were used to determine the heat stability of this venom . Assay procedures Animals were deprived of food for 24 hr prior to decapitation . Tissues were removed promptly and placed in Tyrode's solution . Ileum and colon sections were divided into sections 1-5 cm long . Rat uterine sections-prepared by separating the uterine horns from young virgin rats of a Wistar strain-were obtained from rats which had received 24 hr previously an intramuscular injection of an estrogen preparation (Theelin, 100 Fig/kg, Parke, Davis & Co .) . Either a glass or Plexiglass tube served as a muscle chamber. Tyrode's solution was added or removed through a glass tube inserted through a rubber stopper sealing the lower end of the chamber . Chambers were calibrated to have a volume of 10 ml and were gassed by passing a stream of 5 per cent carbon dioxide in oxygen through a side arm bypass tube . This also rapidly mixed venom and drugs with the chamber contents . Tissues were either anchored in the chamber to a stainless steel pin in the bottom stopper or were tied to a glass rod and lowered into the chamber . Recordings of isometric contractions were obtained by means of Grass FT 03 tension transducers (Grass Instrument Co ., Inc ., Quincy Mass .) . Tissues were stretched with a tension of 1 g and the recording system was calibrated to yield a deflection of 1 cm/g of additional tension . A Grass Polygraph was used to record the events . Chambers as well as the Tyrode's solution reservoir were placed in a water bath . Uterine sections were tested at 33°, while ileum and colon sections were tested at 39°. Ileum and colon sections were tested in Tyrode's solution containing NaCI (8-5 g/l), KC1 (0-2 g/1), CaCla (0-2 g/1), glucose (1 g/l), and buffered with NaHCOa (1 g/l) . Uterine sections were tested in a modified Tyrode's solution containing only 0-05 g of CaC1 Q in place of the usual quantity to reduce spontaneous activity . Commercially obtained acetylcholine chloride, atropine sulfate, histamine phosphate, and Benadryl hydrochloride (Diphcnhydramine hydrochloride, Parke, Davis & Co .) were diluted with glucose-free Tyrode's solution . Concentrations of atropine and Benadryl required to block the action of acetylcholine and histamine were determined by trial tests .
RESULTS Rat uterus Figure 1 illustrates the typical effects of Gila monster venom on normal (A-1) and atropinized (A-2) preparations . Venom was tested on 8 normal and 12 atropinized sections at concentrations of either 2 or 4 leg/ml . Preliminary tests had demonstrated that rat uterine section did not respond uniformly to concentrations of venom less than 2 i`g/ml . Typically, a series of rhythmical contractions started 25 to 35 sec after venom was added . Atropine, though used at concentrations which blocked the action of acetylcholine as tested both before and after the addition of venom, failed to modify the effects of venom . Simultaneous assay techniques were used to compare the action of heated and nonheated samples of venom . Heating of venom on a boiling water bath for 10 min or less
Heloderma Venom-Action on Smooth Muscle
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Do. 1 . EFFECTS of Heloderma suspectum VENOM ON ISOLATED VISCERAL SMOOTH Mvscc .E. Top pair of responses show effects of venom (V, 4 tag/ml) on estronized rat uterine sections . One section (A-2) was treated with enough atropine (1 jig/ml) to block the stimulating effects of 1 Wg of ACH per ml . Response A-1 shows the effect of 1 t`g of ACH before (ACH-1) and after (ACH-2) the addition of venom (V, 4 pg/ml) to the bath. Middle pair of responses show the effect of venom (V, 4 J&g/ml) which had been boiled (B-1) for 10 min. Response B-2 is a control . Tissue is from the estronized rat uterus . Lower response (C) shows the temporary inhibiting effect of this venom (V, 1 pg/ml) on the rat ascending colon. Chamber volume, 10 ml . Test solutions : rat uterus, Tyrode's solution with reduced calcium concentration; rat ascending colon, Tyrode's solution . Temperature used : rat uterus 33°, ascending colon 39°.
failed to alter the typical effects as described above. Four sections were tested with venom heated for 10 min and 8 others were tested with venom heated either 5 or 3 min. Venom concentration used was 4 lyg/ml. However, only 1 of 3 sections of uterus tested with venom heated for 20 min reacted typically. None of the 4 sections tested with venom which had been heat-treated for 30 min responded. All sections which did not react to heattreated venom were subsequently rinsed and treated with non-heated venom to verify that the muscle tissue could be stimulated by the venom . In Fig. 1, line B-1 represents the response of a rat uterine section treated with venom which had been boiled for 10 min . Line B-2 is a simultaneous control test of unboiled venom. Rat ascending colon
This tissue was tested at 33° to reduce the frequency of rhythmic contractions . Venom was found to have an inhibitory effect in 6 of 7 sections tested with venom at a concentration of 1-0 lug/ml . This was followed after a period of from 30 to 35 sec by a resumption of contractile activity. This inhibitory effect was characterized by a reduction in frequency and strength of activity . No change in minimal tension was observed (Fig. 1, C) . Similar effects were found in 2 sections tested with 2-0 jig of venom per ml, and in 2 others tested with 2-5 Wg of venom per ml . Only 1 of 4 sections tested with 0-05 l~g of venom per ml developed any inhibitory response.
14
ROBERT A. PATTERSON
Guinea pig ileum H. suspectum venom at a concentration of either 2 or 4 jzg/ml caused a prolonged con-
traction of this tissue after a short delay (Fig. 2, middle). Sections treated at lower concentrations (1-0 and 0-5 jLg/ml) produced inconsistent results, and sections tested at less than 0-1 kg of venom per ml yielded no response. Simultaneous assay techniques were used to test venom in ileum sections treated with either atropine (0-01 ,ug/ml) or with atropine and Benadryl (0-01 jug/ml). Untreated control sections were tested simultaneously . Preliminary tests had shown that, at these concentrations, atropine blocked the stimulatory effect of 0-01 jig of acetylcholine per ml and that
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FIO. 2. SIMULTANEOUS ASSAYS OF HejOderJ17R SIfSpeCt!l»I VENOM ON SECTIONS OF THE OUINEA PIG ILEUM.
Top response was obtained from a section which responded to histamine (H, 0'02 tag/ml) before-far right-and after the addition of venom (V, 4 4n-d). Response to acetylcholine (ACH, 001 tag/ml) had been blocked by atropine (001 jug/ml) . Middle response was obtained from a control section treated with venom (V, 4 4ml) . Response to acetylcholine (ACH, 001 pg,/orl) is shown before-far right-,and after envenooration. Lower response was obtained from a section which had been blocked by atropine (001 pg/ml) and Benadryl (001 pg/ml) . Response on the far right shows the stimulating effect of histamine (H, 002 fcg/ml) before the muscle had been treated with blocking agents . After venom treatment (V, 4 pg/ml), the section was tested with acetylcholine (ACH, 001 pg/ml) and histamine (H). Bath temperature : 39°. Bath volume : 10 ml . Test media: bicarbonate buffered Tyrode's solution .
Benadryl blocked the stimulating action of002 icg of histamine per ml. Judging from results obtained following 13 tests in which atropine was used alone and the 6 tests in which both atropine and Benadryl were used, venom action was not significantly altered by these agents (Fig. 2). Routinely, acetylcholine was added both before and after test doses of venom in this and other tissues. However, ileum sections were tested more thoroughly . Routinely, acetylcholine was added and then rinsed out several times 3 to 5 min apart before the addition
Helodernm
Venom-Action on Smooth Muscle
15
of venom to verify the uniformity of response of this tissue to this drug. Then acetylcholine was added after the addition of venom. No significant potentiation or inhibition was found. In a few cases the response was increased and in a few the response to acetylcholine was decreased by a factor of 10 to 15 per cent. Most sections exhibited the same response after the addition of venom as developed in the control tests . SUMMARY
Gila monster venom contains a relatively heat stable, non-cholinergic, and nonhistaminic smooth muscle stimulating factor . Heat stability was tested in isolated sections of the estronized rat uterus, while the cholinergic and histaminic actions were tested in isolated sections of guinea pig ileum . Peculiar effects were found in the isolated rat ascending colon. While this venom stimulated the other tissues tested, temporary inhibition was found in sections of isolated colon. REFERENCE Ell PAIN, R. A., Some physiogical effects caused by venom from the Gila monster, Heloderma suspectum. Toxlcon 5,5-10 .
SMOOTH MUSCLE STIMULATING ACTION OF VENOM FROM THE GILA MONSTER, HELODERMA SUSPECTUM* ROBERT A. PATTERSON Department of Zoology, Arizona State University, Tempe, Arizona, U.S.A . (Accepted for publication 17 March 1967)
Abstract-Venom from the Gila monster, Heloderma suspectum Cope, was tested on isolated sections of the guinea pig ileum, rat ascending colon, and rat estronized uterus . A stimulating effect of a delayed nature was found which was neither blocked by atropine nor by the antihistaminic agent Henadryl . While incubation in a boiling water bath for 20 min destroyed this smooth muscle stimulatory effect, shorter incubation periods did not . INTRODUCTION
work [1] has shown that intravenous injections of Gila monster venom caused tachycardia, hypotension, and respiratory distress in anesthetized dogs and rats . Intracardial injections of this venom in non-anesthetized rats produced symptoms indicating similar responses [1]. This study was initiated as part of a survey of effects produced by venom from the Gila monster (Heloderma suspectum Cope) on various smooth muscles. Tissues were selected to test the histaminic and/or cholinergic properties of this venom. These tissues included guinea pig ileum which is sensitive to histamine, rat colon relatively insensitive to histamine, and estronized rat uterine tissue sensitive to various other types of smooth muscle stimulating agents . Atropine and Benadryl were chosen to block the actions of acetylcholine and histamine. In addition, the rat uterus was used to test venom samples which had been heated on a boiling water bath . PREvious
Venom preparation
MATERIALS AND METHODS
Venom was obtained from the Poisonous Animals Research Laboratory of Arizona State University. The venom secreted from the labial glands had been pooled, then lyophilized and weighed. Finally, the venom was diluted in mammalian saline (0-85 per cent NSCI) and subdivided into small quantities in ampoules which were lyophilized, sealed, and stored at freezing temperatures . As needed, the ampoules were opened and the dehydrated venom diluted with distilled water . The final concentration used in the different tests varied but the volume used was always less than 0-5 ml . *This study was supported by the United States Public Health Service Research Grant HE 04923 from the National Heart Institute.
12
ROBERT A . PATTERSON
Heat stability To test the heat stability of the diluted venom, five ampoules of diluted venom were heated on a boiling water bath . At intervals of 3, 5, 10, 20 and 30 min a vial was removed and frozen after replacing any fluid lost by evaporation . Contents of these ampoules were used to determine the heat stability of this venom . Assay procedures Animals were deprived of food for 24 hr prior to decapitation . Tissues were removed promptly and placed in Tyrode's solution . Ileum and colon sections were divided into sections 1-5 cm long . Rat uterine sections-prepared by separating the uterine horns from young virgin rats of a Wistar strain-were obtained from rats which had received 24 hr previously an intramuscular injection of an estrogen preparation (Theelin, 100 Fig/kg, Parke, Davis & Co .) . Either a glass or Plexiglass tube served as a muscle chamber. Tyrode's solution was added or removed through a glass tube inserted through a rubber stopper sealing the lower end of the chamber . Chambers were calibrated to have a volume of 10 ml and were gassed by passing a stream of 5 per cent carbon dioxide in oxygen through a side arm bypass tube . This also rapidly mixed venom and drugs with the chamber contents . Tissues were either anchored in the chamber to a stainless steel pin in the bottom stopper or were tied to a glass rod and lowered into the chamber . Recordings of isometric contractions were obtained by means of Grass FT 03 tension transducers (Grass Instrument Co ., Inc ., Quincy Mass .) . Tissues were stretched with a tension of 1 g and the recording system was calibrated to yield a deflection of 1 cm/g of additional tension . A Grass Polygraph was used to record the events . Chambers as well as the Tyrode's solution reservoir were placed in a water bath . Uterine sections were tested at 33°, while ileum and colon sections were tested at 39°. Ileum and colon sections were tested in Tyrode's solution containing NaCI (8-5 g/l), KC1 (0-2 g/1), CaCla (0-2 g/1), glucose (1 g/l), and buffered with NaHCOa (1 g/l) . Uterine sections were tested in a modified Tyrode's solution containing only 0-05 g of CaC1 Q in place of the usual quantity to reduce spontaneous activity . Commercially obtained acetylcholine chloride, atropine sulfate, histamine phosphate, and Benadryl hydrochloride (Diphcnhydramine hydrochloride, Parke, Davis & Co .) were diluted with glucose-free Tyrode's solution . Concentrations of atropine and Benadryl required to block the action of acetylcholine and histamine were determined by trial tests .
RESULTS Rat uterus Figure 1 illustrates the typical effects of Gila monster venom on normal (A-1) and atropinized (A-2) preparations . Venom was tested on 8 normal and 12 atropinized sections at concentrations of either 2 or 4 leg/ml . Preliminary tests had demonstrated that rat uterine section did not respond uniformly to concentrations of venom less than 2 i`g/ml . Typically, a series of rhythmical contractions started 25 to 35 sec after venom was added . Atropine, though used at concentrations which blocked the action of acetylcholine as tested both before and after the addition of venom, failed to modify the effects of venom . Simultaneous assay techniques were used to compare the action of heated and nonheated samples of venom . Heating of venom on a boiling water bath for 10 min or less
Heloderma Venom-Action on Smooth Muscle
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13
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Do. 1 . EFFECTS of Heloderma suspectum VENOM ON ISOLATED VISCERAL SMOOTH Mvscc .E. Top pair of responses show effects of venom (V, 4 tag/ml) on estronized rat uterine sections . One section (A-2) was treated with enough atropine (1 jig/ml) to block the stimulating effects of 1 Wg of ACH per ml . Response A-1 shows the effect of 1 t`g of ACH before (ACH-1) and after (ACH-2) the addition of venom (V, 4 pg/ml) to the bath. Middle pair of responses show the effect of venom (V, 4 J&g/ml) which had been boiled (B-1) for 10 min. Response B-2 is a control . Tissue is from the estronized rat uterus . Lower response (C) shows the temporary inhibiting effect of this venom (V, 1 pg/ml) on the rat ascending colon. Chamber volume, 10 ml . Test solutions : rat uterus, Tyrode's solution with reduced calcium concentration; rat ascending colon, Tyrode's solution . Temperature used : rat uterus 33°, ascending colon 39°.
failed to alter the typical effects as described above. Four sections were tested with venom heated for 10 min and 8 others were tested with venom heated either 5 or 3 min. Venom concentration used was 4 lyg/ml. However, only 1 of 3 sections of uterus tested with venom heated for 20 min reacted typically. None of the 4 sections tested with venom which had been heat-treated for 30 min responded. All sections which did not react to heattreated venom were subsequently rinsed and treated with non-heated venom to verify that the muscle tissue could be stimulated by the venom . In Fig. 1, line B-1 represents the response of a rat uterine section treated with venom which had been boiled for 10 min . Line B-2 is a simultaneous control test of unboiled venom. Rat ascending colon
This tissue was tested at 33° to reduce the frequency of rhythmic contractions . Venom was found to have an inhibitory effect in 6 of 7 sections tested with venom at a concentration of 1-0 lug/ml . This was followed after a period of from 30 to 35 sec by a resumption of contractile activity. This inhibitory effect was characterized by a reduction in frequency and strength of activity . No change in minimal tension was observed (Fig. 1, C) . Similar effects were found in 2 sections tested with 2-0 jig of venom per ml, and in 2 others tested with 2-5 Wg of venom per ml . Only 1 of 4 sections tested with 0-05 l~g of venom per ml developed any inhibitory response.
14
ROBERT A. PATTERSON
Guinea pig ileum H. suspectum venom at a concentration of either 2 or 4 jzg/ml caused a prolonged con-
traction of this tissue after a short delay (Fig. 2, middle). Sections treated at lower concentrations (1-0 and 0-5 jLg/ml) produced inconsistent results, and sections tested at less than 0-1 kg of venom per ml yielded no response. Simultaneous assay techniques were used to test venom in ileum sections treated with either atropine (0-01 ,ug/ml) or with atropine and Benadryl (0-01 jug/ml). Untreated control sections were tested simultaneously . Preliminary tests had shown that, at these concentrations, atropine blocked the stimulatory effect of 0-01 jig of acetylcholine per ml and that
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FIO. 2. SIMULTANEOUS ASSAYS OF HejOderJ17R SIfSpeCt!l»I VENOM ON SECTIONS OF THE OUINEA PIG ILEUM.
Top response was obtained from a section which responded to histamine (H, 0'02 tag/ml) before-far right-and after the addition of venom (V, 4 4n-d). Response to acetylcholine (ACH, 001 tag/ml) had been blocked by atropine (001 jug/ml) . Middle response was obtained from a control section treated with venom (V, 4 4ml) . Response to acetylcholine (ACH, 001 pg,/orl) is shown before-far right-,and after envenooration. Lower response was obtained from a section which had been blocked by atropine (001 pg/ml) and Benadryl (001 pg/ml) . Response on the far right shows the stimulating effect of histamine (H, 002 fcg/ml) before the muscle had been treated with blocking agents . After venom treatment (V, 4 pg/ml), the section was tested with acetylcholine (ACH, 001 pg/ml) and histamine (H). Bath temperature : 39°. Bath volume : 10 ml . Test media: bicarbonate buffered Tyrode's solution .
Benadryl blocked the stimulating action of002 icg of histamine per ml. Judging from results obtained following 13 tests in which atropine was used alone and the 6 tests in which both atropine and Benadryl were used, venom action was not significantly altered by these agents (Fig. 2). Routinely, acetylcholine was added both before and after test doses of venom in this and other tissues. However, ileum sections were tested more thoroughly . Routinely, acetylcholine was added and then rinsed out several times 3 to 5 min apart before the addition
Helodernm
Venom-Action on Smooth Muscle
15
of venom to verify the uniformity of response of this tissue to this drug. Then acetylcholine was added after the addition of venom. No significant potentiation or inhibition was found. In a few cases the response was increased and in a few the response to acetylcholine was decreased by a factor of 10 to 15 per cent. Most sections exhibited the same response after the addition of venom as developed in the control tests . SUMMARY
Gila monster venom contains a relatively heat stable, non-cholinergic, and nonhistaminic smooth muscle stimulating factor . Heat stability was tested in isolated sections of the estronized rat uterus, while the cholinergic and histaminic actions were tested in isolated sections of guinea pig ileum . Peculiar effects were found in the isolated rat ascending colon. While this venom stimulated the other tissues tested, temporary inhibition was found in sections of isolated colon. REFERENCE Ell PAIN, R. A., Some physiogical effects caused by venom from the Gila monster, Heloderma suspectum. Toxlcon 5,5-10 .