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Effects of Naja haje (Egyptian cobra), Naja haja (hooded cobra), Naja nigricollis (spitting cobra) and Naja mossambica mossambica (Mozambique spitting cobra) venoms on the isolated guinea-pig tracheal muscle
Tmk+a~, Vol . 2A, Nor I1-12, pp. 1162-1165, 1986 . Ptioted io drat Britain .
0041-0101/!6 ß.00+ .00 Perpmoo JaornW Ltd.
EFFECTS OF NAJA HAJE (EGYPTIAN COBRA), NAJA NAJA (HOODED COBRA), NAJA NIGRICOLLIS (SPITTING COBRA) AND NAJA MOSSAMBICA MOSSAMBICA (MOZAMBIQUE SPITTING COBRA) VENOMS ON THE ISOLATED GUINEA-PIG TRACHEAL MUSCLE A. K.
TILMISANY,
A.
ABDEL
Azlz, O. H.
OSMAN
and A. A.
MUSTAFA
Department of Pharmacology, College of Medicine and Allied Sciences, King Abdulaziz University, 21413 Jeddah, P .O . Box 9029, Saudi Arabia (Accepted Jor publication 30 !u!y 1986)
A . K . TILAi1SANY, A . ABnel. Auz, O . H . Osst~uv and A . A. Musr~A. Effects of Ngja hgje (Egyptian cobra), Ngja ngja (hooded cobra), Ngja nigrlcollis (spitting cobra) and Ngja mos5ambka massanlbica (Mozambique spitting cobra) venoms on the isolated guinea-pig tracheal muscle . Taxicon 24, 1162 -1165, 1986. - Venom : from N. hale, N. ngja, N. nigricollLs and N. mossambka were tested on the isolated guinea-pig trachea . The four venoms (1- 30 ~g/ml) contracted the tracheal smooth muscle after a delay of 40-60 sec . A second challenge with the venom : caused eithc no or a much reduced contraction or a rchwnt effect . The contraction could be prevented by prareatmmt with antihistaminic:, but not by atropine, methyaergide or indomethacin, indicating that it is due to hist"Tlns release by the venom: . This release requires extracellular Ca'*, as it could be prevented by pretreatment with verapamil. Under conditions which prevented histamine release or its effort, each of the four venom: resulted in a reproducible relaxant effect which was not blocked by propranolol. It is concluded that the venoms have one or more component(:) causing histamine release which masks the relaxation caused by another component(:) of the venom :.
of the indirect effects of snake venoms may be due to the liberation of pharmacologically active autacoids, such as histamine, S-hydroxytryptamine, kinins and slow reacting substances, which may contribute to the morbidity and mortality of these venoms (DOUGLAS,1975 ; ROTHSCHILD and ROTHSCHILD, 1979) . The present work deals with the effects of some venoms from the spitting cobra (N, nigricollis), the hooded cobra (N. R4%a), the Egyptian cobra (N. hgje) and N. mossambica mossambica on the isolated guinea-pig tracheal smooth muscle . The isolated guinea-pig muscle was prepared by a modification of the method of CASTiLLO and DE BEER (1947) . Male guinea-pigs (350-650 g) were killed by stunning and bleeding and the trachea excised and cut into rings. The final preparations, consisting of six tracheal rings to form a chain, were suspended in Kreb's solution at 37°C and aerated with 95% 02 + 5% C02 using a 10 ml isolated organ bath. One end of the chain was attached to record isometric tension changes using a Bioscience force displacement transducer (Dynanmometer UFI) and a PR 200 Bioscience potentiometric recorder . The SOME
1162
Short Communications
1163
9
HIS FIG .
1 . EFFECT OF ATROPINE AND CHIARPHFNIRAMINE ON THE CONTRACTILE RESPONSE OF THE ISOLATED GUINEA-P10 TRACHEAL MUSCIB TO N. mossambka vENGM .
HIS, at each dot a dose of histamine (10 - ' M) was added ; W, waahill~ ; 1VM, N. mw>~ambica venom (2 ~/ml) ; ATR, atropine (10'` M); CXL chlorpheniramine (10'' M). Note the delayed onset of the contractile response to the venom compared to that due to histamine .
initial resting tension was 0.5 -1 .0 g and the preparation was left for a 90 min equilibration period . Venons from N. mossambica mossambica, N. ngja and N. nigricollis were purchased from Sigrna Chemical Company (St. Louis, MO, U.S.A .) and that of N. hgje was purchased from Latoxan (Rosans, France). Solutions of venons were prepared fresh in Kreb's solution and concentrations are expressed as Etg per ml, while other drug concentrations are expressed as molar concentration of the base . Drugs used were : acetylcholine chloride (B .D .H ., Poole, U .K.), atropine sulphate (B.D.H., Poole, U.K.), chlorpheniramine HCl (Allen and Hanburys, London, U.K .), diphenhydramine (Sigma, St, Louis, MO, U.S.A .), histamine acid phosphate (B.D .H., Poole, U.K .), ethylene glycol-bis (ß-aminoethylether) N,N'-tetraacetic acid (EGTA) (Sigma, St . Louis, U.S.A.), indomethacin (Merck, Sharp and Dohme, Rahway, NJ, U .S.A.) isoprenaline sulphate (Bturoughs Wellcome, London, U .K.), methysergide (Sandoz, Basle, Switzerland), propranolol HCl (LC.L, Macclesfield, U.K .) and verapamil (Isoptin, Knoll, Ludwigschafen, F.R .G .). The four Ngja venons (1-30 Etg/ml) contracted the tracheal smooth muscle . Of the four venons, Ngja hgje was the weakest and in 5 out of 30 preparations and in concentration less than S Etg/ml failed to contract the trachea. The contractile response to the four venons was slow in onset, appearing 40 - 60 sec after addition of the venom, while a contractile response to exogenously addod histamine was almost immediate (Fig . 1). The magnitude of the contractile response depended upon the absence or presence of spontaneous tone of the muscle, as well as on the concentration of the venom. Three or four washings at S min intervals were needed for the preparation to return to the initial basal tone and a second addition of the venom showed tachyphylaxis to the contractile response and in 60 out of 70 preparations the second challenge with the venom caused relaxation of the trachea. The contractile response of the trachea to the four venons was not reversed or prevented by atropine (10 -° M) (Fig.l), methysergide (10-° M) or by the
1164
Short Communications A
0
H FIG . 2 . EFFECT OF VERAPAMIL ON RESPONSE OF THE ISOLATED GUINEA-PIG TRACHEAL MUSCLE TO N. AigriC01/iS VENOM . (A) Control response to histamine (f~ (10 ` M) . (B) Response to histamine (Fl) (10 " M) 30 min after addition of verapamil (10 ' M) . (C) Relaxant effect of N. Aigrlrol(is venom (10 pg/ml) added
-
-
-
30 min after vetapamil (10- ' M) .
addition of indomethacin (10_ e M) 30-60 min before challenge with the venons. The antihistamines chlorpheniramine and diphenhydramine (10- ' M) antagonized the contractile response to the venons (Fig. 1). This contractile response could not be elided in a Cat+-free solution or in the presence of verapamil (10- ' M), but instead relaxation of the tracheal muscle was obselvod . Relaxation of the muscle was also obtained when the venons were added S -10 min following addition of the antihistamines . Verapamil (10-' M) had very little effect on the contractile response to added histamine (Fig. 2). The relaxation caused by the four venons was not affected by propranolol (10 -6 M), which completely blocked relaxation by isoprenaline (10-° M). Relaxation of the tracheal muscle could also be demonstrated following the addition of the venons at the peak of the contractile response to acetylcholine . The isolated guinea-pig tracheal muscle has not been used previously to demonstrate the release of histamine by venons. The present results show that the four venons caused a spasmogenic effect . The significance of the use of the isolated guinea-pig tracheal muscle is that this preparation is generally considered to resemble human airways smooth muscles in its response to drugs and spasmogens (FOSTER, 1974). The release of histamine and other pharmacologically active substances by snake venons has been reported by many workers (for review see ROTHSCHILD and ROTHSCHILD, 1979). The spasmogenic effect of the four venons is due to histamine release and not due to the presence of hi~tam ;ne in the venons . This is supported by the delayed onset of the contractile response to the venous, as compared to the almost immediate contractile effect of exogenously added histamine. Furthermore, pretreatment of tracheal muscle with verapamil, a calcium channel blocker, prevented the contractile effect of the venons and unmasked a relaxant effect of the four venons, while the response to added histamine was
Technology which significantly guinea-pig failure or GtuuN, particular BIn is for RRcomplote PThe caused more which of AW Jand vitro W ln known conditions Load Dis Education G release Cand to AH response of histamine W and Snake (1974) THON, independent M OxArwLUao, (1976) and A isolated blockade (SANCST) study M or with the and referen« conponent(s) would a140, (1975) SCHILD, acalcium Ds Eds) Ct and second affected 5-HT to release reproducible by morbidity Thin SctüLn, Venom, Animal IThe on and ItBt?ea, 272 indomethacin, Ltrachealia RoTHSCHtLn, cause Autacaida the of which effects New mask is(1%1) work histamine toby Research Archs Bantagonists Sof guinea-pig challenge not E1958), four of bronchodilator models HpYork of IW was This histamine these lthe of and histamine 591 Eviden« the prevented O extracellular and Allergy Br due (1947) which compound venons In supported CurHaear, release in(1958) relaxation Macmillan SutAt rather relaxant for D (LEE, Jconfirms Therapeutic contractile The mortality venous to ZPharmtrc The by studying afor appl tracheal indicates process (1979) C cause The Phtrrntacological prostaglandin known release the Rdrugs than tracheal seems content enzymatic by 48/80 Y histamine Communications M Ceffect Iramun effects avenom the Cat+ Liberation bronchodilators Ed (1984) It81, could and Fgrant !athe on can muscle to chain response only Pharmoc spasmogenic inhibitor 499 finding that PATERSON, concomitant of of by the New release Sl, histamine be (UNAS from Antagonism be to calcium other lead the release dependm« Iof 608 similar in Btrs(s this York release, concluded contract the Ato pharmacologically the that first exp preparation caused and to Saudi Jcomponents) added by of release of and contractile In Springer-Verlag structural or W presence to 77rer Theropeuties, of verapamil challenge bronchospasm, prostaglandin THON, effect Evahtatlon pH antihistamines Arabian of as and Ca'' that the its from by the histamine that 90, on itfor effect, SHELLY, trachea and changes compotmds caused process the by mast was 104 1961 the of the National response active other of anaphylactic does pof inducing study cells extracellular not four Bronchodilator Jin each 589 on the KRUGER, might actions by substan«s (FOSTER Hsynthesis rat of F not Centre (GOODMAN, cell prevented which and anaphylaxis, venons venous peritoneal antispasmodics Eda) was such of preparations, ofverapamil CellRes affect be histamine reaction the of not London due by due et as 1976) would Scien« CaZ+ have Drugs snake four The LThe mast al 48/ the by by 23, toJS to
116 5
Short not contractile 1984). acetylcholine histamine pretreatment histamine which (MONGAR 80, The almost Under venons one release, release contribute
.
.,
.
.
.
; . . . .
Acknowledgement and C~srtt.w, . . with Douat.~s, . . and ., FosrEA, .W. (BuRt.eY, . ., Trust FosrEa, . ., in ICauoea, . . cells . temperature MoNaAa, . . Physiol., . ROTHSCHtLD, . . venoms . : ÜvNAS, . 45.
REFERENCES . . . . .
. . .
. .Aat~, .
. . .
. .
.
.
.
.
. ., . .i, . . . . .
.,
. . .
:
:
.
.
. . .
.
. .,
.).
.
. :
.
.
.
. . .
.,
.
:
. .
. . :
.
. :rp .
.
0041-0101/!6 ß.00+ .00 Perpmoo JaornW Ltd.
EFFECTS OF NAJA HAJE (EGYPTIAN COBRA), NAJA NAJA (HOODED COBRA), NAJA NIGRICOLLIS (SPITTING COBRA) AND NAJA MOSSAMBICA MOSSAMBICA (MOZAMBIQUE SPITTING COBRA) VENOMS ON THE ISOLATED GUINEA-PIG TRACHEAL MUSCLE A. K.
TILMISANY,
A.
ABDEL
Azlz, O. H.
OSMAN
and A. A.
MUSTAFA
Department of Pharmacology, College of Medicine and Allied Sciences, King Abdulaziz University, 21413 Jeddah, P .O . Box 9029, Saudi Arabia (Accepted Jor publication 30 !u!y 1986)
A . K . TILAi1SANY, A . ABnel. Auz, O . H . Osst~uv and A . A. Musr~A. Effects of Ngja hgje (Egyptian cobra), Ngja ngja (hooded cobra), Ngja nigrlcollis (spitting cobra) and Ngja mos5ambka massanlbica (Mozambique spitting cobra) venoms on the isolated guinea-pig tracheal muscle . Taxicon 24, 1162 -1165, 1986. - Venom : from N. hale, N. ngja, N. nigricollLs and N. mossambka were tested on the isolated guinea-pig trachea . The four venoms (1- 30 ~g/ml) contracted the tracheal smooth muscle after a delay of 40-60 sec . A second challenge with the venom : caused eithc no or a much reduced contraction or a rchwnt effect . The contraction could be prevented by prareatmmt with antihistaminic:, but not by atropine, methyaergide or indomethacin, indicating that it is due to hist"Tlns release by the venom: . This release requires extracellular Ca'*, as it could be prevented by pretreatment with verapamil. Under conditions which prevented histamine release or its effort, each of the four venom: resulted in a reproducible relaxant effect which was not blocked by propranolol. It is concluded that the venoms have one or more component(:) causing histamine release which masks the relaxation caused by another component(:) of the venom :.
of the indirect effects of snake venoms may be due to the liberation of pharmacologically active autacoids, such as histamine, S-hydroxytryptamine, kinins and slow reacting substances, which may contribute to the morbidity and mortality of these venoms (DOUGLAS,1975 ; ROTHSCHILD and ROTHSCHILD, 1979) . The present work deals with the effects of some venoms from the spitting cobra (N, nigricollis), the hooded cobra (N. R4%a), the Egyptian cobra (N. hgje) and N. mossambica mossambica on the isolated guinea-pig tracheal smooth muscle . The isolated guinea-pig muscle was prepared by a modification of the method of CASTiLLO and DE BEER (1947) . Male guinea-pigs (350-650 g) were killed by stunning and bleeding and the trachea excised and cut into rings. The final preparations, consisting of six tracheal rings to form a chain, were suspended in Kreb's solution at 37°C and aerated with 95% 02 + 5% C02 using a 10 ml isolated organ bath. One end of the chain was attached to record isometric tension changes using a Bioscience force displacement transducer (Dynanmometer UFI) and a PR 200 Bioscience potentiometric recorder . The SOME
1162
Short Communications
1163
9
HIS FIG .
1 . EFFECT OF ATROPINE AND CHIARPHFNIRAMINE ON THE CONTRACTILE RESPONSE OF THE ISOLATED GUINEA-P10 TRACHEAL MUSCIB TO N. mossambka vENGM .
HIS, at each dot a dose of histamine (10 - ' M) was added ; W, waahill~ ; 1VM, N. mw>~ambica venom (2 ~/ml) ; ATR, atropine (10'` M); CXL chlorpheniramine (10'' M). Note the delayed onset of the contractile response to the venom compared to that due to histamine .
initial resting tension was 0.5 -1 .0 g and the preparation was left for a 90 min equilibration period . Venons from N. mossambica mossambica, N. ngja and N. nigricollis were purchased from Sigrna Chemical Company (St. Louis, MO, U.S.A .) and that of N. hgje was purchased from Latoxan (Rosans, France). Solutions of venons were prepared fresh in Kreb's solution and concentrations are expressed as Etg per ml, while other drug concentrations are expressed as molar concentration of the base . Drugs used were : acetylcholine chloride (B .D .H ., Poole, U .K.), atropine sulphate (B.D.H., Poole, U.K.), chlorpheniramine HCl (Allen and Hanburys, London, U.K .), diphenhydramine (Sigma, St, Louis, MO, U.S.A .), histamine acid phosphate (B.D .H., Poole, U.K .), ethylene glycol-bis (ß-aminoethylether) N,N'-tetraacetic acid (EGTA) (Sigma, St . Louis, U.S.A.), indomethacin (Merck, Sharp and Dohme, Rahway, NJ, U .S.A.) isoprenaline sulphate (Bturoughs Wellcome, London, U .K.), methysergide (Sandoz, Basle, Switzerland), propranolol HCl (LC.L, Macclesfield, U.K .) and verapamil (Isoptin, Knoll, Ludwigschafen, F.R .G .). The four Ngja venons (1-30 Etg/ml) contracted the tracheal smooth muscle . Of the four venons, Ngja hgje was the weakest and in 5 out of 30 preparations and in concentration less than S Etg/ml failed to contract the trachea. The contractile response to the four venons was slow in onset, appearing 40 - 60 sec after addition of the venom, while a contractile response to exogenously addod histamine was almost immediate (Fig . 1). The magnitude of the contractile response depended upon the absence or presence of spontaneous tone of the muscle, as well as on the concentration of the venom. Three or four washings at S min intervals were needed for the preparation to return to the initial basal tone and a second addition of the venom showed tachyphylaxis to the contractile response and in 60 out of 70 preparations the second challenge with the venom caused relaxation of the trachea. The contractile response of the trachea to the four venons was not reversed or prevented by atropine (10 -° M) (Fig.l), methysergide (10-° M) or by the
1164
Short Communications A
0
H FIG . 2 . EFFECT OF VERAPAMIL ON RESPONSE OF THE ISOLATED GUINEA-PIG TRACHEAL MUSCLE TO N. AigriC01/iS VENOM . (A) Control response to histamine (f~ (10 ` M) . (B) Response to histamine (Fl) (10 " M) 30 min after addition of verapamil (10 ' M) . (C) Relaxant effect of N. Aigrlrol(is venom (10 pg/ml) added
-
-
-
30 min after vetapamil (10- ' M) .
addition of indomethacin (10_ e M) 30-60 min before challenge with the venons. The antihistamines chlorpheniramine and diphenhydramine (10- ' M) antagonized the contractile response to the venons (Fig. 1). This contractile response could not be elided in a Cat+-free solution or in the presence of verapamil (10- ' M), but instead relaxation of the tracheal muscle was obselvod . Relaxation of the muscle was also obtained when the venons were added S -10 min following addition of the antihistamines . Verapamil (10-' M) had very little effect on the contractile response to added histamine (Fig. 2). The relaxation caused by the four venons was not affected by propranolol (10 -6 M), which completely blocked relaxation by isoprenaline (10-° M). Relaxation of the tracheal muscle could also be demonstrated following the addition of the venons at the peak of the contractile response to acetylcholine . The isolated guinea-pig tracheal muscle has not been used previously to demonstrate the release of histamine by venons. The present results show that the four venons caused a spasmogenic effect . The significance of the use of the isolated guinea-pig tracheal muscle is that this preparation is generally considered to resemble human airways smooth muscles in its response to drugs and spasmogens (FOSTER, 1974). The release of histamine and other pharmacologically active substances by snake venons has been reported by many workers (for review see ROTHSCHILD and ROTHSCHILD, 1979). The spasmogenic effect of the four venons is due to histamine release and not due to the presence of hi~tam ;ne in the venons . This is supported by the delayed onset of the contractile response to the venous, as compared to the almost immediate contractile effect of exogenously added histamine. Furthermore, pretreatment of tracheal muscle with verapamil, a calcium channel blocker, prevented the contractile effect of the venons and unmasked a relaxant effect of the four venons, while the response to added histamine was
Technology which significantly guinea-pig failure or GtuuN, particular BIn is for RRcomplote PThe caused more which of AW Jand vitro W ln known conditions Load Dis Education G release Cand to AH response of histamine W and Snake (1974) THON, independent M OxArwLUao, (1976) and A isolated blockade (SANCST) study M or with the and referen« conponent(s) would a140, (1975) SCHILD, acalcium Ds Eds) Ct and second affected 5-HT to release reproducible by morbidity Thin SctüLn, Venom, Animal IThe on and ItBt?ea, 272 indomethacin, Ltrachealia RoTHSCHtLn, cause Autacaida the of which effects New mask is(1%1) work histamine toby Research Archs Bantagonists Sof guinea-pig challenge not E1958), four of bronchodilator models HpYork of IW was This histamine these lthe of and histamine 591 Eviden« the prevented O extracellular and Allergy Br due (1947) which compound venons In supported CurHaear, release in(1958) relaxation Macmillan SutAt rather relaxant for D (LEE, Jconfirms Therapeutic contractile The mortality venous to ZPharmtrc The by studying afor appl tracheal indicates process (1979) C cause The Phtrrntacological prostaglandin known release the Rdrugs than tracheal seems content enzymatic by 48/80 Y histamine Communications M Ceffect Iramun effects avenom the Cat+ Liberation bronchodilators Ed (1984) It81, could and Fgrant !athe on can muscle to chain response only Pharmoc spasmogenic inhibitor 499 finding that PATERSON, concomitant of of by the New release Sl, histamine be (UNAS from Antagonism be to calcium other lead the release dependm« Iof 608 similar in Btrs(s this York release, concluded contract the Ato pharmacologically the that first exp preparation caused and to Saudi Jcomponents) added by of release of and contractile In Springer-Verlag structural or W presence to 77rer Theropeuties, of verapamil challenge bronchospasm, prostaglandin THON, effect Evahtatlon pH antihistamines Arabian of as and Ca'' that the its from by the histamine that 90, on itfor effect, SHELLY, trachea and changes compotmds caused process the by mast was 104 1961 the of the National response active other of anaphylactic does pof inducing study cells extracellular not four Bronchodilator Jin each 589 on the KRUGER, might actions by substan«s (FOSTER Hsynthesis rat of F not Centre (GOODMAN, cell prevented which and anaphylaxis, venons venous peritoneal antispasmodics Eda) was such of preparations, ofverapamil CellRes affect be histamine reaction the of not London due by due et as 1976) would Scien« CaZ+ have Drugs snake four The LThe mast al 48/ the by by 23, toJS to
116 5
Short not contractile 1984). acetylcholine histamine pretreatment histamine which (MONGAR 80, The almost Under venons one release, release contribute
.
.,
.
.
.
; . . . .
Acknowledgement and C~srtt.w, . . with Douat.~s, . . and ., FosrEA, .W. (BuRt.eY, . ., Trust FosrEa, . ., in ICauoea, . . cells . temperature MoNaAa, . . Physiol., . ROTHSCHtLD, . . venoms . : ÜvNAS, . 45.
REFERENCES . . . . .
. . .
. .Aat~, .
. . .
. .
.
.
.
.
. ., . .i, . . . . .
.,
. . .
:
:
.
.
. . .
.
. .,
.).
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. :
.
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.
. . .
.,
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:
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. . :
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. :rp .
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