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Interaction of Gila monster venom with secretin receptors in rat pancreatic membranes
Pepttde~. Vol. 5, pp 407-409. 1984 AnkhoInternational Inc Pnnted m the U.S A
0196-9781/84 $3 00 + 00
Interaction of Gila Monster Venom With Secretin Receptors in Rat Pancreatic Membranes' L . G I L L E T , P. R O B B E R E C H T , M. W A E L B R O E C K , J. C. C A M U S , P. DE N E E F , W . K O N I G * A N D J. C H R I S T O P H E "
Department o f Biochemisto' and Nutrition, Medical School, Universitt; Libre de Bruxelles Boulevard de Waterloo 115, B-IO00 Brussels, Belgium and *Hoechst Aktiengeseilschaft, Franl~furt/Main, F . R . G .
GILLET, L., P. ROBBERECHT, M. WAELBROECK, J. C. CAMUS, P. DE NEEF, W. KONIG AND J. CHRISTOPHE. interat tion of Gila monster venom with secretin receptors in rat pancreatic membranes. PEP'rIDES 5(2) 407-409, 1984.--The stimulatory effect of Gila monster venom on adenylate cyclase activity in rat pancreatic membranes was compared to that of porcine secretm and porcine VIP. The maximal effect exerted by the venom was identical to that of VIP but s,gnificantly lower than that of secretin. The effect of Gila monster venom could, however, be attributed to its interaction with secretin receptors rather than with VIP receptors, at variance with its previously described action on guinea pig pancreatic acini. Adenylate cyclase activation by both Gila monster venom and secretin in rat pancreatic membranes was, indeed: (I) dose-dependently inhibited by two secretin fragments secretin-(4--27) and secretin-(7-27), and (2) more severely depressed than VIP stimulation, after pretreating pancreatic membranes with dithiothreitoi (DTr). Secretin Vasoactlve intestinal peptide Adenylate cyclase Dithiothreltol
Gila monster venom
Rat pancreatic membranes
were preincubated for 15 min at 25°C in the presence of the mentioned concentrations o f DTT (see legend o f Fig. 1) then diluted with the buffer in which the membranes were stored so as to obtain a maximum concentration o f 0.5 mM DTT in the assay medium. Adenylate cyclase activity was determined with minor modifications of the Salomon et ai. procedure [5]: 5 to 10 p,g membrane protein were incubated in a f'mal volume of 60 p,l containing 30 mM Tris-HCl, 5 mM MgCI2, 0.5 mM E G T A , 1 mM cyclic AMP, 1 mM theophylline, l0 mM phospho(enol) pyruvate, 30 g,g/ml pyruvate kinase, 10/~M GTP, 0.5 mM ( a ~ P ) A T P , at a final pH o f 7.5. The reaction was initiated by addition of membranes and was stopped after 8 minutes at 37°C by adding 0.5 ml o f a 0.5% sodium dodecylsulfate solution containing 1.5 mM ATP, 0.5 mM cyclic A M P and 20,000 cpm o f cyclic (8-all)AMP. Cyclic A M P was separated from A T P by two successive chromatographies on Dowex 50 W x8 and neutral alumina.
CRUDE venoms from Heioderma Suspectum and H e l o d e r m a Horridum (Gila monster venom) were recently found to promote amylase secretion, to increase cyclic A M P levels, and to displace v'~I-VIP from its receptors on guinea pig pancreatic acim, suggesting the presence in these venoms of a peptide of the secretin/VIP/PHI family [l]. The venom component active on guinea pig pancreatic acini was considered as interacting with VIP-preferring receptors rather than with secretin-preferring receptors [1], The aim of the present study was to test the same venoms for their ability to stimulate adenylate cyclase in a preparation of rat pancreatic plasma membranes [7] possessing at least two classes o f secretin receptors and one (independent) class of VIP receptors [3,4]. Using two methodological approaches, we conclude that the venoms interacted mainly, if not exclusively, with secretin-preferring receptors in rat pancreatic membranes. METHOD
Origin of the Drugs and Chemicals Used
Rat pancreatic plasma membranes were prepared as previously described [7]. Pretreatment o f membranes with dithiothreitol (DTT) was performed as follows: membranes
Synthetic VIP was obtained from Peninsula Laboratories Inc. (San Carlos, CA). Synthetic secretin, and secretin-(4--
'Th~s work was supported by a "'Concerted Action" from the Mlnistere de ia Politique Scientifique (Belgmm), Grant 3.4504.81 from the Fonds de la Recherche Scientifique Medicale (Belgium). and Grant 5 R01-AMI7010-7 from the National Institutes of Health (U.S.A.). -'Requests for reprints should be addressed to Dr. J. Chnstophe.
407
408
G I L L E T ET A L
10-6M VlP 100. 0"
o,,
,/ //
::=4
A
E
50.
>(
',D, .10
-9
.8
.7
.(,
-5
_~
.7
_6
.s
oo,
o1~
,b
rO
,oo
E
[ S E C m ~ X l N ] Clog M )
FIG. 1. Dose-effect curves of adenylate cyclase acttvatton m presence of secretin (left panel), VIP (middle panel), and Gila monster venom (right panel). Rat pancreatic membranes were pretreated as described in the METHOD secUon with buffer only (O) or with 2 mM (A). 5 mM ((3). and l0 mM (i'-I) dithiothreitol (DTT). Cyclic AMP formation was expressed in pmoles'min-t'mg membrane protem-L The data of this experiment were representative of 3 other experiments.
$ECR ETIN_( 4_ 27) CONCENTRATION (log M )
I--
~- 100.
v
X
10-6M VIP
uJ u3
<
_J L~
27) and secretin-(7--27) were synthesized in Hoechst Aktiengesellschaft (Frankfurt/Main, F.R.G.). Different batches of vernon from Heioderma Suspectum and H e l o d e r m a Horridum were purchased from Sigma Chemical Co. (St Louis, MO): only small quantitative differences between batches were found. The results shown here were obtained with batch 42F-0747 from Heloderma Suspectum (Gila monster venom). Cyclic (8-3H)AMP (24 Ci/mmoi) and (a-'~"P)ATP (20-30 Ci/mmol) were obtained, respectively, from the Radiochemical Centre (Amersham, Bucks, U.K.) and New England Nuclear Corporation (Boston, MA). RESULTS Comparative EJ)~,cts o f Se¢ retin, 1lIP. and Gila Monster Venom on Adenylate Cyclase Acttvity Secretin, VIP, and Gila monster venom stimulated ndenylate cyclase activity in the presence o f GTP (Fig. I, control curves). As previously described [3,4], the dose-effect curve of secretin stimulation was biphasic but that of VIP stimulation was monophasic. The dose-effect curve for Gila monster venom activation was also monophasic. The maximal activity o f adenylate cyclase was significantly higher in the presence of secretin than in the presence o f VIP and Gila monster venom (with 10 izM secretin: 450-+32 pmoles cyclic A M P . r a i n - L i n g protein -*, mean-+SE o f 6 e x p e r i m e n t s ; with 10 tzM VIP 320-+ 18, and with 3 mg/ml Gila monster venom 350___15). EjJect of Dithiothreitol (DTT) Pretreatment o.t Rat Pancreattc PkL~ma Memhrane~ on the Secretm. VIP. and Gila Mon,~ter Venom Stimulatum ot'Adenvlate Cycht ~e A( tivtty A premcubation of rat pancreatic membranes w~th the reducing agent DTT did not modify significantly the basal, Gpp(NH)p- and NaF-stimulated adenylate cyclase activities (data not shown). By contrast, this pretreatment markedly altered the ability of secretin to stimulate the enzyme (Fig. I, left panel). This effect was relatively selective for secretin as higher DTT concentrations were required to inhibit the VIP stimulation of adenylate cyclase (Fig. I, middle panel): for instance, a 2 mM DTT pretreatment exerted no effect on
I0-7 M S¢crehn
" ~
Monster 0.3 mg/ml
Gila
,,, 50. p-J
z
t~
O.
~1 _7 _g _g _'4 SECRETIN_(7_27)CONCENTRATION(log M )
FIG. 2. Dose-effect curves of adenylate cyclase inhibition in the presence of increasing concentrations of secretin-(4--27) (upper panel) and secretm-(7-27) (lower panel), and m the presence of 0. I t~M secretin (Q). 1 v,M VIP (X) and 0.3 mg/ml Gila monster venom (©). The concentrations of secretin, VIP, and Gila monster venom utilized were approximately those provoking half-maximal activation of adenylate cyctase. The results were expressed in percent of enzyme activity m the absence of secretin fragments and were the mean of 4 experiments.
VlP-stimulated adenylate cyclase but sufficed to provoke a 100-fold shift to the right of the secretin dose-effect curve, Using this 2 mM DTT concentration, the Hofstee analysis of adenylate cyclase stimulation suggests that enzyme activation through high-affinity secretm binding sites was reduced by 80%, the potency and efficacy of low-affinity secretin binding sites decreasing only slightly. Gila monster venom stimulation was also reduced after a pretreatment with 2 mM DTT and was almost completely abolished with 10 mM DDT (Fig. I, right panel), the effect consisting in a large reduction of the intnnsic activity of the venom rather than in a modification of the venom concentration reqmred for half-maximal actwation of adenylate cyclase. ~/l('( t o / S e ( retm-(4-271 and Se( retm-(7-27) on Se( retm-. VIP-. and Gila Monster Vcnoot-Stimtd(lted Adenvlate ('v( ht,se A( tivtty The two synthetic secretin fragments (4-27) and (7-27) were devoid of any detectable capacity to stimulate rat pancreatic adenylate cyclase, they were able, however, to in-
GILA MONSTER VENOM AND SECRETIN RECEPTORS hibit secretin-, VIP-, and Gila monster venom stimulations dose-dependently. When tested in the presence o f agonist concentrations provoking half-maximal activation of adenylate cyclase, the two secretin fragments were equipotent in inhibiting secretin- and Gila monster venom effects but 10- to 30-fold less potent in inhibiting VIP effects (Fig. 2). DISCUSSION Using a crude preparation of rat pancreatic plasma membranes [7] the present data confirm the capacity of at least one active component o f Gila monster venom to stimulate rat pancreatic adenylate cyclase. In the case o f guinea pig pancreatic acini, this factor was previously shown to belong to the secretin/VIP/PHI group of peptides and to interact with " V I P - p r e f e r r i n g " receptors. Our results on rat pancreatic membranes suggest that this factor interacts mainly with "'secretin-preferring'" receptors. Two arguments support this conclusion: (i) Secretin- and VIP-preferring receptors can be discriminated, in mammalian tnssues, by the ability of secretin fragments, deprived of the N-terminal extremity, to inhibit selectively the stimulatory effects of secretin on adenylate cyclase. The inhibitory effect of such secretin fragments is exerted on both high- and low-affinity secretin receptors interacting with adenylate cyclase in rat pancreatic membranes [2, 3, 4, 8]. In the present study, the activation of adenylate cyclase by Gila monster venom was inhibited by secretin(4--27) and secretin-(7-27) as efficiently as that o f secretin but
409 it was not possible to discriminate the interaction o f Gila monster venom with high-affinity secretin receptors from that with low-affinity secretin receptors; (2) SH-groups are involved in the effects o f secretin on the cat pancreas, based on the action of a p-chioromercuribenzoate pretreatment [6]. Our results likewise indicate that the reduction of S-S bounds by DTT markedly impaired the secretin activation of adenylate cyclase. By contrast, a DTT pretreatment was ineffective on basal, Gpp(NH)p-, N a F - and CCK-derivative-stimulated adenylate cyclase activities and only poorly effective on VIP-stimulated enzyme activity (data not shown). The inhibitory effect being highly specific for secretin, it might well take place at the secretin receptor level. Dose-effect curves for secretin, following the pretreatment of pancreatic membranes with increasing concentrations of DTT, suggest that high-affinity secretin receptors were more severely impaired than low-affinity secretin receptors (Fig. 1). The large decrease of the activation of adenylate cyclase by Gila monster venom, occurring after a DTT pretreatment might, thus, reflect a privileged interaction of the venom with high-affinity secretin receptors. Comparing the present data and those o f Raufman et al. [ 1] it appears that: (I) Gila monster venom contains an originai component from the secretin/VIP/PHI peptide family capable to interact with high-affinity secretin receptors in rat pancreas and with high-affinity VIP-receptors in guinea pig pancreas (2) the venom may be used as a tool to classify secretin/VIP/PHI receptors.
REFERENCES
I. Raufman, J P.. R. T. Jensen, V. E. Sutliff, J. J. Pisano and J. D. Gardner, Actions of Gila monster venom on dispersed acini from guinea pig pancreas. A m J Phvsio! 242: G470-G474, 1982. 2. Robberecht. P.. T. P. Conlon and J. D. Gardner. lnteractnon of porcine vasoactive intestinal peptlde with dispersed pancreatic acmar cells from the guinea pig. Structural requnrements for effects of vasoactnve intestinal peptide and secretnn on cellular adenosine 3':5' monophosphate J Biol Chem 251: 4635--4639, 1976. 3 Robberecht. P.. P. Chatelaln. M. Waelbroeck and J. Christophe. Heterogeneity of VIP-recogmzing binding sntes in rat tissues. In: Va.~oa~ nr~" Intestinal PeptMe. ednted by S. I. Said. New York: Raven Press. 1982, pp. 323-332. 4. Robberecht, P.. M. Waelbroeck, M. Noyer, P. Chatelain, P. De Neef, W, Konig and J. Christophe. Characterizatnon of secretin and vasoactnve intestinal peptide receptors m rat pancreauc plasma membranes using the native peptndes, secretm-(7-27) and five secretnn analogues. Digestion 23: 201-210. 1982.
5. Salomon, Y., C, Londos and M. Rodbeli. A highly sensitive adenylate cyclase assay. Anal Biochem 58: 541-548, 1974. 6. Schultz, I. and S. Milutinovi~. High affinity SH-groups on the surface of pancreas cells involved in secretin stimulation. In: Advances Experimental Medicine and Biology, vol 84, edited by M. W. Miller and A. E. Shamoo. New York: Plenum Press, 1977, pp. 209-227. 7. Svoboda, M., P. Robberecht, J. C. Camus, M. DcschodtLanckman and J. Christophe. Subceilular distribution and response to gastrointestinal hormones of adenylate cyclase in the rat pancreas. Partial purification of a stable plasma membrane preparation. E u r J Biochem 69: 185--193, 1976. 8. Van Calker, D., M. Miiller and B. Hamprecht. Regulation by secretin, vasoactive intestinal peptide, and somatostatin of cyclic AMP accumulation in cultured brain cells. Proc Natl Acad Sci USA 77: 6907-6911, 1980.
0196-9781/84 $3 00 + 00
Interaction of Gila Monster Venom With Secretin Receptors in Rat Pancreatic Membranes' L . G I L L E T , P. R O B B E R E C H T , M. W A E L B R O E C K , J. C. C A M U S , P. DE N E E F , W . K O N I G * A N D J. C H R I S T O P H E "
Department o f Biochemisto' and Nutrition, Medical School, Universitt; Libre de Bruxelles Boulevard de Waterloo 115, B-IO00 Brussels, Belgium and *Hoechst Aktiengeseilschaft, Franl~furt/Main, F . R . G .
GILLET, L., P. ROBBERECHT, M. WAELBROECK, J. C. CAMUS, P. DE NEEF, W. KONIG AND J. CHRISTOPHE. interat tion of Gila monster venom with secretin receptors in rat pancreatic membranes. PEP'rIDES 5(2) 407-409, 1984.--The stimulatory effect of Gila monster venom on adenylate cyclase activity in rat pancreatic membranes was compared to that of porcine secretm and porcine VIP. The maximal effect exerted by the venom was identical to that of VIP but s,gnificantly lower than that of secretin. The effect of Gila monster venom could, however, be attributed to its interaction with secretin receptors rather than with VIP receptors, at variance with its previously described action on guinea pig pancreatic acini. Adenylate cyclase activation by both Gila monster venom and secretin in rat pancreatic membranes was, indeed: (I) dose-dependently inhibited by two secretin fragments secretin-(4--27) and secretin-(7-27), and (2) more severely depressed than VIP stimulation, after pretreating pancreatic membranes with dithiothreitoi (DTr). Secretin Vasoactlve intestinal peptide Adenylate cyclase Dithiothreltol
Gila monster venom
Rat pancreatic membranes
were preincubated for 15 min at 25°C in the presence of the mentioned concentrations o f DTT (see legend o f Fig. 1) then diluted with the buffer in which the membranes were stored so as to obtain a maximum concentration o f 0.5 mM DTT in the assay medium. Adenylate cyclase activity was determined with minor modifications of the Salomon et ai. procedure [5]: 5 to 10 p,g membrane protein were incubated in a f'mal volume of 60 p,l containing 30 mM Tris-HCl, 5 mM MgCI2, 0.5 mM E G T A , 1 mM cyclic AMP, 1 mM theophylline, l0 mM phospho(enol) pyruvate, 30 g,g/ml pyruvate kinase, 10/~M GTP, 0.5 mM ( a ~ P ) A T P , at a final pH o f 7.5. The reaction was initiated by addition of membranes and was stopped after 8 minutes at 37°C by adding 0.5 ml o f a 0.5% sodium dodecylsulfate solution containing 1.5 mM ATP, 0.5 mM cyclic A M P and 20,000 cpm o f cyclic (8-all)AMP. Cyclic A M P was separated from A T P by two successive chromatographies on Dowex 50 W x8 and neutral alumina.
CRUDE venoms from Heioderma Suspectum and H e l o d e r m a Horridum (Gila monster venom) were recently found to promote amylase secretion, to increase cyclic A M P levels, and to displace v'~I-VIP from its receptors on guinea pig pancreatic acim, suggesting the presence in these venoms of a peptide of the secretin/VIP/PHI family [l]. The venom component active on guinea pig pancreatic acini was considered as interacting with VIP-preferring receptors rather than with secretin-preferring receptors [1], The aim of the present study was to test the same venoms for their ability to stimulate adenylate cyclase in a preparation of rat pancreatic plasma membranes [7] possessing at least two classes o f secretin receptors and one (independent) class of VIP receptors [3,4]. Using two methodological approaches, we conclude that the venoms interacted mainly, if not exclusively, with secretin-preferring receptors in rat pancreatic membranes. METHOD
Origin of the Drugs and Chemicals Used
Rat pancreatic plasma membranes were prepared as previously described [7]. Pretreatment o f membranes with dithiothreitol (DTT) was performed as follows: membranes
Synthetic VIP was obtained from Peninsula Laboratories Inc. (San Carlos, CA). Synthetic secretin, and secretin-(4--
'Th~s work was supported by a "'Concerted Action" from the Mlnistere de ia Politique Scientifique (Belgmm), Grant 3.4504.81 from the Fonds de la Recherche Scientifique Medicale (Belgium). and Grant 5 R01-AMI7010-7 from the National Institutes of Health (U.S.A.). -'Requests for reprints should be addressed to Dr. J. Chnstophe.
407
408
G I L L E T ET A L
10-6M VlP 100. 0"
o,,
,/ //
::=4
A
E
50.
>(
',D, .10
-9
.8
.7
.(,
-5
_~
.7
_6
.s
oo,
o1~
,b
rO
,oo
E
[ S E C m ~ X l N ] Clog M )
FIG. 1. Dose-effect curves of adenylate cyclase acttvatton m presence of secretin (left panel), VIP (middle panel), and Gila monster venom (right panel). Rat pancreatic membranes were pretreated as described in the METHOD secUon with buffer only (O) or with 2 mM (A). 5 mM ((3). and l0 mM (i'-I) dithiothreitol (DTT). Cyclic AMP formation was expressed in pmoles'min-t'mg membrane protem-L The data of this experiment were representative of 3 other experiments.
$ECR ETIN_( 4_ 27) CONCENTRATION (log M )
I--
~- 100.
v
X
10-6M VIP
uJ u3
<
_J L~
27) and secretin-(7--27) were synthesized in Hoechst Aktiengesellschaft (Frankfurt/Main, F.R.G.). Different batches of vernon from Heioderma Suspectum and H e l o d e r m a Horridum were purchased from Sigma Chemical Co. (St Louis, MO): only small quantitative differences between batches were found. The results shown here were obtained with batch 42F-0747 from Heloderma Suspectum (Gila monster venom). Cyclic (8-3H)AMP (24 Ci/mmoi) and (a-'~"P)ATP (20-30 Ci/mmol) were obtained, respectively, from the Radiochemical Centre (Amersham, Bucks, U.K.) and New England Nuclear Corporation (Boston, MA). RESULTS Comparative EJ)~,cts o f Se¢ retin, 1lIP. and Gila Monster Venom on Adenylate Cyclase Acttvity Secretin, VIP, and Gila monster venom stimulated ndenylate cyclase activity in the presence o f GTP (Fig. I, control curves). As previously described [3,4], the dose-effect curve of secretin stimulation was biphasic but that of VIP stimulation was monophasic. The dose-effect curve for Gila monster venom activation was also monophasic. The maximal activity o f adenylate cyclase was significantly higher in the presence of secretin than in the presence o f VIP and Gila monster venom (with 10 izM secretin: 450-+32 pmoles cyclic A M P . r a i n - L i n g protein -*, mean-+SE o f 6 e x p e r i m e n t s ; with 10 tzM VIP 320-+ 18, and with 3 mg/ml Gila monster venom 350___15). EjJect of Dithiothreitol (DTT) Pretreatment o.t Rat Pancreattc PkL~ma Memhrane~ on the Secretm. VIP. and Gila Mon,~ter Venom Stimulatum ot'Adenvlate Cycht ~e A( tivtty A premcubation of rat pancreatic membranes w~th the reducing agent DTT did not modify significantly the basal, Gpp(NH)p- and NaF-stimulated adenylate cyclase activities (data not shown). By contrast, this pretreatment markedly altered the ability of secretin to stimulate the enzyme (Fig. I, left panel). This effect was relatively selective for secretin as higher DTT concentrations were required to inhibit the VIP stimulation of adenylate cyclase (Fig. I, middle panel): for instance, a 2 mM DTT pretreatment exerted no effect on
I0-7 M S¢crehn
" ~
Monster 0.3 mg/ml
Gila
,,, 50. p-J
z
t~
O.
~1 _7 _g _g _'4 SECRETIN_(7_27)CONCENTRATION(log M )
FIG. 2. Dose-effect curves of adenylate cyclase inhibition in the presence of increasing concentrations of secretin-(4--27) (upper panel) and secretm-(7-27) (lower panel), and m the presence of 0. I t~M secretin (Q). 1 v,M VIP (X) and 0.3 mg/ml Gila monster venom (©). The concentrations of secretin, VIP, and Gila monster venom utilized were approximately those provoking half-maximal activation of adenylate cyctase. The results were expressed in percent of enzyme activity m the absence of secretin fragments and were the mean of 4 experiments.
VlP-stimulated adenylate cyclase but sufficed to provoke a 100-fold shift to the right of the secretin dose-effect curve, Using this 2 mM DTT concentration, the Hofstee analysis of adenylate cyclase stimulation suggests that enzyme activation through high-affinity secretm binding sites was reduced by 80%, the potency and efficacy of low-affinity secretin binding sites decreasing only slightly. Gila monster venom stimulation was also reduced after a pretreatment with 2 mM DTT and was almost completely abolished with 10 mM DDT (Fig. I, right panel), the effect consisting in a large reduction of the intnnsic activity of the venom rather than in a modification of the venom concentration reqmred for half-maximal actwation of adenylate cyclase. ~/l('( t o / S e ( retm-(4-271 and Se( retm-(7-27) on Se( retm-. VIP-. and Gila Monster Vcnoot-Stimtd(lted Adenvlate ('v( ht,se A( tivtty The two synthetic secretin fragments (4-27) and (7-27) were devoid of any detectable capacity to stimulate rat pancreatic adenylate cyclase, they were able, however, to in-
GILA MONSTER VENOM AND SECRETIN RECEPTORS hibit secretin-, VIP-, and Gila monster venom stimulations dose-dependently. When tested in the presence o f agonist concentrations provoking half-maximal activation of adenylate cyclase, the two secretin fragments were equipotent in inhibiting secretin- and Gila monster venom effects but 10- to 30-fold less potent in inhibiting VIP effects (Fig. 2). DISCUSSION Using a crude preparation of rat pancreatic plasma membranes [7] the present data confirm the capacity of at least one active component o f Gila monster venom to stimulate rat pancreatic adenylate cyclase. In the case o f guinea pig pancreatic acini, this factor was previously shown to belong to the secretin/VIP/PHI group of peptides and to interact with " V I P - p r e f e r r i n g " receptors. Our results on rat pancreatic membranes suggest that this factor interacts mainly with "'secretin-preferring'" receptors. Two arguments support this conclusion: (i) Secretin- and VIP-preferring receptors can be discriminated, in mammalian tnssues, by the ability of secretin fragments, deprived of the N-terminal extremity, to inhibit selectively the stimulatory effects of secretin on adenylate cyclase. The inhibitory effect of such secretin fragments is exerted on both high- and low-affinity secretin receptors interacting with adenylate cyclase in rat pancreatic membranes [2, 3, 4, 8]. In the present study, the activation of adenylate cyclase by Gila monster venom was inhibited by secretin(4--27) and secretin-(7-27) as efficiently as that o f secretin but
409 it was not possible to discriminate the interaction o f Gila monster venom with high-affinity secretin receptors from that with low-affinity secretin receptors; (2) SH-groups are involved in the effects o f secretin on the cat pancreas, based on the action of a p-chioromercuribenzoate pretreatment [6]. Our results likewise indicate that the reduction of S-S bounds by DTT markedly impaired the secretin activation of adenylate cyclase. By contrast, a DTT pretreatment was ineffective on basal, Gpp(NH)p-, N a F - and CCK-derivative-stimulated adenylate cyclase activities and only poorly effective on VIP-stimulated enzyme activity (data not shown). The inhibitory effect being highly specific for secretin, it might well take place at the secretin receptor level. Dose-effect curves for secretin, following the pretreatment of pancreatic membranes with increasing concentrations of DTT, suggest that high-affinity secretin receptors were more severely impaired than low-affinity secretin receptors (Fig. 1). The large decrease of the activation of adenylate cyclase by Gila monster venom, occurring after a DTT pretreatment might, thus, reflect a privileged interaction of the venom with high-affinity secretin receptors. Comparing the present data and those o f Raufman et al. [ 1] it appears that: (I) Gila monster venom contains an originai component from the secretin/VIP/PHI peptide family capable to interact with high-affinity secretin receptors in rat pancreas and with high-affinity VIP-receptors in guinea pig pancreas (2) the venom may be used as a tool to classify secretin/VIP/PHI receptors.
REFERENCES
I. Raufman, J P.. R. T. Jensen, V. E. Sutliff, J. J. Pisano and J. D. Gardner, Actions of Gila monster venom on dispersed acini from guinea pig pancreas. A m J Phvsio! 242: G470-G474, 1982. 2. Robberecht. P.. T. P. Conlon and J. D. Gardner. lnteractnon of porcine vasoactive intestinal peptlde with dispersed pancreatic acmar cells from the guinea pig. Structural requnrements for effects of vasoactnve intestinal peptide and secretnn on cellular adenosine 3':5' monophosphate J Biol Chem 251: 4635--4639, 1976. 3 Robberecht. P.. P. Chatelaln. M. Waelbroeck and J. Christophe. Heterogeneity of VIP-recogmzing binding sntes in rat tissues. In: Va.~oa~ nr~" Intestinal PeptMe. ednted by S. I. Said. New York: Raven Press. 1982, pp. 323-332. 4. Robberecht, P.. M. Waelbroeck, M. Noyer, P. Chatelain, P. De Neef, W, Konig and J. Christophe. Characterizatnon of secretin and vasoactnve intestinal peptide receptors m rat pancreauc plasma membranes using the native peptndes, secretm-(7-27) and five secretnn analogues. Digestion 23: 201-210. 1982.
5. Salomon, Y., C, Londos and M. Rodbeli. A highly sensitive adenylate cyclase assay. Anal Biochem 58: 541-548, 1974. 6. Schultz, I. and S. Milutinovi~. High affinity SH-groups on the surface of pancreas cells involved in secretin stimulation. In: Advances Experimental Medicine and Biology, vol 84, edited by M. W. Miller and A. E. Shamoo. New York: Plenum Press, 1977, pp. 209-227. 7. Svoboda, M., P. Robberecht, J. C. Camus, M. DcschodtLanckman and J. Christophe. Subceilular distribution and response to gastrointestinal hormones of adenylate cyclase in the rat pancreas. Partial purification of a stable plasma membrane preparation. E u r J Biochem 69: 185--193, 1976. 8. Van Calker, D., M. Miiller and B. Hamprecht. Regulation by secretin, vasoactive intestinal peptide, and somatostatin of cyclic AMP accumulation in cultured brain cells. Proc Natl Acad Sci USA 77: 6907-6911, 1980.