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Relationship between secretagogue action of vasoactive intestinal peptide (VIP), secretin, Gila Monster venom, carbamylcholine and protein phosphorylation in rat pancreatic acini in vitro
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R e l a t i o n s h i p between secretaBo~ue a c t i o n o f v a s o a c t i v e i n t e s t i n a l peptide (VIP), s e c r e t i n , G i l a Honster venom, carbamylcholJne and p r o t e i n p h o s p h o r y l a t i o n in r a t pancreatic a c i n i in v i t r o . A. VANDERMEERS, r I.C. VANDERMEERS-PIRET, J. RATHE, J.P. DEHAYE, J. WINAND and J. CHRISTOPHE (Dept. o f Biochemistry and N u t r i t i o n , Medical School, U n i v e r s i t ~ L i b r e de B r u x e l l e s , Brusse|s, Belgium). Rat pancreatic a c i n i were incubated with 0.4 mll 32pi f o r 30-45 min at 37°C, washed, and then exposed f o r 15 min to secretagogues known to use d i f f e r e n t messengers. The i n c u b a t i o n was terminated by the a d d i t i o n o f an i c e - c o l d medium c o n s i s t i n g o f 50 mM phosphate b u f f e r (pH 6.5) enriched with 0.3 H sucrose, 2 mM pyrophosphate, 10 mM NaF, 2 mM EDTA, and 0.2 mM EGTA f o l l o w e d by immediate c e n t r i f u g a t i o n at 250 x g f o r 3 min. The sedimented a c i n i were e i t h e r homogenized in the el ectrophoresis b u f f e r or resuspended in the stop s o l u t i o n and f r a c t i o n a t e d by d i f f e r e n t i a ! c e n t r i f u g a t i o n . ~ t e r heating in SDS-B-mercaptoethanol, the p a t t e r n o f e q u i v a l e n t amounts o f P - I a b e l l e d p r o t e i n s was examined under reducing c o n d i t i o n s , by autoradiography o f SDS-PAGE g e l s . IBPIX reduced ~Pi i n c o r p o r a t i o n i n t o several but not a l l phosphoproteJns. A l l secretagogues, when used a t maximal c o n c e n t r a t i o n , s t i m u l a t e d the phosphorylatJon o f a Plr = 32,500 microsoma! p r o t e i n (by 50-100 %) both in the absence and in the presence o f 0.5 mPl IBMX. S e c r e t i n (10 uM), VIP (10 ~M), and Gi!a P1onster venom (180 ~g/ml) also induced the phosphorylation of a Mr = 20,500 p a r t i c u l a t e p r o t e i n and t h i s was more e a s i l y observed in the presence than in the absence o f IBMX, and s t i l l occurred when carbamylcholine was also added t o the medium. By c o n t r a s t , carbamylcholJne alone (5.6 ~M) caused a r e l a t i v e dephosphorylation o f a Plr = 95,900 p r o t e i n in the absence or presence o f IBMX and whether t e s t e d or not in combination with a member o f the s e c r e t i n / V l P / G i ! a monster venom f a m i l y . To conclude, the endogenous p h o s p h o r y l a t i o n o f s p e c i f i c p r o t e i n s wasg± m o d i f i e d in response to various secretagogues a c t i n g through c y c l i c AMP and/or Ca~ in r a t pancreatic a c i n i . The p a t t e r n o f p r o t e i n phosphorylation induced by G i l a Honster venom was s i m i l a r to t h a t observed w i t h s e c r e t i n and VIP. Vasoactlve Intestinal Peptlde In the Adrenal Gland of the Fro9 : Immunohlstochemlcal LocalIzation and Blolo.qlcal Action ; F. LEBOULENGER, P. LEROUX~ P.M. DUBOIS *t D.H. COY**, G. PELLETIER~'~,~ and H. VAUDRY. (GREM, Lab. EndocrlnoLp Fac. Scl. Rouen, 76130 Mt-StAlgnanp France ; * Lab. Hlstol. Embryol.~ Fac. Meal. Lyon-Sudp 69600 Oulllns, France ; • *Dept. Med. t Sch. Med.~ Tulane Unlv.~ New Orleans~ LA 70112 USA ;*** Lab. Endocrlnol. Molec.p CHU Laval, Quebec G1V 4G2j Canada). Although vasoactlve intestinal peptlde (VIP) has been identified In a number of neuronal cells~ there Is no clear evidence for the presence of VIP In the adrenal medulla of mammals, In the green frog (Rana rldlbund.~.), using specific antibodies against VIP and tyroslne hydroxylase, respectlvely~ we have been able to demonstrate that all the paraneuronal cells which produce catecholamlnes contain also V1P-Ilke Immunoreactivity (V1P-LI). In addltlon~ Met-enkephalln and Leu-enkephalln-LI were detected In about 40% of the cells which stained for tyroslne hydroxylase, whereas substance P-LI was observed In sympathetic nerve endings but was totally absent In the chromaffln cells. At the electron microscopic levelj using the peroxldase-antlperoxldase technique we found that VIP-LI and Met-enkephalln-LI were co-localized In 200-400 nm dense-core secretory granules which also stained for dopamlne (~-hydroxylase. Unlike the mammalian adrenal gland which exhibits a clear zonatlon (cortex and medulla)s the anuran Interrenal tissue consists of contiguous Islets of cortlcosteroldogenlc cells and chromaffln cells which are Intermixed. Thus we have examined the possible action of VIP and opiates on cortlcosterold secretion In the frog. In vitr 9 administration of porcine or chicken VIP (10-6 to 10-5 M) elicited a dose-related stlmulatlon of cortlcosterone and aldosterone secretions whereas morphine, Met- and Leu-enkephallns (10-5 M) did not affect the spontaneous or the ACTH-Induced cortlcosterold production. Prostaglandlns were not Involved In the mechanism of action of VIP~as previously demonstrated for ACTH. In contrast~ the Integrity of the cytoskeleton was required for both VIP and ACTH actions. Taken together~ these results suggest that VIP exerts a paracrlne action on the frog adrenocortlcal cells and that VIP and ACTH have similar mechanisms of action on the adrenal cortex. Supported by INSERM grant n ° 81-6031.
R e l a t i o n s h i p between secretaBo~ue a c t i o n o f v a s o a c t i v e i n t e s t i n a l peptide (VIP), s e c r e t i n , G i l a Honster venom, carbamylcholJne and p r o t e i n p h o s p h o r y l a t i o n in r a t pancreatic a c i n i in v i t r o . A. VANDERMEERS, r I.C. VANDERMEERS-PIRET, J. RATHE, J.P. DEHAYE, J. WINAND and J. CHRISTOPHE (Dept. o f Biochemistry and N u t r i t i o n , Medical School, U n i v e r s i t ~ L i b r e de B r u x e l l e s , Brusse|s, Belgium). Rat pancreatic a c i n i were incubated with 0.4 mll 32pi f o r 30-45 min at 37°C, washed, and then exposed f o r 15 min to secretagogues known to use d i f f e r e n t messengers. The i n c u b a t i o n was terminated by the a d d i t i o n o f an i c e - c o l d medium c o n s i s t i n g o f 50 mM phosphate b u f f e r (pH 6.5) enriched with 0.3 H sucrose, 2 mM pyrophosphate, 10 mM NaF, 2 mM EDTA, and 0.2 mM EGTA f o l l o w e d by immediate c e n t r i f u g a t i o n at 250 x g f o r 3 min. The sedimented a c i n i were e i t h e r homogenized in the el ectrophoresis b u f f e r or resuspended in the stop s o l u t i o n and f r a c t i o n a t e d by d i f f e r e n t i a ! c e n t r i f u g a t i o n . ~ t e r heating in SDS-B-mercaptoethanol, the p a t t e r n o f e q u i v a l e n t amounts o f P - I a b e l l e d p r o t e i n s was examined under reducing c o n d i t i o n s , by autoradiography o f SDS-PAGE g e l s . IBPIX reduced ~Pi i n c o r p o r a t i o n i n t o several but not a l l phosphoproteJns. A l l secretagogues, when used a t maximal c o n c e n t r a t i o n , s t i m u l a t e d the phosphorylatJon o f a Plr = 32,500 microsoma! p r o t e i n (by 50-100 %) both in the absence and in the presence o f 0.5 mPl IBMX. S e c r e t i n (10 uM), VIP (10 ~M), and Gi!a P1onster venom (180 ~g/ml) also induced the phosphorylation of a Mr = 20,500 p a r t i c u l a t e p r o t e i n and t h i s was more e a s i l y observed in the presence than in the absence o f IBMX, and s t i l l occurred when carbamylcholine was also added t o the medium. By c o n t r a s t , carbamylcholJne alone (5.6 ~M) caused a r e l a t i v e dephosphorylation o f a Plr = 95,900 p r o t e i n in the absence or presence o f IBMX and whether t e s t e d or not in combination with a member o f the s e c r e t i n / V l P / G i ! a monster venom f a m i l y . To conclude, the endogenous p h o s p h o r y l a t i o n o f s p e c i f i c p r o t e i n s wasg± m o d i f i e d in response to various secretagogues a c t i n g through c y c l i c AMP and/or Ca~ in r a t pancreatic a c i n i . The p a t t e r n o f p r o t e i n phosphorylation induced by G i l a Honster venom was s i m i l a r to t h a t observed w i t h s e c r e t i n and VIP. Vasoactlve Intestinal Peptlde In the Adrenal Gland of the Fro9 : Immunohlstochemlcal LocalIzation and Blolo.qlcal Action ; F. LEBOULENGER, P. LEROUX~ P.M. DUBOIS *t D.H. COY**, G. PELLETIER~'~,~ and H. VAUDRY. (GREM, Lab. EndocrlnoLp Fac. Scl. Rouen, 76130 Mt-StAlgnanp France ; * Lab. Hlstol. Embryol.~ Fac. Meal. Lyon-Sudp 69600 Oulllns, France ; • *Dept. Med. t Sch. Med.~ Tulane Unlv.~ New Orleans~ LA 70112 USA ;*** Lab. Endocrlnol. Molec.p CHU Laval, Quebec G1V 4G2j Canada). Although vasoactlve intestinal peptlde (VIP) has been identified In a number of neuronal cells~ there Is no clear evidence for the presence of VIP In the adrenal medulla of mammals, In the green frog (Rana rldlbund.~.), using specific antibodies against VIP and tyroslne hydroxylase, respectlvely~ we have been able to demonstrate that all the paraneuronal cells which produce catecholamlnes contain also V1P-Ilke Immunoreactivity (V1P-LI). In addltlon~ Met-enkephalln and Leu-enkephalln-LI were detected In about 40% of the cells which stained for tyroslne hydroxylase, whereas substance P-LI was observed In sympathetic nerve endings but was totally absent In the chromaffln cells. At the electron microscopic levelj using the peroxldase-antlperoxldase technique we found that VIP-LI and Met-enkephalln-LI were co-localized In 200-400 nm dense-core secretory granules which also stained for dopamlne (~-hydroxylase. Unlike the mammalian adrenal gland which exhibits a clear zonatlon (cortex and medulla)s the anuran Interrenal tissue consists of contiguous Islets of cortlcosteroldogenlc cells and chromaffln cells which are Intermixed. Thus we have examined the possible action of VIP and opiates on cortlcosterold secretion In the frog. In vitr 9 administration of porcine or chicken VIP (10-6 to 10-5 M) elicited a dose-related stlmulatlon of cortlcosterone and aldosterone secretions whereas morphine, Met- and Leu-enkephallns (10-5 M) did not affect the spontaneous or the ACTH-Induced cortlcosterold production. Prostaglandlns were not Involved In the mechanism of action of VIP~as previously demonstrated for ACTH. In contrast~ the Integrity of the cytoskeleton was required for both VIP and ACTH actions. Taken together~ these results suggest that VIP exerts a paracrlne action on the frog adrenocortlcal cells and that VIP and ACTH have similar mechanisms of action on the adrenal cortex. Supported by INSERM grant n ° 81-6031.