Differential inhibitory effects of serine/threonine phosphatase inhibitors and a calmodulin antagonist on phosphoinositol/calcium- and cyclic adenosine monophosphate-mediated pancreatic amylase secretion.
BACKGROUND
Protein phosphorylation and dephosphorylation events are considered to be key steps in the control of agonist-induced pancreatic enzyme release. This study was designed to characterize the role of serine/threonine phosphatases in phosphoinositol/calcium- and cyclic adenosine monophosphate (cAMP)-mediated stimulus-secretion coupling in rat pancreatic acini.


METHODS
Isolated rat pancreatic acini were incubated with either the serine/threonine phosphatase inhibitors okadaic acid, calyculin A, and cyclosporin A or the calmodulin antagonist W-7. Amylase secretion was stimulated with cholecystokinin (CCK)-8, secretin, vasoactive intestinal polypeptide (VIP) or pituitary adenylate cyclase-activating polypeptide (PACAP), and the intracellular second messengers calcium and cAMP were determined.


RESULTS
Okadaic acid or calyculin A reduced secretagogue-stimulated amylase release to near-basal levels. Inhibition of cAMP-mediated secretion (by VIP, secretin, or PACAP) occurred at lower concentrations than with inositol triphosphate (IP3)/Ca(2+)-dependent enzyme release (via CCK). Cyclosporin A diminished CCK-8-stimulated secretion by 35%, whereas secretion in response to cAMP-mediated secretagogues was not affected. W-7 completely inhibited acinar secretion in response to cAMP-or IP3/Ca(2+)-mediated secretagogues. Binding of 125I-CCK-8- or 125I-PACAP-(1-27) to acini was not influenced by the phosphatase inhibitors or W-7. Okadaic acid and calyculin A affected neither CCK-8-stimulated intracellular Ca2+ release nor PACAP-(1-27)-stimulated cAMP synthesis, whereas W-7 inhibited by 50% and 40%, respectively.


CONCLUSIONS
The inhibitory profiles of okadaic acid, calyculin A, cyclosporin A, and W-7 indicate that phosphatases 1 and 2A play a relevant role in cAMP-mediated enzyme release, whereas phosphatases 1 and 2B are predominantly involved in IP3/Ca(2+)-dependent stimulus-secretion coupling. The calmodulin antagonist W-7 interferes at multiple steps of intracellular signal-transduction pathways.
Meyer-Alber
A
A
1st Dept. of Medicine, Christian-Albrechts University Kiel, Germany.
Höcker
M
M
Fetz
I
I
Fornefeld
H
H
Waschulewski
I H
IH
Fölsch
U R
UR
Schmidt
W E
WE
D000681:Amylases / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000502:physiology*; D002147:Calmodulin / Q000037:antagonists & inhibitors*; D000242:Cyclic AMP / Q000502:physiology*; D016572:Cyclosporine / Q000494:pharmacology; D004988:Ethers, Cyclic / Q000494:pharmacology; D066298:In Vitro Techniques; D015544:Inositol 1,4,5-Trisphosphate / Q000502:physiology*; D008297:Male; D008387:Marine Toxins; D019319:Okadaic Acid; D010080:Oxazoles / Q000494:pharmacology; D010179:Pancreas / Q000187:drug effects* / Q000201:enzymology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D054645:Protein Phosphatase 1; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction; D013449:Sulfonamides / Q000494:pharmacology*
D002147:Calmodulin; D004988:Ethers, Cyclic; D008387:Marine Toxins; D010080:Oxazoles; D013449:Sulfonamides; D019319:Okadaic Acid; C017967:W 7; C059041:calyculin A; D016572:Cyclosporine; D015544:Inositol 1,4,5-Trisphosphate; D000242:Cyclic AMP; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D000681:Amylases; D002118:Calcium