calyculin-a and Insulinoma

Stimulus-induced insulin secretion involves the activation of several protein kinases within the beta cell. Most prominent are protein kinase A, protein kinase C and calcium/calmodulin-dependent protein kinases. Protein kinase action is functionally antagonized by protein phosphatases. The four ubiquious serine/threonine protein phosphatases are termed PP-1, PP-2A, -2B and -2C. PP-1 and PP-2A are in vivo parts of major protein complexes. These complexes presumably regulate the phosphatase activity and direct the enzyme to its site of action. Therefore, PP-1 and -2A could play an important role in controlling intracellular signal transmission. Two different toxins, okadaic acid and calyculin A, both from marine invertebrates, were recently discovered and identified as potent and highly specific inhibitors of PP-1 and PP-2A. Both compounds emerged as very useful tools for studying intracellular phosphorylation events. We took advantage of these substances to investigate the significance of protein phosphatase action in stimulus-induced insulin secretion. To avoid major complexity, we confined our study to the cAMP and the phosphoinositide signal pathway. Okadaic acid alone evoked virtually no secretory response. cAMP-dependent secretion was markedly enhanced by 1 microM okadaic acid. The stimulatory effect of okadaic acid was strongly dependent on the concentration of cAMP analoga. In contrast, insulin release caused by the cholinergic agonist carbachol was not influenced by okadaic acid. Calyculin A (10 nM) slightly increased cAMP-induced secretion, but its high toxicity prohibited accurate interpretation of the data. Our findings support the idea that serine/threonine phosphatases act as important regulators in stimulus response coupling.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Bucladesine; Carbachol; Colforsin; Cyclic AMP; Ethers, Cyclic; Insulin; Insulinoma; Marine Toxins; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Rats; Tumor Cells, Cultured

This study investigates the occurrence and regulation of serine/threonine protein phosphatases (PPases) in insulin-secreting RINm5F insulinoma cells. PPases types 1 and 2A were identified in crude RINm5F cell homogenates by both enzymatic assay and Western blot analysis. We then characterized and compared the inhibitory actions of several compounds isolated from cyanobacteria, marine dinoflagellates and marine sponges, (viz. okadaic acid, microcystin-LR, calyculin-A and nodularin) cation-independent PPase activities in RINm5F cell homogenates. It was found that okadaic acid was the least potent inhibitor (IC50 approximately 10(-9) M, IC100 approximately 10(-6) M), while the other compounds exhibited IC50 values of approximately 5 x 10(-10) M and IC100 approximately 5 x 10(-9) M. The findings indicate that the inhibitory substances employed in this study may be used pharmacologically to investigate the role of serine/threonine PPases in RINm5F cell insulin secretion, a process that is likely to be regulated to a major extent by protein phosphorylation.

Topics: Animals; Ethers, Cyclic; In Vitro Techniques; Insulinoma; Marine Toxins; Microcystins; Okadaic Acid; Oxazoles; Pancreatic Neoplasms; Peptides, Cyclic; Phosphoprotein Phosphatases; Rabbits; Rats; Tumor Cells, Cultured