ABSTRACT
The role of the different isoforms of protein kinase C (PKC) in modulating insulin secretion is still widely unknown. The aim of our studies was to investigate which isoforms are influenced by gastrin-releasing peptide (GRP), a neuropeptide which has been shown to modulate insulin secretion by activating PKC. Presence of PKC isoforms alpha, beta, gamma, delta, epsilon and zeta was tested by immunoblot analysis in whole pancreatic islets of mouse and rat and in the insulinoma cell line RINm5F. Effects of GRP, the truncated peptide GRP1-16 and KCl were also measured on translocation of PKC isoforms. In pancreatic islets of mouse and rat, the PKC isoforms alpha, beta, gamma, delta, epsilon and zeta could be detected. No PKCgamma activity was present in the pancreatic tumor cell line RINm5F. Incubation of mouse or rat islets or of RINm5F cells with GRP induced translocation of the PKC isoforms alpha, beta and zeta. The N-terminal portion of the peptide GRP1-16 induced partial translocation only of the PKC isoforms alpha, beta and zeta in mouse and rat islets in 4 out of 10 cases, but failed to show any effect on PKC isoforms in RINm5F cells. Depolarization of the islets by KCl did not translocate any tested PKC isoform. However, incubation with GRP followed by depolarization with KCl led to translocation of the PKC isoforms alpha, beta and zeta. It is suggested that PKC alpha, beta and/or zeta may play a role in the modulation of insulin secretion by GRP.
Subject(s)
Gastrin-Releasing Peptide/pharmacology , Insulin/metabolism , Islets of Langerhans/enzymology , Isoenzymes/metabolism , Protein Kinase C/metabolism , Animals , Blotting, Western , Electrophoresis, Polyacrylamide Gel , Female , Immunoblotting , Insulin Secretion , Insulinoma , Islets of Langerhans/drug effects , Islets of Langerhans/metabolism , Male , Mice , Pancreatic Neoplasms , Potassium Chloride/pharmacology , Rats , Rats, Wistar , Tumor Cells, CulturedABSTRACT
1. Microcystin-LR, a specific and effective inhibitor of serine/threonine phosphatases type 1/2A which does not permeate cells, was used to distinguish intracellular and extracellular effects of phosphatase inhibitors on insulin secretion by RINm5F cells. 2. Incubation of intact RINm5F cells with microcystin-LR (0.1 - 2 microM) almost doubled basal insulin release at 3 mM glucose but left maximal insulin release induced by KCl (30 mM) unaffected. 3. In parallel, there was an increase in cytosolic Ca(2+) by up to half maximum, which could be suppressed by the Ca(2+)-channel blocker D600. 4. In contrast, microcystin-LR incubation of intact cells did not affect phosphatase activity but significantly reduced phosphatase activity when used in cellular fractions. 5. From these data we conclude that microcystin-LR could affect Ca(2+)-channels and insulin release by inhibiting an extracellular phosphatase-like activity.
Subject(s)
Calcium/metabolism , Enzyme Inhibitors/pharmacology , Insulin/metabolism , Peptides, Cyclic/pharmacology , Phosphoprotein Phosphatases/antagonists & inhibitors , Animals , Calcium Channel Blockers/pharmacology , Cell Membrane Permeability , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacokinetics , Gallopamil/pharmacology , Insulin Secretion , Marine Toxins , Microcystins , Peptides, Cyclic/pharmacokinetics , Potassium Chloride/pharmacology , Subcellular Fractions/drug effects , Subcellular Fractions/enzymology , Tumor Cells, CulturedABSTRACT
Opening of dihydropyridine-sensitive voltage-dependent L-type Ca2+-channels (LTCCs) represents the final common pathway for insulin secretion in pancreatic beta-cells and related cell lines. In insulin-secreting cells their exact subunit composition is unknown. We therefore investigated the subunit structure of (+)-[3H]isradipine-labeled LTCCs in insulin-secreting RINm5F cells. Using subunit-specific antibodies we demonstrate that alpha1C subunits (199 kDa, short form) contribute only a minor portion of the total alpha1 immunoreactivity in membranes and partially purified Ca2+-channel preparations. However, alpha1C forms a major constituent of (+)-[3H]isradipine-labeled LTCCs as 54% of solubilized (+)-[3H]isradipine-binding activity was specifically immunoprecipitated by alpha1C antibodies. Phosphorylation of immunopurified alpha1C with cAMP-dependent protein kinase revealed the existence of an additional 240-kDa species (long form), that remained undetected in Western blots. Fifty seven percent of labeled LTCCs were immunoprecipitated by an anti-beta-antibody directed against all known beta-subunits. Isoform-specific antibodies revealed that these mainly corresponded to beta1b- and beta3-subunits. We found beta2- and beta4-subunits to be major constituents of cardiac and brain L-type channels, respectively, but not part of L-type channels in RINm5F cells. We conclude that alpha1C is a major constituent of dihydropyridine-labeled LTCCs in RINm5F cells, its long form serving as a substrate for cAMP-dependent protein kinase. beta1b- and beta3-Subunits were also found to associate with L-type channels in these cells. These isoforms may therefore represent biochemical targets for the modulation of LTCC activity in RINm5F cells.
