The role of protein kinase C (PKC) as a mediator of glucose-induced insulin secretion has been a subject of controversy. Glucose-induced translocation of PKC has not been reported, and the relevant PKC isoenzymes in islets have not been identified. To address these issues, we developed specific antibodies to the a, /3, and y isoenzymes of PKC. Western blots of homogenates of freshly isolated rat islets probed with these antibodies revealed that the major isoenzyme present is a-PKC. Islets were perifused for 15 min with either 2.75 mM glucose, 20 mM glucose, 20 mM glucose plus 30 mM mannoheptulose, 15 mM a-ketoisocaproate, or a-ketoisocaproate plus mannoheptulose. Quantitative immunoblotting of membrane and cytosol fractions showed that a-PKC translocated from the cytosol to the membrane in freshly isolated rat islets stimulated with either 20 mM glucose or 15 mM ac-ketoisocaproate. Both the secretory response and the translocation of a-PKC were blocked by the addition of mannoheptulose, an inhibitor of glucose metabolism, in islets stimulated with glucose but not in islets stimulated with a-ketoisocaproate. These results support a role for a-PKC in mediating glucoseinduced insulin secretion in pancreatic islets.The protein kinase C (PKC) family of enzymes is thought to play an important role in the calcium-dependent activation of many cellular responses (1). A PKC-like enzyme activity exists in isolated pancreatic islets (2), and there is substantial indirect evidence that the activation of PKC is involved in glucose-induced insulin secretion (3). This evidence includes reports that exposure of isolated islets to stimulatory glucose concentrations leads to the accumulation of diacylglycerol (4,5), a natural activator of PKC; that phorbol esters, specific pharmacological activators of PKC (6), induce a slowly increasing rate of insulin secretion similar to the second phase of glucose-induced insulin secretion (7); that both phorbol esters and glucose induce the phosphorylation of an 80-kDa PKC substrate protein in mouse islets (8); that glyceraldehyde, a fuel secretagogue of insulin secretion in RIN rat insulinoma cells, causes the translocation of PKC from cytosol to membrane in RIN cells (9); and that staurosporine, a specific PKC inhibitor, can block glucose-induced insulin secretion (10). However, direct evidence of PKC activation during glucose-induced insulin secretion is lacking.In many tissues the activation of PKC is associated with its translocation from the cytosol to a membrane-associated state (11). To more closely examine the role of PKC in insulin secretion, we developed a protocol to analyze the membrane/cytosol distribution of PKC in rat islets and correlate any change in distribution with changes in the insulin secretory response. To achieve this we developed specific antibodies to the a, /3, and y isoenzymes of PKC. These antibodies were used in Western blotting experiments to identify the PKC isoenzyme or isoenzymes present in rat islets and then to examine the membrane/cytosol distrib...

Using intact muscle strips from the bovine carotid artery, the time course of translc~ation of protein kinase C (PKC) from the cytosol to the membrane fraction was measured in response to various agonists at induce contractile responses. PKC activity was assessed by Ca2+/phospholipid-dependent phosphorylation of histoiie. Exposure of the muscle strips to phorbol ester (12-deoxyphorbol 1 3-isobutyrate) induced a rapid and sustained translocation of PKC from the cytosol to the membrane fraction, and a slowly developing but sustained contractile response. Histamine induced a comparable initial translocation of PKC to the membrane which then decreased somewhat to a stable plateau significantly above basal values. Histamine also led to a rapid and sustained increase in tension. Angiotensin I, which caused a rapid but transient contraction, induced a rapid initial translocation of PKC to the membrane. The men. brane-associated PKC then declined to a stable plateau significantly lower than that seen after a histamine-induced respon.e, and only slightly above the basal value. Endothelin, which induced a sustained contraction, caused a sustained translocation of PKC from the cytosol to the membrane. In contrast, although exposure to 35 mM-KCI induced a rapid and sustained contraction, it caused only a transient translocation of PKC; the membrane-associated PKC returned to its basal value within 20 min. These results demonstrate that PKC in intact smooth muscle can be rapidly translocated to the membrane and remains membranebound during sustained phorbol ester-or agonist-induced contractions, but that such .t sustained translocation of PKC does not occur during prolonged stimulation with KCI. INTRODUCTIONThe Ca2+/phospholipid-dependent protein kinase, protein kinase C (PKC), is widely distributed in tissues and organs [1]. Over the last few years the enzyme has been implicated in the regulation of an increasing number of cellular processes [1][2][3][4]. In vascular smooth muscle, PKC is present in relatively high concentrations [4], suggesting that it also plays an important role in the control of smooth muscle function. A unique feature of PKC regulation is the spatial translocation of the enzyme. Under basal conditions PKC is thought to be located mainly in the cytosol, but after hormonal stimulation PKC is rapidly translocated to the membrane where it associates with membrane phospholipids [4]. The activation of the enzyme is often closely linked to changes in phosphoinositide (PI) metabolism [3]. When an appropriate agonist activates a specific receptor, an immediate receptor-linked event is the activation of a specific phospholipase C that catalyses the rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to two intracellular messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) [2]. It has been shown that IP3 triggers the release of Ca2+ from intracellular stores in smooth-muscle [5]. Likewise, it has been demonstrated that appropriate agonists induce sustained increases in the DAG cont...

Pharmacologic activation of endogenous protein kinase C (PKC) together with elevation of the intracellular Ca2+ level was previously shown to cause reduction of two voltage-dependent K+ currents (IA and ICa2+-K+) across the soma membrane of the type B photoreceptor within the eye of the mollusc Hermissenda crassicornis. Similar effects were also found to persist for days after acquisition of a classically conditioned response. Also, the state of phosphorylation of a low-molecular-weight protein was changed only within the eyes of conditioned Hermissenda. To examine the role of PKC in causing K+ current changes as well as changes of phosphorylation during conditioning (and possibly other physiologic contexts), we studied here the effects of endogenous PKC activation and exogenous PKC injection on phosphorylation and K+ channel function. Several phosphoproteins (20, 25, 56, and 165 kilodaltons) showed differences in phosphorylation in response to PKC activators applied to intact nervous systems or to isolated eyes. Specific differences were observed for membrane and cytosolic fractions in response to both the phorbol ester 12-deoxyphorbol 13-isobutyrate 20-acetate (DPBA) or exogenous PKC in the presence of Ca2+ and phosphatidylserine/diacylglycerol. Type B cells pretreated with DPBA responded to PKC injection with a persistent reduction of K+ currents. In the absence of DPBA, PKC injection also caused K+ current reduction only following Ca2+ loading conditions. However, the direct effect of PKC injection in the absence of DPBA was only to increase ICa2+-K+. According to a proposed model, the amplitude of the K+ currents would depend on the steady-state balance of effects mediated by PKC within the cytoplasm and membrane-associated PKC. The model further specifies that the effects on K+ currents of cytoplasmic PKC require an intervening proteolytic step. Such a model predicts that increasing the concentration of cytoplasmic protease, e.g., with trypsin, will increase K+ currents, whereas blocking endogenous protease, e.g., with leupeptin, will decrease K+ currents. These effects should be opposed by preexposure of the cells to DPBA. Furthermore, prior injection of leupeptin should block or reverse the effects of subsequent injection of PKC into the type B cell. All of these predictions were confirmed by results reported here. Taken together, the results of this and previous studies suggest that PKC regulation of membrane excitability critically depends on its cellular locus. The implications of such function for long-term physiologic transformations are discussed.

Studies were performed to determine the primary signal transduction mechanism that mediates adenosine stimulation of electrogenic sodium transport in renal epithelial cells. Experiments were performed on cultured amphibian A6 cells with an adenosine analogue that preferentially binds to the A1 receptor, cyclohexyladenosine (CHA). Sodium transport was assessed by the equivalent short circuit current (Ieq). CHA was found to stimulate Ieq via activation of an A1 receptor because (1) the threshold concentration was 1 nM compared to that of 10 microM for the specific A2 agonist CGS21680, (2) CHA inhibited vasopressin (AVP)-stimulated cAMP production by a pertussis toxin-sensitive mechanism, and (3) the action of CHA was inhibited by the A1 antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). CHA increased intracellular Ca2+ ([Ca2+]i) and stimulated phosphoinositide turnover at concentrations that increased Ieq and in a time course that paralleled the increase in Ieq. Ion transport was stimulated by a Ca(2+)-dependent mechanism because the CHA induced increase in Ieq was inhibited by chelating [Ca2+]i with 5,5'dimethyl BAPTA in a dose-dependent manner, with a Ki of approximately 10 microM. The increase in Ieq was also dose-dependently inhibited by the specific PKC inhibitors dihydroxychlorpromazine and chelerythrine, and by trifluoperazine which inhibits PKC and calmodulin. Further studies indicated that CHA-stimulated Ieq was independent of cAMP generation because CHA did not induce an increase in cAMP accumulation parallel to the increase in Ieq in a dose-response analysis, and the adenylate cyclase inhibitor 2',5' dideoxy-adenosine (DDA) did not affect the CHA-induced increase in Ieq.(ABSTRACT TRUNCATED AT 250 WORDS)

Ca2" serves a nearly universal intracellular messenger function in cell activation, but excess Ca2+ is also a cellular toxin. The possibility of Ca2' intoxication is minimized by an elaborate autoregulatory system in which changes in Ca`+ influx rate across the plasma membrane are rapidly compensated for by parallel changes in Ca2' efflux rate. By this mean, cellular Ca21 homestasis is maintained so that minimal changes in total cell calcium and cytosolic Ca2+ concentration occur during sustained Ca2+-mediated responses. Rather than a sustained increase in cytosolic Ca2+ concentration, it is the localized cycling of Ca2+ across the plasma membrane that is the critically important Ca2+ messenger during the sustained phase of cellular responses mediated via surface receptors linked to the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 hydrolysis gives rise to inositol(1,4,5)trisphosphate (IP) and diacylglycerol (DAG). The IP3 acts to release Ca2+ from an intracellular pool, thereby causing a transient rise in cytosolic Ca2+ concentration. This transient Ca2+ signal activates calmodulin-dependent protein kinases transiently, and hence, causes the transient phosphorylation of a subset of cellular proteins that mediate the initial phase of the response. The DAG brings about the association of protein kinase C (PKC) with the plasma membrane where a receptor-mediated increase in Ca2+ cycling across the membrane regulates PKC activity. The sustained phosphorylation of a second subset of proteins by PKC mediates the sustained phase of the response. Hence, Ca2+ serves as a messenger during both phases of the cellular response, but its cellular sites of action, its mechanisms of generation, and its molecular targets differ during the initial and sustained phases of the response. It is likely that this Ca2+ messenger system is a target for many cellular toxins. cellular Ca2' homeostasis over a wide range of circumstances (3). This creates a seeming paradox: changes in Ca2" ion concentration are thought to