SUMMARY1. Intracellular recordings were made from chromaffin cells isolated from adrenal medullae of gerbils to examine the effects, on membrane potential, of changes in the ionic environment that are known, from other experiments, to influence the rate of catecholamine secretion.2. Depolarization in response to acetylcholine fell linearly with the logarithm of the extracellular sodium concentration over the range 154-3 mM and reached a value, in sodium-free medium, of about 30 % of the control value.3. The depolarizing effect of acetylcholine in sodium-free media increased linearly with the logarithm of the extracellular calcium concentration over the range 1-117 mM.It is concluded that depolarization in response to acetylcholine involves inward movement of both sodium and calcium ions.4. Depolarization was also observed in response to the secretagogues, excess potassium and barium, both in sodium-rich and sodium-free media. The effect of barium was antagonized by calcium, and it is suggested that these two cations interact at the level of the plasma membrane.5. Depolarization does not appear to be tightly coupled to secretion, for acetylcholine or excess potassium still depolarized the chromaffin cells when the environment was calcium-free or contained an excess of magnesium, conditions that inhibit secretion. Furthermore, although acetylcholine had some depolarizing effect in sodium-free media, the level to which the membrane potential fell was not below the control 'resting'

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Several reports indicate that protein kinase C (PKC) plays a role in insulin-induced glucose transport in certain cells. The precise effects of insulin on specific PKC isoforms are as yet unknown. Utilizing primary cultures of rat skeletal muscle, we investigated the possibility that insulin may influence the activation state of PKC isoenzymes by inducing their translocation and tyrosine phosphorylation. This, in turn, may mediate insulin effects on glucose transport. We identified and determined the glucose transporters and PKC isoforms affected by insulin and 12-O-tetradecanoylphorbol-13-acetate (TPA). Insulin and TPA each caused an increase in glucose uptake. Insulin translocated GLUT3 and GLUT4 without affecting GLUT1. In contrast, TPA translocated GLUT1 and GLUT3 without affecting GLUT4. Insulin translocated and tyrosine phosphorylated and activated PKC-beta2 and -zeta; these effects were blocked by phosphatidylinositol 3-kinase (PI3K) inhibitors. TPA translocated and activated PKC-alpha, -beta2, and -delta; these effects were not noticeably affected by PI3K inhibitors. Furthermore, wortmannin significantly inhibited both insulin and TPA effects on GLUT translocation and glucose uptake. Finally, insulin-induced glucose transport was blocked by the specific PKC-beta2 inhibitor LY379196. These results indicate that specific PKC isoenzymes, when tyrosine-phosphorylated, are implicated in insulin-induced glucose transport in primary cultures of skeletal muscle.

Insulin stimulates glucose uptake into skeletal muscle tissue mainly through the translocation of glucose transporter 4 (GLUT4) to the plasma membrane. The precise mechanism involved in this process is presently unknown. In the cascade of events leading to insulin-induced glucose transport, insulin activates specific protein kinase C (PKC) isoforms. In this study we investigated the roles of PKC in insulin-stimulated glucose uptake and GLUT4 translocation in primary cultures of rat skeletal muscle. We found that insulin initially caused PKC to associate specifically with the GLUT4 compartments and that PKC together with the GLUT4 compartments were then translocated to the plasma membrane as a complex. PKC and GLUT4 recycled independently of one another. To further establish the importance of PKC in glucose transport, we used adenovirus constructs containing wild-type or kinase-inactive, dominant-negative PKC (DNPKC) cDNA to overexpress this isoform in skeletal muscle myotube cultures. We found that overexpression of PKC was associated with a marked increase in the activity of this isoform. The overexpressed, active PKC coprecipitated with the GLUT4 compartments. Moreover, overexpression of PKC caused GLUT4 translocation to the plasma membrane and increased glucose uptake in the absence of insulin. Finally, either insulin or overexpression of PKC induced serine phosphorylation of the GLUT4-compartment-associated vesicle-associated membrane protein 2. Furthermore, DNPKC disrupted the GLUT4 compartment integrity and abrogated insulin-induced GLUT4 translocation and glucose uptake. These results demonstrate that PKC regulates insulin-stimulated GLUT4 translocation and glucose transport through the unique colocalization of this isoform with the GLUT4 compartments.

SUIMMARY1. A method has been devised for isolating adrenal chromaffin cells (from gerbils) and maintaining them in vitro. Transmembrane potentials of these cells were recorded with intracellular micro-electrodes.2. Acetylcholine depolarized the chromaffin cells and so did various other substances known to evoke catecholamine secretion: nicotine, pilocarpine, histamine, 5-hydroxytryptamine, angiotensin, and bradykinin.3. The depolarizing effect of acetylcholine was partially antagonized by hexamethonium and was blocked completely by hexamethonium in combination with atropine.4. Hexamethonium alone completely blocked the response to nicotine; and atropine alone abolished the response to pilocarpine. Thus both nicotinic and muscarinic receptors are present in gerbil chromaffin cells.5. The experiments demonstrate that the various secretagogues and antagonists tested act on the plasma membrane of the chromaffin cell and raise the question whether depolarization may be an important event in stimulus-secretion coupling.