It is well known that protein kinase C (PKC) plays an important role in mediating insulin secretion in response to cholinergic stimulation. In various cells PKC also mediates a desensitization process. The role of PKC for homologous desensitization of the insulin response to repetitive stimulation with the muscarinic agonist carbachol (CCh) was investigated in perifusion experiments using isolated rat pancreatic islets. Repetitive (six times) stimulation with CCh (100 µM) reduced insulin secretion over time (up to 50% during the second challenge). This was not a toxic effect since the desensitizing effect was mostly washed out after 45 min. When PKC was downregulated by long term preincubation (20 h) with 200 nM phorbol 12-myristate 13-acetate (TPA), the initial stimulation of insulin release by CCh was reduced by 50%, and a desensitization by further CCh stimulation was no longer obvious. In contrast, when other compounds with different mechanisms of actions for inactivating PKC were used, i.e. PKC inhibitors such as staurosporin (100 nM), Ro 31-8220 (5 µM) or PKC peptide(19-31), the insulin secretion in response to CCh was reduced but the desensitization was not abolished. When PKC was downregulated or inhibited by the above methods, the PKC activator phorbol 12-myristate 13-acetate (TPA; 200 nM) was no longer able to evoke an increase in insulin secretion during static incubation, i.e. these control experiments indicate a real PKC inhibition. When heparin (50 µg/ml), an inhibitor of G-protein coupled receptor kinase (GRK), was used, the desensitization of the cholinergic stimulation of insulin release remained unchanged. The data indicate that PKC plays a role in CCh-mediated insulin secretion and also show a desensitization of this effect after repetitive stimulation with CCh. The data further indicate that specific PKC isoenzymes that are inhibited by staurosporin or Ro 31-8220 do not take part in the desensitization process, while isoenzymes that are downregulated by TPA are involved. It may be speculated that a hitherto unknown PKC isoenzyme that is downregulated by TPA but not by the other used PKC inhibitors is involved in the desensitization process, or that a nonspecific effect of TPA is involved. Members of the GRK family are not involved in the desensitization process of CCh.

A technique is established for the role of intracellular proteins to be eliminated and thereby gives information about their specific role in signal transduction within cells. Rat pancreatic islets as well as INS-1 cells (an insulin secreting cell line) were electrically permeabilized in order to introduce high molecular weight compounds. Optimized conditions were five exposures with 15-s intervals, tau = 200 ms, an electric field of 1.36 kV per 0.4 cm in a specific permeabilization buffer at a calculated Ca++ concentration of 5 x 10(-8) M. In electroporation control experiments the spectrophotometrically measured uptake of the cell membrane-impermeable propidium iodide, FITC-labelled dextran (MW approximately 4000) and FITC-labelled antibodies (MW approximately 150,000) was established as being 81.5 +/- 5.0, 82.7 +/- 3.0 and 81.0 +/- 1.0 per cent of maximum, respectively. These data were corroborated qualitatively by visualizing microscopically the fluorescence of the FITC-labelled compounds in islets as well as in INS-1 cells. The cells appear to reseal since control experiments indicated a short-lived outflow of lactate dehydrogenase (MW of 140,000 which is similar to that of antibodies) and of insulin for the first 15-20 min. After electroporation the cells were functionally intact, i.e. responded to the stimulus carbachol (CCh). Only 18.0 +/- 10.1 per cent of cells had not resealed after 2 h (propidium iodide uptake measured at various time intervals after electroporation). As was shown recently the effect of specific compounds such as CCh and CCK8 on insulin release was eliminated selectively by antibodies against specific G proteins thus proving this method to be a valuable tool. In conclusion, adding antibodies to electrically permeabilized cells is a valuable tool for eliminating a specific cell function in order to elucidate the specific role of intracellular compounds. This method can probably be used for testing the specific role of other proteins in cell functions.

A technique is established for the role of intracellular proteins to be eliminated and thereby gives information about their specific role in signal transduction within cells. Rat pancreatic islets as well as INS-1 cells (an insulin secreting cell line) were electrically permeabilized in order to introduce high molecular weight compounds. Optimized conditions were five exposures with 15-s intervals, tau = 200 ms, an electric field of 1.36 kV per 0.4 cm in a specific permeabilization buffer at a calculated Ca++ concentration of 5 x 10(-8) M. In electroporation control experiments the spectrophotometrically measured uptake of the cell membrane-impermeable propidium iodide, FITC-labelled dextran (MW approximately 4000) and FITC-labelled antibodies (MW approximately 150,000) was established as being 81.5 +/- 5.0, 82.7 +/- 3.0 and 81.0 +/- 1.0 per cent of maximum, respectively. These data were corroborated qualitatively by visualizing microscopically the fluorescence of the FITC-labelled compounds in islets as well as in INS-1 cells. The cells appear to reseal since control experiments indicated a short-lived outflow of lactate dehydrogenase (MW of 140,000 which is similar to that of antibodies) and of insulin for the first 15-20 min. After electroporation the cells were functionally intact, i.e. responded to the stimulus carbachol (CCh). Only 18.0 +/- 10.1 per cent of cells had not resealed after 2 h (propidium iodide uptake measured at various time intervals after electroporation). As was shown recently the effect of specific compounds such as CCh and CCK8 on insulin release was eliminated selectively by antibodies against specific G proteins thus proving this method to be a valuable tool. In conclusion, adding antibodies to electrically permeabilized cells is a valuable tool for eliminating a specific cell function in order to elucidate the specific role of intracellular compounds. This method can probably be used for testing the specific role of other proteins in cell functions.