title: Dynamics of Ca2+ and guanosine 5'-[gamma-thio]triphosphate action on insulin secretion from alpha-toxin-permeabilized HIT-T15 cells.
abstract: The time course of Ca2+ and GTP-analogue effects on insulin secretion was investigated in HIT-T15 cells permeabilized with Staphylococcus alpha-toxin. These cells responded to Ca2+ in the range 0.1-10 microM and could be used in a dynamic perifusion system because of the minimal run-down of the secretory response. High Ca2+ (10 microM) elicited a monophasic ATP-dependent stimulation of insulin secretion that reached a peak within 5 min (approximately 20-fold increase) and rapidly decreased during the subsequent 15 min to a plateau remaining above basal rates (0.1 microM Ca2+). The decrease in Ca(2+)-induced insulin secretion with time could not be attributed to decreased capacity to respond to Ca2+ after prolonged perfusion at low Ca2+ (run-down), nor to depletion of a particular secretory-granule pool. It was rather due to desensitization of the secretory machinery to Ca2+ that was not reversed by selective inhibition of the Ca2+/calmodulin-dependent kinase II with KN-62. However, an intermediate Ca2+ concentration (2 microM) increased insulin secretion to stable level without causing any desensitization. Imposed oscillations of Ca2+ (0.1-10 microM) produced phasic oscillations of insulin secretion, but did not prevent desensitization to Ca2+. Poorly hydrolysable GTP analogues increased insulin secretion at low Ca2+, whereas they strongly inhibited Ca(2+)-induced insulin secretion. By contrast, GTP did not affect basal secretion, and slightly increased Ca(2+)-evoked secretion. These results indicate the following. (1) Oscillations of insulin secretion are tightly coupled to cytosolic Ca2+ oscillations. (2) Oscillations of Ca2+ do not prevent high-Ca(2+)-induced desensitization to Ca2+; this result does not support the idea of a greater efficiency of oscillations compared with sustained Ca2+ rises in triggering exocytosis. (3) Activation of G-proteins modulates exocytosis in a bimodal manner.
authors.affiliation: Département de Médecine, Centre Médical Universitaire, Genève, Switzerland.
authors.forename: J C
authors.initials: JC
authors.lastname: Jonas
authors.forename: G
authors.initials: G
authors.lastname: Li
authors.forename: M
authors.initials: M
authors.lastname: Palmer
authors.forename: U
authors.initials: U
authors.lastname: Weller
authors.forename: C B
authors.initials: CB
authors.lastname: Wollheim
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D002460:Cell Line; D002463:Cell Membrane Permeability; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D010879:Piperazines / Q000494:pharmacology; D013211:Staphylococcus aureus; D010738:Type C Phospholipases / Q000494:pharmacology*
chemical_list: D007328:Insulin; D007546:Isoquinolines; D010879:Piperazines; D006165:Guanylyl Imidodiphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C063302:KN 62; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010738:Type C Phospholipases; D002118:Calcium