VASOPRESSIN INDUCES FREQUENCY-MODULATED REPETITIVE CALCIUM TRANSIENTS IN SINGLE INSULIN-SECRETING HIT CELLS

Ca2+ is central to the stimulation of insulin secretion from pancreatic beta-cells. Arginine-vasopressin (AVP) may participate in the modulation of insulin release. In the present study, the AVP-induced changes in cytosolic free Ca2+ ([Ca2+](i)) were investigated in single fura-2 loaded insulin-secreting HIT cells. Stimulation with AVP (0.1-5 nM) caused repetitive Ca2+ transients. The frequency but not the amplitude of the Ca2+ transients was modulated by the concentration of AVP. High concentrations of AVP (10-100 nM) triggered a biphasic rise in [Ca2+](i). In Ca2+-free medium AVP caused only one or two Ca2+ transients. Withdrawal of extracellular Ca2+ rapidly abolished the AVP-induced Ca2+ transients in all cells tested. The Ca2+ channel blocker, verapamil (50 mu M), reduced amplitude and frequency of the Ca2+ transients by about 25% and 60%, respectively, and terminated the Ca2+ transients in 2 of 6 cells. When HF cells were incubated in Ca2+-free medium, and extracellular Ca2+ was restored, there was a small increase in [Ca2+](i). If, however, the agonist-sensitive Ca2+ pool was functionally depleted by repetitive stimulation with high concentrations of AVP or thapsigargin in Ca2+-free medium before extracellular Ca2+ was restored, an agonist-independent increase in [Ca2+](i) was observed, which was transiently larger than in the control cells, and was mainly preserved in the presence of verapamil. Thus, depletion of the agonist-sensitive Ca2+ pool enhances the influx of extracellular Ca2+ through a Ca2+ entry mechanism independent from verapamil-sensitive voltage-dependent Ca2+ channels (VDCC). In conclusion, the AVP-induced Ca2+ response in single HIT cells is periodic in nature with frequency-modulated repetitive Ca2+ transients. AVP mobilizes Ca2+ from intracellular pools, but influx of extracellular Ca2+ partly through verapamil-sensitive VDCC, and partly through an additional, VDCC-independent pathway, which could be controlled by the filling state of the agonist-sensitive Ca2+ pool, is required for maintaining the repetitive nature of the Ca2+ response.