GENERATION OF REPETITIVE CA2-DEPENDENT AND VOLTAGE INDEPENDENT CHANNELS IN SINGLE HIT CELLS( TRANSIENTS BY BOMBESIN REQUIRES INTRACELLULAR RELEASE AND INFLUX OF CA2+ THROUGH VOLTAGE)

In the present study, the bombesin-induced changes in cytosolic free C a2+ ([Ca2+](i)) were investigated in single Fura-2 loaded SV-40 transf ormed hamster beta-cells (HIT). Bombesin (50-500 pM) caused frequency- modulated repetitive Ca2+ transients. The average frequency of the Ca2 + transients induced by bombesin (200 pM) was 0.58 +/- 0.02 min(-1) (n = 121 cells). High concentrations of bombesin (greater than or equal to 2 nM) triggered a large initial Ca2+ transient followed by a sustai ned plateau or by a decrease to basal levels. In Ca2+-free medium, bom besin caused only one or two Ca2+ transients and withdrawal of extrace llular Ca2+ abolished the Ca2+ transients. The voltage-dependent Ca2channel (VDCC) blockers, verapamil (50 mu M) and nifedipine (10 mu M), reduced amplitude and frequency of the Ca2+ transients and stopped th e Ca2+ transients in some cells. Thapsigargin caused a sustained rise in [Ca2+](i) in the presence of extracellular Ca2+ while in its absenc e the rise in [Ca2+](i) was transient. Verapamil (50 mu M) inhibited t he thapsigargin-induced increase in [Ca2+](i) by about 50%. Depletion of intracellular Ca2+ stores by repetitive stimulation with increasing concentrations of bombesin or thapsigargin in Ca2+-free medium caused an agonist-independent increase in [Ca2+](i) when extracellular Ca2was restored, which was larger than in control cells that had been inc ubated in Ca2+-free medium for the same period of time. This rise in [ Ca2+](i) and the thapsigargin-induced increase in [Ca2+](i) were only partly inhibited by VDCC-blockers. Thus, depletion of the agonist-sens itive Ca2+ pool enhances Ca2+ influx through VDCC and voltage-independ ent Ca2+ channels (VICC). In conclusion, the bombesin-induced Ca2+ res ponse in single HIT cells is periodic in nature with frequency-modulat ed repetitive Ca2+ transients. Intracellular Ca2+ is mobilized during each Ca2+ transient, but Ca2+ influx through VDCC and VICC is required for maintaining the sustained nature of the Ca2+ response. Ca2+ influ x in whole or part is activated by a capacitative Ca2+ entry mechanism .