Oscillations of cytosolic free calcium in bombesin-stimulated HIT-T15 cells

Abstract

The mechanism underlying the generation of cytosolic free Ca 2+ ([Ca 2+ i) oscillations by bombesin, a receptor agonist activating phospholipase C, in insulin secreting HIT-T15 cells was investigated. At 25 μM, 61% of cells displayed [Ca 2+] i oscillations with variable patterns. The bombesin-induced [Ca 2+] i oscillations could last more than 1 h and glucose was required for maintaining these [Ca 2+ fluctuations. Bombesin-evoked [Ca 2+] i oscillations were dependent on extracellular Ca 2+ entry and were attenuated by membrane hype rpolarization or by L-type Ca 2+ channel blockers. These [Ca 2+] i oscillations were apparently not associated with fluctuations in plasma membrane Ca 2+ permeability as monitored by the Mn 2+ quenching technique. 2,5-di-( tert-butyl)-1,4-benzohydroquinone (tBuBHQ) and 4-chloro- m-cresol, which interfere with intracellular Ca 2+ stores, respectively, by inhibiting Ca 2+-ATPase of endoplasmic reticulum and by affecting Ca 2+-induced Ca 2+ release, disrupted bombesin-induced [Ca 2+] i oscillations. 4-chloro- m-resol raised [Ca 2+] i by mobilizing an intracellular Ca 2+ pool, an effect not altered by ryanodine. Caffeine exerted complex actions on [Ca 2+] i It raised [Ca 2+] i by promoting Ca 2+ entry while inhibiting bombesin-elicited [Ca 2+] i oscillations. Our results suggest that in bombesin-elicited [Ca 2+] i oscillations in HIT-T15 cells: (i) the oscillations originate primarily from intracellular Ca 2+ stores; and (ii) the Ca 2+ influx required for maintaining the oscillations is in part membrane potential-sensitive and not coordinated with [Ca 2+] i oscillations. The interplay between intracellular Ca 2+ stores and voltage-sensitive and voltage-insensitive extracellular Ca 2+ entry determines the [Ca 2+] i oscillations evoked by bombesin.