Loperamide mobilizes intracellular Ca2+ stores in insulin-secreting HIT-T15 cells

1 We have investigated the effects of loperamide on intracellular Ca2+ stores and membrane K+ channels in insulin-secreting hamster insulinoma (HIT-T15) cells. 2 In cell-attached patch-clamp mode, loperamide (3-250 muM) activated large single-channel currents. The loperamide-activated currents were tentatively identified as Ca2+-activated K+ channel (K-Ca) currents based on their single-channel conductance (145 pS), apparent reversal potential, and insensitivity to tolbutamide. Smaller single-channel currents with a conductance (32 pS) indicative of adenosine triphosphate-sensitive K+ channels (K-ATP channels) were also recorded, but were insensitive to loperamide. 3 Surprisingly, the loperamide-activated currents persisted in the absence of extracellular Ca2+. Yet under these conditions, we still measured loperamide-induced Ca2+ increases. These effects are dose dependent. Loperamide had no effects in the inside-out patch configuration, suggesting that loperamide does not directly activate the channels with large conductance, but does so secondarily to release of Ca2+ from intracellular stores. 4 Carbachol (100 muM), an agonist of muscarinic receptors, which mediates IP3-dependent intracellular Ca2+ release, enhanced the effects of loperamide on KCa channels. 5 Both the putative KCa currents and Ca2+ signals induced by loperamide (with '0' [Ca2+](o)) were abolished when the intracellular Ca2+ stores had been emptied by pretreating the cells with either carbachol or thapsigargin, an endoplasmic reticulum Ca2+-ATPase inhibitor that blocks reuptake of calcium. 6 These data indicate that loperamide in insulin-secreting beta-cells evokes intracellular Ca2+ release from IP3-gated stores and activates membrane currents that appear to be carried by K-Ca, rather than K-ATP channels.