Loperamide mobilizes intracellular Ca ²⁺ stores in insulin-secreting HIT-T15 cells

1. We have investigated the effects of loperamide on intracellular Ca ²⁺ stores and membrane K ⁺ channels in insulin-secreting hamster insulinoma (HIT-T15) cells. 2. In cell-attached patch-clamp mode, loperamide (3-250 μM) activated large single-channel currents. The loperamide-activated currents were tentatively identified as Ca ²⁺-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 Ca ²⁺. Yet under these conditions, we still measured loperamide-induced Ca ²⁺ 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 Ca ²⁺ from intracellular stores. 4. Carbachol (100 μM), an agonist of muscarinic receptors, which mediates IP ₃-dependent intracellular Ca ²⁺ release, enhanced the effects of loperamide on K _Ca channels. 5. Both the putative K _Ca currents and Ca ²⁺ signals induced by loperamide (with '0' [Ca ²⁺] _o) were abolished when the intracellular Ca ²⁺ stores had been emptied by pretreating the cells with either carbachol or thapsigargin, an endoplasmic reticulum Ca ²⁺-ATPase inhibitor that blocks reuptake of calcium. 6. These data indicate that loperamide in insulin-secreting β-cells evokes intracellular Ca ²⁺ release from IP ₃-gated stores and activates membrane currents that appear to be carried by K _Ca, rather than K _ATP channels.