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

摘要

class="enumerated" style="list-style-type:decimal">We have investigated the effects of loperamide on intracellular Ca²⁺ stores and membrane K⁺ channels in insulin-secreting hamster insulinoma (HIT-T15) cells.In cell-attached patch-clamp mode, loperamide (3–250 μM) activated large single-channel currents. The loperamide-activated currents were tentatively identified as Ca²⁺-activated K⁺ channel (KCa) 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 (KATP channels) were also recorded, but were insensitive to loperamide.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.Carbachol (100 μM), an agonist of muscarinic receptors, which mediates IP3-dependent intracellular Ca²⁺ release, enhanced the effects of loperamide on KCa channels.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.These data indicate that loperamide in insulin-secreting β-cells evokes intracellular Ca²⁺ release from IP3-gated stores and activates membrane currents that appear to be carried by KCa, rather than KATP channels.