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

Abstract

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.