Previous studies in pulmonary arterial smooth muscle cells (PASMCs)

Aims/hypothesis: ATP-sensitive K + (K ATP ) channels located on the beta cell plasma membrane play a critical role in regulating insulin secretion and are targets for the sulfonylurea class of antihyperglycaemic drugs. Recent reports suggest that these channels may also reside on insulin-containing dense-core vesicles and mitochondria. The aim of this study was to explore these possibilities and to test the hypothesis that vesicle-resident channels play a role in the control of organellar Ca 2+ concentration or pH. Methods: To quantify the subcellular distribution of the pore-forming subunit Kir6.2 and the sulfonylurea binding subunit SUR1 in isolated mouse islets and clonal pancreatic MIN6 beta cells, we used four complementary techniques: immunoelectron microscopy, density gradient fractionation, vesicle immunopurification and fluorescence-activated vesicle isolation. Intravesicular and mitochondrial concentrations of free Ca 2+ were measured in intact or digitonin-permeabilised MIN6 cells using recombinant, targeted aequorins, and intravesicular pH was measured with the recombinant fluorescent probe pHluorin. Results: SUR1 and Kir6.2 immunoreactivity were concentrated on dense-core vesicles and on vesicles plus the endoplasmic reticulum/Golgi network, respectively, in both islets and MIN6 cells. Reactivity to neither subunit was detected on mitochondria. Glibenclamide, tolbutamide and diazoxide all failed to affect Ca 2+ uptake into mitochondria, and K ATP channel regulators had no significant effect on intravesicular free Ca 2+ concentrations or vesicular pH. Conclusions/Interpretation: A significant proportion of Kir6.2 and SUR1 subunits reside on insulin-secretory vesicles and the distal secretory pathway in mouse beta cells but do not influence intravesicular ion homeostasis. We propose that densecore vesicles may serve instead as sorting stations for the delivery of channels to the plasma membrane.

Previous studies in pulmonary arterial smooth muscle cells (PASMCs) showed that TRPC1 channel mediates capacitative Ca2+ entry (CCE) but the molecular signal(s) that activate TRPC1 in PASMCs remains unknown. The aim of the present study was to determine if TRPC1 mediate CCE through activation of STIM1 protein in mouse PASMCs. In primary cultured PASMCs loaded with fura‐2, 10μM CPA caused a rise in [Ca2+]i but it was abolished when extracellular Ca2+ was removed. Subsequent addition of 2mM Ca2+ elicited a transient rise in [Ca2+]i that was partially inhibited by 10μM nifedipine, leaving a nifedipine‐insensitive transient and nifedipine‐insensitive sustained rise in [Ca2+]i. The nifedipine‐insensitive sustained, but not transient rise in [Ca2+]i was inhibited in cells pretreated with antibodies raised against extracellular epitope of TRPC1 and STIM1. RT‐PCR revealed TRPC1 and STIM1 mRNAs, whereas Western blot analysis identified TRPC1 and STIM1 proteins in cultured mouse PASMCs. Taken together, store‐depletion causes activation of voltage‐operated Ca2+ entry, CCE and another as yet unidentified voltage‐independent Ca2+ entry pathway. These data suggest that CCE is mediated by TRPC1 channel possibly through activation of STIM1 protein in mouse PASMCs. (Supported by HL49254 and NCRR P20RR15581)

The present study aimed to determine if TRPC1 and STIM1 mediate capacitative Ca2+ entry (CCE) in mouse pulmonary arterial smooth muscle cells (PASMCs). In primary cultured PASMCs loaded with fura‐2, cyclopiazonic acid (CPA) caused a transient followed by a sustained rise in intracellular Ca2+ concentration ([Ca2+]i). The transient but not sustained rise in [Ca2+]i was partially inhibited by nifedipine but they were both inhibited by SKF 96365, Ni2+, La3+ and Gd3+. In addition, CPA increased the rate of Mn2+ quench of fura‐2 fluorescence that was also inhibited by these blockers, exhibiting pharmacological properties characteristic of CCE. The nifedipine‐insensitive rise in [Ca2+]i and the increase in Mn2+ quench rate were both inhibited in cells pretreated with antibodies raised against extracellular epitopes of TRPC1 and STIM1. Moreover, overexpression of STIM1 resulted in a marked increase in [Ca2+]i and Mn2+ quench rate caused by CPA, and they were reduced by TRPC1 antibody. RT‐PCR and Western blot analysis revealed TRPC1 and STIM1 mRNAs and proteins. Furthermore, TRPC1 was found to co‐immunoprecipitate with STIM1. Taken together, store‐depletion causes activation of voltage‐operated Ca2+ entry and CCE. These data provide direct evidence that TRPC1 channel mediates CCE through activation of STIM1 in mouse PASMCs. [Supported by HL49254, NCRR P20RR15581 (JR Hume) and AHA Scientist Development Grant (LC Ng)]