Activity of Ca²⁺‐Activated Cl⁻ Channels Contributes to Regulating Receptor‐ and Store‐Operated Ca²⁺ Entry in Human Pulmonary Artery Smooth Muscle Cells

Abstract

Intracellular Ca2+ plays a fundamental role in regulating cell functions in pulmonary arterial smooth muscle cells (PASMCs). A rise in cytosolic Ca2+ concentration ([Ca2+]cyt) triggers pulmonary vasoconstriction and stimulates PASMC proliferation. [Ca2+]cyt is increased mainly by Ca2+ release from intracellular stores and Ca2+ influx through plasmalemmal Ca2+-permeable channels. Given the high concentration of intracellular Cl- in PASMCs, Ca2+-activated Cl-(ClCa) channels play an important role in regulating membrane potential and cell excitability of PASMCs. In this study, we examined whether activity of ClCa channels was involved in regulating [Ca2+]cyt in human PASMCs via regulating receptor- (ROCE) and store- (SOCE) operated Ca2+ entry. The data demonstrated that an angiotensin II (100 nM)-mediated increase in [Ca2+]cyt via ROCE was markedly attenuated by the ClCa channel inhibitors, niflumic acid (100 μM), flufenamic acid (100 μM), and 4,4’-diisothiocyanatostilbene-2,2’-disulfonic acid (100 μM). The inhibition of ClCa channels by niflumic acid and flufenamic acid significantly reduced both transient and plateau phases of SOCE that was induced by passive depletion of Ca2+ from the sarcoplasmic reticulum by 10 μM cyclopiazonic acid. In addition, ROCE and SOCE were abolished by SKF-96365 (50 μM) and 2-aminoethyl diphenylborinate (100 μM), and were slightly decreased in the presence of diltiazem (10 μM). The electrophysiological and immunocytochemical data indicate that ClCa currents were present and TMEM16A was functionally expressed in human PASMCs. The results from this study suggest that the function of ClCa channels, potentially formed by TMEM16A proteins, contributes to regulating [Ca2+]cyt by affecting ROCE and SOCE in human PASMCs.

Self-cleavage of Human CLCA1 Protein by a Novel Internal Metalloprotease Domain Controls Calcium-activated Chloride Channel Activation

“…5C). These data are in agreement with reported biophysical properties of Ca 2ϩ -activated Cl Ϫ channels in native cells and heterologous expression systems (4,(32)(33)(34)(35) and are consistent with a previous report that CLCA1 modulates the activity of endogenous CaCCs in HEK293T cells (9). In cells transfected with CLCA1 variants, in which the metalloprotease activity was abolished (H156A, E157Q) or in which the cleavage site was disrupted (contra), the gluconate-sensitive currents were markedly decreased (Fig.…”

Molecular and Functional Significance of Ca2+‐Activated Cl− Channels in Pulmonary Arterial Smooth Muscle

“…Consistent with the effects of NFA on SOCE and ROCE in human PA myocytes, 239 we also observed a significant relaxation by the TMEM16A inhibitor T16A Inh -A01 of small intralobar rat pulmonary arteries exposed to CPA and nifedipine. This then leads to the following questions: Is E Cl stable and lying where expected (approx.…”

ATP promotes cell survival via regulation of cytosolic [Ca2+] and Bcl-2/Bax ratio in lung cancer cells

“…This result was surprising because aggressive cancer cells would be expected to show stronger Ca 2ϩ signaling intensity in consideration of sustained proliferation and migration. Although it has been demonstrated in proliferative pulmonary artery smooth muscle cells that enhanced SOCE promotes cell proliferation and growth, its effect on tumor growth is less clear (38,46). Several studies have shown that, in cancer, decreased SERCA2 expression leads to low ER Ca 2ϩ capacity, which then lowers the risk of mitochondrial Ca 2ϩ overload that triggers cell apoptosis (27,40 Cell viability results showing that lung cancer cells exhibit resistance to the cytotoxicity of high concentration of ATP in comparison to normal cells are consistent with published data that showed low (nearly undetectable) expression levels of P2X 7 R in lung cancer cells (20,21,39).…”