Role of Na⁺/Ca²⁺ exchange in regulating cytosolic Ca²⁺ in cultured human pulmonary artery smooth muscle cells

Abstract

A rise in cytosolic Ca2+ concentration ([Ca2+]cyt) in pulmonary artery smooth muscle cells (PASMC) is an important stimulus for cell contraction, migration, and proliferation. Depletion of intracellular Ca2+ stores opens store-operated Ca2+ channels (SOC) and causes Ca2+ entry. Transient receptor potential (TRP) cation channels that are permeable to Na+ and Ca2+ are believed to form functional SOC. Because sarcolemmal Na+/Ca2+ exchanger has also been implicated in regulating [Ca2+]cyt, this study was designed to test the hypothesis that the Na+/Ca2+ exchanger (NCX) in cultured human PASMC is functionally involved in regulating [Ca2+]cyt by contributing to store depletion-mediated Ca2+ entry. RT-PCR and Western blot analyses revealed mRNA and protein expression for NCX1 and NCKX3 in cultured human PASMC. Removal of extracellular Na+, which switches the Na+/Ca2+ exchanger from the forward (Ca2+ exit) to reverse (Ca2+ entry) mode, significantly increased [Ca2+]cyt, whereas inhibition of the Na+/Ca2+ exchanger with KB-R7943 (10 μM) markedly attenuated the increase in [Ca2+]cyt via the reverse mode of Na+/Ca2+ exchange. Store depletion also induced a rise in [Ca2+]cyt via the reverse mode of Na+/Ca2+ exchange. Removal of extracellular Na+ or inhibition of the Na+/Ca2+ exchanger with KB-R7943 attenuated the store depletion-mediated Ca2+ entry. Furthermore, treatment of human PASMC with KB-R7943 also inhibited cell proliferation in the presence of serum and growth factors. These results suggest that NCX is functionally expressed in cultured human PASMC, that Ca2+ entry via the reverse mode of Na+/Ca2+ exchange contributes to store depletion-mediated increase in [Ca2+]cyt, and that blockade of the Na+/Ca2+ exchanger in its reverse mode may serve as a potential therapeutic approach for treatment of pulmonary hypertension.

Identification of functional voltage-gated Na+ channels in cultured human pulmonary artery smooth muscle cells

“…Conversely, an enhanced local rise in [Na + ] cyt due to enhanced I Na activity, could have a similar effect on both NCX activity and [Ca 2+ ] cyt . In a recent study, we demonstrated that human PASMC express high levels of NCX-encoding proteins, NCX1 and NCKX3 [37]. Enhanced reverse-mode NCX activity produced not only an increase in [Ca 2+ ] cyt , but also promoted PASMC proliferation [37].…”

“…In a recent study, we demonstrated that human PASMC express high levels of NCX-encoding proteins, NCX1 and NCKX3 [37]. Enhanced reverse-mode NCX activity produced not only an increase in [Ca 2+ ] cyt , but also promoted PASMC proliferation [37]. Preliminary experiments conducted within our lab suggest that TTX-sensitive Na + channel activation does not influence the histamine-induced release of Ca 2+ from the SR in cultured human PASMC (Mauban and Yuan, unpublished observations), or serum-induced PASMC proliferation (Remillard and Yuan, unpublished observations).…”

Na+/Ca2+exchangers regulate the migration and proliferation of human gastric myofibroblasts

“…However, that may not be the mechanism by which HGMs generate oscillation, since CB-DMB does inhibit nor mitochondrial nor ER Ca 2ϩ channels/transporters in applied concentration (37), and no oscillations could be observed after applying it. In pulmonary artery smooth muscle cells, NCX plays an important role in regulating cytosolic Ca 2ϩ levels; however, in these cells Ca 2ϩ increases via the NCX and store-operated entry (43). Although NCX regulates the cytosolic Ca 2ϩ in the HGMs in a similar way, store-operated entry does not play a role in HGMs, since inhibiting the NCX results in Ca 2ϩ depletion, not store depletion-mediated Ca 2ϩ entry.…”

“…Furthermore, without proper Ca 2ϩ signaling, resting cells cannot get over the G 0 phase (1). In pulmonary artery smooth muscle cells, Ca 2ϩ enters the cytosol after store depletion via the reverse mode of NCX, and the NCX inhibitor KB-R7943 inhibited cell proliferation via disorganization of intracellular Ca 2ϩ signaling (43).…”

“…It is known that NCX plays an important role in regulating the [Ca 2ϩ ] i in several cell types (3). NCX alters the migration of rat tendon fibroblasts and migration and proliferation in human pulmonary artery smooth muscle cells (36,43). Moreover, it has been reported that Ca 2ϩ entry-mode operation of NCX is required for des-Arg10-kallidin-and TGF-␤1-stimulated fibrogenesis and participates in the maintenance of the myofibroblast phenotype (35).…”