Intracellular calcium oscillations induced by Hypoxia Induced Mitogenic Factor in pulmonary artery smooth muscle cells

Abstract

BACKGROUND: Hypoxia-induced mitogenic factor (HIMF), a upregulated cytokine in pulmonary endothelium and vascular smooth muscle in a hypoxia-induced pulmonary hypertension, can increase pulmonary arterial pressure and pulmonary vascular resistance in vivo. We now investigate the underlying mechanism by examing if HIMF can directly increase intracellular calcium in smooth muscle cells. METHODS: Human pulmonary artery smooth muscle cells (HPASMC) were seeded on glass coverslips and loaded with Fluo-4-AM for 30 min at room temperature before intracellular calcium was imaged with an confocal microscope. Recombinant HIMF was added directly for stimulation. Various inhibitors pretreated cells for 30 min cells before the calcium image assay. RESULTS: Intracellular calcium oscillations were induced by HIMF in HPASMC dose-dependently, with a maximum response at 60 nM. Pretreatment of cells with U-73122, a PLC inhibitor, completely blocked the HIMF-induced intracellular calcium increase, whereas Gs-specific inhibitors, pertussis toxin and NF449, did not have effect. In addition, HIMF-induced Ca2+ release was abolished by pretreatment with 2-APB, an IP3 receptor antagonist; but not by ryanodine. CONCLUSIONS: These results indicated that HIMF can stimulate intracellular calcium release in HPASMC though the PLC-IP3 signaling pathway.