Contribution of elevated intracellular calcium to pulmonary arterial myocyte alkalinization during chronic hypoxia

In the lung, exposure to chronic hypoxia (CH) causes pulmonary hypertension, a debilitating disease. Development of this condition arises from increased muscularity and contraction of pulmonary vessels, associated with increases in pulmonary arterial smooth muscle cell (PASMC) intracellular pH (pH_i) and Ca²⁺ concentration ([Ca²⁺]_i). In this study, we explored the interaction between pH_i and [Ca²⁺]_i in PASMCs from rats exposed to normoxia or CH (3 weeks, 10% O₂). PASMC pH_i and [Ca²⁺]_i were measured with fluorescent microscopy and the dyes BCECF and Fura-2. Both pH_i and [Ca²⁺]_i levels were elevated in PASMCs from hypoxic rats. Exposure to KCl increased [Ca²⁺]_i and pH_i to a similar extent in normoxic and hypoxic PASMCs. Conversely, removal of extracellular Ca²⁺ or blockade of Ca²⁺ entry with NiCl₂ or SKF 96365 decreased [Ca²⁺]_i and pH_i only in hypoxic cells. Neither increasing pH_i with NH₄Cl nor decreasing pH_i by removal of bicarbonate impacted PASMC [Ca²⁺]_i. We also examined the roles of Na⁺/Ca²⁺ exchange (NCX) and Na⁺/H⁺ exchange (NHE) in mediating the elevated basal [Ca²⁺]_i and Ca²⁺-dependent changes in PASMC pH_i. Bepridil, dichlorobenzamil, and KB-R7943, which are NCX inhibitors, decreased resting [Ca²⁺]_i and pH_i only in hypoxic PASMCs and blocked the changes in pH_i induced by altering [Ca²⁺]_i. Exposure to ethyl isopropyl amiloride, an NHE inhibitor, decreased resting pH_i and prevented changes in pH_i due to changing [Ca²⁺]_i. Our findings indicate that, during CH, the elevation in basal [Ca²⁺]_i may contribute to the alkaline shift in pH_i in PASMCs, likely via mechanisms involving reverse-mode NCX and NHE.