Inhibition of hypoxia-induced calcium responses in pulmonary arterial smooth muscle by acetazolamide is independent of carbonic anhydrase inhibition

Hypoxic pulmonary vasoconstriction ( HPV) occurs with ascent to high altitude and can contribute to development of high altitude pulmonary edema ( HAPE). Vascular smooth muscle contains carbonic anhydrase ( CA), and acetazolamide ( AZ), a CA inhibitor, blunts HPV and might be useful in the prevention of HAPE. The mechanism by which AZ impairs HPV is uncertain. Originally developed as a diuretic, AZ also has direct effects on systemic vascular smooth muscle, including modulation of pH and membrane potential; however, the effect of AZ on pulmonary arterial smooth muscle cells ( PASMCs) is unknown. Since HPV requires Ca2+ influx into PASMCs and can be modulated by pH, we hypothesized that AZ alters hypoxia- induced changes in PASMC intracellular pH ( pHi) or Ca2+ concentration ([Ca2+](i)). Using fluorescent microscopy, we tested the effect of AZ as well as two other potent CA inhibitors, benzolamide and ethoxzolamide, which exhibit low and high membrane permeability, respectively, on hypoxia- induced responses in PASMCs. Hypoxia caused a significant increase in [Ca2+](i) but no change in pHi. All three CA inhibitors slightly decreased basal pHi, but only AZ caused a concentrationdependent decrease in the [Ca2+](i) response to hypoxia. AZ had no effect on the KCl- induced increase in [ Ca2+](i) or membrane potential. N- methyl- AZ, a synthesized compound lacking the unsubstituted sulfonamide group required for CA inhibition, had no effect on pHi but inhibited hypoxia- induced Ca2+ responses. These results suggest that AZ attenuates HPV by selectively inhibiting hypoxia- induced Ca2+ responses via a mechanism independent of CA inhibition, changes in pHi, or membrane potential.