Acetazolamide prevents hypoxia-induced reactive oxygen species generation and calcium release in pulmonary arterial smooth muscle

Upon sensing a reduction in local oxygen partial pressure, pulmonary vessels constrict, a phenomenon known as hypoxic pulmonary vasoconstriction. Excessive hypoxic pulmonary vasoconstriction can occur with ascent to high altitude and is a contributing factor to the development of high-altitude pulmonary edema. The carbonic anhydrase inhibitor, acetazolamide, attenuates hypoxic pulmonary vasoconstriction through stimulation of alveolar ventilation via modulation of acid–base homeostasis and by direct effects on pulmonary vascular smooth muscle. In pulmonary arterial smooth muscle cells (PASMCs), acetazolamide prevents hypoxia-induced increases in intracellular calcium concentration ([Ca(2+)](i)), although the exact mechanism by which this occurs is unknown. In this study, we explored the effect of acetazolamide on various calcium-handling pathways in PASMCs. Using fluorescent microscopy, we tested whether acetazolamide directly inhibited store-operated calcium entry or calcium release from the sarcoplasmic reticulum, two well-documented sources of hypoxia-induced increases in [Ca(2+)](i) in PASMCs. Acetazolamide had no effect on calcium entry stimulated by store-depletion, nor on calcium release from the sarcoplasmic reticulum induced by either phenylephrine to activate inositol triphosphate receptors or caffeine to activate ryanodine receptors. In contrast, acetazolamide completely prevented Ca(2+)-release from the sarcoplasmic reticulum induced by hypoxia (4% O(2)). Since these results suggest the acetazolamide interferes with a mechanism upstream of the inositol triphosphate and ryanodine receptors, we also determined whether acetazolamide might prevent hypoxia-induced changes in reactive oxygen species production. Using roGFP, a ratiometric reactive oxygen species-sensitive fluorescent probe, we found that hypoxia caused a significant increase in reactive oxygen species in PASMCs that was prevented by 100 μM acetazolamide. Together, these results suggest that acetazolamide prevents hypoxia-induced changes in [Ca(2+)](i) by attenuating reactive oxygen species production and subsequent activation of Ca(2+)-release from sarcoplasmic reticulum stores.