Long-term hypoxia uncouples Ca²⁺ and eNOS in bradykinin-mediated pulmonary arterial relaxation

Bradykinin-induced activation of the pulmonary endothelium triggers a rise in intracellular Ca²⁺ that activates nitric oxide (NO)-dependent vasorelaxation. Chronic hypoxia is commonly associated with increased pulmonary vascular tone, which can cause pulmonary hypertension in responsive individuals. In the present study, we tested the hypothesis that long-term high-altitude hypoxia (LTH) diminishes bradykinin-induced Ca²⁺ signals and inhibits endothelial nitric oxide synthase (eNOS), prostacyclin (PGI₂), and large-conductance K⁺ (BK_Ca) channels in sheep, which are moderately responsive to LTH, resulting in decreased pulmonary arterial vasorelaxation. Pulmonary arteries were isolated from ewes kept near sea level (720 m) or at high altitude (3,801 m) for >100 days. Vessel force was measured with wire myography and endothelial intracellular Ca²⁺ with confocal microscopy. eNOS was inhibited with 100 μM N^G-nitro-l-arginine methyl ester (l-NAME), PGI₂ production was inhibited with 10 µM indomethacin that inhibits cyclooxygenase, and BK_Ca channels were blocked with 1 mM tetraethylammonium. Bradykinin-induced endothelial Ca²⁺ signals increased following LTH, but bradykinin relaxation decreased. Furthermore, some vessels contracted in response to bradykinin after LTH. l-NAME sensitivity decreased, suggesting that eNOS dysfunction played a role in uncoupling Ca²⁺ signals and bradykinin relaxation. The Ca²⁺ ionophore A-23187 (10 µM) elicited an enhanced Ca²⁺ response following LTH while relaxation was unchanged although l-NAME sensitivity increased. Additionally, BK_Ca function decreased during bradykinin relaxation following LTH. Western analysis showed that BK_Ca α-subunit expression was increased by LTH while that for the β₁ subunit was unchanged. Overall, these results suggest that those even moderately responsive to LTH can have impaired endothelial function.