| Popis: |
To demonstrate a Ca2+-independent component of hypoxic vasorelaxation and to investigate its mechanism, we utilized permeabilized porcine coronary arteries, in which [Ca2+] could be clamped. Arteries permeabilized with β-escin developed maximum force in response to free Ca2+ (6.6 μm), concomitant with a parallel increase in myosin regulatory light chain phosphorylation (MRLC-Pi), from 0.183 ± 0.023 to 0.353 ± 0.019 MRLC-Pi (total light chain)−1. Hypoxia resulted in a significant decrease in both force (–31.9 ± 4.1% prior developed force) and MRLC-Pi (from 0.353 to 0.280 ± 0.023), despite constant [Ca2+] buffered by EGTA (4 mm). Forces developed in response to Ca2+ (6.6 μm), Ca2+ (0.2 μm) + GTPγS (1 mm), or in the absence of Ca2+ after treatment with ATPγS (1 mm), were of similar magnitude. Hypoxia also relaxed GTPγS contractures but importantly, arteries could not be relaxed after treatment with ATPγS. Permeabilization with Triton X-100 for 60 min also abolished hypoxic relaxation. The blocking of hypoxic relaxation after ATPγS suggests that this Ca2+-independent mechanism(s) may operate through alteration of MRLC-Pi or of phosphorylation of the myosin binding subunit of myosin light chain phosphatase. Treatment with the Rho kinase inhibitor Y27632 (1 μm) relaxed GTPγS and Ca2+ contractures; but the latter required a higher concentration (10 μm) for consistent relaxation. Relaxations to N2 and/or Y27632 averaged 35% and were not additive or dependent on order. Our data suggest that the GTP-mediated, Rho kinase-coupled pathway merits further investigation as a potential site of this novel, Ca2+-independent O2-sensing mechanism. Importantly, these results unambiguously show that hypoxia-induced vasorelaxation can occur in permeabilized arteries where the Ca2+ is clamped at a constant value. |
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