George D. Thorne

Effects of Hypoxia on Isometric Force, Intracellular Ca2+, pH, and Energetics in Porcine Coronary Artery

When exposed to hypoxic conditions, coronary arteries dilate, which is an important protective response. Although vessel sensitivity to oxygen is well documented, the mechanisms are not known with certainty. To further characterize the mechanisms of oxygen sensing in the coronary artery, we tested the major classes of hypotheses by measuring the effects of hypoxia on energetics, [Ca(2+)](i), K(+) channel function, and pH(i). Hypoxia relaxes porcine coronary arteries stimulated with either KCl or U46619. The extent of relaxation is dependent on both the degree and kind of stimulation. [Ca(2+)](i) was measured in endothelium-denuded arteries using fura 2-AM and ratiometric fluorescent techniques. At lower stimulus levels, hypoxia decreased both force and [Ca(2+)](i). Inhibitor studies suggest that K(Ca) and K(ATP) channels are not involved in the hypoxic relaxation, whereas K(V) channels may play a minor role, if any. Despite the hypoxia-mediated decrease in force, [Ca(2+)](i) was unchanged or increased at high levels of stimulation. Despite a marked increase in lactate content, pH(i) (measured with the ratiometric fluorescent dye BCECF) was also little affected by hypoxia. Measurement of the phosphagen and metabolite profile of freeze-clamped arteries with analytical isotachophoresis indicated that hypoxia increased lactate content by 4-fold and decreased phosphocreatine to 60% of control. However, neither ATP nor P(i) was affected by hypoxia. Interestingly, additional stimulation under hypoxia increased force but not ATP utilization, as estimated from measurements of anaerobic lactate production. Thus, surprisingly, the economy of force maintenance is increased under hypoxia. In porcine coronary artery, both Ca(2+)-dependent and, importantly, Ca(2+)-independent mechanisms are involved in hypoxic vasodilatation. For the latter, mechanisms involving either ATP, [Ca(2+)](i), pH(i), or P(i) cannot be invoked. This novel oxygen sensing mechanism involves a decreased Ca(2+) sensitivity.

Ca2+‐independent hypoxic vasorelaxation in porcine coronary artery

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.