In precontracted proximal intrapulmonary arteries, severe hypoxia caused a triphasic response: (1) early transient contraction, which was endothelium-dependent and reduced by L-NAME; (2) relaxation, which was endothelium-independent, reduced by glibenclamide, and associated with decreased smooth muscle energy state and intracellular pH; and (3) late sustained contraction, which was endothelium-dependent, unaffected by L- NAME or indomethacin, and associated with recovery of smooth muscle energy state and intracellular pH. In pulmonary arterial myocytes, severe hypoxia caused depolarization and decreased outward potassium currents, which were inhibited by 4-aminopyridine, but not TEA or charybdotoxin. Based on these results, we propose that the direct effect of hypoxia on pulmonary arterial smooth muscle is depolarization, which occurs because delayed rectifier potassium (Kdr) channels are inhibited, perhaps secondary to desaturation of a sarcolemmal heme protein. If and when this direct effect is expressed as increased vasomotor tone, however, depends on many factors, including baseline membrane potential, intracellullar calcium concentration ({ca2+}i), energy state, and intracellular pH in smooth muscle, as well as the presence or absence of endothelial influences. With respect to proximal intrapulmonary arteries, we hypothesize, first, that early hypoxic contraction is caused by inhibition of basal endothelium-derived NO activity. Although hypoxia inhibits Kdr channels in smooth muscle, leading to depolarization, activation of voltage-dependent calcium channels, and calcium influx, these events do not contribute to early hypoxic contraction because the depolarization is too small and the rise in {Ca2+}i too slow to trigger contraction before the onset of hypoxic relaxation. Second, hypoxic relaxation is caused by inhibition of receptor-linked vasoconstrictor transduction pathways and activation of ATP-dependent potassium (KATP) channels in smooth muscle, both of which result from energy state deterioration and intracellular acidosis secondary to decreased oxidative phosphorylation. Third, late hypoxic contraction is caused by endothelial factors which upregulate glycolysis and improve energy state in smooth muscle, leading to normalization of intracellular pH via enhanced Na-H exchange; improved transduction of receptor-linked vasoconstrictor stimuli, including an endothelium-derived contractile factor; and depolarization secondary to inactivation of KATP channels and direct hypoxic inhibition of Kdr channels. To test these hypotheses, we will perform experiments in proximal intrapulmonary and systemic arteries of the pig, measuring membrane potential, ion currents, {Ca2+}i, and intracellular pH in arterial myocytes; isometric tension, 31P nuclear magnetic resonance spectroscopy, glucose utilization, lactate production, and concentrations of inositol phosphates and diacylglycerol in arterial rings; and transmural pressure-diameter relations and bioassays of endothelial factors in perfused arteries. By elucidating the direct effects of hypoxia on pulmonary arterial smooth muscle, and how these effects are altered by endothelium, we hope to improve understanding of in vivo pulmonary vasomotor responses to hypoxia, which have been difficult to study mechanistically in intact animals or isolated lungs. Understanding these responses is important, because they optimize oxygen exchange in normal lungs and cause pulmonary hypertension, cor pulmonale, and increased mortality in patients with chronic lung disease.
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