calcimycin and Persistent-Fetal-Circulation-Syndrome

Increased pulmonary artery endothelial cell (PAEC) endothelium-dependent nitric oxide synthase (eNOS) activity mediates perinatal pulmonary vasodilation. Compromised eNOS activity is central to the pathogenesis of persistent pulmonary hypertension of the newborn (PPHN). Voltage-derived anion channel (VDAC)-1 was recently demonstrated to bind eNOS in the systemic circulation. We hypothesized that VDAC isoforms modulate eNOS activity in the pulmonary circulation, and that decreased VDAC expression contributes to PPHN. In PAECs derived from an ovine model of PPHN: (1) there is eNOS activity, but not expression; and (2) VDAC1 and -2 proteins are decreased. Immunocytochemistry, coimmunoprecipitation, and in situ proximity ligation assays in human PAECs (hPAECs) demonstrate binding between eNOS and both VDAC1 and -2, which increased upon stimulation with NO agonists. The ability of agonists to increase the eNOS/VDAC interaction was significantly blunted in hypertensive, compared with normotensive, ovine PAECs. Depletion of VDAC2, but not VDAC1, blocked the agonist-induced increase in eNOS activity in hPAECs. Overexpression of VDAC2 in hypertensive PAECs increased eNOS activity. Binding of VDAC2 enhances eNOS activity in the pulmonary circulation, and diminished VDAC2 constrains eNOS in PAECs derived from fetal lambs with chronic intrauterine pulmonary hypertension. We speculate that decreases in VDAC2 may contribute to the limited eNOS activity that characterizes pulmonary hypertension.

Topics: Animals; Calcimycin; Cells, Cultured; Endothelial Cells; Endothelium, Vascular; Gene Expression; Histamine; Humans; Infant, Newborn; Nitric Oxide Synthase Type III; Persistent Fetal Circulation Syndrome; Protein Binding; Protein Interaction Mapping; Pulmonary Artery; Sheep; Voltage-Dependent Anion Channel 1; Voltage-Dependent Anion Channel 2

Acute hypoxia causes pulmonary hypertension in the fetus and newborn that is contrasted by systemic hypotension or normotension. To better understand the role of nitric oxide (NO) in this specific pulmonary vascular response, we determined the acute effects of decreased oxygenation on NO production in ovine fetal pulmonary and systemic (mesenteric) endothelial cells. NO was assessed by measuring cGMP accumulation in fetal vascular smooth muscle (VSM) cells during co-culture incubations of endothelium and VSM (40 s) in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. Changes in cGMP were dependent on the endothelium and on NO synthase and guanylate cyclase activity. At high O2 (680 mm Hg), basal NO was detectable and NO increased 6- to 10-fold with bradykinin or A23187. In pulmonary endothelium, basal NO fell 58% at pO2 = 150 mm Hg and 51% at 40 mm Hg versus 680 mm Hg, while NO with bradykinin fell 56% and 63%, respectively. NO with A23187, however, was unchanged at 150 mm Hg, but it fell 56% at 40 mm Hg. In contrast, in systemic endothelium basal and stimulated NO production were not altered at lower O2. Findings were similar using pulmonary or systemic detector VSM cells, and exogenous L-arginine had no effect. Thus, decreased O2 acutely attenuates NO production specifically in fetal pulmonary endothelial cells. This process is not related to changes in O2 or L-arginine availability as substrates for NO synthase; alternatively, it may be partially mediated by specific effects of O2 on pulmonary endothelial cell calcium homeostasis.

Topics: 1-Methyl-3-isobutylxanthine; Amino Acid Oxidoreductases; Animals; Bradykinin; Calcimycin; Cells, Cultured; Cyclic GMP; Disease Models, Animal; Endothelium, Vascular; Female; Guanylate Cyclase; Humans; Hypoxia; Infant, Newborn; Lung; Mesenteric Arteries; Muscle, Smooth, Vascular; Nitric Oxide; Nitric Oxide Synthase; Nitroprusside; Oxygen; Persistent Fetal Circulation Syndrome; Pregnancy; Pulmonary Artery; Sheep