Chronic intrauterine pulmonary hypertension selectively modifies pulmonary artery smooth muscle cell gene expression

Abstract

. Chronic intrauterine pulmonary hypertension selectively modifies pulmonary artery smooth muscle cell gene expression. Am J Physiol Lung Cell Mol Physiol 290: L426 -L432, 2006; doi:10.1152/ajplung.00281.2005.-Pulmonary artery smooth muscle cell (PASMC) relaxation at birth results from an increase in cytosolic cGMP, cGMP-dependent and kinase-mediated activation of the Ca 2ϩ -sensitive K ϩ channel (KCa), and closure of voltage-operated Ca 2ϩ channels (VOCC). How chronic intrauterine pulmonary hypertension compromises perinatal pulmonary vasodilation remains unknown. We tested the hypothesis that chronic intrauterine pulmonary hypertension selectively modifies gene expression to mitigate perinatal pulmonary vasodilation mediated by the cGMP kinase-KCa-VOCC pathway. PASMC were isolated from late-gestation fetal lambs that had undergone either ligation of the ductus arteriosus (hypertensive) or sham operation (control) at 127 days of gestation and were maintained under either hypoxic (ϳ25 Torr) or normoxic (ϳ120 Torr) conditions in primary culture. We studied mRNA levels for cGMP kinase I␣ (PKG-1␣), the ␣-chain of VOCC (Cav1.2), and the ␣-subunit of the KCa channel. Compared with control PASMC, hypertensive PASMC had decreased VOCC, KCa, and PKG-1␣ expression. In response to sustained normoxia, expression of VOCC and KCa channel decreased and expression of PKG-1␣ increased. In contrast, sustained normoxia had no effect on PKG-1␣ levels and an attenuated effect on VOCC and KCa channel expression in hypertensive PASMC. Protein expression of PKG-1␣ was consistent with the mRNA data. We conclude that chronic intrauterine pulmonary hypertension decreases PKG expression and mitigates the genetic effects of sustained normoxia on pulmonary vasodilation, because gene expression remains compromised even after sustained exposure to normoxia. fetal; oxygen sensing; nitric oxide IN UTERO, oxygen tension is low and pulmonary vascular resistance is greater than systemic vascular resistance (36). At birth, the pulmonary circulation undergoes an unprecedented and unparalleled transition, given that pulmonary blood flow increases 8-to 10-fold and arterial pressure decreases by 50% within 24 h, concomitant with an increase in oxygen tension, establishment of an air-liquid interface, and rhythmic distention of the lung (9,15,44).Recent data suggest that activation of the large-conductance Ca 2ϩ -sensitive K ϩ channel (K Ca , also known as BK Ca or MaxiK) plays a critically important role in mediating the response to perinatal pulmonary vasodilator stimuli such as oxygen (11) ] i ) and promoting vasodilation (20). PKG also may directly activate the K Ca channel by phosphorylation of its ␣-chain (42). Activation of the channel results in membrane hyperpolarization, closure of voltage-gated Ca 2ϩ channels, a decrease in cytosolic Ca 2ϩ , and vasodilation. Recent data indicate the existence of several different isoforms of cGMP-dependent protein kinase. In vascular smooth muscle cells, relaxation is contingent on activation of the type I ...

Hydrogen Peroxide Regulates Extracellular Superoxide Dismutase Activity and Expression in Neonatal Pulmonary Hypertension

“…We isolated fifth-generation pulmonary artery SMCs from control and PPHN lambs and confirmed that the PPHN cells maintained their phenotype in culture by demonstrating a decrease in expression of PKG-1a (data not shown), in agreement with the previous study of Resnik et al (35). We then exposed the cells to 95% O 2 for 24 h to mimic the effects of hyperoxia on lambs ventilated with 100% O 2 .…”

Developmental regulation of hypoxia-inducible factor 1 and prolyl-hydroxylases in pulmonary vascular smooth muscle cells

“…The techniques used for the isolation of ovine PA SMC have been described (27). Distal PAs were quickly excised from pentobarbital-anesthetized ovine fetuses and placed in physiological saline solution (120 mM NaCl/5.9 mM KCl/11.5 mM dextrose/25 mM NaHCO 3 /1.2 mM NaH 2 PO 4 /1.…”

“…The dispersed cell suspension was aliquoted onto 25-mm 2 glass coverslips coated with poly-L-lysine (Sigma) and into 25-cm 2 tissue culture flasks (Falcon Plastics, Oxnard, CA) at a density of 5 Ϫ10 ϫ 10 3 cells/cm 2 . The cells were incubated at 37°C in either a humidified 5% oxygen, 5% CO 2 balance nitrogen atmosphere or a humidified air, 5% CO 2 atmosphere (27,28). After 18-24 h the cultures were washed once with Hanks' balanced salt solution to remove nonadherent cells and debris and refed with fresh medium.…”

“…For Western blotting and ELISAs, fetal and PA SMC were incubated under either normoxic or hypoxic conditions as described (27,28). Eight micrograms of nuclear extract was used in ELISAs (Active Motif, Carlsbad CA), and results were standardized to COS7 nuclear protein.…”

cGMP-Dependent Protein Kinase in the Regulation of Cardiovascular Functions

“…PKG expression and/or activity are/is reduced in animal models of pulmonary arterial hypertension (PAH) induced by ligation of the ductus arteriosus of fetal lambs (Resnik et al, 2006) and in caveolin-1 (Cav-1) knockout mice (Zhao et al, 2009). Cav-1, a 21-kDa integral membrane protein, is an intracellular physiological inhibitor of eNOS activity.…”