Sustained therapeutic hypercapnia attenuates pulmonary arterial Rho-kinase activity and ameliorates chronic hypoxic pulmonary hypertension in juvenile rats

Abstract

Jankov RP. Sustained therapeutic hypercapnia attenuates pulmonary arterial Rho-kinase activity and ameliorates chronic hypoxic pulmonary hypertension in juvenile rats. Am J Physiol Heart Circ Physiol 302: H2599 -H2611, 2012. First published April 13, 2012; doi:10.1152/ajpheart.01180.2011.-Sustained therapeutic hypercapnia prevents pulmonary hypertension in experimental animals, but its rescue effects on established disease have not been studied. Therapies that inhibit Rho-kinase (ROCK) and/or augment nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) signaling can reverse or prevent progression of chronic pulmonary hypertension. Our objective in the present study was to determine whether sustained rescue treatment with inhaled CO 2 (therapeutic hypercapnia) would improve structural and functional changes of chronic hypoxic pulmonary hypertension. Spontaneously breathing pups were exposed to normoxia (21% O 2) or hypoxia (13% O 2) from postnatal days 1-21 with or without 7% CO2 (PaCO 2 elevated by ϳ25 mmHg) or 10% CO2 (PaCO 2 elevated by ϳ40 mmHg) from days 14 to 21. Compared with hypoxia alone, animals exposed to hypoxia and 10% CO 2 had significantly (P Ͻ 0.05) decreased pulmonary vascular resistance, right-ventricular systolic pressure, right-ventricular hypertrophy, and medial wall thickness of pulmonary resistance arteries as well as decreased lung phosphodiesterase (PDE) V, RhoA, and ROCK activity. Rescue treatment with 10% CO 2, or treatment with a ROCK inhibitor (15 mg/kg ip Y-27632 twice daily from days 14 to 21), also increased pulmonary arterial endothelial nitric oxide synthase and lung NO content. In contrast, cGMP content and cGMP-dependent protein kinase (PKG) activity were increased by exposure to 10% CO 2, but not by ROCK inhibition with Y-27632. In vitro exposure of pulmonary artery smooth muscle cells to hypercapnia suppressed serum-induced ROCK activity, which was prevented by inhibition of PKG with Rp-8-Br-PETcGMPS. We conclude that sustained hypercapnia dose-dependently inhibited ROCK activity, augmented NO-cGMP-PKG signaling, and led to partial improvements in the hemodynamic and structural abnormalities of chronic hypoxic PHT in juvenile rats. Increased PKG content and activity appears to play a major upstream role in CO 2-induced suppression of ROCK activity in pulmonary arterial smooth muscle. carbon dioxide; hypoxia; nitric oxide; RhoA; protein kinase G CHRONIC PULMONARY HYPERTENSION (PHT) is a debilitating disease characterized by a progressive increase in pulmonary arterial pressure and resistance, secondary to sustained pulmonary vasoconstriction and arterial wall remodeling. In early life, chronic PHT is most frequently observed in the context of severe bronchopulmonary dysplasia (51, 64), vascular hypoplasia syndromes, including congenital diaphragmatic hernia (43), and in a subset of cases of idiopathic persistent PHT of the newborn (46). Efforts to treat chronic PHT in this population are frequently hampered by nonresponsiveness to pulmonary vasodilators, such as inhaled nit...

Sex-dependent changes in the pulmonary vasoconstriction potential of newborn rats following short-term oxygen exposure

“…We have recently shown that chronic exposure to hypoxia promotes pulmonary hypertension in newborn rats via the Rho kinases pathway (30). In our study, we observed significant ROCK activation in hyperoxia-treated male pup lungs but not their third-generation pulmonary arteries.…”

“…Lung tissue and third-generation intrapulmonary arteries were lysed in radioimmunoprecipitation assay buffer, and protein samples (50 μg/ lane) were blotted, as previously described (30). Myosin phosphatase target subunit 1 (MYPT-1) is one of the substrates of ROCK.…”

“…Myosin phosphatase target subunit 1 (MYPT-1) is one of the substrates of ROCK. ROCK activity was quantified by the measurement of phosphothreonine 850 normalized to pan-MYPT-1, as previously described (30). Protein bands were identified using enhanced chemiluminescent substrate (Immobilon; Millipore) and images were digitally captured using a MicroChemi chemiluminescent image analysis system (DNR Bioimaging Systems, Jerusalem, Israel).…”

Leukotriene B4mediates macrophage influx and pulmonary hypertension in bleomycin-induced chronic neonatal lung injury

“…Histological studies. Lungs from six animals from each group (3 from each of 2 separate litters) were air-inflated and perfusion-fixed at constant pressure, embedded in paraffin, sectioned, and immunostained for CD68 (to identify macrophages), or were stained with hematoxylin-eosin, or for elastin, as previously described (23,29,33,35). For all analyses, measurements were carried out on four noncontiguous left lung sections per animal by an observer blinded to group identity.…”

Arginase inhibition prevents bleomycin-induced pulmonary hypertension, vascular remodeling, and collagen deposition in neonatal rat lungs

“…Paraffin-embedded inflated lung tissue sections were stained with hematoxylin and eosin (for lung morphometry), Hart's elastin (for the arterial medial wall area), or Picrosirius Red (to identify collagen), as previously described (23,26,33,42,43). To localize NOS2 or to assess an indicator of oxidative/ nitrative stress, sections were immunostained for NOS2 or 3-nitrotyrosine, respectively (22,23).…”