Hypoxic Pulmonary Artery Smooth Muscle Research Articles

Reciprocal regulation of Na+/H+ exchanger isoform 1 (NHE1) and Na+/H+ exchange regulatory factor 1 (NHERF1) in Hypoxic Pulmonary Arterial Smooth Muscle Cells (PASMCs)

Increased PASMC contraction and growth during chronic hypoxia (CH) may be due to cytoskeletal rearrangement involving transmembrane and cytosolic proteins. We found that the ion transporter, NHE1, is upregulated in chronically hypoxic rats (10% O2; 3 wk) and is required for development of hypoxic pulmonary hypertension. In addition to regulating pH, NHE1 was recently found to bind phosphorylated ezrin (p‐ezrin), an actin filament binding protein. NHERF1 also binds p‐ezrin, at the same site as NHE1. Since the transmembrane spanning NHE1 can act as an anchor, but cytosolic NHERF1 cannot, we hypothesized that changes in NHE1/ezrin interactions during hypoxia enables actin filaments to be tethered to the cell membrane, promoting PASMC contraction and growth. Confocal images of immunofluorescent stained chronically hypoxic mouse lung sections confirmed co‐localization of p‐ezrin and smooth muscle cell‐specific α‐actin (SM‐actin). Immunoblots showed increased NHE1, increased p‐ezrin and decreased NHERF1 levels in response to hypoxia. Co‐immunoprecipitation studies showed increased NHE1 binding to p‐ezrin and SM‐actin with hypoxia (4% O2; 24 hr), and decreased NHERF1 binding. These results suggest reciprocal regulation of NHERF1 and NHE1 expression in PASMC during hypoxia, with both proteins competing to bind p‐ezrin. Increased NHE1/ezrin interactions may contribute to hypoxic pulmonary hypertension.

Nitric oxide modulates hypoxic pulmonary smooth muscle cell proliferation and apoptosis by regulating carbon monoxide pathway

To explore the role of carbon monoxide (CO) in the regulation of hypoxic pulmonary artery smooth muscle cell (PASMC) proliferation and apoptosis by nitric oxide (NO). PASMC of Wistar rats was cultured in vitro in the presence of a NO donor, sodium nitroprusside, or an inhibitor of heme oxygenase (HO), zinc protoporphyrin-IX, or under both normoxic and hypoxic conditions. Nitrite and carboxyhemoglobin in PASMC medium were detected with spectrophotometry. The proliferating and apoptotic percentage of PASMC was measured by flow cytometry. The expression of HO-1 mRNA in PASMC was analyzed by fluorescent real-time quantitative PCR, and the proliferating cell nuclear antigen and caspase-3 were examined by immunocytochemical analysis. The results showed that hypoxia suppressed NO generation from PASMC, which promoted hypoxic PASMC proliferation and induced apoptosis. Meanwhile, hypoxia induced HO-1 expression in PASMC and promoted CO production from PASMC, which inhibited PASMC proliferation and regulated PASMC apoptosis. NO upregulated the expression of HO-1 mRNA in hypoxic PASMC; NO also inhibited proliferation and promoted apoptosis of hypoxic PASMC, possibly by regulating the production of CO. The results indicated that CO could inhibit proliferation and regulate apoptosis of PASMC, and NO inhibited proliferation and promoted apoptosis of hypoxic PASMC, possibly by regulating the production of CO.

Point:Counterpoint: Hypoxic pulmonary vasoconstriction is/is not mediated by increased production of reactive oxygen species

This series of debates was initiated for the Journal of Applied Physiology because we believe an important means of searching for truth is through debate where contradictory viewpoints are put forward. This dialectic process whereby a thesis is advanced, then opposed by an antithesis, with a

Free radical production in hypoxic pulmonary artery smooth muscle cells

This study used an inexpensive and versatile environmental exposure system to test the hypothesis that hypoxia promoted free radical production in primary cultures of rat main pulmonary artery smooth muscle cells (PASMCs). Production of reactive species was detected by fluorescence microscopy with the probe 2', 7'-dichlorodihydrofluorescein, which is converted to the fluorescent dichlorofluorescein (DCF) in the presence of various oxidants. Flushing the airspace above the PASMC cultures with normoxic gas (20% O(2), 75% N(2), and 5% CO(2)) resulted in stable PO(2) values of approximately 150 Torr, whereas perfusion of the airspace with hypoxic gas (0% O(2), 95% N(2), and 5% CO(2) ) was associated with a reduction in PO(2) values to stable levels of approximately 25 Torr. Hypoxic PASMCs became increasingly fluorescent at approximately 500% above the normoxic baseline after 60 min. Hypoxia-induced DCF fluorescence was attenuated by the addition of the antioxidants dimethylthiourea and catalase. These findings show that PASMCs acutely exposed to hypoxia exhibit a marked increase in intracellular DCF fluorescence, suggestive of reactive oxygen or nitrogen species production.

Polyamine transport and ornithine decarboxylase activity in hypoxic pulmonary artery smooth muscle cells.

Hypoxia causes remodeling of the pulmonary circulation that is dependent on increases in lungs polyamine contents. Mechanisms by which polyamines are regulated in hypoxic lung cells are unknown, but ornithine decarboxylase (ODC) activity, the initial enzyme in de novo biosynthesis, is depressed and polyamine transport is augmented in lungs from hypoxic rats (R.-T. Shiao et al. 1990. Am. J. Physiol. 259:L351-L358). To determine if hypoxia directly influences polyamine regulatory mechanisms in pulmonary vascular cells, we examined [14C]spermidine (SPD) transport and ODC activity in bovine main pulmonary artery smooth muscle cells (PASMCs) cultured under standard (culture medium Po2: greater than 100 mm Hg), "normoxic" (culture medium Po2: 50 to 70 mm Hg), or "hypoxic" (culture medium Po2: 18 to 30 mm Hg) conditions. Uptake of [14C]SPD in cells cultured under standard conditions was temperature- and concentration-dependent, exhibited saturation kinetics, and was abolished by metabolic inhibition. Modeling of transport according to Michaelis-Menten kinetics revealed that [14C]SPD uptake in cells cultured under standard conditions was characterized by Km and Vmax values of 0.78 microM and 4.5 pmol/min/10(6) cells, respectively. In comparison to cells cultured under standard conditions, Km was unaffected by culture under normoxic or hypoxic conditions while Vmax was increased to 18 pmol/min/10(6) cells in normoxic cells and to 33 pmol/min/10(6) cells in preparations cultured under hypoxic conditions. Inhibition of ODC with alpha-difluoromethylornithine (DFMO) also induced SPD transport, as evidenced by an increase in the Vmax to 65 pmol/min/10(6) cells. Both hypoxia- and DFMO-induced increases in [14C]SPD transport were suppressed by cycloheximide and actinomycin D, thus highlighting the importance of protein and RNA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)