Altered Lipid Domains Facilitate Enhanced Pulmonary Vasoconstriction after Chronic Hypoxia.

Chronic hypoxia (CH) augments depolarization-induced pulmonary vasoconstriction through superoxide-dependent, Rho kinase-mediated Ca2+ sensitization. Nicotinamide adenine dinucleotide phosphate oxidase and EGFR (epidermal growth factor receptor) signaling contributes to this response. Caveolin-1 regulates the activity of a variety of proteins, including EGFR and nicotinamide adenine dinucleotide phosphate oxidase, and membrane cholesterol is an important regulator of caveolin-1 protein interactions. We hypothesized that derangement of these membrane lipid domain components augments depolarization-induced Ca2+ sensitization and resultant vasoconstriction after CH. Although exposure of rats to CH (4 wk, ∼380 mm Hg) did not alter caveolin-1 expression in intrapulmonary arteries or the incidence of caveolae in arterial smooth muscle, CH markedly reduced smooth muscle membrane cholesterol content as assessed by filipin fluorescence. Effects of CH on vasoreactivity and superoxide generation were examined using pressurized, Ca2+-permeabilized, endothelium-disrupted pulmonary arteries (∼150 µm inner diameter) from CH and control rats. Depolarizing concentrations of KCl evoked greater constriction in arteries from CH rats than in those obtained from control rats, and increased superoxide production as assessed by dihydroethidium fluorescence only in arteries from CH rats. Both cholesterol supplementation and the caveolin-1 scaffolding domain peptide antennapedia-Cav prevented these effects of CH, with each treatment restoring membrane cholesterol in CH arteries to control levels. Enhanced EGF-dependent vasoconstriction after CH similarly required reduced membrane cholesterol. However, these responses to CH were not associated with changes in EGFR expression or activity, suggesting that cholesterol regulates this signaling pathway downstream of EGFR. We conclude that alterations in membrane lipid domain signaling resulting from reduced cholesterol content facilitate enhanced depolarization- and EGF-induced pulmonary vasoconstriction after CH.

Augmented Pulmonary Vasoconstrictor Reactivity after Chronic Hypoxia Requires Src Kinase and Epidermal Growth Factor Receptor Signaling.

Chronic hypoxia augments pressure- and agonist-induced pulmonary vasoconstriction through myofilament calcium sensitization. NADPH oxidases contribute to the development of pulmonary hypertension, and both epidermal growth factor receptor and Src kinases can regulate NADPH oxidase. We tested the hypothesis that Src-epidermal growth factor receptor (EGFR) signaling mediates enhanced vasoconstrictor sensitivity after chronic hypoxia through NADPH oxidase-derived superoxide generation. Protocols employed pharmacological inhibitors in isolated, pressurized rat pulmonary arteries to examine the contribution of a variety of signaling moieties to enhanced vascular tone after chronic hypoxia. Superoxide generation in pulmonary arterial smooth muscle cells was assessed using the fluorescent indicator dihydroethidium. Indices of pulmonary hypertension were measured in rats treated with the EGFR inhibitor gefitinib. Inhibition of NADPH oxidase, Rac1 (Ras-related C3 botulinum toxin substrate 1), and EGFR abolished pressure-induced pulmonary arterial tone and endothelin-1 (ET-1)-dependent calcium sensitization and vasoconstriction after chronic hypoxia. Consistently, chronic hypoxia augmented ET-1-induced superoxide production through EGFR signaling, and rats treated chronically with gefitinib displayed reduced right ventricular pressure and diminished arterial remodeling. Src kinases were also activated by ET-1 after chronic hypoxia and contributed to enhanced basal arterial tone and vasoconstriction in response to ET-1. A role for matrix metalloproteinase 2 to mediate Src-dependent EGFR activation is further supported by our findings. Our studies support a novel role for an Src kinase-EGFR-NADPH oxidase signaling axis to mediate enhanced pulmonary vascular smooth muscle Ca2+ sensitization, vasoconstriction, and pulmonary hypertension after chronic hypoxia.

Reduced membrane cholesterol limits pulmonary endothelial Ca2+ entry after chronic hypoxia.

Chronic hypoxia (CH)-induced pulmonary hypertension is associated with diminished production of endothelium-derived Ca2+-dependent vasodilators such as nitric oxide. Interestingly, ATP-induced endothelial Ca2+ entry as well as membrane cholesterol (Chol) are decreased in pulmonary arteries from CH rats (4 wk, barometric pressure = 380 Torr) compared with normoxic controls. Store-operated Ca2+ entry (SOCE) and depolarization-induced Ca2+ entry are major components of the response to ATP and are similarly decreased after CH. We hypothesized that membrane Chol facilitates both SOCE and depolarization-induced pulmonary endothelial Ca2+ entry and that CH attenuates these responses by decreasing membrane Chol. To test these hypotheses, we administered Chol or epicholesterol (Epichol) to acutely isolated pulmonary arterial endothelial cells (PAECs) from control and CH rats to either supplement or replace native Chol, respectively. The efficacy of membrane Chol manipulation was confirmed by filipin staining. Epichol greatly reduced ATP-induced Ca2+ influx in PAECs from control rats. Whereas Epichol similarly blunted endothelial SOCE in PAECs from both groups, Chol supplementation restored diminished SOCE in PAECs from CH rats while having no effect in controls. Similar effects of Chol manipulation on PAEC Ca2+ influx were observed in response to a depolarizing stimulus of KCl. Furthermore, KCl-induced Ca2+ entry was inhibited by the T-type Ca2+ channel antagonist mibefradil but not the L-type Ca2+ channel inhibitor diltiazem. We conclude that PAEC membrane Chol is required for ATP-induced Ca2+ entry and its two components, SOCE and depolarization-induced Ca2+ entry, and that reduced Ca2+ entry after CH may be due to loss of this key regulator.NEW & NOTEWORTHY This research is the first to examine the direct role of membrane cholesterol in regulating pulmonary endothelial agonist-induced Ca2+ entry and its components. The results provide a potential mechanism by which chronic hypoxia impairs pulmonary endothelial Ca2+ influx, which may contribute to pulmonary hypertension.

Central role of T helper 17 cells in chronic hypoxia-induced pulmonary hypertension.

Inflammation is a prominent pathological feature in pulmonary arterial hypertension, as demonstrated by pulmonary vascular infiltration of inflammatory cells, including T and B lymphocytes. However, the contribution of the adaptive immune system is not well characterized in pulmonary hypertension caused by chronic hypoxia. CD4+ T cells are required for initiating and maintaining inflammation, suggesting that these cells could play an important role in the pathogenesis of hypoxic pulmonary hypertension. Our objective was to test the hypothesis that CD4+ T cells, specifically the T helper 17 subset, contribute to chronic hypoxia-induced pulmonary hypertension. We compared indices of pulmonary hypertension resulting from chronic hypoxia (3 wk) in wild-type mice and recombination-activating gene 1 knockout mice (RAG1-/-, lacking mature T and B cells). Separate sets of mice were adoptively transferred with CD4+, CD8+, or T helper 17 cells before normoxic or chronic hypoxic exposure to evaluate the involvement of specific T cell subsets. RAG1-/- mice had diminished right ventricular systolic pressure and arterial remodeling compared with wild-type mice exposed to chronic hypoxia. Adoptive transfer of CD4+ but not CD8+ T cells restored the hypertensive phenotype in RAG1-/- mice. Interestingly, RAG1-/- mice receiving T helper 17 cells displayed evidence of pulmonary hypertension independent of chronic hypoxia. Supporting our hypothesis, depletion of CD4+ cells or treatment with SR1001, an inhibitor of T helper 17 cell development, prevented increased pressure and remodeling responses to chronic hypoxia. We conclude that T helper 17 cells play a key role in the development of chronic hypoxia-induced pulmonary hypertension.

Role of ASIC1 in the development of chronic hypoxia-induced pulmonary hypertension.

Chronic hypoxia (CH) associated with respiratory disease results in elevated pulmonary vascular intracellular Ca(2+) concentration, which elicits enhanced vasoconstriction and promotes vascular arterial remodeling and thus has important implications in the development of pulmonary hypertension (PH). Store-operated Ca(2+) entry (SOCE) contributes to this elevated intracellular Ca(2+) concentration and has also been linked to acute hypoxic pulmonary vasoconstriction (HPV). Since our laboratory has recently demonstrated an important role for acid-sensing ion channel 1 (ASIC1) in mediating SOCE, we hypothesized that ASIC1 contributes to both HPV and the development of CH-induced PH. To test this hypothesis, we examined responses to acute hypoxia in isolated lungs and assessed the effects of CH on indexes of PH, arterial remodeling, and vasoconstrictor reactivity in wild-type (ASIC1(+/+)) and ASIC1 knockout (ASIC1(-/-)) mice. Restoration of ASIC1 expression in pulmonary arterial smooth muscle cells from ASIC1(-/-) mice rescued SOCE, confirming the requirement for ASIC1 in this response. HPV responses were blunted in lungs from ASIC1(-/-) mice. Both SOCE and receptor-mediated Ca(2+) entry, along with agonist-dependent vasoconstrictor responses, were diminished in small pulmonary arteries from control ASIC(-/-) mice compared with ASIC(+/+) mice. The effects of CH to augment receptor-mediated vasoconstrictor and SOCE responses in vessels from ASIC1(+/+) mice were not observed after CH in ASIC1(-/-) mice. In addition, ASIC1(-/-) mice exhibited diminished right ventricular systolic pressure, right ventricular hypertrophy, and arterial remodeling in response to CH compared with ASIC1(+/+) mice. Taken together, these data demonstrate an important role for ASIC1 in both HPV and the development of CH-induced PH.

Enhanced depolarization-induced pulmonary vasoconstriction following chronic hypoxia requires EGFR-dependent activation of NAD(P)H oxidase 2.

AIMS: Chronic hypoxia (CH) enhances depolarization-induced myofilament Ca(2+) sensitization and resultant pulmonary arterial constriction through superoxide (O(2)(-))-dependent stimulation of RhoA. Because NAD(P)H oxidase (NOX) has been implicated in the development of pulmonary hypertension, we hypothesized that vascular smooth muscle (VSM) depolarization increases NOX-derived O(2)(-) production leading to myofilament Ca(2+) sensitization and augmented vasoconstrictor reactivity following CH. As epidermal growth factor receptor (EGFR) mediates Rac1-dependent NOX activation in renal mesangial cells, we further sought to examine the role EGFR plays in this response. RESULTS: Vasoconstrictor responses to depolarizing concentrations of KCl were greater in lungs isolated from CH (4 wk, 0.5 atm) rats compared to normoxic controls, and this effect of CH was abolished by the general NOX inhibitor, apocynin. CH similarly augmented KCl-induced vasoconstriction and O(2)(-) generation (assessed using the fluorescent indicator, dihydroethidium) in Ca(2+)-permeabilized, pressurized small pulmonary arteries. These latter responses to CH were prevented by general inhibition of NOX isoforms (apocynin, diphenylene iodonium), and by selective inhibition of NOX 2 (gp91ds-tat), Rac1 (NSC 23766), and EGFR (AG 1478). Consistent with these observations, CH increased KCl-induced EGFR phosphorylation, and augmented depolarization-induced Rac1 activation in an EGFR-dependent manner. INNOVATION: This study establishes a novel signaling axis in VSM linking membrane depolarization to contraction that is independent of Ca(2+) influx, and which mediates myofilament Ca(2+) sensitization in the hypertensive pulmonary circulation. CONCLUSION: CH augments membrane depolarization-induced pulmonary VSM Ca(2+) sensitization and vasoconstriction through EGFR-dependent stimulation of Rac1 and NOX 2.

Chronic hypoxia upregulates pulmonary arterial ASIC1: a novel mechanism of enhanced store-operated Ca2+ entry and receptor-dependent vasoconstriction.

Acid-sensing ion channel 1 (ASIC1) is a newly characterized contributor to store-operated Ca(2+) entry (SOCE) in pulmonary vascular smooth muscle (VSM). Since SOCE is implicated in elevated basal VSM intracellular Ca(2+) concentration ([Ca(2+)](i)) and augmented vasoconstriction in chronic hypoxia (CH)-induced pulmonary hypertension, we hypothesized that ASIC1 contributes to these responses. To test this hypothesis, we examined effects of the specific pharmacologic ASIC1a inhibitor, psalmotoxin 1 (PcTX1), on vasoconstrictor and vessel wall [Ca(2+)](i) responses to UTP and KCl (depolarizing stimulus) in fura-2-loaded, pressurized small pulmonary arteries from control and CH (4 wk at 0.5 atm) Wistar rats. PcTX1 had no effect on basal vessel wall [Ca(2+)](i), but attenuated vasoconstriction and increases in vessel wall [Ca(2+)](i) to UTP in arteries from control and CH rats; normalizing responses between groups. In contrast, responses to the depolarizing stimulus, KCl, were unaffected by CH exposure or PcTX1. Upon examining potential Ca(2+) influx mechanisms, we found that PcTX1 prevented augmented SOCE following CH. Exposure to CH resulted in a significant increase in pulmonary arterial ASIC1 protein. This study supports a novel role of ASIC1 in elevated receptor-stimulated vasoconstriction following CH which is likely mediated through increased ASIC1 expression and SOCE.

Altered membrane lipid domains limit pulmonary endothelial calcium entry following chronic hypoxia.

Agonist-induced Ca(2+) entry into the pulmonary endothelium depends on activation of both store-operated Ca(2+) (SOC) entry and receptor-operated Ca(2+) (ROC) entry. We previously reported that pulmonary endothelial cell SOC entry and ROC entry are reduced in chronic hypoxia (CH)-induced pulmonary hypertension. We hypothesized that diminished endothelial Ca(2+) entry following CH is due to derangement of caveolin-1 (cav-1) containing cholesterol-enriched membrane domains important in agonist-induced Ca(2+) entry. To test this hypothesis, we measured Ca(2+) influx by fura-2 fluorescence following application of ATP (20 µM) in freshly isolated endothelial cells pretreated with the caveolar-disrupting agent methyl-ß-cyclodextrin (mßCD; 10 mM). Cholesterol depletion with mßCD attenuated agonist-induced Ca(2+) entry in control endothelial cells to the level of that from CH rats. Interestingly, endothelial membrane cholesterol was lower in cells isolated from CH rats compared with controls although the density of caveolae did not differ between groups. Cholesterol repletion with a cholesterol:mßCD mixture or the introduction of the cav-1 scaffolding peptide (AP-cav; 10 µM) rescued ATP-induced Ca(2+) entry in endothelia from CH arteries. Agonist-induced Ca(2+) entry assessed by Mn(2+) quenching of fura-2 fluorescence was also significantly elevated by luminal AP-cav in pressurized intrapulmonary arteries from CH rats to levels of controls. Similarly, patch-clamp experiments revealed diminished inward current in response to ATP in cells from CH rats compared with controls that was restored by AP-cav. These data suggest that CH-induced pulmonary hypertension leads to reduced membrane cholesterol that limits the activity of ion channels necessary for agonist-activated Ca(2+) entry.

Altered protein kinase C regulation of pulmonary endothelial store- and receptor-operated Ca2+ entry after chronic hypoxia.

Chronic hypoxia (CH)-induced pulmonary hypertension is associated with decreased basal pulmonary artery endothelial cell (EC) Ca(2+), which correlates with reduced store-operated Ca(2+) (SOC) entry. Protein kinase C (PKC) attenuates SOC entry in ECs. Therefore, we hypothesized that PKC has a greater inhibitory effect on EC SOC and receptor-operated Ca(2+) entry after CH. To test this hypothesis, we assessed SOC in the presence or absence of the nonselective PKC inhibitor GF109203X [2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)maleimide] in freshly isolated, Fura-2-loaded ECs obtained from intrapulmonary arteries of control and CH rats (4 weeks at 0.5 atm). We found that SOC entry and 1-oleoyl-2-acetyl-sn-glycerol (OAG)- and ATP-induced Ca(2+) influx were attenuated in ECs from CH rats versus controls, and GF109203X restored SOC and OAG responses to the level of controls. In contrast, nonselective PKC inhibition with GF109203X or the selective PKC(epsilon) inhibitor myristoylated V1-2 attenuated ATP-induced Ca(2+) entry in ECs from control but not CH pulmonary arteries. ATP-induced Ca(2+) entry was also attenuated by the T-type voltage-gated Ca(2+) channel (VGCC) inhibitor mibefradil in control cells. Consistent with the presence of endothelial T-type VGCC, we observed depolarization-induced Ca(2+) influx in control cells that was inhibited by mibefradil. This response was largely absent in ECs from CH arteries. We conclude that CH enhances PKC-dependent inhibition of SOC- and OAG-induced Ca(2+) entry. Furthermore, these data suggest that CH may reduce the ATP-dependent Ca(2+) entry that is mediated, in part, by PKCepsilon and mibefradil-sensitive Ca(2+) channels in control cells.

Regulatory role of G protein-coupled estrogen receptor for vascular function and obesity.

We found that the selective stimulation of the intracellular, transmembrane G protein-coupled estrogen receptor (GPER), also known as GPR30, acutely lowers blood pressure after infusion in normotensive rats and dilates both rodent and human arterial blood vessels. Stimulation of GPER blocks vasoconstrictor-induced changes in intracellular calcium concentrations and vascular tone, as well as serum-stimulated cell proliferation of human vascular smooth muscle cells. Deletion of the GPER gene in mice abrogates vascular effects of GPER activation and is associated with visceral obesity. These findings suggest novel roles for GPER in protecting from cardiovascular disease and obesity.

Gender differences in mesenteric vasoconstrictor reactivity following chronic hypoxia.

Male rats demonstrate persistent endothelium-dependent attenuation of vasoconstrictor reactivity following chronic hypoxia (CH). Since estrogen may interfere with hypoxia-induced gene expression, we hypothesized that gender differences exist in this response to CH. However, in conscious, instrumented rats, we found that CH resulted in a similar persistent reduction of pressor/total peripheral resistance responses to phenylephrine (PE) in rats of both genders. In contrast, although previous studies show mesenteric vascular responses to PE are reduced in CH males, we found that mesenteric reactivity was maintained in CH females. Since normoxic females demonstrate greater nitric oxide (NO) production, we hypothesized that the failure of CH to further diminish mesenteric reactivity in females was due to the inhibition of NO-dependent vasodilation. To test this hypothesis, constrictor reactivity of mesenteric arteries from male and female rats was examined. NO synthase (NOS) inhibition augmented constrictor responses to PE in arteries from both normoxic and CH males and normoxic females. In contrast, NOS inhibition had no effect in CH female vessels. Endothelial NOS (eNOS) levels were not different in arteries from control and CH females. Endothelial [Ca2+]i was greater in arterioles from CH females. Thus, CH reduces NO-dependent mesenteric dilation in females; this effect is not due to altered eNOS levels or diminished endothelial [Ca2+]i.

Maturational changes in the regulation of pulmonary vascular tone by nitric oxide in neonatal rats.

Nitric oxide (NO) is an important regulator of vasomotor tone in the pulmonary circulation. We tested the hypothesis that the role NO plays in regulating vascular tone changes during early postnatal development. Isolated, perfused lungs from 7- and 14-day-old Sprague-Dawley rats were studied. Baseline total pulmonary vascular resistance (PVR) was not different between age groups. The addition of KCl to the perfusate caused a concentration-dependent increase in PVR that did not differ between age groups. However, the nitric oxide synthase (NOS) inhibitor N(omega)-nitro-L-arginine augmented the K(+)-induced increase in PVR in both groups, and the effect was greater in lungs from 14-day-old rats vs. 7-day-old rats. Lung levels of total endothelial, inducible, and neuronal NOS proteins were not different between groups; however, the production rate of exhaled NO was greater in lungs from 14-day-old rats compared with those of 7-day-old rats. Vasodilation to 0.1 microM of the NO donor spermine NONOate was greater in 14-day lungs than in 7-day lungs, and lung levels of both soluble guanylyl cyclase and cGMP were greater at 14 days than at 7 days. Vasodilation to 100 microM of the cGMP analog 8-(4-chlorophenylthio)guanosine-3',5'-cyclic monophosphate was greater in 7-day lungs than in 14-day lungs. Our results demonstrate that the pulmonary vascular bed depends more on NO production to modulate vascular tone at 14 days than at 7 days of age. The observed differences in NO sensitivity may be due to maturational increases in soluble guanylyl cyclase protein levels.

Impaired NO-dependent inhibition of store- and receptor-operated calcium entry in pulmonary vascular smooth muscle after chronic hypoxia.

We have recently demonstrated that chronic hypoxia (CH) attenuates nitric oxide (NO)-mediated decreases in pulmonary vascular smooth muscle (VSM) intracellular free calcium concentration ([Ca2+]i) and promotes NO-dependent VSM Ca2+ desensitization. The objective of the current study was to identify potential mechanisms by which CH interferes with regulation of [Ca2+]i by NO. We hypothesized that CH impairs NO-mediated inhibition of store-operated (capacitative) Ca2+ entry (SOCE) or receptor-operated Ca2+ entry (ROCE) in pulmonary VSM. To test this hypothesis, we examined effects of the NO donor, spermine NONOate, on SOCE resulting from depletion of intracellular Ca2+ stores with cyclopiazonic acid, and on UTP-induced ROCE in isolated, endothelium-denuded, pressurized pulmonary arteries (213 +/- 8 microm inner diameter) from control and CH (4 wk at 0.5 atm) rats. Arteries were loaded with fura-2 AM to continuously monitor VSM [Ca2+]i. We found that the change in [Ca2+]i associated with SOCE and ROCE was significantly reduced in vessels from CH animals. Furthermore, spermine NONOate diminished SOCE and ROCE in vessels from control, but not CH animals. We conclude that NO-mediated inhibition of SOCE and ROCE is impaired after CH-induced pulmonary hypertension.

Intratracheal adenoviral-mediated delivery of iNOS decreases pulmonary vasoconstrictor responses in rats.

We hypothesized that adenovirus-mediated inducible nitric oxide synthase (iNOS) gene transduction of the lung would result in time-dependent iNOS overexpression and attenuate the vascular constrictor responses to a thromboxane mimetic, U-46619. Rats were treated via the trachea with surfactant alone (sham), surfactant containing an adenoviral construct with a cytomegalovirus promoter-regulated human iNOS gene (Adeno-iNOS), or an adenoviral construct without a gene insert (Adeno-Control). Adeno-iNOS-transduced rats demonstrated human iNOS mRNA and increased iNOS protein levels only in the lungs. Immunohistochemistry of lungs from Adeno-iNOS-treated animals demonstrated transgene expression in alveolar wall cells. In the lungs from Adeno-iNOS-transduced rats, the expression of iNOS protein and exhaled nitric oxide concentrations were increased on days 1-4 and 7 but returned to baseline values by day 14. The administration of the selective iNOS inhibitor L-N6-(1-iminoethyl)lysine dihydrochloride (L-NIL) decreased exhaled nitric oxide concentrations to levels found in Adeno-Control-transduced lungs. In a second group of rats, the segmental vasoconstrictor responses to U-46619 were determined in isolated, perfused lungs 3 days after transduction. Lungs from rats transduced with Adeno-iNOS had reduced total, arterial, and venous vasoconstrictor responses to U-46619 compared with sham, Adeno-Control, and control groups. In a third set of experiments, the response to 400 nM U-46619 in the presence of 10 microM L-NIL was not different in the isolated lungs from Adeno-Control- and Adeno-iNOS-transduced rats. We conclude that adenovirus-mediated iNOS gene transduction of the lung results in time-dependent iNOS overexpression, which attenuates the vascular constrictor responses to the thromboxane mimetic U-46619.

17-beta estradiol attenuates hypoxic induction of HIF-1alpha and erythropoietin in Hep3B cells.

Hypoxia inducible factor-1 (HIF-1) is a heterodimeric transcription factor that regulates expression of several hypoxia-inducible genes, including erythropoietin (EPO), by binding to hypoxia response elements (HREs) in their promoters/enhancers. Previously, we have shown that 17-beta estradiol (E2-beta) attenuates hypoxic induction of EPO in rats. We hypothesized that this response is mediated by E2-beta-induced attenuation of HIF-1alpha activity/expression. To test this hypothesis, we performed reporter gene assays in Hep3B cells to assess E2-beta effects on hypoxia-induced activity of a reporter gene driven by the HRE from a cloned EPO-enhancer element. Immunocytochemistry and Western blots were additionally used to determine effects of E2-beta on hypoxic increases in HIF-1alpha and EPO immunoreactivity. Finally, we examined potential influences of E2-beta on HIF-1alpha mRNA levels by real-time PCR. Consistent with our hypothesis, E2-beta (100 pM) inhibited hypoxic increases in HRE-mediated reporter gene activity. Furthermore, the estrogen-receptor antagonist ICI 182,780 (25 microM) eliminated these inhibitory effects of E2-beta. E2-beta similarly attenuated hypoxic induction of both EPO and HIF-1alpha protein in an estrogen-receptor dependent manner, but was without effect on HIF-1alpha mRNA expression. These findings suggest a role for E2-beta to attenuate EPO expression by interfering with hypoxic increases in HIF-1alpha protein through an estrogen receptor-dependent mechanism.

17-beta estradiol independently regulates erythropoietin synthesis and NOS activity during hypoxia.

We reported previously that 17-beta estradiol (E2-beta) attenuates hypoxic induction of erythropoietin (EPO) synthesis in rats. We hypothesized this attenuation is mediated by increased nitric oxide (NO) bio-availability. To investigate this hypothesis, ovariectomized estrogen-depleted rats were instrumented with arterial and venous catheters and treated with either E2-beta (20 microg/24 hrs) or vehicle (polypropylene glycol) for 7 days. Rats were placed in Plexiglas boxes and administered a bolus of either the NO synthase inhibitor, Nomega-nitro-L-arginine (l-NNA, 15 mg/kg) or saline. Following this bolus, saline or l-NNA was continuously infused (15 mg/kg/h) throughout the 8 hours of hypoxic exposure (12% O2). Hypoxia increased plasma NO metabolites (NOx) in both saline groups but more in E2-beta-treated rats. l-NNA prevented this increase in both groups. Renal endothelial NO synthase (NOS) expression was unaltered by hypoxia, l-NNA, or E2-beta. Despite preventing increases in plasma NOx during hypoxia, l-NNA did not affect E2-beta attenuation of EPO synthesis. We conclude that E2-beta independently attenuates hypoxic induction of EPO and augments hypoxic increases in NO synthesis.

Pulmonary PKG-1 is upregulated following chronic hypoxia.

Recent studies from our laboratory indicate that pulmonary vasodilatory responses to exogenous nitric oxide (NO) are attenuated following chronic hypoxia (CH) and that this NO-dependent vasodilation is mediated by cGMP. Similarly, we have demonstrated that CH attenuates vasodilatory responses to the cGMP analog 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP). We hypothesized that attenuated pulmonary vasodilation to 8-BrcGMP following CH is mediated by decreased protein kinase G-1 (PKG-1) expression/activity. Therefore, we examined vasodilatory responses to 8-BrcGMP (1 microM) in isolated, saline-perfused lungs from control and CH (4 wk at barometric pressure of 380 mmHg) rats in the presence of the competitive PKG inhibitor Rp-beta-phenyl-1, N2-etheno-8-bromoguanosine 3',5'-cyclic monophosphorothionate (30 microM) or the highly specific PKG inhibitor KT-5823 (10 microM). PKG-1 expression and activity were determined in whole lung homogenates from each group, and vascular PKG-1 levels were assessed by quantitative immunohistochemistry. PKG inhibition with either Rp-8-Br-PET-cGMPS or KT-5823 diminished vasodilatory responses to 8-BrcGMP in lungs from both control and CH rats, thus indicating a role for PKG in mediating reactivity to 8-BrcGMP in each group. However, in contrast to our hypothesis, PKG-1 levels were approximately twofold greater in lungs from CH rats vs. controls, and furthermore, this upregulation was localized to the vasculature. This correlates with an increase in PKG activity following CH. We conclude that PKG-1 is involved in 8-BrcGMP-mediated vasodilation; however, attenuated pulmonary vasodilation following CH is not associated with decreased expression/activity of PKG-1.

17Beta-estradiol decreases hypoxic induction of erythropoietin gene expression.

Exposure to chronic hypoxia induces erythropoietin (EPO) production to facilitate oxygen delivery to hypoxic tissues. Previous studies from our laboratory found that ovariectomy (OVX) exacerbates the polycythemic response to hypoxia and treatment with 17beta-estradiol (E2-beta) inhibits this effect. We hypothesized that E2-beta decreases EPO gene expression during hypoxia. Because E2-beta can induce nitric oxide (NO) production and NO can attenuate EPO synthesis, we further hypothesized that E2-beta inhibition of EPO gene expression is mediated by NO. These hypotheses were tested in OVX catheterized rats treated with E2-beta (20 microg/day) or vehicle for 14 days and exposed to 8 or 12 h of hypoxia (12% O(2)) or normoxia. We found that E2-beta treatment significantly decreased EPO synthesis and gene expression during hypoxia. E2-beta treatment did not induce endothelial NO synthase (eNOS) expression in the kidney but potentiated hypoxia-induced increases in plasma nitrates. We conclude that E2-beta decreases hypoxic induction of EPO. However, this effect does not appear to be related to changes in renal eNOS expression.

Estradiol attenuates hypoxia-induced pulmonary endothelin-1 gene expression.

The ovarian hormone 17beta-estradiol (E2beta) attenuates chronic hypoxia-induced pulmonary hypertension. We hypothesized that E2beta attenuates this response to hypoxia by decreasing pulmonary expression of the vasoactive and mitogenic peptide endothelin-1 (ET-1). To test this hypothesis, we measured preproET-1 mRNA and ET-1 peptide levels in the lungs of adult female normoxic and hypoxic (24 h or 4 wk at barometric pressure = 380 mmHg) rats with intact ovaries and in hypoxic ovariectomized (OVX) rats administered E2beta or vehicle via subcutaneous osmotic pumps. Hypoxic exposure increased lung preproET-1 mRNA levels in OVX vehicle-treated rats, but not in rats with intact ovaries. In addition, E2beta replacement prevented hypoxia-mediated increases in preproET-1 mRNA and ET-1 peptide expression. Considering that hypoxic induction of ET-1 gene expression is mediated by a hypoxia-inducible transcription factor(s) (HIF), we further hypothesized that E2beta-induced attenuation of pulmonary ET-1 expression during hypoxia results from decreased HIF activity. We found that E2beta abolished HIF-dependent increases in reporter gene activity. Further experiments demonstrated that overexpression of the transcriptional coactivator cAMP response element binding protein (CREB) binding protein (CBP)/p300, a factor common to both the estrogen receptor and HIF pathways, eliminated E2beta-mediated attenuation of hypoxia-induced ET-1 promoter activity. We conclude that E2beta inhibits hypoxic induction of ET-1 gene expression by interfering with HIF activity, possibly through competition for limiting quantities of CBP/p300.