sq-23377 and Hypertension--Pulmonary

The nitric oxide/soluble guanylyl cyclase (sGC) signal transduction pathway plays an important role in smooth muscle relaxation and phenotypic regulation. However, the transcriptional regulation of sGC gene expression is largely unknown. It has been shown that sGC expression increases in pulmonary arteries from chronic hypoxia-induced pulmonary hypertensive animals. Since the transcription factor NFATc3 is required for the upregulation of the smooth muscle hypertrophic/differentiation marker alpha-actin in pulmonary artery smooth muscle cells from chronically hypoxic mice, we hypothesized that NFATc3 is required for the regulation of sGC-alpha1 expression during chronic hypoxia. Exposure to chronic hypoxia for 2 days induced a decrease in sGC-alpha1 expression in mouse pulmonary arteries. This reduction was independent of NFATc3 but mediated by nuclear accumulation of the mRNA-stabilizing protein human antigen R (HuR). Consistent with our hypothesis, chronic hypoxia (21 days) upregulated pulmonary artery sGC-alpha1 expression, bringing it back to the level of the normoxic controls. This response was prevented in NFATc3 knockout and cyclosporin (calcineurin/NFATc inhibitor)-treated mice. Furthermore, we identified effective binding sites for NFATc in the mouse sGC-alpha1 promoter. Activation of NFATc3 increased sGC-alpha1 promoter activity in human embryonic derived kidney cells, rat aortic-derived smooth muscle cells, and human pulmonary artery smooth muscle cells. Our results suggest that NFATc3 and HuR are important regulators of sGC-alpha1 expression in pulmonary vascular smooth muscle cells during chronic hypoxia-induced pulmonary hypertension.

Topics: Animals; Antigens, Surface; Base Sequence; Binding Sites; Calcineurin; Cell Line; Cell Nucleus; Chronic Disease; ELAV Proteins; ELAV-Like Protein 1; Gene Deletion; Guanylate Cyclase; Humans; Hypertension, Pulmonary; Hypoxia; Ionomycin; Male; Mice; NFATC Transcription Factors; Point Mutation; Promoter Regions, Genetic; Protein Binding; Protein Isoforms; Pulmonary Artery; Rats; Receptors, Cytoplasmic and Nuclear; RNA-Binding Proteins; Soluble Guanylyl Cyclase; Transfection

Intermittent hypoxia (IH) resulting from sleep apnea can lead to pulmonary hypertension (PH) and right heart failure, similar to chronic sustained hypoxia (CH). Supplemental CO(2), however, attenuates hypoxic PH. We therefore hypothesized that, similar to CH, IH elicits PH and associated increases in arterial endothelial nitric oxide synthase (eNOS) expression, ionomycin-dependent vasodilation, and receptor-mediated pulmonary vasoconstriction. We further hypothesized that supplemental CO(2) inhibits these responses to IH. To test these hypotheses, we measured eNOS expression by Western blot in intrapulmonary arteries from CH (2 wk, 0.5 atm), hypocapnic IH (H-IH) (3 min cycles of 5% O(2)/air flush, 7 h/day, 2 wk), and eucapnic IH (E-IH) (3 min cycles of 5% O(2), 5% CO(2)/air flush, 7 h/day, 2 wk) rats and their respective controls. Furthermore, vasodilatory responses to the calcium ionophore ionomycin and vasoconstrictor responses to the thromboxane mimetic U-46619 were measured in isolated saline-perfused lungs from each group. Hematocrit, arterial wall thickness, and right ventricle-to-total ventricle weight ratios were additionally assessed as indexes of polycythemia, arterial remodeling, and PH, respectively. Consistent with our hypotheses, E-IH resulted in attenuated polycythemia, arterial remodeling, RV hypertrophy, and eNOS upregulation compared with H-IH. However, in contrast to CH, neither H-IH nor E-IH increased ionomycin-dependent vasodilation. Furthermore, H-IH and E-IH similarly augmented U-46619-induced pulmonary vasoconstriction but to a lesser degree than CH. We conclude that maintenance of eucapnia decreases IH-induced PH and upregulation of arterial eNOS. In contrast, increases in pulmonary vasoconstrictor reactivity following H-IH are unaltered by exposure to supplemental CO(2).

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Carbon Dioxide; Chronic Disease; Disease Models, Animal; Dose-Response Relationship, Drug; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypocapnia; Hypoxia; Ionomycin; Ionophores; Male; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Oxygen; Polycythemia; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Vasoconstriction; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents

Female rats develop less severe pulmonary hypertension (PH) in response to chronic hypoxia compared with males, thus implicating a potential role for ovarian hormones in mediating this gender difference. Considering that estrogen upregulates endothelial nitric oxide (NO) synthase (eNOS) in systemic vascular tissue, we hypothesized that estrogen inhibits hypoxic PH by increasing eNOS expression and activity. To test this hypothesis, we examined responses to the endothelium-derived NO-dependent dilator ionomycin and the NO donors S-nitroso-N-acetylpenicillamine and spermine NONOate in U-46619-constricted, isolated, saline-perfused lungs from the following groups: 1) normoxic rats with intact ovaries, 2) chronic hypoxic (CH) rats with intact ovaries, 3) CH ovariectomized rats given 17 beta-estradiol (E(2)beta), and 4) CH ovariectomized rats given vehicle. Additional experiments assessed pulmonary eNOS levels in each group by Western blotting. Our findings indicate that E(2)beta attenuated chronic hypoxia-induced right ventricular hypertrophy, pulmonary arterial remodeling, and polycythemia. Furthermore, although CH augmented vasodilatory responsiveness to ionomycin and increased pulmonary eNOS expression, these responses were not potentiated by E(2)beta. Finally, responses to S-nitroso-N-acetylpenicillamine and spermine NONOate were similarly attenuated in all CH groups compared with normoxic control groups. We conclude that the inhibitory influence of E(2)beta on chronic hypoxia-induced PH is not associated with increased eNOS expression or activity.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Chronic Disease; Endothelium, Vascular; Enzyme Inhibitors; Estradiol; Female; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Ionomycin; Ionophores; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Nitroarginine; Nitrogen Oxides; Ovariectomy; Penicillamine; Polycythemia; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; Spermine; Vascular Resistance; Vasoconstrictor Agents; Vasodilation

Pulmonary hypertension is characterized by increased vascular resistance due to smooth muscle cell hyper-activity and excess deposition of extracellular matrix (ECM) in the vessel wall. We investigated the possibility that changes in cell-ECM interactions may play an active role in this process by modifying the contractile response of pulmonary vascular smooth muscle (PVSM) cells. Contractility was measured within individual cultured PVSM cells, when resting or stimulated with vasoactive agents, by quantitating changes in stiffness of the cytoskeleton (CSK) using magnetic twisting cytometry (N. Wang, J. P. Butler, and D. E. Ingber. Science 260: 1124-1127, 1993). Control studies confirmed that changes in CSK stiffness closely paralleled alterations in cell contraction and relaxation as measured in response to endothelin-1 (ET-1) and dibutyryl guanosine 3',5'-cyclic monophosphate (cGMP), respectively, in a collagen gel contraction assay. CSK stiffness and contractile tone in cultured PVSM cells increased in direct proportion as the density of fibronectin (FN) coating was raised from 10 to 500 ng/well in 96-well plates. Dibutyryl cGMP had no effect in cells on low FN, although it completely inhibited the FN-dependent increase in CSK stiffness on higher ECM densities. In contrast, ET-1 induced the greatest increase in CSK stiffness on the intermediate FN density (100 ng/well). The reduced sensitivity to ET-1 on high FN was not due to dysfunction of the contractile apparatus nor to changes in protein tyrosine phosphorylation. Taken together, these results show that ECM can modulate PVSM cell contractility and suggest that the changes in ECM observed in hypertensive vessels could play an important role in the etiology of this disease.

Topics: Animals; Animals, Newborn; Cattle; Cells, Cultured; Collagen; Cytoskeleton; Dibutyryl Cyclic GMP; Endothelin-1; Extracellular Matrix; Fibronectins; Hypertension, Pulmonary; Ionomycin; Isometric Contraction; Kinetics; Muscle, Smooth, Vascular; Phosphoproteins; Phosphorylation; Phosphotyrosine; Pulmonary Artery; Stress, Mechanical

We have previously demonstrated that arterial, but not venous, vasodilatory responses to endothelium-derived nitric oxide (EDNO)-dependent agonists are enhanced in lungs isolated from rats with chronic hypoxia (CH)-induced pulmonary arterial hypertension. These data suggest that CH is associated with increased endothelial nitric oxide synthase (eNOS) activity within the pulmonary arterial vasculature. In addition, the correlation of increased pulmonary arterial pressure with selectively enhanced arterial responsiveness to EDNO-mediated agonists suggests that arterial hypertension, rather than hypoxia per se, is a contributing factor in this response. Therefore, we hypothesized that 1) CH selectively upregulates eNOS within the pulmonary arterial vasculature and 2) monocrotaline (MC)-induced pulmonary arterial hypertension selectively enhances pulmonary arterial dilation to EDNO-dependent dilators and upregulates arterial eNOS. We examined the responses to the EDNO-dependent dilators arginine vasopressin and ionomycin in U-46619-constricted isolated perfused lungs from control and MC-treated rats. Microvascular pressure was assessed by the double-occlusion technique, allowing calculation of segmental resistances. Lungs from MC-treated rats exhibited augmented arterial dilation to arginine vasopressin compared with control lungs. However, the responses to ionomycin were not different between the two groups. Quantitative immunocytochemistry was used to compare pulmonary eNOS immunoreactivity in vessels from control, CH, and MC-treated rats. eNOS staining was more intense in the arteries of CH and MC-treated rats compared with those of control animals, whereas CH and MC treatment had no effect on eNOS staining in veins. We conclude that pulmonary arterial hypertension, or altered vascular mechanical forces associated with hypertension, may be responsible for the augmented EDNO-dependent arterial dilation and upregulation of arterial eNOS in lungs from CH and MC-treated rats.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Arginine Vasopressin; Blood Pressure; Chronic Disease; Endothelium, Vascular; Hypertension, Pulmonary; Hypoxia; In Vitro Techniques; Ionomycin; Lung; Male; Monocrotaline; Nitric Oxide Synthase; Prostaglandin Endoperoxides, Synthetic; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Thromboxane A2; Vascular Resistance; Vasoconstrictor Agents

1. The effect of the nitric oxide synthase inhibitor, NG-nitro-L-arginine (L-NOARG), on endothelium-dependent relaxation to a receptor-independent agent, ionomycin, was examined in isolated pulmonary arteries and veins from control, short-term and chronic pulmonary hypertensive sheep. All vessel segments were contracted to optimal levels of active force with endothelin-1 to record endothelium-dependent relaxation. 2. Pulmonary hypertension was induced by continuous pulmonary artery air embolization for 1 day (short-term) and 14 days (chronic) and was associated with a 2 and 3 fold increase in pulmonary vascular resistance respectively. 3. L-NOARG (0.1 mM) reduced the maximum relaxation (Rmax) to ionomycin in large and medium-sized pulmonary arteries from control sheep by approximately 70%. By contrast, L-NOARG (0.1 mM) did not inhibit the Rmax to ionomycin in matched vessels from short-term and chronic pulmonary hypertensive sheep. 4. Resistance of ionomycin-induced relaxations to inhibition by L-NOARG, was confined to the arterial vasculature in chronic pulmonary hypertensive animals, as relaxations to ionomycin in large and medium-sized chronic pulmonary hypertensive veins were, like those in control veins, abolished by L-NOARG. Both large and medium-sized pulmonary veins from short-term pulmonary hypertensive sheep, however, were resistant to block by L-NOARG. 5. Neither sensitivity (pEC50) nor Rmax to ionomycin in large, short-term pulmonary hypertensive arteries was affected when the extracellular concentration of K+ was increased isotonically to 30 mM. Nifedipine (0.3 microM) was present throughout to prevent high K(+)-induced smooth muscle contraction. In the presence of this high extracellular K+, however, L-NOARG (0.1 mM) caused complete inhibition of the relaxation to ionomycin, whereas in normal extracellular K+ (4.7 mM), L-NOARG only weakly inhibited ionomycin relaxations. 6. In conclusion, the onset of pulmonary hypertension in sheep following air embolization, is associated with the development of resistance of endothelium-dependent relaxations to block by L-NOARG. The mechanism of L-NOARG resistance appears to be due to the up-regulation of a K+ channel-mediated backup vasodilator mechanism which can compensate for the loss of nitric oxide (NO)-mediated relaxation. Although this mechanism remains functionally 'silent' in the presence of NO it is able to maintain adequate endothelium-dependent vasodilatation during pulmonary hypertension if

Topics: Animals; Arginine; Blood Gas Analysis; Endothelium, Vascular; Enzyme Inhibitors; Female; Hemodynamics; Hypertension, Pulmonary; In Vitro Techniques; Ionomycin; Ionophores; Muscle Relaxation; Muscle, Smooth, Vascular; Nitric Oxide Synthase; Nitroarginine; Nitroprusside; Potassium Channels; Potassium Chloride; Pulmonary Artery; Pulmonary Veins; Sheep; Up-Regulation; Vasodilator Agents