Chronic hypoxic decreases in soluble guanylate cyclase protein and enzyme activity are age dependent in fetal and adult ovine carotid arteries

The present study tests the hypothesis that chronic hypoxia enhances reactivity to nitric oxide (NO) through age-dependent increases in soluble guanylate cyclase (sGC) and protein kinase G (PKG) activity. In term fetal and adult ovine carotids, chronic hypoxia had no significant effect on mRNA levels for the β1-subunit of sGC, but depressed sGC abundance by 16% in fetal and 50% in adult arteries, through possible depression of rates of mRNA translation (15% in fetal and 50% in adult) and/or increased protein turnover. Chronic hypoxia also depressed the catalytic activity of sGC, but only in fetal arteries (63%). Total sGC activity was reduced by chronic hypoxia in both fetal (69%) and adult (37%) carotid homogenates, but this effect was not observed in intact arteries when sGC activity was measured by timed accumulation of cGMP. In intact arteries treated with 300 μM 3-isobutyl-1-methylxanthine (IBMX), chronic hypoxia dramatically enhanced sGC activity in fetal (186%) but not adult (89%) arteries. This latter observation suggests that homogenization either removed an sGC activator, released an sGC inhibitor, or altered the phosphorylation state of the enzyme, resulting in reduced activity. In the absence of IBMX, chronic hypoxia had no significant effect on rates of cGMP accumulation. Chronic hypoxia also depressed the ability of the cGMP analog, 8-( p-chlorophenylthio)-cGMP, to promote vasorelaxation in both fetal (8%) and adult (12%) arteries. Together, these results emphasize the fact that intact and homogenized artery studies of sGC activity do not always yield equivalent results. The results further suggest that enhancement of reactivity to NO by chronic hypoxia must occur upstream of PKG and can only be possible if changes in cGMP occurred in functional compartments that afforded either temporal or chemical protection to the actions of phosphodiesterase. The range and age dependence of hypoxic effects observed also suggest that some responses to hypoxia must be compensatory and homeostatic, with reactivity to NO as the primary regulated variable.

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Hypoxic depression of PKG-mediated inhibition of serotonergic contraction in ovine carotid arteries

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00212.2012 ◽

2013 ◽

Vol 304 (9) ◽

pp. R734-R743 ◽

Cited By ~ 5

Author(s):

Richard B. Thorpe ◽

Sara L. Stockman ◽

James M. Williams ◽

Thomas M. Lincoln ◽

William J. Pearce

Keyword(s):

Smooth Muscle ◽

Guanylate Cyclase ◽

Vascular Function ◽

Carotid Arteries ◽

Chronic Hypoxia ◽

Soluble Guanylate Cyclase ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Maternal Hypoxia ◽

Endothelial Nitric Oxide

Chronic hypoxia attenuates soluble guanylate cyclase-induced vasorelaxation in serotonin (5-HT)-contracted ovine carotid arteries. Because protein kinase G (PKG) mediates many effects of soluble guanylate cyclase activation through phosphorylation of multiple kinase targets in vascular smooth muscle, we tested the hypothesis that chronic hypoxia reduces the ability of PKG to phosphorylate its target proteins, which attenuates the ability of PKG to induce vasorelaxation. We also tested the hypothesis that hypoxia attenuates PKG expression and/or activity. Arteries from normoxic and chronically hypoxic (altitude of 3,820 m for 110 days) fetal and adult sheep were denuded of endothelium and equilibrated with 95% O2-5% CO2 in the presence of nitro-l-arginine methyl ester (l-NAME) and NG-nitro-l-arginine (l-NNA) to inhibit residual endothelial nitric oxide synthase. Concentration-response relations for 5-HT were determined in the presence of prazosin to minimize activation of α-adrenergic receptors. The PKG activator 8-( p-chlorophenylthio)-guanosine 3′,5′-cyclic monophosphate (8-pCTP-cGMP) reduced agonist binding affinity of the 5-HT receptor in a concentration-dependent manner that was attenuated by hypoxia. Expression and activity of PKG-I was not significantly affected by chronic hypoxia in either fetal or adult arteries, although PKG-I abundance was greater in fetal arteries. Pretreatment with the large conductance calcium-sensitive potassium channel (BK) inhibitor iberiotoxin attenuated the vasorelaxation induced by 8-pCPT-cGMP in normoxic but not chronically hypoxic arteries. These results support the hypothesis that hypoxia attenuates the vasorelaxant effects of PKG through suppression of the ability of PKG to activate large conductance calcium-sensitive potassium channels in arterial smooth muscle. The results also reveal that this hypoxic effect is greater in fetal than adult arteries and that chronic maternal hypoxia can profoundly affect fetal vascular function.

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Effects of chronic hypoxia on soluble guanylate cyclase activity in fetal and adult ovine cerebral arteries

Journal of Applied Physiology ◽

10.1152/japplphysiol.00233.2009 ◽

2009 ◽

Vol 107 (1) ◽

pp. 192-199 ◽

Cited By ~ 14

Author(s):

William J. Pearce ◽

James M. Williams ◽

Charles R. White ◽

Thomas M. Lincoln

Keyword(s):

Guanylate Cyclase ◽

Chronic Hypoxia ◽

Soluble Guanylate Cyclase ◽

Specific Activity ◽

Cerebral Arteries ◽

Cerebrovascular Reactivity ◽

Pulmonary Vasculature ◽

General Hypothesis ◽

Cgmp Analog ◽

Broad Variety

A broad variety of evidence obtained largely in pulmonary vasculature suggests that chronic hypoxia modulates vasoreactivity to nitric oxide (NO). The present study explores the general hypothesis that chronic hypoxia also modulates cerebrovascular reactivity to NO, and does so by modulating the activity of soluble guanylate cyclase (sGC), the primary target for NO in vascular smooth muscle. Pregnant and nonpregnant ewes were maintained at either sea level or at 3,820 m for the final 110 days of gestation, at which time middle cerebral arteries from term fetal lambs and nonpregnant adults were harvested. In both fetal and adult arteries, NO-induced vasodilatation was attenuated by chronic hypoxia and completely inhibited by 10 μM 1 H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a selective inhibitor of sGC. sGC abundance (in ng sGC/mg protein) measured via Western immunoblots was ∼10-fold greater in fetal (17.6 ± 1.6) than adult (1.7 ± 0.3) arteries but was not affected by chronic hypoxia. The specific activity of sGC (in pmol cGMP·μg sGC−1·min−1) was similar in fetal (255 ± 64) and adult (280 ± 75) arteries and was inhibited by chronic hypoxia in both fetal (120 ± 10) and adult (132 ± 26) arteries. Rates of cGMP degradation (in pmol cGMP·mg protein−1·min−1) were similar in fetal (159 ± 59) and adult (134 ± 36) arteries but were not significantly depressed by chronic hypoxia in either fetal (115 ± 25) or adult (108 ± 25) arteries. The cGMP analog 8-( p-chlorophenylthio)-cGMP was a more potent vasorelaxant in fetal (pD2 = 4.7 ± 0.1) than adult (pD2 = 4.3 ± 0.1) arteries, but its ability to promote vasodilatation was not affected by chronic hypoxia in either age group. Together, these results reveal that hypoxic inhibition of NO-induced vasodilatation is attributable largely to attenuation of the specific activity of sGC and does not involve significant changes in sGC abundance, cGMP-phosphodiesterase activity, or the vasorelaxant activity of protein kinase G.

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Nitric oxide decreases endothelin-1 secretion through the activation of soluble guanylate cyclase

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00224.2003 ◽

2004 ◽

Vol 286 (5) ◽

pp. L984-L991 ◽

Cited By ~ 57

Author(s):

Lisa K. Kelly ◽

Stephen Wedgwood ◽

Robin H. Steinhorn ◽

Stephen M. Black

Keyword(s):

Nitric Oxide ◽

Guanylate Cyclase ◽

Soluble Guanylate Cyclase ◽

Primary Cultures ◽

No Donor ◽

Endothelin 1 ◽

Cgmp Analog ◽

Pulmonary Arterial ◽

Long Acting ◽

Arterial Endothelial Cells

The use of exogenous nitric oxide (NO) has been shown to alter the regulation of other endothelially derived mediators of vascular tone, such as endothelin-1 (ET-1). However, the interaction between NO and ET-1 appears to be complex and remains incompletely understood. One of the major actions of NO is the activation of soluble guanylate cyclase (sGC) with the subsequent generation of cGMP. Therefore, we undertook this study to test the hypothesis that NO regulates ET-1 production via the activation of the sGC/cGMP pathway. The results obtained indicated that the exposure of primary cultures of 4-wk-old ovine pulmonary arterial endothelial cells (4-wk PAECs) to the long-acting NO donor DETA NONOate induced both a dose- and time-dependent decrease in secreted ET-1. This decrease in ET-1 secretion occurred in the absence of changes in endothelin-converting enzyme-1 or sGC expression but in conjunction with a decrease in prepro-ET-1 mRNA. The changes in ET-1 release were inversely proportional to the cellular cGMP content. Furthermore, the NO-independent activator of sGC, YC-1, or treatment with a cGMP analog also produced significant decreases in ET-1 secretion. Conversely, pretreatment with the sGC inhibitor ODQ blocked the NO-induced decrease in ET-1. Therefore, we conclude that exposure of 4-wk PAECs to exogenous NO decreases secreted ET-1 resulting from the activation of sGC and increased cGMP generation.

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Pulmonary soluble guanylate cyclase, a nitric oxide receptor, is increased during the perinatal period

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1997.272.3.l400 ◽

1997 ◽

Vol 272 (3) ◽

pp. L400-L406 ◽

Cited By ~ 26

Author(s):

K. D. Bloch ◽

G. Filippov ◽

L. S. Sanchez ◽

M. Nakane ◽

S. M. de la Monte

Keyword(s):

Nitric Oxide ◽

Smooth Muscle ◽

Smooth Muscle Cells ◽

Guanylate Cyclase ◽

Soluble Guanylate Cyclase ◽

Muscle Cells ◽

Perinatal Period ◽

Mrna Levels ◽

Old Rats ◽

Beta1 Subunit

Nitric oxide (NO) has an important role in the pulmonary vasodilatation associated with the transition from fetal to neonatal life. NO activates pulmonary soluble guanylate cyclase (sGC), an obligate heterodimer composed of alpha1- and beta1-subunits, increasing synthesis of guanosine 3',5'-cyclic monophosphate (cGMP) and leading to vasodilation. In this study, regulation of sGC subunit expression during pulmonary development was examined. RNA blot hybridization revealed abundant alpha1- and beta1-subunit mRNA in lungs of late-gestation fetal and neonatal Sprague-Dawley rats, with markedly reduced levels detected in adult lungs. Pulmonary sGC enzyme activity in the presence of 1 mM sodium nitroprusside, a NO-donor compound, was approximately sevenfold greater in 1- and 8-day-old rats than in adult rats (P < 0.03). With the use of immunoblot techniques, pulmonary alpha1-subunit concentrations closely correlated with mRNA levels. With in situ hybridization, alpha1- and beta1-subunit mRNAs were readily detected in pulmonary vascular and bronchial smooth muscle cells as well as alveolar and serosal epithelial cells in lungs of 1-day-old rats. In adult lungs, sGC subunit mRNAs were present at low levels and were found nearly exclusively in bronchial and vascular smooth muscle cells. These results demonstrate that abundant pulmonary sGC is available to respond to the increased NO produced during the perinatal period. High-level expression of sGC subunit genes outside the vasculature of lungs of 1-day-old rats suggests an important role for NO-cGMP signal transduction in the perinatal regulation of pulmonary epithelial function and bronchial tone.

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Pharmacological stimulation of soluble guanylate cyclase modulates hypoxia-inducible factor-1α in rat heart

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00503.2009 ◽

2009 ◽

Vol 297 (4) ◽

pp. H1274-H1280 ◽

Cited By ~ 4

Author(s):

Toshihiro Tsuruda ◽

Kinta Hatakeyama ◽

Hiroyuki Masuyama ◽

Yoko Sekita ◽

Takuroh Imamura ◽

...

Keyword(s):

Guanylate Cyclase ◽

Soluble Guanylate Cyclase ◽

Mechanical Load ◽

Hypoxia Inducible Factor ◽

Pressure Overload ◽

Hypoxia Inducible Factor 1Α ◽

Hypoxic Conditions ◽

Cgmp Analog ◽

Male Wistar Rats ◽

Cultured Cardiomyocytes

Mechanical load and ischemia induce a series of adaptive physiological responses by activating the expression of O2-regulated genes, such as hypoxia inducible factor-1α (HIF-1α). The aim of this study was to explore the interaction between HIF-1α and soluble guanylate cyclase (sGC) and its second messenger cGMP in cultured cardiomyocytes exposed to hypoxia and in pressure-overloaded heart. In cultured cardiomyocytes of neonatal rats, either sGC stimulator BAY 41-2272 or cGMP analog 8-bromo-cGMP decreased the hypoxia (1% O2/5% CO2)-induced HIF-1α expression, whereas the inhibition of protein kinase G by KT-5823 reversed the effect of BAY 41-2272 on the expression under hypoxic conditions. In pressure-overloaded heart induced by suprarenal aortic constriction (AC) in 7-wk-old male Wistar rats, the administration of BAY 41-2272 (2 mg·kg−1·day−1) for 14 days significantly suppressed the protein expression of HIF-1α ( P < 0.05), vascular endothelial growth factor ( P < 0.01), and the number of capillary vessels ( P < 0.01) induced by pressure overload. This study suggests that the pharmacological sGC-cGMP stimulation modulates the HIF-1α expression in response to hypoxia or mechanical load in the heart.

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The soluble guanylate cyclase activator HMR1766 reverses hypoxia-induced experimental pulmonary hypertension in mice

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00189.2009 ◽

2009 ◽

Vol 297 (4) ◽

pp. L658-L665 ◽

Cited By ~ 26

Author(s):

Norbert Weissmann ◽

Sascha Hackemack ◽

Bhola Kumar Dahal ◽

Soni Savai Pullamsetti ◽

Rajkumar Savai ◽

...

Keyword(s):

Pulmonary Hypertension ◽

Guanylate Cyclase ◽

Vascular Remodeling ◽

Chronic Hypoxia ◽

Soluble Guanylate Cyclase ◽

Pressor Response ◽

Acute Hypoxia ◽

Heart Hypertrophy ◽

Right Heart ◽

Pulmonary Vascular Remodeling

Severe pulmonary hypertension (PH) is a disabling disease with high mortality, characterized by pulmonary vascular remodeling and right heart hypertrophy. In mice with PH induced by chronic hypoxia, we examined the acute and chronic effects of the soluble guanylate cyclase (sGC) activator HMR1766 on hemodynamics and pulmonary vascular remodeling. In isolated perfused mouse lungs from control animals, HMR1766 dose-dependently inhibited the pressor response of acute hypoxia. This dose-response curve was shifted leftward when the effects of HMR1766 were investigated in isolated lungs from chronic hypoxic animals for 21 days at 10% oxygen. Mice exposed for 21 or 35 days to chronic hypoxia developed PH, right heart hypertrophy, and pulmonary vascular remodeling. Treatment with HMR1766 (10 mg·kg−1·day−1), after full establishment of PH from day 21 to day 35, significantly reduced PH, as measured continuously by telemetry. In addition, right ventricular (RV) hypertrophy and structural remodeling of the lung vasculature were reduced. Pharmacological activation of oxidized sGC partially reverses hemodynamic and structural changes in chronic hypoxia-induced experimental PH.

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