8-bromocyclic-gmp and Hypoxia

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Apoptosis; Caspase 3; Cell Line, Tumor; Colonic Neoplasms; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Drug Resistance, Neoplasm; Drug Synergism; Drugs, Chinese Herbal; Humans; Hypoxia; Male; Mice, Inbred BALB C; Nitroso Compounds; Oxadiazoles; Oxazines; Phosphodiesterase 5 Inhibitors; Plant Extracts; Reactive Oxygen Species; Serum Albumin, Human; Soluble Guanylyl Cyclase; Vardenafil Dihydrochloride

An increase in Rho kinase (ROCK) activity is implicated in chronic hypoxia-induced pulmonary hypertension. In the present study, we determined the role of ROCKs in cGMP-dependent protein kinase (PKG)-mediated pulmonary vasodilation of fetal lambs exposed to chronic hypoxia. Fourth generation pulmonary arteries were isolated from near-term fetuses ( approximately 140 days of gestation) delivered from ewes exposed to chronic high altitude hypoxia for approximately 110 days and from control ewes. In vessels constricted to endothelin-1, 8-bromoguanosine-cGMP (8-Br-cGMP) caused a smaller relaxation in chronically hypoxic (CH) vessels compared with controls. Rp-8-Br-PET-cGMPS, a PKG inhibitor, attenuated relaxation to 8-Br-cGMP in control vessels to a greater extent than in CH vessels. Y-27632, a ROCK inhibitor, significantly potentiated 8-Br-cGMP-induced relaxation of CH vessels and had only a minor effect in control vessels. The expression of PKG was increased but was not accompanied with an increase in the activity of the enzyme in CH vessels. The expression of type II ROCK and activity of ROCKs were increased in CH vessels. The phosphorylation of threonine (Thr)696 and Thr850 of the regulatory subunit MYPT1 of myosin light chain phosphatase was inhibited by 8-Br-cGMP to a lesser extent in CH vessels than in controls. The difference was eliminated by Y-27632. These results suggest that chronic hypoxia in utero attenuates PKG-mediated relaxation in pulmonary arteries, partly due to inhibition of PKG activity and partly due to enhanced ROCK activity. Increased ROCK activity may inhibit PKG action through increased phosphorylation of MYPT1 at Thr696 and Thr850.

Topics: Amides; Animals; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Enzyme Inhibitors; Female; Fetus; Gene Expression Regulation, Developmental; Gene Expression Regulation, Enzymologic; Hypoxia; Intracellular Signaling Peptides and Proteins; Male; Muscle, Smooth, Vascular; Myosin-Light-Chain Phosphatase; Oxygen; Pregnancy; Protein Serine-Threonine Kinases; Pulmonary Artery; Pulmonary Circulation; Pyridines; rho-Associated Kinases; RNA, Messenger; Sheep; Vasodilation

The activation of ATP-sensitive potassium (K ATP) ion channels in the heart is thought to exert a cardioprotective effect under low oxygen conditions, possibly enhancing tolerance of environmental hypoxia in aquatic vertebrates. The purpose of this study was to examine the possibility that hypoxia-induced activation of cardiac K ATP channels, whether in the sarcolemma (sarcK ATP) or mitochondria (mitoK ATP), enhances viability in cardiac muscle cells from a species highly tolerant of low oxygen environments, the goldfish Carassius auratus. During moderate hypoxia (6-7 kPa), the activation of sarcK ATP channels was indicated by a reduction in transmembrane action potential duration (APD). This response to hypoxia was mimicked by the NO-donor SNAP (100 micromol l(-1)) and the stable cGMP analog 8-Br-cGMP, but abolished by glibenclamide or l-NAME, an inhibitor of NO synthesis. The mitoK ATP channel opener diazoxide did not affect APD. Isolated ventricular muscle cells were then incubated under normoxic and hypoxic conditions. Cell viability was decreased in hypoxia; however, the negative effects of low oxygen were reduced during simultaneous exposure to SNAP, 8-Br-cGMP, and diazoxide. The cardioprotective effect of diazoxide, but not 8-Br-cGMP, was reduced by the mitoK ATP channel blocker 5-HD. These data suggest that hypoxia-induced activation of sarcK ATP or mitoK ATP channels could enhance tolerance of low-oxygen environments in this species, and that sarcK ATP activity is increased through a NO and cGMP-dependent pathway.

Topics: Acclimatization; Action Potentials; Analysis of Variance; Animals; Cyclic GMP; Diazoxide; Glyburide; Goldfish; Hypoxia; Mitochondria; Models, Biological; Myocardium; NG-Nitroarginine Methyl Ester; Penicillamine; Potassium Channels; Sarcolemma

To investigate the effects of long-term nitric oxide (NO) inhalation on the recovery process of right ventricular hypertrophy (RVH) and functional alterations in the NO-cyclic guanosine monophosphate (cGMP) relaxation pathway in rat conduit pulmonary arteries (PAs) in established chronic hypoxic pulmonary hypertension.. A total of 35 rats were exposed to chronic hypobaric hypoxia (380 mm Hg, 10% oxygen), and 39 rats were exposed to air for 10 days. Both groups were then exposed to 3 or 10 days of NO 10 ppm, NO 40 ppm, or air (control groups for each NO concentration), resulting in a total of 16 groups. Acetylcholine- and sodium nitroprusside (SNP)-induced relaxation were evaluated in precontracted PA rings. RVH was assessed by heart weight ratio of right ventricle to left ventricle plus septum.. NO inhalation had no effect on either the regression of RVH or the recovery process of impaired relaxation induced by acetylcholine or SNP in a endothelium-intact hypertensive conduit extrapulmonary artery or intrapulmonary artery (IPA). In a normal endothelium-intact conduit IPA, 40 ppm NO inhalation for 10 days partially augmented SNP-induced relaxation, but not that induced by acetylcholine.. Continuous NO inhalation did not affect the regression process of either established RVH or the impaired endogenous NO-cGMP relaxation cascade in a conduit PA in rats during the recovery period after chronic hypoxia.

Topics: Acetylcholine; Administration, Inhalation; Animals; Chronic Disease; Cyclic GMP; Endothelium-Dependent Relaxing Factors; Endothelium, Vascular; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; In Vitro Techniques; Male; Nitric Oxide; Nitroprusside; Pulmonary Artery; Rats; Rats, Wistar; Vasodilation; Vasodilator Agents

Hypoxia-evoked vasodilatation is a fundamental regulatory mechanism that is often attributed to adenosine. The identity of the O(2) sensor is unknown. Nitric oxide (NO) inhibits endothelial mitochondrial respiration and ATP generation by competing with O(2) for its binding site on cytochrome oxidase. We proposed that in vivo this interaction allows endothelial cells to release adenosine when O(2) tension falls or NO concentration increases. Using anaesthetised rats, we confirmed that the increase in femoral vascular conductance (FVC, hindlimb vasodilatation) evoked by systemic hypoxia is attenuated by NO synthesis blockade with L-NAME, but restored when baseline FVC is restored by infusion of NO donor. This "restored" hypoxic response, like the control hypoxic response, is inhibited by the adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. Similarly, the FVC increase evoked by adenosine infusion was attenuated by L-NAME but restored by infusion of NO donor. However, when baseline FVC was restored after L-NAME with 8-bromo-cGMP, the FVC increase evoked by adenosine infusion was restored, but not in response to systemic hypoxia, suggesting that adenosine was no longer released by hypoxia. Infusion of NO donor at a given rate after treatment with L-NAME evoked a greater FVC increase during systemic hypoxia than during normoxia, both responses being reduced by 8-cyclopentyl-1,3-dipropylxanthine. Finally, both bradykinin and NO donor released adenosine from superfused endothelial cells in vitro; L-NAME attenuated only the former response. We propose that in vivo, shear-released NO increases the apparent K(m) of endothelial cytochrome oxidase for O(2), allowing the endothelium to act as an O(2) sensor, releasing adenosine in response to moderate falls in O(2).

Topics: Adenosine; Animals; Cyclic GMP; Endothelium, Vascular; Enzyme Inhibitors; Hindlimb; Hypoxia; In Vitro Techniques; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Purinergic P1 Receptor Antagonists; Rats; Rats, Wistar; S-Nitroso-N-Acetylpenicillamine; Vasodilation; Vasodilator Agents; Xanthines

Chronic hypoxia (CH) augments endothelium-derived nitric oxide (NO)-dependent pulmonary vasodilation; however, responses to exogenous NO are reduced following CH in female rats. We hypothesized that CH-induced attenuation of NO-dependent pulmonary vasodilation is mediated by downregulation of vascular smooth muscle (VSM) soluble guanylyl cyclase (sGC) expression and/or activity, increased cGMP degradation by phosphodiesterase type 5 (PDE5), or decreased VSM sensitivity to cGMP. Experiments demonstrated attenuated vasodilatory responsiveness to the NO donors S-nitroso-N-acetylpenicillamine and spermine NONOate and to arterial boluses of dissolved NO solutions in isolated, saline-perfused lungs from CH vs. normoxic female rats. In additional experiments, the sGC inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, blocked vasodilation to NO donors in lungs from each group. However, CH was not associated with decreased pulmonary sGC expression or activity as assessed by Western blotting and cGMP radioimmunoassay, respectively. Consistent with our hypothesis, the selective PDE5 inhibitors dipyridamole and T-1032 augmented NO-dependent reactivity in lungs from CH rats, while having little effect in lungs from normoxic rats. However, the attenuated vasodilatory response to NO in CH lungs persisted after PDE5 inhibition. Furthermore, CH similarly inhibited vasodilatory responses to 8-bromoguanosine 3'5'-cyclic monophosphate. We conclude that attenuated NO-dependent pulmonary vasodilation after CH is not likely mediated by decreased sGC expression, but rather by increased cGMP degradation by PDE5 and decreased pulmonary VSM reactivity to cGMP.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Blotting, Western; Chronic Disease; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Enzyme Inhibitors; Female; Guanylate Cyclase; Hypertrophy, Right Ventricular; Hypoxia; In Vitro Techniques; Lung; Muscle, Smooth, Vascular; Nitric Oxide; Nitric Oxide Donors; Nitroarginine; Polycythemia; Radioimmunoassay; Rats; Rats, Sprague-Dawley; Receptors, Cytoplasmic and Nuclear; Soluble Guanylyl Cyclase; Vasodilation

We tested the hypothesis that chronic high-altitude (3,820 m) hypoxia during pregnancy was associated with the upregulation of endothelial nitric oxide (NO) synthase (eNOS) protein and mRNA in ovine uterine artery endothelium and enhanced endothelium-dependent relaxation. In pregnant sheep, norepinephrine-induced dose-dependent contractions were increased by removal of the endothelium in both control and hypoxic uterine arteries. The increment was significantly higher in hypoxic tissues. The calcium ionophore A23187-induced relaxation of the uterine artery was significantly enhanced in hypoxic compared with control tissues. However, sodium nitroprusside- and 8-bromoguanosine 3',5'-cyclic monophosphate-induced relaxations were not changed. Accordingly, chronic hypoxia significantly increased basal and A23187-induced NO release. Chronic hypoxia increased eNOS protein and mRNA levels in the endothelium from uterine but not femoral or renal arteries. In nonpregnant animals, chronic hypoxia increased eNOS mRNA in uterine artery endothelium but had no effects on eNOS protein, NO release, or endothelium-dependent relaxation. Chronic hypoxia selectively augments pregnancy-associated upregulation of eNOS gene expression and endothelium-dependent relaxation of the uterine artery.

Topics: Altitude Sickness; Animals; Calcimycin; Chronic Disease; Cyclic GMP; Endothelium, Vascular; Female; Femoral Artery; Gene Expression Regulation, Enzymologic; Hypoxia; Ionophores; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Nitroprusside; Norepinephrine; Pregnancy; Pregnancy, Animal; Renal Artery; RNA, Messenger; Sheep; Umbilical Arteries; Uterus; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents

Chronic myocardial hypoxia results in elevated nitric oxide (NO) production and increased current through the sarcolemmal K(ATP) channel. We hypothesized these two processes are related and determined whether NO alters the electrophysiology of Purkinje fibers obtained from rabbits (n=12/group) raised in a normoxic (F(I)O2=0.21) or hypoxic (F(I)O2=0.12) environment from birth to 9 days of age. Action potential duration (APD)(90) was shorter (112+/-3 ms v 126+/-3 ms) and maximum diastolic potential (MDP) was more negative (-84+/-2 mV v-80+/-1 mV) in hypoxic hearts compared with normoxic controls. In normoxic hearts the NO donors, S-nitrosoglutathione (GSNO) 50 microM and spermine NONOate (50 microM) shortened APD(90) and increased MDP to levels present in chronically hypoxic hearts. This effect was completely abolished by the K(ATP) channel blocker glibenclamide (3 microM) and by a nitric oxide trap, Carboxy-PTIO (100 microM). The NO carrier glutathione (50 microM) and decomposed spermine NONOate had no effect on APD(90) or MDP. GSNO had no effect in hypoxic hearts; however, when GSNO was combined with glibenclamide APD(90) increased, and MDP decreased to normoxic values. 8-Bromo cGMP (100 microM) shortened APD(90) and increased MDP to levels present in chronically hypoxic hearts. This effect was abolished by glibenclamide. A soluble guanylyl cyclase inhibitor, ODQ (10 microM), had no effect on action potentials in normoxic hearts but in hypoxic hearts resulted in an increase in APD(90) to levels present in normoxic hearts and a decrease in MDP. The effect of ODQ could not be reversed by GSNO. We conclude that NO activates the sarcolemmal K(ATP) channel in normoxic and chronically hypoxic hearts by a cyclic GMP-dependent mechanism.

Topics: Action Potentials; Animals; Animals, Newborn; Blood Pressure; Cyclic GMP; Electron Spin Resonance Spectroscopy; Electrophysiology; Female; Glutathione; Glyburide; Hypoglycemic Agents; Hypoxia; Male; Models, Biological; Neuroprotective Agents; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; Nitroso Compounds; Oxygen; Potassium Channels; Rabbits; S-Nitrosoglutathione; Sarcolemma; Spermine; Time Factors

Nitric oxide (NO) is thought to play an important role in the regulation of neonatal pulmonary vasculature. It has been suggested that neonates with pulmonary hypertension have a defective NO pathway. Therefore, we measured in 1-day-old piglets exposed to hypoxia (fraction of inspired O(2) = 0.10) for 3 or 14 days to induce pulmonary hypertension 1) the activity of NO synthase (NOS) via conversion of L-arginine to L-citrulline and the concentration of the NO precursor L-arginine in isolated pulmonary vessels, 2) the vasodilator response to the NO donor 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1) and the cGMP analog 8-bromo-cGMP in isolated perfused lungs, and 3) the production of cGMP in response to SIN-1 in isolated perfused lungs. After 3 days of exposure to hypoxia, endothelial NOS (eNOS) activity was unaffected, whereas, after 14 days of hypoxia, eNOS activity was decreased in the cytosolic fraction of pulmonary artery (P < 0.05) but not of pulmonary vein homogenates. Inducible NOS activity was decreased in the cytosolic fraction of pulmonary artery homogenates after both 3 (P < 0.05) and 14 (P < 0.05) days of hypoxia but was unchanged in pulmonary veins. Pulmonary artery levels of L-arginine were unaffected by hypoxic exposure. After 3 days of exposure to hypoxia, the reduction in the dilator response to SIN-1 (P < 0.05) coincided with a decrease in cGMP production (P < 0.005), suggesting that soluble guanylate cyclase activity may be altered. When the exposure was prolonged to 14 days, dilation to SIN-1 remained decreased (P < 0.05) and, although cGMP production normalized, the dilator response to 8-bromo-cGMP decreased (P < 0.05), suggesting that, after prolonged exposure to hypoxia, cGMP-dependent mechanisms may also be impaired. In conclusion, neonatal hypoxia-induced pulmonary hypertension is associated with multiple disruptions in the NO pathway.

Topics: Animals; Animals, Newborn; Arginine; Blood Vessels; Cyclic GMP; Hypertension, Pulmonary; Hypoxia; In Vitro Techniques; Molsidomine; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Pulmonary Circulation; Swine; Vasodilation; Vasodilator Agents

Nitric oxide (NO) and dopamine (DA) have similar effects on renal function, with both having natriuretic and diuretic effects mediated by vascular and tubular mechanisms. Renal ischaemia or hypoxia have been shown to influence the activity of both systems. However, it is not known whether there is any crosstalk between the NO and dopaminergic systems in the kidney. Here using the porcine proximal tubule-like renal epithelial LLC-PK1 cell line as a model system, we determined whether exposure of cells to chemical hypoxia altered the steady-state levels of D1A receptor mRNA and whether the changes involved the NO system. Exposure of LLC-PK1 cells to chemical hypoxia resulted in a marked increase in D1A receptor mRNA levels as measured by reverse transcription-polymerase chain reaction (RT-PCR). The increased levels of D1A receptor mRNA following hypoxia were blocked by the NO synthase inhibitors NG-nitro-L-arginine methylester (L-NAME) or NG-monomethyl-L-arginine (L-NMMA). Further evidence that the NO system exerted positive effects on D1A receptor gene expression came from finding that the NO donor sodium nitroprusside, the NO precursor L-arginine and the guanosine 3', 5'-cyclic monophosphate (cyclic GMP) analogue 8-Br-cGMP all increased D1A receptor mRNA levels in LLC-PK1 cells. These results indicate that expression of the D1A receptor in LLC-PK1 cells can be positively regulated by the NO system. Such an interaction between the renal NO and DA systems may contribute to the reported protective effects that NO and DA exert upon the kidney under conditions of ischaemia.

Topics: Animals; Arginine; Cyclic GMP; Deoxyglucose; Enzyme Inhibitors; Epithelial Cells; Hypoxia; Kidney; LLC-PK1 Cells; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; omega-N-Methylarginine; Receptors, Dopamine D1; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sodium Cyanide; Swine

Activation of ATP sensitive K+ channels (K(ATP)) and the NO-cGMP pathway have both been implicated in reducing norepinephrine (NE) release from cardiac sympathetic nerves during stimulation. Our aim was to test whether these pathways could interact and modulate cardiac excitability during sympathetic nerve stimulation (SNS).. The effect of inhibitors and activators of K(ATP) channels and the NO-cGMP pathway on the heart rate (HR) response to cardiac SNS in the isolated guinea pig (Cavia porcellus) double atrial/right stellate ganglion preparation was studied (n=48).. The K(ATP) channel activator, diazoxide (100 microM, n=6) or hypoxia (0% O2/5% CO2, n=6) significantly attenuated the HR response to 3 Hz SNS by -10+/-4% and -27+/-6% respectively; an effect that was reversed by the K(ATP) channel inhibitor, glibenclamide (30 microM). Glibenclamide (n=6) on its own enhanced the HR response to SNS by 20+/-8%. Bath applied NE (0.1-0.7 microM, n=6) did not affect the HR response to diazoxide, although an increased response to glibenclamide was observed at 0.3 and 0.5 microM NE. In the presence of 8-Br-cGMP (0.5 mM, n=7), diazoxide further decreased the HR response SNS (19+/-3%). The NO synthase inhibitor, N-omega-nitro-L-arginine (100 microM) significantly increased the HR response (13+/-3%) to SNS in the presence of diazoxide (100 microM, n=6). This effect was reversed with excess (1 mM) L-arginine. Conversely, the NO donor, sodium nitroprusside (SNP, 20-100 microM) significantly attenuated the HR response to SNS. The addition of glibenclamide (30 microM, n=10) could still enhance the HR response (42+/-15%) to SNS. Similar results were seen with the cyclic GMP analogue, 8-Br-cGMP (0.5 mM, n=12).. Our results indicate that NO and sulphonlyurea-sensitive channels act in a complementary fashion, but appear to be independent of each other in the regulation of HR during cardiac SNS activation.

Topics: Analysis of Variance; Animals; Cyclic GMP; Diazoxide; Electric Stimulation; Enzyme Inhibitors; Glyburide; Guinea Pigs; Heart Atria; Heart Rate; Hypoxia; In Vitro Techniques; Male; Nitric Oxide; Norepinephrine; Potassium Channel Blockers; Potassium Channels; Stellate Ganglion; Sympathetic Nervous System; Tolbutamide

This study was designed to investigate the potential role of endogenous peroxynitrite (ONOO-) formation in the inhibition of cardiac muscle contractility and mitochondrial respiration during posthypoxic reoxygenation. Isometric contraction of isolated rat left ventricular posterior papillary muscle was virtually eliminated at the end of an exposure to 15 minutes of hypoxia and remained 40+/-5% depressed an hour after the reintroduction of O2. O2 uptake by rat left ventricular cardiac muscle, measured by a Clark-type O2 electrode, was also inhibited by 24+/-2% at 10 minutes after reoxygenation. The inhibition of contractility and respiration during posthypoxic reoxygenation was markedly attenuated by the NO synthase inhibitor nitro-L-arginine, exogenous superoxide dismutase, and the ONOO- scavenger urate but not by the hydroxyl radical scavenger mannitol. Generation of ONOO- with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) plus the superoxide-releasing agent pyrogallol caused an irreversible inhibition of cardiac contractile and respiratory function. Unlike ONOO-, exogenous (SNAP) and endogenous (bradykinin) sources of NO inhibited contractility in a reversible manner. Under conditions of comparable amounts of respiratory inhibition in unstimulated incubated muscle, the NO-dependent agents and the mitochondrial antagonist NaCN produced a smaller degree of suppression of contractility compared with ONOO- and posthypoxic reoxygenation. These results are consistent with a contributing role for endogenous ONOO- formation in the inhibition of cardiac muscle contractility and mitochondrial respiration during posthypoxic reoxygenation.

Topics: Animals; Bradykinin; Cyclic GMP; Free Radical Scavengers; Hydroxyl Radical; Hypoxia; In Vitro Techniques; Kinetics; Mannitol; Mitochondria, Heart; Myocardial Contraction; Nitrates; Nitric Oxide; Nitroarginine; Oxidants; Oxygen Consumption; Penicillamine; Rats; S-Nitroso-N-Acetylpenicillamine; Sodium Cyanide; Superoxide Dismutase

Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis and blood vessel remodeling. Its expression is up-regulated in vascular smooth muscle cells by a number of conditions, including hypoxia. Hypoxia increases the transcriptional rate of VEGF via a 28-base pair enhancer located in the 5'-upstream region of the gene. The gas molecules nitric oxide (NO) and carbon monoxide (CO) are important vasodilating agents. We report here that these biological molecules can suppress the hypoxia-induced production of VEGF mRNA and protein in smooth muscle cells. In transient expression studies, both NO and CO inhibited the ability of the hypoxic enhancer we have previously identified to activate gene transcription. Furthermore, electrophoretic mobility shift assays indicated decreased binding of hypoxia-inducible factor 1 (HIF-1) to this enhancer by nuclear proteins isolated from CO-treated cells, although HIF-1 protein levels were unaffected by CO. Given that both CO and NO activate guanylyl cyclase to produce cGMP and that a cGMP analog (8-Br-cGMP) showed a similar suppressive effect on the hypoxic induction of the VEGF enhancer, we speculate that the suppression of VEGF by these two gas molecules occurs via a cyclic GMP-mediated pathway.

Topics: Animals; Carbon Monoxide; Cattle; Cells, Cultured; Cyclic GMP; DNA-Binding Proteins; Endothelial Growth Factors; Enhancer Elements, Genetic; Gene Expression Regulation; Guanylate Cyclase; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Lymphokines; Muscle, Smooth, Vascular; Nitric Oxide; Nuclear Proteins; RNA, Messenger; Transcription Factors; Transcription, Genetic; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors; Vasodilator Agents

It has been previously observed that nitric oxide (NO) contributes to hypoxic pial artery dilation and that both sodium nitroprusside (SNP), a releaser of NO, and hypoxia elicit dilation via activation of ATP-sensitive K+ channels in the newborn pig. Other studies, however, have shown that NO activates calcium-sensitive K+ (K(Ca)) channels. The present study, therefore, was designed to investigate the role of K(Ca)-channel activation in NO and hypoxic dilation and to relate this mechanism to the previously observed role of NO in hypoxic dilation in newborn pigs equipped with closed cranial windows. SNP (10(-8) and 10(-6) M) elicited pial artery dilation that was unchanged in the presence of the K(Ca)-channel antagonist iberiotoxin (10(-7) M; 10 +/- 1 and 20 +/- 1 vs. 9 +/- 1 and 20 +/- 2% for 10(-8) and 10(-6) M SNP in the absence and presence of iberiotoxin, respectively). Responses to S-nitroso-N-acetylpenicillamine and 8-bromoguanosine 3',5'-cyclic monophosphate were similarly unchanged by iberiotoxin. In contrast, iberiotoxin attenuated the dilation resulting from moderate and severe hypoxia (arterial PO2 approximately 35 and 25 mmHg, respectively; 27 +/- 1 vs. 21 +/- 2 and 34 +/- 1 vs. 16 +/- 2% for moderate and severe hypoxia in the absence and presence of iberiotoxin, respectively). Iberiotoxin blocked responses to the K(Ca)-channel agonist NS-1619, whereas responses to the ATP-sensitive K+ agonist cromakalim were unchanged (8 +/- 1 and 15 +/- 1 vs. 1 +/- 1 and 1 +/- 1% for 10(-8) and 10(-6) M NS-1619 in the absence and presence of iberiotoxin, respectively). These data show that NO and guanosine 3',5'-cyclic monophosphate do not elicit dilation via K(Ca)-channel activation. However, activation of K(Ca) channels does contribute to hypoxic pial dilation. Finally, these data suggest that substances other than NO are involved in the contribution of K(Ca)-channel activation to hypoxic pial artery dilation.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Animals, Newborn; Arterioles; Benzimidazoles; Benzopyrans; Cerebral Arteries; Cromakalim; Cyclic GMP; Female; Hypoxia; Male; Muscle, Smooth, Vascular; Nitric Oxide; Nitroprusside; Penicillamine; Pia Mater; Potassium Channels; Pyrroles; S-Nitroso-N-Acetylpenicillamine; Swine; Vasodilation; Vasodilator Agents

1. Previous studies in our laboratory revealed the presence of atrial natriuretic peptide (ANP) in preneural chemosensory type I cells of the cat carotid body, and demonstrated that submicromolar concentrations of the peptide inhibited carotid sinus nerve (CSN) activity evoked by hypoxia. In the present study, we have evaluated the role of the cyclic nucleotide second messenger, cyclic GMP (cGMP), and the involvement of type I cells in rabbit chemosensory inhibition. 2. Submicromolar concentrations of the potent ANP analogue, APIII, greatly elevated both the content and release of cGMP from the carotid body. Denervation experiments confirmed earlier immunocytochemical studies which suggested that APIII-induced cGMP production occurs almost exclusively in type I cells; these experiments also indicate that both the sympathetic and sensory innervation to the carotid body exert a trophic influence on the metabolism of this second messenger. 3. Submicromolar concentrations of APIII inhibited the CSN activity evoked by hypoxia (79.8 +/- 3.2% (mean +/- S.E.M.) inhibition with 100 nM APIII) and nicotine (74.5 +/- 3.6% inhibition with 100 nM APIII), but did not affect basal CSN activity established in 100% O2-equilibrated superfusion solutions. 4. The biologically inactive analogue of ANP, C-ANP, failed to produce CSN inhibition; however, the inhibitory effects of APIII were mimicked by cell-permeant analogues of cGMP (dibutyryl-cGMP and 8-bromo-cGMP, 2 mM), which likewise did not alter basal CSN activity. Because we found that unmodified cGMP was an ineffective inhibitor of CSN activity, our data suggest that APIII inhibition is mediated intracellularly by cGMP produced within the type I cells. 5. APIII does not inhibit the CSN activity produced by 20 mM K+ (in zero Ca2+ media), which very probably results from direct depolarization of the sensory nerve terminals. 6. Catecholamine release from the carotid body evoked by hypoxia is likewise not altered by APIII (100 nM). 7. The data are consistent with the notion that APIII and analogues of cGMP alter the release of excitatory and/or inhibitory transmitters from chemosensory type I cells in the carotid body.

Topics: Animals; Atrial Natriuretic Factor; Carotid Body; Chemoreceptor Cells; Cyclic GMP; Denervation; Depression, Chemical; Dibutyryl Cyclic GMP; Dose-Response Relationship, Drug; Hypoxia; Nicotine; Peptide Fragments; Potassium; Rabbits

ANP increases cellular cGMP content in cultured hepatocytes and decreases Ca2(+)-inflow in a concentration- and time-dependent manner which explains a beneficial effect on hypoxia cell injury (25). Both observations are mimicked by SNP and 8-Br-cGMP and blocked by Ly 83583 indicating a cGMP-mediated mechanism. The protective effect was also inhibited by Pertussis Toxin (PT) without lowering the elevated cGMP-level. But PT in combination with ANP leads to a higher Ca2(+)-inflow. Stimulated Na(+)-inflows are also be lowered by ANP. Here, neither SNP can mimick nor PT can inhibit this effect. Our results now indicate that the beneficial effect by ANP at the cellular level is mediated through cGMP which decreases calcium-inflow. ANP seems to control Ca2(+)-channels direct via a PT-sensitive G-protein and indirect by a cGMP-mediated mechanism and Na(+)-channels cGMP-independent through a PT-insensitive G-protein, thus preventing cells on hypoxia and oxygen radicals.

Topics: Aminoquinolines; Animals; Atrial Natriuretic Factor; Calcium; Cells, Cultured; Cyclic GMP; Free Radicals; GTP-Binding Proteins; Hypochlorous Acid; Hypoxia; Kinetics; Liver; Nitroprusside; Pertussis Toxin; Rats; Virulence Factors, Bordetella

The effects of endothelium-dependent vasodilation on pulmonary vascular hemodynamics were evaluated in a variety of in vivo and in vitro models to determine 1) the comparability of the hemodynamic effects of acetylcholine (ACh), bradykinin (BK), nitric oxide (NO), and 8-bromo-guanosine 3',5'-cyclic monophosphate (cGMP), 2) whether methylene blue is a useful inhibitor of endothelium-dependent relaxing factor (EDRF) activity in vivo, and 3) the effect of monocrotaline-induced pulmonary hypertension on the responsiveness of the pulmonary vasculature to ACh. In isolated rat lungs, which were preconstricted with hypoxia, ACh, BK, NO, and 8-bromo-cGMP caused pulmonary vasodilation, which was not inhibited by maximum tolerable doses of methylene blue. Methylene blue did not inhibit EDRF activity in any model, despite causing increased pulmonary vascular tone and responsiveness to various constrictor agents. There were significant differences in the hemodynamic characteristics of ACh, BK, and NO. In the isolated lung, BK and NO caused transient decreases of hypoxic vasoconstriction, whereas ACh caused more prolonged vasodilation. Pretreatment of these lungs with NO did not significantly inhibit ACh-induced vasodilation but caused BK to produce vasoconstriction. Tachyphylaxis, which was agonist specific, developed with repeated administration of ACh or BK but not NO. Tachyphylaxis probably resulted from inhibition of the endothelium-dependent vasodilation pathway proximal to NO synthesis, because it could be overcome by exogenous NO. Pretreatment with 8-bromo-cGMP decreased hypoxic pulmonary vasoconstriction and, even when the hypoxic pressor response had largely recovered, subsequent doses of ACh and NO failed to cause vasodilation, although BK produced vasoconstriction. These findings are compatible with the existence of feedback inhibition of the endothelium-dependent relaxation by elevation of cGMP levels. Responsiveness to ACh was retained in lungs with severe monocrotaline-induced pulmonary hypertension. Many of these findings would not have been predicted based on in vitro studies and illustrate the importance for expanding studies of EDRF to in vivo and ex vivo models.

Topics: Acetylcholine; Animals; Bradykinin; Cyclic GMP; Endothelium, Vascular; Hemodynamics; Hypertension, Pulmonary; Hypoxia; Lung; Male; Methylene Blue; Monocrotaline; Nitric Oxide; Pulmonary Circulation; Pyrrolizidine Alkaloids; Rats; Rats, Inbred Strains; Vasodilation

We have previously reported that the isolated rat branch pulmonary artery (PA) contracts when made hypoxic and that the contraction is dependent in large part on the presence of a functioning endothelium. This study tested if the hypoxic contraction was caused by reduced endothelium-derived relaxing factor (EDRF) activity. To do so we tested if chemical inhibitors of EDRF mimicked the effect of hypoxia, if PA guanosine 3',5'-cyclic monophosphate (cGMP) fell during hypoxic contraction, and if stimulation of smooth muscle cGMP attenuated hypoxic contraction. We found that the EDRF inhibitors hemoglobin and methylene blue caused a concentration-dependent increase in PA force that equaled that produced by hypoxia. PA cGMP decreased in endothelium-intact rings from 105 +/- 14 pM/g (wet wt) during normoxia to 41 +/- 9 pM/g during hypoxia. In endothelium-denuded rings normoxic cGMP was reduced to 32 +/- 10 pM/g with no further decrease during hypoxia. The endothelium-independent stimulators of cGMP, nitric oxide, and 8-bromo-cGMP, reduced maximum hypoxic contraction by 80 +/- 11 and 93 +/- 3%, respectively, whereas the endothelium-dependent stimulator acetylcholine did not. PA adenosine 3',5'-cyclic monophosphate (cAMP) fell only slightly during hypoxia and cAMP inhibitors failed to mimic the hypoxic contraction. We conclude that the hypoxic contraction of isolated rat PA is caused largely by decreased EDRF activity.

Topics: Acetylcholine; Adenosine Monophosphate; Animals; Cyclic AMP; Cyclic GMP; Dideoxyadenosine; Endothelium, Vascular; Hemoglobins; Hypoxia; In Vitro Techniques; Isomerism; Kinetics; Male; Methylene Blue; Muscle, Smooth, Vascular; Nitric Oxide; Phenylephrine; Pulmonary Artery; Rats; Rats, Inbred Strains; Thionucleotides; Vasodilation

We examined, in isolated blood perfused rat lungs, the effect of the cell permeable 8-bromo derivative of cGMP on pulmonary vasoconstriction induced by either alveolar hypoxia or angiotensin II. 8-Bromo cGMP dose-dependently reduced both hypoxia-(IC50 = 2.2 X 10(-5) M) and angiotensin-II-induced pulmonary vasoconstriction (IC50 = 5.0 X 10(-5) M). This effect of 8-bromo cGMP on pulmonary vasoconstriction was not affected by cyclooxygenase blockade. M & B 22948 (0.1 mM), an inhibitor of cGMP-phosphodiesterase, reduced synergistically with 8-bromo cGMP the hypoxia or angiotensin-II-induced vasoconstriction. The cGMP-phosphodiesterase inhibitor M & B 22948, by itself, selectively reduced hypoxia-induced vasoconstriction, suggesting a modulating effect of endogenous cGMP during hypoxic vasoconstriction.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Angiotensin II; Animals; Cyclic GMP; Dose-Response Relationship, Drug; Hypoxia; Lung; Male; Meclofenamic Acid; Purinones; Rats; Vasoconstriction

The effects of 8-bromo-cAMP (10(-4)M) and 8-bromo-cGMP (10(-4)M) on tissue lactate, NADH, creatine phosphate (CP), ATP, ADP and AMP were studied in hypoxic (50% oxygen saturation) spontaneously beating rat atria. CP/ATP ratio and energy charge (EC) were also calculated. In hypoxic rat atria there was a significant increase in tissue lactate, NADH, ADP and AMP and a decrease in CP, CP/ATP ratio and EC. 8-bromo-cGMP abolished these hypoxia-induced changes. The levels of lactate and NADH decreased and those of CP, ATP, ADP and EC increased after administration of 8-bromo-cGMP. 8-bromo-cAMP increased the level of lactate during hypoxia, but did not affect the other hypoxia-induced changes. The present work indicates that cyclic GMP can change the redox state and energy state in a more favourable direction for the hypoxic rat heart.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenine Nucleotides; Animals; Cyclic GMP; Energy Metabolism; Heart Atria; Hypoxia; Lactates; Lactic Acid; Male; Myocardium; NAD; Oxidation-Reduction; Phosphocreatine; Rats; Rats, Inbred Strains