calcimycin and Hypoxia

In the lung, nitric oxide (NO) derives from several cellular sources, forming networks of paracrine communication. In pulmonary vessels, NO produced by endothelial cells is a powerful vasodilator. In the airways, NO originates from epithelial cells and from adventitial nerve endings to induce smooth muscle relaxation. Activated macrophages can also produce large quantities of NO during lung immunological reactions. In the normal pulmonary circulation, NO not only mediates vasodilation, but also opposes vasoconstriction, prevents platelet adhesion, controls growth of smooth muscle and influences the composition of the extracellular matrix. During exposure to chronic hypoxia, impaired endothelial NO production contributes to the increased vasomotor tone and vascular remodelling leading to sustained pulmonary hypertension. Exogenous NO gas delivered via the airspaces is a selective pulmonary vasodilator. Inhaled NO is now used as a therapy to treat various forms of pulmonary hypertension and to improve arterial oxygenation during lung injury.

Topics: Animals; Calcimycin; Endothelium, Vascular; Humans; Hypertension, Pulmonary; Hypoxia; Lung; Nitric Oxide; Nitric Oxide Synthase; Pulmonary Circulation; Vasodilation

Topics: Adenosine; Animals; Calcimycin; Carbon Dioxide; Cerebrovascular Circulation; Cyclic GMP; Endothelium, Vascular; Fetus; Humans; Hydrogen-Ion Concentration; Hypoxia; Infant, Newborn; Oxygen; Penicillamine; Prostaglandins; S-Nitroso-N-Acetylpenicillamine; Sheep; Vascular Resistance

Topics: 5,8,11,14-Eicosatetraynoic Acid; Acetophenones; Acetylcholine; Adenosine Diphosphate; Adenosine Triphosphate; Animals; Arachidonic Acid; Arachidonic Acids; Bradykinin; Calcimycin; Catechols; Endothelium; Histamine; Hydroquinones; Hypoxia; Masoprocol; Methylene Blue; Muscle Relaxation; Muscle, Smooth, Vascular; Prostaglandins; Quinacrine; Serotonin; Substance P; Thrombin

Reduced NO levels due to the deficiency of tetrahydrobiopterin (BH(4)) contribute to impaired vasodilation in pulmonary hypertension. Due to the chemically unstable nature of BH(4), it was hypothesised that oxidatively stable analogues of BH(4) would be able to support NO synthesis to improve endothelial dysfunction in pulmonary hypertension. Two analogues of BH(4), namely 6-hydroxymethyl pterin (HMP) and 6-acetyl-7,7-dimethyl-7,8-dihydropterin (ADDP), were evaluated for vasodilator activity on precontracted rat pulmonary artery rings. ADDP was administered to pulmonary hypertensive rats, followed by measurement of pulmonary vascular resistance in perfused lungs and eNOS expression by immunohistochemistry. ADDP and HMP caused significant relaxation in vitro in rat pulmonary arteries depleted of BH(4) with a maximum relaxation at 0.3μM (both P<0.05). Vasodilator activity of ADDP and HMP was completely abolished following preincubation with the NO synthase inhibitor, L-NAME. ADDP and HMP did not alter relaxation induced by carbachol or spermine NONOate. BH(4) itself did not produce relaxation. In rats receiving ADDP 14.1mg/kg/day, pulmonary vasodilation induced by calcium ionophore A23187 was augmented and eNOS immunoreactivity was increased. In conclusion, ADDP and HMP are two analogues of BH(4), which can act as oxidatively stable alternatives to BH(4) in causing NO-mediated vasorelaxation. Chronic treatment with ADDP resulted in improvement of NO-mediated pulmonary artery dilation and enhanced expression of eNOS in the pulmonary vascular endothelium. Chemically stable analogues of BH(4) may be able to limit endothelial dysfunction in the pulmonary vasculature.

Topics: Animals; Biopterins; Calcimycin; Calcium; Endothelium, Vascular; Gene Expression Regulation, Enzymologic; Hypertension, Pulmonary; Hypoxia; In Vitro Techniques; Ionophores; Male; Nitric Oxide; Nitric Oxide Synthase Type III; Pterins; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Vasodilation

The present study tested the hypothesis that chronic hypoxia alters pregnancy-mediated adaptation of Ca2+ homeostasis and contractility in the uterine artery. Uterine arteries were isolated from nonpregnant and near-term pregnant ewes of normoxic control or high-altitude (3820 m) hypoxic (oxygen pressure in the blood [PaO2], 60 mm Hg) treatment for 110 days. Contractions and intracellular-free Ca2+ concentration ([Ca2+]i) were measured simultaneously in the same tissue. In normoxic animals, pregnancy increased norepinephrine (NE), but not 5-hydroxy-thymide (5-HT) or KCl, contractile sensitivity in the uterine artery. Chronic hypoxia significantly attenuated NE-induced contractions in the pregnant, but not nonpregnant, uterine arteries. Similarly, 5-HT-mediated contractions of nonpregnant arteries were not changed. In the pregnant uterine artery, chronic hypoxia significantly increased NE-mediated Ca2+ mobilization, but decreased the Ca2+ sensitivity. In addition, hypoxia increased the calcium ionophore A23187-induced relaxation in pregnant, but not nonpregnant, uterine arteries. However, the A23187-mediated reduction of [Ca2+]i was significantly impaired in hypoxic arteries. In contrast, hypoxia significantly increased the slope of the [Ca2+]i-tension relationship of A23187-induced reductions in [Ca2+]i and tension in the pregnant uterine artery. The results suggest that the contractility of nonpregnant uterine artery is insensitive to moderate chronic hypoxia, but the adaptation of sympathetic tone that normally occurs in the uterine artery during pregnancy is inhibited by chronic hypoxia. In addition, changes in Ca2+ sensitivity of myofilaments play a predominant role in the adaptation of uterine artery contractility to pregnancy and chronic hypoxia.

Topics: Animals; Arteries; Calcimycin; Calcium; Chronic Disease; Female; Homeostasis; Hypoxia; Intracellular Membranes; Ionophores; Norepinephrine; Osmolar Concentration; Potassium Chloride; Pregnancy; Pregnancy, Animal; Serotonin; Sheep; Uterus; Vasoconstriction; Vasoconstrictor Agents; Vasodilation

Tissue damage after stroke is partly due to disruption of the blood-brain barrier (BBB). Little is known about the role of calcium in modulating BBB disruption. We investigated the effect of hypoxic and aglycemic stress on BBB function and intracellular calcium levels. Bovine brain microvessel endothelial cells were treated with A-23187 to increase intracellular calcium without hypoxia or treated with a calcium chelator (BAPTA) or calcium channel blockers (nifedipine or SKF-96365) and 6 h of hypoxia. A-23187 alone did not increase paracellular permeability. Hypoxia increased intracellular calcium, and hypoxia or hypoxia-aglycemia increased paracellular permeability. Treatment with nifedipine and SKF-96365 increased intracellular calcium under normoglycemic conditions, instead of blocking calcium influx, and was protective against hypoxia-induced BBB disruption under normoglycemia. Protection by nifedipine and SKF-96365 was not due to antioxidant properties of these compounds. These data indicate that increased intracellular calcium alone is not enough to disrupt the BBB. However, increased intracellular calcium after drug treatment and hypoxia suggests a potential mechanism for these drugs in BBB protection; nifedipine and SKF-96365 plus hypoxic stress may trigger calcium-mediated signaling cascades, altering BBB integrity.

Topics: Animals; Antioxidants; Blood-Brain Barrier; Calcimycin; Calcium; Calcium Channel Blockers; Cattle; Cell Membrane Permeability; Cells, Cultured; Chelating Agents; Egtazic Acid; Endothelium, Vascular; Hypoxia; Imidazoles; Intracellular Fluid; Ionophores; Nifedipine

Postmitotic neurons were generated from the human NT-2 teratocarcinoma cell line in a novel rapid differentiation procedure. These neurons were used to establish an in vitro assay system that allows the investigation of hypoxic/ischaemic cell damage and the development of neuroprotective strategies. In experiments of simulated ischaemia, the neurons were subjected to anoxia and hypoglycaemia. The viability of NT-2 neuronal cells was significantly reduced by anoxia especially in the presence of glutamate, reflecting the cellular vulnerability to excitotoxic conditions. The addition of the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 reduced glutamate-induced neuronal damage. Calcium imaging showed that NT-2 neurons increased cytosolic calcium levels in response to stimulation with glutamate or NMDA, an effect that was abolished in calcium free medium and at low pH values. The NMDA receptor antagonists MK-801, AP 5 and ketamine reduced the NMDA-induced response, suggesting the presence of functional NMDA receptors in the human neuronal cells. The mitochondrial potential of neurons was estimated using the fluorescent dye rhodamine 123 (R123). The fluorescence imaging experiments indicated an energetic collapse of mitochondrial functions during anoxia, suggesting that the human NT-2 neurons can be used to investigate subcellular processes during the excitotoxic cascade.

Topics: Aging; Calcimycin; Calcium; Cell Death; Cell Line, Tumor; Cell Survival; Dizocilpine Maleate; Dose-Response Relationship, Drug; Drug Interactions; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Fluorescein; Glutamic Acid; Humans; Hypoxia; Ionophores; Mitochondria; N-Methylaspartate; Neurons; Teratocarcinoma; Time Factors

Cysteinyl-leukotrienes (cys-LTs), potent mediators in inflammatory diseases, are produced by nervous tissue, but their cellular source and role in the brain are not very well known. In this report we have demonstrated that rat cultured astrocytes express the enzymes (5'-lipoxygenase and LTC(4) synthase) required for cys-LT production, and release cys-LTs in resting condition and, to a greater extent, in response to calcium ionophore A23187, 1 h combined oxygen-glucose deprivation or 2-methyl-thioATP, a selective P2Y(1)/ATP receptor agonist. MK-886, a LT synthesis inhibitor, prevented basal and evoked cys-LT release. In addition, 2-methyl-thioATP-induced cys-LT release was abolished by suramin, a P2 receptor antagonist, or by inhibitors of ATP binding cassette proteins involved in cys-LT release. We also showed that astrocytes express cys-LT(1) and not cys-LT(2) receptors. The stimulation of these receptors by LTD(4) activated the mitogen-activated protein kinase (MAPK) pathway. This effect was: (i) insensitive to inhibitors of receptor-coupled Gi protein (pertussis toxin) or tyrosine kinase receptors (genistein); (ii) abolished by MK-571, a cys-LT(1) selective receptor antagonist, or PD98059, a MAPK inhibitor; (iii) reduced by inhibitors of calcium/calmodulin-dependent kinase II (KN-93), Ca(2+)-dependent and -independent (GF102903X) or Ca(2+)-dependent (Gö6976) protein kinase C isoforms. LTD(4) also increased astrocyte proliferation and glial fibrillary acidic protein content, which are considered hallmarks of reactive astrogliosis. Both effects were counteracted by cell pretreatment with MK-571 or PD98059. Thus, cys-LTs released from astrocytes might play an autocrine role in the induction of reactive astrogliosis that, in brain injuries, contributes to the formation of a reparative glial scar.

Topics: Adenosine Triphosphate; Animals; Animals, Newborn; Astrocytes; Blotting, Northern; Blotting, Western; Calcimycin; Cell Division; Cell Survival; Cells, Cultured; Cerebral Cortex; Cysteine; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Glial Fibrillary Acidic Protein; Glucose; Glutathione Transferase; Hypoxia; Immunoenzyme Techniques; Ionophores; Leukotrienes; Lipoxygenase; Membrane Proteins; Mitogen-Activated Protein Kinases; Rats; Receptors, Leukotriene; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Thionucleotides; Thymidine; Time Factors; Tritium

The present study was designed to test the hypothesis that lack of oxygen in severely hypoxic tissue may inhibit arachidonic acid oxygenation and thereby result in an inhibition of eicosanoid synthesis. Hypoxia was induced in the isolated rabbit ear, and arachidonate metabolism and peripheral resistance of the preparation were monitored simultaneously. Severe hypoxia completely inhibited the biosynthesis of prostaglandin I(2) induced by ionophore A23187 and converted the vasodilatory response observed under normoxia into vasoconstriction. The cyclooxygenase 1 inhibitor SC560 (1 micromol/l) effectively inhibited the normoxic prostaglandin I(2) biosynthesis, while the cyclooxygenase 2 inhibitor DFU (1 micromol/l) did not. Neither SC560 nor DFU affected normoxic vasodilatory responses, indicating no involvement of prostanoids. The nitric oxide (synthase inhibitor Nomega-nitro-L-arginine methyl ester (100 micromol/l) converted the vasodilation into vasoconstriction, similar to what was observed under hypoxia, suggesting that the hypoxia-mediated conversion might occur due to the inhibition of nitric oxide.

Topics: Animals; Calcimycin; Ear; Epoprostenol; Female; Hypoxia; Ionophores; Male; Muscle, Smooth, Vascular; Rabbits; Vasodilation

Pulmonary hypertension and blunted pulmonary vascular responses to ACh develop when newborn pigs are exposed to chronic hypoxia for 3 days. To determine whether a cyclooxygenase (COX)-dependent contracting factor, such as thromboxane, is involved with altered pulmonary vascular responses to ACh, newborn piglets were raised in 11% O(2) (hypoxic) or room air (control) for 3 days. Small pulmonary arteries (100-400 microm diameter) were cannulated and pressurized, and their responses to ACh were measured before and after either the COX inhibitor indomethacin; a thromboxane synthesis inhibitor, dazoxiben or feregrelate; or the thromboxane-PGH(2)-receptor antagonist SQ-29548. In control arteries, indomethacin reversed ACh responses from dilation to constriction. In contrast, hypoxic arteries constricted to ACh before indomethacin and dilated to ACh after indomethacin. Furthermore, ACh constriction in hypoxic arteries was nearly abolished by either dazoxiben, feregrelate, or SQ-29548. These findings suggest that thromboxane is the COX-dependent contracting factor that underlies the constrictor response to ACh that develops in small pulmonary arteries of piglets exposed to 3 days of hypoxia. The early development of thromboxane-mediated constriction may contribute to the pathogenesis of chronic hypoxia-induced pulmonary hypertension in newborns.

Topics: Acetylcholine; Animals; Animals, Newborn; Calcimycin; Chronic Disease; Endothelium, Vascular; Female; Hypoxia; Male; Muscle Tonus; Muscle, Smooth, Vascular; Prostaglandin-Endoperoxide Synthases; Pulmonary Circulation; S-Nitroso-N-Acetylpenicillamine; Swine; Thromboxanes

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

1. Tonic contraction in response to K+ is well known to be highly dependent on aerobic metabolism in ileal muscle. The ionophore A23187 (10(-5) M) induced an initial contraction and successive rhythmic contraction in ileal muscle, but did not induce tonic contraction. This study, therefore, was performed to investigate the metabolic dependency during contraction induced by A23187. 2. Under hypoxic conditions, A23187 (10(-5) M) induced an initial contraction accompanied by an increase in lactate release. However, it induced only small rhythmic contractions and decreases in ATP, phosphocreatinine (PCr) and glycogen contents. 3. In glucose-free medium, A23187 (10(-5) M) induced an initial contraction and concomitant significant decrease in the ATP and glycogen contents. However, it did not induce successive rhythmic contractions. In 'glycogen-depleted muscle' stimulated repeatedly with 60 mM K+ in glucose-free medium, 60 mM K+ induced a phasic contraction without tonic contraction. A23187 induced no contraction under these conditions. 4. These results suggested that the initial contraction induced by A23187 was dependent on endogenous glycogen, while successive rhythmic contractions were dependent on aerobic metabolism after supply of external glucose to the ileal muscle.

Topics: Adenosine Triphosphate; Animals; Calcimycin; Electric Stimulation; Glucose; Glycogen; Guinea Pigs; Hypoxia; Ileum; Ionophores; Lactose; Male; Muscle Contraction; Muscle, Smooth; Phosphocreatine; Potassium

Chronic hypoxic pulmonary hypertension (PH) is associated with vasoconstriction and structural remodeling of pulmonary vessels including narrowing of the arterial lumen and loss of distal functional arteries. To test whether lung overexpression of the angiogenic factor vascular endothelial growth factor (VEGF) is beneficial in hypoxic PH, recombinant adenovirus encoding the human VEGF 165 gene under the control of a cytomegalovirus promoter (Ad. VEGF) or control vector containing no gene in the expression cassette (Ad.Null) was administered intratracheally to rats. With Ad. VEGF (10(8) plaque-forming units [pfu]), VEGF protein was present in bronchoalveolar lavage fluid as early as 2 d and until 17 d after gene transfer, but was not detected in serum. Only small patchy areas of mononuclear cells without cell damage, edema, or hemorrhage were observed on lung histology with no significant change in lung permeability. In rats pretreated with Ad.VEGF (10(8) pfu) 2 d before a 2-wk exposure to hypoxia (10% O(2)), lower values versus Ad. Null-pretreated controls were found for pulmonary artery pressure (25 +/- 1 versus 30 +/- 2 mm Hg, P < 0.05), right ventricular over left ventricular-plus-septum weight (0.37 +/- 0.01 versus 0.47 +/- 0. 02, P < 0.001), normalized wall thickness of 50- to 200-microm vessels (P < 0.001), and muscularization of distal vessels (P < 0. 001). Pretreatment with Ad.VEGF (10(8) pfu) increased endothelial nitric oxide synthase activity in lung tissue and partially restored endothelium-dependent vasodilation in isolated lungs from chronically hypoxic rats, as assessed by improvement of ionophore A23187-induced vasodilation and attenuation of endothelin-1 (300 pmol)-induced vasoconstriction, an effect abolished in the presence of nitro-L-arginine methylester. We conclude that adenoviral-mediated VEGF overexpression in the lungs attenuates development of hypoxic PH, in part by protecting endothelium-dependent function.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenoviridae; Animals; Bronchoalveolar Lavage Fluid; Calcimycin; Capillary Permeability; DNA, Recombinant; Dose-Response Relationship, Drug; Endothelial Growth Factors; Endothelin-1; Gene Expression Regulation; Gene Transfer, Horizontal; Humans; Hypertension, Pulmonary; Hypoxia; In Vitro Techniques; Ionophores; Lung; Lymphokines; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Rats; Rats, Wistar; Time Factors; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors; Vasodilation

Acute cell injury can activate intracellular phospholipase A2 (PLA2) and can inhibit plasma membrane aminophospholipid translocase(s). The latter maintains inner/outer plasma membrane phospholipid (PL) asymmetry. The mechanistic importance of PLA2-mediated PL breakdown and possible PL redistribution ("flip flop") to lethal tubule injury has not been well defined. This study was performed to help clarify these issues.. Proximal tubule segments (PTS) from normal CD-1 mice were subjected to either 30 minutes of hypoxia, Ca2+ ionophore (50 microM A23187), or oxidant attack (50 microM Fe). Lethal cell injury [the percentage of lactate dehydrogenase (LDH) release], plasma membrane PL expression [two-dimensional thin layer chromatography (TLC)], and free fatty acid (FFA) levels were then assessed. "Flip flop" was gauged by preferential decrements in phosphatidylserine (PS) versus phosphatidylcholine (PC; PS/PC ratios) in response to extracellular (Naja) PLA2 exposure.. Hypoxia induced approximately 60% LDH release, but no PL losses were observed. FFA increments suggested, at most 3% or less PL hydrolysis. Naja PLA2 reduced PLs in hypoxic tubules, but paradoxically, mild cytoprotection resulted. In contrast to hypoxia, Ca2+ ionophore and Fe each induced significant PL losses (6 to 15%) despite minimal FFA accumulation or cell death (26 to 27% LDH release). Arachidonic acid markedly inhibited PLA2 activity, potentially explaining an inverse correlation (r = -0.91) between tubule FFA accumulation and PL decrements. No evidence for plasma membrane "flip flop" was observed. In vivo ischemia reperfusion and oxidant injury (myohemoglobinuria) induced 0 and 24% cortical PL depletion, respectively, validating these in vitro data.. (a) Plasma membrane PLs are well preserved during acute hypoxic/ischemic injury, possibly because FFA accumulation (caused by mitochondrial inhibition) creates a negative feedback loop, inhibiting intracellular PLA2. (b) Exogenous PLA2 induces PL losses during hypoxia, but decreased cell injury can result. Together these findings suggest that PL loss may not be essential to hypoxic cell death. (c) Oxidant/Ca2+ overload injury induces early PL losses, perhaps facilitated by ongoing mitochondrial FFA metabolism, and (d) membrane "flip flop" does not appear to be an immediate mediator of acute necrotic tubular cell death.

Topics: Animals; Calcimycin; Cell Membrane; Hypoxia; Ionophores; Iron; Kidney Tubules, Proximal; Male; Membrane Lipids; Mice; Mice, Inbred Strains; Oxidants; Oxidative Stress; Phospholipases A; Phospholipases A2; Phospholipids

These studies tested whether fetal artery reactivity is sensitive to both acute changes in oxygen levels (in vitro) and chronic changes (in utero).. Pregnant guinea pigs near term were exposed to either normoxia or hypoxia (12% oxygen) for 4 or 7 days. The effect of decreasing PO (2 ) in vitro (acute hypoxia) on relaxation in response to acetylcholine, A23187, sodium nitroprusside, and 8-bromo-cyclic guanosine monophosphate was measured in isolated carotid arteries from normoxic fetuses. In separate experiments relaxation in response to acetylcholine and sodium nitroprusside of endothelially intact and denuded fetal arteries from fetuses exposed to normoxic conditions and long-term (4 and 7 days) hypoxic conditions was measured in the presence and absence of nitro-L -arginine (10(-4) mol/L).. Acute hypoxia inhibited endothelium-dependent relaxation in response to acetylcholine and A23187, increased sensitivity to sodium nitroprusside, but had no effect on relaxation in response to 8-bromo-cyclic guanosine monophosphate. Chronic hypoxia (4 but not 7 days) inhibited maximal relaxation of arteries in response to acetylcholine but not relaxation of arteries in response to sodium nitroprusside with respect to relaxation seen in arteries from normoxic fetuses. Nitro-L -arginine attenuated the differences between normoxic and hypoxic fetuses in acetylcholine response.. Hypoxia may alter relaxation of fetal arteries by decreasing the availability of oxygen for nitric oxide production and causing vascular adaptations related to altered nitric oxide release.

Topics: Acetylcholine; Acute Disease; Animals; Arteries; Calcimycin; Chronic Disease; Cyclic GMP; Disease Models, Animal; Female; Guinea Pigs; Hypoxia; Muscle, Smooth, Vascular; Nitric Oxide; Nitroprusside; Pregnancy; Vasodilator Agents

The management of sick newborn infants who have sustained a hypoxic insult is a common clinical problem but relatively little is known about the recovery process. The aim of this study was to investigate this process in newborn piglets.. Thirty five newborn piglets were exposed to chronic hypobaric hypoxia for three days, either from birth, three or 14 days of age, and were allowed to recover for one, three, or six days. Control animals of relevant age were also studied. The heart weight ratio and pulmonary arterial muscularity were measured. Endothelial dependent and independent relaxation of the isolated intrapulmonary conduit arteries was determined in classical organ chamber studies, together with measurement of basal and stimulated cGMP accumulation.. After six days of recovery the hypoxia induced right ventricular hypertrophy and pulmonary arterial medial hypertrophy had decreased in all animals but values were still abnormal in the two younger age groups. Relaxation was still impaired during the first three days of recovery in all groups, had normalised by six days in the two youngest groups, but relaxation (both endothelium dependent and independent) remained impaired in older animals. In these older animals basal nitric oxide (NO) production and basal and stimulated cGMP accumulation was normal.. The recovery of the smooth muscle cells lags behind that of the endothelial cells. A normal stimulated increase in cGMP with reduced relaxation suggests an altered threshold for cGMP effected relaxation. These findings help to explain why some hypoxic infants require protracted NO therapy.

Topics: Acetylcholine; Animals; Animals, Newborn; Calcimycin; Cyclic GMP; Enzyme Inhibitors; Hypertension, Pulmonary; Hypertrophy; Hypertrophy, Right Ventricular; Hypoxia; Nitric Oxide; omega-N-Methylarginine; Phosphodiesterase Inhibitors; Pulmonary Artery; Purinones; Swine; Tunica Intima; Vasoconstriction; Vasodilator Agents

We reported previously that ATP depletion induced by glycolytic inhibition or cellular hypoxia increases the permeability of intestinal epithelial monolayers [N. Unno, M. J. Menconi, A. L. Salzman, M. Smith, S. Hagen, Y. Ge, R. M. Ezzell, and M. P. Fink. Am. J. Physiol. 270 (Gastrointest. Liver Physiol. 33): G1010-G1021, 1996]. In the present study, we examined the effects of the Ca2+ ionophore A-23187 or the intracellular ionized Ca2+ concentration ([Ca2+]i) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM on the permeability of enterocytic (T84) monolayers depleted of ATP by metabolic inhibition. Permeability was assessed by measuring transepithelial electrical resistance and the transepithelial flux of fluorescein sulfonic acid. Although neither A-23187 nor BAPTA-AM affected ATP depletion, A-23187 augmented, whereas BAPTA-AM ameliorated, chemical hypoxia-induced hyperpermeability. BAPTA-AM ameliorated chemical hypoxia-induced cytoskeletal derangements. Monolayers subjected to chemical hypoxia but incubated in a low (i.e., 100 microM) [Ca2+] environment showed preservation of junctional integrity, whereas voltage-dependent Ca2+ channel blockers (NiCl2 or verapamil) failed to ameliorate chemical hypoxia-induced hyperpermeability. ATP depletion induces hyperpermeability in intestinal epithelial monolayers via a [Ca2+]i-dependent mechanism. Increased [Ca2+]i under these conditions reflects leakage of Ca2+ from the extracellular milieu via a mechanism unrelated to voltage-dependent Ca2+ channels.

Topics: Adenosine Triphosphate; Calcimycin; Calcium; Calcium Channel Blockers; Cytosol; Egtazic Acid; Enzyme Inhibitors; Epithelial Cells; Humans; Hypoxia; Intestinal Mucosa; Intracellular Membranes; Ions; Microscopy, Fluorescence; Osmolar Concentration; Permeability; Phospholipases A; Tumor Cells, Cultured

In light of evidence that hypoxia inhibits cerebrovascular calcium influx and that the contractile importance of calcium influx is greater in neonatal than adult cerebral arteries, the present studies evaluate the hypothesis that the mechanisms involved in hypoxic cerebral vasodilation are different in newborn and adult cerebral arteries. Intact segments of ovine common carotid (COM) and middle cerebral (MCA) arteries from 3- to 7-day-old newborn lambs and nonpregnant adults were mounted in tissue baths, contracted with 1 microM 5-HT, and exposed to hypoxia for 20 min (PO2 approximately 12 Torr). In endothelium-intact arteries, the magnitude of relaxation was similar and near maximal in all groups (range 82-92%) and did not vary with either age or artery type. However, the magnitude of hypoxic relaxation after endothelium removal was attenuated more in adult (COM 37 +/- 6%, MCA 53 +/- 7%) than in newborn (COM 75 +/- 5%, MCA 93 +/- 1%) arteries. In addition, endothelium-dependent responses to A23187, which like hypoxia increases endothelial cell concentrations of calcium, were attenuated in immature compared to mature arteries. Reductions of extracellular calcium concentration from 1.6 to 0.8 mM eliminated all age-dependent differences in relaxation responses to hypoxia. Together these data demonstrate that in newborn arteries, the endothelium-dependent component of hypoxic vasodilation is minimal, especially in intracerebral arteries, and robust relaxant responses to hypoxia depend on the high sensitivity of immature smooth muscle cells to decreases in PO2. In contrast, in adult arteries the smooth muscle sensitivity to decreases in PO2 is minimal and the endothelial component of hypoxic relaxation is particularly important in responses to hypoxia.

Topics: Adenosine Diphosphate; Aging; Animals; Animals, Newborn; Calcimycin; Calcium; Carotid Arteries; Cerebral Arteries; Endothelium, Vascular; Hypoxia; Ionophores; Receptors, Purinergic P2; Sheep; Vasodilation

Neonatal pulmonary hypertension is associated with increased pulmonary vascular reactivity. We studied the responses of isolated porcine intrapulmonary arteries after exposure of piglets to chronic hypobaric hypoxia (CHH) from 0 to 2.5, 3 to 6, or 14 to 17 days of age. CHH inhibited the postnatal development of endothelium-dependent vasorelaxation to acetylcholine (ACh) and the calcium ionophore A-23187. Basal accumulation of guanosine 3', 5'-cyclic monophosphate (cGMP) was unaffected, but cGMP response to ACh was inhibited. Endothelium-independent relaxation to nitric oxide (NO) and zaprinast (a phosphodiesterase inhibitor) was also inhibited, but cGMP accumulation in response to these agonists was normal. The ability of sodium nitroprusside (SNP) to cause vasorelaxation and increase cGMP accumulation was unaffected. Contractile responses to potassium chloride and prostaglandin F2 alpha (PGF2 alpha) were similar to normal after exposure from birth and 3 days and were decreased in the older group, but the ability of NG-monomethyl-L-arginine acetate to increase PGF2 alpha-induced contractions decreased. Thus exposure of newborn piglets to CHH causes 1) no increase in contractile responses and 2) impairment of endothelium-dependent and -independent relaxation by impairing signal transduction mechanisms involved in the release of NO and the effectiveness of cGMP.

Topics: Acetylcholine; Animals; Animals, Newborn; Atmospheric Pressure; Calcimycin; Chronic Disease; Cyclic GMP; Dinoprost; Endothelium, Vascular; Heart; Hypertension, Pulmonary; Hypoxia; In Vitro Techniques; Nitric Oxide; omega-N-Methylarginine; Potassium Chloride; Pulmonary Artery; Purinones; Swine; Vasodilation; Vasomotor System

The Bcl-2 family and the ICE family of cysteine proteases play important roles in regulating cell death. We show here that induction of cell death by a Ca2+ ionophore or hypoxia results in increased levels and activity of active ICE(-like) proteases and the subsequent activation of CPP32/Yama(-like) proteases, and that inhibition of these protease activities reduces the extent of cell death. Overexpression of the anti-apoptotic proteins Bcl-2 or Bcl-xL inhibits the cell death and the activation of ICE(-like) and CPP32/Yama(-like) proteases, indicating that Bcl-2 and Bcl-xL act upstream of these proteases. We also show that specific inhibition of ICE(-like) proteases in vivo prevents activation of CPP32/Yama(-like) proteases, whereas inhibition of CPP32/Yama(-like) proteases does not prevent activation of ICE(-like) proteases, suggesting the existence of a protease cascade in vivo that requires ICE(-like) proteases for activation of CPP32/Yama(-like) proteases. Induction of necrotic cell death by KCN also induces activation of ICE(-like) proteases but not of CPP32/Yama(-like) proteases, and Bcl-2 and Bcl-xL inhibit the activation and the cell death, suggesting that the functional site of Bcl-2 and Bcl-xL is also upstream of ICE(-like) proteases in at least some forms of necrosis.

Topics: Adrenal Gland Neoplasms; Animals; Apoptosis; Calcimycin; Caspase 1; Caspase 3; Caspases; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Enzyme Activation; Humans; Hypoxia; Ionophores; Oligopeptides; Pheochromocytoma; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Tumor Cells, Cultured

The effects of glucose deprivation, hypoxia and glucose-free hypoxia conditions on phosphoinositide (PI) hydrolysis were studied in cortical slices from 8-day-old rats. Only glucose-free hypoxia induced a significant increase of inositol phosphate formation. The inositol phosphate formation induced by noradrenaline, carbachol and several excitatory amino acid receptor agonists, but not the Ca2+ ionophore A23187-induced stimulation, was blocked by glucose-free hypoxia and differentially reduced by glucose and oxygen deprivation depending on the neurotransmitter receptor agonist. The stimulatory effect of glucose-free hypoxia was not reduced by the muscarinic receptor antagonist atropine or by the inhibitors of the excitatory amino acid-stimulated PI hydrolysis DL-2-amino-3-phosphono-propionic acid and L-aspartate-beta-hydroxamate, and neither by the voltage-sensitive Na+ channel tetrodotoxin. The effect of glucose-free hypoxia was partially dependent on extracellular Ca2+ and it was blocked by verapamil and amiloride, but not by nifedipine, Co2+ and neomycin. These results suggest that Ca2+ influx through the Na(+)-Ca2+ exchanger underlies the PI hydrolysis stimulation induced by combined glucose and oxygen deprivation in neonatal cerebral cortical slices.

Topics: Animals; Animals, Newborn; Calcimycin; Calcium Channels; Carrier Proteins; Cerebral Cortex; Glucose; GTP-Binding Proteins; Hydrolysis; Hypoxia; In Vitro Techniques; Phosphatidylinositols; Potassium Channels; Rats; Rats, Wistar; Receptors, Glutamate; Sodium-Calcium Exchanger

The effects of hypoxia followed by reoxygenation on endothelium-dependent relaxation in isolated rat aorta were investigated. Acetylcholine (ACh, 3 nM-10 microM) and calcium ionophore A-23187 (3 nM-300 nM)-induced endothelium-dependent vasorelaxation of isolated rate aortic vessel rings was impaired after 15 min of hypoxia followed by 30 min of reoxygenation. Impairment of ACh-induced relaxation was prevented by pretreatment with the combination of superoxide dismutase (200 U/ml) and catalase (1,000 U/ml). Hypoxia-reoxygenation did not affect sodium nitroprusside (0.1 nM-1 microM)-induced endothelium-independent relaxation nor the dissociation constant of ACh to endothelial M3 muscarinic receptors. Propidium iodide staining of the vascular endothelium revealed a significant increase in the number of dead endothelial cells on the aortic vessel rings following hypoxia-reoxygenation, but not on those pretreated with superoxide dismutase and catalase. These results suggest that hypoxia-reoxygenation impairs endothelium-dependent relaxation of rat aorta by a mechanism that involves oxidant-mediated endothelial cell death.

Topics: Acetylcholine; Animals; Aorta; Binding, Competitive; Calcimycin; Catalase; Cell Survival; Endothelium, Vascular; Hypoxia; In Vitro Techniques; Male; Nitroprusside; Oxygen; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; Superoxide Dismutase; Vasodilation

The present study utilized a monoculture of vascular endothelium to: (1) determine if nitric oxide (NO) production was decreased during hypoxia, (2) ascertain if specific prostaglandins were released in response to hypoxia, and (3) determine if cyclo-oxygenase inhibition would modulate hypoxia-induced decreases in NO production.. Bovine aortic endothelial cells (BAE cells) were grown to confluence on microcarrier beads. NO released in response to the receptor-independent agonist, A23187 calcium ionophore, was directly and continuously measured using a sensitive and specific chemiluminescence method. Cells were exposed to either "hypoxia" (pO2 = 10 mmHg) or "normoxia" (pO2 = 160 mmHg) for 30 min. NO was quantitatively measured with and without indomethacin (1.7 microM) in the incubation medium, and also following incubation with the prostacyclin analog, iloprost. The prostaglandins PGI2 and PGE2 released in response to hypoxia were quantitated using an enzyme immunoassay.. Hypoxia significantly decreased NO production, resulting in a 22.8(2.1)% reduction in NO from 94.3(5.3) nmol/micrograms protein (during normoxia) to 73.5(2) nmol/micrograms protein (during hypoxia). Hypoxia significantly stimulated the production of PGI2 and PGE2, in excess of that released in response to A23187 when compared with normoxia. Following cyclo-oxygenase inhibition, the hypoxia-induced decrease in NO production was abolished (0.13 [2.7] % change relative to controls). Furthermore, iloprost (10 nM) directly inhibited NO production.. The results demonstrate that ionophore-stimulated NO production is sensitive to oxygen tension, decreasing in response to hypoxia. Inhibition of prostaglandin synthesis restores NO production during hypoxia, while iloprost directly suppresses NO production. Thus, endothelium-derived prostanoids produced in response to hypoxia may modulate NO production via an autocrine negative feedback mechanism.

Topics: Animals; Calcimycin; Cattle; Cyclooxygenase Inhibitors; Dinoprostone; Endothelium, Vascular; Epoprostenol; Feedback; Hypoxia; Iloprost; Indomethacin; Ionophores; Nitric Oxide; Vasodilator Agents

Acute hypoxia causes pulmonary hypertension in the fetus and newborn that is contrasted by systemic hypotension or normotension. To better understand the role of nitric oxide (NO) in this specific pulmonary vascular response, we determined the acute effects of decreased oxygenation on NO production in ovine fetal pulmonary and systemic (mesenteric) endothelial cells. NO was assessed by measuring cGMP accumulation in fetal vascular smooth muscle (VSM) cells during co-culture incubations of endothelium and VSM (40 s) in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. Changes in cGMP were dependent on the endothelium and on NO synthase and guanylate cyclase activity. At high O2 (680 mm Hg), basal NO was detectable and NO increased 6- to 10-fold with bradykinin or A23187. In pulmonary endothelium, basal NO fell 58% at pO2 = 150 mm Hg and 51% at 40 mm Hg versus 680 mm Hg, while NO with bradykinin fell 56% and 63%, respectively. NO with A23187, however, was unchanged at 150 mm Hg, but it fell 56% at 40 mm Hg. In contrast, in systemic endothelium basal and stimulated NO production were not altered at lower O2. Findings were similar using pulmonary or systemic detector VSM cells, and exogenous L-arginine had no effect. Thus, decreased O2 acutely attenuates NO production specifically in fetal pulmonary endothelial cells. This process is not related to changes in O2 or L-arginine availability as substrates for NO synthase; alternatively, it may be partially mediated by specific effects of O2 on pulmonary endothelial cell calcium homeostasis.

Topics: 1-Methyl-3-isobutylxanthine; Amino Acid Oxidoreductases; Animals; Bradykinin; Calcimycin; Cells, Cultured; Cyclic GMP; Disease Models, Animal; Endothelium, Vascular; Female; Guanylate Cyclase; Humans; Hypoxia; Infant, Newborn; Lung; Mesenteric Arteries; Muscle, Smooth, Vascular; Nitric Oxide; Nitric Oxide Synthase; Nitroprusside; Oxygen; Persistent Fetal Circulation Syndrome; Pregnancy; Pulmonary Artery; Sheep

Chronic hypoxic exposure elicits pulmonary vascular remodeling and may alter normal pulmonary endothelial function. We examined the vasodilatory response to the receptor-mediated endothelium-dependent dilator arginine vasopressin (AVP), the non-receptor-mediated endothelium-dependent dilator A-23187, and the nitric oxide (NO) donor sodium nitroprusside in lungs isolated from control or chronically hypoxic rats. Lungs were isolated from male Sprague-Dawley rats and perfused with a physiological saline solution containing 4% albumin. Arterial and venous pressures were monitored and microvascular pressure was estimated by double occlusion, allowing assessment of segmental resistances. After equilibration, lungs were constricted with the thromboxane mimetic U-46619. Upon development of a stable pressor response, lungs were dilated with one of the above agents. A series of doses of AVP was administered to separate groups of lungs from control or chronically hypoxic rats. Lungs from chronically hypoxic rats exhibited an augmented dilatory response to AVP compared with control lungs, and this effect was due to enhanced dilation of precapillary segments. The total and segmental vasodilatory responses to A-23187 and sodium nitroprusside were not different between the two groups of lungs, suggesting that chronic hypoxia did not upregulate the enzyme NO synthase or enhance the vascular smooth muscle responsiveness to NO. Thus our data suggest that the augmented total and pulmonary arterial dilation to AVP after chronic hypoxia is most likely due to altered receptor-mediated processes of the hormone.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Amino Acid Oxidoreductases; Animals; Arginine Vasopressin; Calcimycin; Capillary Resistance; Dose-Response Relationship, Drug; Hypoxia; Male; Nitric Oxide; Nitric Oxide Synthase; Nitroprusside; Perfusion; Prostaglandin Endoperoxides, Synthetic; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Receptors, Vasopressin; Thromboxane A2; Vasodilation

Rat embryonic hippocampal neurons cultured on astrocyte feeder-layers were sensitive to different excitotoxic stimuli after 10-12 DIV. Almost all neurons (approximately 95%) died within 20 h after a transient exposure for 10 min to 50 microM glutamate, a continuous exposure to either 25 microM NMDA or 250 microM kainate or after a 15-min deprivation of glucose and oxygen. Dizocilpine at 10 microM protected neurons against the glutamate- and NMDA-mediated toxicity as well as against 30 min glucose and oxygen deprivation. However, it failed to protect against kainate toxicity and prolonged glucose/oxygen deprivation (60 min). An additional treatment with CNQX (100 microM) protected neurons even under the latter two conditions. This indicates that the vast majority of neurons was sensitive to different excitotoxic stimuli acting through different types of glutamate receptors leading to calcium overload of the cells which might be the common denominator of triggering cell death under these conditions. Expression of calcium-binding proteins, such as calbindin D28K or calretinin, might increase the intracellular calcium buffer capacity of neurons, thus, rendering them more resistant to calcium overload. Therefore, we analysed whether neurons expressing these calcium-binding proteins would survive these toxic stimuli. Indeed, a small population of the neurons (3-5%) survived, including a subpopulation of calretinin-positive but not calbindin D28K-positive neurons. This implies that the expression of calcium-binding proteins per se does not render neurons more resistant towards these excitotoxic stimuli. Moreover, most of the surviving calretinin-positive neurons showed morphological damage as indicated by loss of neurites. When cytotoxicity due to calcium overload was induced by an exposure of the cells to the calcium ionophore 4-bromo-A23187 rather than by activation of glutamate receptors, calretinin-positive cells were found not to be significantly more resistant than the vast majority of neurons. This may indicate that the lower sensitivity of a subpopulation of calretinin-positive neurons to excitotoxic stimuli may be due to a lower expression of glutamate receptors.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Calbindin 1; Calbindin 2; Calbindins; Calcimycin; Cell Death; Cells, Cultured; Dizocilpine Maleate; Glucose; Glutamic Acid; Hippocampus; Hypoxia; Immunohistochemistry; Kainic Acid; Mice; Nerve Tissue Proteins; Neurons; Oxygen Consumption; Rats; S100 Calcium Binding Protein G

The objective of this study was to investigate the effect of hypoxia on the adhesiveness of endothelial cells for granulocytes. Human umbilical vein endothelial cells (HUVEC) were exposed to a PO2 of 7.5 mmHg (1.0 kPa), and the adherence of granulocytes was assessed under continuous hypoxia by means of a hypoxic incubator room. After 2 h of hypoxia the adherence of granulocytes decreased to 50% of the normoxic control, which was not due to a decreased viability of the endothelial cells nor to an increased generation of the antiadhesive factors nitric oxide, prostacyclin, and adenosine. Hypoxia also had no effect on the expression of intercellular adhesion molecule (ICAM)-1 or ICAM-2 on the endothelium. Although the mechanism of the action of hypoxia on the adhesiveness of endothelial cells remains unclear as yet, our data suggest that HUVEC possess a protective mechanism that prevents granulocyte adherence to endothelial cells under extreme hypoxic conditions. The decreased adherence seems paradoxical to the in vivo situation for which the increased margination of granulocytes within the vascular compartment of the ischemic tissue has been observed. However, hypoxia did not impair the potential adhesiveness of HUVEC, since stimulation of endothelial cells under hypoxic conditions with calcium ionophore or lipopolysaccharide increased the adherence of granulocytes in a similar fashion as under normoxic conditions. We therefore conclude that the increased margination of granulocytes during ischemia may be accomplished by the additional stimulation of hypoxic endothelial cells.

Topics: Adenosine; Calcimycin; Cell Adhesion; Cell Adhesion Molecules; Cells, Cultured; Culture Media; Endothelium, Vascular; Epoprostenol; Granulocytes; Humans; Hypoxia; Leukocytes; Lipopolysaccharides; Nitric Oxide; Superoxides

We have previously shown alterations in cerebrovascular composition, contractility, and endothelial function in normoxic pregnant (P) and chronically hypoxic nonpregnant (HNP) adult sheep compared with nonpregnant normoxic controls (NP). This study focuses on a fourth group, pregnant sheep exposed to chronic high-altitude hypoxia (HP) (110 days at 3,820 m). The combined challenges of pregnancy and high-altitude hypoxia resulted in significant alterations in cerebrovascular function that were not simply the summation of the responses seen in the P and HNP animals. Compared with NP, HP arteries had increased protein content and increased intracranial arterial wall thickness. Both P and HP arteries showed increased contractility, while HNP artery maximum tensions were depressed. Endothelial function was depressed in the P common carotid and was enhanced in all HNP arteries. In contrast, an increased endothelial response was shown only in the HP common carotid. Thus, for contractility, the effects of pregnancy predominated in the HP arteries. For endothelial function, hypoxia effects were dominant in the common carotid but not in the intracranial arteries. Clearly, cerebrovascular characteristics are dynamic in nature, with artery-specific responses to both pregnancy and hypoxia.

Topics: Adaptation, Physiological; Altitude; Animals; Blood Vessels; Calcimycin; Cerebrovascular Circulation; Female; Hypoxia; Penicillamine; Pregnancy; Pregnancy Complications; S-Nitroso-N-Acetylpenicillamine; Sheep; Vasoconstriction; Vasodilation

Hypoxic pulmonary hypertension complicates many primary respiratory and cardiac conditions. To define the potential role of endothelial nitric oxide (NO) further in both the acute and chronic forms of this disorder, we determined the effects of acute changes in O2 in vitro and prolonged variations in O2 in vivo on endothelial NO production in rat main pulmonary arteries. NO production was assessed by measuring segment cyclic GMP synthesis, which was dependent on the presence of the endothelium and on NO synthase and soluble guanylate cyclase activity. With an acute decrease in pO2 in vitro from 150 to 40 mm Hg, basal endothelial NO production was attenuated by 52%. NO production stimulated by acetylcholine (ACh) or A23187, however, was not altered, suggesting that the underlying mechanism involves acute changes in endothelial intracellular calcium homeostasis or in the production or action of a local activator of endothelial NO synthase. Although prolonged hypoxia in vivo (7 days) also caused a 52% decrease in basal endothelial NO production, ACh- and A23187-stimulated production were diminished as well, by 69 and 73%, respectively; the attenuation in NO production was evident when tested at high pO2 in vitro, was not altered by exogenous L-arginine, and was reversed by 3 days of normoxic recovery, indicating that the chronic process may involve diminished availability of cofactor(s) required for NO synthase activity. Parallel studies of aortic segments showed that these effects are specific to the pulmonary endothelium. Thus, both acute and prolonged hypoxia selectively attenuate pulmonary endothelial NO production by different mechanisms.

Topics: Acetylcholine; Amino Acid Oxidoreductases; Animals; Arginine; Calcimycin; Cyclic GMP; Dose-Response Relationship, Drug; Endothelium, Vascular; Hypoxia; In Vitro Techniques; Male; Nitric Oxide; Nitric Oxide Synthase; Oxygen Consumption; Pulmonary Artery; Rats; Rats, Sprague-Dawley

To explore endothelium-dependent relaxation and the L-arginine (L-ARG)-nitric oxide (NO) pathway during chronic hypoxia, we examined isolated rings from large conduit pulmonary arteries and aorta from rats exposed to either room air (N), 3-week hypoxia (H), or 3-week H followed by 72-h recovery to normoxia (room air). We examined the vasodilatory actions of acetylcholine (ACh), ionophore A23187, and endothelin-3 (ET-3) on extrapulmonary left and right branches of pulmonary arteries and thoracic aorta precontracted by phenylephrine (PE 10(-6) M). Endothelium-dependent relaxation of N rat pulmonary arteries and aorta to ACh and A23187 was abolished in the presence of L-NG nitroarginine methyl ester (L-NAME 10(-4) M) or methylene blue (MB 10(-5) M) but was suppressed only partially by NG-monomethyl-L-arginine (L-NMMA 5 x 10(-4) M). In pulmonary arteries but not in aorta, ET-3 induced endothelium-dependent relaxation that was suppressed by L-NAME, MB, and L-NMMA. Pulmonary arteries from H rats did not relax with ET-3. As compared with those of N rats, they exhibited less relaxation to ACh and A23187, (47 +/- 3 vs. 89 +/- 2 and 53 +/- 2 vs. 85 +/- 4%, p < 0.001, respectively) but exhibited similar relaxation to the nonendothelium-dependent vasodilator linsidomine. In contrast, endothelial-relaxation did not differ between N and H rat aorta.2+ pretreatment with L-ARG.

Topics: Acetylcholine; Animals; Arginine; Calcimycin; Endothelins; Endothelium, Vascular; Hypoxia; In Vitro Techniques; Male; Molsidomine; Muscle Relaxation; Muscle, Smooth, Vascular; Phenylephrine; Pulmonary Artery; Rats; Rats, Wistar; Vasodilation; Vasodilator Agents

The human hepatoma cell line, Hep 3B, produces biologically active erythropoietin (Epo) in response to normal physiologic stimuli and thus provides a model system for the study of Epo regulation. The addition of phorbol 12-myristate 13-acetate (PMA) to Hep 3B cells subsequently grown under hypoxic conditions resulted in a dose-dependent inhibition of hypoxia-induced Epo production by as much as 95 +/- 1% with half-maximal inhibition at 8 ng/mL. By Northern blot analysis, Epo mRNA levels were correspondingly decreased after treatment with PMA. Direct measurement of both membrane and cytosolic protein kinase C activity in Hep 3B cells following treatment with PMA demonstrated a biphasic response as a function of time. Membrane-associated protein kinase C activity initially increased but subsequently decreased to baseline levels by 12 hours. The PMA-induced inhibition of hypoxia-induced Epo production was shown to occur as early as 3 hours after PMA addition, suggesting that the initial activation, rather than the subsequent decrease in protein kinase C activity, is of primary importance. The relative specificity of the PMA-induced inhibition of Epo production was demonstrated by 1) the finding that overall protein and RNA synthesis were not similarly decreased as measured by 3H-leucine and 3H-uridine pulse labeling studies and 2) the observation that the biologically inactive phorbol ester, 4 alpha-phorbol didecanoate, failed to have any effect on hypoxia-induced Epo production. In addition, the synthetic analog of diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG) and the calcium ionophore, A23187, inhibited hypoxia-induced Epo production up to 85 +/- 3% and 82 +/- 4%, respectively, in a dose-dependent manner. Taken together, these findings suggest that hypoxia-induced Epo production may be negatively regulated by activators of a protein kinase C-mediated pathway.

Topics: Blotting, Northern; Calcimycin; Carcinoma, Hepatocellular; Diglycerides; DNA; Dose-Response Relationship, Drug; Erythropoietin; Gene Expression Regulation, Neoplastic; Humans; Hypoxia; Leucine; Liver Neoplasms; Protein Kinase C; Radioimmunoassay; RNA; Tetradecanoylphorbol Acetate; Tritium; Tumor Cells, Cultured; Uridine

Hypoxia has major effects on endothelium-dependent relaxation. To further understand the underlying mechanism(s), we investigated the O2 dependence of the endothelium-dependent relaxations elicited by ionophore A-23187 or agonists substance P (SP) or thrombin (TB) in porcine coronary arteries. A-23187 elicits an endothelium-dependent relaxation of KCl- or U-46619-induced contractures that can be described in terms of a rapid and slow phase. The duration of the relaxation was dose dependent. SP (10 nM) and TB (0.1 U/ml) also elicited endothelium-dependent relaxations that were rapid but transient. Hypoxic conditions (95% N2-5% CO2 instead of 95% O2-5% CO2; PO2 < 1%) abolished the A-23187 rapid phase and the SP and TB transient relaxation but not the A-23187 slow phase. Threshold PO2 for the rapid phase was approximately 35 mmHg. Pretreatment with cyanide (5 mM), to inhibit respiration, or 2-deoxy-D-glucose, to inhibit glycolysis, had little effect. Similarly, propranolol (10 microM) or indomethacin (10 microM) had no effect on the relaxation to A-23187, TB, or SP. In contrast, both NO synthesis inhibitors and ouabain blunted all endothelium-dependent relaxations studied. Our results suggest that the rapid relaxations to A-23187, SP, and TB are sensitive to O2 but not mitochondrial respiration. The slow sustained relaxation induced by A-23187, however, is characterized by a sensitivity to O2 that is distinct from that of the rapid phase, yet is dependent on an intact endothelium and is affected by NO synthesis inhibitors. Thus the endothelium-dependent relaxation to A-23187 is probably mediated by NO, but its sensitivity to O2 suggests that two distinct mechanisms may be involved.

Topics: Animals; Calcimycin; Coronary Vessels; Culture Media; Endothelium, Vascular; Hypoxia; In Vitro Techniques; Nitric Oxide; Nitroprusside; Oxygen; Oxygen Consumption; Vasodilation

Primary cultures of rat Kupffer cells liberated significant amounts of prostaglandin (PG) D2, PGE2, and thromboxane (measured as thromboxane B2) when exposed to reoxygenation after 4 h of hypoxia. After a delayed onset, prostanoids were released at high rates for at least 8 h and after that time 700 pmol PGD2, 280 pmol PGE2, and 200 pmol thromboxane per 10(6) cells had been liberated. Unlike prostanoid release, leukotriene B4 production in reoxygenated cell cultures was only twice as much as in aerobic controls. Superoxide dismutase and catalase had no effect on PGD2, PGE2, and thromboxane production, indicating that prostanoid formation was independent of reactive oxygen species generated extracellularly and of cell injury. On the other hand, diphenyliodonium, as well as amiloride, blocked hypoxia-reoxygenation-induced PGD2, PGE2, and thromboxane release. The elevated prostanoid synthesis was preceded by increases in intracellular pH (from 7.23 to 7.38) and in intracellular Ca2+ (from 55 nM to a maximum level of 807 nM). These observations suggest a participation of NADPH oxidase and a related Na(+)-H+ exchange in the enhanced prostanoid synthesis, probably through the induction of an increased intracellular Ca2+ concentration.

Topics: Amiloride; Animals; Anti-Infective Agents; Biphenyl Compounds; Calcimycin; Calcium; Carrier Proteins; Cell Survival; Cells, Cultured; Dinoprostone; Hydrogen-Ion Concentration; Hypoxia; Kupffer Cells; Leukotriene B4; Male; NADH, NADPH Oxidoreductases; NADPH Oxidases; Onium Compounds; Oxidation-Reduction; Oxygen; Prostaglandin D2; Rats; Rats, Wistar; Sodium-Hydrogen Exchangers; Tetradecanoylphorbol Acetate; Thromboxane B2

Isolated hepatocytes, suspended in an organ preservation solution, can be preserved at 4 degrees C for up to 6 days. After preservation, normothermic-normoxic incubation causes loss of hepatocyte viability. The addition of 3 mmol/L glycine to the rewarming medium prevents the loss of viability. In this study we investigated the cytoprotective effects of glycine under many conditions known to cause hepatocellular injury to understand the mechanism of cold-induced injury in the liver. Hepatocytes were suspended in modified Krebs-Henseleit buffer with or without 3 mmol/L glycine and exposed to agents or conditions known to induce cell death. Hepatocyte viability was assessed by measuring the percentage of lactate dehydrogenase leakage from the cells and the concentration of ATP during incubation at 37 degrees C under room air for up to 90 min. Mitochondrial inhibitors (potassium cyanide and carbonyl cyanide m-chlorophenylhydrazone); calcium ionophores (ionomycin and A23187); an oxidizing agent, tert-butyl hydroperoxide; and anoxia were all used to cause cell injury. Hepatocytes were also isolated from fasted rats and hypothermically preserved as another model of cell death. Other amino acids were also tested in the hypothermic preservation model to study the specificity of the amino acid requirement for prevention of lactate dehydrogenase leakage. Of the amino acids tested, only alanine (10 mmol/L) and the combination of alanine (3 mmol/L) and serine (3 mmol/L) were as effective as glycine in preventing lactate dehydrogenase release in the hypothermic preservation model.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acids; Animals; Calcimycin; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cold Temperature; Glycine; Hypoxia; Ionomycin; Ischemia; L-Lactate Dehydrogenase; Liver; Liver Circulation; Mitochondria; Potassium Cyanide; Rats; Rats, Sprague-Dawley

In the fetus and infant, high-altitude hypoxemia is associated with increased cerebrovascular morbidity. To test the hypothesis that this increased morbidity involves changes in cerebrovascular endothelial and smooth muscle function, we examined middle cerebral, posterior communicating, basilar, and common carotid arteries obtained from 23 normoxic fetuses, 19 hypoxemic fetuses maintained at high altitude (3,820 m) from 30 days gestation to near term (approximately 143 days), 55 normoxic non-pregnant adults, and 24 hypoxemic nonpregnant adults maintained at the same altitude and duration as the hypoxemic fetuses. Long-term hypoxemia was associated with several significant changes in both fetal and adult arteries, including a generalized increase in base-soluble protein (5-50%), a depression of the maximum potassium-induced tensions (16-49%), and a depression of the relaxation responses to S-nitroso-N-acetylpenicillamine (1-11%), which releases nitric oxide into solution upon hydration. Altitude acclimatization significantly enhanced amine-to-potassium ratios (the ratio of tension produced by 10 microM serotonin with 20 microM histamine to that produced by 122 mM potassium) only in adult cerebral arteries (51-87%) and significantly depressed potassium-induced stresses (up to 41%) and serotonin/histamine-induced tensions (20-37%) only in fetuses. Endothelium-dependent relaxations to A23187 were significantly depressed in hypoxemic fetuses (4-11%) but were significantly enhanced in hypoxemic adults (2-14%). We conclude that chronic hypoxemia depresses both vascular smooth muscle and endothelial function to a greater extent in fetal than in adult cerebral arteries and that this effect could contribute to the greater postnatal vulnerability to asphyxic and hypertensive insults seen in hypoxemic neonates.

Topics: Acclimatization; Aging; Altitude; Analysis of Variance; Animals; Body Water; Calcimycin; Cerebral Arteries; Cerebrovascular Circulation; Fetus; Hypoxia; In Vitro Techniques; Proteins; Rest; Sheep; Vasoconstriction

We have previously demonstrated that arginine vasopressin (AVP) dilates the preconstricted pulmonary vasculature via the release of nitric oxide (NO). However, recent evidence suggests that NO release in response to other agents may be suppressed in lungs from animals that have been chronically exposed to hypoxia. The purpose of the present experiment was to determine whether vasopressinergic pulmonary vasodilation is similarly affected by chronic exposure to hypoxia (barometric pressure = 380 Torr for 4 wk). Inhibition of NO synthesis with N omega-nitro-L-arginine (L-NNA) had no effect on baseline perfusion pressure in isolated salt-perfused lungs from either control or chronically hypoxic rats. Similarly, pulmonary vasodilatory responses to AVP and the calcium ionophore A23187 were unaffected by chronic hypoxic exposure. Pretreatment with the cyclooxygenase inhibitor meclofenamate did not alter vasopressinergic pulmonary vasodilation in lungs from either control or chronically hypoxic animals, ruling out involvement of vasodilator prostaglandins in the response to AVP. In contrast, vasodilatory responses to both AVP and A23187 were inhibited by L-NNA pretreatment not only in lungs from control animals but also in lungs from chronically hypoxic rats, suggesting the involvement of NO in the vasodilatory response. The inhibition by L-NNA was reversible by prior addition of excess L-arginine but not by D-arginine. In addition, vasodilatory responses to the endothelium-independent vasodilators sodium nitroprusside and isoproterenol were unaffected by chronic hypoxic exposure. We conclude that endothelium-dependent vasodilation remains intact in male Sprague-Dawley rats after chronic hypoxic exposure.

Topics: Air Pressure; Animals; Arginine; Arginine Vasopressin; Calcimycin; Chronic Disease; Endothelium, Vascular; Hypoxia; In Vitro Techniques; Isoproterenol; Male; Meclofenamic Acid; Nitroarginine; Nitroprusside; Prostaglandins; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; Vasodilation; Vasodilator Agents

The role that second messengers play in pulmonary vasoconstriction is not understood. The purpose of this study was to directly measure inositol phosphates in isolated pulmonary arterial preparations before and during norepinephrine (NE) stimulation and acute hypoxia. Rat main pulmonary arteries were isolated and incubated with myo-[3H]-inositol. After incubation, control tissue was stimulated with 0.5 microM NE or 30 mM KCl. Test preparations were precontracted with 30 mM KCl and then exposed to hypoxia. Samples were homogenized and applied to a high-pressure liquid chromatography column for analysis of inositol phosphates. Results show that inositol trisphosphate (IP3) increases twofold at 5 s following NE stimulation. Thirty micromolars of KCl results in a slight but significant increase in IP3 formation at 5 min following the stimulation. Phentolamine inhibits the KCl-induced increase in IP3 formation, whereas A23187 has no effect on IP3 levels. Hypoxia caused a biphasic contraction in the precontracted isolated rat pulmonary artery. IP3 levels did not change during the hypoxic period. In conclusion, NE causes a rapid increase in IP3 formation consistent with the time course of production of an excitation-contraction coupling second messenger. However, inositol trisphosphate is not involved in the signal transduction pathway leading to pulmonary arterial contraction induced by hypoxia.

Topics: Acute Disease; Animals; Calcimycin; Chromatography, High Pressure Liquid; Hypoxia; Inositol 1,4,5-Trisphosphate; Male; Norepinephrine; Potassium Chloride; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Vasoconstriction

Eicosanoid products of arachidonic acid are suspected modulators of hypoxic vasoconstriction in the pulmonary vasculature. Vascular endothelial cells (EC) release several eicosanoids, but there is disagreement regarding the effect of hypoxia on EC eicosanoid release. We postulated that the oxygen level of growth in culture might influence the release of eicosanoids during acute hypoxia. We studied EC cultured from the main pulmonary arteries of pigs and grown at either 5% or near 20% oxygen, representing the normal limits of oxygen exposure to endothelium in normal lungs. Although cultures grown in 5% oxygen grew slightly faster by 4 days, the confluent cell number, protein content, and baseline eicosanoid release were no different compared with paired cultures grown in 20% oxygen. However, with an acute decrease in oxygen level, cultures grown in 5% oxygen released less prostaglandin E2, F2 alpha, and 6-ketoprostaglandin F1 alpha compared with amounts released at the growth oxygen level. In contrast, cultures grown in 20% oxygen released increased amounts of these eicosanoids compared with release at the growth oxygen level. Release of thromboxane B2 was not significantly different during hypoxia between cultures grown at 5% vs. 20% oxygen. In other experiments, cyclooxygenase activity, stimulated arachidonic acid release by calcium ionophore A23187, and uptake of arachidonic acid were no different in cultures grown at 5% vs. 20% oxygen. However, arachidonic acid release during hypoxia was reduced in 5% cultures and increased in 20% cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Arachidonic Acid; Calcimycin; Cell Division; Eicosanoids; Endothelium, Vascular; Hypoxia; L-Lactate Dehydrogenase; Oxygen; Pulmonary Circulation; Swine

Hypoxia-induced stimulation of the rate of von Willebrand factor (vWF) release from human umbilical vein endothelial cells in culture, and the influence of the calcium antagonist verapamil, was studied using a system in which a pO2 of 20 mm Hg was achieved over 60 min. The mean +/- SEM rate of vWF release over 60 min in normoxia was 0.42 +/- 0.07% pooled plasma standard. The addition of verapamil did not alter the basal rate of vWF release, but after 60 min of hypoxia, the rate was increased to 0.71 +/- 0.03% plasma standard (p < 0.01). Verapamil at a concentration of 0.1 microgram/ml of media had no influence on the stimulated rate of release (0.78 +/- 0.07%), but increasing concentrations of verapamil up to a maximum of 1 microgram/ml attenuated the hypoxia induced stimulation (0.48 +/- 0.07%; p = 0.04 vs. hypoxic control). Thus, verapamil at this concentration completely blocked the hypoxia-induced stimulation.

Topics: Calcimycin; Cells, Cultured; Endothelium, Vascular; Humans; Hypoxia; Verapamil; von Willebrand Factor

We investigated whether loss of endothelial-derived relaxing factor (EDRF) activity in the pulmonary vessels of chronically hypoxic rats could be restored by pretreatment with L-arginine. We measured vasodilation to acetylcholine (ACh), calcium ionophore A23187, or linsidomine (Sin-1) under conditions of increased vascular tone induced by U-46619 (50 pmol/min), as well as vasoconstriction to endothelin-1 (ET) in isolated lungs pretreated with meclofenamate (3 microM). In lungs from normoxic (N) rats, in vitro L- or D-arginine (10(-3) M) did not alter vasodilation to the endothelium-dependent agents ACh (10(-9)-10(-6) M) and A23187 (10(-9)-10(-7) M), but NG-monomethyl-L-arginine (10(-3) M) completely abolished it. In lungs from rats exposed to 3 wk of hypoxia (H), vasodilation to ACh or A23187 was fully restored after in vitro L-arginine (10(-3) M) or N alpha-benzoyl-L-arginine (5 x 10(-5) M) but remained abolished after D-arginine, L-citrulline, L-ornithine, or L-argininosuccinic acid. In vivo pretreatment of H rats with L-arginine (300 mg/kg iv) 30 min before isolating the lung also restored vasodilation to A23187. Vasodilation to the endothelium-independent agent Sin-1 was similar in both groups of lungs and was not altered by in vitro L-arginine. L-arginine attenuated the increased pressor response to ET (300 pmol) of H rat lungs but had no effect in N rats. Our results demonstrate that loss of EDRF activity associated with hypoxic pulmonary hypertension may be reversed by supplying L-arginine.

Topics: Acetylcholine; Animals; Arginine; Calcimycin; Chronic Disease; Endothelins; Endothelium, Vascular; Hypoxia; Male; Pulmonary Circulation; Rats; Rats, Inbred Strains; Reference Values; Vasodilation

We have studied the metabolism of endogenous arachidonic acid by isolated perfused lungs of ferrets when stimulated with calcium ionophore A23187 at two ages, neonatal (2- to 3-week-old) and adult (greater than 6 months). We have also determined the effect of hypoxia on lung metabolism of arachidonic acid in the two age groups. Lungs were perfused with Kreb's bicarbonate buffer in a recirculating system and eicosanoid production was stimulated by addition of A23187 (5 microM). Eicosanoids produced were measured in perfusate and lung homogenate supernatant. Cyclooxygenase metabolites; PGE2, TxB2 and 6-keto-PGF1 alpha were measured by radioimmunoassay and 5-lipoxygenase metabolites, leukotrienes; LTB4, LTC4, LTD4 and LTE4 by high performance liquid chromatography. During normoxia, leukotriene production, LTC4 and LTB4, was 10- to 20-fold higher in neonatal than in adult lungs, but total amount of cyclooxygenase products measured was similar. Compared with adult lungs, neonatal lungs produced more 6-keto-PGF1 alpha, but produced less PGE2. There was no difference in TxB2 production by neonatal and adult lungs. Hypoxia increased the production of 5-lipoxygenase metabolites in adult lungs, with production of cyclooxygenase metabolites remaining unchanged, whereas hypoxia decreased all eicosanoid production in neonatal lungs. In summary, there are age-related differences in endogenous arachidonic acid metabolism by isolated ferret lungs when stimulated with calcium ionophore A23187 as well as age related differences in the effect of hypoxia on endogenous arachidonic acid metabolism in ferret lungs.

Topics: Aging; Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Ferrets; Hypoxia; In Vitro Techniques; Leukotrienes; Lung; Perfusion; Prostaglandins; Stimulation, Chemical; Thromboxanes

The contribution of metabolic energy to the degradation of intracellular proteins in skeletal muscle was investigated. Isolated chick skeletal muscles deprived of oxygen and muscles incubated in buffer under nonphysiological conditions containing inhibitors of glycolysis and mitochondrial respiration had lower concentrations or undetectable levels of ATP and faster rates of proteolysis. Both total protein breakdown and the breakdown of myofibrillar proteins were stimulated 35-124% in ATP-depleted tissues. However, ATP-depleted muscles incubated in buffer to which no Ca2+ was added showed slower rates of total protein breakdown and no significant change in myofibrillar proteolysis compared with control muscles. Trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64), a compound that inhibits the calpains and the lysosomal cysteine proteases, completely blocked the Ca(2+)-stimulated breakdown of nonmyofibrillar and myofibrillar proteins in ATP-depleted muscles. However, Ca(2+)-stimulated proteolysis was not inhibited in ATP-depleted muscles incubated with weak bases to prevent lysosome function. These data suggest that intracellular proteins can be degraded in skeletal muscle in the absence of metabolic energy and that the calpains play a major role in the enhanced proteolysis in skeletal muscles depleted of ATP.

Topics: Adenosine Triphosphate; Animals; Animals, Newborn; Calcimycin; Calcium; Calpain; Chickens; Cysteine Proteinase Inhibitors; Energy Metabolism; Hypoxia; Ionomycin; Male; Muscle Proteins; Peptide Hydrolases

We have investigated overlapping activation pathways for two families of stress genes that are expressed in cells exposed to hypoxia. The growth arrest and DNA damage (gadd) genes are induced by DNA damage and irradiation, and their expression is associated with growth arrest. The glucose-regulated proteins (GRPs) are induced by chemical agents that disrupt protein trafficking in the endoplasmic reticulum such as tunicamycin and A23187 and by hypoxia. Here, we demonstrate that the treatment of NIH-3T3 cells with chemical inducers of GRPs results in increased levels of gadd45 and gadd153 mRNA as well as GRP78 mRNA. In addition, hypoxia was also able to increase gadd45, gadd153, and GRP78 mRNA. Therefore the GRP and gadd genes can be activated by similar stimuli (e.g., hypoxia and chemical inducers). However, the mechanisms leading to increased levels of GRP78 and gadd gene mRNA are different and may involve distinct protein kinases. Increased expression of GRPs after treatment with chemical inducers is sensitive to cycloheximide and the protein kinase inhibitors genistein, 2-aminopurine, and H7, whereas the increase in gadd gene mRNA could be blocked by the protein kinase inhibitors H7 and 2-aminopurine but not by genistein or cycloheximide. GRP78 induction occurs by a pathway that requires protein synthesis and is sensitive to genistein, H7, and 2-aminopurine, whereas gadd gene induction is independent of protein synthesis and is inhibited by H7 and 2-aminopurine only.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Calcimycin; DNA Damage; Endoplasmic Reticulum Chaperone BiP; HSP70 Heat-Shock Proteins; Hypoxia; Isoquinolines; Membrane Proteins; Mice; Piperazines; RNA, Messenger; Terpenes; Thapsigargin; Tunicamycin

The pulmonary vascular reactivity to endothelin-1 (ET-1) was assessed in rats previously exposed to 11% O2 (hypoxic) or room air (controls) for 3 weeks. In isolated control lung preparations studied during conditions of increased tone by U46619 (50 pmol/min) and treated with meclofenamate (3 microM), low doses of ET-1 (30 and 100 pM) reduced the pressor response to U46619 by 58 +/- 5% (p less than 0.01). Vasodilation induced by ET-1 was not abolished by the antagonist of endothelium-dependent relaxing factor (EDRF) NG-monomethyl-L-arginine (5 x 10(-4) M), which suppressed vasodilator response to ionophore A23187 (10(-8)-10(-7) M). Higher doses of ET-1 (300 and 1,000 pM) induced vasoconstriction during conditions of basal tone, and the pressor response to 300 pM ET-1 was enhanced by EDRF antagonists. Administration of ET-1 to lungs from hypoxic rats failed to cause pulmonary vasodilation and instead induced a greater pulmonary pressor response (300 pM) than in control rat lungs (7 +/- 1.5 vs. 1.6 +/- 0.5 mm Hg, p less than 0.01), which was not further potentiated by EDRF antagonists. Infusion of 300 pM ET to conscious catheterized animals induced a sustained increase in pulmonary resistance only in the hypoxic group (from 305 +/- 37 to 389 +/- 55 mm Hg/L/min, p less than 0.01) (n = 7). The results suggest that depending on the dose, ET-1 can cause pulmonary vasodilation (independent of EDRF release) or vasoconstriction (opposed by EDRF). During chronic hypoxic pulmonary hypertension, ET-1 behaves only as a pulmonary vasoconstrictor.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Arginine; Calcimycin; Endothelins; Hemodynamics; Hypertension, Pulmonary; Hypoxia; In Vitro Techniques; Male; Muscle Tonus; Nitric Oxide; omega-N-Methylarginine; Prostaglandin Endoperoxides, Synthetic; Pulmonary Circulation; Rats; Rats, Inbred Strains; Vasodilation

To determine whether exposure to chronic hypoxia and subsequent development of pulmonary hypertension induces alterations of endothelium-dependent relaxation in rat pulmonary vascular bed, we studied isolated lung preparations from rats exposed to either room air (controls) or hypoxia (H) during 1 wk (1W-H), 3 wk (3W-H), or 3W-H followed by 48 h recovery to room air (3WH + R). In lungs pretreated with meclofenamate (3 microM), the endothelium-dependent vasodilator responses to acetylcholine (10(-9)-10(-6) M) and ionophore A23187 (10(-9)-10(-7) M) were examined during conditions of increased tone by U46619 (50 pmol/min). Acetylcholine or A23187 produced dose-dependent vasodilation in control lungs, this response was reduced in group 1W-H (P less than 0.02), abolished in group 3W-H (P less than 0.001), and restored in group 3WH + R. In contrast, the endothelium-independent vasodilator agent sodium nitroprusside remained fully active in group 3W-H. The pressor response to 300 pM endothelin was greater in group 3W-H than in controls (6.8 +/- 0.5 mmHg vs. 1.6 +/- 0.2 mmHg, P less than 0.001) but was not potentiated by the endothelium-dependent relaxing factor (EDRF) antagonists: hydroquinone (10(-4) M); methylene blue (10(-4) M); and pyrogallol (3 x 10(-5) M) as it was in controls. It was similar to controls in group 3W-H + R. Our results demonstrate that hypoxia-induced pulmonary hypertension is associated with a loss of EDRF activity in pulmonary vessels, with a rapid recovery on return to a normoxic environment.

Topics: Acetylcholine; Animals; Blood Pressure; Calcimycin; Chronic Disease; Endothelins; Endothelium, Vascular; Hypertension, Pulmonary; Hypoxia; Male; Nitric Oxide; Nitroprusside; Pulmonary Circulation; Rats; Rats, Inbred Strains

Human umbilical vein endothelial cells subjected to 24 h of anoxia followed by reoxygenation released less prostacyclin (PGI2) in response to thrombin, calcium ionophore A23187, or arachidonic acid. This was associated with a substantial increase in stimulated platelet adherence. Increased lactate dehydrogenase and 51Cr release occurred after 1 h of reoxygenation, but the high rate of release did not persist during the subsequent 23 h of reoxygenation. The changes in platelet adherence and PGI2 release partially resolved over 24 h. PGI2 formation from prostaglandin H2 was not reduced, suggesting that cyclooxygenase activity, but not prostacyclin synthase, is affected by reoxygenation. A decrease in arachidonic acid release from cellular lipids also occurred. The reduction in cyclooxygenase activity, but not arachidonic acid release, was prevented by the presence of ibuprofen during reoxygenation. Addition of catalase or superoxide dismutase during reoxygenation increased PGI2 release but did not completely overcome the reduction relative to control cultures. These findings suggest that the increase in platelet adherence during reoxygenation may be mediated in part by a change in cyclooxygenase activity. This is only partly overcome by extracellular oxygen species scavengers but is prevented by the presence of a reversible cyclooxygenase inhibitor during reoxygenation.

Topics: Aerobiosis; Arachidonic Acid; Arachidonic Acids; Blood Platelets; Calcimycin; Cells, Cultured; Endothelium, Vascular; Epoprostenol; Humans; Hydrogen-Ion Concentration; Hypoxia; Kinetics; Platelet Adhesiveness; Superoxide Dismutase; Thrombin

Because leukotrienes may mediate hypoxic pulmonary vasoconstriction (HPV), we examined the influence of two lipoxygenase inhibitors on HPV in anesthetized pigs. HPV was induced by ventilation with a hypoxic gas mixture (FIO2 at 0.095), resulting in a fall in PaO2 to 23 +/- 2 mm Hg and a rise in pulmonary vascular resistance from 285 +/- 15 to 595 +/- 30 dyne/s/cm-5. After infusion of either U-60,257B (50 mg/kg, n = 13) or BW 755c (20 mg/kg, n = 8), the responses to repeated hypoxic challenges were recorded. After U-60,257B infusion the hypoxic pressor response was eliminated at 10 and 30 min and remained significantly (p less than 0.01) attenuated at 50 min. The pulmonary pressor response to angiotensin II infusion (0.2 micrograms/kg/min) was also ablated, whereas the systemic response was unchanged. In contrast, after BW 755c infusion there was a modest but sustained augmentation of HPV, maximum at 30 min (pulmonary vascular resistance, 158 +/- 23% control, p less than 0.01), and no alteration of the responses to angiotensin II. BW 755c inhibited the A23187-induced release of leukotrienes, but not histamine, from isolated porcine lung cells (IC50, 6.3 x 10(-5) M), whereas U-60,257B inhibited the release of both leukotriene (IC50, 1.1 x 10(-4) M) and histamine. These findings indicate that reduction of HPV by lipoxygenase inhibitors is not necessarily a consequence of inhibition of leukotriene synthesis.

Topics: 4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-amine; Angiotensin II; Animals; Calcimycin; Epoprostenol; Female; Histamine Release; Hypoxia; Lipoxygenase Inhibitors; Lung; Male; Pulmonary Circulation; Pyrazoles; SRS-A; Swine; Vascular Resistance; Vasoconstriction

The induction of glucose-regulated proteins by a variety of specific inducers leads to an increase in resistance to Adriamycin (Shen et al., Proc. Natl., Acad. Sci. USA, 84: 3278, 1987). In this study we examine several additional agents for cross-resistance induced during a glucose-regulated response in an attempt to better define the mechanism through which this phenomenon occurs. When anoxia, calcium ionophore A23187, or 2-deoxyglucose are used, a substantial resistance is obtained against the topoisomerase II-targeted agent, etoposide. Partial resistance is induced against vincristine and actinomycin D. Glucose-regulated protein inducers do not substantially alter cellular response to either bleomycin or radiation. In the case of mitomycin C there is a cellular sensitization with anoxia and 2-deoxyglucose while calcium ionophore A23187 had no effect on survival. This study suggests that the resistance obtained during a glucose-regulated response against etoposide and Adriamycin may involve topoisomerase II.

Topics: Animals; Calcimycin; Cricetinae; Cricetulus; Dactinomycin; Deoxyglucose; Doxorubicin; Drug Resistance; Etoposide; Female; HSP70 Heat-Shock Proteins; Hypoxia; Membrane Proteins; Ovarian Neoplasms; Tumor Cells, Cultured; Vincristine

Arachidonic acid metabolites, notably leukotrienes (LTs), have been postulated to play a role in hypoxic pulmonary vasoconstriction. In the present study, we examined the contribution of arachidonic acid metabolites, via the cyclooxygenase, 5-lipoxygenase and cytochrome P-450 monooxygenase pathways, to the hypoxia (25 +/- 3 torr)- and anoxia (0 +/- 2 torr)-induced contractions of isolated pulmonary venules. Neither the cyclooxygenase inhibitors indomethacin (5 microM) or ibuprofen (10 microM) nor the 5-lipoxygenase inhibitors nordihydroguaiaretic acid (5 microM) or U 60257B (10 microM) affected the contractile responses. Similarly, the LT receptor antagonists FPL 57231 (3 microM) or LY 163443 (1 microM), at concentrations that inhibited LT-induced venular contractions, did not significantly affect the responses to hypoxia or anoxia. In fact, anoxia suppressed spontaneous LT release from the venules. The cytochrome P-450 inhibitor SKF-525A (500 microM) nonselectively depressed venular contractions induced by decreased PO2 and pharmacological agents. Induction of the cytochrome P-450 monooxygenase system with beta-naphthoflavone did not alter venular contractions induced by hypoxia or anoxia. Contractions of isolated guinea pig pulmonary venules elicited by decreased PO2 are not mediated by 5-lipoxygenase or cyclooxygenase metabolites. Furthermore, the data do not support a role for cytochrome P-450 metabolites of endogenous substrates in these contractions.

Topics: Acetophenones; Animals; Benzoflavones; beta-Naphthoflavone; Calcimycin; Chromones; Cytochrome P-450 Enzyme System; Epoprostenol; Guinea Pigs; Hypoxia; Leukotrienes; Lipoxygenase; Lipoxygenase Inhibitors; Lung; Masoprocol; Muscle Contraction; Muscles; Prostaglandin-Endoperoxide Synthases; Pyridines; Veins; Venules

The effect of hypoxia on endothelium-dependent and endothelium-independent vasodilation was studied in phenylephrine-precontracted, isolated rings of rabbit first-branch pulmonary artery. Concentration-dependent relaxation responses to the endothelium-dependent dilators methacholine, ATP, and the calcium ionophore (A23187) as well as to the endothelium-independent dilators sodium nitroprusside and isoproterenol were obtained before, during, and after exposure to hypoxia (PO2 = 42 +/- 1 mm Hg) in the presence of indomethacin (2.8 x 10(-5) M). This moderate degree of hypoxia inhibited (p less than 0.05) endothelium-dependent but not endothelium-independent relaxation responses without producing irreversible vascular damage. In parallel experiments, cyclic GMP accumulation in pulmonary vascular rings in response to maximal doses of the above vasodilators was measured in the presence and absence of hypoxia. Cyclic GMP accumulation in response to endothelium-dependent dilators (methacholine, ATP, and A23187) was inhibited (p less than 0.05) by hypoxia while cyclic GMP accumulation in response to the endothelium-independent dilator sodium nitroprusside was not. When phenylephrine precontracted vessels were exposed to hypoxia in the absence of vasodilators, a small, transient increase in tension occurred, which was greater in endothelium-intact than in endothelium-denuded vessels (0.70 +/- 0.12 vs. 0.09 +/- 0.03 g, respectively; p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine Triphosphate; Animals; Calcimycin; Cyclic GMP; Endothelium, Vascular; Hypoxia; Isoproterenol; Methacholine Chloride; Methacholine Compounds; Nitric Oxide; Nitroprusside; Pulmonary Artery; Rabbits; Receptors, Purinergic; Vasodilation

The mechanism for hypoxic pulmonary vasoconstriction (HPVC) was investigated in human pulmonary arterial strips. Hypoxia in the presence of histamine (10(-6) M) caused marked pulmonary arterial contraction, which was reversed by O2. The hypoxic contraction in the presence of histamine was inhibited by diphenhydramine, but not by cimetidine. The hypoxic histamine-mediated contraction was attenuated but still present in the absence of extracellular Ca2+, or by the inhibitors of voltage-dependent Ca2+ influx. However, it was inhibited significantly by a further depletion of intracellular Ca2+, or by HA 1004, an intracellular calcium antagonist. A low concentration (10(-7) M) of a calcium ionophore, A23187, enhanced the hypoxic contraction in the presence of histamine, whereas procaine completely inhibited it. W-7, a calmodulin inhibitor, significantly decreased the hypoxic histamine-mediated contraction, but 12-O-tetradecanoylphorbol-13-acetate (TPA), a C-kinase promotor, had no effect. The hypoxic contractile response was also observed in the presence of both A23187 and KCl instead of histamine, but the hypoxia-induced contraction with KCl alone was much smaller than that. These results indicate that hypoxia in the presence of certain other vasoactive agents has a potent contractile effect on the human pulmonary artery and that the response is dependent on Ca2+. Enhancement of both Ca2+ influx and Ca2+ release from intracellular storage sites by hypoxia, which interacts with calmodulin, were suggested to be involved in the mechanism of HPVC.

Topics: Aged; Calcimycin; Calcium; Female; Histamine; Humans; Hypoxia; In Vitro Techniques; Male; Middle Aged; Potassium Chloride; Pulmonary Artery; Vasoconstriction

There is evidence both from in vivo and in vitro studies which suggests that hypoxia stimulates the synthesis of prostanoids in some tissues. In the present study, the in vitro production of prostaglandin E2 (PGE2) was studied in three different renal cell lines incubated at various PO2 values between 143 and 3 mm Hg for 24 h. In rat kidney mesangial cell cultures, PGE2 production increased up to 99 ng PGE2/mg protein at 7 mm Hg O2, compared to 52 ng/mg at 143 mm Hg O2, but was lowered at 26 ng/mg at 3 mm Hg O2. PGE2 production by the pig kidney tubule cell lines LLC-PK1 and PK-15 was insensitive to PO2 changes. Because PGE2 production is known to be Ca2+-dependent and was indeed stimulated by the Ca-ionophore A 23187, effects of hypoxia on 45Ca2+-fluxes were also studied. In none of the 3 cell lines, net 45Ca-influx was altered after incubation at low PO2. However, net 45Ca-efflux increased during hypoxic incubation of mesangial cells possibly as a result of intracellular Ca-mobilization. These results indicate that hypoxia stimulates PGE2 synthesis in mesangial but not in tubule cell cultures. However, at very low PO2 values, or anoxia, the formation of cyclic endoperoxides from arachidonic acid may be lowered. Since mesangiocytes are smooth muscle-like cells, the hypoxia-induced synthesis of relaxing prostanoids could play a role in the regulation of smooth muscle tone.

Topics: Animals; Arachidonic Acids; Calcimycin; Calcium; Cell Line; Epithelium; Glomerular Mesangium; Hypoxia; Ion Channels; Kidney; Male; Oxygen; Prostaglandins E; Rats; Rats, Inbred Strains; Swine

The ability of hypoxia (PO2 57 Torr) and anoxia (PO2 0 Torr) to induce the release of histamine or sulfidopeptide leukotrienes from dispersed porcine parenchymal lung cells was examined. Spontaneous release of histamine (9.2 +/- 1.3%) was not significantly increased during hypoxia or anoxia, and spontaneous leukotriene release was not detected under any conditions. The release of leukotriene induced by A23187 (78 +/- 11 pmol leukotriene D4 equivalent/10(7) parenchymal lung cells) was unchanged during hypoxia and was significantly reduced (55.4 +/- 7.7% control leukotriene release) during anoxia, whereas A23187-induced histamine release (63.2 +/- 4.2% total cell histamine) was unaffected by reduced oxygenation. Reduction of final buffer pH from 7.4 to 7.0 did not affect mediator release. High-pressure liquid chromatographic analysis of the released leukotrienes revealed a mixture of leukotrienes C4 and D4, with a symmetrical reduction in product during anoxia. Although leukotriene release in response to hypoxia was not demonstrated, the findings do not preclude limited local release of leukotrienes, perhaps in association with increased smooth muscle responsiveness.

Topics: Animals; Buffers; Calcimycin; Chromatography, High Pressure Liquid; Histamine Release; Hypoxia; In Vitro Techniques; Lung; SRS-A; Swine

Synaptosomal glycolysis is stimulated eight- to 10-fold when the respiratory chain is inhibited by cyanide or by anoxia. However, the stimulation is transient and after 15 min declines toward the preanoxic rate. The decline is not seen when Ca2+ is absent or when the respiratory chain is inhibited by rotenone. The decline in glycolysis is reversible, is not due to substrate exhaustion, and is the cause, rather than the effect, of lowered synaptosomal ATP/ADP ratios. The failure to maintain glycolysis when the terminal oxidase of the respiratory chain is inhibited may have relevance to the sensitivity of the brain to anoxic damage.

Topics: Adenine Nucleotides; Animals; Calcimycin; Calcium; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Glycolysis; Guinea Pigs; Hypoxia; Nerve Endings; Respiration; Rotenone; Sodium Cyanide; Synaptosomes

Our study was designed to evaluate the effects of the K+ channel blocker tetraethylammonium TEA (20 mM), Ca++ ionophore A23187 (6.9 microM), the Ca++ channel blocker verapamil (2 microM), and CaCl2 (6 mM) on a model of heart damage, hypoxia/reoxygenation of isolated guinea-pig hearts. The right ventricular papillary muscles maintained at 37 degrees C and paced at 60 beats/min were perfused with Ringer solution equilibrated with 95% O2-5% CO2 (normoxia) or 95% N2-5% CO2 (hypoxia). After stabilization of the electrical and mechanical activity, 60 min of hypoxia was induced and then followed by 15 min of reoxygenation. During hypoxia, there was a significant decrease in the action potential duration (APD) and the contractile force (CF). The decrease in APD in the TEA-treated muscle was significantly less than that in the non-treated muscle. However, CF declined at the same rate in both groups. Neither A23187, CaCl2 nor verapamil prevented the decline in both APD and CF. To study the slow responses, the fast Na+ current was first voltage-inactivated by partial depolarization to about -40 mM using an elevated K+ solution. The rate of hypoxia-induced blockade of the slow response was not slower in the presence of 20 mM TEA. These data demonstrate that TEA significantly prevented the decline of APD in hypoxia, but this resistance in APD did not affect the rate of inhibition of mechanical activity.

Topics: Action Potentials; Animals; Calcimycin; Calcium Chloride; Guinea Pigs; Heart; Hypoxia; In Vitro Techniques; Male; Myocardial Contraction; Tetraethylammonium; Tetraethylammonium Compounds; Time Factors; Verapamil

[Ca2+]i was raised experimentally in mammalian and amphibian skeletal and cardiac muscles by A23187, DNP, anoxia or the Ca2+ -paradox. Trifluoperazine (TFP) at 10(-5) M failed to protect against the characteristic and rapid damage triggered by elevated [Ca2+]i in any of the preparations. It is concluded that calmodulin is not implicated in this rapid ultrastructural damage. TFP alone also causes identical patterns of damage. It may be acting to raise [Ca2+]i in skeletal and cardiac muscle cells.

Topics: Animals; Calcimycin; Calcium; Coronary Disease; Dinitrophenols; Hypoxia; Mice; Muscular Diseases; Rana temporaria; Subcellular Fractions; Trifluoperazine

5 mM KCN as well as anoxia have corresponding effects on Physarum plasmodia: Both induce a disintegration of the plasmalemma invaginations, an increase in cytoplasmic vacuoles, formation of a thick cortical actomyosin layer and an increase of cytoplasmic actomyosin fibrils. Both KCN treatment and anoxia cause a temporary increase in the level of the force oscillations and a reversible prolongation of the periods of the contraction-relaxation cycle of cytoplasmic actomyosin. The normal pattern of oscillation can be restored by the addition of 10 mM alpha-ketoglutarate +5 mM AMP to the solution containing 5 mM KCN. A combination of the Ca2+ ionophore A-23187 and KCN induces a strong contracture, whereas a combination of KCN, ionophore, alpha-ketoglutarate and AMP prevents this effect. The state of contracture is characterised by a pronounced increase in the Young's modulus and an increased fibrillogenesis of cytoplasmic actomyosin. The isometrically contracted state during the high force output represents a strict parallel arrangement of F-actin. The capability of alpha-ketoglutarate in combination with AMP to restore the normal pattern of oscillation as well as to inhibit contractures is interpreted as a stimulation of the alternate pathway of respiration. It is suggested that this stimulation leads to a restoration of cellular Ca2+-homeostasis originally disturbed by the impediment of cell respiration.

Topics: Adenosine Monophosphate; Calcimycin; Calcium; Cyanides; Hypoxia; Ketoglutaric Acids; Microscopy, Electron; Mitochondria; Oxygen Consumption; Physarum; Potassium Cyanide