ryanodine and Hypoxia

Intracellular taurine is abundant in many animals and it influences an array of physiological processes, including osmoregulation, metabolism, and cardiac contractility. Taurine is an important osmolyte in teleost hearts, but its role in stress tolerance, cardiac metabolism, and contractility has not been assessed. The goal of this study was to determine if ventricular taurine concentration changes in response to environmental stress and to characterize its influence on contractility. Cardiac taurine concentrations varied in killifish (Fundulus heteroclitus) but were generally maintained following acute environmental challenges. In isometrically contracting ventricular strips, supplemental taurine (40 mmol L

Topics: Animals; Calcium; Cardiotonic Agents; Female; Fundulidae; Hypoxia; Male; Myocardial Contraction; Myocardium; Ryanodine; Sarcoplasmic Reticulum; Taurine; Ventricular Function

We examined whether exogenous glucose affects contractile performance of electrically paced ventricle strips from rainbow trout under conditions known to alter cardiomyocyte performance, ion regulation and energy demands. Physiological levels of d-glucose did not influence twitch force development for aerobic preparations (1) paced at 0.5 or 1.1 Hz, (2) at 15 or 23°C, (3) receiving adrenergic stimulation or (4) during reoxygenation with or without adrenaline after severe hypoxia. Contractile responses to ryanodine, an inhibitor of Ca(2+) release from the sarcoplasmic reticulum, were also not affected by exogenous glucose. However, glucose did attenuate the fall in twitch force during severe hypoxia. Glucose uptake was assayed in non-contracting ventricle strips using 2-[(3)H] deoxy-d-glucose (2-DG) under aerobic and hypoxic conditions, at different incubation temperatures and with different inhibitors. Based upon a lack of saturation of 2-DG uptake and incomplete inhibition of uptake by cytochalasin B and d-glucose, 2-DG uptake was mediated by a combination of facilitated transport and simple diffusion. Hypoxia stimulated lactate efflux sixfold to sevenfold with glucose present, but did not increase 2-DG uptake or reduce lactate efflux in the presence of cytochalasin B. Increasing temperature (14 to 24°C) also did not increase 2-DG uptake, but decreasing temperature (14 to 4°C) reduced 2-DG uptake by 45%. In conclusion, exogenous glucose improves mechanical performance under hypoxia but not under any of the aerobic conditions applied. The extracellular concentration of glucose and cold temperature appear to determine and limit cardiomyocyte glucose uptake, respectively, and together may help define a metabolic strategy that relies predominantly on intracellular energy stores.

Topics: Animals; Biomechanical Phenomena; Cytochalasin B; Deoxyglucose; Diffusion; Epinephrine; Female; Glucose; Heart Ventricles; Hypoxia; In Vitro Techniques; Kinetics; Male; Myocardial Contraction; Myocardium; Oncorhynchus mykiss; Oxygen; Ryanodine; Temperature

  Hypoxic pulmonary vasoconstriction (HPV) maintains blood oxygenation during acute hypoxia but contributes to pulmonary hypertension during chronic hypoxia. The mechanisms of HPV remain controversial, in part because HPV is usually studied in the presence of agonist-induced preconstriction ('pretone'). This potentiates HPV but may obscure and distort its underlying mechanisms. We therefore carried out an extensive assessment of proposed mechanisms contributing to HPV in isolated intrapulmonary arteries (IPAs) in the absence of pretone by using a conventional small vessel myograph. Hypoxia elicited a biphasic constriction consisting of a small transient (phase 1) superimposed upon a sustained (phase 2) component. Neither phase was affected by the L-type Ca2+ channel antagonists diltiazem (10 and 30 μm) or nifedipine (3 μm). Application of the store-operated Ca2+ entry (SOCE) blockers BTP2 (10 μm) or SKF96365 (50 μm) attenuated phase 2 but not phase 1, whereas a lengthy (30 min) incubation in Ca2+-free physiological saline solution similarly reduced phase 2 but abolished phase 1. No further effect of inhibition of HPV was observed if the sarco/endoplasmic reticulum Ca2+-ATPase inhibitor cyclopiazonic acid (30 μm) was also applied during the 30 min incubation in Ca2+-free physiological saline solution. Pretreatment with 10 μm ryanodine and 15 mm caffeine abolished both phases, whereas treatment with 100 μm ryanodine attenuated both phases. The two-pore channel blocker NED-19 (1 μm) and the nicotinic acid adenine dinucleotide phosphate (NAADP) antagonist BZ194 (200 μm) had no effect on either phase of HPV. The lysosomal Ca2+-depleting agent concanamycin (1 μm) enhanced HPV if applied during hypoxia, but had no effect on HPV during a subsequent hypoxic challenge. The cyclic ADP ribose antagonist 8-bromo-cyclic ADP ribose (30 μm) had no effect on either phase of HPV. Neither the Ca2+-sensing receptor (CaSR) blocker NPS2390 (0.1 and 10 μm) nor FK506 (10 μm), a drug which displaces FKBP12.6 from ryanodine receptor 2 (RyR2), had any effect on HPV. HPV was virtually abolished by the rho kinase blocker Y-27632 (1 μm) and attenuated by the protein kinase C inhibitor Gö6983 (3 μm). Hypoxia for 45 min caused a significant increase in the ratio of oxidised to reduced glutathione (GSSG/GSH). HPV was unaffected by the NADPH oxidase inhibitor VAS2870 (10 μm), whereas phase 2 was inhibited but phase 1 was unaffected by the antioxidants ebselen (100 μm) and TEMPOL (3 mm). W

Topics: Animals; Antioxidants; Calcium; Calcium Channel Blockers; Calcium Signaling; Calcium-Transporting ATPases; Glutathione; Hypoxia; Male; Myocytes, Smooth Muscle; Protein Kinase Inhibitors; Pulmonary Artery; Rats; Rats, Wistar; Receptors, Calcium-Sensing; Ryanodine; Sarcoplasmic Reticulum; Vasoconstriction

There is a growing body of evidence indicating that transient receptor potential (TRP) channels are implicated in calcium signaling and various cellular functions in the pulmonary vasculature. The aim of this study was to investigate the expression, functional role, and coupling to reticulum calcium channels of the type 4 vanilloid TRP subfamily (TRPV4) in the pulmonary artery from both normoxic (Nx) and chronically hypoxic (CH) rats. Activation of TRPV4 with the specific agonist 4α-phorbol-12,13-didecanoate (4α-PDD, 5 μM) increased the intracellular calcium concentration ([Ca(2+)](i)). This effect was significantly reduced by a high concentration of ryanodine (100 μM) or chronic caffeine (5 mM) that blocked ryanodine receptor (RyR) but was insensitive to xestospongin C (10 μM), an inositol trisphosphate receptor antagonist. Inhibition of RyR1 and RyR3 only with 10 μM of dantrolene did not attenuate the 4α-PDD-induced [Ca(2+)](i) increase. Western blotting experiments revealed the expression of TRPV4 and RyR2 with an increase in both receptors in pulmonary arteries from CH rats vs. Nx rats. Accordingly, the 4α-PDD-activated current, measured with patch-clamp technique, was increased in pulmonary artery smooth muscle cells (PASMC) from CH rats vs. Nx rats. 4α-PDD increased isometric tension in artery rings, and this response was also potentiated under chronic hypoxia conditions. 4α-PDD-induced calcium response, current, and contraction were all inhibited by the selective TRPV4 blocker HC-067047. Collectively, our findings provide evidence of the interplay between TRPV4 and RyR2 in the Ca(2+) release mechanism and contraction in PASMC. This study provides new insights onto the complex calcium signaling in PASMC and point out the importance of the TRPV4-RyR2 signaling pathway under hypoxic conditions that may lead to pulmonary hypertension.

Topics: Animals; Caffeine; Calcium Channel Agonists; Calcium Signaling; Cell Hypoxia; Cells, Cultured; Dantrolene; Hypertension, Pulmonary; Hypoxia; In Vitro Techniques; Macrocyclic Compounds; Male; Morpholines; Muscle Contraction; Muscle Relaxants, Central; Myocytes, Smooth Muscle; Oxazoles; Patch-Clamp Techniques; Phorbols; Pulmonary Artery; Pyrroles; Rats; Rats, Wistar; Ryanodine; Ryanodine Receptor Calcium Release Channel; TRPV Cation Channels

The contribution of sympathetic nerves arising from the superior cervical ganglia (SCG) toward the growth and function of cerebral blood vessels is pertinent throughout maturation as well as in response to cardiovascular stress imposed by high-altitude long-term hypoxia (LTH). The function of SCG sympathetic neurons is dependent on intracellular Ca2+ concentration ([Ca2+]i) signaling, which is strongly influenced by a process known as Ca(2+)-induced Ca2+ release (CICR) from the smooth endoplasmic reticulum (SER). In this study, we used the sheep SCG neuronal model to test the hypotheses that maturation decreases CICR and high-altitude LTH depresses CICR in fetal SCG neurons but not in those of the adult. We found that the contribution of CICR to electric field stimulation (EFS)-evoked [Ca2+]i transients was greatest in SCG cells from normoxic fetuses and was abolished by LTH. The decline in CICR was associated with a reduction in sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) function in fetal SCG cells during LTH, reducing SER Ca2+ levels below the threshold needed for the coupling of Ca2+ influx and CICR. With respect to the maturation from the fetus to adult, the decrease in CICR may reflect both a reduction in the levels of ryanodine receptor isoforms 2 and 3 and SERCA function. In response to LTH and in contrast to the fetus, CICR function in adult SCG cells is maintained and may reflect alterations in other mechanisms that modulate the CICR process. As CICR is instrumental in the function of sympathetic neurons within the cerebrovasculature, the loss of this signaling mechanism in the fetus may have consequences for the adaptation to LTH in terms of fetal susceptibility to vascular insults.

Topics: Age Factors; Aging; Animals; Caffeine; Calcium; Calcium Signaling; Cerebral Arteries; Cyclic ADP-Ribose; Disease Models, Animal; Electric Stimulation; Enzyme Inhibitors; Fetal Hypoxia; Hypoxia; Indoles; Nitric Oxide Synthase Type I; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sheep; Superior Cervical Ganglion; Sympathetic Fibers, Postganglionic; Time Factors

PPHN, caused by perinatal hypoxia or inflammation, is characterized by an increased thromboxane-prostacyclin ratio and pulmonary vasoconstriction. We examined effects of hypoxia on myocyte thromboxane responsiveness. Myocytes from 3rd-6th generation pulmonary arteries of newborn piglets were grown to confluence and synchronized in contractile phenotype by serum deprivation. On the final 3 days of culture, myocytes were exposed to 10% O2 for 3 days; control myocytes from normoxic piglets were cultured in 21% O2. PPHN was induced in newborn piglets by 3-day hypoxic exposure (Fi(O2) 0.10); pulmonary arterial myocytes from these animals were maintained in normoxia. Ca2+ mobilization to thromboxane mimetic U-46619 and ATP was quantified using fura-2 AM. Three-day hypoxic exposure in vitro results in increased basal [Ca2+]i, faster and heightened peak Ca2+ response, and decreased U-46619 EC50. These functional changes persist in myocytes exposed to hypoxia in vivo but cultured in 21% O2. Blockade of Ca2+ entry and store refilling do not alter peak U-46619 Ca2+ responses in hypoxic or normoxic myocytes. Blockade of ryanodine-sensitive or IP3-gated intracellular Ca2+ channels inhibits hypoxic augmentation of peak U-46619 response. Ca2+ response to ryanodine alone is undetectable; ATP-induced Ca2+ mobilization is unaltered by hypoxia, suggesting no independent increase in ryanodine-sensitive or IP3-linked intracellular Ca2+ pool mobilization. We conclude hypoxia has a priming effect on neonatal pulmonary arterial myocytes, resulting in increased resting Ca2+, thromboxane hypersensitivity, and hyperreactivity. We postulate that hypoxia increases agonist-induced TP-R-linked IP3 pathway activation. Myocyte thromboxane hyperresponsiveness persists in culture after removal from the initiating hypoxic stimulus, suggesting altered gene expression.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Animals, Newborn; Calcium; Calcium Channel Blockers; Cells, Cultured; Disease Models, Animal; Heart Ventricles; Humans; Hypoxia; Infant, Newborn; Macrocyclic Compounds; Muscle Cells; Nifedipine; Organ Size; Oxazoles; Persistent Fetal Circulation Syndrome; Pulmonary Artery; Receptors, Thromboxane; Ryanodine; Swine; Vasoconstriction

Apelin, a ligand for apelin-angiotension receptor-like 1 (APJ), has recently been shown to be a potent positive inotropic agent in normal hearts. In humans, levels of apelin have been shown to rise in early-stage heart failure and to fall in late-stage heart failure. In this study, we tested the hypothesis that apelin augments contraction directly in failing rat cardiac muscle. Right ventricular heart failure secondary to pulmonary hypertension was induced by exposing the rats to hypoxia (10% O(2) inhaled air) for 14-16 weeks. Trabeculae were dissected and mounted between a force transducer and a motor arm, superfused with Krebs-Henseleit (K-H) solution (pH 7.4, 22 degrees C), and loaded with fura-2. Both force development and [Ca(2+)](i) transient amplitude increased in a dose-dependent manner in the presence of Apelin-12 (10 approximately 70 nM, [Ca(2+)](o)=0.5 mM) in failing muscles as compared to control (36+/-7% vs. 7.4+/-5% at 70 nM, P<0.05). Also, [Ca(2+)](i) transients increased up to 18.4+/-9.5% as compared to control (4.5+/-1.9%, P<0.05). The increases in contraction in the presence of apelin were also maintained over a range of external Ca(2+) (0.5-2.0 mM). Steady-state force-[Ca(2+)](i) relation of the failing muscles reveals decreased maximal Ca(2+)-activated force (F(max)) (51.45+/-5.3 vs. 98.5+/-11.5 mN/mm(2), P<0.001), with no changes in Ca(2+) required for 50% of maximal activation (Ca(50)) (0.45+/-0.07 vs. 0.30+/-0.04 muM, P>0.05) and Hill coefficient (4.60+/-0.73 vs. 3.17+/-0.92, P>0.05). Apelin (70 nM) had no effect on the steady-state force-[Ca(2+)](i) relation in failing muscles (F(max): 63.03+/-3.5 mN/mm(2); Ca(50): 0.50+/-0.08 microM; Hill coefficient: 4.73+/-0.89). These results indicate that apelin exerts a selective positive inotropic action in failing myocardium. The increased force development is the result of increased [Ca(2+)](i) transients rather than changes in myofilament calcium responsiveness.

Topics: Actin Cytoskeleton; Algorithms; Animals; Antihypertensive Agents; Apelin; Calcium; Calcium Signaling; Cardiotonic Agents; Carrier Proteins; Chronic Disease; Heart Failure; Hypoxia; Intercellular Signaling Peptides and Proteins; Myocardial Contraction; Rats; Ryanodine; Sarcomeres; Stimulation, Chemical

Specialized O2-sensing cells exhibit a particularly low threshold to regulation by O2 supply and function to maintain arterial pO2 within physiological limits. For example, hypoxic pulmonary vasoconstriction optimizes ventilation-perfusion matching in the lung, whereas carotid body excitation elicits corrective cardio-respiratory reflexes. It is generally accepted that relatively mild hypoxia inhibits mitochondrial oxidative phosphorylation in O2-sensing cells, thereby mediating, in part, cell activation. However, the mechanism by which this process couples to Ca2+ signaling mechanisms remains elusive, and investigation of previous hypotheses has generated contrary data and failed to unite the field. We propose that a rise in the cellular AMP/ATP ratio activates AMP-activated protein kinase and thereby evokes Ca2+ signals in O2-sensing cells. Co-immunoprecipitation identified three possible AMP-activated protein kinase subunit isoform combinations in pulmonary arterial myocytes, with alpha1 beta2 gamma1 predominant. Furthermore, their tissue-specific distribution suggested that the AMP-activated protein kinase-alpha1 catalytic isoform may contribute, via amplification of the metabolic signal, to the pulmonary selectivity required for hypoxic pulmonary vasoconstriction. Immunocytochemistry showed AMP-activated protein kinase-alpha1 to be located throughout the cytoplasm of pulmonary arterial myocytes. In contrast, it was targeted to the plasma membrane in carotid body glomus cells. Consistent with these observations and the effects of hypoxia, stimulation of AMP-activated protein kinase by phenformin or 5-aminoimidazole-4-carboxamide-riboside elicited discrete Ca2+ signaling mechanisms in each cell type, namely cyclic ADP-ribose-dependent Ca2+ mobilization from the sarcoplasmic reticulum via ryanodine receptors in pulmonary arterial myocytes and transmembrane Ca2+ influx into carotid body glomus cells. Thus, metabolic sensing by AMP-activated protein kinase may mediate chemotransduction by hypoxia.

Topics: Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; AMP-Activated Protein Kinases; Animals; Antibodies; Calcium; Carotid Arteries; Catalysis; Dose-Response Relationship, Drug; Hypoxia; Immunohistochemistry; Male; Mitochondria; Models, Biological; Multienzyme Complexes; Myocytes, Smooth Muscle; Oxidative Phosphorylation; Oxygen; Phosphorylation; Protein Isoforms; Protein Serine-Threonine Kinases; Pulmonary Artery; Rats; Rats, Wistar; Ribose; Ryanodine; Sarcoplasmic Reticulum; Signal Transduction; Spectrometry, Fluorescence

Hypoxia-induced pulmonary vasoconstriction (HPV) is an important adaptive process that remains incompletely understood. In preconstricted rat pulmonary arteries (inner diameter, 250 to 400 microm), hypoxia (pO2 approximately 10 mm Hg) induces an initial transient phase and a more slowly developing sustained phase of vasoconstriction. Since the release of calcium ions (Ca2+) from intracellular stores by redox-sensitive intracellular Ca2+ release channels known as ryanodine receptors (RyRs) in pulmonary arterial smooth-muscle cells (PASMCs) may play a role in HPV, and considerable evidence now supports that levels of reactive oxygen species (ROS) are paradoxically increased in PASMC under hypoxia, we investigated whether redox activation of RyRs by ROS may transduce HPV. By reverse transcriptase-polymerase chain reaction, we found that all three RyR isoforms are expressed in rat pulmonary arteries and in PASMCs. The sustained phase, but not the transient phase, of HPV can be prevented by pretreating pulmonary arteries with RyR inhibitors ryanodine (200 micromol/L) or dantrolene (50 micromol/L). The addition of dantrolene, ryanodine or the thiol-reducing agent dithiothreitol (1 mmol/L) during the sustained phase of HPV reversed the hypoxic vasoconstriction. In contrast, the superoxide scavenger nitroblue tetrazolium (500 nmol/L) prevented further hypoxic pulmonary vasoconstriction during the sustained phase of HPV but did not reverse it. Taken together, our data suggest that redox activation of RyRs by ROS has an important role in transducing the sustained contraction of pulmonary arteries under hypoxia.

Topics: Animals; Calcium; Dantrolene; Hypoxia; In Vitro Techniques; Lung; Male; Nitroblue Tetrazolium; Oxidation-Reduction; Pulmonary Artery; Rats; Rats, Wistar; Ryanodine; Ryanodine Receptor Calcium Release Channel; Vasoconstriction

Previous studies showed that long-term hypoxia (LTH) during pregnancy alters myometrial contractility. The present study was designed to test the hypothesis that LTH during pregnancy suppresses myometrial contractility in sheep by affecting the calcium signaling cascade. Pregnant sheep were maintained at high altitude (3820 m) from Day 30 to Day 139 of gestation, when the animals were killed for collection of myometrial tissue. Tissue was also collected from age-matched, normoxic controls. Circular and longitudinal layers were separated, and strips from each layer were mounted in a muscle bath. After pretreatment with 10(-8) M oxytocin, the strips were exposed to increasing half- or quarter-log doses of nifedipine (L-type calcium-channel blocker), ruthenium red, ryanodine (blockers of inositol 1,4,5-trisphosphate-insensitive calcium stores), or 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC; phospholipase C inhibitor). Area under the contraction curve was analyzed, and pD(2) (log of concentration yielding 50% of maximum response) values and maximum relaxation responses were calculated. The maximum relaxation response to nifedipine was increased in both longitudinal (P < 0.01) and circular (P < 0.05) myometrial layers from LTH compared to control tissue, whereas no difference was observed in response to ruthenium red or ryanodine. The maximum relaxation response to NCDC was lower in the LTH circular layer (P < 0.05). Together, these data are indicative of an increase in the dependence of ovine uterine smooth muscle on extracellular calcium influx through the L-type, voltage-gated calcium channels following LTH. This appears to occur not through an increase in L-type calcium channels but, rather, through a possible decline in importance of the oxytocin-induced, phospholipase C-mediated pathway, resulting in a greater proportion of extracellular calcium contributing to contraction. Layer-dependent differences also exist between the circular and longitudinal myometrium in response to phospholipase C inhibition.

Topics: Animals; Calcium; Calcium Channel Blockers; Chronic Disease; Female; Gestational Age; Hypoxia; Myometrium; Nifedipine; Phenylcarbamates; Pregnancy; Pregnancy, Animal; Protease Inhibitors; Ruthenium Red; Ryanodine; Sheep; Type C Phospholipases

This study further investigates the mechanisms responsible for the effects of acute and severe hypoxia, and subsequent reoxygenation, on the contractility of isolated rat mesenteric arteries. In noradrenaline (NA)-contracted arteries, hypoxia caused a relaxation to near baseline levels. Reoxygenation resulted in an immediate transient contraction before tension returned more slowly to prehypoxia levels. Similar responses to hypoxia were observed in tissues precontracted by addition of KCl (60 mM) or U46619 (10 microM); however, the transient contraction upon reoxygenation was absent (KCl) or reduced (U46619). Responses to hypoxia were independent of changes in intracellular calcium ([Ca2+]i), while those to reoxygenation were accompanied by corresponding changes in [Ca2+]i and were completely abolished by ryanodine. In NA-contracted tissues, all responses were unaffected by endothelial removal or by inhibitors of nitric oxide synthase and cyclooxygenase. The K+ channel blockers triethylamine (TEA), glibenclamide, and 4-aminopyridine (4-AP) had no effect on the responses to hypoxia. The transient contractile response to reoxygenation was, however, significantly reduced in the presence of 4-AP. The response to reoxygenation, but not that to hypoxia, was inhibited by the antioxidant dithiothreitol (DTT) and the NAD(P)H-oxidase inhibitor diphenyliodonium (DPI). These data suggest that hypoxic vasodilation occurs independently of reductions in [Ca2+]i. Alternatively, transient contractions on reoxygenation are dependent upon the generation of reactive oxygen species and the release of stored Ca2+.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; 4-Aminopyridine; Adenosine Triphosphate; Animals; Anti-Arrhythmia Agents; Calcium; Endothelium, Vascular; Ethylamines; Glyburide; Hypoxia; Male; Mesenteric Arteries; Muscle Contraction; Norepinephrine; Oxygen; Potassium; Potassium Channel Blockers; Potassium Channels; Rats; Rats, Wistar; Reperfusion Injury; Ryanodine

Organ culture specifically inhibits vasorelaxation to acute hypoxia and preferentially decreases specific voltage-dependent K(+) channel expression over other K(+) and Ca(2+) channel subtypes. To isolate further potential oxygen-sensing mechanisms correlated with altered gene expression, we performed differential display analysis on RNA isolated from control and cultured coronary arterial rings. We hypothesize that organ culture results in altered gene expression important for vascular smooth muscle contractility important to the mechanism of hypoxia-induced relaxation. Our results indicate a milieu of changes suggesting both up- and downregulation of several genes. The altered expression pattern of two positive clones was verified by Northern analysis. Subsequent screening of a porcine cDNA library indicated homology to the ryanodine receptor (RyR). RT-PCR using specific primers to the three subtypes of RyR shows an upregulation of RyR2 and RyR3 after organ culture. Additionally, the caffeine- and/or ryanodine-sensitive intracellular Ca(2+) store was significantly more responsive to caffeine activation after organ culture. Our data indicate that organ culture increases expression of specific RyR subtypes and inhibits hypoxic vasorelaxation. Importantly, ryanodine blunted hypoxic relaxation in control coronary arteries, suggesting that upregulated RyR might play a novel role in altered intracellular Ca(2+) handling during hypoxia.

Topics: Animals; Arteries; Base Sequence; Caffeine; Calcium; DNA, Complementary; Gene Expression; Gene Expression Profiling; Gene Library; Hypoxia; Intracellular Membranes; Molecular Sequence Data; Organ Culture Techniques; Protein Isoforms; Reference Values; Reverse Transcriptase Polymerase Chain Reaction; Ryanodine; Ryanodine Receptor Calcium Release Channel; Swine; Vasodilation

The contribution of alterations in mitochondrial K(ATP) channel activity and the sarcoplasmic reticulum (SR) to anaerobic cardiac function in the anoxia tolerant armored catfish Liposarcus pardalis were assessed. K(ATP) channels contribute to hypoxic cardioprotection in mammals, but little is known of their action in more hypoxia tolerant animals. Anoxia resulted in a decrease in force in isometrically contracting ventricle strips to approximately 40% of the pre-anoxic level. This was maintained for at least 2 h. Upon reoxygenation, hearts recovered to the same level as control preparations. Treatment with 5-hydroxydecanoic acid (5HD), a specific mitochondrial K(ATP) blocker significantly increased force in preparations during anoxia and caused hypercontracture at reoxygenation. Ryanodine, a specific inhibitor of SR function, significantly increased force loss in ventricle preparations under anoxia. Results show that mitochondrial K(ATP) channel activity and SR function are important in anaerobic and post-anaerobic contractility in armored catfish heart.

Topics: Adenosine Triphosphate; Animals; Catfishes; Decanoic Acids; Hydroxy Acids; Hypoxia; In Vitro Techniques; Mitochondria, Heart; Myocardial Contraction; Oxygen; Potassium Channel Blockers; Potassium Channels; Ryanodine; Sarcoplasmic Reticulum

The relationship between nitric oxide (NO) and intracellular Ca2+ in hypoxic-ischemic brain damage is not known in detail. Here we used rat striatal slices perfused under low-oxygen and Ca2+-free conditions and cultured human astrocytoma cells incubated under similar conditions as models to study the dynamics of intracellular NO and Ca2+ in hypoxia-induced tissue damage. Exposure of rat striatal slices for 70 min to low oxygen tension elicited a delayed and sustained increase in the release of 45Ca2+. This was potentiated by the NO donors sodium nitroprusside (SNP) and spermine-NO and inhibited by N-omega-nitro-L-arginine methyl ester (L-NAME) or by the NO scavenger 2-phenyl-4,4,5,5 tetramethylimidazoline-1-oxyl-3-oxide (PTIO). A membrane-permeant form of heparin in combination with either ruthenium red (RR) or ryanodine (RY) also inhibited 45Ca2+ release. In human astrocytoma U-373 MG cells, hypoxia increased intracellular Ca2+ concentration ([Ca2+]i) by 67.2 +/- 13.1% compared to normoxic controls and this effect was inhibited by L-NAME, PTIO or heparin plus RR. In striatal tissue, hypoxia increased NO production and LDH release and both effects were antagonized by L-NAME. Although heparin plus RR or RY antagonized hypoxia-induced increase in LDH release they failed to counteract increased NO production. These data therefore indicate that NO contributes to hypoxic damage through increased intracellular Ca2+ mobilization from endoplasmic reticulum and suggest that the NO-Ca2+ signalling might be a potential therapeutic target in hypoxia-induced neuronal degeneration.

Topics: Animals; Anticoagulants; Astrocytoma; Calcium; Cell Line, Tumor; Corpus Striatum; Cyclic N-Oxides; Dose-Response Relationship, Drug; Drug Combinations; Drug Interactions; Enzyme Inhibitors; Free Radical Scavengers; Fura-2; Heparin; Humans; Hydro-Lyases; Hypoxia; Imidazoles; In Vitro Techniques; Intracellular Space; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Perfusion; Rats; Rats, Sprague-Dawley; Ruthenium; Ryanodine

To test the hypothesis that chronic intrauterine pulmonary hypertension (PHTN) compromises pulmonary artery (PA) smooth muscle cell (SMC) O2 sensing, fluorescence microscopy was used to study the effect of an acute increase in Po2 on the cytosolic Ca2+ concentration ([Ca2+]i) of chronically hypoxic subconfluent monolayers of PA SMC in primary culture. PA SMCs were derived from fetal lambs with PHTN due to intrauterine ligation of the ductus arteriosus. Acute normoxia decreased [Ca2+]i in control but not PHTN PA SMC. In control PA SMC, [Ca2+]i increased after Ca2+-sensitive (KCa) and voltage-sensitive (Kv) K+ channel blockade and decreased after diltiazem treatment. In PHTN PA SMC, KCa blockade had no effect, whereas Kv blockade and diltiazem increased [Ca2+]i. Inhibition of sarcoplasmic reticulum Ca2+ ATPase activity caused a greater increase in [Ca2+]i in controls compared with PHTN PA SMC. Conversely, ryanodine caused a greater increase of [Ca2+]i in PHTN compared with control PA SMC. KCa channel mRNA is decreased and Kv channel mRNA is unchanged in PHTN PA SMC compared with controls. We conclude that PHTN compromises PA SMC O2 sensing, alters intracellular Ca2+ homeostasis, and changes the predominant ion channel that determines basal [Ca2+]i from KCa to Kv.

Topics: Animals; Blood Proteins; Calcium; Calcium-Transporting ATPases; Cells, Cultured; Cytoplasm; Enzyme Inhibitors; Female; Fetal Diseases; Fetus; Hypertension, Pulmonary; Hypoxia; Muscle, Smooth, Vascular; Oxygen; Peptides; Potassium; Potassium Channels; Pregnancy; Pulmonary Artery; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Ryanodine; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sheep; Thapsigargin

Intermittent hypoxia (IH) adaptation has been shown to exert beneficial effects on the functions of hearts that had been subjected to insult by ischemia or ischemia/reperfusion. To understand whether calcium release channels/ryanodine receptors (RyRs) were involved, the effects of IH and continuous hypoxia (CH) on [3H]ryanodine binding to homogenates of rat hearts were investigated. Similar studies were performed on rat skeletal muscle. The main results on cardiac muscle were as follows: 1) Ischemia for up to 45 min in normal rat hearts had no obvious effect on the equilibrium ryanodine binding constant (K(d)), while the maximum number of ryanodine binding sites (B(max)) was affected in a time-dependent manner. B(max) was significantly increased with 15 min ischemia, which then returned to control levels upon prolonging the ischemia to 30 min. After 45 min ischemia, a small decrease of B(max) was observed. 2) IH adaptation for up to 28 days did not change B(max), but a significant decrease of B(max) was apparent after longer IH adaptation or after CH exposure. Although B(max) was not altered by 30 min ischemia, 30 min reperfusion following 30 min ischemia induced an evident decrease of B(max). After either IH or CH adaptation, the ischemia/reperfusion- induced decrease of B(max) was abolished. 3) Several effects on K(d) of ischemia and ischemia/reperfusion, with and without IH or CH adaptation, were observed. The most distinct and consistent finding was that a clear increase of K(d) was induced by ischemia or ischemia/reperfusion in CH adapted rats. [3H]Ryanodine binding to homogenates of rat skeletal muscle was also affected by IH and CH adaptation. In contrast to that found in cardiac muscle, a decrease of B(max) in skeletal muscle appeared only after CH adaptation. The physiological significance of these effects is discussed.

Topics: Animals; Binding Sites; Hypoxia; In Vitro Techniques; Male; Muscle, Skeletal; Myocardial Reperfusion Injury; Myocardium; Rats; Rats, Sprague-Dawley; Ryanodine; Ryanodine Receptor Calcium Release Channel; Time Factors

Studies of thapsigargin, cyclopiazonic acid, and ryanodine in isolated pulmonary arteries and smooth muscle cells suggest that release of Ca(2+) from inositol 1,4,5-trisphosphate (IP(3))- and/or ryanodine-sensitive sarcoplasmic reticulum Ca(2+) stores is a component of the mechanism of acute hypoxic pulmonary vasoconstriction (HPV). However, the actions of these agents on HPV in perfused lungs have not been reported. Thus we tested effects of thapsigargin and cyclopiazonic acid, inhibitors of sarcoplasmic reticulum Ca(2+)-ATPase, and of ryanodine, an agent that either locks the ryanodine receptor open or blocks it, on HPV in salt solution-perfused rat lungs. After inhibition of cyclooxygenase and nitric oxide synthase, thapsigargin (10 nM) and cyclopiazonic acid (5 microM) augmented the vasoconstriction to 0% but not to 3% inspired O(2). Relatively high concentrations of ryanodine (100 and 300 microM) blunted HPV in nitric oxide synthase-inhibited lungs. The results indicate that release of Ca(2+) from the ryanodine-sensitive, but not the IP(3)-sensitive, store, contributes to the mechanism of HPV in perfused rat lungs and that Ca(2+)-ATPase-dependent Ca(2+) buffering moderates the response to severe hypoxia.

Topics: Animals; Calcium; Enzyme Inhibitors; Hypoxia; Indoles; Male; Nitric Oxide Synthase; Nitroarginine; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; Ryanodine; Thapsigargin; Vasoconstriction

To determine the role of endothelium in hypoxic pulmonary vasoconstriction (HPV), we measured vasomotor responses to hypoxia in isolated seventh-generation porcine pulmonary arteries < 300 microm in diameter with (E+) and without endothelium. In E+ pulmonary arteries, hypoxia decreased the vascular intraluminal diameter measured at a constant transmural pressure. These constrictions were complete in 30-40 min; maximum at PO(2) of 2 mm Hg; half-maximal at PO(2) of 40 mm Hg; blocked by exposure to Ca(2+)-free conditions, nifedipine, or ryanodine; and absent in E+ bronchial arteries of similar size. Hypoxic constrictions were unaltered by indomethacin, enhanced by indomethacin plus N(G)-nitro-L-arginine methyl ester, abolished by BQ-123 or endothelial denudation, and restored in endothelium-denuded pulmonary arteries pretreated with 10(-10) M endothelin-1 (ET-1). Given previous demonstrations that hypoxia caused contractions in isolated pulmonary arterial myocytes and that ET-1 receptor antagonists inhibited HPV in intact animals, our results suggest that full in vivo expression of HPV requires basal release of ET-1 from the endothelium to facilitate mechanisms of hypoxic reactivity in pulmonary arterial smooth muscle.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Bronchial Arteries; Caffeine; Calcium; Cell Survival; Endothelin-1; Endothelium, Vascular; Enzyme Inhibitors; Hypoxia; In Vitro Techniques; Lung; Male; Pulmonary Artery; Receptors, Endothelin; Ryanodine; Swine; Vasoconstriction; Vasoconstrictor Agents; Vasodilator Agents

The hypoxic constriction of isolated pulmonary vessels is composed of an initial transient phase (phase 1) followed by a slowly developing increase in tone (phase 2). We investigated the roles of the endothelium and of intracellular Ca2+ stores in both preconstricted and unpreconstricted intrapulmonary rabbit arteries when challenged with hypoxia (PO2 16-21 Torr). Removing the endothelium did not affect phase 1, but phase 2 appeared as a steady plateau. Removing extracellular Ca2+ had essentially the same effect as removing the endothelium. Depletion of sarcoplasmic reticulum Ca2+ stores with caffeine and ryanodine abolished the hypoxic response. Omitting preconstriction reduced the amplitude of the hypoxic response but did not qualitatively affect any of the above responses. We conclude that hypoxia releases intracellular Ca2+ from ryanodine-sensitive stores by a mechanism intrinsic to pulmonary vascular smooth muscle without the need for Ca2+ influx across the plasmalemma or an endothelial factor. Our results also suggest that extracellular Ca2+ is required for the release of an endothelium-derived vasoconstrictor.

Topics: Animals; Caffeine; Calcium; Extracellular Space; Hypoxia; In Vitro Techniques; Intracellular Membranes; Male; Muscle, Smooth, Vascular; Osmolar Concentration; Pulmonary Artery; Rabbits; Ryanodine; Sarcoplasmic Reticulum; Vasoconstriction

Hypoxic vasoconstriction (HV) is an intrinsic response of mammalian pulmonary and cyclostome aortic vascular smooth muscle. The present study examined the utilization of calcium during HV in dorsal aortas (DA) from sea lamprey and New Zealand hagfish. HV was temporally correlated with increased free cytosolic calcium (Ca2+c) in lamprey DA. Extracellular calcium (Ca2+o) did not contribute significantly to HV in lamprey DA, but it accounted for 38.1 +/- 5.3% of HV in hagfish DA. Treatment of lamprey DA with ionomycin, ryanodine, or caffeine added to thapsigargin-reduced HV, whereas HV was augmented by BAY K 8644. Methoxyverapamil (D600) in zero Ca2+o did not affect HV in lamprey DA, nor did it prevent further constriction when Ca2+o was restored during hypoxia in hagfish DA. Removal of extracellular sodium (Na+o) caused a constriction in both species. Lamprey DA relaxed to prehypoxic tension following return to normoxia in zero Na+o, whereas relaxation was inhibited in hagfish DA. Relaxation following HV was inhibited in lamprey DA when Na+o and Ca2+o were removed. These results show that HV is correlated with [Ca2+]c in lamprey DA and that Na+/Ca2+ exchange is used during HV in hagfish but not lamprey DA. Multiple receptor types appear to mediate stored intracellular calcium release in lamprey DA, and L-type calcium channels do not contribute significantly to constriction in either cyclostome.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Aorta; Caffeine; Calcium; Calcium Channel Agonists; Calcium Channel Blockers; Enzyme Inhibitors; Gallopamil; Hagfishes; Hypoxia; In Vitro Techniques; Indoles; Ionomycin; Ionophores; Lampreys; Muscle, Smooth, Vascular; Norepinephrine; Oxygen; Ryanodine; Thapsigargin; Vasoconstriction; Vasodilator Agents

Mammalian hearts experience calcium overload during extreme and prolonged hypoxia and the calcium overload may lead to enzyme activation and cell death. Several calcium transport systems were examined in muskrat hearts and compared to those found in rat hearts to determine if there is a species difference that might be related to the muskrats' superior ability to survive hypoxia. Radiolabeled nitredendipine binding was determined in rat and muskrat hearts to estimate the density of voltage gated calcium channels in surface membranes. There were no species differences. Calcium release channel density in the sarcoplasmic reticulum was estimated by the determination of radiolabeled ryanodine binding in muskrat and rat heart SR membranes. No differences were revealed between species. The SR uptake of calcium was measured in SR membranes from the hearts of the two species. No differences were found in the B(max) values, however, the muskrat SR membranes did have a slightly lower K(m) value. There were large species differences in Na(+)/Ca(2+) exchange in SL membranes with the muskrat heart having approximately 3.5 times the transport capacity of rat SL membranes. During hypoxic conditions in which there is extensive ATP depletion leading to [Na(+)](i) accumulation and discharge of cellular membrane potential, the Na(+)/Ca(2+) exchanger may operate in the reverse mode and import calcium into the cell and accelerate hypoxic damage. Prior to reaching this state a robust Na(+)/Ca(2+) exchange would facilitate the maintenance of normal diastolic calcium levels and calcium cycling. Muskrats hearts are hypoxia tolerant by virtue of their ability to reduce metabolic demand and generate ATP anaerobically thus, maintaining a favorable ATP balance. Therefore, the relative overexpression of Na(+)/Ca(2+) exchangers in muskrat hearts may be beneficial in the preservation of contractile function and calcium homeostasis in this freshwater diving mammal.

Topics: Adenosine Triphosphate; Animals; Arvicolinae; Biological Transport; Body Weight; Calcium; Calcium Channel Agonists; Calcium Channels, L-Type; Cell Membrane; Dose-Response Relationship, Drug; Hypoxia; Kinetics; Myocardium; NADH Dehydrogenase; Protein Binding; Rats; Rats, Sprague-Dawley; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcolemma; Sarcoplasmic Reticulum; Sodium; Species Specificity; Time Factors

A change in the intracellular Ca(2+) ([Ca(2+)](i)) level induced by hypoxia was detected in rat adrenal slices by use of fura-2/AM. After hypoxic stress, an increase in [Ca(2+)](i) was observed only in the adrenal medulla. This increase was inhibited by nifedipine, but not modified by the cholinergic receptor blockers. The hypoxia-induced increase in [Ca(2+)](i) was observed in all postnatal developmental stages to a similar extent, whereas the nicotine and high K(+) sensitivities increased along with postnatal development. A 10 nM ryanodine enhanced the hypoxia-induced [Ca(2+)](i) increase in adult but not in neonatal rat slices. These results suggest the existence of an oxygen-sensing mechanism in adult rat adrenals even after sympathetic innervation. Hypoxic responses seemed to be similar both in neonate and in adult rat adrenals and were triggered by the influx of Ca(2+) via L-type voltage-sensitive Ca(2+) channels. However, the sustained [Ca(2+)](i) increase caused by hypoxia might depend on postnatal development and be triggered by Ca(2+)-induced Ca(2+) release (CICR).

Topics: Adrenal Glands; Animals; Animals, Newborn; Calcium; Cells, Cultured; Chromaffin Cells; Hypoxia; In Vitro Techniques; Male; Nicotine; Potassium; Rats; Rats, Wistar; Ryanodine; Sympathetic Nervous System

Striatal large aspiny interneurons were recorded from a slice preparation using a combined electrophysiologic and microfluorometric approach. The role of intracellular Ca2+ stores was analyzed during combined oxygen/glucose deprivation (OGD). Before addressing the role of the stores during energy deprivation, the authors investigated their function under physiologic conditions. Trains of depolarizing current pulses caused bursts of action potentials coupled to transient increases in intracellular calcium concentration ([Ca2+]i). In the presence of cyclopiazonic acid (30 micromol/L), a selective inhibitor of the sarcoendoplasmic reticulum Ca2+ pumps, or when ryanodine receptors were directly blocked with ryanodine (20 [micromol/L), the [Ca2+]i transients were progressively smaller in amplitude, suggesting that [Ca2+]i released from intracellular stores helps to maintain a critical level of [Ca2+]i during physiologic firing activity. As the authors have recently reported, brief exposure to combined OGD induced a membrane hyperpolarization coupled to an increase in [Ca2+]i. In the presence of cyclopiazonic acid or ryanodine, the hyperpolarization and the rise in [Ca2+]i induced by OGD were consistently reduced. These data support the hypothesis that Ca2+ release from ryanodine-sensitive Ca2+ pools is involved not only in the potentiation of the Ca2+ signals resulting from cell depolarization, but also in the amplification of the [Ca2+]i rise and of the concurrent membrane hyperpolarization observed in course of OGD in striatal large aspiny interneurons.

Topics: Animals; Calcium; Calcium Channel Blockers; Corpus Striatum; Electrophysiology; Glucose; Hypoxia; In Vitro Techniques; Indoles; Interneurons; Intracellular Membranes; Male; Osmolar Concentration; Rats; Rats, Wistar; Ryanodine

It has been proposed that hypoxic pulmonary vasoconstriction (HPV) is mediated via K+ channel inhibition and Ca2+ influx through voltage-gated channels. HPV depends strongly on the degree of preconstriction, and we therefore examined the effect of Ca2+ channel blockade on tension and intracellular [Ca2+] ([Ca2+]i) during HPV in rat intrapulmonary arteries (IPAs), whilst maintaining preconstriction constant. We also investigated the role of intracellular Ca2+ stores. HPV demonstrated a transient constriction (phase I) superimposed on a sustained constriction (phase II). Nifedipine (1 microM) partially inhibited phase I, but did not affect phase II. In arteries exposed to 80 mM K+ and nifedipine or diltiazem the rises in tension and [Ca2+]i were blunted during phase I, but were unaffected during phase II. At low concentrations (< 3 microM), La3+ almost abolished the phase I constriction and rise in [Ca2+]i, but had no effect on phase II, or constriction in response to 80 mM K+. Phase II was inhibited by higher concentrations of La3+ (IC50 approximately 50 microM). IPA treated with thapsigargin (1 microM) in Ca2+-free solution to deplete Ca2+ stores showed sustained constriction upon re-exposure to Ca2+ and an increase in the rate of Mn2+ influx, suggesting capacitative Ca2+ entry. The concentration dependency of the block of constriction by La3+ was similar to that for phase I of HPV. Pretreatment of IPA with 30 microM CPA reduced phase I by > 80 %, but had no significant effect on phase II. We conclude that depolarization-mediated Ca2+ influx plays at best a minor role in the transient phase I constriction of HPV, and is not involved in the sustained phase II constriction. Instead, phase I appears to be mainly dependent on capacitative Ca2+ entry related to release of thapsigargin-sensitive Ca2+ stores, whereas phase II is supported by Ca2+ entry via a separate voltage-independent pathway.

Topics: Animals; Caffeine; Calcium; Calcium Channel Blockers; Calcium Channels; Chelating Agents; Diltiazem; Dinucleoside Phosphates; Egtazic Acid; Electric Stimulation; Enzyme Inhibitors; Hypoxia; Imidazoles; In Vitro Techniques; Ion Channel Gating; Lanthanum; Male; Membrane Potentials; Nifedipine; Phosphodiesterase Inhibitors; Pulmonary Artery; Pulmonary Circulation; Rats; Rats, Wistar; Ryanodine; Thapsigargin; Vasoconstriction; Verapamil

Perturbations of the trans-sarcolemmal and sarcoplasmic Ca2+ transport contribute to the abnormal myocardial activity provoked by anoxia and reoxygenation. Whether Ca2+ pools of the extracellular compartment and sarcoplasmic reticulum (SR) are involved to the same extent in the dysfunction of the anoxic-reoxygenated immature heart has not been investigated. Spontaneously contracting hearts isolated from 4-day-old chick embryos were submitted to repeated anoxia (1 min) followed by reoxygenation (5 min). Heart rate, atrioventricular propagation velocity, ventricular shortening, velocities of contraction and relaxation, and incidence of arrhythmias were studied, recorded continuously. Addition of verapamil (10 nM), which blocks selectively sarcolemmal L-type Ca2+ channels, was expected to protect against excessive entry of extracellular Ca2+, whereas addition of ryanodine (10 nM), which opens the SR Ca2+ release channel, was expected to increase cytosolic Ca2+ concentration. Verapamil (a) had no dromotropic effect by contrast to adult heart, (b) attenuated ventricular contracture induced by repeated anoxia, (c) shortened cardioplegia induced by reoxygenation, and (d) had remarkable antiarrhythmic properties during reoxygenation specially. On the other hand, ryanodine potentiated markedly arrhythmias both during anoxia and at reoxygenation. Thus despite its immaturity, the SR seems to be functional early in the developing chick heart and involved in the reversible dysfunction induced by anoxia-reoxygenation. Moreover, Ca2+ entry through L-type channels appears to worsen arrhythmias especially during reoxygenation. These findings show that the Ca2+-handling systems involved in irregular activity in immature heart, such as the embryonic chick heart, may differ from those in the adult.

Topics: Animals; Anti-Arrhythmia Agents; Arrhythmias, Cardiac; Calcium Channel Blockers; Chick Embryo; Contracture; Heart; Heart Arrest; Heart Rate; Hypoxia; In Vitro Techniques; Myocardial Contraction; Oxygen; Ryanodine; Verapamil

We studied myocardial contractility in fetal sheep from ewes exposed to approximately 112 days of hypoxia at high altitude (3,820 m). We measured the inotropic response to extracellular Ca2+ concentration ([Ca2+]o, 0.2-10 mM) and ryanodine (10(-10) to 10(-4) M) in isometrically contracting papillary muscles and quantified dihydropyridine (DHPR) and ryanodine (RyR) receptors. In hypoxic fetuses, curves describing the force-[Ca2+]o relationship were shifted left, and the top plateaus were decreased by approximately 35% in both left and right ventricles. In normoxic and hypoxic fetuses, ryanodine (10(-4) M) reduced maximum active tension (Tmax) to approximately 25-40% of baseline values, indicating that the sarcoplasmic reticulum was the chief source of activator Ca2+ and that Ca2+ influx alone was not sufficient to activate a contraction of normal amplitude. Hypoxia resulted in a lower Tmax in the right ventricle and a lower maximum rate of rise in the left ventricle after treatment with ryanodine. DHPR number did not change, but RyR number and the RyR/DHPR in both ventricles were higher in hypoxic fetuses. We conclude that hypoxia decreases contractility, possibly by reducing the availability of activator Ca2+. Further studies are needed to directly measure the Ca2+ current and intracellular Ca2+ transient and to examine myofilament protein and adenosinetriphosphatase activity.

Topics: Altitude; Animals; Calcium; Calcium Channels; Calcium Channels, L-Type; Electric Stimulation; Female; Fetal Heart; Hypoxia; In Vitro Techniques; Isradipine; Muscle Proteins; Myocardial Contraction; Papillary Muscles; Pregnancy; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sheep; Ventricular Function, Left; Ventricular Function, Right

The effects of hypoxia on norepinephrine-induced contraction to explain why rabbit pulmonary arteries must be precontracted to observe a hypoxic response were studied. A force transducer was used to record the tone of isolated rabbit intrapulmonary artery rings placed in an organ chamber perfused with a physiological solution at 37 degrees C. Norepinephrine (10(-7) M) induced a phasic followed by a tonic contraction, and hypoxia increased the former and decreased the latter. Removal of external calcium (zero calcium solution) abolished the tonic contraction but left the phasic contraction intact. In the zero calcium solution, hypoxia increased the amplitude of the phasic contraction (9.8 +/- 7.4 vs. 13.3 +/- 11.9 mN) and decreased the 50% relaxation time (59 +/- 38 vs. 48 +/- 22 s). Hypoxia also increased the caffeine (5 mM)-induced contraction. This hypoxic increase in amplitude was abolished by ryanodine (100 microM). The hypoxic decrease in the 50% relaxation time was reduced by cyclopiazonic acid (1-10 microM). Therefore, hypoxia increases the reuptake of calcium by calcium pumps sensitive to cyclopiazonic acid in the caffeine- and ryanodine-sensitive stores.

Topics: Animals; Caffeine; Calcium; Hypoxia; In Vitro Techniques; Indoles; Muscle Contraction; Muscle, Smooth, Vascular; Norepinephrine; Pulmonary Artery; Rabbits; Ryanodine; Sarcoplasmic Reticulum; Time Factors; Vasodilator Agents

A reduction in oxygen tension in the lungs is believed to inhibit a voltage-dependent K+ (Kv) current, which is thought to result in membrane depolarization leading to hypoxic pulmonary vasoconstriction (HPV). However, the direct mechanism by which hypoxia inhibits Kv current is not understood.. Experiments were performed on rat pulmonary artery resistance vessels and single smooth muscle cells isolated from these vessels to examine the role of Ca2+ release from intracellular stores in initiating HPV. In contractile experiments, hypoxic challenge of endothelium-denuded rat pulmonary artery resistance vessels caused either a sustained or transient contraction in Ca2+-containing or Ca2+-free solution, respectively (n=44 vessels from 11 animals). When the ring segments were treated with either thapsigargin (5 micromol/L), ryanodine (5 micromol/L), or cyclopiazonic acid (5 micromol/L) in Ca2+-containing or Ca2+-free solution, a significant increase in pulmonary arterial tone was observed (n=44 vessels from 11 animals). Subsequent hypoxic challenge in the presence of each agent produced no further increase in tone (n=44 vessels from 11 animals). In isolated pulmonary resistance artery cells loaded with fura 2, hypoxic challenge, thapsigargin, ryanodine, and cyclopiazonic acid resulted in a significant increase in [Ca2+]i (n=18 cells from 6 animals) and depolarization of the resting membrane potential (n=22 cells from 6 animals). However, with prior application of thapsigargin, ryanodine, or cyclopiazonic acid, a hypoxic challenge produced no further change in [Ca2+]i (n=18 from 6 animals) or membrane potential (n=22 from 6 animals). Finally, application of an anti-Kv1.5 antibody increased [Ca2+]i and caused membrane depolarization. Subsequent hypoxic challenge resulted in a further increase in [Ca2+]i with no effect on membrane potential (n=16 cells from 4 animals).. In rat pulmonary artery resistance vessels, an initial event in HPV is a release of Ca2+ from intracellular stores. This rise in [Ca2+]i causes inhibition of voltage-dependent K+ channels (possibly Kv1.5), membrane depolarization, and an increase in pulmonary artery tone.

Topics: Animals; Calcium; Hypoxia; In Vitro Techniques; Indoles; Inositol 1,4,5-Trisphosphate; Intracellular Membranes; Male; Membrane Potentials; Pulmonary Artery; Rats; Ryanodine; Thapsigargin; Vascular Resistance; Vasoconstriction; Vasoconstrictor Agents; Vasodilator Agents

We previously reported that a brief period of hypoxic perfusion (BHP) prior to ischemia in rat hearts improved functional recovery upon reperfusion with reduced Ca2+ overload. The present study was designed to determine whether the effect of BHP would be associated with a reduction in reperfusion arrhythmias and a preservation of function of the sarcoplasmic reticulum (SR).. Hearts were subjected to 40 min of global ischemia and 30 min of reperfusion after a 20 min period of oxygenated perfusion (oxygenated group: OG), or a 10 min period of oxygenation and 10 min of hypoxic perfusion (hypoxic group: HG). We evaluated the release of Ca2+ by SR blocked by ryanodine, the recovery of left ventricular function, and the reperfusion induced ventricular tachycardia/fibrillation (VT/VF).. Functional recovery improved and the incidence and duration of reperfusion VT/VF were reduced in HG. In HG the uptake of Ca2+ in SR decreased during ischemia, but this decrease was less than that in OG. However, recovery of Ca2+ uptake after reperfusion did not differ between groups. The release of Ca2+ by SR blocked by ryanodine was inhibited in HG throughout the ischemia-reperfusion sequence.. Observations suggest that the benefits of BHP on recovery of function and reperfusion arrhythmias were associated with a decrease in release of Ca2+ by SR blocked by ryanodine.

Topics: Animals; Anti-Arrhythmia Agents; Calcium; Hypoxia; In Vitro Techniques; Male; Myocardial Reperfusion Injury; Myocardium; Rats; Rats, Sprague-Dawley; Ryanodine; Sarcoplasmic Reticulum

The effect of stretch on cardiac muscle contraction and the Ca2+ transient was studied during hypoxia and acidosis in isolated ferret ventricular muscles. In control conditions, a maintained stretch produced an immediate increase in tension followed by a slow increase in tension and the Ca2+ transient. A stretch between contractions (diastolic stretch) caused only a slow increase in tension and the Ca2+ transient, whereas a stretch during the period of contraction (systolic stretch) produced an immediate increase in tension followed by a small slow increase in tension and the Ca2+ transient. In hypoxia, the immediate percent increase in tension was the same as in control. However, the slow increase was smaller during all three types of stretch. In acidosis, the immediate percent increase in tension was larger than in control. The slow change was the same during maintained stretch. However, the slow increase in tension was smaller during diastolic stretch and larger during systolic stretch. Thus the stretch-dependent increase in contraction is inhibited during hypoxia and modulated by acidosis.

Topics: Acidosis; Aequorin; Animals; Calcium; Female; Ferrets; Hypoxia; Male; Muscle Contraction; Papillary Muscles; Physical Stimulation; Ryanodine; Ventricular Function

Acute systemic hypoxia induces mesenteric venoconstriction in intact rabbits in part because of an increase in chemoreflex-mediated sympathetic efferent nerve activity. Inhaled anesthetics attenuate this reflex response. The direct effects of hypoxia on mesenteric veins are unknown. The purpose of the current study was to examine the effects of hypoxia on isolated rabbit mesenteric capacitance veins and to determine the effects of halothane, isoflurane, and enflurane on the responses to hypoxia.. Isometric tension was measured before, during, and after 10 min of hypoxia in the rings of either quiescent or norepinephrine contracted veins, with or without endothelium. Effects of various pharmacologic agents and volatile anesthetics on the responses to hypoxia were examined.. Hypoxia augmented contractions to norepinephrine and phenylephrine only in endothelium-intact veins. The hypoxic response was inhibited by phentolamine (alpha-adrenoceptor antagonist) and abolished in the absence of extracellular Ca2+. There were no effects of propranolol (beta-adrenoceptor antagonist), ryanodine (a sarcoplasmic reticulum Ca2+ depleter), indomethacin (cyclooxygenase inhibitor), or nordihydroguaiaretic acid (lipoxygenase inhibitor). L-NAME (an inhibitor of nitric oxide synthase) enhanced basal sensitivity of veins to norepinephrine but had no effect on the response to hypoxia. Nicardipine (a blocker of voltage-gated calcium channels) depressed the hypoxic contraction by 86 +/- 5%, phosphoramidon (an inhibitor of endothelin-converting enzyme) by 82 +/- 8%, and BQ-123 (a specific endothelin-1 receptor antagonist) by 47 +/- 10%. Volatile anesthetics (1.0 MAC) inhibited responses to hypoxia in the absence as well as presence of L-NAME.. These results suggest that in mesenteric capacitance veins of rabbits an intrinsic vascular mechanism contributes to endothelium-dependent hypoxic augmentation of contraction to alpha-adrenergic agonists that involve activation of endothelin-1, an endothelium-derived constricting factor. Inhibition of hypoxic contraction by volatile anesthetics is not mediated by endothelium relaxing factor.

Topics: Acetylcholine; Animals; Arginine; Endothelium, Vascular; Enflurane; Glycopeptides; Halothane; Hypoxia; In Vitro Techniques; Indomethacin; Isoflurane; Male; Masoprocol; NG-Nitroarginine Methyl Ester; Nicardipine; Norepinephrine; Peptides, Cyclic; Rabbits; Ryanodine; Vasoconstriction

Changes in oxygen tension in the perinatal period contribute to high pulmonary vascular tone in the fetus and the decline in resistance that occurs at birth. Distal pulmonary artery smooth muscle cells (PASMC) isolated from late-gestation ovine fetuses respond to acute hypoxia with an increase in cytosolic calcium concentration ([Ca2+]i) dependent on Ca2+ entry. The purpose of this study is to determine 1) whether acute hypoxia results in PASMC membrane depolarization, 2) whether Ca2+ entry was through voltage-operated calcium channels (VOCC), 3) the contribution of Ca(2+)-induced Ca2+ release (CICR) to the hypoxic response, and 4) whether a subset of K+ channels might serve as oxygen sensors in fetal PASMC. We used microfluorimetry on subconfluent monolayers of PASMC in primary culture loaded with either a membrane potential-sensitive dye, bis(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4; DPASMC), to estimate membrane potential, or the Ca(2+)-sensitive fluorophore, fura 2, to measure [Ca2+]i. Hypoxia increased fluorescence from PASMC loaded with DiBAC4, consistent with membrane depolarization. Verapamil (an inhibitor of VOCC) attenuated, and BAY K 8644 (a VOCC facilitator) potentiated, the hypoxia-induced increase in [Ca2+]i, respectively. The hypoxic response was transient after treatment with ryanodine (10(-7) M), a blocker of calcium release from intracellular stores. Charybdotoxin (10(-7) M), an inhibitor of Ca(2+)-activated K+ channels, almost doubled [Ca2+]i, whereas glibenclamide (10(-5) M), an ATP-sensitive K(+)-channel antagonist, had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Acute Disease; Animals; Calcium; Cell Membrane; Cells, Cultured; Electrophysiology; Fetus; Fluorescence; Hypoxia; Manganese; Muscle, Smooth; Pulmonary Artery; Ryanodine; Sheep; Verapamil

The acute changes of muscle tone and membrane current upon occlusion of oxygenation (O2 occlusion) were studied in vitro in mouse diaphragms. O2 occlusion immediately produced a contraction and a relaxation, respectively, in ryanodine- and high K(+)-contracted muscles while a biphasic change (an initial decrease then a late increase) of muscle tone was produced in muscles contracted with caffeine. The O2 occlusion effects were reversed after reoxygenation. CN- produced similar acute changes of muscle tone and abolished O2 occlusion effects. The O2 occlusion-induced relaxation in high K+ medium was converted into a contraction by 3,4-diaminopyridine and by low Cl- Tyrode's. O2 occlusion induced a small outward current and membrane hyperpolarization at a rate slower than the changes of muscle tone. Glybenclamide inhibited all of the changes induced by O2 occlusion. It is possible that the K+ and Cl- permeabilities of sarcoplasmic reticulum are highly sensitive to hypoxic challenge and related to the immediate changes of muscle tone after O2 occlusion.

Topics: Animals; Caffeine; Cyanides; Electric Stimulation; Glyburide; Hypoxia; In Vitro Techniques; Mice; Mice, Inbred ICR; Muscle Contraction; Muscle Relaxation; Potassium; Respiratory Muscles; Ryanodine; Sodium

In the presence of 1 microM ryanodine, muscles loaded with the calcium indicator aequorin were stimulated at 15-20 Hz to produce steady levels of force and intracellular Ca2+ concentrations [( Ca2+]i) at various extracellular Ca2+ concentration ([Ca2+]o). After 5, 10, and 15 min of hypoxia and 3 min of reoxygenation, tetani were initiated. Force vs. [Ca2+]i relation was shifted to the right 0.11, 0.18, and 0.24 pCa units, and maximal force was down 66, 48, and 37% after start of hypoxia. During reoxygenation, the relationship was shifted up by 26%. In skinned fiber preparations, an increase in inorganic phosphate ion concentration from 0 to 10 mM and 15 mM decreased maximal force development by 32 and 53%, respectively, and shifted the pCa-force curve to the right by 0.08 and 0.14 pCa. A decrease in pH from 7.1 to 6.8 shifted the pCa-force curve to the right by 0.20 pCa units without affecting maximal force. These changes indicate that during hypoxia, a decrease in the sensitivity of the myofilaments to Ca2+ and a depression of maximal Ca2(+)-activated force occur, whereas during reoxygenation, there is an increase in maximal Ca2(+)-activated force.

Topics: Animals; Calcium; Ferrets; Histological Techniques; Hydrogen-Ion Concentration; Hypoxia; In Vitro Techniques; Myocardial Contraction; Myocardium; Myofibrils; Osmolar Concentration; Oxygen Consumption; Phosphates; Ryanodine

We tested the hypothesis that accumulation of H+ or inorganic phosphate (Pi) is responsible for the early contractile failure of hypoxia by measuring maximal Ca2+-activated pressure and 31P nuclear magnetic resonance spectra in Langendorff-perfused ferret hearts at 30 degrees C. Maximal Ca2+-activated pressure was identified by the saturation of pressure with respect to [Ca2+]o observed during tetani as [Ca2+]o was increased to 15 mM in HEPES-buffered, 100% O2-bubbled perfusate and during hypoxia induced by bubbling with room air or with 100% N2. Tetani were produced by pacing at 8-12 Hz following exposure to ryanodine (1-5 microM), an inhibitor of Ca2+ release from the sarcoplasmic reticulum, and were elicited once a minute to measure maximal Ca2+-activated pressure during acquisition of nuclear magnetic resonance spectra. An inverse correlation was observed between [Pi] and maximal Ca2+-activated pressure (r = -0.87 mean, n = 12), with an average decline of 8.6% in pressure per 1 mumol/g wet wt. increase in [Pi]. Intracellular pH (pHi) showed no significant correlation with maximal Ca2+-activated pressure (r = 0.49 mean, n = 12). Two other protocols, pacing at variable rates and gated measurements at two different times during the tetanus, were also used to correlate [Pi], pHi, and maximal Ca2+-activated pressure. These protocols confirmed the highly significant correlation between [Pi] and maximal Ca2+-activated pressure, as well as the lack of correlation with pHi. Acidosis induced by NH4Cl (20 mM) or by bubbling with 95% O2/5% CO2 was associated with less than 20% depression of maximal Ca2+-activated pressure in the pHi range down to 6.8, but much greater depression at lower pHi. The data are consistent with depression of maximal Ca2+-activated force during the early phase of hypoxia by Pi but not by H+.

Topics: Animals; Calcium; Cardiac Pacing, Artificial; Carnivora; Ferrets; Heart; Hydrogen-Ion Concentration; Hypoxia; Ion Channels; Magnetic Resonance Spectroscopy; Myocardial Contraction; Phosphates; Ryanodine