h-89 and Hypoxia

Glucagon-like peptide-1 (GLP-1), an incretin hormone, is secreted from L cells located in the intestinal epithelium. It is known that intestinal oxygen tension is decreased postprandially. In addition, we found that the expression of hypoxia-inducible factor-1α (HIF-1α), which accumulates in cells under hypoxic conditions, was significantly increased in the colons of mice with food intake, indicating that the oxygen concentration is likely reduced in the colon after eating. Therefore, we hypothesized that GLP-1 secretion is affected by oxygen tension. We found that forskolin-stimulated GLP-1 secretion from GLUTag cells, a model of intestinal L cells, is suppressed in hypoxia (1% O2). Forskolin-stimulated elevations of preproglucagon (ppGCG) and proprotein convertase 1/3 (PC1/3) mRNA expression were decreased under hypoxic conditions. The finding that H89, a protein kinase A (PKA) inhibitor, inhibited the forskolin-stimulated increase of ppGCG and PC1/3 indicated that the cAMP-PKA pathway is involved in the hypoxia-induced suppression of the genes. Hypoxia decreased hexokinase 2 mRNA and protein expression and increased lactate dehydrogenase A mRNA and protein expression. Concomitantly, lactate production was increased and ATP production was decreased. Together, the results indicate that hypoxia decreases glucose utilization for ATP production, which probably causes a decrease in cAMP production and in subsequent GLP-1 production. Our findings suggest that the postprandial decrease in oxygen tension in the intestine attenuates GLP-1 secretion.

Topics: Adenosine Triphosphate; Animals; Cell Line; Colforsin; Colon; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Eating; Glucagon-Like Peptide 1; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Isoquinolines; Lactic Acid; Male; Mice; Mice, Inbred C57BL; Proglucagon; Proprotein Convertase 1; Protein Kinase Inhibitors; RNA, Messenger; Sulfonamides

Urocortin-1 (UCN1) exerts protective effects on hypoxia/reoxygenation injury in the heart. Serum- and glucocorticoid- responsive kinase-1 (SGK1), a serine-threonine kinase, has been shown to be crucial for cardiomyocyte survival. The purpose of the present study was to investigate whether SGK1 is involved in UCN1-induced cardioprotection.. Cardiomyocytes were obtained from neonatal rats and used as a model to investigate UCN1 regulation of SGK1. Specific small interfering RNA targeting SGK1 was used to knock down SGK1 expression. The messenger RNA (mRNA) level of SGK1 was measured using quantitative real time reverse transcription polymerase chain reaction, and the protein levels of SGK1 and phosphorylated SGK1 were determined using Western blot analysis.. SGK1 knockdown attenuated the protective effects of UCN1 against hypoxia/reoxygenation injury in cardiomyocytes. Treatment of cardiomyocytes with UCN1 stimulated SGK1 mRNA and protein expression and time-dependently increased phosphorylated SGK1 level. These effects were completely reversed with corticotrophin-releasing hormone receptor type 2 antagonist. Adenylate cyclase and protein kinase A inhibitors abolished the stimulatory effect of UCN1 on SGK1 expression. SGK1 phosphorylation induced by UCN1 was blocked by phosphorinositide-3-kinase inhibitor.. SGK1 is involved in the cardioprotective effects of UCN1 in cardiomyocytes. UCN1 stimulates SGK1 phosphorylation via the phosphorinositide-3-kinase signalling pathway and it induces SGK1 expression via the adenylate cyclase/protein kinase A pathway.

Topics: Animals; Animals, Newborn; Blotting, Western; Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; Chromones; Enzyme Inhibitors; Flavonoids; Gene Knockdown Techniques; Hypoxia; Immediate-Early Proteins; Isoquinolines; Models, Animal; Morpholines; Myocytes, Cardiac; Peptide Fragments; Peptides, Cyclic; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Rats; Real-Time Polymerase Chain Reaction; Receptors, Corticotropin-Releasing Hormone; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Sulfonamides; Urocortins

Non-steroidal anti-inflammatory drugs (NSAIDs), which inhibit COX activity, reduce the production of retinal VEGF and neovascularization in relevant models of ocular disease. We hypothesized that COX-2 mediates VEGF production in retinal Müller cells, one of its primary sources in retinal neovascular disease. The purpose of this study was to determine the role of COX-2 and its products in VEGF expression and secretion. These studies have more clearly defined the role of COX-2 and COX-2-derived prostanoids in retinal angiogenesis. Müller cells derived from wild-type and COX-2 null mice were exposed to hypoxia for 0-24 h. COX-2 protein and activity were assessed by western blot analysis and GC-MS, respectively. VEGF production was assessed by ELISA. Wild-type mouse Müller cells were treated with vehicle (0.1% DMSO), 10 microM PGE(2), or PGE(2) + 5 microM H-89 (a PKA inhibitor), for 12 h. VEGF production was assessed by ELISA. Hypoxia significantly increased COX-2 protein (p < 0.05) and activity (p < 0.05), and VEGF production (p < 0.0003). COX-2 null Müller cells produced significantly less VEGF in response to hypoxia (p < 0.05). Of the prostanoids, PGE(2) was significantly increased by hypoxia (p < 0.02). Exogenous PGE(2) significantly increased VEGF production by Müller cells (p < 0.0039), and this effect was inhibited by H-89 (p < 0.055). These data demonstrate that hypoxia induces COX-2, prostanoid production, and VEGF synthesis in Müller cells, and that VEGF production is at least partially COX-2-dependent. Our study suggests that PGE(2), signaling through the EP(2) and/or EP(4) receptor and PKA, mediates the VEGF response of Müller cells.

Topics: Animals; Animals, Newborn; Blotting, Western; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Cyclooxygenase 2; Dinoprostone; Enzyme-Linked Immunosorbent Assay; Gas Chromatography-Mass Spectrometry; Gene Deletion; Hypoxia; Isoquinolines; Mice; Mice, Inbred C57BL; Mice, Knockout; Microglia; Prostaglandins; Protein Kinase Inhibitors; Retinal Neurons; Sulfonamides; Vascular Endothelial Growth Factor A

We have investigated the effects of hypoxia and myocardial ischemia/reperfusion on the structure and function of cytochrome c oxidase (CcO). Hypoxia (0.1% O(2) for 10 h) and cAMP-mediated inhibition of CcO activity were accompanied by hyperphosphorylation of subunits I, IVi1, and Vb and markedly increased reactive O(2) species production by the enzyme complex in an in vitro system that uses reduced cytochrome c as an electron donor. Both subunit phosphorylation and enzyme activity were effectively reversed by 50 nm H89 or 50 nm myristoylated peptide inhibitor (MPI), specific inhibitors of protein kinase A, but not by inhibitors of protein kinase C. In rabbit hearts subjected to global and focal ischemia, CcO activity was inhibited in a time-dependent manner and was accompanied by hyperphosphorylation as in hypoxia. Additionally, CcO activity and subunit phosphorylation in the ischemic heart were nearly completely reversed by H89 or MPI added to the perfusion medium. Hyperphosphorylation of subunits I, IVi1, and Vb was accompanied by reduced subunit contents of the immunoprecipitated CcO complex. Most interestingly, both H89 and MPI added to the perfusion medium dramatically reduced the ischemia/reperfusion injury to the myocardial tissue. Our results pointed to an exciting possibility of using CcO activity modulators for controlling myocardial injury associated with ischemia and oxidative stress conditions.

Topics: Animals; Carbon Monoxide; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Electron Transport Complex IV; Hypoxia; Immunoblotting; Immunoprecipitation; Ischemia; Isoquinolines; Macrophages; Male; Mice; Mitochondria; Monocytes; Myocardial Ischemia; Myocardium; Oxidative Stress; Oxygen; Peptides; Perfusion; Phosphorylation; Protein Kinase C; Rabbits; Reactive Oxygen Species; Reperfusion Injury; Sulfonamides; Time Factors

Cardioprotection by intermittent high-altitude (IHA) hypoxia against ischemia-reperfusion (I/R) injury is associated with Ca(2+) overload reduction. Phospholamban (PLB) phosphorylation relieves cardiac sarcoplasmic reticulum (SR) Ca(2+)-pump ATPase, a critical regulator in intracellular Ca(2+) cycling, from inhibition. To test the hypothesis that IHA hypoxia increases PLB phosphorylation and that such an effect plays a role in cardioprotection, we compared the time-dependent changes in the PLB phosphorylation at Ser(16) (PKA site) and Thr(17) (CaMKII site) in perfused normoxic rat hearts with those in IHA hypoxic rat hearts submitted to 30-min ischemia (I30) followed by 30-min reperfusion (R30). IHA hypoxia improved postischemic contractile recovery, reduced the maximum extent of ischemic contracture, and attenuated I/R-induced depression in Ca(2+)-pump ATPase activity. Although the PLB protein levels remained constant during I/R in both groups, Ser(16) phosphorylation increased at I30 and 1 min of reperfusion (R1) but decreased at R30 in normoxic hearts. IHA hypoxia upregulated the increase further at I30 and R1. Thr(17) phosphorylation decreased at I30, R1, and R30 in normoxic hearts, but IHA hypoxia attenuated the depression at R1 and R30. Moreover, PKA inhibitor H89 abolished IHA hypoxia-induced increase in Ser(16) phosphorylation, Ca(2+)-pump ATPase activity, and the recovery of cardiac performance after ischemia. CaMKII inhibitor KN-93 also abolished the beneficial effects of IHA hypoxia on Thr(17) phosphorylation, Ca(2+)-pump ATPase activity, and the postischemic contractile recovery. These findings indicate that IHA hypoxia mitigates I/R-induced depression in SR Ca(2+)-pump ATPase activity by upregulating dual-site PLB phosphorylation, which may consequently contribute to IHA hypoxia-induced cardioprotection against I/R injury.

Topics: Altitude; Animals; Calcium-Binding Proteins; Disease Models, Animal; Hypoxia; In Vitro Techniques; Isoquinolines; Male; Myocardial Reperfusion Injury; Phosphorylation; Rats; Rats, Sprague-Dawley; Sarcoplasmic Reticulum; Sulfonamides

At birth, pulmonary vasodilation occurs as air-breathing life begins. The mechanism of O2-induced pulmonary vasodilation is unknown. We proposed that O2 causes fetal pulmonary vasodilation through activation of a calcium-dependent potassium channel (KCa) via a cyclic nucleotide-dependent kinase. We tested this hypothesis in hemodynamic studies in acutely prepared fetal lambs and in patch-clamp studies on resistance fetal pulmonary artery smooth muscle cells. Fetal O2 tension (PaO2) was increased by ventilating the ewe with 100% O2, causing fetal total pulmonary resistance to decrease from 1.18 +/- 0.14 to 0.41 +/- 0.03 mmHg per ml per min. Tetraethylammonium and iberiotoxin, preferential KCa-channel inhibitors, attenuated O2-induced fetal pulmonary vasodilation, while glibenclamide, an ATP-sensitive K+-channel antagonist, had no effect. Treatment with either a guanylate cyclase antagonist (LY83583) or cyclic nucleotide-dependent kinase inhibitors (H-89 and KT 5823) significantly attenuated O2-induced fetal pulmonary vasodilation. Under hypoxic conditions (PaO2 = 25 mmHg), whole-cell K+-channel currents (Ik) were small and were inhibited by 1 mM tetraethylammonium or 100 nM charybdotoxin (CTX; a specific KCa-channel blocker). Normoxia (PaO2 = 120 mmHg) increased Ik by more than 300%, and this was reversed by 100 nM CTX. Nitric oxide also increased Ik. Resting membrane potential was -37.2 +/- 1.9 mV and cells depolarized on exposure to CTX, while hyperpolarizing in normoxia. We conclude that O2 causes fetal pulmonary vasodilation by stimulating a cyclic nucleotide-dependent kinase, resulting in KCa-channel activation, membrane hyperpolarization, and vasodilation.

Topics: Alkaloids; Aminoquinolines; Animals; Carbazoles; Charybdotoxin; Endothelium, Vascular; Enzyme Inhibitors; Female; Fetus; Glyburide; Guanylate Cyclase; Hypoxia; Indoles; Isoquinolines; Membrane Potentials; Models, Cardiovascular; Muscle, Smooth, Vascular; Nitric Oxide; Nitric Oxide Synthase; Oxygen; Patch-Clamp Techniques; Peptides; Potassium Channel Blockers; Potassium Channels; Pregnancy; Protein Kinase Inhibitors; Pulmonary Artery; Sheep; Sulfonamides; Tetraethylammonium; Tetraethylammonium Compounds; Vasodilation