ucn-1028-c and Hypoxia

Desflurane triggers post-conditioning in the diabetic human myocardium. We determined whether protein kinase C (PKC), mitochondrial adenosine triphosphate-sensitive potassium (mitoK(ATP)) channels, Akt, and glycogen synthase kinase-3β (GSK-3β) were involved in the in vitro desflurane-induced post-conditioning of human myocardium from patients with type 2 diabetes.. The isometric force of contraction (FoC) of human right atrial trabeculae obtained from patients with type 2 diabetes was recorded during 30 min of hypoxia followed by 60 min of reoxygenation. Desflurane (6%) was administered during the first 5 min of reoxygenation either alone or in the presence of calphostin C (PKC inhibitor) or 5-hydroxydecanoate (5-HD) (mitoK(ATP) channel antagonist). Phorbol 12-myristate 13-acetate (PKC activator) and diazoxide (a mitoK(ATP) channel opener) were superfused during early reoxygenation. The FoC at the end of the 60 min reoxygenation period was compared among treatment groups (FoC(60); mean and sd). The phosphorylation of Akt and GSK-3β was studied using western blotting.. Desflurane enhanced the recovery of force [FoC(60): 79 (3)% of baseline] after 60 min of reoxygenation when compared with the control group (P>0.0001). Calphostin C and 5-HD abolished the beneficial effect of desflurane-induced post-conditioning (both P<0.0001). Phorbol 12-myristate 13-acetate and diazoxide enhanced the FoC(60) when compared with the control group (both P<0.0001). Desflurane increased the level of phosphorylation of Akt and GSK-3β (P<0.0001).. Desflurane-induced post-conditioning in human myocardium from patients with type 2 diabetes was mediated by the activation of PKC, the opening of the mitoK(ATP) channels, and the phosphorylation of Akt and GSK-3β.

Topics: Aged; Anesthetics, Inhalation; Blotting, Western; Decanoic Acids; Desflurane; Diabetes Mellitus, Type 2; Diazoxide; Enzyme Inhibitors; Female; Glycated Hemoglobin; Glycogen Synthase Kinase 3; Heart; Heart Atria; Humans; Hydroxy Acids; Hypoxia; Ischemic Postconditioning; Isoflurane; KATP Channels; Male; Middle Aged; Mitochondria, Heart; Myocardial Reperfusion Injury; Naphthalenes; Protein Kinase C; Proto-Oncogene Proteins c-akt; Stroke Volume; Tetradecanoylphorbol Acetate

Although hypoxic late preconditioning (LPC) limits ischemia-reperfusion injury in vitro, its cardioprotective effect is not established in vivo.. In part 1, rats were exposed to 4 h of hypoxia (16%, 12%, 8% oxygen) before 24 h of reoxygenation. In part 2, normoxic rats received early preconditioning with sevoflurane (1 minimum alveolar concentration [MAC] for 3 × 5 min), continuous administration of 1 MAC sevoflurane, or 11 mg · kg · h propofol. Thereafter, all rats underwent 25 min of regional myocardial ischemia and 120 min of reperfusion. After reperfusion, hearts were excised for infarct staining. The expression of protein kinase C (PKC)α and PKCε was assessed by Western blot analysis and the expression of heme oxygenase-1 and vascular endothelial growth factor by reverse transcriptase polymerase chain reaction.. In normoxic control rats, infarct size was 62 ± 6% of the area at risk. Hypoxic LPC reduced infarct size (LPC16: 36 ± 11%, LPC12: 38 ± 10%, LPC8: 39 ± 11%; each P < 0.001) to approximately the same magnitude as sevoflurane-preconditioning (40 ± 8%; P < 0.001). Combined LPC16 and sevoflurane preconditioning was not superior to either substance alone. Continuous sevoflurane or propofol was not protective. The PKC inhibitor calphostin C abolished the cardioprotective effects of LPC16. PKCε, but not PKCα, expression was increased 6 and 28 h after hypoxic LPC. Heme oxygenase-1 and vascular endothelial growth factor were transiently up-regulated after 6 h.. Hypoxic LPC at 8%, 12%, and 16% oxygen reduces infarct size in the rat heart in vivo. This effect is as powerful as sevoflurane-preconditioning. PKCε is a key player in mediating hypoxic LPC.

Topics: Anesthetics, Inhalation; Animals; Blotting, Western; Enzyme Inhibitors; Heart; Heme Oxygenase (Decyclizing); Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Ischemic Preconditioning, Myocardial; Methyl Ethers; Myocardial Infarction; Myocardial Reperfusion Injury; Naphthalenes; Oxygen; Protein Kinase C-alpha; Protein Kinase C-epsilon; Rats; Reverse Transcriptase Polymerase Chain Reaction; Sevoflurane; Vascular Endothelial Growth Factor A

The current study was done to test the hypothesis that protein kinase C (PKC) inhibitors prevent the increase in pulmonary vascular resistance and compliance that occurs in isolated, blood-perfused dog lungs during hypoxia. Pulmonary vascular resistances and compliances were measured with vascular occlusion techniques. Hypoxia significantly increased pulmonary arterial resistance, pulmonary venous resistance, and pulmonary capillary pressure and decreased total vascular compliance by decreasing both microvascular and large-vessel compliances. The nonspecific PKC inhibitor staurosporine (10(-7) M), the specific PKC blocker calphostin C (10(-7) M), and the specific PKC isozyme blocker Gö-6976 (10(-7) M) inhibited the effect of hypoxia on pulmonary vascular resistance and compliance. In addition, the PKC activator thymeleatoxin (THX; 10(-7) M) increased pulmonary vascular resistance and compliance in a manner similar to that in hypoxia, and the L-type voltage-dependent Ca(2+) channel blocker nifedipine (10(-6) M) inhibited the response to both THX and hypoxia. These results suggest that PKC inhibition blocks the hypoxic pressor response and that the pharmacological activation of PKC by THX mimics the hypoxic pulmonary vasoconstrictor response. In addition, L-type voltage-dependent Ca(2+) channel blockade may prevent the onset of the hypoxia- and PKC-induced vasoconstrictor response in the canine pulmonary vasculature.

Topics: Animals; Calcium Channel Blockers; Calcium Channels, L-Type; Carbazoles; Dogs; Enzyme Inhibitors; Female; Hypoxia; In Vitro Techniques; Indoles; Male; Naphthalenes; Perfusion; Phorbol Esters; Protein Kinase C; Pulmonary Circulation; Staurosporine; Vascular Resistance; Vasoconstriction

Macrophages can adapt to the absence of oxygen by switching to anaerobic glycolysis. In this study, we investigated (a) the roles of fructose 2,6-bisphosphate (Fru-2,6-P2) and ribose 1,5-bisphosphate (Rib-1,5-P2), potent activators of phosphofructokinase, (b) the enzymes responsible for the synthesis of Rib-1,5-P2, and (c) the mechanisms of regulation of these enzymes in H36.12j macrophages during the initial phase of hypoxia. Within 1 min after initiating hypoxia, glycolysis was activated through activation of phosphofructokinase. Over the same period, Fru-2,6-P2 decreased 50% and recovered completely upon reoxygenation. Similar changes in cAMP levels were observed. In contrast, the Rib-1,5-P2 concentration rapidly increased to a maximum level of 8.0 +/- 0.9 nmol/g cell 30 s after hypoxia. Thus, Rib-1,5-P2 was the major factor increasing the rate of glycolysis during the initial phase of hypoxia. Moreover, we found that Rib-1,5-P2 was synthesized by two steps: the ribose-phosphate pyrophosphokinase (5-phosphoribosyl-1-pyrophosphate synthetase; PRPP synthetase) reaction (EC ) catalyzing the reaction, Rib-5-P + ATP --> PRPP + AMP and a new enzyme, "PRPP pyrophosphatase" catalyzing the reaction, PRPP --> Rib-1,5-P2 + P(i). Both PRPP synthetase and PRPP pyrophosphatase were significantly activated 30 s after hypoxia. Pretreatment with 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine and calphostin C prevented the activation of ribose PRPP synthetase and PRPP pyrophosphatase as well as increase in Rib-1,5-P2 and activation of phosphofructokinase 30 s after hypoxia. These data suggest that the activation of the above enzymes was mediated by protein kinase C acting via activation of phosphatidylinositol specific phospholipase C in the macrophages during hypoxia.

Topics: Adenosine Monophosphate; Adenosine Triphosphate; Animals; Cell Line; Cyclic AMP; Enzyme Activation; Enzyme Inhibitors; Fructosediphosphates; Hot Temperature; Hypoxia; Kinetics; Macrophages; Mice; Models, Biological; Naphthalenes; Oxygen; Pentosephosphates; Phospholipid Ethers; Protein Kinase C; Ribose-Phosphate Pyrophosphokinase; Temperature; Time Factors

We have investigated whether hypoxia and muscle contractions stimulate glucose transport in perfused rat muscle to the same extent, additively and with the same sensitivity to the microbial products calphostin C and wortmannin. Hindlimb glucose uptake increased gradually from 3.4+/-0.5 to a maximal level of 12.7+/-0.6 micromol g-1 h-1 (n=11) after 50 min of hypoxia. Compared with hypoxia, the effect of maximal electrical stimulation of the sciatic nerve on muscle glucose uptake was more than two-fold higher (27+/-2 micromol g-1 h-1 (n=14)). This was due to a higher contraction- vs. hypoxia-induced glucose transport rate in oxidative fibers. The stimulatory effect of hypoxia and electrical stimulation was not additive. Contraction-induced muscle glucose transport was inhibitable by both calphostin C and wortmannin in the micromolar range, whereas the effect of hypoxia was totally insensitive to these drugs. Our data suggest that diacylglycerol/phorbol ester-sensitive protein kinase C is involved in stimulation of muscle glucose transport by contractions and that in contrast to the prevailing concept, hypoxia and contractions do not stimulate muscle glucose transport by the same signaling mechanism.

Topics: Androstadienes; Animals; Biological Transport; Electric Stimulation; Glucose; Hypoxia; Infusions, Intra-Arterial; Male; Muscle Contraction; Muscle, Skeletal; Naphthalenes; Perfusion; Rats; Rats, Wistar; Signal Transduction; Wortmannin

Glucose transport in skeletal muscle can be stimulated by insulin and also by contractions and hypoxia. Activation of protein kinase C (PKC) stimulates glucose transport in muscle and other insulin-responsive cells. This study was performed to determine if the diacylglycerol (DAG)/phorbol ester-sensitive PKC isoforms participate in insulin and/or hypoxia-stimulated glucose transport in skeletal muscle. The phorbol ester 12-deoxyphorbol 13-phenylacetate 20-acetate (dPPA) induced a three- to fourfold increase in glucose transport in rat epitrochlearis muscle. The effects of dPPA on glucose transport and on cell surface GLUT-4 were completely additive to the maximal effects of insulin or hypoxia. Phorbol ester treatment induced 5- to 10-fold increases in phosphorylation of the myristoylated alanine-rich C kinase substrate protein in muscle, whereas insulin and hypoxia had negligible effects. Calphostin C, an inhibitor of DAG-sensitive PKC isoforms, decreased glucose transport stimulation by dPPA but not by insulin or hypoxia. These results provide evidence that activation of DAG/phorbol ester-sensitive PKCs is not involved in the pathways by which either insulin or hypoxia stimulates muscle glucose transport. They also show that activation of this group of PKCs increases glucose transport by a mechanism that is independent of and additive to the effects of insulin or hypoxia.

Topics: Animals; Biological Transport; Enzyme Activation; Enzyme Inhibitors; Glucose; Glucose Transporter Type 4; Hypoxia; Intracellular Signaling Peptides and Proteins; Inulin; Isoenzymes; Kinetics; Male; Membrane Proteins; Monosaccharide Transport Proteins; Muscle Contraction; Muscle Proteins; Muscle, Skeletal; Myristoylated Alanine-Rich C Kinase Substrate; Naphthalenes; Phorbol Esters; Protein Kinase C; Proteins; Rats; Rats, Wistar; Tetradecanoylphorbol Acetate

We wished to determine whether the cytokine-inducible nitric oxide synthase (iNOS) pathway is modulated by chronic hypoxia in vitro.. We investigated the effects of the proinflammatory cytokine interleukin (IL)-1beta on expression of iNOS mRNA, iNOS protein, and NO production in cultured neonatal rat cardiomyocytes subjected to 1% O2 for 48 hours. Among several cytokines tested, IL-1beta was the most effective in stimulating NO production, which was maximum at 48 hours. In parallel, IL-1beta induced expression of both iNOS mRNA and protein. Hypoxia alone had no effect on NO production, iNOS gene expression, or protein induction. However, chronic hypoxia decreased IL-1beta-stimulated NO production, mRNA expression, and protein level in cardiac myocytes. Radioligand binding and electrophoretic mobility shift assays showed that during chronic hypoxia, IL-1 receptor density and activity of the transcription factor NF-kappaB induced by IL-1beta were decreased, which may account at least in part for the decrease in iNOS expression.. These data indicate that IL-1beta induces iNOS gene expression, de novo synthesis of iNOS protein, and NO generation in neonatal rat cardiomyocytes and that chronic hypoxia appears to be a potent negative regulator of iNOS expression in these cells.

Topics: Animals; Anti-Inflammatory Agents; Cells, Cultured; Chronic Disease; Dexamethasone; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Genistein; Hypoxia; Interleukin 1 Receptor Antagonist Protein; Interleukin-1; Isoflavones; Muscle Fibers, Skeletal; Myocardium; Naphthalenes; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase; omega-N-Methylarginine; Rats; Rats, Sprague-Dawley; Receptors, Interleukin-1; RNA, Messenger; Sialoglycoproteins; Signal Transduction

The signal transduction pathway of hypoxic pulmonary arterial contraction has not been elucidated. Phosphorylation of the 20-kDa myosin light chain (MLC20) is thought to be essential for vascular muscle contraction. However, there are reports that smooth muscle will contract in response to nonphysiological stimuli such as phorbol esters without the involvement of MLC20 phosphorylation. The purpose of this study was to determine if hypoxia-induced pulmonary arterial contraction is dependent on MLC20 phosphorylation. Isolated rat pulmonary and carotid (for comparative purposes) arterial strips were contracted with 80 mM KCl to establish maximum active tension in response to membrane depolarization. The strips were then stimulated with one of the following: 30 mM KCl, 1 microM phenylephrine, 0.01 microM angiotensin II, 1 microM phorbol 12-myristate 13-acetate (PMA), or hypoxia (95% N2-5% CO2). In some experiments ML-9, a myosin light chain kinase inhibitor, or calphostin C, a protein kinase C (PKC) inhibitor, was introduced into the bath before hypoxia. Isometric tension was recorded as a function of time. Muscle strips were freeze-clamped (liquid N2) at various time points during the course of responses to the various stimuli. MLC20 phosphorylation levels were measured by ureaglycerol gel electrophoresis followed by Western blot procedure. Results show that increased MLC20 phosphorylation correlates with initiation of pulmonary arterial smooth muscle contraction in response to all agonists with the exception of PMA, a known activator of PKC. The MLC20 phosphorylation levels correlate with tension development in response to hypoxia, and ML-9 abolished the hypoxic contractions. In contrast, hypoxia relaxed carotid arterial muscle, and there was a corresponding decrease in the MLC20 phosphorylation level. In conclusion, hypoxia appears to result in MLC20 phosphorylation-mediated contraction in conduit pulmonary arterial muscle and in MLC20 dephosphorylation-mediated relaxation in systemic arterial muscle.

Topics: Angiotensin II; Animals; Azepines; Enzyme Inhibitors; Hypoxia; In Vitro Techniques; Kinetics; Male; Muscle Contraction; Muscle, Smooth, Vascular; Myosin Light Chains; Myosin-Light-Chain Kinase; Naphthalenes; Phenylephrine; Phosphorylation; Potassium Chloride; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Tetradecanoylphorbol Acetate; Time Factors