kn-93 and Hypoxia

Intermittent high-altitude (IHA) hypoxia-induced cardioprotection against ischemia-reperfusion (I/R) injury is associated with the preservation of sarcoplasmic reticulum (SR) function. Although Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) and phosphatase are known to modulate the function of cardiac SR under physiological conditions, the status of SR CaMKII and phosphatase during I/R in the hearts from IHA hypoxic rats is unknown. In the present study, we determined SR and cytosolic CaMKII activity during preischemia and I/R (30 min/30 min) in perfused hearts from normoxic and IHA hypoxic rats. The left ventricular contractile recovery, SR CaMKII activity as well as phosphorylation of phospholamban at Thr(17), and Ca(2+)/CaM-dependent SR Ca(2+)-uptake activity were depressed in the I/R hearts from normoxic rats, whereas these changes were prevented in the hearts from IHA hypoxic rats. Such beneficial effects of IHA hypoxia were lost by treating the hearts with a specific CaMKII inhibitor, KN-93. I/R also depressed cytosolic CaMKII and SR phosphatase activity, but these alterations remained unchanged in IHA hypoxic group. Furthermore, we found that the autophosphorylation at Thr(287), which confers Ca(2+)/CaM-independent activity, was not altered by I/R in both groups. These findings indicate that preservation of SR CaMKII activity plays an important role in the IHA hypoxia-induced cardioprotection against I/R injury via maintaining SR Ca(2+)-uptake activity.

Topics: Animals; Benzylamines; Calcium; Calcium-Binding Proteins; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cytoplasmic Vesicles; Cytosol; Hypoxia; Male; Myocardial Contraction; Myocardial Reperfusion Injury; Myocardium; Phosphorylation; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Sarcoplasmic Reticulum; Sulfonamides; Threonine; Ventricular Dysfunction, Left; Ventricular Function, Left

The present study investigated the possible regulatory mechanisms of redox agents and hypoxia on the K(ATP) current (I(KATP)) in acutely isolated rat ventricular myocytes.. Single-channel and whole-cell patch-clamp techniques were used to record the K(ATP) current (I(KATP)) in acutely isolated rat ventricular myocytes.. Oxidized glutathione (GSSG, 1 mmol/L) increased the I(KATP), while reduced glutathione (GSH, 1 mmol/L) could reverse the increased I(KATP) during normoxia. To further corroborate the effect of the redox agent on the K(ATP) channel, we employed the redox couple DTT (1 mmol/L)/H2O2 (0.3, 0.6, and 1 mmol/L) and repeated the previous processes, which produced results similar to the previous redox couple GSH/GSSG during normoxia. H2O2 increased the I(KATP) in a concentration dependent manner, which was reversed by DTT (1 mmol/L). In addition, our results have shown that 15 min of hypoxia increased the I(KATP), while GSH (1 mmol/L) could reverse the increased I(KATP). Furthermore, in order to study the signaling pathways of the I(KATP) augmented by hypoxia and the redox agent, we applied a protein kinase C(PKC) inhibitor bisindolylmaleimide VI (BIM), a protein kinase G(PKG) inhibitor KT5823, a protein kinase A (PKA) inhibitor H-89, and Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitors KN-62 and KN-93. The results indicated that BIM, KT5823, KN-62, and KN-93, but not H-89, inhibited the I(KATP) augmented by hypoxia and GSSG; in addition, these results suggest that the effects of both GSSG and hypoxia on K(ATP) channels involve the activation of the PKC, PKG, and CaMK II pathways, but not the PKA pathway.. The present study provides electrophysiological evidence that hypoxia and the oxidizing reaction are closely related to the modulation of I(KATP).

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinases; Carbazoles; Cells, Cultured; Cyclic GMP-Dependent Protein Kinases; Dithiothreitol; Dose-Response Relationship, Drug; Electrophysiology; Female; Glutathione; Glutathione Disulfide; Heart Ventricles; Hydrogen Peroxide; Hypoxia; Indoles; Male; Maleimides; Myocytes, Cardiac; Oxidation-Reduction; Patch-Clamp Techniques; Potassium Channels, Inwardly Rectifying; Protein Kinase C; Protein Kinase Inhibitors; Rats; Sulfonamides; Time Factors

Intermittent hypoxia (IH) occurs in many pathological conditions. However, very little is known about the molecular mechanisms associated with IH. Hypoxia-inducible factor 1 (HIF-1) mediates transcriptional responses to continuous hypoxia. In the present study, we investigated whether IH activates HIF-1 and, if so, which signaling pathways are involved. PC12 cells were exposed to either to 20% O2 (non-hypoxic control) or to 60 cycles consisting of 30 s at 1.5% O2, followed by 4 min at 20% O2 (IH). Western blot analysis revealed significant increases in HIF-1alpha protein in nuclear extracts of cells subjected to IH. Expression of a HIF-1-dependent reporter gene was increased 3-fold in cells subjected to IH. Although IH induced the activation of ERK1, ERK2, JNK, PKC-alpha, and PKC-gamma, inhibitors of these kinases and of phosphatidylinositol 3-kinase did not block HIF-1-mediated reporter gene expression induced by IH, indicating that signaling via these kinases was not required. In contrast, addition of the intracellular Ca2+ chelator BAPTA-AM or the Ca2+/calmodulin-dependent (CaM) kinase inhibitor KN93 blocked reporter gene activation in response to IH. CaM kinase activity was increased 5-fold in cells subjected to IH. KN 93 prevented IH-induced transactivation mediated by HIF-1alpha, and its coactivator p300, which was phosphorylated by CaM kinase II in vitro. Expression of the HIF-1-regulated gene encoding tyrosine hydroxylase was induced by IH and this effect was blocked by KN93. These observations suggest that IH induces HIF-1 transcriptional activity via a novel signaling pathway involving CaM kinase.

Topics: Animals; Asparagine; Benzylamines; Blotting, Northern; Blotting, Western; Calcium; Calcium-Calmodulin-Dependent Protein Kinases; Cell Membrane; Cell Nucleus; DNA; Dose-Response Relationship, Drug; E1A-Associated p300 Protein; Enzyme Activation; Gene Expression Regulation; Genes, Reporter; Glutathione Transferase; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Immunoblotting; MAP Kinase Signaling System; Nuclear Proteins; Oligonucleotides; Oxygen; PC12 Cells; Phosphatidylinositol 3-Kinases; Phosphorylation; Plasmids; Protein Kinase C; Rats; RNA, Messenger; Signal Transduction; Sulfonamides; Time Factors; Trans-Activators; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Transfection; Tyrosine 3-Monooxygenase

Calcium/calmodulin-dependent kinase II (CaMKII) is an ubiquitous second messenger that is highly expressed in neurons, where it has been implicated in some of the pathways regulating neuronal discharge as well as N-methyl-d-aspartate receptor-mediated synaptic plasticity. The full expression of the mammalian hypoxic ventilatory response (HVR) requires intact central relays within the nucleus of the solitary tract (NTS), and neural transmission of hypoxic afferent input is mediated by glutamatergic receptor activity, primarily through N-methyl-D-aspartate receptors. To examine the functional role of CaMKII in HVR, KN-93, a highly selective antagonist of CaMKII, was microinjected in the NTS via bilaterally placed osmotic pumps in freely behaving adult male Sprague-Dawley rats for 3 days. Vehicle-loaded osmotic pumps were surgically placed in control animals, and adequate placement of cannulas was ascertained for all animals. HVR was measured using whole body plethysmography during exposure to 10% O(2)-balance N(2) for 20 min. Compared with control rats, KN-93 administration elicited marked attenuations of peak HVR (pHVR) but did not modify normoxic minute ventilation. Differences in pHVR were primarily attributable to diminished respiratory frequency recruitments during pHVR without significant differences in tidal volume. These findings indicate that CaMKII activation in the NTS mediates respiratory frequency components of the ventilatory response to acute hypoxia; however, CaMKII activity does not appear to underlie components of normoxic ventilation.

Topics: Animals; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Hypoxia; Male; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Respiration; Solitary Nucleus; Sulfonamides