h-89 and Reperfusion-Injury

To determine the effects of astragaloside IV on cerebral ischemic-reperfusion injury in rats and to explore underlying mechanisms of brain protection.. Sixty Sprague-Dawley rats were randomized into four groups: Sham, cerebral ischemia-reperfusion (I/R group), I/R+astragaloside IV (I/R+AST-IV group) and I/R+astragaloside IV+PKA kinase inhibitor H-89 (I/R+AST-IV+H-89 group). All I/R rats were subjected to 2 h cerebral ischemia, followed by 24 h reperfusion and scored for neurobehavior. Cerebral infarct volume, pathomorphological changes and brain apoptosis, in addition to changes in expression of Cx36, PKA, Bax and Bcl-2 proteins, were assessed.. Astragaloside IV treatment reduced neurobehavioral score and percentage volume of cerebral infarct, reducing pathomorphological injury and brain apoptosis. Expressions of Cx36 and PKA protein were increased and the Bax/Bcl-2 ratio decreased. All astragaloside IV effects were reversed by the PKA inhibitor and H-89.. Astragaloside IV attenuated cerebral I/R injury in rats by increasing Cx36 and PKA protein expression and reducing the Bax/Bcl-2 ratio.

Topics: Animals; bcl-2-Associated X Protein; Brain Ischemia; Cerebral Infarction; Isoquinolines; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Saponins; Sulfonamides; Triterpenes; Up-Regulation

We aim to investigate the effects of sevoflurane on the ATPase activity of the hippocampal neurons in rats with cerebral ischemia-reperfusion injury (IRI) via the cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) signaling pathway. Sixty rats were assigned into the normal, model and sevoflurane groups (n = 20, the latter two groups were established as focal cerebral IRI models). The ATPase activity was detected using an ultramicro Na (+)-K (+)-ATP enzyme kit. Immunohistochemical staining was used to detect the positive protein expression of cAMP and PKA. The hippocampal neurons were assigned to the normal, IRI, IRI + sevoflurane, IRI + forskolin, IRI + H89 and IRI + sevoflurane + H89 groups. qRT-PCR and Western blotting were performed for the expressions of cAMP, PKA, cAMP-responsive element-binding protein (CREB) and brain derived neurotrophic factor (BDNF). The normal and sevoflurane groups exhibited a greater positive protein expression of cAMP and PKA than the model group. Compared with the normal group, the expressions of cAMP, PKA, CREB and BDNF all reduced in the IRI, model and IRI + H89 groups. The sevoflurane group showed higher cAMP, PKA, CREB and BDNF expressions than the model group. Compared with the IRI group, ATPase activity and expressions of cAMP, PKA, CREB and BDNF all increased in the normal, IRI + sevoflurane and IRI + forskolin groups but decreased in the IRI + H89 group. It suggests that sevoflurane could enhance ATPase activity in hippocampal neurons of cerebral IRI rats through activating cAMP-PKA signaling pathway.

Topics: Animals; Brain-Derived Neurotrophic Factor; Colforsin; CREB-Binding Protein; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Disease Models, Animal; Gene Expression Regulation; Germ-Free Life; Hippocampus; Humans; Infarction, Middle Cerebral Artery; Isoquinolines; Male; Neurons; Neuroprotective Agents; Platelet Aggregation Inhibitors; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sevoflurane; Sodium-Potassium-Exchanging ATPase; Sulfonamides

Myocardial cAMP elevation confers cardioprotection against ischaemia/reperfusion (I/R) injury. cAMP activates two independent signalling pathways, PKA and Epac. This study investigated the cardiac effects of activating PKA and/or Epac and their involvement in cardioprotection against I/R.. Hearts from male rats were used either for determination of PKA and PKC activation or perfused in the Langendorff mode for either cardiomyocyte isolation or used to monitor functional activity at basal levels and after 30 min global ischaemia and 2 h reperfusion. Functional recovery and myocardial injury during reperfusion (LDH release and infarct size) were evaluated. Activation of PKA and/or Epac in perfused hearts was induced using cell permeable cAMP analogues in the presence or absence of inhibitors of PKA, Epac and PKC. H9C2 cells and cardiomyocytes were used to assess activation of Epac and effect on Ca. Selective activation of either PKA or Epac was found to trigger a positive inotropic effect, which was considerably enhanced when both pathways were simultaneously activated. Only combined activation of PKA and Epac induced marked cardioprotection against I/R injury. This was accompanied by PKCε activation and repressed by inhibitors of PKA, Epac or PKC.. Simultaneous activation of both PKA and Epac induces an additive inotropic effect and confers optimal and marked cardioprotection against I/R injury. The latter effect is mediated by PKCε activation. This work has introduced a new therapeutic approach and targets to protect the heart against cardiac insults.

Topics: Animals; Cardiotonic Agents; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Enzyme Activation; Guanine Nucleotide Exchange Factors; Heart; Hydrazones; Isoquinolines; Isoxazoles; Male; Rats; Rats, Wistar; Reperfusion Injury; Structure-Activity Relationship; Sulfonamides

The cyclic AMP-dependent protein kinase (PKA), which activates prosurvival signaling proteins, has been implicated in the expression of long-term potentiation and hippocampal long-term memory. It has come to light that H89 commonly known as the PKA inhibitor have diverse roles in the nervous system that are unrelated to its role as a PKA inhibitor. We have investigated the role of H89 in ischemic and reperfusion injury. First, we examined the expression of postsynaptic density protein 95 (PSD95), microtubule-associated protein 2 (MAP2), and synaptophysin in mouse brain after middle cerebral artery occlusion injury. Next, we examined the role of H89 pretreatment on the expression of brain-derived neurotrophic factor (BDNF), PSD95, MAP2, and the apoptosis regulators Bcl2 and cleaved caspase-3 in cultured neuroblastoma cells exposed to hypoxia and reperfusion injury. In addition, we investigated the alteration of AKT activation in H89 pretreated neuroblastoma cells under hypoxia and reperfusion injury. The data suggest that H89 may contribute to brain recovery after ischemic stroke by regulating neuronal death and proteins related to synaptic plasticity.

Topics: Animals; Apoptosis Regulatory Proteins; Brain Ischemia; Brain-Derived Neurotrophic Factor; Cell Death; Cyclic AMP-Dependent Protein Kinases; Disks Large Homolog 4 Protein; Guanylate Kinases; Hypoxia, Brain; Infarction, Middle Cerebral Artery; Isoquinolines; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Microtubule-Associated Proteins; Neurons; Protein Kinase Inhibitors; Reperfusion Injury; Sulfonamides; Synapses

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

Hepatic ischemia/reperfusion injury (IRI) occurs in multiple clinical settings, including liver transplantation. The cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) pathway inhibits hepatocellular apoptosis and regulates toll-like receptor 4-triggered inflammation responses in vitro. Here we examined the function and therapeutic potential of cAMP-PKA activation in a murine (C57/BL6) model of liver warm ischemia (90 minutes) followed by reperfusion. Liver IRI triggered cAMP-PKA activation, whereas the administration of its specific inhibitor, H89, exacerbated hepatocellular damage. Conversely, forskolin therapy, which activates PKA by elevating cAMP levels, protected livers from IRI; this was evidenced by diminished serum alanine aminotransferase levels and well-preserved tissue architecture. Liver protection due to cAMP-PKA stimulation was accompanied by diminished neutrophil and macrophage infiltration/activation, reduced hepatocyte necrosis/apoptosis, and increased cAMP response element-binding protein (CREB) expression and augmented interleukin-10 (IL-10) expression. The neutralization of IL-10 restored liver damage in otherwise ischemia/reperfusion-resistant, forskolin-treated mice. In vitro, cAMP-PKA activation diminished macrophage tumor necrosis factor α, IL-6, and IL-12 in an IL-10-dependent manner and prevented necrosis/apoptosis in primary mouse hepatocyte cultures. Our novel findings in a mouse model of liver IRI document the importance of cAMP-PKA signaling in hepatic homeostasis and cytoprotection in vivo. The activation of cAMP-PKA signaling differentially regulates local inflammation and prevents hepatocyte death, and this provides a rationale for novel therapeutic approaches to combating liver IRI in transplant recipients.

Topics: Animals; Apoptosis; Cells, Cultured; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Disease Models, Animal; Enzyme Inhibitors; Hepatocytes; Interleukin-10; Isoquinolines; Liver; Liver Transplantation; Macrophages; Male; Mice; Mice, Inbred C57BL; Necrosis; Peroxidase; Reperfusion Injury; Signal Transduction; Sulfonamides; Temperature

Ischemic episodes lead to profound functional and structural alterations of the gastrointestinal tract which may contribute to disorders of intestinal motility. Enhancement of glutamate overflow and the consequent activation of NMDA (N-methyl-D-aspartate) receptors may participate to such changes by modulating different enteric neurotransmitter systems, including cholinergic motor pathways.. The molecular mechanism/s underlying activation of NMDA receptors in the guinea pig ileum were investigated after glucose/oxygen deprivation (in vitro ischemia) and during reperfusion.. The number of ileal myenteric neurons positive for NR1, the functional subunit of NMDA receptors, and its mRNA levels were unchanged after in vitro ischemia/reperfusion. In these conditions, the protein levels of NR1, and of its phosphorylated form by protein kinase C (PKC), significantly increased in myenteric neurons, whereas, the levels of NR1 phosphorylated by protein kinase A (PKA) did not change, with respect to control values. Spontaneous glutamate overflow increased during in vitro ischemia/reperfusion. In these conditions, the NMDA receptor antagonists, D(-)-2-amino-5-phosphonopentanoic acid [(D)-AP5] (10 μmol L(-1)) and 5,7-dichlorokynurenic acid (5,7-diClKyn acid) (10 μmol L(-1)) and the PKC antagonist, chelerythrine (1 μmol L(-1)), but not the PKA antagonist, H-89 (1 μmol L(-1)), were able to significantly depress the increased glutamate efflux.. The present data suggest that in the guinea pig ileum during in vitro ischemia/reperfusion, NR1 protein levels increase. Such event may rely upon posttranscriptional events involving NR1 phosphorylation by PKC. Increased NR1 levels may, at least in part, explain the ability of NMDA receptors to modulate a positive feedback on ischemia/reperfusion-induced glutamate overflow.

Topics: 2-Amino-5-phosphonovalerate; Animals; Benzophenanthridines; Guinea Pigs; Ileum; In Vitro Techniques; Isoquinolines; Kynurenic Acid; Male; Models, Animal; Myenteric Plexus; Phosphorylation; Protein Kinase C; Receptors, N-Methyl-D-Aspartate; Reperfusion Injury; Sulfonamides

Diadenosine tetraphosphate (AP4A) is a vasoactive mediator that may be released from platelet granules and that may reach higher plasma concentrations during coronary ischemia-reperfusion. The objective of this study was to analyze its coronary effects in such conditions. To this, rat hearts were perfused in a Langendorff preparation and the coronary response to Ap4A (10(-7)-10(-5) M) was recorded. In control hearts, Ap4A produced concentration-dependent vasodilatation both at the basal coronary resting tone and after precontracting coronary vasculature with 11-dideoxy-1a,9a-epoxymethanoprostaglandin F2α (U46619), and this vasodilatation was reduced by reactive blue 2 (2×10(-6) M), glibenclamide (10(-5) M), H89 (10(-6) M), U73122 (5×10(-6) M) and endothelin-1 (10(-9) M), but not by L-NAME (10(-4) M), isatin (10(-4) M), GF109203x (5×10(-7) M), or wortmannin (5×10(-7) M). After ischemia-reperfusion, the vasodilatation to Ap4A diminished, both in hearts with basal or increased vascular tone, and in this case the relaxation to Ap4A was not modified by reactive blue 2, L-NAME, glibenclamide, isatin, H89, GF109203x or wortmannin, although it was reduced by U73122 and endothelin-1. UTP produced coronary relaxation that was also reduced after ischemia-reperfusion. These results suggest that the coronary relaxation to Ap4A is reduced after ischemia-reperfusion, and that this reduction may be due to impaired effects of KATP channels and to reduced response of purinergic P2Y receptors.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Androstadienes; Animals; Coronary Vessels; Dinucleoside Phosphates; Endothelin-1; Estrenes; Glyburide; Heart; In Vitro Techniques; Indoles; Isatin; Isoquinolines; Male; Maleimides; NG-Nitroarginine Methyl Ester; Perfusion; Pyrrolidinones; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Rest; Sulfonamides; Triazines; Vasoconstriction; Wortmannin

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