h-89 and Brain-Ischemia

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

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

Corticotrophin-releasing factor receptor 2β (CRFR2β) is expressed in the myocardium. In the present study we explore whether acute treatment with the neuropeptide corticotrophin-releasing factor (CRF) could induce cytoprotection against a lethal ischemic insult in the heart (isolated murine neonatal cardiac myocytes and the isolated Langendorff perfused rat heart) by activating CRFR2. In vitro, CRF offered cytoprotection when added prior to lethal simulated ischemic stress by reducing apoptotic and necrotic cell death. Ex vivo, CRF significantly reduced infarct size from 52.1±3.1% in control hearts to 35.3±3.1% (P<0.001) when administered prior to a lethal ischemic insult. The CRF peptide did not confer cytoprotection when administered at the point of hypoxic reoxygenation or ischemic reperfusion. The acute effects of CRF treatment are mediated by CRF receptor type 2 (CRFR2) since the cardioprotection ex vivo was inhibited by the CRFR2 antagonist astressin-2B. Inhibition of the mitogen activated protein kinase-ERK1/2 by PD98059 failed to inhibit the effect of CRF. However, both protein kinase A and protein kinase C inhibitors abrogated CRF-mediated protection both ex vivo and in vitro. These data suggest that the CRF peptide reduces both apoptotic and necrotic cell death in cardiac myocytes subjected to lethal ischemic induced stress through activation of PKA and PKC dependent signaling pathways downstream of CRFR2.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Brain Ischemia; Cell Death; Corticotropin-Releasing Hormone; Cyclic AMP-Dependent Protein Kinases; Heart; Isoquinolines; Mice; Mice, Inbred C57BL; Myocardium; Myocytes, Cardiac; Protein Kinase C; Receptors, Corticotropin-Releasing Hormone; Signal Transduction; Structure-Activity Relationship; Sulfonamides

MCT1 (monocarboxylic acid transporter 1) facilitates bidirectional monocarboxylic acid transport across membranes. MCT1 function and regulation have not been characterized previously in cerebral endothelial cells but may be important during normal cerebral energy metabolism and during brain diseases such as stroke. Here, by using the cytoplasmic pH indicator 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein-acetoxymethyl ester, the initial rates of monocarboxylate-dependent cytoplasmic acidification were measured as an indication of MCT1 kinetic function in vitro using the rat brain endothelial cell (RBE4) model of blood-brain transport. The initial rate of L-lactate-dependent acidification was significantly inhibited by 5-10-min incubations with agonists of intracellular cAMP-dependent cell signaling pathways as follows: dibutyryl cAMP, forskolin, and isoproterenol. Isoproterenol reduced V(max) but did not affect K(m) values. The effects of forskolin were completely reversed by the protein kinase A inhibitor H89, whereas H89 alone increased transport rates. Cytoplasmic cAMP levels, measured by radioimmunoassay, were increased by forskolin or isoproterenol, and the effect of isoproterenol was inhibited by propranolol. MCT1-independent intracellular pH control mechanisms did not contribute to the forskolin or H89 effects on MCT1 kinetic function as determined with amiloride, monocarboxylate-independent acid loading, or the transport inhibitor alpha-cyano-4-hydroxycinnamate. The data demonstrate the direct modulation of MCT1 kinetic function in cerebral endothelial cells by agents known to affect the beta-adrenergic receptor/adenylyl cyclase/cAMP/protein kinase A intracellular signaling pathway.

Topics: Animals; Brain; Brain Ischemia; Calibration; Colforsin; Coumaric Acids; Cyclic AMP; Cytoplasm; Endothelial Cells; Enzyme Inhibitors; Hydrogen-Ion Concentration; Immunohistochemistry; Isoproterenol; Isoquinolines; Kinetics; Microscopy, Fluorescence; Models, Biological; Monocarboxylic Acid Transporters; Radioimmunoassay; Rats; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Sulfonamides; Symporters