anisomycin and Ischemia

In previous studies, inhibition of mitogen-activated protein kinase (MAP) p38 significantly improved recovery and attenuated apoptosis after retinal ischemia in rats. Yet, ischemic preconditioning (IPC) attenuated the ischemia-induced increase in p38 expression. We hypothesized that p38 was required for induction of ischemic tolerance by IPC. We examined the mechanisms of involvement of p38 in IPC neuroprotection. IPC or ischemia was induced in rat retina in vivo. Recovery after ischemia performed 24h after IPC was assessed functionally (electroretinography) and histologically at 7d after ischemia in the presence or absence of inhibition of p38. We examined the role of p38alpha in the mimicking of IPC produced by opening mitochondrial KATP channels using diazoxide, or stimulation of p38 activation by anisomycin. The importance of adenosine receptors in p38 activation after IPC was assessed using specific blockers of adenosine A1 and A2a receptors. Interfering RNA (siRNA) or SB203580 was used to block p38alpha. Phosphorylated p38 levels were measured. Phosphorylated p38 protein increased with IPC. Interfering RNA (siRNA) to p38alpha prior to IPC, or inhibiting p38 activation with SB203580, with ischemia following 24h later, significantly attenuated the neuroprotective effect of IPC. Anisomycin administered to increase p38 mimicked IPC, an effect blocked by SB203580. IPC-mimicking with diazoxide, an opener of mitochondrial KATP channels, was diminished with p38alpha siRNA. Adenosine receptor blockade did not decrease the elevated levels of phosphorylated p38 after IPC. Specific inhibition of p38alpha suggests that this MAPK is involved in the protective effects of IPC, and that p38 is downstream of mitochondrial KATP channels, but not adenosine receptors, in this neuroprotection.

Topics: Adenosine A1 Receptor Antagonists; Adenosine A2 Receptor Antagonists; Animals; Anisomycin; Diazoxide; Disease Models, Animal; Electroretinography; Enzyme Activators; Imidazoles; Intraocular Pressure; Ischemia; Ischemic Preconditioning; Mitogen-Activated Protein Kinase 14; Phosphorylation; Potassium Channels; Protein Kinase Inhibitors; Pyridines; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A1; Receptor, Adenosine A2A; Reperfusion Injury; Retinal Neurons; Retinal Vessels; RNA Interference; Signal Transduction; Time Factors

We have established an in vitro model of ischemia incorporating the combination of anoxia with glucose deprivation, which is toxic to PC12 cells. In this model, nerve growth factor (NGF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) improve PC12 cell survival. K252a, a specific inhibitor of NGF-induced trk p140 autophosphorylation, did not alter the neuroprotection provided by EGF or bFGF, yet it completely abolished the protection provided by NGF. Activation of protein kinase A (PKA) with dibutyryl-cAMP also protected during ischemia, although it was not additive with the effect provided by growth factors. Furthermore, growth factors protected a PKA-deficient mutant as effectively as the parental cell line; thus, activation of PKA is protective against ischemia but is not necessary for the action of peptide growth factors. Neither the stimulation of protein kinase C (PKC) with acute phorbol ester treatment nor the downregulation of PKC with chronic high-dose phorbol ester treatment resulted in an altered response to growth factors in either the PC12 wild type or PKA-deficient mutant. Thus, protection by peptide growth factors depends on neither PKA nor PKC. Furthermore, downregulation of PKC alone was protective, indicating that PKC may contribute to toxicity. Interestingly, treatment with the kinase inhibitor H-7 was neuroprotective and may have enhanced the neuroprotective effect of NGF. In contrast, staurosporine, a broadly acting kinase inhibitor, inhibited the neuroprotective effect of NGF, but not of EGF or FGF.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Alkaloids; Animals; Anisomycin; Cell Hypoxia; Cell Survival; Cyclic AMP-Dependent Protein Kinases; Epidermal Growth Factor; Fibroblast Growth Factor 2; Ischemia; Isoquinolines; Kinetics; Models, Biological; Neoplasm Proteins; Nerve Growth Factors; PC12 Cells; Phosphorylation; Piperazines; Protein Kinase C; Staurosporine; Time Factors