okadaic-acid and Myocardial-Ischemia

We report that okadaic acid (OA), a known inhibitor of Ser/Thr phosphatases, protects pig myocardium against ischemic injury in an in vivo model and stimulates the activities of stress-activated protein kinases/c-Jun N-terminal kinases (SAPKs/JNKs). When OA was directly infused into the subsequently ischemic myocardium for 60 min before a 60-min period of coronary occlusion followed by reperfusion, infarct size was reduced from a control value of 83.4 +/- 2.8% of the risk region to 40.7 +/- 9.1%. When OA was infused for 10 min before a 5-min occlusion and during 45 min thereafter, infarct size was reduced to 26.5%. In a separate set of similar experiments, we pretreated pig hearts in vivo with the protein-synthesis inhibitor and known activator of SAPK/JNK, anisomycin (AN), and found that this compound also significantly reduced infarct size from 83.4 +/- 2.8.1% to 48.1 +/- 5.1%. For in vitro assays, OA (600 nM), AN (500 microM), or solvent (KHB) were locally infused into the left ventricular myocardium, and biopsies from in situ beating hearts were obtained after 10, 30, and 60 min of infusion. The activities of Ser/Thr phosphatases (PPases), especially PP-2A, were significantly decreased after OA infusion. OA infusion increased the activity (in-gel phosphorylation of N-terminal c-Jun1-135) of both 46- and 55-kDa SAPK/JNKs (twofold to threefold, 30 and 60 min of infusion), and this increase correlated well with the observed decrease of PPase activities. Western blot analysis with a phosphospecific SAPK/JNK (Thr 183/Tyr 185) antibody showed an increased content of the phosphorylated forms after OA treatment. We observed significant stimulation of SAPK/JNK activity also after AN treatment (threefold to fourfold, after 30 min of infusion). In contrast to the SAPK/JNKs, the infusion of both OA and AN did not significantly change the activities and phosphorylation of extracellular signal-related kinases (ERKs) and p38-MAPK. The findings that the protective effect of both OA and AN correlates with increased activity of SAPK/JNKs suggest the involvement of these enzymes in the mechanism of cardioprotection.

Topics: Animals; Anisomycin; Calcium-Calmodulin-Dependent Protein Kinases; Hemodynamics; JNK Mitogen-Activated Protein Kinases; Male; MAP Kinase Kinase 4; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Myocardial Infarction; Myocardial Ischemia; Okadaic Acid; p38 Mitogen-Activated Protein Kinases; Phosphoprotein Phosphatases; Protein Kinases; Swine

Isolated adult rat myocytes were subjected to 180 min of metabolic inhibition or incubated in ischaemic pellets, in the presence and absence of 10 microM okadaic acid (OA) or calyculin A (CL-A). Contracture and viability was determined by light microscopic analysis of trypan blue-stained preparations and ATP levels by HPLC. Osmotic fragility was assessed by brief hypotonic swelling of cells in 170 or 85 mOsm media prior to determination of viability. Neither drug significantly affected the relatively rapid rates of contracture of myocytes during metabolic inhibition, and both afforded significant protection from development of trypan blue permeability and osmotic fragility. Both OA and CL-A significantly accelerated the rates of contracture and ATP depletion of myocytes during ischaemic incubations. Despite an enhanced rate of ATP depletion, which would be expected to accelerate development of injury, neither drug accelerated development of loss of viability or development of osmotic fragility as measured by 170 mOsm swelling. Mathematical compensation for different rates of ATP depletion confirmed that a protective effect of the drugs, during ischaemic incubation, was masked by their enhancement of the rate of injury, following swelling at 170 mOsm. When the effects of CL-A on ischaemic cells were examined at 85 mOsm, a more stringent test for osmotic fragility, protection was found without compensation for differing rates of ATP depletion. A dose/response curve for CL-A showed some effect at 100 nM and a nearly full effect during metabolic inhibition at 1 microM concentrations. It is concluded that protein phosphatase inhibitors reduce the rates of development of osmotic fragility of metabolically inhibited cells and reduces the rate of injury relative to the rate of ATP depletion of ischaemic cardiomyocytes. Phosphorylation mechanisms may be important to development of irreversible myocardial cell injury.

Topics: Adenosine Triphosphate; Animals; Cell Survival; Ethers, Cyclic; Heart; In Vitro Techniques; Marine Toxins; Myocardial Contraction; Myocardial Ischemia; Myocardium; Okadaic Acid; Osmotic Fragility; Oxazoles; Phosphoprotein Phosphatases; Rats