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Protein-synthesis rates in freshly isolated cardiac myocytes from adult rats were acutely stimulated by 20-30% by 1 microM-adrenaline, by 1 microM-noradrenaline or by 1 microM-phenylephrine, but were not stimulated by 1 microM-isoprenaline. Stimulation by 1 microM-adrenaline was completely prevented by 100 nM-prazosin. Yohimbine was much less effective in preventing stimulation, and 20 microM-DL-propranolol was completely ineffective. The stimulation of protein synthesis by adrenaline was still observed after inhibition of transcription by actinomycin D. None of these manipulations affected myocyte ATP contents. In anterogradely perfused hearts, protein-synthesis rates were stimulated by 1-2 microM-adrenaline in the presence of 10 microM-DL-propranolol (to decrease the beta-adrenergic effects of adrenaline). ATP contents were not altered, but phosphocreatine contents were increased. These observations lead us to conclude that cardiac protein synthesis can be stimulated acutely at the level of translation by alpha 1-adrenergic stimulation. We discuss possible roles for protein kinase C and intracellular alkalinization in the mediation of this effect.

Increasing the extracellular pH over the range pH 7.4-8.9 stimulated protein synthesis by about 60% in the rat heart preparation anterogradely perfused in vitro. Protein degradation was inhibited by this pH increase. The magnitudes of the effects at pH 8.9 on protein synthesis and degradation were similar to those of high concentrations of insulin. Cardiac outputs were increased, as were cardiac phosphocreatine contents, indicating that the alterations in extracellular pH did not adversely affect the physiological viability of the preparation. ATP contents were unaltered. The creatine kinase equilibrium was used to assess the magnitude of the change in intracellular pH induced by these treatments. The increase in intracellular pH was about 0.2 for a 1-unit increase in extracellular pH. Thus small changes in intracellular pH have dramatic effects on cardiac protein turnover. INTRODUCTION Recently, interest in the regulation of intracellular processes by intracellular pH (pHi) has increased (for reviews, see [1][2][3][4][5] Perfusion buffersMost perfusions were carried out with modified Tyrode's solution equilibrated with 02 When Hepes was buffer, a solution containing 10 mM-Hepes, 120 mMNaCl, 6 mM-KCl, 1 MM-MgCl2, 2 mM-CaCl2 and 5 mMglucose was adjusted at 22-25°C to the desired pH by using 10 M-NaOH and a pH-meter calibrated between 7.00 and 9.00. Solid NaCl was then added to give a total Na+ concentration of 140 mm. When Tris was buffer, a solution containing 10 mM-Tris base, 140 mM-NaCl, 6 mM-KCl, 1 mM-MgCl2, 2 mM-CaCl2 and 5 mM-glucose was adjusted at 22-25°C to the desired pH with 12 M-HCI. Additionally, some perfusions were carried out with Krebs-Henseleit bicarbonate-buffered saline [9] (119 mM-NaCl, 25 mM-NaHCO3, 4.7 mM-KCl, 2.5 mMCaCl2, 1.2 mM-MgSO4, 1.2 mM-KH2PO4 and 5 mMglucose) equilibrated with 02: CO2 (19: 1) with a measured pH of 7.40 at 37 'C. Heart perfusionsAfter a retrograde pre-perfusion during which cannulation was completed, hearts from 275-325 g fed male rats were perfused anterogradely essentially as described previously [10,11]. The buffer volume was 120 ml for protein-synthesis measurements and 100 ml for protein-degradation measurements. The filling pressure was 0.5 kPa and the aortic pressure was 7.0 kPa. S. J. Fuller, C. J. Gaitanaki and P. H. Sugden measurements, pre-perfusion buffers did not contain amino acids but were otherwise identical with the perfusion buffers. For protein-degradation measurements, both buffers were the same. When required, insulin and/or cycloheximide were added to both the pre-perfusion and the perfusion buffers. Ventricular protein synthesis was measured as described in detail in [12,13]. Amino acids were added after 10 min of anterograde perfusion.[U-14C]Phenylalanine concentration was 0.4 mm (sp. radioactivity 0.04 Ci/mol). At this concentration, extracellular [U-'4C]phenylalanine specific radioactivity rapidly equilibrates with that of phenylalanyl-tRNA [14]. The remaining amino acids required for protein synthesis were each present at 0.2 mm. Under t...

SUMMARYWe investigated the activation of three subfamilies of MAPKs (ERK, JNKs and p38-MAPK) by oxidative stress in the isolated perfused amphibian heart. Activation of p43-ERK by 100 μmol l-1 H2O2was maximally observed within 5 min, remained elevated for 30 min and was comparable with the effect of 1 μmol l-1 PMA. p43-ERK activation by H2O2 was inhibited by PD98059 but not by SB203580. The p46 and p52 species of JNKs were maximally activated by 2.5- and 2.1-fold,respectively, by 100 μmol l-1 H2O2 within 2 min. JNK activation was still detectable after 15 min, reaching control values at 30 min of treatment. p38-MAPK was maximally activated by 9.75-fold by 100 μmol l-1 H2O2 after 2 min and this activation progressively declined thereafter, reaching control values within 45 min of treatment. The observed dose-dependent profile of p38-MAPK activation by H2O2 revealed that 30 μmol l-1 H2O2 induced maximal phosphorylation,whereas 100–300 μmol l-1 H2O2induced considerable activation of the kinase. Our studies also showed that the phosphorylation of MAPKAPK2 by H2O2 followed a parallel time-dependent pattern and that SB203580 abolished this phosphorylation. Furthermore, our experiments clearly showed that 30 μmol l-1 H2O2 induced a strong, specific phosphorylation of HSP27. Our immunohistochemical studies showed that immune complexes of phosphorylated forms of both p38-MAPK and HSP27 were strongly enhanced by 30 μmol l-1 H2O2 in the perinuclear region as well as dispersedly in the cytoplasm of ventricular cells and that SB203580 abolished this phosphorylation. These data indicate that oxidative stress is a powerful activator of all three MAPK subfamilies in the amphibian heart. Stimulation of p38-MAPK and the consequent phosphorylation of HSP27 may be important in cardioprotection under such conditions.