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The p21-activated protein kinases (PAKs) may participate in signalling from Cdc42/Rac1 to the stress-regulated MAPKs (SAPKs/JNKs and p38-/HOG-1-related-MAPKs). We characterized the expression and regulation of alpha PAK in cultured ventricular myocytes. alpha PAK was specifically immunoprecipitated from myocyte extracts. High basal alpha PAK activity was detected in unstimulated myocytes. Its activity was increased rapidly (<30 s) by hyperosmotic shock in the presence of okadaic acid, and was maximal by 3 min (187 +/- 7% relative to unstimulated cells). Endothelin-1 and interleukin-1beta, which also activate SAPKs/JNKs, did not increase alpha PAK activity and presumably act through different PAK isoforms or other mechanisms.

It has recently been recognized that cellular stresses activate certain members of the mitogen-activated protein kinase (MAPK) superfamily. One role of these "stress-activated" MAPKs is to increase the transactivating activity of the transcription factors c-Jun, Elk1, and ATF2. These findings may be particularly relevant to hearts that have been exposed to pathological stresses. Using the isolated perfused rat heart, we show that global ischemia does not activate the 42- and 44-kD extracellular signal-regulated (protein) kinase (ERK) subfamily of MAPKs but rather stimulates a 38-kD activator of MAPK-activated protein kinase-2 (MAPKAPK2). This activation is maintained during reperfusion. The molecular characteristics of this protein kinase suggest that it is a member of the p38/reactivating kinase (RK) group of stress-activated MAPKs. In contrast, stress-activated MAPKs of the c-Jun N-terminal kinase (JNK/SAPKs) subfamily are not activated by ischemia alone but are activated by reperfusion following ischemia. Furthermore, transfection of ventricular myocytes with activated protein kinases (MEKK1 and SEK1) that may be involved in the upstream activation of JNK/ SAPKs induces increases in myocyte size and transcriptional changes typical of the hypertrophic response. We speculate that activation of multiple parallel MAPK pathways may be important in the responses of hearts to cellular stresses.

An antisense oligodeoxynucleotide (ODN) approach was used to investigate whether mitogen-activated protein kinase (MAPK) is necessary for the hypertrophic response in cardiac myocytes. A phosphorothioate-protected 17-mer directed against the initiation of translation sites of the p42 and p44 MAPK isoform mRNAs was introduced into cultured cardiac myocytes by liposomal transfection. At an antisense ODN concentration of 0.2 mumol/L, p42 MAPK protein was reduced by 82% (immunoblot) after 48 hours, and p42 and p44 MAPK activities were reduced by 44% and 60%, respectively. The same concentration of anti-MAPK ODN inhibited development of the morphological features of hypertrophy (sarcomerogenesis, increased cell size) in myocytes exposed to phenylephrine. Phenylephrine-induced activation of the atrial natriuretic factor (ANF) promoter (measured by the activity of a transfected ANF promoter/luciferase reporter gene) and induction of ANF mRNA (measured by RNase protection assay) were also attenuated. We conclude that MAPK is important for the development of the hypertrophic phenotype in this model of hypertrophy.

Background: Endothelin-1 (ET-1) has potent effects on cell growth and induces hypertrophy of cultured ventricular myocytes. Catecholamines increase expression of ET-1 mRNA by cultured myocytes. We investigated the role of endogenous ET-1 in catecholamine-induced hypertrophy in vivo by studying the effects of continuous norepinephrine infusion on physical and molecular markers of ventricular hypertrophy, ventricular and noncardiac expression of ET-1 mRNA, and the acute effects of bosentan, an orally active ETA and ETB receptor antagonist., Methods and Results: Seventy male Sprague-Dawley rats (175 to 200 g) were divided into four groups: (1) sham-operated rats, (2) norepinephrine-infused rats (600 micrograms.kg-1.h-1 by subcutaneous osmotic pump, up to 7 days), (3) sham-operated rats given bosentan, and (4) norepinephrine-infused rats given bosentan. Bosentan (100 mg/kg once daily) was administered by gavage for 6 days starting 1 day before operation. Norepinephrine caused increases in absolute ventricular weight and ratios of ventricular weight to body weight and ventricular RNA to protein. Ventricular expression of mRNAs for atrial natriuretic factor, skeletal alpha-actin, and beta-myosin heavy chain, which in adult rat ventricle are indicators of hypertrophy, also increased. Ventricular expression of ET-1 mRNA was elevated in the norepinephrine group at 1, 2, and 3 days. By 5 days, this had fallen to control levels. In lung, kidney, and skeletal muscle, norepinephrine did not significantly increase expression of ET-1 mRNA. Bosentan attenuated norepinephrine-induced increases in ventricular weight, ratio of RNA to protein, and expression of skeletal alpha-actin mRNA and beta-myosin heavy chain mRNA at 5 days, but it did not attenuate increased ventricular expression of atrial natriuretic factor mRNA., Conclusions: These data suggest that endogenous ET-1 plays a direct role in mediating norepinephrine-induced ventricular hypertrophy in vivo.

Maximally effective concentrations of endothelin-1 (ET-1), acidic FGF (aFGF), or 12-O-tetradecanoylphorbol-13-acetate (TPA) activated mitogen-activated protein kinase (MAPK) by 3-4-fold in crude extracts of myocytes cultured from neonatal rat heart ventricles. Maximal activation was achieved after 5 min. Thereafter, MAPK activity stimulated by ET-1 or aFGF declined to control values within 1-2 h, whereas activation by TPA was more sustained. Two peaks of MAPK activity (a 42- and a 44-kDa MAPK) were resolved in cells exposed to ET-1 or aFGF by fast protein liquid chromatography on a Mono Q column. One major and one minor peak of MAPK kinase (MAPKK) was stimulated by ET-1 or aFGF. Cardiac myocytes expressed protein kinase C (PKC)-alpha, -delta, -epsilon and -zeta as shown immunoblotting. Exposure to 1 microM TPA for 24 h down-regulated PKC-alpha, -delta, and -epsilon, but not PKC-zeta. This maneuver wholly abolished the activation of MAPK on re-exposure to TPA but did not affect the response to aFGF. The effect of ET-1 was partially down-regulated. ET-1 stimulated phospho[3H]inositide hydrolysis 18-fold, whereas aFGF stimulated by only 30%. Agonists which initially utilize dissimilar signaling pathways may therefore converge at the level of MAPKK/MAPK and this may be relevant to the hypertrophic response of the heart.

The aim of the experiments was to compare the effects of endothelin and alpha-adrenoceptor stimulation on the contraction and inositol phosphate turnover of cardiomyocytes enzymatically isolated from rat and guinea-pig hearts. The effects of agonists on the contraction amplitude of warmed (32 degrees C), electrically stimulated (0.5 Hz) myocytes was recorded using a video-edge detection system. Phosphoinositide hydrolysis was measured in suspensions of myocytes prelabelled with myo-[2(-3)H]-inositol. A doubling of contraction amplitude was observed in rat ventricular myocytes in response to maximally effective concentrations of either endothelin-1 (10 nM) or phenylephrine (1 mM). In rat myocytes, prazosin prevented the effect of phenylephrine but not the effect of endothelin-1. Reversal of the maximal inotropic effect of endothelin was slow (halftime for reversal 11.5 +/- 4.5 min) compared with phenylephrine (3.4 +/- 1.1 min). Endothelin (10 nM) added at the peak effect of phenylephrine produced no further increase in contraction amplitude. The half-time for the reversal of the effect of phenylephrine plus endothelin in these experiments was not significantly different from that with endothelin alone (12.8 +/- 4.0 min). This indicates that phenylephrine did not interact with endothelin binding. Phosphoinositide hydrolysis was increased in rat myocytes by either endothelin or phenylephrine. In guinea-pig myocytes, endothelin-1 stimulated phosphoinositide hydrolysis but did not induce an inotropic response, whereas phenylephrine gave neither an increase in phosphoinositide hydrolysis nor an inotropic effect. We conclude that the observations in rat myocytes are consistent with different receptors for endothelin-1 and phenylephrine, but a common final pathway through the inositol phosphate system for the inotropic effect.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of endothelin-1 (ET-1) on protein synthesis and phosphoinositide (PI) hydrolysis were investigated in ventricular myocytes isolated by collagenase digestion of adult rat hearts. The maximum stimulation of protein synthesis by ET-1 was about 35% and the EC50 value was about 0.3 nM. The stimulation was exerted at the translational stage since it was insensitive to inhibition by actinomycin D. The maximum stimulation of PI hydrolysis by ET-1 as measured by the formation of [3H]inositol phosphates was about 11-fold and the EC50 value was about 0.7 nM. The ET-1 analogue sarafotoxin-6b stimulated protein synthesis by a maximum of 27% and stimulated PI hydrolysis about 8- to 9-fold. The EC50 values were 1.6 nM and 0.6 nM, respectively. Other endothelins stimulated protein synthesis and PI hydrolysis in the following order of potency: ET-1 approximately ET-2 > ET-3. This order of potency suggests that the stimulation of both protein synthesis and PI hydrolysis is mediated through the ETA receptor. Although both angiotensin II and [Arg]vasopressin stimulated PI hydrolysis significantly, the stimulation was less than 60%, i.e., much less than the stimulation by ET-1 and its analogues. Neither insulin nor substance P stimulated PI hydrolysis. Stimulation of protein synthesis by ET-1 and its analogues correlated strongly with the stimulation of PI hydrolysis and we suggest that the stimulation of protein synthesis may be dependent on the stimulation of PI hydrolysis. We hypothesize that the mechanism may involve a protein kinase C-mediated increase in intracellular pH.

In the presence of 5 microM-DL-propranolol and in HCO3(-)-containing buffers, 1 microM-adrenaline acutely stimulated protein synthesis by about 25% in the anterogradely perfused rat heart. This stimulation was opposed by low (1-10 nM) concentrations of prazosin, but not by similar concentrations of yohimbine, suggesting involvement of the alpha 1-adrenoceptor. Under the same conditions, adrenaline raised intracellular pH (pHi) by about 0.1 unit. The increase in pHi induced by adrenaline was prevented by 5 nM-prazosin, but not by 5 nM-yohimbine, again suggesting involvement of the alpha 1-adrenoceptor. Since an increase in pHi stimulates protein synthesis in the heart [Sugden & Fuller (1991) Biochem. J. 273, 339-346], the increase in pHi induced by adrenaline may be involved in its stimulation of protein synthesis. Adrenaline also increased phosphocreatine concentrations. As discussed, the increase in pHi induced by adrenaline may be responsible for this effect. Using second-order polynomial regression analysis, we showed that rates of protein synthesis were significantly correlated (P less than 0.0001) with phosphocreatine concentrations. We discuss two possible reasons for this correlation: (i) increases in pHi stimulate protein synthesis and separately raise phosphocreatine concentrations, or (ii) the increase in protein synthesis rates is a consequence of the raised phosphocreatine concentrations induced by the increase in pHi. Rates of protein synthesis were not significantly correlated with ATP/ADP concentration ratios, nor with any of the following: ATP, ADP, AMP or total adenine nucleotide concentrations. In freshly isolated adult rat cardiomyocytes, the protein kinase inhibitor staurosporine (1 microM) prevented stimulation of protein synthesis by 0.3 microM-adrenaline (and by 1 microM-phorbol 12-myristate 13-acetate or 1 m-unit of insulin/ml). The results are discussed within a mechanistic framework initiated by stimulation of the hydrolysis of membrane phospholipids by alpha 1-adrenergic agonists.

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.

In animals the pyruvate dehydrogenase reaction is mainly responsible for the irreversible loss of glucose carbon by oxidation. Regulation of this reaction is shown to be a major determinant of glucose conservation in starvation and diabetes. Estimates of conservation in man in starvation and diabetes are reviewed. The pyruvate dehydrogenase complex is inhibited by products of its reactions; it is also regulated by a phosphorylation-dephosphorylation cycle catalysed by a kinase intrinsic to the complex and by a more loosely associated phosphatase. Inactivation is largely accomplished by phosphorylation of the tetrameric decarboxylase component (alpha2beta2) to alpha2Pbeta2. Complete phosphorylation produces the (alpha2P3)beta2 form. Both forms are completely reactivated by phosphatase action but the initial rate of reactivation of a complex containing alpha2Pbeta2 is approximately three times that of (alpha2P3)beta2. The proportion of active (dephosphorylated) complex is decreased in rat tissues by starvation and diabetes and in perfused rat heart by oxidation of fatty acids and ketone bodies. In adipose tissue in vitro, insulin increases the proportion of active complex and lipolytic hormones may decrease this proportion. It is suggested that rates of oxidation of lipid fuels may be a major determinant of the activity of pyruvate dehydrogenase in tissues in relation to the actions of insulin and lipolytic hormones and the effects of diabetes and starvation. Phosphorylation and inactivation of the complex are enhanced by high mitochondrial ratios of [acetyl-CoA]/[CoA], [ATP]/[ADP], [NADH]/[NAD+] and low concentrations of pyruvate, Mg2+ and Ca2+, and vice versa.

In animal tissues the pyruvate dehydrogenase complex is regulated by product inhibition and by a phosphorylation-dephosphorylation cycle catalysed by a kinase and a phosphatase. Physiologic and molecular aspects of this regulation are reviewed, and the results of recent studies are described. Insulin deficiency in the rat (diabetes or starvation) is shown to inhibit the conversion of inactive (phospho-) complex into active (dephospho-) complex by the phosphatase by an effect on the substrate for the phosphatase (phosphorylated complex). This change is stable and persists during isolation, incubation, and extraction of mitochondria or purification of phosphorylated complex. The subunit ratios in the purified pig heart pyruvate dehydrogenase complex and the stoichiometry of phosphorylations have been determined by radioamidination and incorporation of 32P. The ratios of decarboxylase tetramer (alpha 2, beta 2) : dihydrolipoyl acetyltransferase monomer : dihydrolipoly dehydrogenase monomer were 1:1:0.5. Inactivation of the complex was accomplished by incorporation of a single phosphate into one alpha subunit of the decarboxylase tetramer. Two further phosphates are then incorporated and these additional phosphorylations inhibit reactivation of the complex by the phosphate. It is suggested that multisite phosphorylations may inhibit reactivation of the complex by the phosphatase in diabetes and in starvation.

An isotope-dilution method is described for the measurement of N tau-methylhistidine release from the perfused rat heart. We argue that release of N tau-methylhistidine is indicative of cardiac actin degradation. N tau-Methylhistidine release is compared with phenylalanine release in the presence of cycloheximide (phenylalanine release being a measure of degradation of mixed proteins). In hearts perfused with glucose plus acetate, the rate of actin degradation was increased by starvation and was not inhibited by insulin. In contrast, the rate of mixed-protein degradation was decreased by starvation and was inhibited by insulin. The fractional rate of degradation of mixed proteins in hearts from fed or starved rats was greater than that for actin. It is suggested that there are at least two pools of intracellular protein, the degradation rates of which differ in terms of their response to insulin and starvation.