Abstract: Gq-protein-coupled receptor (GqPCR) signalling is associated with the induction of cardiac myocyte hypertrophy, which is characterized by an increase in expression of immediate early genes via activation of pre-existing transcription factors. Here, we explore the role of MSK1 and MAPK signalling pathways in the regulation of the immediate early gene c-jun. The results provide further support for the role of MSK1 in cardiac myocyte hypertrophy and indicate that PE activates distinct signalling mechanisms which culminate with a complex activation of c-jun. ERK1/2 and JNKs are the principal kinases responsible for phosphorylation of c-Jun, whereas c-jun mRNA and protein up-regulation by PE is mediated by multiple signalling pathways that include MSK1, ERK1/2, p38-MAPK and JNKs. These signalling mechanisms seem to be critical to the phenotypic changes of cardiac myocytes in response to hypertrophic stimulation.
Abstract: The present study set out to investigate the thermal limits of the Mediterranean bivalve Modiolus barbatus, acclimated to various temperatures, and includes a comparison of laboratory determined limits with its temperature-dependent restriction to deeper water layers in its natural habitat. Thermal responses and limits were determined by integrating information from various levels of biological organization, including the expression of Hsp70 and Hsp90, the phosphorylation of stress-activated protein kinases, p38 mitogen-activated protein kinase (p38 MAPK) and cJun-N-terminal kinases (JNKs) as well as metabolic adjustments. The latter were assessed by examining temperature effects on the activity of the key glycolytic enzyme pyruvate kinase (PK). The expression of Hsp70 and Hsp90 was activated when mussels were acclimated to temperatures above 20 degrees C. Increased phosphorylation of p38 MAPK and JNKs at about the same temperatures indicate activation of MAPK signaling cascades and their potential involvement in the induction of Hsp genes. As indicated by the activity of PK, Modiolus barbatus maintains some aerobic capacity when acclimated to temperatures up to 24 degrees C, while further warming probably caused metabolic depression and a shift from aerobic to anaerobic metabolism. An increase in mortality occurred in parallel, during acclimation to temperatures above 24 degrees C. Our results indicate that both the biochemical stress indicators and metabolic status respond in parallel once hypoxemia becomes extreme. Comparison with our previous study of thermal limits and vertical distribution in M. galloprovincialis dwelling in shallow waters emphasizes the relevance of maintained aerobic scope over that of passive tolerance for permanent vertical zonation at higher temperatures in the field. These findings and conclusions are in line with the concept of oxygen and capacity limited thermal tolerance and the associated systemic to molecular hierarchy of thermal limitation.
Abstract: The present study aimed to determine the thermal response of the Mediterranean mussel Mytilus galloprovincialis by integrating information from various levels of biological organization including behavior, metabolic adjustments, heat shock protein expression, and protein kinase activity. Behavioral responses were determined by examining the effect of warming on valve closure and opening. Metabolic impacts were assessed by examining the activity of the key glycolytic enzyme pyruvate kinase (PK). Molecular responses were addressed through the expression of Hsp70 and Hsp90 and the phosphorylation of stress-activated protein kinases, p38 mitogen-activated protein kinase (p38 MAPK) and cJun-N-terminal kinases (JNKs). Mussels increased the duration of valve closure by about sixfold when acclimated to 24 degrees C rather than to 17 degrees C. As indicated by the activity of PK, such behavior caused metabolic depression and probably a shift from aerobic to anaerobic metabolism. Acclimation to temperatures higher than 24 degrees C caused an increase in mortality and induced the expression of Hsp72. Increased phosphorylation of p38 MAPK and JNKs indicated activation of MAPK signaling cascades. The potential involvement of MAPKs in the induction of Hsp genes in the tissues of M. galloprovincialis is discussed. In conclusion, it seems that M. galloprovincialis lives close to its acclimation limits and incipient lethal temperature and that a small degree of warming will elicit stress responses at whole organism and molecular levels.
Abstract: H2O2, as an example of oxidative stress, induces cardiac myocyte apoptosis. Bcl-2 family proteins are key regulators of the apoptotic response while their functions can be regulated by post-translational modifications including phosphorylation, dimerization or proteolytic cleavage. In this study, we examined the role of various protein kinases in regulating total BAD protein levels in adult rat cardiac myocytes undergoing apoptosis. Stimulation with 0.1 mM H2O2, which induces apoptosis, resulted in a marked down-regulation of BAD protein, which is attributed to cleavage by caspases since it can be restored in the presence of a general caspase inhibitor. Inhibition of PKC, p38-MAPK, ERK1/2 and PI-3-K did not influence the reduced BAD protein levels observed after stimulation with H2O2. On the contrary, inhibition of PKA or specifically PKCdelta resulted in up-regulation of BAD. Decreased caspase 3 activity was observed in H2O2 treated cells after inhibition of PKA or PKCdelta whereas inhibition of PKA also resulted in improved cell survival. Furthermore, addition of okadaic acid to inhibit selected phosphatases resulted in enhanced BAD cleavage. These data suggest that, during oxidative stress-induced cardiac myocyte apoptosis, there is a caspase-dependent down-regulation of BAD protein, which seems to be regulated by coordinated action of PKA, PKCdelta and phosphatases.
Abstract: The phenomenon of ischemic preconditioning has been recognized as one of the most potent mechanisms to protect against myocardial ischemic injury. In experimental animals and humans, a brief period of ischemia has been shown to protect the heart from more prolonged episodes of ischemia, reducing infarct size, attenuating the incidence, and severity of reperfusion-induced arrhythmias, and preventing endothelial cell dysfunction. Although the exact mechanism of ischemic preconditioning remains obscure, several reports indicate that this phenomenon may be a form of receptor-mediated cardiac protection and that the underlying intracellular signal transduction pathways involve activation of a number of protein kinases, including protein kinase C, and mitochondrial K(ATP) channels. Apoptosis, a genetically programmed form of cell death, has been associated with cardiomyocyte cell loss in a variety of cardiac pathologies, including cardiac failure and those related to ischemia/reperfusion injury. While ischemic preconditioning significantly reduces DNA fragmentation and apoptotic myocyte death associated with ischemia-reperfusion, the potential mechanisms underlying this effect have not been fully clarified. A comprehensive understanding of these mechanisms and application to clinical scenarios will provide new directions in research and translate this information into new treatment approaches for reducing the extent of ischemia/reperfusion injury.
Abstract: Previous studies have shown that the cardioprotective effect of ischemic preconditioning (IPC) can be mimicked pharmacologically with clinically relevant agents, including nitric oxide (NO) donors. However, whether pharmacological preconditioning shares the same molecular mechanism with IPC is not fully elucidated. The present study aimed to determine the activation of mitogen-activated protein kinases (MAPKs) (ERK1/2, p38 MAPK and p46/p54 JNKs) during ischemia and at reperfusion in nitroglycerin-induced preconditioning as compared to IPC and to correlate this with the conferred cardioprotection in anesthetized rabbits. Sixty minutes of intravenous administration of nitroglycerin was capable of inducing both early and late phase preconditioning in anesthetized rabbits, as it was expressed by the reduction of infarct size. Despite the cardioprotective effect conferred by both ischemic and nitroglycerin-induced preconditioning, there was a differential phosphorylation of MAPKs between the studied groups. p38 MAPK was activated early in ischemia in both ischemic and the early nitroglycerin-induced preconditioning while JNKs were markedly increased only after IPC. Furthermore, in these groups, ERK1/2 were activated during reperfusion. A different profile was observed in the late preconditioning induced by nitroglycerin with increased p38 MAPK and ERK1/2 phosphorylation during late ischemia. No activation of JNKs was observed at any time point in this group. It seems that activation of individual MAPK subfamilies depends on the nature of preconditioning stimulus.
Abstract: OBJECTIVE: Recent studies suggest that ischemic preconditioning (IPC) inhibits myocardial apoptosis after ischemia and reperfusion. This study aimed first, to examine whether short mechanical stretch with acute pressure overload (MPC), which has been shown to reduce infarct size after ischemia/reperfusion, mimics IPC in attenuating myocardial apoptosis and second, to evaluate whether induced cardioprotection involves modulation of the expression of the Bcl-2 family proteins and phosphorylation of prosurvival kinases. METHODS AND RESULTS: A model of anaesthetized rabbit was used and the preconditioning protocol included one cycle of short ischemia/reperfusion, or short mechanical stretch with acute pressure overload. Preconditioning stimuli were equally effective in reducing the infarct size, determined after 4 h reperfusion. However, IPC but not MPC attenuated myocardial apoptosis. IPC restored the decreased expression of Bcl-2 and Bcl-xL observed in hearts subjected to ischemia and reperfusion only. Bax levels were not different among the groups. ERK1/2 were activated during reperfusion in both IPC and MPC groups. CONCLUSIONS: The data provide further evidence that apoptosis and necrosis contribute independently to infarct size after ischemia and reperfusion. Inhibition of the myocardial apoptotic processes by IPC may involve modulation of the expression of anti-apoptotic proteins, Bcl-2 and Bcl-xL. ERK1/2 may be involved in the inhibition of both apoptosis and necrosis.
Abstract: The aim of the present study was to clarify whether pharmacological preconditioning with dopamine protects the heart against ischemia and whether this effect is mediated through dopaminergic receptors (D1 and D2) or alpha1-adrenoceptors. Isolated perfused rat hearts were either non-preconditioned, preconditioned with 5 min ischemia, or treated for 5 min with dopamine (1, 5 or 10 microM) before being subjected to 45 min of sustained ischemia followed by 60 min reperfusion. Postischemic functional recovery and infarct size were used as indices of the effects of ischemia. Treatment with the lower concentration of dopamine (1 microM), did not provide any protection to the ischemic myocardium. On the other hand, treatment with 5 microM dopamine resulted in significantly improved functional recovery, whereas administration of dopamine (10 microM) resulted in significantly improved functional recovery as well as reduction of infarct size. Pretreatment with the mixed D1/D2 dopaminergic receptor antagonist haloperidol or the beta-adrenoceptor selective antagonist propranolol did not attenuate the protective effect of pharmacological preconditioning with 10 microM dopamine with respect to both functional recovery and infarct size reduction. On the other hand, the cardioprotective effect of dopamine was blocked when the alpha1-adrenoceptor selective antagonist, prazosin, was administered. In conclusion, pharmacological preconditioning with dopamine protects the myocardium against ischemia and this effect seems to be mediated through activation of alpha1-adrenoceptors.
Abstract: AIM: Experimental studies have demonstrated that an exogenous supply of glutamate improves mechanical function and recovery of ischemic myocardium. The aim of the present study was to investigate the effect of myocardial pre-bypass loading with glutamate on myocardial protection during global ischemia and reperfusion of patients undergoing coronary artery bypass grafting (CABG). METHODS: The study was double blinded. Twenty patients undergoing elective CABG were randomized to receive L-glutamate (n = 10) or normal saline (n = 10). Intracellular levels of glutamate, ATP and lactate were measured in left ventricular biopsies collected 10 min after aortic clamp release. Hemodynamic data, and postoperative release of CK-MB and troponin T were also measured. RESULTS: Pre-bypass administration of glutamate resulted in myocardial glutamate loading since glutamate levels were significantly higher in the glutamate group of patients than in controls (18.6 +/- 3.1 versus 8.7 +/- 1.2 microg/g tissue, P < 0.001). In the same group ATP levels were also significantly higher (2.4 +/- 0.7 versus 1.5 +/- 0.4 microg/g tissue, P < 0.05) and lactate levels significantly less than in controls (6.9 +/- 1.9 versus 12.0 +/- 2.1 microg/g tissue, P < 0.001). Glutamate patients had statistically significantly superior post-bypass hemodynamic performance (cardiac index, left ventricular stroke work index, systemic vascular resistance and pulmonary vascular resistance). Statistically significantly lower levels of CK-MB (6 h postoperative), total and peak CK-MB, troponin T (24 h postoperative), and total troponin T were found in the glutamate group. CONCLUSIONS: The results of this preliminary study indicate that pre-bypass intravenous administration of glutamate in patients undergoing CABG has a supportive effect on myocardial metabolism during global ischemia and reperfusion, improves patients' postoperative hemodynamic performance and reduces postoperative cardiac enzyme release.
Abstract: cAMP responsive element binding protein (CREB) is a stimulus induced transcription factor with possible relevance for the pathophysiology of the heart. In the present study, we provide evidence that the hypertrophic agonist, phenylephrine (PE), promotes phosphorylation of CREB in adult rat cardiac myocytes through alpha(1)- and beta-adrenergic receptors. PE-induced phosphorylation of CREB was partially inhibited by Ro318220 and H89, which were shown to be potent inhibitors of mitogen- and stress-activated protein kinase-1 (MSK1) activation, implicating the involvement of this kinase in the response. Similar results were obtained when cardiac myocytes were treated with the inhibitors of ERK1/2 and p38 MAPK pathways. In addition, inhibition of protein kinase A by RpcAMP reduced phosphorylation of CREB, suggesting that this pathway is also involved. Furthermore, PE stimulation was accompanied by an increase in CRE-binding activity, which was reduced by drugs that prevented phosphorylation of CREB. An enhanced CBP/phospho-CREB complex formation was also observed, suggesting recruitment of CBP to phosphorylated CREB. These results suggest that PE stimulates phosphorylation and DNA binding activity of CREB in adult rat ventricular myocytes through multiple signaling pathways involving ERK1/2, p38 MAPK, MSK1 and PKA. The same pathways seem to regulate atrial natriuretic peptide (ANF) mRNA expression, a highly conserved marker gene of cardiac hypertrophy, suggesting that the PE-stimulated activation of CREB is likely to play an important role in the hypertrophic response.
Abstract: We investigated the activation of mitogen-activated protein kinases (MAPKs) pathways by purinergic stimulation in cardiac myocytes from adult rat hearts. ATPgammaS increased the phosphorylation (activation) of the extracellular signal regulated kinase 1 and 2 (ERK1/2) and p38 MAPK. ERK1/2 and p38 MAPK activation was differential, ERK1/2 being rapid and transient while that of p38 MAPK slow and sustained. Using selective inhibitors, activation of ERK1/2 was shown to involve protein kinase C and MEK1/2 while that of p38 MAPK was regulated by both protein kinase C and protein kinase A. Furthermore, we show that purinergic stimulation induces the phosphorylation of the MAPK downstream target, mitogen- and stress-activated protein kinase 1 (MSK1), in cardiac myocytes. The time course of MSK1 phosphorylation closely follows that of ERK activation. Inhibitors of the ERK and p38 MAPK pathways were tested on the phosphorylation of MSK1 at two different time points. The results suggest that ERKs initiate the response but both ERKs and p38 MAPK are required for the maintenance of the complete phosphorylation of MSK1. The temporal relationship of MSK1 phosphorylation and cPLA2 translocation induced by purinergic stimulation, taken together with previous findings, is an indication that cPLA2 may be a downstream target of MSK1.
Abstract: 1. We tested the hypothesis that ischaemic preconditioning of the rat heart activates cardiovascular adenosine formation to provide enhanced cardioprotection. 2. Rat isolated perfused hearts were either non-preconditioned, preconditioned with 5 min ischaemia or treated for 5 min with the alpha1-adrenoceptor agonist phenylephrine (50 micro mol/L) before being subjected to 30 min sustained ischaemia followed by 30 min reperfusion. Isolated cardiomyocytes were either non-preconditioned, subjected to 10 min simulated ischaemia or treated for 10 min with phenylephrine (50 micro mol/L) before being subjected to 30 min simulated ischaemia. Functional recovery of hearts and cell viability were used as indices of the effects of ischaemia. 3. Myocardial adenosine, as well as intracellular pH, was determined at the end of the preconditioning period and at 10, 20 and 30 min of sustained ischaemia. Intracellular pH was also determined during the reperfusion. 4. Ischaemic or pharmacological preconditioning with phenylephrine correlated with an improved functional recovery of perfused hearts during reperfusion and increased cell viability during ischaemia. 5. In perfused hearts, ischaemic preconditioning resulted in increased adenosine production in the myocardium during the following sustained ischaemia. However, in isolated cardiomyocytes, adenosine levels during sustained ischaemia were lower in ischaemically preconditioned cells compared with the respective non-preconditioned cardiomyocytes. 6. The increase in adenosine production was not observed in hearts preconditioned with phenylephrine instead of transient ischaemia. Similarly, pharmacological preconditioning resulted in decreased adenosine levels during sustained ischaemia in isolated cardiomyocytes. 7. Intracellular pH was preserved during ischaemia to the same extent in both ischaemically or pharmacologically preconditioned hearts and cardiomyocytes, indicating that less acidosis during ischaemia is related to protection. 8. Taken together, the results suggest that cardioprotection does not necessarily correlate with increased adenosine production. Thus, adenosine concentration is not crucial to the beneficial effects of preconditioning in rat heart.
Abstract: 1. The aim of the present study was to clarify the role of alpha1-adrenoceptors in the mechanism of ischaemic preconditioning (IP). 2. Rat isolated perfused hearts were either non- preconditioned, preconditioned with 5 min ischaemia or treated for 5 min with alpha1-adrenoceptor agonists (50 micromol/L phenylephrine; 0.1, 0.5 and 1 micromol/L methoxamine) before being subjected to 45 min of sustained ischaemia followed by 60 min reperfusion. 3. Within each of the above protocols, hearts were divided into groups to which alpha1-adrenoceptor antagonists (prazosin, 5'-methyl urapidil and chloroethylclonidine (CEC)) were administered. Functional recovery and infarct size were used as indices of the effects of ischaemia. Ischaemic contracture characteristics and maximal diastolic pressure during reflow were also assessed. 4. Blockade of alpha(1)-adrenoceptors with prazosin or the subtype-selective antagonists 5'-methyl urapidil and CEC did not abolish the protective effect of IP with respect to both functional recovery and infarct size reduction. 5. Protection afforded by phenylephrine was attenuated in hearts treated with prazosin or the alpha(1B)-adrenoceptor- selective antagonist CEC, but not in those treated with the alpha(1A)-adrenoceptor-selective antagonist 5'-methyl urapidil. 6. Treatment with low concentrations of methoxamine, considered to be alpha(1A)-adrenoceptor selective, did not confer any protection to the ischaemic myocardium. 7. A close relationship between accelerated ischaemic contracture and enhanced cardioprotection was observed. 8. The results suggest that alpha1-adrenoceptor stimulation mimics IP, but it is not an essential component in the mechanism behind the protective effect of IP in rat heart. In addition, the present study demonstrates that stimulation of the alpha(1B)- but not the alpha(1A)-adrenoceptor subtype is responsible for the catecholamine-induced protection of ischaemic myocardium in rat.
Abstract: G-protein-coupled receptor agonists are powerful stimulators of mitogen-activated protein kinase (MAPK) cascades in cardiac myocytes. However, little is known regarding the physiological activation of enzymes downstream of MAPKs. We examined the activation of mitogen- and stress-activated protein kinase-1 (MSK1), a downstream target of MAPKs, in adult rat cardiac myocytes by phenylephrine and endothelin-1. Both agonists induced the phosphorylation of MSK1 at Thr-581 and Ser-376 but not at Ser-360. Maximal phosphorylation was observed at 10-15 min after stimulation and it correlated with increased activity. Maximal activation of MSK1 in adult cardiomyocytes temporally coincided with maximal p38 MAPK activation while activation of the extracellular-signal-regulated kinase (ERK) cascade was more rapid. Phosphorylation and activation of MSK1 was completely inhibited by either PD98059 (ERK1/2 pathway inhibitor) or SB203580 (p38 MAPK inhibitor) alone. These data demonstrate that MSK1 activation in adult rat cardiac myocytes by G-protein-coupled receptor agonists requires the simultaneous activation of both the ERK and p38 MAPK pathways. However, the lack of phosphorylation at Ser-360, an identified phosphorylation site targeted by MAPKs, may indicate that MSK1 is not a direct substrate of ERK1/2 and p38 MAPK in adult rat cardiomyocytes.
Abstract: We assessed the activation of p38-MAPK (mitogen-activated protein kinase) by osmotic and thermal stresses in the isolated perfused amphibian (Rana ridibunda) heart. Hyperosmotic stress induced the rapid activation of the kinase. In particular, in the presence of 0.5 mol l(-1) sorbitol, p38-MAPK was maximally phosphorylated (by approximately twelvefold) at 15 min, while excess of NaCl (206 mmol l(-1) final concentration) or KCl (16 mmol l(-1) final concentration) stimulated a less potent activation, maximised (by approximately eightfold and fourfold) within 2 min and 30 s, respectively, relative to control values. The effect of all three compounds examined was reversible, since the kinase phosphorylation levels decreased upon reperfusion of the heart with normal bicarbonate-buffered saline. Conversely, hypotonicity did not induce any p38-MAPK activation. Furthermore, both hypothermia and hyperthermia induced considerable phosphorylation of the kinase, by four- and 7.5-fold, respectively, relative to control values. Immunohistochemical studies elucidated the localisation pattern of phospho-p38-MAPK and also revealed enhanced atrial natriuretic peptide (ANP) immunoreactivity in osmotically stressed hearts. Interestingly, SB 203580 (1 micromol l(-1)) not only completely blocked the activation of p38-MAPK by all these interventions, but also abolished the enhanced ANP immunoreactivity induced by 0.5 mol l(-1) sorbitol. These findings indicate the possible involvement of ANP in the mechanisms regulating responses under such stressful conditions.
Abstract: The aim of the present study was to examine and compare the role of the stress-activated protein kinases in ischemic and stretch-induced preconditioning. A model of anesthetized rabbits was used, and the preconditioning protocol included one or three cycles of short ischemia/reperfusion, or short mechanical stretch with acute pressure overload without or with the addition of the stretch blocker gadolinium. Infarct size was determined after 2h reperfusion and p38 MAPK and JNKs phosphorylation was determined after 20 min of prolonged ischemia. Preconditioning stimuli were equally effective in reducing the infarct size (14.2+/-3.4%, 12.9+/-3.0%, 15.9+/-3.3%, P<0.01 vs control). The addition of the stretch channel blocker gadolinium abrogated the effect of stretch preconditioning only, without any effect on ischemic preconditioning. Comparing p38-MAPK and p46/p54 JNKs phosphorylation in the ischemic and non-ischemic regions of the heart at the time of sustained ischemia, activation was observed in the ischemic or mechanically preconditioned groups compared with the control. The addition of gadolinium abolished this activation. The above results indicate that the phosphorylation of p38-MAPK and p46/p54 JNKs is increased in preconditioning but this effect can be dissociated from the protective effect of ischemic preconditioning. Activation of the stress-activated protein kinases may be related to the increased contracture, a characteristic of ischemic preconditioning.
Abstract: The aim of this study was to characterize alpha(1)-adrenergic receptors in frog heart and to examine their related signal transduction pathway. alpha(1)-Adrenergic binding sites were studied in purified heart membranes using the specific alpha(1)-adrenergic antagonist [(3)H]prazosin. Analysis of the binding data indicated one class of binding sites displaying a K(d) of 4.19 +/- 0.56 nM and a B(max) of 14.66 +/- 1.61 fmol/mg original wet weight. Adrenaline, noradrenaline, or phenylephrine, in the presence of propranolol, competed with [(3)H]prazosin binding with a similar potency and a K(i) value of about 10 microM. The kinetics of adrenaline binding was closely related to its biological effect. Adrenaline concentration dependently increased the production of inositol phosphates in the heart in the presence or absence of propranolol. Maximal stimulation was about 8.5-fold, and the half-maximum effective concentration was 30 and 21 microM in the absence and presence of propranolol, respectively. These data clearly show that alpha(1)-adrenergic receptors are coupled to the phosphoinositide hydrolysis in frog heart. To our knowledge, this is the first direct evidence supporting the presence of functional alpha(1)-adrenergic receptors in the frog heart.
Abstract: The role of PKC isoforms in the protection of ischemic preconditioning remains controversial. The aim of the present study was to compare PKC translocation in ischemic and pharmacological preconditioning and to test the hypothesis that induction of the preconditioned state results in a sustained translocation of PKC during the following ischemic period. Isolated rat cardiac myocytes were subjected to established pre-conditioning protocols using either transient ischemia or alpha(1)-adrenergic stimulation. Translocation of PKC isoforms, -alpha, -delta and -epsilon to the particulate fraction during induction of preconditioning, post incubation or final sustained ischemia was assessed by immunoblotting. Ischemia alone caused the translocation of PKC-alpha and -epsilon from the soluble to the particulate fraction. All three PKC isoforms examined were translocated to the particulate fraction in response to stimulation with alpha(1)-adrenergic agonists or phorbol esters. Ischemic preconditioning resulted in the translocation of only the PKC-epsilon isoform while pharmacological precondi-tion-ing did not affect any of the isoforms. During the following sustained ischemic period, increased percentage of PKC-alpha and -epsilon isoforms associated with the particulate fraction was observed only for the pharmacologically preconditioned cells. It is concluded that PKC translocation during preconditioning or the following ischemic period is not essential for the mediation of protection of rat cardiomyocytes in vitro.
Abstract: We investigated the activation of p38-MAPK by various adrenergic agents in the perfused Rana ridibunda heart. Phenylephrine (50 micromol l(-1)) rapidly induced the differential activation of all three mitogen-activated protein kinase (MAPK) subfamilies (ERK, JNKs and p38-MAPK) in this experimental system. Focusing on p38-MAPK response to phenylephrine, we found that the kinase phosphorylation reached maximal values at 30 s, declining thereafter to basal values at 15 min. p38-MAPK activation by phenylephrine was verified as exclusively alpha(1)-AR-mediated. Furthermore, SB203580 (1 micromol l(-1)) abolished the kinase phosphorylation by phenylephrine. Isoproterenol (50 micromol l(-1)) was also shown to activate p38-MAPK in a time- and temperature-dependent manner. A marked, sustained p38-MAPK activation profile was observed at 25 degrees C, while at 18 degrees C the kinase response to isoproterenol was modest. Isoproterenol effect on p38-MAPK stimulation was beta-AR-mediated. Immunohistochemical studies revealed the enhanced presence of phosphorylated p38-MAPK and atrial natriuretic peptide (ANP) in both phenylephrine- and isoproterenol-stimulated hearts, a reaction completely blocked by the respective specific antagonists, or the specific p38-MAPK inhibitor SB203580. These findings indicate a functional correlation between p38-MAPK activation and ANP accumulation in the perfused amphibian heart.
Abstract: We have used the alpha(1D)-adrenoceptor selective antagonist, BMY 7378, to investigate the presence of alpha(1D)-adrenoceptor subtype in adult rat heart by radioligand binding assays. We also determined the role of this subtype in stimulating phosphoinositide (PI) hydrolysis in adult rat cardiac myocytes. BMY 7378 inhibited [(3)H]prazosin binding to cardiac membranes in a biphasic mode with a pK(i) of 9.19 +/- 0.26 for high affinity sites and 6.64 +/- 0.09 for low affinity sites. The inhibition of the adrenaline-induced stimulation of PI hydrolysis by BMY 7378 fitted a one-site model and the calculated pK(b) value (6.92 +/- 0.28) was consistent with the involvement of alpha(1A) and alpha(1B) adrenoceptors. In addition, BMY 7378, at concentrations up to 100 nM, did not significantly affect the concentration-response curves for the adrenaline-induced stimulation of PI hydrolysis. Taken together, these data suggest that alpha(1D)-adrenoceptors are expressed in adult rat heart but this subtype is not involved in the adrenaline-induced stimulation of PI hydrolysis.
Abstract: OBJECTIVES: There is controversy concerning the beneficial effects of ischaemic preconditioning during short periods of ischaemia (stunning). The aim of the study was to investigate post-ischaemic myocardial performance after various periods of ischaemia in both non-preconditioned and preconditioned hearts and to compare these results with infarct volume estimation. METHODS: Isolated perfused rat hearts were subjected to various periods of sustained ischaemia (15, 20, 30, and 45 min). Haemodynamic parameters, infarct size and lactate dehydrogenase (LDH) leakage were recorded in both preconditioned and non-preconditioned hearts. RESULTS: After 15 min of ischaemia, preconditioned hearts revealed significantly lower developed pressure than non-preconditioned hearts (80+/-4.1 vs. 95+/-0.3%, P=0.02). In the 20 min ischaemia group, preconditioning resulted in non-significantly lower developed pressure (76+/-3.1% in preconditioned hearts vs. 87+/-5.3% in non-preconditioned hearts, P=0.11). In these groups infarct volume was small and not different between non-preconditioned and preconditioned hearts. After 30 min of ischaemia, preconditioning significantly improved developed pressure (66+/-3.1% in preconditioned and 44+/-5% in non-preconditioned hearts, P=0.002). LDH leakage was significantly higher in non-preconditioned hearts compared with preconditioned hearts (16+/-2.3 vs. 9.0+/-1.3, P=0.04), whereas infarct volume was not (12.5+/-0.8 and 9.8+/-1.5, respectively, P=0.1). Non-preconditioned hearts of this group, subjected to inotropic stimulation at the end of reperfusion, responded poorly. Significantly higher developed pressure was attained by preconditioned hearts (150+/-3.1 vs. 123+/-7.5%, P=0.01). After 45 min of ischaemia, preconditioning resulted in 69% limitation of infarct volume (P<0.0001) and 53% reduction in LDH release (P=0.009). Developed pressure was 57+/-8.5% in preconditioned hearts and 32+/-4.5% in non-preconditioned hearts (P=0.02). CONCLUSIONS: When ischaemic insult results in minimally lethal injuries, preconditioned hearts do not have the advantage of not being prone to stunning rather than non-preconditioned. If ischaemic insult is potentially able to produce extensive infarction, improvement in post-ischaemic myocardial function is mainly due to infarct size limitation evoked by preconditioning.
Abstract: We investigated the expression and activation of three MAPK subfamilies in the isolated perfused amphibian heart. ERK was detected as a 43 kDa band; p38-MAPK was detected as a band corresponding to 38 kDa and JNKs were detected as two bands corresponding to 46 and 52 kDa, respectively. PMA induced the activation of the ERK pathway as assessed by determining the phosphorylation state of ERK and the upstream component MEK1/2. PD98059 abolished this activation. p38-MAPK was phosphorylated by sorbitol (almost 12-fold, maximal within 10-15 min) and JNKs were phosphorylated and activated by sorbitol or anoxia/reoxygenation (approximately 4- and 2.5-fold, respectively). SB203580 completely blocked the activation of p38-MAPK by sorbitol. These results indicate that the MAPK pathways activated by phorbol esters, hyperosmotic stress or anoxia/ reoxygenation in the amphibian heart may have an important role in this experimental system.
Abstract: We studied the effects of increased Ca(2+) influx on alpha(1)-adrenoceptor-stimulated InsP formation in adult rat cardiac myocytes. We further examined if such effects could be mediated through a specific alpha(1)-adrenoceptor subtype. [(3)H]InsP responses to adrenaline were dependent on extracellular Ca(2+) concentration, from 0.1 microM to 2 mM, and were completely blocked by Ca(2+) removal. However, in cardiac myocytes preloaded with BAPTA, a highly selective calcium chelating agent, Ca(2+) concentrations higher than 1 microM had no effect on adrenaline-stimulated [(3)H]InsP formation. Taken together these results suggest that [(3)H]InsP formation induced by alpha(1)-adrenergic stimulation is in part mediated by increased Ca(2+) influx. Consistent with this, ionomycin, a calcium ionophore, stimulated [(3)H]InsP formation. This response was additive with the response to adrenaline stimulation implying that different signaling mechanisms may be involved. In cardiac myocytes treated with the alpha(1B)-adrenoceptor alkylating agent, CEC, [(3)H]InsP formation remained unaffected by increased Ca(2+) concentrations, a pattern similar to that observed when intracellular Ca(2+) was chelated with BAPTA. In contrast, addition of the alpha(1A)-subtype antagonist, 5'-methyl urapidil, did not affect the Ca(2+) dependence of [(3)H]InsP formation. Neither nifedipine, a voltage-dependent Ca(2+) channel blocker nor the inorganic Ca(2+) channel blockers, Ni(2+) and Co(2+), had any effect on adrenaline stimulated [(3)H]InsP, at concentrations that inhibit Ca(2+) channels. The results suggest that in adult rat cardiac myocytes, in addition to G protein-mediated response, alpha(1)-adrenergic-stimulated [(3)H]InsP formation is activated by increased Ca(2+) influx mediated by the alpha(1B)-subtype.
Abstract: The mitogen-activated protein kinase (MAPK) signal transduction pathway activated by mechanical stress was investigated in the isolated perfused amphibian (Rana ridibunda) heart. High perfusion pressure induced the rapid (30 s) and prolonged (30 min) phosphorylation of a p43-extracellular regulated kinase, a response almost completely inhibited by 25 microM PD-98059. c-Jun NH2-terminal kinase (JNK) was also phosphorylated with maximal values attained at 15 min and remained elevated over 30 min. In-gel kinase assays verified that phosphorylated JNKs are active, phosphorylating the transcription factor c-Jun. Furthermore, pressure overload rapidly stimulated the p38-MAPK phosphorylation (30 s), a transient process (5 min) abolished by 1 microM SB-203580. In-gel kinase assays revealed that with phosphorylation, active p38-MAPKs phosphorylate their substrate MAP kinase-activated protein kinase 2. Biochemical analysis along with immunohistochemical studies showed that with activation, the three MAPK subfamily members examined are localized not only in the cytoplasm but in the nucleus as well. Present results therefore demonstrate for the first time in an amphibian species the involvement of multiple MAPK pathways in the mechanical overload-induced adaptive responses of the heart as well as their possible physiological roles.
Abstract: 1. The effect of calcium paradox on oxidative status and the activity of anti-oxidant enzymes were studied in the rat isolated heart. Glutathione status, sulphydryl group contents and lipid peroxidation in the myocardium, as well as the release of oxidized and reduced glutathione from the heart, were taken as indices of oxidative events. 2. Reperfusion with calcium after calcium-free perfusion induced a significant decrease in the myocardial content of reduced and oxidized glutathione and non-protein sulphydryl groups. At the same time, a significant release of both forms of glutathione from the heart was observed. However, the ratio of oxidized to reduced glutathione remained unchanged and was not different from control. Increased lipid peroxidation was observed only after 30 min of reperfusion with calcium. 3. Increased anti-oxidant activity during the reperfusion period was observed. Mitochondrial Mn-superoxide dismutase (SOD) activity was increased throughout the reperfusion period, while cytoplasmic Cu,Zn-SOD and glutathione peroxidase activity showed a transient increase at 5 min reperfusion. 4. The results do not support an important role of oxygen free radicals in cell damage observed during calcium paradox in the rat isolated heart. Production of oxygen free radicals may occur during the reperfusion period, but the quantity produced is insufficient to exceed the anti-oxidant capacity of the heart.
Abstract: We investigated the ability of phenylephrine (PE), an alpha-adrenergic agonist and promoter of hypertrophic growth in the ventricular myocyte, to activate the three best-characterized mitogen-activated protein kinase (MAPK) subfamilies, namely p38-MAPKs, SAPKs/JNKs (i.e. stress-activated protein kinases/c-Jun N-terminal kinases) and ERKs (extracellularly responsive kinases), in perfused contracting rat hearts. Perfusion of hearts with 100 microM PE caused a rapid (maximal at 10 min) 12-fold activation of two p38-MAPK isoforms, as measured by subsequent phosphorylation of a p38-MAPK substrate, recombinant MAPK-activated protein kinase 2 (MAPKAPK2). This activation coincided with phosphorylation of p38-MAPK. Endogenous MAPKAPK2 was activated 4-5-fold in these perfusions and this was inhibited completely by the p38-MAPK inhibitor, SB203580 (10 microM). Activation of p38-MAPK and MAPKAPK2 was also detected in non-contracting hearts perfused with PE, indicating that the effects were not dependent on the positive inotropic/chronotropic properties of the agonist. Although SAPKs/JNKs were also rapidly activated, the activation (2-3-fold) was less than that of p38-MAPK. The ERKs were activated by perfusion with PE and the activation was at least 50% of that seen with 1 microM PMA, the most powerful activator of the ERKs yet identified in cardiac myocytes. These results indicate that, in addition to the ERKs, two MAPK subfamilies, whose activation is more usually associated with cellular stresses, are activated by the Gq/11-protein-coupled receptor (Gq/11PCR) agonist, PE, in whole hearts. These data indicate that Gq/11PCR agonists activate multiple MAPK signalling pathways in the heart, all of which may contribute to the overall response (e.g. the development of the hypertrophic phenotype).
Abstract: Recent studies have demonstrated enhanced myocardial protection during ischemia using the oxygen free radical scavenger, deferoxamine. This effect of deferoxamine may be related either to its iron-chelating property or to intervention in an iron-independent mechanism. We tested the latter by determining the rate of superoxide anion production and the degree of lipid peroxidation in human myocardial tissue after including deferoxamine in cardioplegic solution. Fourteen patients who underwent aortic, mitral, or double valve replacement were included in the study. The mean value for superoxide radical production was 59.8 +/- 17.0 nmol.min-1.g-1 for the control group (group C; n = 7) and 21.3 +/- 8.1 (p < 0.001) for the deferoxamine-treated group (group D; n = 7). The mean value for thiobarbituric reactive substances was 80.00 +/- 23.4 in group C and 38.7 +/- 23.8 nmol.min-1.g-1 in group D (p < 0.01). In conclusion, deferoxamine appears to have a moderating effect on the biochemical markers of ischemia reperfusion injury. Its scavenging effect on superoxide anion could play a role in the cellular defense against oxygen radicals during cardiac operations.
Abstract: The expression of protein kinase C (PKC) isoforms (PKC-alpha, PKC-beta 1, PKC-delta, PKC-epsilon, and PKC-zeta) was studied by immunoblotting in whole ventricles of rat hearts during postnatal development (1-26 days) and in the adult. PKC-alpha, PKC-beta 1, PKC-delta, PKC-epsilon, and PKC-zeta were detected in ventricles of 1-day-old rats, although PKC-alpha and PKC-beta 1 were only barely detectable. All isoforms were rapidly downregulated during development, with abundances relative to total protein declining in the adult to < 25% of 1-day-old values. PKC-beta 1 was not detectable in adult ventricles. The specific activity of PKC was also downregulated. The rat ventricular myocyte becomes amitotic soon after birth but continues to grow, increasing its protein content 40- to 50-fold between the neonate and the 300-g adult. An important question is thus whether the amount of PKC per myocyte is downregulated. With the use of isolated cells, immunoblotting showed that the contents per myocyte of PKC-alpha and PKC-epsilon increased approximately 10-fold between the neonatal and adult stages. In rat ventricles, the rank of association with the particulate fraction was PKC-delta > PKC-epsilon > PKC-zeta. Association of these isoforms with the particulate fraction was less in the adult than in the neonate. In primary cultures of ventricular myocytes prepared from neonatal rat hearts, 1 microM 12-O-tetradecanoylphorbol-13-acetate (TPA) elicited translocation of PKC-alpha, PKC-delta, and PKC-epsilon from the soluble to the particulate fraction in < 1 min, after which time no further translocation was observed. Prolonged exposure (16 h) of myocytes to 1 microM TPA caused essentially complete downregulation of these isoforms, although downregulation of PKC-epsilon was slower than for PKC-delta. In contrast, PKC-zeta was neither translocated nor downregulated by 1 microM TPA. Immunoblotting of human ventricular samples also revealed downregulation of PKC relative to total protein during fetal/postnatal development.
Abstract: The regulation of mitogen-activated protein kinase (MAPK) and MAPK kinase (MEK) was studied in freshly isolated adult rat heart preparations. In contrast to the situation in ventricular myocytes cultured from neonatal rat hearts, stimulation of MAPK activity by 1 mumol/L phorbol 12-myristate 13-acetate (PMA) was not consistently detectable in crude extracts. After fast protein liquid chromatography, MAPK isoforms p42MAPK and p44MAPK and two peaks of MEK were shown to be activated > 10-fold in perfused hearts or ventricular myocytes exposed to 1 mumol/L PMA for 5 minutes. The identities of MAPK or MEK were confirmed by immunoblotting and, for MAPK, by the "in-gel" myelin basic protein phosphorylation assay. In retrogradely perfused hearts, high coronary perfusion pressure (120 mm Hg for 5 minutes), norepinephrine (50 mumol/L for 5 minutes), or isoproterenol (50 mumol/L for 5 minutes) stimulated MAPK and MEK approximately 2- to 5-fold. In isolated myocytes, endothelin 1 (100 nmol/L for 5 minutes) also stimulated MAPK, but stimulation by norepinephrine or isoproterenol was difficult to detect. Immunoblotting showed that the relative abundances of MAPK and MEK protein in ventricles declined to < 20% of their postpartal abundances after 50 days. This may explain the difficulties encountered in assaying the activity of MAPK in crude extracts from adult hearts. We conclude that potentially hypertrophic agonists and interventions stimulate the MAPK cascade in adult rats and suggest that the MAPK cascade may be an important intracellular signaling pathway in this response.
Abstract: 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.
Abstract: The coupling of the pharmacologically defined alpha 1A- and alpha 1B-adrenoceptors to the hydrolysis of phospho[3H]inositides (PI) was investigated in ventricular myocytes freshly isolated from adult rat hearts. The alpha 1-adrenoceptor population in the heart was characterized by competitive binding experiments using [3H]prazosin and the alpha 1A-adrenoceptor-selective antagonist 5-methyl urapidil. It was heterogeneous with approximately 25% being pharmacologically of the alpha 1A-adrenoceptor subtype and 75% being of the alpha 1B-adrenoceptor subtype. Epinephrine, norepinephrine, or phenylephrine stimulated PI hydrolysis in the presence or absence of propranolol. The greatest stimulation (7-fold) was with epinephrine. The half-maximum effective concentrations for agonists were approximately 0.5-3.5 and 0.2 microM in the absence and presence of propranolol, respectively. The inhibition by 5-methyl urapidil of the stimulation of PI hydrolysis by a fixed concentration of epinephrine fitted a two-site competition curve. The distribution between high-affinity (25%) and low-affinity (75%) sites suggested that both the alpha 1A- and alpha 1B-adrenoceptors were coupled to PI hydrolysis in proportion to their relative abundance. Equally, the stimulation of PI hydrolysis by epinephrine in the presence of a fixed concentration of 5-methyl urapidil was biphasic. In addition, chloroethylclonidine, an irreversible inhibitor of the alpha 1B-adrenoceptor, inhibited the epinephrine stimulation of PI hydrolysis by 35%. We conclude that the pharmacologically defined alpha 1A- and alpha 1B-adrenoceptor subtypes are both coupled to PI hydrolysis in the ventricular myocyte.
Abstract: The plasma membrane protein pattern of Rana ridibunda embryos subjected to lithium (Li) treatment at various stages of development was examined by two-dimensional gel electrophoresis. Differences were observed at the neurula stage not only as compared to controls but among lithium-treated embryos as well. Of particular interest was the presence of proteins, specific for the gastrula stage, in lithium-treated embryos. The results are discussed in relation to the well-known effect of lithium on amphibian morphogenesis.
Abstract: Plasma membranes of fully-grown oocytes and early developmental stage embryos of Rana ridibunda were isolated by differential and density gradient centrifugation; they were purified 18-fold as indicated by 5'-nucleotidase. The plasma membrane protein pattern of five different developmental stages was studied by two-dimensional polyacrylamide gel electrophoresis. More than 60 protein species could be detected by silver staining. Most of them are largely conserved during development. The rest either show precise stage specificity or exhibit stronger staining intensity at particular stages. The number of proteins specific for each stage is small, neurula being exempted. The changes observed in the plasma membrane profile during development are mostly prominent in a group of proteins with similar molecular weights (40-45 kDa) but with different pI values. The differences observed in the plasma membrane patterns are discussed in relation to the significance of each stage during development.
Abstract: The effect of adenosine on the calcium paradox in the isolated frog heart was studied. Addition of adenosine during calcium depletion protected the frog heart against a calcium paradox. This protective effect was indicated by reduced protein and creatine kinase release, maintenance of electrical activity, and recovery of mechanical activity during reperfusion. Tissue calcium determination results showed that adenosine protected frog myocardial cells by reducing the massive calcium influx during reperfusion possibly through an action on calcium channels. Adenosine exerted its action in a dose-dependent manner; a concentration of 10 microM adenosine provided maximum protection of myocardial cells against the calcium paradox damage. Higher concentrations of adenosine produced side effects on both electrical and mechanical activity. These results are discussed in terms of the possible mechanism involved in the protective effect of adenosine.
Abstract: A method for eliminating artifactual bands due to the presence of 2-mercaptoethanol in two-dimensional gels is described. The method is based on a modification of the procedure of application of the first dimension gel to the SDS slab gel. 2-Mercaptoethanol is removed during equilibration and replaced by iodoacetamide. The use of iodoacetamide improves the recovery of proteins and results in a better detection of them.
Abstract: The effects of adenosine were examined on the isolated perfused heart of the frog Rana ridibunda. Adenosine produced negative chronotropic and inotropic effects on frog ventricle in a concentration-dependent manner. The effects of adenosine on cardiac metabolism were also investigated by measuring the tissue content of adenine nucleotides, lactate, pyruvate, adenosine and inorganic phosphate, during adenosine perfusion. Adenosine had no effect on the tissue content of metabolites. No net synthesis of adenine nucleotides was observed during perfusion with increasing concentrations of adenosine. Lactate output from the heart decreased significantly with adenosine perfusion. Correlation of adenosine effects on cardiac muscle with the effects of hypoxia are discussed.
Abstract: Activities of four lysosomal proteolytic enzymes, namely cathepsins B, D, H and L are 1.8 to 2.8 times greater in atria of 360 g rats compared with ventricles. Activities of acid phosphatase and beta-acetylglucosaminidase (both lysosomal enzymes) are not significantly different in atria and ventricles. Catheptic activities thus reflect the greater protein turnover rate in atria.
