Differential response of cardiac aquaporins to hyperosmotic stress; salutary role of AQP1 against the induced apoptosis.

OBJECTIVE: Multiple pathophysiological conditions are associated with disturbance of myocardial osmotic equilibrium, exerting detrimental effects on cardiac performance. Cardiac myocytes may encounter hyperosmotic stress during hyperglycemia, ischemia/reperfusion injury, myocardial infarction, diabetes mellitus, severe dehydration, hypoxia or heat stress. Aquaporins (AQPs) constitute transmembrane channels that facilitate water transport in response to osmotic gradients. Therefore, the present study aimed at probing into AQPs mode of response and potential role as effector molecules and sensors, under hyperosmotic stress. MATERIALS AND METHODS: H9c2 cardiac myoblasts were left untreated (control) or were exposed to 0.5 M sorbitol so as to induce hyperosmotic stress conditions. After the experimental treatments, MTT assay was performed to assess cell viability. Endogenous mRNA levels of AQP1 and AQP7 were assessed by ratiometric RT-PCR. Their subcellular localization pattern was revealed by immunofluorescence microscopy. Protein levels of AQP1 and AQP7, as well as of apoptotic markers (cleaved caspase-3 and PARP), were detected by immunoblot analysis. RESULTS: Hyperosmotic stress (0.5 M sorbitol) induced a time-dependent upregulation of AQP7 (but not of AQP1) mRNA in H9c2 cells. Of note, biochemical and immunocytochemical analyses revealed the increased membrane-associated protein expression of AQP1, under these conditions, while AQP7 respective levels remained unchanged. Interestingly, inhibition of AQP1 by HgCl2, aggravated the sorbitol-induced apoptosis in H9c2 cells, as evidenced by chromatin condensation and fragmentation of caspase-3 and PARP. CONCLUSIONS: AQP1 and AQP7 are differentially regulated under hyperosmotic stress conditions in H9c2 cells. AQP1, acting as an osmotic stress sensor and response factor, exerts a beneficial effect against the sorbitol-induced apoptosis, potentially favoring preservation of cardiac function.

ERKs and JNKs mediate hydrogen peroxide-induced Egr-1 expression and nuclear accumulation in H9c2 cells.

One of the most significant insults that jeopardize cardiomyocyte homeostasis is a surge of reactive oxygen species (ROS) in the failing myocardium. Early growth response factor-1 (Egr-1) has been found to act as a transcriptional regulator in multiple biological processes known to exert deleterious effects on cardiomyocytes. We thus investigated the signaling pathways involved in its regulation by H2O2. Egr-1 mRNA levels were found to be maximally induced after 2 h in H2O2-treated H9c2 cells. Egr-1 respective response at the protein level, was found to be maximally induced after 2 h of treatment with 200 microM H2O2, remaining elevated for 6 h, and declining thereafter. H2O2-induced upregulation of Egr-1 mRNA and protein levels was ablated in the presence of agents inhibiting ERKs pathway (PD98059) and JNKs (SP600125, AS601245). Immunofluorescent experiments revealed H2O2-induced Egr-1 nuclear sequestration to be also ERK- and JNK-dependent. Overall, our results show for the first time the fundamental role of ERKs and JNKs in regulating Egr-1 response to H2O2 treatment in cardiac cells at multiple levels: mRNA, protein and subcellular distribution. Nevertheless, further studies are required to elucidate the specific physiological role of Egr-1 regarding the modulation of gene expression and determination of cell fate.

Hyperthermia-induced Hsp70 and MT20 transcriptional upregulation are mediated by p38-MAPK and JNKs in Mytilus galloprovincialis (Lamarck); a pro-survival response.

In the present study we investigated the signal transduction cascades triggered by acute thermal stress in Mytilus galloprovincialis gills. This particular species has been reported to exhibit a significant tolerance to high temperatures; thus, it was intriguing to examine the molecular mechanisms responsible for this extraordinary trait. In particular, exposure to 30 degrees C was found to cause a significant and sustained stimulation of p38-MAPK phosphorylation while the activation profile of JNKs was transient and relatively moderate. We also observed that hyperthermia induced apoptosis as a delayed response, with both MAPK subfamilies rapidly translocating to the nucleus. The phosphorylation of cJun, ATF2 and NFkappaB was detected next. Using selective inhibitors, phosphorylation of these transcription factors was established to be dependent on p38-MAPK or JNKs. Subsequently, potential changes in gene expression were assessed. In this context, hyperthermia resulted in the transcriptional upregulation of Hsp70 and MT20 genes with a widely known salutary effect, preserving mussel fitness and performance under adverse environmental conditions. Interestingly, p38-MAPK and JNKs were found to mediate the hyperthermia-induced Hsp70 and MT20 upregulation as well as the delayed induction of apoptosis under the interventions studied. Overall this is, to our knowledge, the first time that an insight into the compensatory survival ;programme' initiated in Mytilus galloprovincialis gills, contributing to this organism's exceptional tolerance to thermal stress, has been gained. In particular, we provide evidence demonstrating the principal role of p38-MAPK and JNKs in transducing the stress signal via mobilization of specific transcription factors and the transcriptional upregulation of cytoprotective genes.

Dopamine mimics the cardioprotective effect of ischemic preconditioning via activation of alpha1-adrenoceptors in the isolated rat heart.

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.

Hyperosmotically induced accumulation of a phosphorylated p38-like MAPK involved in protoplast volume regulation of plasmolyzed wheat root cells.

A 46 kDa protein resembling immunochemically to the mammalian dually phosphorylated p38-MAPK was detected in wheat root cells under hyperosmotic conditions, using Western blot analysis. This protein accumulated in a time- and dose-dependent fashion and exhibited pharmacological sensitivity similar to the activated p38-MAPK. The application of a highly specific p38-MAPK inhibitor revealed that the p38-like MAPK is probably implicated in hyperosmotically induced tubulin cytoskeleton reorganization as well as in protoplast volume regulation and osmotic tolerance of wheat root cells. As far as we know, the p38-MAPK has not been previously reported in higher plants.

Various divalent cations protect the isolated perfused pigeon heart against a calcium paradox.

The protective effects of various divalent cations against the irreversible damage of myocardium, a phenomenon termed "the Ca2+-paradox", were examined in the isolated perfused pigeon heart. All cations examined were added at a concentration of 200 micromol l(-1) in the "calcium-free" medium. In hearts perfused with low calcium, upon normal calcium repletion, the maximal recovery of the contractile tension (in the 2nd minute) was approximately 115% and the recovery obtained at the end of reperfusion was 81.5% (compared to the equilibration period value). From the other divalent cations examined, the presence of cobalt, nickel, manganese or barium during calcium depletion powerfully protected the pigeon heart. Upon calcium repletion, the maximal recovery of contractile tension was approximately 60%, 76.5%, 100% and 85%, the recovery estimated at the end of reperfusion was 40%, 12%, 70% and 53%, and the resting tension estimated at the end of reperfusion was 2.69+/-0.18 g, 6.40+/-0.50 g, 1.20+/-0.10 g and 1.90+/-0.10 g for cobalt, nickel, manganese and barium, respectively. On the contrary, strontium exerted no protective effects. The protective effects were also indicated by reduced total protein and lactate dehydrogenase activity release into the effluent perfusate and maintenance of electrical activity. The effectiveness of the added divalent cations (with the exception of strontium) showed a strong dependence upon their ionic radius. The most potent inhibitors of this phenomenon in the pigeon heart were the divalent cations having an ionic radius closer to the ionic radius of calcium. These results are discussed in terms of the possible mechanisms involved in the protective effects of these cations.

Dissociation of stress-activated protein kinase (p38-MAPK and JNKs) phosphorylation from the protective effect of preconditioning in vivo.

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.

Novel potent inhibitors of lipid peroxidation with protective effects against reperfusion arrhythmias.

A series of new compounds that contain lipoic acid and trolox connected through spacers were synthesized and examined for their antioxidant activity and their protective effects against reperfusion arrhythmias in isolated heart preparations. All compounds tested are strong inhibitors of lipid peroxidation in rat liver microsomal membranes induced by ferrous ions and ascorbate. N-(3,4-Dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-carbonyl)-N'-(1,2-dithiolane-3-pentanoyl)-1,2-phenylenediamine (13) exhibits anti-lipid peroxidation activity at the nanomolar range. N-(3,4-Dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-carbonyl)-N'-(1,2-dithiolane-3-pentanoyl)ethylenediamine (10) and 13 totally suppressed reperfusion arrhythmias.

Stimulation of multiple MAPK pathways by mechanical overload in the perfused amphibian heart.

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.

Activation of multiple MAPK pathways (ERKs, JNKs, p38-MAPK) by diverse stimuli in the amphibian heart.

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.

alpha(1D)-Adrenoceptors do not contribute to phosphoinositide hydrolysis in adult rat cardiac myocytes.

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.

Degradation of myofibrillar proteins by a calpain-like proteinase in the arm muscle of Octopus vulgaris.

The effects of a calpain-like proteinase (CaDP) isolated from the arm muscle of Octopus vulgaris on the myofibrils and myofibrillar proteins isolated from the same tissue were examined. Our studies clearly showed that treatment of intact myofibrils with CaDP in the presence of 5 mM Ca2+ results in the degradation of the major myofibrillar proteins myosin, paramyosin, and actin. From the isolated alpha- and beta-paramyosins only beta-paramyosin is degraded by CaDP in the presence of 5 mM Ca2+ producing three groups of polypeptides of 80, 75, and 60 kDa, respectively. The degradation rate depends on the proteinase to substrate ratio, temperature, and time of proteolysis and is inhibited by the endogenous CaDP inhibitory factor (CIF), as well as by various known cysteine proteinase inhibitors (E-64, leupeptin, and antipain). From the other myofibrillar proteins examined myosin, but not actin, is degraded by CaDP; myosin heavy chain (MHC, 200 kDa) is degraded by CaDP producing four groups of polypeptides of lower molecular masses (155, 125, 115, and 102 kDa, respectively); the degradation rate depends on the incubation time and the proteinase to substrate ratio. Furthermore, CaDP undergoes limited autolysis in the presence of both the exogenous casein and the endogenous beta-paramyosin producing two large active fragments of 52 and 50.6 kDa, respectively; CIF reversibly inhibits this CaDP autolysis.

Studies on the autolysis of m-calpain from the skeletal muscle of the amphibian Rana ridibunda.

The autolytic mechanisms responsible for the regulation of m-calpain purified from the skeletal muscle of the amphibian Rana ridibunda were examined. Both subunits of the calpain molecule were found to undergo autolysis in the presence of Ca2+. Various divalent cations were examined for their ability to induce calpain autolysis. The concentrations of these cations required for the complete calpain autolysis were: 500 microM Ca2+, 800 microM Mn2+, 2 mM Sr2+, 10 mM Ba2+, whereas Mg2+, even at 10 mM did not induce any autolysis. Calpain autolysis induced by the above divalent cations is a temperature dependent process. Presence of Mn2+ or Sr2+ reduces the Ca2+ requirement of calpain for autolysis. The rate of autolysis depends on the protease concentration; protease inhibitors such as E-64, leupeptin, antipain, and iodoacetic acid inhibit the autolysis of calpain; E-64 inhibits irreversibly while leupeptin inhibits reversibly the autolysis; and irreversibly inactivated by E-64 calpain is fully digested by native calpain. Autolysis of calpain in the presence of alkali denatured casein increases the Ca2+ sensitivity of the protease for its half maximal and maximal caseinolytic activity. Limited autolysis of calpain is also induced in the presence of the endogenous substrate G-actin, and the rate of autolysis is slower than that obtained in the absence of substrates.

Purification and characterization of m-calpain from the skeletal muscle of the amphibian Rana ridibunda.

Calpain was purified to apparent homogeneity from the skeletal muscle of the amphibian Rana ridibunda. It is composed of two subunits of 78 and 28 kDa, respectively. The enzyme exhibits kinetic properties similar to those of mammalian and avian skeletal muscle m-calpains. Ca2+ requirements for half and maximum activities are 400 microM and 1.5 mM, respectively. It is strongly inhibited by thiol protease inhibitors such as leupeptin, E-64, and antipain and by alkylating thiol group agents such as iodoacetic acid (IAA), iodoacetamide (IAM), and N-ethylmaleimide (NEM). Its activity is enhanced by reduced thiols such as dithiothreitol (DTT), cysteine, and 2-mercaptoethanol. The enzyme is stable in the absence of Ca2+ at 55 degrees C, it displays maximum activity at 25 degrees C, and it shows a broad pH optimum between 6.5 and 7.8. In the absence of Ca2+, various divalent cations such as Sr2+, Mn2+, and Ba2+ strongly activate, while other divalent cations such as Ni2+, Co2+, Cd2+, Zn2+, and Cu2+ have no effect on its activity. In the presence of Ca2+, the cations Sr2+, Mn2+, and Ba2+ show a synergistic effect, while the cations of the other group strongly inhibit the calpain activity. The above data demonstrate that calpain from the skeletal muscle of the amphibian Rana ridibunda is a neutral, Ca(2+)-activated thiol protease and that it belongs to the class of m-calpains.

Acute alpha 1-adrenergic stimulation of cardiac protein synthesis may involve increased intracellular pH and protein kinase activity.

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.

Effects of catecholamines on protein synthesis in cardiac myocytes and perfused hearts isolated from adult rats. Stimulation of translation is mediated through the alpha 1-adrenoceptor.

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.

Stimulation of protein synthesis by raised extracellular pH in cardiac myocytes and perfused hearts.

Protein synthesis was stimulated in freshly-isolated rats cardiac myocytes by increasing the extracellular pH of Hepes-buffered Tyrode's solutions over the range pH 7.4-8.4. The maximal stimulation was about 45%. Protein synthesis in anterogradely-perfused rat hearts was stimulated by 11% by increasing the pH of the bicarbonate-containing perfusion medium from pH 7.4 to 7.8. This manoeuvre increased intracellular pH by 0.12 units. A concomitant increase in phosphocreatine concentration was observed. These findings are consistent with the hypothesis that intracellular pH may exert profound effects on tissue protein synthesis rates.

Pyruvate kinase isoenzymes in marine invertebrates: a comparative study by the use of monoclonal antibodies.

1. Six monoclonal antibodies specific to the pyruvate kinase from the foot muscle of the common limpet P. caerulea were produced. 2. They also exhibited specificity against the mouse liver where the L-type isoenzyme of pyruvate kinase is present. They did not react with the mouse skeletal muscle, heart or red blood cells isoenzymes of pyruvate kinase (PK). One of these, the monoclonal antibody B did not react with any PK isoenzymes of the mouse tissues. 3. The presence of the isoenzymic type of PK which was recognized by the monoclonals, (type L), was traced in five phyla of marine invertebrates by the application of the monoclonal antibodies A, B and C. 4. In two phyla the majority of the animals were found to possess an L-type PK isoenzyme in their muscles while in quite a few of them a different isoenzymic type was present in the other tissues. The results of this study are compared with the existing literature, and the use of monoclonal antibodies in the study of enzymic systems is considered in the discussion.

Effects of increasing extracellular pH on protein synthesis and protein degradation in the perfused working rat heart.

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.