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Biomedical subjects

D Mochly-Rosen

Publications and source records attributed to D Mochly-Rosen.

At least 19 recordsLinked to original sources
PubMed2005

DeltaPKC-mediated activation of epsilonPKC in ethanol-induced cardiac protection from ischemia.

Previous studies have demonstrated that acute ethanol exposure induces activation of delta protein kinase C (deltaPKC) and epsilonPKC, and mimics ischemic preconditioning via epsilonPKC activation. However, the role of deltaPKC isozyme in ischemia and reperfusion is still controversial. Here, we investigated the role of deltaPKC in ethanol-induced cardioprotection using a selective deltaPKC activator (psideltaRACK), or inhibitor (deltaV1-1), and a selective epsilonPKC inhibitor (epsilonV1-2) in isolated mouse hearts. Mice were injected intraperitoneally or by gavage with ethanol, regulators of delta and epsilonPKC or an adenosine A1 receptor blocker (DPCPX). Isolated perfused mouse hearts were subjected to a 30-min global ischemia and a 120-min reperfusion, ex vivo. Injection of 0.5 g/kg ethanol 1 h, but not 10 min, before ischemia reduced infarct size and CPK release. Pretreatment with epsilonV1-2 abolished this ethanol-induced cardioprotection. Pretreatment with deltaV1-1 induced cardioprotection when injected with ethanol (0.5 g/kg) 10 min before ischemia, but deltaV1-1 partly inhibited ethanol-induced cardioprotection when injected with ethanol 1-h before the onset of ischemia. psideltaRACK injection 1 h, but not 10 min, before ischemia induced cardioprotection and translocation of epsilonPKC from the cytosol to the particulate fraction. Pretreatment with DPCPX or epsilonV1-2 inhibited psideltaRACK-induced cardioprotection and translocation of epsilonPKC. Therefore, activation of deltaPKC-induced by ethanol or by the deltaPKC activator is cardioprotective, provided that sufficient time passes to allow deltaPKC-induced activation of epsilonPKC, an A1 adenosine receptor-dependent process.

Animals↗
PubMed2001

Adaptor proteins in protein kinase C-mediated signal transduction.

Spatial and temporal organization of signal transduction is essential in determining the speed and precision by which signaling events occur. Adaptor proteins are key to organizing signaling enzymes near their select substrates and away from others in order to optimize precision and speed of response. Here, we describe the role of adaptor proteins in determining the specific function of individual protein kinase C (PKC) isozymes. These isozyme-selective proteins were called collectively RACKs (receptors for activated C-kinase). The role of RACKs in PKC-mediated signaling was determined using isozyme-specific inhibitors and activators of the binding of each isozyme to its respective RACK. In addition to anchoring activated PKC isozymes, RACKs anchor other signaling enzymes. RACK1, the anchoring protein for activated betaIIPKC, binds for example, Src tyrosine kinase, integrin, and phosphodiesterase. RACK2, the epsilonPKC-specific RACK, is a coated-vesicle protein and thus is involved in vesicular release and cell-cell communication. Therefore, RACKs are not only adaptors for PKC, but also serve as adaptor proteins for several other signaling enzymes. Because at least some of the proteins that bind to RACKs, including PKC itself, regulate cell growth, modulating their interactions with RACKs may help elucidate signaling pathways leading to carcinogenesis and could result in the identification of novel therapeutic targets.

Animals↗
PubMed2001

Opposing cardioprotective actions and parallel hypertrophic effects of delta PKC and epsilon PKC.

Conflicting roles for protein kinase C (PKC) isozymes in cardiac disease have been reported. Here, deltaPKC-selective activator and inhibitor peptides were designed rationally, based on molecular modeling and structural homology analyses. Together with previously identified activator and inhibitor peptides of epsilonPKC, deltaPKC peptides were used to identify cardiac functions of these isozymes. In isolated cardiomyocytes, perfused hearts, and transgenic mice, deltaPKC and epsilonPKC had opposing actions on protection from ischemia-induced damage. Specifically, activation of epsilonPKC caused cardioprotection whereas activation of deltaPKC increased damage induced by ischemia in vitro and in vivo. In contrast, deltaPKC and epsilonPKC caused identical nonpathological cardiac hypertrophy; activation of either isozyme caused nonpathological hypertrophy of the heart. These results demonstrate that two related PKC isozymes have both parallel and opposing effects in the heart, indicating the danger in the use of therapeutics with nonselective isozyme inhibitors and activators. Moreover, reduction in cardiac damage caused by ischemia by perfusion of selective regulator peptides of PKC through the coronary arteries constitutes a major step toward developing a therapeutic agent for acute cardiac ischemia.

Amino Acid Sequence↗
PubMed2001

Binding specificity for RACK1 resides in the V5 region of beta II protein kinase C.

Identification of selective anchoring proteins responsible for specialized localization of specific signaling proteins has led to the identification of new inhibitors of signal transduction, inhibitors of anchoring protein-ligand interactions. RACK1, the first receptor for activated C kinase identified in our lab, is a selective anchoring protein for betaII protein kinase C (betaIIPKC). We previously found that at least part of the RACK1-binding site resides in the C2 domain of betaIIPKC (Ron, D., Luo, J., and Mochly-Rosen, D. (1995) J. Biol. Chem. 270, 24180-24187). Here we show that the V5 domain also contains part of the RACK1-binding site in betaIIPKC. In neonatal rat cardiac myocytes, the betaIIV5-3 peptide (amino acids 645-650 in betaIIPKC) selectively inhibited phorbol 12-myristate 13-acetate (PMA)-induced translocation of betaIIPKC and not betaIPKC. In addition, the betaIIV5-3 peptide inhibited cardiac myocyte hypertrophy in PMA-treated cells. Interestingly, betaIV5-3 (646-651 in betaIPKC), a selective translocation inhibitor of betaIPKC, also inhibited PMA-induced cardiac myocyte hypertrophy, demonstrating that both betaI- and betaIIPKC are essential for this cardiac function. Therefore, the betaIIV5 domain contains part of the RACK1-binding site in betaIIPKC; a peptide corresponding to this site is a selective inhibitor of betaIIPKC and, hence, enables the identification of betaIIPKC-selective functions.

Amino Acid Sequence↗
PubMed2001

Opposing effects of delta and xi PKC in ethanol-induced cardioprotection.

Low amounts of ethanol reduce cardiac damage induced by ischemia. The protection from ischemic damage by acute exposure to low amounts of ethanol in isolated myocytes and intact heart have been attributed to activation of protein kinase C (PKC). We previously found that two PKC isozymes, delta and xi, are activated by ethanol in several cell models. Here, we perfused isozyme-selective agonist and antagonist peptides that we have generated into intact heart to determine the role of these two isozymes in ethanol-induced protection from transient ischemia. Whereas xi PKC activation was required for ethanol-induced protection, delta PKC activation led to further damage. These data explain the conflicting reports on the role of acute exposure to ethanol in protection from cardiac ischemia. The clinical implications of these findings are also discussed.

Animals↗
PubMed2001

Localization, anchoring, and functions of protein kinase C isozymes in the heart.

Although protein kinase C (PKC) was identified more than 20 years ago, and is involved in a wide variety of essential cellular processes, assigning specific roles to each PKC isozyme has proved difficult. Results over the last few years have suggested that much of the specificity of activated PKC isozymes is attributed to their subcellular localization bringing them into close proximity to a subset of substrates. Our laboratory has taken advantage of the importance of PKC localization and studied the way in which PKC isozymes are anchored. We have identified PKC anchoring proteins (RACKs or Receptors for Activated C Kinase) and used information about interaction sites between PKC isozymes and their respective RACKs to design peptides which modulate translocation of specific PKC isozymes to the functional site. These isozyme-specific peptides can be delivered into isolated or cultured cells or expressed in transgenic mice to determine the role of specific PKC isozymes in particular functions. Here we will describe the isozymes-specific peptide activators and inhibitors that we have developed and the specific functions of each isozyme in cardiac ventricular tissue.

Animals↗
PubMed2001

Arachidonic acid protects neonatal rat cardiac myocytes from ischaemic injury through epsilon protein kinase C.

OBJECTIVES: Arachidonic acid is a second messenger which activates protein kinase C (PKC) and is released from the heart during ischaemic preconditioning. The purpose of this study was to examine the effect of arachidonic acid on activation of PKC in cardiac myocytes and the cellular consequences. METHODS: Neonatal rat cardiac myocytes were isolated and maintained in culture. Arachidonic acid-induced activation of PKC was examined by cell fractionation and western blot analysis. Contraction frequency was measured by visual inspection under a microscope. Ischaemia was simulated by subjecting cells to an atmosphere of lower than 0.5% oxygen in the absence of glucose and cell damage determined by release of cytosolic lactate dehydrogenase or direct cell viability assay. RESULTS: Arachidonic acid resulted in translocation of delta and epsilonPKC but not alpha, betaII, eta or zetaPKC isozymes, indicating activation of only delta and epsilonPKC. Arachidonic acid induced a dose-dependent decrease in spontaneous contraction rate of cardiac myocytes which was blocked by a selective peptide translocation inhibitor of epsilonPKC. Pretreatment with arachidonic acid partially protected cardiac myocytes against ischaemia. Down-regulation of PKC with 24 h 4beta-phorbol,12-myristate,13-acetate treatment, inhibition of PKC by chelerythrine and selective inhibition of epsilonPKC translocation all decreased the protective effect of arachidonic acid. Pretreatment with eicosapentaenoic acid or oleic acid also protected cardiac myocytes against ischaemia. CONCLUSIONS: These results demonstrate that arachidonic acid selectively activates delta and epsilonPKC in neonatal rat cardiac myocytes, leading to protection from ischaemia. We suggest this is a potential mechanism of PKC activation during PC. In addition, our results suggest that different classes of free fatty acid directly exert cardioprotection from ischaemic injury in cardiac myocytes.

Animals↗
PubMed2001

Molecular transporters for peptides: delivery of a cardioprotective epsilonPKC agonist peptide into cells and intact ischemic heart using a transport system, R(7).

BACKGROUND: Recently, we reported a novel oligoguanidine transporter system, polyarginine (R(7)), which, when conjugated to spectroscopic probes (e.g., fluorescein) and drugs (e.g., cyclosporin A), results in highly water-soluble conjugates that rapidly enter cells and tissues. We report herein the preparation of the first R(7) peptide conjugates and a study of their cellular and organ uptake and functional activity. The octapeptide (psi)(epsilon)RACK was selected for this study as it is known to exhibit selective epsilon protein kinase C isozyme agonist activity and to reduce ischemia-induced damage in cardiomyocytes. However, (psi)(epsilon)RACK is not cell-permeable. RESULTS: Here we show that an R(7)-(psi)(epsilon)RACK conjugate readily enters cardiomyocytes, significantly outperforming (psi)(epsilon)RACK conjugates of the transporters derived from HIV Tat and from Antennapedia. Moreover, R(7)-(psi)(epsilon)RACK conjugate reduced ischemic damage when delivered into intact hearts either prior to or after the ischemic insult. CONCLUSIONS: Our data suggest that R(7) converts a peptide lead into a potential therapeutic agent for the ischemic heart.

Animals↗
PubMed2001

Evidence for functional role of epsilonPKC isozyme in the regulation of cardiac Na(+) channels.

Investigation of the role of individual protein kinase C (PKC) isozymes in the regulation of Na(+) channels has been largely limited by the lack of isozyme-selective modulators. Here we used a novel peptide-specific activator (epsilonV1-7) of epsilonPKC and other peptide isozyme-specific inhibitors in addition to the general PKC activator phorbol 12-myristate 13-acetate (PMA) to dissect the role of individual PKCs in the regulation of the human cardiac Na(+) channel hH1, heterologously expressed in Xenopus oocytes. Peptides were injected individually or in combination into the oocyte. Whole cell Na(+) current (I(Na)) was recorded using two-electrode voltage clamp. epsilonV1-7 (100 nM) and PMA (100 nM) inhibited I(Na) by 31 +/- 5% and 44 +/- 8% (at -20 mV), respectively. These effects were not seen with the scrambled peptide for epsilonV1-7 (100 nM) or the PMA analog 4alpha-phorbol 12,13-didecanoate (100 nM). However, epsilonV1-7- and PMA-induced I(Na) inhibition was abolished by epsilonV1-2, a peptide-specific antagonist of epsilonPKC. Furthermore, PMA-induced I(Na) inhibition was not altered by 100 nM peptide-specific inhibitors for alpha-, beta-, delta-, or etaPKC. PMA and epsilonV1-7 induced translocation of epsilonPKC from soluble to particulate fraction in Xenopus oocytes. This translocation was antagonized by epsilonV1-2. In native rat ventricular myocytes, PMA and epsilonV1-7 also inhibited I(Na); this inhibition was antagonized by epsilonV1-2. In conclusion, the results provide evidence for selective regulation of cardiac Na(+) channels by epsilonPKC isozyme.

Animals↗
PubMed2000

Chronic hypersensitivity for inflammatory nociceptor sensitization mediated by the epsilon isozyme of protein kinase C.

We have identified a mechanism, mediated by the epsilon isozyme of protein kinase C (PKCepsilon) in peripheral neurons, which may have a role in chronic inflammatory pain. Acute inflammation, produced by carrageenan injection in the rat hindpaw, produced mechanical hyperalgesia that resolved by 72 hr. However, for up to 3 weeks after carrageenan, injection of the inflammatory mediators prostaglandin E(2) or 5-hydroxytryptamine or of an adenosine A(2) agonist into the same site induced a markedly prolonged hyperalgesia (>24 hr compared with 5 hr or less in control rats not pretreated with carrageenan). A nonselective inhibitor of several PKC isozymes and a selective PKCepsilon inhibitor antagonized this prolonged hyperalgesic response equally. Acute carrageenan hyperalgesia could be inhibited by PKA or PKG antagonists. However, these antagonists did not inhibit development of the hypersensitivity to inflammatory mediators. Our findings indicate that different second messenger pathways underlie acute and prolonged inflammatory pain.

Adenosine↗
PubMed2000

Cardiotrophic effects of protein kinase C epsilon: analysis by in vivo modulation of PKCepsilon translocation.

Protein kinase C (PKC) is a key mediator of many diverse physiological and pathological responses. Although little is known about the specific in vivo roles of the various cardiac PKC isozymes, activation-induced translocation of PKC is believed to be the primary determinant of isozyme-specific functions. Recently, we have identified a catalytically inactive peptide translocation inhibitor (epsilonV1) and translocation activator (psiepsilonRACK [receptors for activated C kinase]) specifically targeting PKCepsilon. Using cardiomyocyte-specific transgenic expression of these peptides, we combined loss- and gain-of-function approaches to elucidate the in vivo consequences of myocardial PKCepsilon signaling. As expected for a PKCepsilon RACK binding peptide, confocal microscopy showed that epsilonV1 decorated cross-striated elements and intercalated disks of cardiac myocytes. Inhibition of cardiomyocyte PKCepsilon by epsilonV1 at lower expression levels upregulated alpha-skeletal actin gene expression, increased cardiomyocyte cell size, and modestly impaired left ventricular fractional shortening. At high expression levels, epsilonV1 caused a lethal dilated cardiomyopathy. In contrast, enhancement of PKCepsilon translocation with psiepsilonRACK resulted in selectively increased beta myosin heavy chain gene expression and normally functioning concentric ventricular remodeling with decreased cardiomyocyte size. These results identify for the first time a role for PKCepsilon signaling in normal postnatal maturational myocardial development and suggest the potential for PKCepsilon activators to stimulate "physiological" cardiomyocyte growth.

Actins↗
PubMed2000

Involvement of a p38 mitogen-activated protein kinase phosphatase in protecting neonatal rat cardiac myocytes from ischemia.

Our recent results showed that extended p38 mitogen-activated protein kinase (p38) activation during ischemia leads to cell death, at least partly through apoptosis, in neonatal rat cardiomyocytes. However, other studies have shown that p38 activation during a short preconditioning treatment protects cardiomyocytes from ischemic cell death. This suggests that the duration of p38 activation determines its cellular function and therefore inactivation of p38 by phosphatases may play an important role. In neonatal rat cardiomyocytes, we used the tyrosine phosphatase inhibitor, vanadate, to prevent p38 inactivation, thus extending the strength and length of p38 activation during ischemia. This resulted in higher susceptibility to cell death from ischemia in a dose-dependent manner and over time; the additional damage induced by vanadate was inhibited by SB203580, a selective inhibitor of p38. We conclude that a tyrosine phosphatase is inactivated during ischemia, resulting in prolonged p38 activation which causes cell death.

Animals↗
PubMed2000

Pharmacological regulation of network kinetics by protein kinase C localization.

Protein kinase C (PKC) is a conserved family of 11 serine/threonine kinases. Most cell types express multiple members of the family. Because the catalytic sites are homologous, and able to accommodate a broad range of substrates in vitro, specificity in function is dependent on subcellular localization of each isozyme in each cell type. Physiological stimulation can result in major changes in localization of individual PKC isozymes, mediated through binding to specific anchoring proteins. We describe data demonstrating that disruption of such translocations of PKC isozymes by pharmacological agents, peptides, or antibodies, causes profound effects on T cell functions. The pharmacological opportunity provided by distinct kinetic properties of complex assembly is also discussed.

Animals↗
PubMed2000

Evidence for functional role of epsilonPKC isozyme in the regulation of cardiac Ca(2+) channels.

Limited information is available regarding the effects of protein kinase C (PKC) isozyme(s) in the regulation of L-type Ca(2+) channels due to lack of isozyme-selective modulators. To dissect the role of individual PKC isozymes in the regulation of cardiac Ca(2+) channels, we used the recently developed novel peptide activator of the epsilonPKC, epsilonV1-7, to assess the role of epsilonPKC in the modulation of L-type Ca(2+) current (I(Ca,L)). Whole cell I(Ca,L) was recorded using patch-clamp technique from rat ventricular myocytes. Intracellular application of epsilonV1-7 (0.1 microM) resulted in a significant inhibition of I(Ca,L) by 27.9 +/- 2.2% (P < 0.01, n = 8) in a voltage-independent manner. The inhibitory effect of epsilonV1-7 on I(Ca,L) was completely prevented by the peptide inhibitor of epsilonPKC, epsilonV1-2 [5.2 +/- 1.7%, not significant (NS), n = 5] but not by the peptide inhibitors of cPKC, alphaC2-4 (31.3 +/- 2.9%, P < 0.01, n = 6) or betaC2-2 plus betaC2-4 (26.1 +/- 2.9%, P < 0.01, n = 5). In addition, the use of a general inhibitor (GF-109203X, 10 microM) of the catalytic activity of PKC also prevented the inhibitory effect of epsilonV1-7 on I(Ca,L) (7.5 +/- 2.1%, NS, n = 6). In conclusion, we show that selective activation of epsilonPKC inhibits the L-type Ca channel in the heart.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed2000

Protein kinase C isozymes and the regulation of diverse cell responses.

Individual protein kinase C (PKC) isozymes have been implicated in many cellular responses important in lung health and disease, including permeability, contraction, migration, hypertrophy, proliferation, apoptosis, and secretion. New ideas on mechanisms that regulate PKC activity, including the identification of a novel PKC kinase, 3-phosphoinositide-dependent kinase-1 (PDK-1), that regulates phosphorylation of PKC, have been advanced. The importance of targeted translocation of PKC and isozyme-specific binding proteins (like receptors for activated C-kinase and caveolins) is well established. Phosphorylation state and localization are now thought to be key determinants of isozyme activity and specificity. New concepts on the role of individual PKC isozymes in proliferation and apoptosis are emerging. Opposing roles for selected isozymes in the same cell system have been defined. Coupling to the Wnt signaling pathway has been described. Phenotypes for PKC knockout mice have recently been reported. More specific approaches for studying PKC isozymes and their role in cell responses have been developed. Strengths and weaknesses of different experimental strategies are reviewed. Future directions for investigation are identified.

Animals↗
PubMed1999

Cardioprotection from ischemia by a brief exposure to physiological levels of ethanol: role of epsilon protein kinase C.

Recent epidemiological studies indicate beneficial effects of moderate ethanol consumption in ischemic heart disease. Most studies, however, focus on the effect of long-term consumption of ethanol. In this study, we determined whether brief exposure to ethanol immediately before ischemia also produces cardioprotection. In addition, because protein kinase C (PKC) has been shown to mediate protection of the heart from ischemia, we determined the role of specific PKC isozymes in ethanol-induced protection. We demonstrated that (i) brief exposure of isolated adult rat cardiac myocytes to 10-50 mM ethanol protected against damage induced by prolonged ischemia; (ii) an isozyme-selective epsilonPKC inhibitor developed in our laboratory inhibited the cardioprotective effect of acute ethanol exposure; (iii) protection of isolated intact adult rat heart also occurred after incubation with 10 mM ethanol 20 min before global ischemia; and (iv) ethanol-induced cardioprotection depended on PKC activation because it was blocked by chelerythrine and GF109203X, two PKC inhibitors. Consumption of 1-2 alcoholic beverages in humans leads to blood alcohol levels of approximately 10 mM. Therefore, our work demonstrates that exposure to physiologically attainable ethanol levels minutes before ischemia provides cardioprotection that is mediated by direct activation of epsilonPKC in the cardiac myocytes. The potential clinical implications of our findings are discussed.

Adhesins, Bacterial↗
PubMed1999

Sustained in vivo cardiac protection by a rationally designed peptide that causes epsilon protein kinase C translocation.

Brief periods of cardiac ischemia trigger protection from subsequent prolonged ischemia (preconditioning). epsilon Protein kinase C (epsilonPKC) has been suggested to mediate preconditioning. Here, we describe an epsilonPKC-selective agonist octapeptide, psiepsilon receptor for activated C-kinase (psiepsilonRACK), derived from an epsilonPKC sequence homologous to its anchoring protein, epsilonRACK. Introduction of psiepsilonRACK into isolated cardiomyocytes, or its postnatal expression as a transgene in mouse hearts, increased epsilonPKC translocation and caused cardio-protection from ischemia without any deleterious effects. Our data demonstrate that epsilonPKC activation is required for protection from ischemic insult and suggest that small molecules that mimic this epsilonPKC agonist octapeptide provide a powerful therapeutic approach to protect hearts at risk for ischemia.

Amino Acid Sequence↗