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A M Samarel

Publications and source records attributed to A M Samarel.

At least 19 recordsLinked to original sources
PubMed2001

Differential activation of mitogen-activated protein kinase cascades and apoptosis by protein kinase C epsilon and delta in neonatal rat ventricular myocytes.

Protein kinase C (PKC) epsilon and PKCdelta translocation in neonatal rat ventricular myocytes (NRVMs) is accompanied by subsequent activation of the ERK, JNK, and p38(MAPK) cascades; however, it is not known if either or both novel PKCs are necessary for their downstream activation. Use of PKC inhibitors to answer this question is complicated by a lack of isoenzyme specificity, and the fact that many PKC inhibitors stimulate JNK and p38(MAPK) activity. Therefore, replication-defective adenoviruses (Advs) encoding constitutively active (ca) mutants of PKCepsilon and PKCdelta were used to test if either or both of these PKCs are sufficient to activate ERKs, JNKs, and/or p38(MAPK) in NRVMs. Adv-caPKCepsilon infection (1 to 25 multiplicities of viral infection (MOI); 4 to 48 hours) increased total PKCepsilon levels in a time- and dose-dependent manner, with maximal expression observed 8 hours after Adv infection. Adv-caPKCepsilon induced a time- and dose-dependent increase in phosphorylated p42 and p44 ERKs, as compared with a control Adv encoding beta-galactosidase (Adv-nebetagal). Maximal ERK phosphorylation occurred 8 hours after Adv infection. In contrast, JNK was only minimally activated, and p38(MAPK) was relatively unaffected. Adv-caPKCdelta infection (1 to 25 MOI, 4 to 48 hours) increased total PKCdelta levels in a similar fashion. Adv-caPKCdelta (5 MOI) induced a 29-fold increase in phosphorylated p54 JNK, and a 15-fold increase in phosphorylated p38(MAPK) 24 hours after Adv infection. In contrast, p42 and p44 ERK were only minimally activated. Whereas neither Adv induced NRVM hypertrophy, Adv-caPKCdelta, but not Adv-caPKCepsilon, induced NRVM apoptosis. We conclude that the novel PKCs differentially regulate MAPK cascades and apoptosis in an isoenzyme-specific and time-dependent manner.

Adenoviridae↗
PubMed2001

Pyk2 expression and phosphorylation in neonatal and adult cardiomyocytes.

A. L. Bayer, A. G. Ferguson, P. A. Lucchesi and A. M. Samarel. PYK2 Expression and Phosphorylation in Neonatal and Adult Cardiomyocytes. Journal of Molecular and Cellular Cardiology (2001) 33, 1017-1030. Proline-rich tyrosine kinase (PYK2) is a Ca(2+)-dependent, non-receptor protein tyrosine kinase involved in growth factor signaling. Although PYK2 is expressed in a variety of tissues, it has not yet been identified in cardiac muscle. Therefore, immunocytochemical and Western blotting techniques were used to examine PYK2 expression and phosphorylation in neonatal and adult rat ventricular cardiomyocytes (NRVM and ARVM, respectively). PYK2 concentration was much greater in neonatal, than in adult ventricular tissue and cardiomyocytes. In cultured cells, PYK2 expression was highly dependent on [Ca(2+)](i)transients and contractile activity. Non-contracting, low-density NRVM in serum-free culture expressed very low levels of PYK2, while high-density, spontaneously contracting NRVM showed a approximately 12-fold increase in PYK2 expression. Conversely, high-density NRVM treated with nifedipine (10 microM, 48 h) to block spontaneous [Ca(2+)](i)transients and contractile activity resulted in a 2.6-fold decrease in PYK2 levels. Similarly, overnight culture of quiescent ARVM markedly reduced PYK2 levels. Chronic treatment (48 h) of cultured NRVM with the hypertrophic agonist endothelin-1 (ET) (10-300 n M) did not significantly increase PYK2 levels, but strongly shifted the ratio of phosphorylated to total PYK2, indicating that PYK2 phosphorylation accompanies cardiomyocyte hypertrophy. Endothelin-1 also acutely activated PYK2 in both cultured NRVM, and in freshly isolated ARVM. These results suggest that PYK2 is involved in the generation of certain aspects of cardiomyocyte hypertrophy.

Animals↗
PubMed2001

Combined antiretroviral therapy causes cardiomyopathy and elevates plasma lactate in transgenic AIDS mice.

Highly active antiretroviral therapy (HAART) is implicated in cardiomyopathy (CM) and in elevated plasma lactate (LA) in AIDS through mechanisms of mitochondrial dysfunction. To determine mitochondrial events from HAART in vivo, 8-week-old hemizygous transgenic AIDS mice (NL4-3Delta gag/pol; TG) and wild-type FVB/n littermates were treated with the HAART combination of zidovudine, lamivudine, and indinavir or vehicle control for 10 days or 35 days. At termination of the experiments, mice underwent echocardiography, quantitation of abundance of molecular markers of CM (ventricular mRNA encoding atrial natriuretic factor [ANF] and sarcoplasmic calcium ATPase [SERCA2]), and determination of plasma LA. Myocardial histologic features were analyzed semiquantitatively and results were confirmed by transmission electron microscopy. After 35 days in the TG + HAART cohort, left ventricular mass increased 160% by echocardiography. Molecularly, ANF mRNA increased 250% and SERCA2 mRNA decreased 57%. Biochemically, LA was elevated (8.5 +/- 2.0 mM). Pathologically, granular cytoplasmic changes were found in cardiac myocytes, indicating enlarged, damaged mitochondria. Findings were confirmed ultrastructurally. No changes were found in other cohorts. After 10 days, only ANF was elevated, and only in the TG + HAART cohort. Results show that cumulative HAART caused mitochondrial CM with elevated LA in AIDS transgenic mice.

Acquired Immunodeficiency Syndrome↗
PubMed2001

Brief rapid pacing depresses contractile function via Ca(2+)/PKC-dependent signaling in cat ventricular myocytes.

The purpose of this study is to determine the effects of brief rapid pacing (RP; approximately 200-240 beats/min for approximately 5 min) on contractile function in ventricular myocytes. RP was followed by a sustained inhibition of peak systolic cell shortening (-44 +/- 4%) that was not due to changes in diastolic cell length, membrane voltage, or L-type Ca(2+) current (I(Ca,L)). During RP, baseline and peak intracellular Ca(2+) concentration ([Ca(2+)](i)) increased markedly. After RP, Ca(2+) transients were similar to control. The effects of RP on cell shortening were not prevented by 1 microM calpain inhibitor I, 25 microM L-N(5)-(1-iminoethyl)-orthinthine, or 100 microM N(G)-monomethyl-L-arginine. However, RP-induced inhibition of cell shortening was prevented by lowering extracellular [Ca(2+)] (0.5 mM) during RP or exposure to chelerythrine (2-4 microM), a protein kinase C (PKC) inhibitor, or LY379196 (30 nM), a selective inhibitor of PKC-beta. Exposure to phorbol ester (200 nM phorbol 12-myristate 13-acetate) inhibited cell shortening (-46 +/- 7%). Western blots indicated that cat myocytes express PKC-alpha, -delta, and -epsilon as well as PKC-beta. These findings suggest that brief RP of ventricular myocytes depresses contractility at the myofilament level via Ca(2+)/PKC-dependent signaling. These findings may provide insight into the mechanisms of contractile dysfunction that follow paroxysmal tachyarrhythmias.

Action Potentials↗
PubMed2001

Role of protein kinase C-epsilon in hypertrophy of cultured neonatal rat ventricular myocytes.

Using adenovirus (Adv)-mediated overexpression of constitutively active (ca) and dominant-negative (dn) mutants, we examined whether protein kinase C (PKC)-epsilon, the major novel PKC isoenzyme expressed in the adult heart, was necessary and/or sufficient to induce specific aspects of the hypertrophic phenotype in low-density, neonatal rat ventricular myocytes (NRVM) in serum-free culture. Adv-caPKC-epsilon did not increase cell surface area or the total protein-to-DNA ratio. However, cell shape was markedly affected, as evidenced by a 67% increase in the cell length-to-width ratio and a 17% increase in the perimeter-to-area ratio. Adv-caPKC-epsilon also increased atrial natriuretic factor (ANF) and beta-myosin heavy chain (MHC) mRNA levels 2.5 +/- 0.3- and 2.1 +/- 0.2-fold, respectively, compared with NRVM infected with an empty, parent vector (P < 0.05 for both). Conversely, Adv-dnPKC-epsilon did not block endothelin-induced increases in cell surface area, the total protein-to-DNA ratio, or upregulation of beta-MHC and ANF gene expression. However, the dominant-negative inhibitor markedly suppressed endothelin-induced extracellular signal-regulated kinase (ERK) 1/2 activation. Taken together, these results indicate that caPKC-epsilon overexpression alters cell geometry, producing cellular elongation and remodeling without a significant, overall increase in cell surface area or total protein accumulation. Furthermore, PKC-epsilon activation and downstream signaling via the ERK cascade may not be necessary for cell growth, protein accumulation, and gene expression changes induced by endothelin.

Adenoviridae↗
PubMed2000

Focal adhesion kinase is involved in angiotensin II-mediated protein synthesis in cultured vascular smooth muscle cells.

The rate of vascular smooth muscle cell protein synthesis and cellular hypertrophy in response to angiotensin II (Ang II) is dependent on activation of protein tyrosine kinases (PTKs) and both the extracellular signal-regulated kinase (ERK) 1/2 and p70(S6K) pathways. One potential PTK that may regulate these signaling cascades is focal adhesion kinase (FAK), a nonreceptor PTK associated with focal adhesions. We used an actin depolymerizing agent, cytochalasin D (Cyt-D), and a replication-defective adenovirus encoding FAK-related nonkinase (FRNK), an inhibitor of FAK-dependent signaling, as tools to assess whether FAK was upstream of the ERK1/2 and/or the p70(S6K) pathways. Cyt-D reduced basal FAK phosphorylation and blocked Ang II-dependent FAK phosphorylation in a dose-dependent manner. Confocal microscopy indicated that Cyt-D induced actin filament disruption and FAK delocalization from focal adhesions. Cyt-D also reduced Ang II-induced ERK1/2 activation, but p70(S6K) activation was relatively unaffected. Cyt-D reduced basal protein synthetic rate and substantially reduced the Ang II-induced increase in protein synthesis. Similarly, FRNK overexpression blocked Ang II-induced FAK phosphorylation and ERK1/2 activation, but not p70(S6K) phosphorylation, and markedly inhibited protein synthesis. This is the first report to demonstrate that FAK is a critical component of the signal transduction pathways that mediate Ang II-induced ERK1/2 activation, c-fos induction, and enhanced protein synthesis in vascular smooth muscle cells.

Actins↗
PubMed2000

Laminin binding to beta1-integrins selectively alters beta1- and beta2-adrenoceptor signalling in cat atrial myocytes.

1. Perforated patch recordings were used to determine how plating atrial cells on laminin alters beta-adrenergic receptor (beta-AR) regulation of L-type Ca2+ current (ICa,L). 2. Isoproterenol (isoprenaline; ISO; 0.01 microM), a non-selective beta-AR agonist, elicited a greater stimulation of ICa,L in cells plated on laminin (+79 +/- 16 %; n = 17) than on glass (+33 +/- 5 %; n = 23). Also, desensitization to ISO was greater in cells on laminin (-16 +/- 2 %) than on glass (-3 +/- 1 %). Atenolol (0.1 microM), a selective beta1-AR antagonist, inhibited the effects of ISO in cells on glass but not laminin. Conversely, 0.1 microM ICI 118,551, a selective beta2-AR antagonist, inhibited the effects of ISO in cells on laminin but not glass. With beta2-ARs blocked, ISO-induced stimulation of ICa,L was greater in cells on glass than laminin. 3. Zinterol (0.01-0.1 microM), a selective beta2-AR agonist, elicited a greater stimulation of ICa,L in cells on laminin than on glass. The effects of zinterol were blocked by ICI 118,551. 4. ISO-induced stimulation of ICa,L was greater in cells plated on an alphabeta1-integrin antibody than on glass. Also, addition of 20 microM cytochalasin D to cells on laminin prevented the enhanced effects of ISO typically elicited in cells on laminin alone. 5. We conclude that laminin binding to alphabeta1-integrins, in conjunction with the actin cytoskeleton, reduces beta1-AR and enhances beta2-AR signalling which regulates ICa,L. This novel mechanism may contribute to remodelling of beta-AR signalling in the failing heart.

Actins↗
PubMed2000

Laminin acts via beta 1 integrin signalling to alter cholinergic regulation of L-type Ca(2+) current in cat atrial myocytes.

A perforated patch recording method was used to determine how plating cells on laminin (20 microg ml(-1); >2 h) alters cholinergic regulation of L-type Ca(2+) current (I(Ca,L)) in atrial myocytes. Acetylcholine (ACh; 1 microm)-induced inhibition of basal I(Ca,L) was not different between cells on glass and laminin. However, stimulation of I(Ca,L) elicited by ACh withdrawal was significantly smaller in cells on laminin (10 +/- 2 %) than on glass (48 +/- 5 %) (P < 0.001). Stimulation of I(Ca,L) induced by either spermine-NO (200 microm), milrinone (10 microm), IBMX (100 microm) or forskolin (1 microm) was significantly smaller in cells plated on laminin than on glass. However, stimulation of I(Ca,L) by 100 microm 8-CPT-cAMP or intracellular dialysis with 50 microM cAMP was not different between cells plated on laminin or glass. Basal, forskolin- and IBMX-stimulated cAMP content was significantly smaller in cells plated on laminin than on glass. Stimulation of I(Ca,L) by ACh withdrawal was significantly smaller in cells plated on an alpha beta 1-integrin antibody (10 +/- 4 %) than on glass (3 +/- 6 %; P < 0.001). In cells on laminin, prior exposure to 100 microg ml-1 YIGSR, a laminin receptor-binding peptide, restored ACh-induced stimulation of I(Ca,L) (58 +/- 14 %)laminin alone (7 +/- 2 %; P < 0. 05). Addition of 20 microm cytochalasin D or 1 microM latrunculin A, agents that prevent actin polymerization, to cells on laminin restored ACh-induced stimulation of I(Ca,L). We conclude that laminin binding to beta 1 integrins acts in association with the actin-based cytoskeleton to attenuate adenylate cyclase activity. As a result, laminin inhibits NO-mediated stimulation of I(Ca,L) elicited by ACh withdrawal. Laminin-integrin signalling may be relevant to changes in autonomic regulation that occur during cardiac development and/or disease.

1-Methyl-3-isobutylxanthine↗
PubMed2000

Isoenzyme-specific protein kinase C and c-Jun N-terminal kinase activation by electrically stimulated contraction of neonatal rat ventricular myocytes.

Previous studies from our laboratory and others indicate that contraction-induced mechanical loading of cultured neonatal rat ventricular myocytes produces many of the phenotypic changes associated with cardiomyocyte hypertrophy in vivo, and that these changes occur via the activation of serine-threonine protein kinases. These may include the extracellular regulated protein kinases (ERK1 and ERK2), the c-Jun N-terminal kinases (JNK1, JNK2, and JNK3), and one or more isoenzymes of protein kinase C. In this study, we assessed whether one or more of these kinases are activated by stimulated contraction, and whether activation was isoenzyme-specific. Low-density, quiescent cultures of neonatal rat ventricular myocytes were maintained in serum-free medium, or electrically stimulated to contract (3 Hz) for up to 48 h. ERK and JNK activation was assessed by Western blotting with polyclonal antibodies specific for the phosphorylated forms of both kinases. PKC activation was analysed by subcellular fractionation, detergent extraction, and Western blotting using isoenzyme-specific monoclonal antibodies. Stimulated contractile activity produced myocyte hypertrophy, as indicated by increased cell size, a 15+/-5% increase in total protein/DNA ratio, and induction of ANF and beta MHC gene transcription. Electrical pacing did not cause ERK1/2 or JNK1 activation, but increased JNK2 and JNK3 phosphorylation by;two-fold. Subcellular fractionation revealed a time-dependent increase in PKC delta, and to a much lesser extent PKC xi, in a Triton X-100-soluble membrane fraction within 5 min of the onset of stimulated contraction. PKC alpha was not activated by electrical pacing. These results indicate that contraction-induced mechanical loading acutely activates some but not all of the specific isoenzymes of JNKs and PKCs in cardiomyocytes.

Animals↗
PubMed2000

Cardiac dysfunction occurs in the HIV-1 transgenic mouse treated with zidovudine.

Cardiomyopathy in AIDS is an increasingly important clinical problem. Mechanisms of AIDS cardiomyopathy were explored using AIDS transgenic mice that express replication-incompetent HIV-1 (NL4-3delta gag/pol). Transgenic and FVB/n mice (n = 3 to 6 per cohort) received water ad libitum with and without zidovudine (3'-azido-2',3'-deoxythymidine; AZT; 0.7 mg/ml) for 21 or 35 days. After 21 days, echocardiographic studies were performed and abundance of mRNA for cardiac sarcoplasmic reticulum calcium ATPase (SERCA2), sodium calcium exchanger (NCX1), and atrial natriuretic factor were determined individually using Northern analysis of extracts of left ventricles. After 35 days, contractile function and relaxation were analyzed in isolated work-performing hearts. Histopathological and ultrastructural (transmission electron microscopy) changes were identified. After 21 days, molecular indicators of cardiac dysfunction were found. Depressed SERCA2 and increased atrial natriuretic factor mRNA abundance occurred in left ventricles from AZT-treated transgenic mice. NCX1 abundance was unchanged. Eccentric left ventricle hypertrophy was determined echocardiographically. After 35 days, cardiac dysfunction was worst in AZT-treated and AZT-untreated transgenic mice. Decreases in the first derivative of the maximal change in left ventricle systolic pressure with respect to time (+dP/dt) occurred in transgenic mice with and without AZT. Increased half-time of relaxation and ventricular relaxation (-dP/dt) occurred in AZT-treated and -untreated transgenic mice. Increased time to peak pressure was found only in AZT-treated transgenic mice. In AZT-treated FVB/n mice, -dP/dt was decreased. Ultrastructurally, mitochondrial destruction was most pronounced in AZT-treated transgenic mice, but also was found in AZT-treated FVB/n mice. Transgenic mice that express HIV-1 demonstrate cardiac dysfunction. AZT treatment of FVB/n mice causes mitochondrial ultrastructural alterations that are similar to those in other species. In transgenic mice, AZT treatment worsens molecular and ultrastructural features of cardiomyopathy. HIV-1 constructs and AZT each contribute to cardiac dysfunction in this murine model of AIDS cardiomyopathy.

Acquired Immunodeficiency Syndrome↗
PubMed2000

Endothelin-induced cardiac myocyte hypertrophy: role for focal adhesion kinase.

Endothelin-1 (ET) produces neonatal rat ventricular myocyte (NRVM) hypertrophy and activates focal adhesion kinase (FAK) in other cell types. In the present study, we examined whether ET activated FAK in NRVM and whether FAK was necessary and/or sufficient for ET-induced NRVM hypertrophy. Chronic ET-1 stimulation (100 nM, 48 h) increased protein-to-DNA and myosin heavy chain (MHC)-to-DNA ratios and stimulated the assembly of newly synthesized MHC into sarcomeres. ET-1 also induced the assembly of focal adhesions and costameres, as evidenced by increased phosphotyrosine, FAK, and paxillin immunostaining. Acutely, ET treatment rapidly increased tyrosine phosphorylation of FAK and paxillin. FAK was also activated by phorbol 12-myristate 13-acetate (2 microM, 5 min). Pretreatment with chelerythrine (5 microM) or rottlerin (10 microM) completely blocked ET-induced FAK phosphorylation, indicating that protein kinase C activation was upstream of ET-induced FAK activation. In contrast, ET-induced FAK activation was not affected by blocking calcium influx via L-type voltage-gated calcium channels. Adenoviruses (Adv) containing FAK and FAK-related nonkinase (FRNK) were used to specifically define the role of FAK in ET-induced hypertrophy. ET stimulation failed to increase total protein-to-DNA or MHC-to-DNA ratios or to stimulate sarcomeric assembly in myocytes infected with Adv-FRNK. However, Adv-FAK alone did not increase total protein-to-DNA or MHC-to-DNA ratios and failed to increase the number or size of myofibrils as evidenced by double immunofluorescence labeling for MHC and FAK. Thus, although FAK is necessary for ET-induced NRVM hypertrophy, other ET-generated signals are also required to elicit the hypertrophic phenotype.

Adenoviridae↗
PubMed1999

Upregulation of Na(+)/Ca(2+) exchanger expression and function in an arrhythmogenic rabbit model of heart failure.

Three-dimensional cardiac mapping in rabbits with nonischemic cardiomyopathy has shown that ventricular arrhythmias initiate by a nonreentrant mechanism that may be due to triggered activity from delayed afterdepolarizations. Delayed afterdepolarizations are thought to be due to spontaneous release of Ca(2+) from the sarcoplasmic reticulum (SR) and consequent activation of an inward Na(+)/Ca(2+) exchange (NaCaX) current. The goal of this study was to determine whether there is enhanced NaCaX gene expression and functional activity that may contribute to nonreentrant activation. Heart failure (HF) was induced in rabbits by combined aortic insufficiency and aortic constriction. HF rabbits had left ventricular enlargement (left ventricular end-diastolic dimension increased from 1.43+/-0.03 to 1.97+/-0.05 cm) and severely depressed function (fractional shortening reduced from 37% to 26%, P<0.02). Heart-to-body weight was increased by 79% in HF. Western blots showed a 93% increase in NaCaX protein in HF (P<0.04). NaCaX mRNA (7-kb transcript) was increased by 104% relative to the 18S rRNA in HF. A 14-kb NaCaX transcript was also seen in the HF rabbits, raising total NaCaX mRNA to 2.7-fold compared with controls. The amplitude of caffeine-induced contractures, used to assess SR Ca(2+) load, was not significantly different in HF. Relaxation and [Ca(2+)](i) decline during caffeine-induced contractures is attributable to Ca(2+) transport by NaCaX and was 61% and 45% faster in HF (P<0.05), respectively. NaCaX current measured under controlled voltage clamp conditions was also 2-fold higher in HF cells. SR Ca(2+)-ATPase mRNA and protein levels and Ca(2+) current density were not significantly altered in HF. Twitch amplitudes from HF myocytes were 26% smaller compared with control (P<0.02), but twitch relaxation and [Ca(2+)](i) decline (due largely to SR Ca(2+)-ATPase) were not altered. Thus myocytes and myocardium from HF rabbits exhibit enhanced NaCaX expression and function. The enhanced NaCaX activity may contribute to depressed contractions, increased transient inward current (for a given SR Ca(2+) release), delayed afterdepolarizations, and nonreentrant initiation of ventricular tachycardia in this arrhythmogenic model of HF.

Animals↗
PubMed1999

Sarcomeric myosin heavy chain is degraded by the proteasome.

Cardiac myofibrillar proteins, like all other intracellular proteins, are in a dynamic state of continual degradation and resynthesis. The balance between these opposing metabolic processes ultimately determines the number of functional contractile units within each cardiac muscle cell. Although alterations in myofibrillar protein degradation have been shown to contribute to cardiac growth and remodeling, the intracellular proteolytic systems responsible for degrading myofibrillar proteins to their constitutive amino acids are currently unknown. Lactacystin, a recently developed, highly specific proteasome inhibitor, was used in this study to examine the role of the proteasome in myosin heavy chain (MHC) degradation in cultured neonatal rat ventricular myocytes. Cells were treated with growth medium alone or with lactacystin (1-50 microM) for up to 48 h. Lactacystin significantly increased the total protein/DNA ratio and markedly prolonged MHC half-life. Other proteasome inhibitors, namely carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (10 microM) and N-acetyl-L-leucyl-L-leucyl-norleucinal (100 microM), were also effective in suppressing MHC degradation. Lactacystin and other proteasome inhibitors also suppressed the markedly accelerated MHC degradation associated with Ca2+ channel blockade but did not prevent the disassembly and loss of myofibrils that accompanied contractile arrest. Thus, sarcomere disassembly precedes the degradation of MHC, which is at least in part mediated by the proteasome.

Acetylcysteine↗
PubMed1998

Calcium- and protein kinase C-dependent activation of the tyrosine kinase PYK2 by angiotensin II in vascular smooth muscle.

Angiotensin II (Ang II) induces vascular smooth muscle cell (VSMC) growth by activating Gq-protein-coupled AT1 receptors, which leads to elevation of cytosolic Ca2+ ([Ca2+]i) and activation of protein kinase C (PKC) and mitogen-activated protein kinases. To assess the link between these Ang II-induced signaling events, we examined the effect of Ang II on the proline-rich tyrosine kinase (PYK2), previously found to be activated by a variety of stimuli that increase [Ca2+]i or activate PKC. PYK2 distribution was demonstrated in rat aortic tissue and in cultured VSMC by immunohistochemistry, revealing a cytosolic distribution distinct from smooth muscle alpha-actin, focal adhesion kinase, or paxillin. The involvement of PYK2 in Ang II signaling was measured by immunoprecipitation and immune complex kinase assays. Treatment of quiescent VSMC with Ang II resulted in a concentration- and time-dependent increase in PYK2 tyrosine phosphorylation and kinase activity in PYK2 immunoprecipitates. PYK2 phosphorylation was inhibited by AT1 receptor blockade and was attenuated by downregulation of PKC or the chelation of [Ca2+]i. Treatment with either phorbol ester or Ca2+ ionophore also increased PYK2 phosphorylation, suggesting that PKC activation and/or increased [Ca2+]i are both necessary and sufficient to activate PYK2. Activation of PYK2 by Ang II was also associated with increased PYK2-src complex formation, suggesting that PYK2 activation represents a potential link between Ang II-stimulated [Ca2+]i and PKC activation with downstream signaling events such as mitogen-activated protein kinase activation involved in the regulation of VSMC growth.

Actins↗
PubMed1998

Hydrogen peroxide activates mitogen-activated protein kinases and Na+-H+ exchange in neonatal rat cardiac myocytes.

Reperfusion of cardiac tissue after an ischemic episode is associated with metabolic and contractile dysfunction, including reduced tension development and activation of the Na+-H+ exchanger (NHE). Oxygen-derived free radicals are key mediators of reperfusion abnormalities, although the cellular mechanisms involved have not been fully defined. In the present study, the effects of free radicals on mitogen-activated protein (MAP) kinase function were investigated using cultured neonatal rat ventricular myocytes. Acute exposure of spontaneously beating myocytes to 50 micromol/L hydrogen peroxide (H2O2) caused a sustained decrease in contraction amplitude (80% of control). MAP kinase activity was measured by in-gel kinase assays and Western blot analysis. Acute exposure to H2O2 (100 micromol/L, 5 minutes) resulted in sustained MAP kinase activation that persisted for 60 minutes. Catalase, but not superoxide dismutase, completely inhibited MAP kinase activation by H2O2. Pretreatment with chelerythrine (10 micromol/L, 45 minutes), a protein kinase C inhibitor, or genistein (75 micromol/L, 45 minutes) or herbimycin A (3 micromol/L, 45 minutes), tyrosine kinase inhibitors, caused significant inhibition of H2O2-stimulated MAP kinase activity (51%, 78%, and 45%, respectively, at 20 minutes). Brief exposure to H2O2 also stimulated NHE activity. This effect was completely abolished by pretreatment with the MAP kinase kinase inhibitor PD 98059 (30 micromol/L, 60 minutes). These results suggest that low doses of H2O2 induce MAP kinase-dependent pathways that regulate NHE activity during reperfusion injury.

Animals↗
PubMed1998

Contractile activity modulates atrial natriuretic factor gene expression in neonatal rat ventricular myocytes.

[Ca2+]i transients, and the activation of Ca(2+)-sensitive kinases have been considered potential signaling mechanisms regulating ANF gene expression in cultured neonatal rat ventricular myocytes (NRVM). However, it is unclear whether [Ca2+]i is directly involved, or is indirectly involved by generating additional mechanical signals via contractile activity. Primary cultures of spontaneously contracting NRVM (CON), and NRVM treated for 48 h with verapamil (V, 10 microM), KCl (50 mM), or 2,3-butanedione monoxime (BDM, 7.5 mM) were used to delineate the affects of contractile activity v [Ca2+]i. Verapamil, a calcium, channel blocker, inhibits contraction and decreases [Ca2+]i. High [K+]o causes membrane depolarization, loss of contraction, and elevates [Ca2+]i; whereas BDM strongly inhibits contractile activity but only modestly reduces [Ca2+]i transients. ANF production, as assessed by radioimmunoassay, was significantly reduced upon contractile arrest independently of [Ca2+]i levels. Northern blotting analysis demonstrated that contractile arrest also reduced ANF mRNA levels. Transient transfection of a 3003 bp ANF promoter-luciferase expression plasmid in CON, V, KCl, and BDM-treated NRVM demonstrated marked down-regulation of ANF promoter activity in all of the contractile arrested myocytes. These results indicate that the activation of Ca(2+)-sensitive processes alone are insufficient to maintain high levels of ANF gene expression and peptide production in NRVM.

Animals↗