Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We studied changes in myofibrillar function and protein profiles after complete global ischemia with anoxia in rat hearts. Hearts were exposed to global ischemia and anoxia (CGI) for 30 or 60 minutes at 37 degrees C, and myofibrils were prepared for measurement of Ca(2+)-dependent Mg(2+)-ATPase activity at pH 7.0 and 6.5. Hearts incubated in cold saline (1 +/- 1 degrees C) and nonincubated hearts served as controls. Maximum ATPase activity was unchanged at pH 7.0 and pH 6.5 in myofibrils from hearts treated with 30 or 60 minutes of CGI. At pH 7.0, the Hill coefficient, which is an index of cooperative interactions among thin-filament proteins, was unchanged after 30 minutes of CGI but was significantly increased after 60 minutes of CGI. A similar trend for increased cooperativity was observed when myofibrillar ATPase activity was measured at pH 6.5 in myofibrils from rat hearts made ischemic for 30 or 60 minutes. Both 30 and 60 minutes of CGI resulted in increased pCa50 values (half-maximally activating free [Ca2+]) at pH 7.0 and pH 6.5. Densitometric analysis of myofibrillar proteins separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that troponin I and troponin T were degraded during 60 minutes of CGI. Two new protein bands appearing in ischemia-treated myofibrils were identified as partially degraded troponin I and troponin T with Western blots. The troponin I fragment could be phosphorylated by cAMP-dependent protein kinase. In addition, we observed phosphorylation of a protein band that corresponded to myosin light chain-2 in myofibrils from CGI-treated hearts. These results suggest that degradation of thin-filament proteins may contribute to the changes in cooperativity of Ca2+ regulation of ATPase activity observed in the myofibrils from rat hearts exposed to CGI.
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PMID:Alterations in myofibrillar function and protein profiles after complete global ischemia in rat hearts. 153 Nov 86

Hearts isolated from 1-yr-old non-insulin-dependent diabetic rats exhibited reduced responsiveness to the beta-adrenergic agonist isoproterenol. Over a concentration range of 3 x 10(-9) to 10(-7) M, isoproterenol-mediated stimulation in the rate of left ventricular pressure decline, a measure of myocardial relaxation, and the rate of left ventricular pressure rise, a measure of myocardial contractility, were significantly depressed in the diabetic hearts. To clarify the basis for this defect, individual steps involved in the actions of the beta-adrenergic agonists were examined. Dihydroalprenolol binding assays revealed that neither beta-adrenergic receptor number nor binding affinity was affected by the diabetic condition. Also unaffected by diabetes was isoproterenol-mediated stimulation of adenylate cyclase activity, myocyte accumulation of adenosine 3',5'-cyclic monophosphate (cAMP), or the increase in cAMP-dependent protein kinase activity ratio. However, it was found that both in the presence and absence of cAMP-dependent protein kinase, activity of the sarcolemmal calcium transporter was significantly depressed in the diabetic heart. Also attenuated was protein kinase-induced enhancement of sarcoplasmic reticular calcium transport. The likelihood that these abnormalities contribute to alterations in calcium homeostasis and myocardial contractile function is discussed.
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PMID:Defective response to cAMP-dependent protein kinase in non-insulin-dependent diabetic heart. 165 26

The protein phosphatases which dephosphorylate native, sarcoplasmic reticulum (SR)-associated phospholamban were studied in cardiac muscle extracts and in a Triton fraction prepared by detergent extraction of myofibrils, the latter fraction containing 70-80% of the SR-associated proteins present in the tissue. At physiological concentrations of free Mg2+ (1 mM), protein phosphatase 1 (PP1) accounted for approximately 70% of the total phospholamban phosphatase activity in these fractions towards either Ser-16 (the residue labelled by cAMP-dependent protein kinase, PK-A) or Thr-17 (the residue phosphorylated by an SR-associated Ca2+/calmodulin-dependent protein kinase). Protein phosphatase 2A (PP2A) and protein phosphatase 2C (PP2C) accounted for the remainder of the activity. A major form of cardiac PP1, present in comparable amounts in both the extract and Triton fraction, was similar, if not identical, to skeletal muscle protein phosphatase 1G (PP1G), which is composed of the PP1 catalytic (C) subunit complexed to a G subunit of approximately 160 kDa, responsible for targeting PP1 to both the SR and glycogen particles of skeletal muscle. This conclusion was based on immunoblotting experiments using antibody to the G subunit, ability to bind to glycogen and the release of PP1 activity from glycogen upon incubation with PK-A and MgATP. PP1 accounted for approximately 90% of the phospholamban (Ser-16 or Thr-17) phosphatase activity in the material sedimented by centrifugation at 45,000 x g, a fraction prepared from cardiac extracts which is enriched in SR membranes. The G subunit in this fraction could be solubilised by Triton X-100, but not with 0.5 M NaCl or digestion with alpha-amylase, indicating that it is bound to membranes and not to glycogen. By analogy with the situation in skeletal muscle, the PK-A catalysed phosphorylation of the G subunit, with ensuing release of the C subunit from the SR, may prevent PP1 from dephosphorylating SR-bound substrates and represent one of the mechanisms by which adrenalin increases the phosphorylation of cardiac phospholamban (Ser-16 and Thr-17) in vivo. Hearts left in situ post mortem lose 85-95% of their PP1 activity within 20-30 min. This remarkable disappearance of PP1 may partly explain why the importance of this enzyme in cardiac muscle metabolism has not been recognized previously.
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PMID:Identification of the major protein phosphatases in mammalian cardiac muscle which dephosphorylate phospholamban. 184 81

The effects of forskolin on phosphorylation of proteins of a 100,000 X g fraction was examined in isolated beating guinea pig hearts. Hearts were perfused with [32P] inorganic phosphate to label intracellular adenine nucleotides. Forskolin was injected into the coronary circulation and after freeze-clamping, phosphorylated proteins in a fraction were separated by sodium dodecyl sulfate-polyacrylamide gel electro-phoresis. Forskolin increased the incorporation into a 25,000 Mr protein approximately 15 fold over control. Incorporation of label was time and dose dependent and was temporally coincident with increases in developed tension. A sarcolemmal fraction prepared from perfused hearts contained a similar 25,000 Mr protein. The data provides evidence that forskolin induced inotropy is accompanied by cAMP-dependent protein kinase mediated phosphorylation. The phosphorylation may be of the same protein whose phosphorylation is associated with epinephrine-induced increase in contractility.
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PMID:Effect of forskolin on phosphorylation of a 25,000 Mr protein in perfused guinea pig hearts. 404 May 33

The effects of beta and alpha-adrenergic stimulation in amphibian superfused hearts and ventricular strips were studied. Superfusion with 3 x 10(-8) M isoproterenol produced a positive inotropic effect, as detected by a 92 +/- 24% increase in the maximal rate of contraction (+T) and a positive lusitropic effect characterized by a decrease in both the ratio +T/-T (23 +/- 5%) and the half relaxation time (t1/2) (19 +/- 4%). The mechanical behavior induced by the beta-agonist was associated with an increase in the intracellular cAMP levels from control values of 173 +/- 19 to 329 +/- 28 nmol/mg wet tissue. Hearts superfused with 32P in the presence of isoproterenol showed a significant increase in Tn 1 phosphorylation (from 151 +/- 13 to 240 +/- 44 pmol 32P/mg MF protein) without consistent changes in phosphorylation of C-protein. In sarcoplasmic reticulum membrane vesicles, no phospholamban phosphorylation was detected either by beta-adrenergic stimulation of superfused hearts or when phosphorylation conditions were optimized by direct treatment of the vesicles with cAMP-dependent protein kinase (PKA) and [gamma 32P] ATP. The effect of alpha-adrenergic stimulation on ventricular strips was studied at 30 and 22 degrees C. At 30 degrees C, the effects of 10(-5) to 10(-4) M phenylephrine on myocardial contraction and relaxation were diminished to non significant levels by addition of propranolol. At 22 degrees C, blockage with propranolol left a remanent positive inotropic effect (10% of the total effect of phenylephrine) and changed the phenylephrine-induced positive lusitropic effect into a negative lusitropic action.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Lusitropic effects of alpha- and beta-adrenergic stimulation in amphibian heart. 789 75

The multifunctional Ca++/calmodulin-dependent protein kinase II (CaM kinase) mediates Ca++-induced augmentation of L-type Ca++ current (ICa); therefore it may act as a proarrhythmic signaling molecule during early afterdepolarizations (EADs) due to ICa. To investigate the hypothesis that ICa-dependent EADs are favored by CaM kinase activation EADs were induced with clofilium in isolated rabbit hearts. All EADs were rapidly terminated with ICa antagonists. Hearts were pretreated with the CaM kinase inhibitor KN-93 or the inactive analog KN-92 (0.5 microM) for 10 min before clofilium exposure. EADs were significantly suppressed by KN-93 (EADs present in 4/10 hearts) compared to KN-92 (EADs present in 10/11 hearts) (P =.024). There were no significant differences in parameters favoring EADs such as monophasic action potential duration or heart rate in KN-93- or KN-92-treated hearts. CaM kinase activity in situ increased 37% in hearts with EADs compared to hearts without EADs (P =.015). This increase in CaM kinase activity was prevented by pretreatment with KN-93. In vitro, KN-93 potently inhibited rabbit myocardial CaM kinase activity (calculated Ki </= 2.58 microM), but the inactive analog KN-92 did not (Ki > 100 microM). The actions of KN-93 and KN-92 on ICa and other repolarizing K+ currents did not explain preferential EAD suppression by KN-93. These data show a novel association between CaM kinase activation and EADs and are consistent with the hypothesis that the ICa and CaM kinase activation both contribute to EADs in this model.
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PMID:KN-93, an inhibitor of multifunctional Ca++/calmodulin-dependent protein kinase, decreases early afterdepolarizations in rabbit heart. 986 85

Recent studies have demonstrated that Ca(2+)/calmodulin-dependent protein kinase phosphorylates the Ca(2+)-pumping ATPase of cardiac sarcoplasmic reticulum (SR) in vitro. Also, evidence from in vitro studies suggested that this phosphorylation, occurring at Ser(38), results in stimulation of Ca(2+) transport. In the present study, we investigated whether serine phosphorylation of the SR Ca(2+)-ATPase occurs in the intact functioning heart. Hearts removed from anesthetized rabbits were subjected to retrograde aortic perfusion of the coronary arteries with oxygenated mammalian Ringer solution containing (32)P(i) and contractions were monitored by recording systolic left ventricular pressure development. Following 45-50 min of (32)P perfusion, the hearts were freeze-clamped, SR isolated, and analyzed for protein phosphorylation. SDS-polyacrylamide gel electrophoresis and autoradiography showed phosphorylation of several peptides including the Ca(2+)-ATPase and Ca(2+) release channel (ryanodine receptor). The identity of Ca(2+)-ATPase as a phosphorylated substrate was confirmed by Western immunoblotting as well as immunoprecipitation using a cardiac SR Ca(2+)-ATPase-specific monoclonal antibody. The Ca(2+)-ATPase showed immunoreactivity with a phosphoserine monoclonal antibody indicating that the in situ phosphorylation occurred at the serine residue. Quantification of Ca(2+)-ATPase phosphorylation in situ yielded a value of 208 +/- 12 pmol (32)P/mg SR protein which corresponded to the phosphorylation of approximately 20% of the Ca(2+) pump units in the SR membrane. Since this phosphorylation occurred under basal conditions (i.e., in the absence of any inotropic intervention), a considerable steady-state pool of serine-phosphorylated Ca(2+)-ATPase likely exists in the normally beating heart. These findings demonstrate that serine phosphorylation of the Ca(2+)-ATPase is a physiological event which may be important in the regulation of SR function.
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PMID:Serine phosphorylation of the sarcoplasmic reticulum Ca(2+)-ATPase in the intact beating rabbit heart. 1052 72

Although beta-adrenoceptor (beta-AR) blockers are used for the treatment of ischemic heart disease, the mechanisms of their beneficial actions have not been fully elucidated. In view of the role of sarcoplasmic reticular (SR) abnormalities in cardiac dysfunction due to ischemia-reperfusion (I/R), we examined the effects of beta-AR blockers on the I/R-induced changes in SR Ca(2+) uptake and release, as well as the protein contents and gene expression of ryanodine receptor, SR Ca(2+)-pump, phospholamban, and calsequestrin. I/R in isolated rat hearts was induced by stopping the perfusion for 30 min and then reperfusing the ischemic hearts for 60 min. Hearts were treated with or without 10 microM atenolol, a beta(1)-specific blocker, or 10 microM propranolol, a nonspecific beta-blocker, 10 min before inducing ischemia as well as during the reperfusion period. I/R depressed cardiac performance, SR Ca(2+) uptake, and Ca(2+) release activities, protein contents, as well as Ca(2+)/calmodulin-dependent protein kinase and cAMP-dependent protein kinase-mediated phosphorylations, significantly. The mRNA levels for SR Ca(2+) pump, ryanodine receptors, phospholamban, and calsequestrin were also reduced by I/R. All these changes due to I/R were partially prevented by beta-AR blocker treatment. The results indicate that the beneficial effects of beta-AR blockers on cardiac performance in the I/R hearts may be related to the prevention of changes in SR Ca(2+) uptake and release activities, protein contents, as well as Ca(2+)/calmodulin-dependent protein kinase and cAMP-dependent protein kinase phosphorylations of SR proteins. On the other hand, the protection of I/R-induced alterations in mRNA levels for SR proteins by beta-AR blockers suggests cardiac SR gene expression as a molecular site of their cardioprotective action.
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PMID:Effect of beta-adrenoceptor blockers on sarcoplasmic reticular function and gene expression in the ischemic-reperfused heart. 1073 48

Three major mammalian mitogen-activated protein kinases, extracellular signal-regulated kinase (ERK), p38, and c-Jun NH(2)-terminal protein kinase (JNK), have been identified in the cardiomyocyte, but their respective roles in the heart are not well understood. The present study explored their functions and cross talk in ischemia/reoxygenation (I/R)-induced cardiac apoptosis. Exposing rat neonatal cardiomyocytes to ischemia resulted in a rapid and transient activation of ERK, p38, and JNK. On reoxygenation, further activation of all 3 mitogen-activated protein kinases was noted; peak activities increased (fold) by 5.5, 5.2, and 6.2, respectively. Visual inspection of myocytes exposed to I/R identified 18.6% of the cells as showing morphological features of apoptosis, which was further confirmed by DNA ladder and terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling (TUNEL). Myocytes treated with PD98059, a MAPK/ERK kinase (MEK1/MEK2) inhibitor, displayed a suppression of I/R-induced ERK activation, whereas p38 and JNK activities were increased by 70.3% and 55.0%, respectively. In addition, the number of apoptotic cells was increased to 33.4%. With pretreatment of cells with SB242719, a selective p38 inhibitor, or SB203580, a p38 and JNK2 inhibitor, I/R+PD98059-induced apoptotic cells were reduced by 42.8% and 63.3%, respectively. Hearts isolated from rats treated with PD98059 and subjected to global ischemia (30 minutes)/reoxygenation (1 hour) showed a diminished functional recovery compared with the vehicle group. Coadministration of SB203580 attenuated the detrimental effects of PD98059 and significantly improved cardiac functional recovery. The data taken together suggest that ERK plays a protective role, whereas p38 and JNK mediate apoptosis in cardiomyocytes subjected to I/R, and the dynamic balance of their activities is critical in determining cardiomyocyte fate subsequent to reperfusional injury.
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PMID:Inhibition of extracellular signal-regulated kinase enhances Ischemia/Reoxygenation-induced apoptosis in cultured cardiac myocytes and exaggerates reperfusion injury in isolated perfused heart. 1074 92

Multiple intracellular signaling pathways have been shown to regulate the hypertrophic growth of cardiomyocytes. Both necessary and sufficient roles have been described for the mitogen activated protein kinase(1) (MAPK) signaling pathway, specific protein kinase C (PKC) isoforms, and calcineurin. Here we investigate the interdependence between calcineurin, MAPK, and PKC isoforms in regulating cardiomyocyte hypertrophy using three separate approaches. Hearts from hypertrophic calcineurin transgenic mice were characterized for PKC and MAPK activation. Transgenic hearts demonstrated activation of c-Jun NH(2)-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK1/2), but not p38 MAPK factors. Calcineurin transgenic hearts demonstrated increased activation of PKCalpha, beta(1), and theta, but not of epsilon, beta(2), or lambda. In a second approach, cultured cardiomyocytes were infected with a calcineurin adenovirus to induce hypertrophy and the effects of pharmacologic inhibitors or co-infection with a dominant negative adenovirus were examined. Calcineurin-mediated hypertrophy was prevented with PKC inhibitors, Ca(2+) chelation, and attenuated with a dominant negative SEK-1 (MKK4) adenovirus, but inhibitors of ERK or p38 activation had no effect. In a third approach, we examined the activation of MAPK factors and PKC isoforms during the progression of load-induced hypertrophy in aortic banded rats with or without cyclosporine. We determined that inhibition of calcineurin activity with cyclosporine prevented PKCalpha, theta, and JNK activation, but did not affect PKCepsilon, beta, lambda, ERK1/2, or p38 activation. Collectively, these data indicate that calcineurin hypertrophic signaling is interconnected with PKCalpha, theta, and JNK in the heart, while PKCepsilon, beta, lambda, p38, and ERK1/2 are not involved in calcineurin-mediated hypertrophy.
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PMID:Calcineurin promotes protein kinase C and c-Jun NH2-terminal kinase activation in the heart. Cross-talk between cardiac hypertrophic signaling pathways. 1078 73


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