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)

Sarcoplasmic reticulum (SR) membrane vesicles derived from human atrium were characterized by specific ryanodine binding assay and fused into planar lipid bilayers. The tritiated form of the alkaloid bound to its receptor with a K(D) of 2.2 nM and a Bmax of 268 fmol/mg protein respectively. Special emphasis was placed on an anion-selective channel present in the SR membrane, which exhibited a mean conductance value of 67 pS when recorded in asymmetrical 50 mM trans/250 mM cis CsCl buffer system and a sensitivity to SITS (1 to 100 microM). Single and multiple channel activities displayed low voltage sensitivity and variability in its gating behavior which might result in spontaneous channel inactivation. However, the majority of the recordings (60%) resulted in a steady-state high open probability. The inactivated channel could be transiently reactivated with depolarizing voltage steps. This behavior is very similar, if not identical, to that observed for the SR Cl- channel in ventricular cells. The inactivation process is probably not directly related to a phosphorylation/dephosphorylation mechanism since PKA and PKG in presence of an adequate phosphorylation cocktail failed to reactivate the SR Cl- channel. In contrast, the use of a monoclonal anti-phospholamban antibody allowed the inhibition of the activity of the anionic channels. These results suggest that the regulation of the human atrial SR Cl- channel is dependent upon an interaction with phospholamban, which was clearly identified in our atrial preparations by Western blot analysis using monoclonal antibody.
J Mol Cell Cardiol 1996 Apr
PMID:Biochemical regulation of sarcoplasmic reticulum Cl- channel from human atrial myocytes: involvement of phospholamban. 873 4

The role of cyclic AMP-dependent protein kinase (PKA) and systolic function during the development of left ventricular hypertrophy (LVH) still remain uncertain. The aim of this work is to study PkA activity and mechanical heart function in two experimental heart hypertrophy models: specifically, one induced by pressure overload (Goldblatt model: two kidneys, one clamped, Gb); and another secondary to myocardial infarction (MI) generated by ligation of the left coronary artery. Hypertension in the Gb group becomes evident by the third and fourth week after surgery without any significant change in the corresponding sham group. The myocardial infarction group did not show any change in systolic pressure. Different degrees of LVH for the two experimental models were observed. Relative cardiac mass (RCM) and relative ventricular mass (RVM) increased 23 and 16%, respectively, above the sham-operated rats in MI group (P < 0.05). For the pressure overload model, the increase values were 42 and 44%, respectively (P < 0.05). Left ventricular hypertrophy was also evaluated through quantitative changes in cardiac beta-myosin heavy chain which agreed with morphometric studies in Goldblatt rats. Ventricular PKA activity did not show any significant difference with respect to the sham-operated group after induction of pressure overload. For the MI model, ventricular PKA activity changed only at day 7 post-infarction with a 289% increase above the sham-operated group (P < 0.05). The absence of activation of ventricular PKA after constriction of renal artery or myocardial infarction was also corroborated by the patterns of PKA-dependent phosphorylated proteins. While force-generating capacity was increased, there was no change in ventricular PKA activity, indicating that there is no relation between this enzyme and systolic stress-strain regression lines in either pressure overload or myocardial infarction conditions. Cyclic AMP-dependent protein kinase activity had no relation with development of cardiac hypertrophy in the two experimental models of LVH. These findings contribute to the hypothesis for a multifactorial interaction of different intracellular biochemical and molecular mechanisms in the genesis of cardiac hypertrophy.
J Mol Cell Cardiol 1996 May
PMID:Cyclic AMP-dependent protein kinase and mechanical heart function in ventricular hypertrophy induced by pressure overload or secondary to myocardial infarction. 876 44

The focal adhesion and microfilament-associated protein VASP is a major substrate of both cAMP- and cGMP-dependent protein kinase in intact human platelets. The recent elucidation of the primary VASP structure and identity of VASP binding proteins suggest that VASP is an important component of focal contacts which link signal transduction pathways and elements controlling cell motility. In this study, the high expression of VASP in vascular smooth muscle and endothelial cells of human blood vessels is reported. Western blot and immunofluorescence analysis detected VASP in human heart, femoral artery and a uterine leiomyosarcoma. Within these tissues, smooth muscle cells, small capillaries and the endothelial cell layer were strongly stained by the VASP antiserum. In human heart, an overlapping cellular distribution of the cGMP-dependent protein kinase I (cGK I) and its substrate VASP was noted. Immunoelectron microscopy experiments with vascular smooth muscle cells of the vessel wall revealed that VASP is localized in close proximity to microfilaments, dense plaques and dense bodies. The data of this study and the properties of VASP as a major target of inhibitory vasoactive agents suggest that VASP is an important component which participates in the regulation of cell motility of human vessel wall cells in vivo.
Basic Res Cardiol
PMID:High expression of the focal adhesion- and microfilament-associated protein VASP in vascular smooth muscle and endothelial cells of the intact human vessel wall. 892 50

In the adult myocardium the Ca2+ uptake and release functions of the sarcoplasmic reticulum (SR) are known to be regulated by a membrane-associated Ca2+-calmodulin-dependent protein kinase (CaM kinase) which phosphorylates the Ca2+-pumping ATPase (Ca2+ pump), Ca2+ release channel (ryanodine receptor) and the Ca2+ pump-regulatory protein, phospholamban. The role of CaM kinase during development, however, has not been examined previously. The present study investigated the ontogenetic expression of SR-associated CaM kinase in the rabbit myocardium as well as development-related changes in CaM kinase-mediated phosphorylation of the SR proteins (Ca2+ pump, Ca2+ release channel and phospholamban) involved in transmembrane Ca2+ cycling. For these experiments, cardiac muscle homogenate and SR-enriched membrane fraction derived from fetal (21- and 28-days gestation), newborn (2 days postnatal) and adult New Zealand White rabbits were used. Western immunoblotting analysis detected the presence of phospholamban, Ca2+ pump and Ca2+ release channel in homogenate and SR at all ages tested. The amount of these proteins in the SR increased substantially during fetal and postnatal development. Phosphorylation studies revealed the presence of CaM kinase-dependent phosphorylation of the Ca2+ pump, Ca2+ release channel and phospholamban as early as 21-days gestation. This phosphorylation could be elicited with the addition of only Ca2+ and calmodulin indicating the presence of a SR-associated CaM kinase as early as 21-days gestation. This was confirmed using a delta-CaM kinase II-specific antibody. Phosphorylation per unit amount of each substrate was greater in the fetus and newborn compared to adult. Phosphorylation of phospholamban could be elicited by exogenous cAMP-dependent protein kinase (PKA) at all developmental stages studied. Activation of SR CaM kinase with Ca2+ and calmodulin, or induction of phospholamban phosphorylation by exogenous PKA, resulted in stimulation of the Ca2+ uptake activity of SR in fetal, newborn and adult heart. These results demonstrate early ontogenetic expression of the Ca2+ cycling proteins and CaM kinase in the SR and the concurrent development of phosphorylation-dependent regulation of SR Ca2+ cycling.
J Mol Cell Cardiol 1997 Jan
PMID:Ontogeny of sarcoplasmic reticulum protein phosphorylation by Ca2+--calmodulin-dependent protein kinase. 904 54

Partial inhibition of cardiac Na/K-ATPase by digitalis drugs such as ouabain is the initial event leading to positive inotropy in the heart. We showed recently that exposure of rat cardiac myocytes to ouabain concentrations that produce positive inotropy, but no overt toxicity, caused inductions of some early response genes and hypertrophy of these myocytes. The aim of this work was to determine if ouabain also affects the expressions of certain late response genes that are regulated by other hypertrophic stimuli. Non-toxic concentrations of ouabain (5-100 microM) increased mRNAs of skeletal alpha-actin, atrial natriuretic factor, myosin light chain 2, and transforming growth factor beta: indicating that ouabain's effects on these marker genes are similar to those of hypertrophic stimuli that mimic the effects of pressure overload. Expression of skeletal alpha-actin was more sensitive to ouabain than that of atrial natriuretic factor, suggesting significant differences in the ouabain-specific pathways of the induction of these fetal genes. The effects of ouabain on skeletal alpha-actin gene were transcriptional, and required an increase in net influx of extracellular Ca2+. Protein kinase C and Ca(2+)-calmodulin kinases, but not protein kinase A, were involved in the signal pathways leading to the induction of skeletal alpha-actin gene. These data and our prior findings indicate that an increase in net influx of Ca2+ through partial inhibition of Na/K-ATPase initiates protein kinase-dependent pathways resulting in alterations in cardiac growth and expressions of both early and late response genes.
J Mol Cell Cardiol 1997 Feb
PMID:Ouabain-induced hypertrophy in cultured cardiac myocytes is accompanied by changes in expression of several late response genes. 914 Aug 3

In atrial myocytes, an initial exposure to acetylcholine (ACh1) exerts a short-term conditioning effect such that a second ACh exposure (ACh2) activates ATP-sensitive K+ current (IK,ATP). The purpose of the present study was to determine the mechanism underlying the short-term conditioning induced by ACh that results in subsequent ACh-induced activation of IK.ATP. Cat atrial myocytes were studied using a nystatin-perforated patch whole cell recording method. Changes in L-type Ca2+ current (ICa,L) amplitude were used as an index of relative changes in cyclic AMP (cAMP). The results show that when atrial myocytes are treated with two consecutive exposures to 10 microM ACh separated by a recovery interval, ACh2 activates a larger increase in potassium conductance (gK+) than ACh1. The additional ACh2-induced increase in gK+ is selectively blocked by 10 microM glibenclamide, identifying the current as IK,ATP. Moreover, ICa,L activated immediately after the withdrawal of ACh1 exhibited a transient increase in amplitude above control (+ 76%), consistent with rebound stimulation of cAMP. Rp-cAMPs (50 microM), a selective antagonist of cAMP-dependent protein kinase A, blocked the rebound stimulation of ICa,L and abolished ACh2-induced activation of IK,ATP. Thapsigargin (5 microM), an inhibitor of Ca2+ ATPase in the sarcoplasmic reticulum (SR), abolished ACh2-induced activation of IK,ATP without decreasing rebound stimulation of ICa,L. Rebound stimulation of ICa,L and ACh2-induced activation of IK,ATP both varied as a function of ACh1 duration. We conclude that withdrawal of an initial ACh exposure elicits a rebound cAMP-mediated stimulation of SR Ca2+ uptake. This mechanism induces a short-term conditioning in atrial myocytes such that a subsequent ACh exposure activates IK,ATP. The present results demonstrate novel cholinergic signaling mechanisms in the regulation of IK,ATP.
J Mol Cell Cardiol 1997 Mar
PMID:Cholinergic short-term conditioning and activation of ATP-sensitive K+ current in cat atrial myocytes. 915 51

In order to advance our previous findings that the macroscopic slow Ca2+ currents of vascular smooth muscle (VSM) cells are regulated by cyclic nucleotides, the effects of cAMP and cGMP on the activity of single slow (L-type) Ca2+ channels were investigated using cell-attached patch clamp (22-25 degrees C). Freshly isolated VSM cells were obtained from adult male rat portal vein. For the single-channel recordings, the pipette was filled with a solution containing 90 mM Ba2+ and 1 microM Bay-K-8644 solution, and the bath contained 140 mM KCl to "zero" the membrane potential. Depolarizing pulses to 0 mV, from a holding potential (HP) of -80 mV, elicited inward unitary currents. The activity of these channels was completely blocked by superfusion of 10 microM nifedipine. Extracellular perfusion of the single cells with membrane-permeable cGMP and cAMP analogs (8Br-cGMP and 8Br-cAMP) at 1 mM caused a slight inhibition, but higher doses (3 mM), clearly showed an inhibitory effect on the single-channel activity. cAMP (100 microM) stimulated one out of five patches tested, and 100 microM cGMP showed no effect in three patches tested. Compared with control, both cyclic nucleotides at 3 mM decreased the ensemble-averaged currents by 26.7 +/- 4.1% and 37.3 +/- 2.1%, respectively. Unit amplitude and slope conductance were not changed. The normal conductance of the Ca2+ channel was 20.8 +/- 0.04 pS (n = 9), and the conductances in the presence of cAMP (n = 5) and cGMP (n = 6) were 19.3 +/- 0.04 and 20.5 +/- 0.05 pS, respectively. Single-channel kinetic analysis showed that cAMP did not affect the mean open-time, and cGMP slightly decreased the mean open-time. However, both cAMP and cGMP increased the mean closed-time. In addition, cAMP decreased the open probability (NPo) by a factor of 1.7, from 0.26 +/- 0.04 to 0.15 +/- 0.03 (P < 0.05, Student's t-test) and cGMP decreased NPo by a factor of 2.5, from 0.24 +/- 0.08 to 0.10 +/- 0.02 (P < 0.05). H-7, a non-specific protein kinase inhibitor, prevented the inhibitory effects of both cAMP and cGMP on the activity of single Ca2+ channels in rat portal vein cells. The results demonstrate that both cAMP and cGMP inhibit L-type Ca2+ channel activities in VSM cells from rat portal vein. This inhibition may be mediated by the cAMP and cGMP-dependent protein kinase phosphorylation of the L-type Ca2+ channels (or an associated regulatory protein).
J Mol Cell Cardiol 1997 May
PMID:Cyclic nucleotides regulate the activity of L-type calcium channels in smooth muscle cells from rat portal vein. 920 26

Phospholamban (PLB), the regulator of the cardiac sarcoplasmic reticulum (SR) Ca2+ pump is specifically phosphorylated at Ser16 and Thr17 by cAMP-dependent protein kinase (PKA) and Ca2+/calmodulin-dependent protein kinase (CaMK), respectively. The regulation of this dual-site phosphorylation of amino acid residues in direct proximity is only poorly understood. In order to study the site-specific phosphorylation of PLB, we used a synthetic peptide (PLB-24) corresponding to the cytosolic part of the PLB monomer with the phosphorylation sites as a model substrate. PLB-24 possesses substrate properties as the native PLB as demonstrated by phosphorylation with exogenous, purified PKA, cGMP-dependent protein kinase (PKG) and a type II CaMK (CaMKII). In isolated vesicles of cardiac SR there was a rapid phosphorylation of the peptide by the endogenous PKA (SR-PKA) and CaMK (SR-CaMK), but not under conditions that activate PKG. Both SR-PKA and SR-CaMK incorporated the same amount of 32P into PLB-24, 0.60 +/- 0.01 nmol 32P/mg SR protein and 0.61 +/- 0.03 nmol 32P/mg SR protein, respectively. Phosphorylation by SR-PKA was abolished by the specific PKA inhibitor (IC50 = 0.2 microM), whereas SR-CaMK phosphorylation was inhibited by calmidazolium (IC50 = 1.6 microM) and a CaMKII-specific inhibitor peptide (IC50 = 2.5 microM). Phosphorylation by SR-PKA was exclusively at Ser, whereas SR-CaMK phosphorylated only Thr. After simultaneous activation of both SR-kinases 32P incorporation into PLB-24 was additive and occurred at Ser as well as at Thr. Sequential activation of SR-PKA and SR-CaMK also caused the additive phosphorylation of PLB-24 independently of which kinase was activated first. Thus, at the monomeric level of PLB the respective phosphorylation site appears to be accessible to its related SR protein kinase in vitro even when the adjacent site is phosphorylated.
Basic Res Cardiol 1997
PMID:Site-specific phosphorylation of a phospholamban peptide by cyclic nucleotide- and Ca2+/calmodulin-dependent protein kinases of cardiac sarcoplasmic reticulum. 920 42

The plasma membrane Ca-pump (134 kDa) is stimulated by calmodulin and by other treatments (exposure to acidic phospholipids, treatments with proteases, phosphorylation by protein kinases A or C, self-association to form oligomers). It is the product of four genes (in humans), but additional isoforms originate through alternative mRNA spicing. Most of the pump mass protrudes into the cytoplasm with three main units. The calmodulin binding domain is located in the C-terminal protruding unit. The domain is a positively charged segment of about 25 residues. The calcium-activated protease calpain activates the pump by removing its calmodulin binding domain and the portion C-terminal to it. The-resulting 124 KDa fragment has been used to test the suggestion of an autoinhibitory function of the calmodulin binding domain. The latter interacts with two domains of the pump, one located close to the active site in the mid-cytoplasmic protruding unit, the other in the first (N-terminal) protruding unit. The isoforms of the pump show variations in the regulatory domains, e.g., alternative mRNA splicing can eliminate the domain phosphorylated by protein kinase A, or alter the sensitivity of the pump to calmodulin. This occurs by inserting sequences rich in His between calmodulin binding subdomains A and B. The inserted domain(s) confer pH sensitivity to the binding of calmodulin. Calcium binding sites have been found in acidic regions preceding and following the calmodulin binding domain.
Basic Res Cardiol 1997
PMID:Plasma membrane calcium pump: structure, function and relationships. 920 45

The report is a discussion of previously published and newly analyzed results concerning the association between heart diseases and alterations in the force-frequency relation (FFR). The optimum stimulation frequency of the FFR is measured and compared in isolated left ventricular myocardium from non-failing hearts with atrial septal defect, coronary artery disease (without and with insulin dependent diabetes mellitus) and from failing hearts with mitral regurgitation, or idiopathic dilated cardiomyopathy. Specifically, we examine the role of altered control of the excitation-contraction coupling system in blunting the force-frequency relation. We use the percent slope of the FFR as a measure of changes in the frequency sensitivity of this control. Our finding of a linear, direct relation between optimum stimulation frequency and % slope across all disease types suggests both parameters are coupled to the same underlying mechanism. To investigate the possible role of altered control of the calcium pump in this mechanism, we analyzed the detailed relation between isometric twitch relaxation kinetics and stimulation frequency in mitral regurgitation myocardium (MR). In the presence of 0.5 microM forskolin the depressed slope and optimum frequency of the FFR and the prolonged half-time of twitch relaxation were all restored to values found in non-failing myocardium. We use the kinetics of isometric twitch relaxation as an index of changes in pumping rate that occur in response to changes in stimulation frequency or in intracellular cyclic adenosine monophosphate concentration. A mathematical model based on the Hill relations for calcium pump uptake rate and for isometric tension as a function of intracellular pCa is developed to simulate isometric twitch relaxation in MR and non-failing myocardium. The success of this model in simulating non-failing and failing twitch relaxation supports a proposed mechanism for the prolonged relaxation time and depressed FFR in MR involving depressed protein kinase-A activity (due to lowered cAMP or to a defect in the Ser16 site of phospholamban) as a mechanism of altered control of the calcium pump in MR heart disease.
Basic Res Cardiol 1997
PMID:Role of cAMP in modulating relaxation kinetics and the force-frequency relation in mitral regurgitation heart failure. 920 49


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