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P Karczewski

Publications and source records attributed to P Karczewski.

At least 37 records · Page 2Linked to original sources
PubMed1997

Site-specific phosphorylation of a phospholamban peptide by cyclic nucleotide- and Ca2+/calmodulin-dependent protein kinases of cardiac sarcoplasmic reticulum.

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.

Animals↗
PubMed1995

Unchanged protein levels of SERCA II and phospholamban but reduced Ca2+ uptake and Ca(2+)-ATPase activity of cardiac sarcoplasmic reticulum from dilated cardiomyopathy patients compared with patients with nonfailing hearts.

BACKGROUND: The aim of the present study was to investigate whether Ca2+ uptake into the sarcoplasmic reticulum (SR) is altered in failing human myocardium resulting from dilated cardiomyopathy. METHODS AND RESULTS: Ca(2+)-ATPase (SERCA II) activity and Ca(2+)-dependent 45Ca2+ uptake (oxalate supported, steady state) in isolated vesicles from the SR (VSR) and in crude membrane preparations (CSR) (free Ca2+, 0.01 to 100 mumol/L) from nonfailing (donor hearts, n = 13) and terminally failing (heart transplants, dilated cardiomyopathy, n = 17) human myocardium were studied. In the same hearts, protein levels (Western blot analysis) and mRNA levels (Northern blot analysis) of SERCA II and phospholamban were measured. Increasing concentrations of Ca2+ were followed by an increased Ca(2+)-ATPase activity and Ca2+ uptake. Ca2+ uptake activity and Ca(2+)-ATPase activity in CSR preparations from failing myocardium were significantly reduced compared with nonfailing hearts (Ca(2+)-ATPase, 163 +/- 8 and 125 +/- 7 nmol ATP/mg protein per minute for nonfailing tissue and failing tissue in New York Heart Association [NYHA] class IV, respectively; Ca2+ uptake, 7.1 +/- 0.8 and 3.5 +/- 0.3 nmol/mg protein per minute in CSR from nonfailing and NYHA class IV hearts, respectively P < .05). In contrast, no significant difference was measured in VSR. In the same preparations (CSR and VSR), both SERCA II and phospholamban levels (Western blot technique with monoclonal antibodies) were unchanged in failing compared with nonfailing tissue. mRNA expression relative to GAPDH mRNA for SERCA IIa and for phospholamban was significantly reduced in failing human myocardium (P < .05). CONCLUSIONS: These findings provide evidence that in failing human myocardium caused by dilated cardiomyopathy, protein levels of SERCA II and phospholamban are unchanged even though mRNA levels for SERCA II and phospholamban and the SERCA II function are reduced compared with nonfailing myocardium.

Adenosine Triphosphatases↗
PubMed1995

The cardiac sarcoplasmic reticulum phospholamban kinase is a distinct delta-CaM kinase isozyme.

Phospholamban is the regulator of the Ca(2+)-ATPase in cardiac sarcoplasmic reticulum (SR). It is phosphorylated by a Ca2+/calmodulin-dependent protein kinase (SRCaM kinase) which is closely associated with cardiac SR membrane preparations. We found that, upon renaturation of pig cardiac SR proteins, blotted onto PVDF membrane, two polypeptides of 54 and 52 kDa showed Ca2+/calmodulin-dependent autophosphorylation. In Western blots of SR proteins, the 54/52 kDa polypeptides were recognized by an antibody specific for the delta-CaM kinase isoforms, but not by an anti-alpha-CaM kinase. The two polypeptides were selectively immunoprecipitated from solubilized SR vesicles with the anti-delta-CaM kinase. The CaM kinase inhibitors KN-62 and peptide CaMK-(281-302) inhibited the activity of the SRCaM kinase with IC50 values in the same range with those obtained for the brain isozyme. In addition, initial autophosphorylation (Ca(2+)-dependent) produced a partially Ca(2+)-independent enzyme while further autophosphorylation (Ca(2+)-independent) made the enzyme completely Ca(2+)-independent. Based on these results we suggest that the SRCaM kinase is a distinct delta-CaM kinase isozyme.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1995

Cyclic GMP-mediated phospholamban phosphorylation in intact cardiomyocytes.

The cGMP-mediated phospholamban phosphorylation was investigated in cardiomyocytes in response to receptor-dependent (atrial natriuretic peptide, ANP) and -independent (sodium nitroprusside; SNP) cGMP generation. ANP (1 nM-1 microM) induced phospholamban phosphorylation in a concentration-dependent fashion (EC50: 5.0 +/- 0.09 nM). Concomitantly, an elevation in cGMP levels was observed. Phospholamban was also dose-dependently phosphorylated in response to SNP, but it required about three orders of magnitude higher concentrations (EC50: 2.9 +/- 0.03 microM) than ANP. Treatment of the cells with 8Br-cGMP (10 microM) or with the specific activator of cGMP-protein kinase 8-pCPT-cGMP (1-100 microM) mimicked these effects. The results demonstrate for the first time that a ANP/cGMP signaling pathway exists in neonatal cardiomyocytes which may contribute to modulation of heart contractility.

Animals↗
PubMed1994

Cardiac troponin I and tension generation of skinned fibres in the developing rat heart.

During development of the myocardium the troponin I (TNI) isoform expression is switched from a cAMP-insensitive, slow skeletal muscle TNI to a cAMP-sensitive, cardiac TNI isoform (cTNI). To study the functional consequence of alterations in cTNI expression in the rat heart we investigated the cAMP-controlled cTNI phosphorylation in comparison with alterations of functional properties of isolated cardiac myofibrils during the first postnatal month. cTNI was identified by Western blot analysis followed by a semiquantitative assessment. From the third to the 28th postnatal day the relative concentrations of the cardiac isoform of TNI increased 2.9 +/- 0.3-fold. In the same period the amount of phosphate incorporated into cTNI in the presence of exogenous cAMP-dependent protein kinase (PKA) and 32P[gamma]-ATP was increased 5.8 +/- 0.2-fold (24.2 +/- 3.5 v 140.2 +/- 7.6 pmolP/mg protein loaded onto the gel) whereas the phosphorylation of C-protein was only increased 1.6 +/- 0.2-fold. Ca(2+)-activated isometric tension generation of skinned heart fibres measured in the range of pCa from 6 to 4.5 was not affected by PKA at day 3. However, isometric tension generation of fibres prepared from 28-day-old rats was suppressed by incubation with PKA which was accompanied by a rightward shift in the force/pCa relation. Under these conditions half-maximal tension development was found at pCa 5.38 v 5.52 (p < 0.05) in the absence of PKA. The Ca2+ sensitivity of the contractile apparatus was not affected by PKA-induced phosphorylation of C-protein. These data give direct evidence for the physiological relevance of the onset of cAMP-induced phosphorylation of cTNI for the Ca(2+)-activated tension generation in cardiac myofibrils during postnatal development.

Animals↗
PubMed1993

Phosphorylation of the L-type calcium channel beta subunit is involved in beta-adrenergic signal transduction in canine myocardium.

Cyclic AMP-mediated phosphorylation of calcium channel subunits was studied in vitro and in vivo in preparations from dog heart. Calcium channels in native cardiac membranes were phosphorylated by cAMP-dependent protein kinase (PKA) solubilized with digitonin and subsequently immunoprecipitated using a polyclonal antibody generated against the deduced carboxy-terminal sequence of the cardiac beta subunit. A 62 kDa protein was identified as the major PKA-substrate in the immunoprecipitates. In the intact myocardium, this putative beta subunit was found to be phosphorylated in response to cAMP elevating agents. In contrast, no phosphorylation of a protein with an electrophoretic mobility similar to the alpha 1 subunit was detected, although 1,4-dihydropyridine receptor sites were recovered in the immunoprecipitates. Thus, we suggest that PKA-mediated phosphorylation of the beta subunit is the major mechanism for beta-adrenergic regulation of cardiac L-type calcium channel activity.

Amino Acid Sequence↗
PubMed1993

Protein phosphorylation and cardiac function: cholinergic-adrenergic interaction.

OBJECTIVE: The muscarinic inhibition of cyclic AMP (cAMP) mediated contractile effects may not only be the result of a reduction in cAMP levels, but may also involve cAMP linked protein phosphorylation in the heart. To show that a cholinergic agonist may antagonise the effects of agents that induce the cAMP signalling sequence at the level of protein phosphorylation, the effect of carbachol was investigated on the in vivo phosphorylation of phospholamban, troponin I, and a 15 kDa protein during positive inotropic intervention with isoprenaline, forskolin, and a phosphodiesterase inhibitor in the isolated rat heart. METHODS: The contractile activity of the heart was monitored during drug administration, and the hearts were freeze clamped for assay of protein phosphorylation and metabolites [cAMP, cyclic guanosine monophosphase (cGMP), cAMP dependent protein kinase (cA-PK), and phosphorylase a]. For estimation of the amount of in vivo phosphorylation, a "back phosphorylation" method was used. RESULTS: Carbachol attenuated in vivo phosphorylation, induced by cAMP increasing drugs, of all the phosphoproteins studied, whereas the accumulation of cAMP was not affected or was decreased to 61% after exposure to isoprenaline alone. In addition, the cAMP mediated phosphorylase a activation was completely reversed by carbachol. similarly, sodium nitroprusside reduced phosphorylation as well as the contractile force of the investigated phosphoproteins. CONCLUSIONS: These data confirm the reduction of cAMP mediated cardiac protein phosphorylation by carbachol. The cholinergic antagonism may primarily involve cellular target systems distal to sites of cAMP generation. The observed similarity in the cardiac response to carbachol and sodium nitroprusside indicates a role of cGMP in the antiadrenergic action of carbachol in the myocardium.

Animals↗
PubMed1993

Protein phosphorylation in the regulation of cardiac contractility and vascular smooth muscle tone.

This review discusses phosphorylation of target proteins in the plasma membrane, the sarcoplasmic reticulum, and the myofilaments of cardiac and vascular smooth muscle cells known to be important in regulating contraction and relaxation. Substantial data have been obtained on the regulation of calcium uptake mechanisms of the sarcoplasmic reticulum in cardiac as well as vascular smooth muscle, suggesting a central role of phospholamban for relaxation in the cardiovascular system. As shown for cardiac muscle, the calcium release from the sarcoplasmic reticulum is also under beta-adrenergic control via phosphorylation of the ryanodine receptor. The developmental switching from the skeletal to the cardiac isoform of the cardiac thin-filament protein troponin I marks the onset of beta-adrenergic regulation of myofilaments through phosphorylation. Further examples for the genetic control of systems regulated by protein phosphorylation are presented. The review emphasizes the power of molecular-biologic approaches in combination with traditional methods to achieve significant progress in this field of cardiovascular research.

Animals↗
PubMed1992

Role of phospholamban in NO/EDRF-induced relaxation in rat aorta.

The role of endothelium-derived nitric oxide (NO) to cause smooth muscle phospholamban (PLB) phosphorylation was studied in the isolated perfused rat aorta precontracted with norepinephrine using a back-phosphorylation technique. NO-induced relaxation was associated with increased PLB-phosphorylation while norepinephrine as such was ineffective. Removal of endothelium significantly reduced PLB-phosphorylation in indomethacin treated vessels. Stimulation of NO-formation by ATP augmented PLB-phosphorylation in intact vessels but was ineffective in denuded aortas. The results indicate that PLB-phosphorylation of vascular smooth muscle plays an important role in mediating NO-dependent relaxation by enhancing Ca(++)-uptake into sarcoplasmic reticulum.

Adenosine Triphosphate↗
PubMed1990

Differential sensitivity to isoprenaline of troponin I and phospholamban phosphorylation in isolated rat hearts.

Phosphorylation of phospholamban (PLB), a membrane-bound 15 kDa protein and troponin I (TNI) was studied in isolated perfused rat hearts by using the back-phosphorylation technique with [32P]ATP catalysed by an excess of exogenous catalytic subunit of cyclic AMP (cAMP)-dependent protein kinase, followed by protein separation. This standardized method allows the quantitative detection of protein phosphorylation specifically stimulated by cAMP. In control hearts the extent of specific phosphorylation was equivalent to 3.3 nmol of PLB and 11.0 mumol of TNI per g of cardiac tissue. In hearts freeze-clamped 30 s after exposure to isoprenaline (10 pM-10 microM), there was a dose-dependent decrease in phosphate incorporation in vitro, indicating a phosphorylation of the respective proteins in vivo. A differential sensitivity of TNI and PLB phosphorylation towards the beta-adrenergic agonist and the subsequent increase in tissue cAMP was found, favouring TNI phosphorylation. K0.5 values for isoprenaline were 2.94 +/- 0.04 nM and 4.46 +/- 0.24 nM for PLB and the 15 kDa protein, but 0.13 +/- 0.01 nM for TNI phosphorylation in the intact tissue. At an isoprenaline-induced increase in cAMP less than 3 pmol/mg of protein there was no or only a small increase in PLB phosphorylation, whereas TNI phosphorylation was nearly maximal. By plotting phosphorylation data against changes in contractile parameters a strong correlation was obtained for TNI (r = 0.95), assuming a linear relationship. For PLB a complex relationship is likely to exist. Our data (i) indicate a functional compartmentalization of the cAMP signal cascade and (ii) confirm that phosphorylation of TNI rather than of PLB is related to changes in mechanical myocardial responses.

Adenosine Triphosphate↗
PubMed1990

Studies of the effect of glyceryl trinitrate and cyclic GMP on calcium turnover in bovine mesenteric artery.

It was recently observed that the relaxation induced by glyceryl trinitrate (GTN) showed a biphasic concentration-response curve; a high-sensitivity component represented by concentrations less than 1 nM and a low-sensitivity component represented by concentrations greater than 1 nM. The effect of two glyceryl trinitrate concentrations (0.1 nM and 1 microM) were tested on the uptake of 45Ca2+ to tissue pieces of bovine mesenteric arteries (BMA) as well as on the uptake of 45Ca2+ to a microsomal preparation of BMA. The effect of GTN and 8-Br-cGMP was also studied on the IP3-induced release of Ca2+ from the microsomal preparation preloaded with 45Ca2+. The influence of IP3 and GTN on the activity of Ca2(+)-ATPase in the microsomal preparation was tested as well. The phenylephrine-stimulated uptake of Ca2+ to tissue pieces of BMA was significantly reduced by the high GTN-concentration (1 microM) but not by the lower concentration. The uptake of Ca2+ to the microsomal preparation was significantly stimulated by the two GTN-concentrations tested, as well as by 8-Br-cGMP (0.1 mM). The calcium release induced by IP3 (1 microM) from the microsomal preparation was inhibited by both the low and the high GTN-concentration and by 8-Br-cGMP (0.1 mM). The Ca2(+)-ATPase activity was stimulated by both GTN-concentrations tested while it was inhibited by IP3. It is concluded that GTN is able to induce a reduction of the free intracellular Ca2+ by several mechanisms, which are of importance for the relaxation represented by the high-affinity component. The low-affinity component in addition reduces the inflow of Ca2+ over the plasma membrane.

Animals↗
PubMed1989

Phosphorylation of phospholamban and troponin I in the ischemic and reperfused heart: attenuation and restoration of isoprenaline responsiveness.

Acute myocardial ischemia maintained for 30 and 60 min with subsequent reperfusion did not induced alterations in the cyclic AMP-mediated phosphorylation capacity of phospholamban and troponin I. Inotropic stimulation of the normal heart with 0.1/uM isoprenaline for 2 min resulted in a simultaneous P-incorporation into phospholamban and troponin I to 44.4 +/- 7.5 pmoles P/mg protein and 42.4 +/- 2.9 pmoles P/mg protein, respectively, assayed by a standardized back-phosphorylation procedure. The adrenergic responsiveness, however, was markedly reduced in the time course of ischemia. After an ischemic period of 60 min the adrenergic-stimulated phosphorylation of phospholamban was diminished to 41 per cent of the control value, whereas the increase of troponin I phosphorylation was completely lost. This differential effect can be discussed in terms of the existence of cytosolic compartments for cA, possessing different lability to ischemic injury of cardiac cells. After post-ischemic reperfusion the isoprenaline responsiveness of the phosphorylation of phospholamban and troponin I was found to be normal demonstrating a reversibility at the level of two important regulator proteins, if the transient ischemia do not exceed 60 min period.

Adenosine Triphosphatases↗
PubMed1987

Isoproterenol induces both cAMP- and calcium-dependent phosphorylation of phospholamban in canine heart in vivo.

The phosphorylation state of phospholamban in hearts of dogs depleted from catecholamines or treated with isoproterenol has been characterized using a sensitive back-phosphorylation method that allows to distinguish between cAMP-dependent and Ca2+/calmodulin-dependent phosphorylation occurring at the protein in situ. The data obtained demonstrate that in response to the beta-adrenergic agonist isoproterenol both the cAMP-dependent and the Ca2+/calmodulin-dependent phosphorylatable site are phosphorylated suggesting a physiological significance also for Ca2+-dependent phosphorylation of phospholamban in canine heart in vivo.

Animals↗
PubMed1986

Metabolic and contractile changes in ischaemic rat hearts after isoproterenol administration: effect of reperfusion.

The effect of isoproterenol and of ischaemia followed by reperfusion was studied in isolated perfused rat heart. Whereas after an interval of 5 min of ischaemia the mechanical and biochemical responses to catecholamine were drastically reduced, reperfusion for 1 min normalized the cardiac reactivity. After prolonged ischaemia and reperfusion the hormone-induced rise in cyclic AMP and activation of cyclic AMP dependent protein kinase remains reduced. It is suggested that these changes might be of importance for cardiac protection during a transient ischaemic period.

Animals↗
PubMed1986

Indirect technique for the estimation of cAMP-dependent and Ca2+/calmodulin-dependent phospholamban phosphorylation state in canine heart in vivo.

An indirect technique was employed to estimate the in vivo phosphorylation state of phospholamban in preparations from dog hearts depleted from catecholamines and from dog hearts treated with isoproterenol. This method allows the separate detection of cAMP-dependent and Ca2+/calmodulin-dependent phospholamban phosphorylation. The data obtained demonstrate the phosphorylation of both the cAMP-dependent and the Ca2+/calmodulin-dependent phosphorylatable site of phospholamban in response to the beta-adrenergic agonist isoproterenol in canine heart in vivo.

Animals↗
PubMed1983

Cyclic nucleotides and changes in protein kinase activity ratio in the ischemic and nonischemic myocardium.

Following coronary artery ligation (CAL), levels of cAMP and the activity ratio of cAMP-dependent protein kinase, of phosphorylase kinase, and of phosphorylase are significantly elevated in both ischemic and nonischemic areas of the canine left ventricle. The aerobic level of cAMP was found to be 0.4 to 0.6 pmol/mg myocardium only after a precooled clamp or a cryobiopsy device was employed to guarantee tissue freezing in situ. Maximal changes in response to ischemia are observed within 2 min in both parts of the heart. Twenty minutes after the onset of ischemia, different responses have been found in the nonischemic and ischemic tissue. Whereas the levels of cAMP and the activity ratio of protein kinase, of phosphorylase kinase, and of phosphorylase returned to aerobic values in the nonischemic area, these parameters remained elevated in the ischemic area. The changes in the levels of myocardial cAMP and in the cAMP-dependent protein kinase activity ratio following CAL could be prevented by propranolol.

Animals↗
PubMed1982

Inhibition of soluble guanylate cyclase activity by citrate.

Soluble guanylate cyclase activity from guinea pig heart is inhibited by increasing concentrations of sodium citrate. The Ki value was found to be 2.83 +/- 0.05 mM in the presence of 3 mM Mn2+ and 0.6 mM GTP. Citrate acts by lowering Vmax and increasing the apparent values of Km for GTP and K0.5 for Mn2+ and Mg2+. The soluble guanylate cyclase, activated by sodium nitroprusside, was also inhibited by citrate. This inhibitory action of citrate was not restricted to soluble guanylate cyclase activity of the heart and has been demonstrated also in the supernatant of lung, liver, diencephalon and in the homogenate of blood platelets. Since citrate is known to be an important intermediate of metabolism, its intracellular concentration may be also of relevance for guanylate cyclase activity.

Animals↗