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E G Krause

Publications and source records attributed to E G Krause.

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

The endogenous cardiac sarcoplasmic reticulum Ca2+/calmodulin-dependent kinase is activated in response to beta-adrenergic stimulation and becomes Ca2+-independent in intact beating hearts.

We investigated the effects of beta-adrenergic stimulation on the activity of the endogenous cardiac sarcoplasmic reticulum Ca2+/calmodulin-dependent protein kinase (SRCaM kinase) in Langendorff-perfused rat hearts. We found that isoproterenol induced generation of autonomous (Ca2+-independent) SRCaM kinase activity to 28 +/- 4.4% of the total activity. Moreover, dephosphorylation of the autonomous SRCaM kinase with protein phosphatase 2A resulted in an enzyme that was again dependent on Ca2+ and calmodulin for its activity. Activation of SRCaM kinase was coupled to phospholamban phosphorylation and activation of the cAMP-signaling system. Our results suggest that the cardiac SRCaM kinase is activated in response to beta-adrenoceptor stimulation. This activation stimulates autophosphorylation at its regulatory domain and converts it to an active Ca2+-independent species that may be the basis for potentiation of Ca2+ transients in the heart.

Adenosine Triphosphatases↗
PubMed1997

Effects of zinc on phospholamban phosphorylation.

The effects of zinc on the phosphorylation of phospholamban (PLB) were studied in sarcoplasmic reticulum (SR) membranes prepared from swine ventricular muscle. Zinc produced a dose dependent inhibition of PLB phosphorylation. With the use of phosphorylation site specific antibodies, it was shown that this inhibition was specific for the PLB phosphorylation at Thr-17. Since phosphorylation of this site is known to be mediated by the Ca2+/calmodulin-dependent protein kinase endogenous to the cardiac SR (SRCaM kinase), the action of zinc on SRCaM kinase was investigated. It was found that (i) zinc inhibited the activity of SRCaM kinase (IC50: 15 microM) and (ii) zinc concentrations, at the millimolar range, stimulated Ca(2+)-independent SRCaM kinase autophosphorylation. This ability of zinc to differentiate between autophosphorylation and substrate phosphorylation activities of SRCaM kinase raises the possibility that zinc mediated independent regulation of these processes can occur in the intact heart.

Animals↗
PubMed1997

Altered expression of PDE1 and PDE4 cyclic nucleotide phosphodiesterase isoforms in 7-oxo-prostacyclin-preconditioned rat heart.

The stable prostacyclin derivative, 7-oxo-prostacyclin, exhibits a delayed, long-lasting cardioprotective effect, which is accompanied by an increase in cyclic nucleotide phosphodiesterase (PDE) activities restricted to the Ca2+-calmodulin-dependent (PDE1) and cyclic AMP-specific phosphodiesterase (PDE4) activities. Mammalian PDEs form a large multi-gene family with differential expression occurring in a cell- and tissue-specific manner. The aim of this study was to identify which isoforms of PDE1 and PDE4 are present in the hearts of control and 7-oxo-prostacyclin treated rats. Using RT-PCR analysis, we were able to identify in control rat hearts transcripts for PDE1C, but not for either PDE1A or PDE1B within the three-gene PDE1 family. Within the four-gene PDE4 family we detected, by generic RT-PCR analysis, transcripts for PDE4A, PDE4B and PDE4D, but not PDE4C. Using RT-PCR primers for specific splice variants, we identified transcripts for PDE4B1, PDE4B2, PDE4B3, PDE4D1, PDE4D2 and PDE4D3 in hearts from the control animals. Immunoblotting of hearts from the control animals for PDE4 forms allowed us to identify a 98-kDa PDE4A species, 68-kDa band representing PDE4D1/2 and a 95-kDa species indicative of PDE4D3. In the hearts of treated animals, 48 h after a single 50 microgram/kg dose of 7-oxo-prostacyclin, a profound increase in transcript levels was seen for both PDE1C and PDE4B3 together with a slight elevation for PDE4B1. No change in PDE4A transcripts occurred, which was consistent with a lack of change indicated in immunoblotting analyses. In contrast, 7-oxo-prostacyclin treatment caused decrease in transcript levels for PDE4D, which was confirmed by immunoblotting and shown to be due to a reduction in the levels of PDE4D3 and also in PDE4D1/D2. Thus, treatment of animals with 7-oxo-prostacyclin initiated profound isoform-specific changes in PDE expression in the myocardium which, presumably, underpin the increased PDE activity.

3',5'-Cyclic-AMP Phosphodiesterases↗
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↗
PubMed1996

Relation between contractile function and regulatory cardiac proteins in hypertrophied hearts.

The aim of this study was to examine the mechanism(s) underlying the reduced isoproterenol-induced positive inotropic and lusitropic effects in hypertrophied hearts. Chronic beta-adrenergic stimulation (2.4 mg isoproterenol.kg-1. day-1 for 4 days) induced cardiac hypertrophy by 33 +/- 2% in rats. A parallel downregulation of phospholamban (PLB) and sarcoplasmic reticulum Ca2(+)-ATPase (SERCA2) protein expression by 49 and 40%, respectively, was observed, whereas troponin I (TNI) and C protein remained unchanged. In papillary muscles from chronically beta-adrenergically stimulated rats, the isoproterenol-induced positive inotropic and lusitropic effects, as well as adenosine 3',5'-cyclic monophosphate (cAMP) accumulation, were attenuated compared with those in control animals. Acute exposure to isoproterenol induced phosphate incorporation into PLB, TNI, and C protein of 48 +/- 4.6, 55 +/- 5.0, and 27 +/- 4.9 pmol/mg homogenate protein, respectively, in control animals. In the hypertrophied hearts, phosphate incorporation into PLB was reduced by 76%, whereas phosphate incorporation into TNI or C protein remained unchanged. In conclusion, chronic beta-adrenergic stimulation reduced the isoproterenol-stimulated positive inotropic and lusitropic effects in papillary muscles, which were accompanied by 1) diminished cAMP formation, 2) attenuation of cAMP-mediated PLB phosphorylation, and 3) downregulation of PLB and SERCA2 protein.

Adenosine Triphosphatases↗
PubMed1996

Activation of cyclic AMP-dependent protein kinase in cardiomyocytes by anti-beta 1-adrenoceptor autoantibodies from patients with idiopathic dilated cardiomyopathy.

The serum of patients with idiopathic dilated cardiomyopathy contains agonistic autoantibodies that bind to beta 1-adrenoceptor and exert a positive chronotropic effect in cultured neonatal rat cardiomyocytes. In this study it is shown that these antibodies like isoproterenol increase the activity of cyclic AMP-dependent protein kinase (PKA) in the challenged myocytes. Cytosolic and particulate PKA activity was found to be increased as indicated by a rise in the PKA activity ratio from control values of 0.20 +/- 0.02 to 0.33 +/- 0.03 (p < 0.001), respectively, after exposure to the autoantibodies. Increases in PKA activity ratio by the autoantibodies as well as by isoproterenol were blocked in the presence of (-) propranolol. These findings complete the pathway by which the autoantibodies realize their chronotropic effects via beta-adrenoceptor-binding, cyclic AMP generation and activation of particulate and cytosolic cyclic AMP-dependent protein kinase.

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↗
PubMed1995

Effects of the phosphatase inhibitor calyculin A on the phosphorylation of C-protein in mammalian ventricular cardiomyocytes.

The effects of inhibitors of protein phosphatase activity on C-protein phosphorylation were studied in preparations from mammalian ventricles. Calyculin A (CyA), an inhibitor of type 1 and 2A protein phosphatases, was studied. CyA concentration- and time-dependency increased the phosphorylation state of C-protein in isolated 32P-labelled guinea pig ventricular cardiomyocytes. C-protein was identified by its reaction with a polyclonal antibody and immunoprecipitation. It is concluded that C-protein in intact cardiomyocytes could be a substrate for type 1 and 2A protein phosphatases.

Animals↗
PubMed1995

Cardioprotective potency of the radical scavenger S-2-(3 aminopropylamino) ethylphosphorothioic acid in the post-ischaemic rat heart.

S-2-(3 aminopropylamino) ethylphosphorothioic acid (WR-2721) shown to surpass radical scavenging thiols in their radioprotective efficacy in cancer-type diseases has been tested for its protective potential in the reperfused heart. We investigated the radical scavenger properties of the compound in a radical generating system in vitro as well as in isolated rat hearts subjected to 30 min ischaemia and 30 min reperfusion with the electron-paramagnetic resonance spin trap technique. The action on high-energy phosphates is observed by means of phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy while its influence on left ventricular systolic segmental length change (SSLC) during 60 min reperfusion following 60 min regional ischaemia was assessed with a fibreoptic system in anaesthetized open-chest rats. WR-2721 (0.1 mM) reduced the vascular concentration of radical adduct in isolated hearts by up to 78% (275 +/- 84% of pre-ischaemic baseline values vs 1260 +/- 413%, p < 0.05) between 5 and 12.5 min reperfusion. This was accompanied by a reduction of the left ventricular end diastolic pressure to pre-ischaemic values at 30 min of reperfusion (9 +/- 6 mmHg vs 42 +/- 8 mmHg in the absence of WR-2721, p < 0.02). An accelerated recovery of creatine phosphate levels (78 +/- 5% of pre-ischaemia values vs 41 +/- 5% within 60 min reperfusion: p < 0.05) was observed under similar conditions with NMR-spectroscopy, although the post-ischaemic tissue content of adenosine triphosphate was not affected.(ABSTRACT TRUNCATED AT 250 WORDS)

Amifostine↗
PubMed1995

4-(4-Guanidinobenzoyl)-2-imidazolones and related compounds: phosphodiesterase inhibitors and novel cardiotonics with combined histamine H2 receptor agonist and PDE III inhibitor activity.

A series of new positive inotropic agents was synthesized with the aim of combining the pharmacophores of the imidazolone-type phosphodiesterase (PDE) inhibitor enoximone and guanidine-type histamine H2 receptor agonists such as arpromidine. All compounds are para-substituted 4-benzoyl-5-alkyl-2-imidazolones. H2 agonism was incorporated by p-(hetero)arylalkyl substituents, in particular by an imidazolylpropyl guanidine group. In addition analogous ureas, cyanoguanidines, alkyl guanidine carboxylates, and amides were prepared. These functional groups were either directly attached to the phenyl ring or linked by an appropriate spacer. The compounds were screened for positive inotropic activity in the isolated electrically stimulated guinea pig papillary muscle and for inhibition of PDE III (cGMP-inhibited cAMP PDE, isolated from guinea pig heart). The cardiotonics obtained proved to be either PDE III inhibitors, some of them surmounting up to 3-fold the potency of enoximone, or pharmacological hybrids combining both PDE III inhibitor and histamine H2 receptor agonist activities. These hybrids were the most potent positive inotropic substances at the papillary muscle, probably due to their synergistic mechanism of action. The participation of histamine H2 receptors could be demonstrated in the papillary muscle preparation by pretreatment with the H2 antagonist famotidine (10 microM) as well as by further pharmacological experiments using isolated perfused hearts of guinea pigs and rats, isolated guinea pig right atria, adenylyl cyclase and H2 receptor binding assays. At equieffective concentrations the moderate PDE III inhibitor and histamine H2 agonist N1-(4-[(1,3-dihydro-5-methyl-2-oxo-3H-imidazol-4-yl)-carbonyl]phenyl)-N2 - [3-(1H-imidazol-4-yl)propyl]guanidine 65 and the 5-ethyl homologue 66 were about 2 and 10 times more potent than enoximone at the papillary muscle. Moreover, both compounds produced a 2.5-fold higher maximal response than the reference compound.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1995

Interaction of beta-adrenoceptor and adenosine receptor agonists on phosphorylation. Identification of target proteins in mammalian ventricles.

The influence of the adenosine derivative (--)-N6-phenylisopropyladenosine (R-PIA, 1 micron) and 5'N-ethylcarbox-amidoadenosine (NECA, 1 micron) on beta-adrenergic stimulated (isoproterenol, 10 nM) phosphorylation of sarcolemmal (15 kDa protein), sarcoplasmic reticular (phospholamban) and myofibrillar proteins (troponin I, C-protein) was studied in isolated 32P-labeled guinea-pig ventricles. The identification of the 15 kDa protein, phospholamban, troponin I and C-protein was based on their reaction with specific antibodies. Isoproterenol increased contractile parameters (developed tension, rate of tension development, rate of relaxation) and stimulated the phosphorylation state of a 15 kDa protein (now named phospholemman), of phospholamban, troponin I and C-protein (regarded as regulatory proteins). Isoproterenol concomitantly increased myocardial cyclic AMP levels. R-PIA and NECA attenuated the effects of isoproterenol on contractile parameters as well as on the phosphorylation of the regulatory proteins without affecting cyclic AMP levels. The effects of 1 microM R-PIA and 1 microM NECA on the isoproterenol-stimulated phosphorylation of regulatory proteins were blocked by the adenosine receptor antagonist 1.3-dipropyl-8-cyclopentylxanthine (DPCPX, 1 microM). Therefore, it is concluded that adenosine derivatives acting via adenosine receptors can reduce the isoproterenol-stimulated phosphorylation state of the following regulatory proteins: phospholemman, phospholamban, troponin I and C-protein.

Adenosine↗
PubMed1995

Immunoenzymometric assay of human glycogen phosphorylase isoenzyme BB in diagnosis of ischemic myocardial injury.

With a new immunoenzymometric assay we measured human glycogen phosphorylase isoenzyme BB (GPBB) in 116 healthy individuals, 14 patients with stable angina, 107 nontraumatic chest pain patients on admission to the emergency department [45 acute myocardial infarction (AMI), 49 unstable angina, 13 other diseases], and in serial samples from 41 AMI patients. GPBB was compared with creatine kinase (CK), CKMB mass, myoglobin, and cardiac troponin T. Receiver-operating characteristic plots demonstrated the significantly greater (P < or = 0.012) discriminatory power of GPBB to detect acute ischemic coronary syndromes compared with all other tested markers. GPBB was the most sensitive marker for detection of AMI during the first 4 h after onset of chest pain, and only GPBB was increased above the upper reference limit (7 micrograms/L) on admission in patients who had unstable angina at rest and reversible ST-T alterations. This and the high early sensitivity of GPBB are most likely explained by its function as a key enzyme of glycogenolysis.

Adult↗
PubMed1994

[Basic research in cardiology].

Phosphorylation of phospholamban and troponin I of dog hearts was measured depending on the cardiac cycle. Myocardium biopsies were obtained with the help of an ECG-triggered cryobiopsy device. A standardized back phosphorylation technique was performed with (32P)ATP and an excess of exogenically applied c-AMP dependent protein kinase, which was followed by electrophoretic fractionation. This standardized method enables quantitative measuring especially of the c-AMP-stimulated protein phosphorylation. A dichotomous behaviour in the phosphorylation of troponin I and phospholamban was detected in the cardiac cycle. Like c-AMP and the c-AMP depended protein kinase troponin I reach a maximum phosphorylation in the systole. In contrast to this, phospholamban in vivo shows the highest degree of phosphorylation in the diastole and drops to a significant lower value (by approximately 25%) in the systole. The surprising dichotomy support the assumption that the Ca-detensivation of the contractile proteins by phosphorylation of troponin I was a dynamic counterregulation in the presence of a high Ca-level takes effect before the Ca-accumulation which can be stimulated by phospholamban. The results show that covalent modifications of regulatory proteins come to pass in the ms-range and have to be considered in distinctive conditions of myocardial function.

Adenosine Triphosphate↗
PubMed1994

Long lasting anti-adrenergic effect of 7-oxo-prostacyclin in the heart: a cycloheximide sensitive increase of phosphodiesterase isoform I and IV activities.

Evidence is accumulating that 7-oxo-prostacyclin (7-oxo-PGI2) induces a delayed indirect anti-adrenergic and cytoprotective effect on the myocardium, the mechanism of which is still unclear. To demonstrate that a single application of 7-oxo-PGI2 (50 micrograms/kg i.m.) 48 h prior to starting experiments attenuates the isoprenaline inducible inotropic response and accumulation of cAMP, isolated hearts of pretreated animals were perfused in the Langendorff mode with and without isoprenaline (1 to 100 nM). The late anti-adrenergic effect of the drug was manifested by a significant attenuation in the elevation of cAMP levels as well as in contractile force development. This effect was not due to changes in cAMP generation as there were identical beta 1-adrenoceptor densities and affinities (as calculated from [3H]-CGP binding studies), Gi and G alpha s protein patterns (as taken from Western blots) as well as adenylyl cyclase activity measurements in the hearts studied. The anti-adrenergic potency of 7-oxo-PGI2, however, was found to be related to a significant rise in cyclic nucleotide hydrolysis by phosphodiesterase (PDE). Using the fast-performance liquid chromatographic separation for PDE isoforms, a significant increase in the activity of PDE isoforms I and IV (260 +/- 28 vs 110 +/- 12 pmol cGMP/min x enzyme fraction and 77 +/- 11 vs 34 +/- 3 pmol cAMP/min x enzyme fraction, respectively) was found in the solubilized fraction of cardiac membranes in comparison to untreated controls; PDE IV activity was also increased in the cytosolic fraction (106 +/- 14 vs 65 +/- 6 pmol cAMP/min x enzyme fraction). The hypothesis that the delayed anti-adrenergic effect of 7-oxo-PGI2 is initiated by an induction and accelerated synthesis of PDE I and IV in the heart is underlined by the fact that cycloheximide suppresses completely both the rise in PDE activities and the anti-adrenergic effects studied. It is suggested that an inducible predominance of cAMP degradation over its generation may be of relevance in processes related to heart protection.

Adenylyl Cyclases↗