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1.
Biochem Biophys Res Commun ; 477(1): 14-19, 2016 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-27237977

RESUMO

In cardiac tissue, regulatory light chain (RLC, myosin light chain 2) phosphorylation (Ser(15)) leads to modulation of muscle contraction through Ca(2+)-sensitization. To elucidate which kinases that are involved in the basal (diastolic phase) RLC phosphorylation, we studied non-contracting adult rat cardiomyocytes. RLC kinase activities in situ were unmasked by maximally inhibiting myosin light chain phosphatase (MLCP) by calyculin A in the absence and presence of various protein kinase inhibitors. Surprisingly MLCK did not contribute to the phosphorylation of RLC in the non-contracting cardiomyocytes. Two kinase activity groups were revealed by different sensitivities to staurosporine. The fraction with the highest sensitivity to staurosporine was inhibited by KN-93, a selective CaMKII inhibitor, producing a 23% ± 7% reduction in RLC phosphorylation. Calmodulin antagonism (W7) and reduction in Ca(2+) (EGTA) combined with low concentration of staurosporine caused a larger decrease in RLC phosphorylation than staurosporine alone. These data strongly suggest that in addition to CaMKII, there is another Ca(2+)/calmodulin-dependent kinase and a Ca(2+)/calmodulin-independent kinase phosphorylating RLC. Thus the RLC phosphorylation seems to be ensured by redundant kinase activities.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Miócitos Cardíacos/enzimologia , Proteínas Quinases/metabolismo , Animais , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Calmodulina/metabolismo , Masculino , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Wistar
2.
Biochem Biophys Res Commun ; 471(1): 219-25, 2016 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-26809094

RESUMO

The aim was to identify kinase activities involved in the phosphorylation of regulatory light chain (RLC) in situ in cardiomyocytes. In electrically stimulated rat cardiomyocytes, phosphatase inhibition by calyculin A unmasked kinase activities evoking an increase of phosphorylated RLC (P-RLC) from about 16% to about 80% after 80 min. The phosphorylation rate in cardiomyocytes was reduced by about 40% by the myosin light chain kinase (MLCK) inhibitor, ML-7. In rat ventricular muscle strips, calyculin A induced a positive inotropic effect that correlated with P-RLC levels. The inotropic effect and P-RLC elevation were abolished by ML-7 treatment. The kinase activities phosphorylating RLC in cardiomyocytes were reduced by about 60% by the non-selective kinase inhibitor staurosporine and by about 50% by the calmodulin antagonist W7. W7 eliminated the inhibitory effect of ML-7, suggesting that the cardiac MLCK is Ca(2+)/calmodulin (CaM)-dependent. The CaM-dependent kinase II (CaMKII) inhibitor KN-93 attenuated the calyculin A-induced RLC phosphorylation by about 40%, indicating a contribution from CaMKII. The residual phosphorylation in the presence of W7 indicated that also CaM-independent kinase activities might contribute. RLC phosphorylation was insensitive to protein kinase C inhibition. In conclusion, in addition to MLCK, CaMKII phosphorylates RLC in cardiomyocytes. Involvement of other kinases cannot be excluded.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Contração Miocárdica/fisiologia , Miócitos Cardíacos/fisiologia , Cadeias Leves de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Animais , Células Cultivadas , Ativação Enzimática , Regulação da Expressão Gênica/fisiologia , Masculino , Fosforilação/fisiologia , Ratos , Ratos Wistar
3.
Eur J Med Chem ; 64: 629-37, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23711770

RESUMO

5-HT4 receptor antagonists have been suggested to have clinical potential in treatment of atrial fibrillation, diarrhea-prone irritable bowel syndrome and urinary incontinence. Recently, the use of 5-HT4 antagonists has been suggested to have a therapeutic benefit in heart failure. Affinity for the hERG potassium ion channel and increased risk for prolonged QT intervals and arrhythmias has been observed for several 5-HT4 ligands. Serotonin may also have beneficial effects in the central nervous system (CNS) through stimulation of the 5-HT4 receptor, and reduced distribution of 5-HT4 antagonists to the CNS may therefore be an advantage. Replacing the amide and N-butyl side chain of the 5-HT4 receptor antagonist SB207266 with an ester and a benzyl dimethyl acetic acid group led to compound 9; a hydrophilic 5-HT4 antagonist with excellent receptor binding and low affinity for the hERG potassium ion channel. To increase oral bioavailability of carboxylic acid 9, two different prodrug approaches were applied. The tert-butyl prodrug 11 did not improve bioavailability, and LC-MS analysis revealed unmetabolized prodrug in the systemic circulation. The medoxomil ester prodrug 10 showed complete conversion and sufficient bioavailability of 9 to advance into further preclinical testing for treatment of heart failure.


Assuntos
Receptores 5-HT4 de Serotonina/metabolismo , Antagonistas do Receptor 5-HT4 de Serotonina/farmacologia , Administração Oral , Animais , Humanos , Interações Hidrofóbicas e Hidrofílicas , Masculino , Estrutura Molecular , Ratos , Ratos Sprague-Dawley , Antagonistas do Receptor 5-HT4 de Serotonina/administração & dosagem , Antagonistas do Receptor 5-HT4 de Serotonina/sangue , Relação Estrutura-Atividade
4.
Eur J Pharmacol ; 686(1-3): 66-73, 2012 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-22546232

RESUMO

Prostanoid-modulatory approaches in heart failure patients have displayed effects which may seem to be mutually incompatible. Both treatment with prostanoids and inhibition of prostanoid synthesis have resulted in increased mortality in heart failure patients. Currently, it is unknown if prostanoids mediate contractile effects in failing human heart and if this can explain some of the clinical effects seen after prostanoid modulatory treatments. Therefore, the objectives of this study were to determine if prostanoids could elicit direct inotropic responses in human ventricle, and if so to determine if they are modified in failing ventricle. Contractile force was measured in left ventricular strips from non-failing or failing human and rat hearts. The ratio of phosphorylated to non-phosphorylated myosin light chain 2 (MLC-2) was measured by Western blotting in myocardial strips, and the levels of prostanoid FP receptor mRNA and protein were measured in rat by real-time RT-PCR and receptor binding assays. In non-failing human hearts, prostanoids evoked a positive inotropic effect and an increase of MLC-2 phosphorylation which was absent in failing human hearts. In failing rat heart, the prostanoid FP receptor-mediated inotropic response and prostanoid FP receptor-density was reduced by ~40-50% compared to non-failing rat heart. Prostanoids mediate a sustained positive inotropic response in non-failing heart, which appears to be down regulated in failing heart. The pathophysiological significance of changes in prostanoid-mediated inotropic support in the failing heart remains to be determined.


Assuntos
Alprostadil/farmacologia , Insuficiência Cardíaca/fisiopatologia , Ventrículos do Coração/fisiopatologia , Iloprosta/farmacologia , Prostaglandinas F Sintéticas/farmacologia , Receptores de Prostaglandina/fisiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Miosinas Cardíacas/fisiologia , Criança , Modelos Animais de Doenças , Feminino , Ventrículos do Coração/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Contração Miocárdica/fisiologia , Cadeias Leves de Miosina/fisiologia , Ratos , Função Ventricular/efeitos dos fármacos
5.
Cardiovasc Res ; 85(4): 763-72, 2010 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-19900965

RESUMO

AIMS: Whereas natriuretic peptides increase cGMP levels with beneficial cardiovascular effects through protein kinase G, we found an unexpected cardio-excitatory effect of C-type natriuretic peptide (CNP) through natriuretic peptide receptor B (NPR-B) stimulation in failing cardiac muscle and explored the mechanism. METHODS AND RESULTS: Heart failure was induced in male Wistar rats by coronary artery ligation. Contraction studies were performed in left ventricular muscle strips. Cyclic nucleotides were measured by radio- and enzyme immunoassay. Apoptosis was determined in isolated cardiomyocytes by Annexin-V/propidium iodide staining and phosphorylation of phospholamban (PLB) and troponin I was measured by western blotting. Stimulation of NPR-B enhanced beta1-adrenoceptor (beta1-AR)-evoked contractile responses through cGMP-mediated inhibition of phosphodiesterase 3 (PDE3). CNP enhanced beta1-AR-mediated increase of cAMP levels to the same extent as the selective PDE3 inhibitor cilostamide and increased beta1-AR-stimulated protein kinase A activity, as demonstrated by increased PLB and troponin I phosphorylation. CNP promoted cardiomyocyte apoptosis similar to inhibition of PDE3 by cilostamide, indicative of adverse effects of NPR-B signalling in failing hearts. CONCLUSION: An NPR-B-cGMP-PDE3 inhibitory pathway enhances beta(1)-AR-mediated responses and may in the long term be detrimental to the failing heart through mechanisms similar to those operating during treatment with PDE3 inhibitors or during chronic beta-adrenergic stimulation.


Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Insuficiência Cardíaca/metabolismo , Peptídeo Natriurético Encefálico/farmacologia , Receptores Adrenérgicos beta 1/metabolismo , Transdução de Sinais/fisiologia , Animais , Apoptose/fisiologia , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Insuficiência Cardíaca/patologia , Masculino , Contração Miocárdica/fisiologia , Miócitos Cardíacos/metabolismo , Peptídeo Natriurético Encefálico/metabolismo , Peptídeo Natriurético Tipo C/metabolismo , Peptídeo Natriurético Tipo C/farmacologia , Inibidores da Fosfodiesterase 3 , Inibidores de Fosfodiesterase/farmacologia , Quinolonas/farmacologia , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos
6.
Cardiovasc Res ; 80(3): 407-15, 2008 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-18703533

RESUMO

AIMS: The aims of this study were to determine if the prostanoid F receptor (FPR)-mediated inotropic effect in rat ventricle is mediated by increased phosphorylation of myosin light chain-2 (MLC-2) and to elucidate the signalling pathway(s) activated by FPRs to regulate MLC-2 phosphorylation. METHODS AND RESULTS: Contractility was measured in left ventricular strips from adult male rats. Strips were also snap-frozen, and changes in the phosphorylation level of both MLC-2 and myosin phosphatase targeting subunit-2 (MYPT-2) were quantified. FPR stimulation with fluprostenol increased contractility by approximately 100% above basal and increased phosphorylation of both MLC-2 (by approximately 30%) and MYPT-2 (by approximately 50%). The FPR-mediated inotropic effect and MLC-2 phosphorylation were reduced by a similar magnitude in the presence of the myosin light chain kinase (MLCK) inhibitor ML-7 (approximately 60-70%) and an inhibitor of Ca(2+)/calmodulin, W-7 (approximately 35%). Inhibition of Rho-associated kinase by Y-27632 reduced the FPR-mediated inotropic effect and MLC-2 phosphorylation by approximately 40-45% and MYPT-2 phosphorylation by approximately 70%. ML-7 and Y-27632 together reduced contractility and MLC-2 phosphorylation by approximately 70-80%. The FPR-mediated inotropic effect was only modestly affected by high concentrations of the inositol tris-phosphate (IP(3)) receptor blocker 2-APB, but not by the protein kinase C (PKC) inhibitor bisindolylmaleimide. CONCLUSION: The FPR-evoked inotropic effect is mediated by increasing the phosphorylation of MLC-2 through regulation of both MLCK and myosin light chain phosphatase activities. The second messenger IP(3) and PKC are unlikely to be involved in the signalling cascade of the FPR-mediated positive inotropic effect. Therefore, FPR signalling mechanism(s) regulating MLC-2 phosphorylation likely extend beyond those classically established for G(q/11)-coupled receptors.


Assuntos
Miosinas Cardíacas/metabolismo , Ventrículos do Coração/metabolismo , Cadeias Leves de Miosina/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Receptores de Prostaglandina/metabolismo , Animais , Calmodulina/antagonistas & inibidores , Calmodulina/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/antagonistas & inibidores , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Contração Miocárdica/fisiologia , Quinase de Cadeia Leve de Miosina/antagonistas & inibidores , Quinase de Cadeia Leve de Miosina/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/antagonistas & inibidores , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Fosforilação , Proteína Quinase C/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais/fisiologia , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismo
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