RESUMO
OBJECTIVE: The aim was to identify the major ATP source controlling the activity of sarcolemmal K(ATP) channels in ventricular cardiomyocytes. METHODS: K(ATP)-channel current (I(KATP)) was measured with the patch-clamp technique in either the whole-cell (glycogenolysis blocked by 10 mmol/l EGTA), cell-attached, or inside-out configuration. RESULTS: In the absence of any substrate, I(KATP) (amplitude 31+/-4 nA; n=5) appeared spontaneously 520+/-160 s (n=6) after whole-cell access. This latency was shortened by exposure to anoxia (117+/-33 s, n=32) and even more by uncoupling (1-10 micromol/l FCCP; 25+/-3 s; n=13) while the amplitude was unchanged. During metabolic inhibition the latency was remarkably prolonged when the F1F0-ATPase was blocked by oligomycin, suggesting that under those conditions the F1F0-ATPase is the major ATP consumer. Glucose (5.5-20.0 mmol/l) in the bath solution did not affect the amplitude of I(KATP) but prolonged its latency compared to respective substrate-free conditions. However, I(KATP) was blocked immediately by mitochondrial substrates. FCCP also induced large I(KATP) in cell-attached measurements in either the absence or presence of glucose and oligomycin. CONCLUSIONS: The activity of K(ATP) channels in cardiomyocytes of mice is controlled by a cytosolic [ATP] pool for which oxidative phosphorylation is the predominant ATP source.
Assuntos
Trifosfato de Adenosina/metabolismo , Citosol/metabolismo , Hipóxia/metabolismo , Mitocôndrias Cardíacas/metabolismo , Miocárdio/metabolismo , Canais de Potássio/metabolismo , Animais , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Ácido Egtázico/farmacologia , Inibidores Enzimáticos/farmacologia , Glucose/metabolismo , Glucose/farmacologia , Glicogênio/metabolismo , Ventrículos do Coração , Camundongos , Oligomicinas/farmacologia , Fosforilação Oxidativa , Técnicas de Patch-Clamp , Canais de Potássio/efeitos dos fármacos , ATPases Translocadoras de Prótons/antagonistas & inibidores , Ácido Pirúvico/farmacologia , Desacopladores/farmacologiaRESUMO
In ventricular myocardial cells of the guinea pig and the mouse, anoxia caused after a mean latency of 439 +/- 141 s and 129 +/- 23 s (mean +/- S.E.M.), respectively, a large current through KATP-channels. This current disappeared within several seconds when reoxygenating the cells but decayed also completely at maintained anoxia. The kinetics of the latter process, however, were much slower and obeyed an approximately monoexponential time course with time constants in the range of 30 s. The results suggest that in the ischaemic myocardium KATP-channels contribute only to the initial phase of extracellular K+ accumulation.
Assuntos
Trifosfato de Adenosina/metabolismo , Miocárdio/metabolismo , Canais de Potássio/metabolismo , Animais , Hipóxia Celular , Cobaias , Técnicas In Vitro , Potenciais da Membrana , Camundongos , Miocárdio/citologiaRESUMO
Ventricular myocytes of the mouse ventricle were voltage clamped with a patch-clamp technique in the whole-cell configuration. At depolarizing voltage pulses, these myocytes develop a large voltage-dependent K+ outward current. Application of the drug dibenzylamine (DBA) to the bath solution blocked the voltage-dependent K+ current. The concentration/response relationship for the peak current at +40 mV indicates a 1:1 binding of the drug to the receptor with a concentration of half maximum effect of 43.1 micromol/l. The block did not require activation of the channels by depolarizing pulses. At concentrations causing partial block (25 micromol/l), the block was independent of voltage. At the same concentration, DBA completely blocked the slow component of the recovery from inactivation (-80 mV) whereas steady-state inactivation was not altered. It is concluded that DBA is a novel blocker of the voltage-dependent K+ current in mouse cardiac myocytes which preferentially affects the current component generating the slow recovery from inactivation.
Assuntos
Benzilaminas/farmacologia , Coração/efeitos dos fármacos , Miocárdio/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/metabolismo , Animais , Células Cultivadas , Ventrículos do Coração , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Técnicas de Patch-Clamp , Potássio/metabolismoRESUMO
The interaction of quinine with free hemin is of importance for the antimalarial effect of the drug in infected erythrocytes. We have investigated the kinetics of the complex formation of quinine with deuterohemin using the temperature jump relaxation method. We use ethyleneglycol-water mixtures as a solvent, where sufficient solubility for both species is provided and dimerization of the hemins, which involves mu-oxo bridges, can be controlled. Equilibrium and kinetic data for the dimerization of deuterohemin are given at 30 and 50 vol% ethyleneglycol. Binding of quinine is significantly slower than dimerization. Both processes are well separated on the time axis and yield a relaxation progress curve which is described with high accuracy by two exponential terms. The slow relaxation process is analyzed with respect to a 1:1 complex formation. This is the simplest mechanism which accounts for the present data, leading at 30 vol% ethyleneglycol, pH 7.5 to a binding constant of 10(4) M-1 and rate constants of 4.4 x 10(5) M-1 s-1 for the association and 44 s-1 for the dissociation step. However, there is evidence from the fast relaxation process that monomeric and dimeric hemin exhibit different reactivity. There is a strong decrease in rate with increasing pH. The implication of the results with respect to the proposed mechanisms of complex formation with quinoline drugs is discussed.
Assuntos
Hemina/análogos & derivados , Quinina/metabolismo , Etilenoglicóis/química , Hemina/química , Hemina/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Ligação Proteica , Quinina/química , Solubilidade , Espectrofotometria Ultravioleta , Temperatura , Água/químicaRESUMO
1. Mouse ventricular myocytes develop a large transient K+ outward current (I(TO)) which accelerates repolarization and is a crucial determinant for the regulation of the action potential duration at various heart rates. The effect of 3-hydroxybutyrate on I(TO) was investigated under voltage- and current-clamp conditions. 2. The drug blocked I(TO) in a stereoselective manner with the L-enantiomer being the effective and the D-enantiomer, the ineffective form. The blocking action of the L-enantiomer was established immediately and it could be completely washed out within several tens of seconds. 3. The dose-response characteristic for the peak I(TO) showed a strict 1:1 binding of the drug to the receptor with a concentration of half-maximum effect of 2.1 mmol l(-1). 4. The action of L-hydroxybutyrate was voltage independent, did not need channel opening and preferentially affected the slow component of both inactivation and recovery from inactivation. 5. At the high concentration of 20 mmol l(-1) the optically inactive form, the racemate, did not affect I(TO), indicating that the ineffective D-enantiomer interacts with the channels at much lower concentrations. 6. At a concentration of 10 mmol l(-1), L-hydroxybutyrate prolonged the action potential by 218 +/- 26 and 127 +/- 10% (means +/- S.E.M.) at 50 and 90% repolarization, respectively. 7. It is concluded that the non-physiological L-hydroxybutyrate is a novel type of blocker of I(TO), It interacts either with the channel itself, or with a receptor protein closely related to the channel and preferentially affects slow inactivation.