RESUMO
Zinc exists in biological systems as bound and histochemically reactive free Zn(2+) in the nanomolar range. Zinc is required as either structural or catalytic component for a large number of enzymes. It also modulates current passage through many ion channels. Here, we reinvestigated the effects of extracellular and intracellular Zn(2+) on the L-type Ca(2+) current (I (CaL)) and its modulation by ß-adrenergic stimulation in rat ventricular cardiomyocytes. In the absence of Ca(2+) ions, Zn(2+) could permeate through the L-type channel at much lower concentrations and at a more positive voltage range, but with a lower permeability than Ca(2+). In the presence of Ca(2+), extracellular Zn(2+) demonstrated strong bimodal inhibitory effects on the I (CaL), with half-inhibition occurring around 30 nM, i.e., in the range of concentrations found in the plasma. Intracellular Zn(2+) also significantly inhibited the I (CaL) with a half-inhibitory effect at 12.7 nM. Moreover, ß-adrenergic stimulation was markedly reduced by intracellular Zn(2+) at even lower concentrations (<1 nM) as a consequence of Zn(2+)-induced inhibition of the adenylyl cyclase. All these effects appeared independent of redox variations and were not affected by dithiothreitol. Thus, both basal intracellular and extracellular Zn(2+) modulate transmembrane Ca(2+) movements and their regulation by ß-adrenergic stimulation. Considering that, in many pathological situations, including diabetes, the extracellular Zn(2+) concentration is reduced and the intracellular one is increased, our results help to explain both Ca(2+) overload and marked reduction in the ß-adrenergic stimulation in these diseases.
Assuntos
Canais de Cálcio Tipo L/fisiologia , Miócitos Cardíacos/fisiologia , Zinco/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L/efeitos dos fármacos , Cardiopatias/metabolismo , Ventrículos do Coração/citologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Ratos , Zinco/farmacologiaRESUMO
Left ventricular remodeling after myocardial infarction is accompanied by electrical abnormalities that might predispose to rhythm disturbances. To get insight into the ionic mechanisms involved, we studied myocytes isolated from four different regions of the rat ventricles, 4-6 months after ligation of the left coronary artery. Using the whole-cell patch-clamp technique, we never observed T-type Ca(2+)current in both diseased and control hearts. In contrast, in 41 out of 78 cells isolated from 16 post-myocardial infarcted rats, analysed in the presence of 30 m m Na(+)ions, we found a tetrodotoxin (TTX)-resistant Na(+)current with quite variable amplitude in every investigated region. Albeit being resistant to 100 microM TTX, this Na(+)-dependent current was highly sensitive to lidocaine since 3 microM lidocaine induced about 65% tonic block. It was also inhibited by 5 microM nifedipine and 2 m m Co(2+), but was insensitive to 100 microM Ni(2+). The TTX-resistant Na(+)channel availability was shifted rightward by 25-30 mV with respect to TTX-sensitive Na(+)current; therefore, a large "window current" might flow in the voltage range from -70 to -20 mV. In conclusion, in late post-myocardial infarction, a Na(+)current with specific kinetics and pharmacology may provide inward charges in a critical range of membrane voltages that are able to alter action potential time course and trigger ventricular arrhythmia. These apparent new characteristics of the Na(+)channel might result in part from environmental changes during heart remodeling.
Assuntos
Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Sódio/metabolismo , Tetrodotoxina/farmacologia , Animais , Arritmias Cardíacas , Células Cultivadas , Íons , Cinética , Masculino , Técnicas de Patch-Clamp , Ratos , Ratos Wistar , Fatores de TempoRESUMO
Sodium ions have been reported to alter the permeation properties of L- and N-type Ca2+ channels. Here in frog atrial cardiomyocytes under whole-cell patch-clamp conditions, we have examined the effects of lowering the external Na+ concentration on the amplitude of T-type Ca2+ current, ICaT, and on the relief of its steady-state inactivation by large depolarizing prepulses, ICaT facilitation. A partial reduction in Na+ ion concentration did not significantly alter ICaT amplitude elicited at -50 mV. However, after a large depolarization, low- Na+ solutions enhanced the relief of inactivation and induced ICaT facilitation. This facilitation occurred independently of the divalent charge carrier, high intracellular Ca2+ buffering or the intracellular Na+ content. Its effects were additional to the beta-adrenergic effects mediated by a decrease of Gi/o-protein inhibitory tone. In Ca2+-free solution the very large T-type current, then carried by Na+ ions, showed only a weak relief of inactivation. In conclusion, ICaT facilitation--which, as previously reported, is modulated by the transient voltage-dependent relief of Gi-protein inhibitory tone--is further enhanced in a low-Na+ solution. In Ca2+-free solution, relief of inactivation due to re-openings dependent on the divalent charge carrier is improbable. It thus appears that for a short while after a large depolarization, external Na+ compete with Ca2+ ions on permeation-controlling sites, so as to modulate channel re-openings and thus the amplitude of voltage-facilitated ICaT independently of the control exerted by the inhibitory G-protein.
Assuntos
Função Atrial , Canais de Cálcio Tipo T/efeitos dos fármacos , Canais de Cálcio Tipo T/fisiologia , Sódio/farmacologia , Animais , Bário/metabolismo , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Condutividade Elétrica , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Átrios do Coração/efeitos dos fármacos , Cinética , Potenciais da Membrana , Fosforilação , Rana catesbeiana , Receptores Adrenérgicos beta/fisiologia , Estrôncio/metabolismoRESUMO
The two novel dihydropyridines, oxodipine and elgodipine greatly depressed the KCl-induced contraction of rabbit aorta and decreased the cardiac force of contraction of rat ventricular strips with lower potency. Both compounds markedly shortened cardiac action potentials. In rat cultured neonatal ventricular myocytes, oxodipine and elgodipine decreased the L-type Ca2+ current (I(CaL)) with IC50 of 0.24 and 0.33 microM respectively while oxodipine was slightly more potent on the T-type Ca2+ current (I(CaT)) than elgodipine (IC50 = 0.41 vs. 2.18 microM). Both compounds were less potent in inhibiting I(CaL) of adult cardiomyocytes. Oxodipine exhibited mostly a tonic block of both currents while elgodipine induced mainly a use-dependent block. Oxodipine and elgodipine increased by at least one order of magnitude their inhibitory potency on I(CaT) and I(CaL) when the cells were partially depolarized. We conclude that the mechanisms of inhibition of Ca2+ channels by these two dihydropyridines are different and suggest that the underlying mechanism of vascular selectivity is the voltage-dependent block of I(CaL), with the use-dependent inhibition of Ca2+ currents by elgodipine further contributing to this selectivity.
Assuntos
Potenciais de Ação/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/farmacologia , Di-Hidropiridinas/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Aorta/citologia , Células Cultivadas , Ventrículos do Coração/citologia , Técnicas de Patch-Clamp , Coelhos , Ratos , Ratos Wistar , Vasoconstrição/efeitos dos fármacosRESUMO
1. The properties of the low-threshold calcium current, ICa,T, were investigated in bullfrog isolated atrial cardiomyocytes using the whole-cell, patch-clamp technique under control conditions and during beta-adrenergic stimulation. 2. The intracellular application of GTP gamma S or adenosine-5'-O-3-thiotriphosphate (ATP gamma S), poorly hydrolysable analogues of GTP and ATP, respectively, barely affected ICa,T amplitude in control conditions. beta-Adrenergic stimulation effects were more marked in the presence of ATP gamma S. 3. The intracellular application of GDP beta S and ADP reduced ICa,T amplitude. In cells pretreated with pertussis toxin, ICa,T amplitude was significantly increased. In both conditions, the addition of isoprenaline was without effect. 4. Under both control and beta-adrenergic-stimulated conditions, a conditioning prepulse to +70 mV did not fully inactivate ICa,T; rather ICa,T facilitation often occurred after beta-adrenergic stimulation. 5. In GTP gamma S- and ATP gamma S-dialysed cells, ICa,T facilitation was generally observed after a prepulse; it was larger in the ATP gamma S dialysis. Facilitation was sustained but ended immediately upon cessation of conditioning prepulses. After beta-adrenergic stimulation, facilitation was more marked in GTP gamma S- than in ATP gamma S-dialysed cells. 6. ICa,T facilitation was prevented by the intracellular application of GDP beta S and by pertussis toxin pretreatment. 7. ICa,T facilitation developed markedly in the presence of intracellular cyclic AMP. This effect was prevented by pertussis toxin pretreatment of the cells. 8. It is thus proposed that ICa,T is under a double antagonistic control by both a Gs and a Gi protein. Furthermore, the double-pulse-induced facilitation of ICa,T results from a voltage-dependent relief of the Gi protein inhibitory tone. Such an effect is increased by protein kinase A-dependent phosphorylation, presumably of the Gi protein.
Assuntos
Canais de Cálcio/metabolismo , Proteínas de Ligação ao GTP/fisiologia , Átrios do Coração/metabolismo , Animais , Canais de Cálcio/efeitos dos fármacos , AMP Cíclico/farmacologia , Átrios do Coração/efeitos dos fármacos , Isoproterenol/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Técnicas de Patch-Clamp , Rana catesbeianaRESUMO
The properties of the low threshold Ca current (ICaT) in bullfrog (Rana catesbeiana) isolated atrial cardiomyocytes were studied using the whole-cell recording patch-clamp technique and compared with those of the high threshold Ca current (ICaL). In 91% of atrial cells we observed both ICaT and ICaL when collagenase and trypsin were used to dissociate the cells. But when pronase was used, only 30% of the cells exhibited ICaT. ICaT was never found in ventricular cells. ICaT could be investigated more easily when ICaL was inhibited by Cd ions (50 microM). Its kinetics were unchanged by substituting Ba for Ca, or in the presence of high concentrations of Ba. Both ICaT and ICaL exhibited reduced inactivation after high depolarizing prepulses. ICaT was found to be sensitive to dihydropyridines: 1 microM nifedipine decreased this current while 1 microM BAY K 8644 increased it; this occurred without significant variations in the steady-state inactivation curve. ICaT was more sensitive than ICaL to alpha 1-adrenergic and P2-purinergic stimulations, while ICaL was more sensitive to beta-adrenergic stimulation. Isoproterenol was still able to increase ICaT in the presence of high intracellular cAMP. Both currents were increased by 1 microM ouabain (although ICaL only transiently) and decreased by 10 microM ouabain. It is concluded that the two types of Ca channels can be observed in bullfrog atrial cells and that they are specifically altered by pharmacological agents and neuromediators. This may have implications for cardiac behavior.
Assuntos
Canais de Cálcio/fisiologia , Átrios do Coração/citologia , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Animais , Função Atrial , Bário/farmacologia , Cádmio/farmacologia , Cálcio/farmacocinética , Canais de Cálcio/ultraestrutura , Células Cultivadas , Relação Dose-Resposta a Droga , Átrios do Coração/química , Isoproterenol/farmacologia , Níquel/farmacologia , Nifedipino/farmacologia , Ouabaína/farmacologia , Fenilefrina/farmacologia , Propranolol/farmacologia , Rana catesbeianaRESUMO
The propyl derivative of ajmaline, N-n-propylajamaline (prajmalium), is an antiarrhythmic compound that lacks the commonly reported negative inotropic effects of all others under clinical use. The present study was undertaken to establish and understand its effects at the cellular level in mammalian preparations. Electrical and mechanical activities were recorded from right ventricular strips and Na and L-type Ca currents (INa and ICaL, respectively) were recorded with the whole-cell patch-clamp technique in right ventricular myocytes freshly dissociated from rabbit hearts. Prajmalium decreased the maximal rate of depolarization of the action potential in a dose-dependent manner with an EC50 of 3 microM. This effect was use and frequency dependent. Action potential duration was increased by 1 microM prajamalium but decreased on applying higher concentrations. The force of contraction was slightly (15%) increased at 0.1 microM, not affected at all at 1 microM and depressed by 30% at 20 microM. In single cardiomyocytes maintained at negative holding potentials, INa was slightly depressed by prajmalium at 10 nM and reduced by 75% at 10 microM. ICaL was increased by 30 and 20% on applying prajmalium at 1 and 10 microM, respectively; on the other hand, ICaL was reduced by these two concentrations of prajmalium at less negative holding potentials. A higher prajmalium concentration (100 microM) decreased ICaL at all holding potentials studies and this effect was enhanced with depolarization. The increase in ICaL induced by prajmalium (1 microM) was also observed after ICaL had been fully beta-adrenergic and P2-purinergic stimulated by isoproterenol (1 microM) in the presence of IBMX (100 microM) and ATP (10 microM). It is concluded that prajmalium is able to increase ICaL in rabbit ventricular cells in a voltage-dependent manner, an effect that could account in part for the observed lack of negative inotropism of this antiarrhythmic in clinics.
Assuntos
Coração/efeitos dos fármacos , Contração Miocárdica/efeitos dos fármacos , Prajmalina/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Cálcio/metabolismo , Relação Dose-Resposta a Droga , Eletrofisiologia , Coração/fisiologia , Técnicas In Vitro , Miocárdio/citologia , Coelhos , Sódio/metabolismo , Estimulação QuímicaRESUMO
The effects of N-n-propylajmaline (prajmalium) on the Na and Ca currents of single frog atrial and ventricular cells were studied by means of the whole-cell patch-clamp technique. Prajmalium (10(-9) to 10(-6) M) depressed the Na current (INa) in a dose- and use-dependent manner. In the same range of concentrations, prajmalium induced a dual effect on the high (ICaL) and low (ICaT) threshold Ca currents (the latter being only present in atrial cells). At a low concentration (10(-9) M), prajmalium increased both Ca currents while at high concentrations (10(-6) M) it depressed them. Prajmalium appeared very potent on ICaT although this current is generally reported to be barely sensitive to agonists and drugs. The action of the drug was also accompanied by a shortening in the half-time of inactivation of the Ca currents and a slight hyperpolarizing shift of their availability curves. The increase in ICaL by prajmalium was not prevented by prazosin (10(-7) M) nor by propranolol (10(-6) M), and it was also observed after ICaL had been fully stimulated by isoproterenol (10(-7)M). Nifedipine (10(-6) M), however, was able to prevent or block the prajmalium-induced increase in ICaL. Some similarities between the actions of prajmalium and dihydropyridine agonists on Ca currents are discussed.