RESUMO
Angiotensin II (AngII) is a circulating peptide that produces a positive inotropic effect in the heart in several species, including humans. The subcellular mechanisms involved in producing this effect have been the focus of numerous studies; however, the results of these studies have generated considerable controversy. Although part of the controversy might arise from species and developmental differences, conflicting results have also been reported in the same species. To further complicate the understanding of the cardiac actions of AngII, the binding of the peptide to its transmembrane G-protein-coupled receptors has been shown to activate signalling cascades that involve numerous second messengers. Among these, inositol 1,4,5-triphosphate (IP3) and protein kinase C (PKC) have been shown to have the potential to modulate either one or both of the two basic mechanisms known to increase contractility: (i) an increase in the intracellular Ca2+ concentration ([Ca2+]i); or (ii) an increase in myofilament responsiveness to Ca2+. The aim of this review is to examine the effect of AngII on the fundamental components of cardiac excitation-contraction coupling: calcium currents, Na+/Ca2+ exchange, sarcoplasmic reticulum (SR)-CaZ+ release, calcium transients and contractile proteins. An answer to the following question is sought: Is the positive inotropic effect of AngII due to an increase in [Ca2+]i, to an increase in myofilament responsiveness to Ca2+, or to both?
RESUMO
Previous studies from our laboratory demonstrated the up-regulation of cardiac dihydropyridine (DHP) receptors in rabbits chronically treated with nifedipine (NIFE). The goal of the present study was to further examine the functionality of this increased number of receptors by analysing different steps of excitation contraction coupling mechanism in adult rats chronically treated with NIFE (a single 10-mg oral dose/kg/day for 28 days). Ca2+ channel density was assessed by specific binding at the DHP receptors with [methyl-(3)H]PN 200-110 in rat ventricular membranes. Chronic NIFE treatment produced up-regulation of Ca2+ channels, being the maximal binding capacities 222+/-19 fmol/mg protein (n=14) and 310+/-21 fmol/mg protein (n=11) in untreated and treated animals, respectively (P<0.05). The functional consequences of this up-regulation of Ca2+ channels were determined in isolated ventricular myocytes by measuring L-type Ca2+ currents (I(Ca)) with the whole-cell configuration of patch-clamp technique and by intracellular Ca2+ (Ca2+(i)) transients estimated by the Indo-1/AM fluorescence ratio (410/482) simultaneously monitored with cell shortening. Peak I(Ca) density recorded at 0 mV was 32% greater in myocytes isolated from the treated group than in those obtained from the untreated group (-10.43+/-0.73 pA/pF (n=13) vs-7.10+/-0.59 pA/pF (n=12) P<0.05). Ca2+(i) transient amplitude and cell shortening, explored at 1 and 2 mM extracellular calcium ([Ca]0) were significantly higher in ventricular myocytes obtained fom NIFE-treated rats than in myocytes isolated from untreated animals. At 2 mM [Ca]0, the values of Ca2+(i) transient and shortening were 460+/-61 nM and 11+/-1 % of resting length (L(0)) in myocytes from treated rats (n=9) and 212+/-22 nM and 5.3+/-0.5% of L(0) in myocytes from control rats (n=6, P<0.05). The results demonstrate an up-regulation of functionally-active cardiac Ca2+ channels after NIFE treatment, and offer a possible explanation for a "withdrawal effect" at myocardial level after the suppression of the treatment with this drug.
Assuntos
Canais de Cálcio Tipo L/fisiologia , Coração/fisiologia , Contração Miocárdica/fisiologia , Miocárdio/metabolismo , Nifedipino/farmacologia , Animais , Cálcio/farmacologia , Cálcio/fisiologia , Canais de Cálcio Tipo L/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Diástole/efeitos dos fármacos , Coração/efeitos dos fármacos , Ventrículos do Coração , Técnicas In Vitro , Isradipino/farmacocinética , Cinética , Masculino , Potenciais da Membrana/efeitos dos fármacos , Contração Miocárdica/efeitos dos fármacos , Técnicas de Patch-Clamp , Coelhos , Ensaio Radioligante , Ratos , Ratos Wistar , Sístole/efeitos dos fármacos , Trítio , Regulação para CimaRESUMO
1. The perforated whole-cell configuration of patch clamp and the pH fluorescent indicator SNARF were used to determine the electrogenicity of the Na+-HCO3- cotransport in isolated rat ventricular myocytes. 2. Switching from Hepes buffer to HCO3- buffer at constant extracellular pH (pHo) hyperpolarized the resting membrane potential (RMP) by 2.9 +/- 0.4 mV (n = 9, P < 0.05). In the presence of HCO3-, the anion blocker SITS depolarized RMP by 2.6 +/- 0.5 mV (n = 5, P < 0.05). No HCO3--induced hyperpolarization was observed in the absence of extracellular Na+. The duration of the action potential measured at 50 % of repolarization time (APD50) was 29.2 +/- 6.1 % shorter in the presence of HCO3- than in its absence (n = 6, P < 0.05). 3. Quasi-steady-state currents were evoked by voltage-clamped ramps ranging from -130 to +30 mV, during 8 s. The development of a novel component of Na+-dependent and Cl--independent steady-state outward current was observed in the presence of HCO3-. The reversal potential (Erev) of the Na+-HCO3- cotransport current (INa,Bic) was measured at four different levels of extracellular Na+. A HCO3-:Na+ ratio compatible with a stoichiometry of 2:1 was detected. INa,Bic was also studied in isolation in standard whole-cell experiments. Under these conditions, INa,Bic reversed at -96.4 +/- 1.9 mV (n = 5), being consistent with the influx of 2 HCO3- ions per Na+ ion through the Na+-HCO3- cotransporter. 4. In the presence of external HCO3-, after 10 min of depolarizing the membrane potential (Em) with 45 mM extracellular K+, a significant intracellular alkalinization was detected (0.09 +/- 0. 03 pH units; n = 5, P < 0.05). No changes in pHi were observed when the myocytes were pre-treated with the anion blocker DIDS (0.001 +/- 0.024 pH units; n = 5, n.s.), or when exposed to Na+-free solutions (0.003 +/- 0.037 pH units; n = 6, n.s.). 5. The above results allow us to conclude that the cardiac Na+-HCO3- cotransport is electrogenic and has an influence on RMP and APD of rat ventricular cells.
Assuntos
Proteínas de Transporte/metabolismo , Coração/fisiologia , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-dissulfônico/farmacologia , Animais , Benzopiranos , Bicarbonatos/metabolismo , Células Cultivadas , Corantes Fluorescentes , HEPES , Ventrículos do Coração , Concentração de Íons de Hidrogênio , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Miocárdio/citologia , Miocárdio/metabolismo , Técnicas de Patch-Clamp , Ratos , Sódio/metabolismo , Sódio/farmacologia , Simportadores de Sódio-Bicarbonato , Espectrometria de FluorescênciaRESUMO
Myocardial contractility falls quickly during respiratory acidosis but if acidosis is maintained a slow gradual return towards control state is detected. In cat papillary muscle, changes in developed tension (DT) during isometric contractions (pacing rate 0.2 Hz) and intracellular pH (pHi) were continuously monitored before and during hypercapnia to study the contribution of pHi recovery to the recovery of contractility. On exposure to hypercapnia (extracellular pH [pHo] = 6.90) DT fell to 50.33 +/- 2.20% of control and pHi decreased from 7.21 +/- 0.05 to 6.90 +/- 0.02. After 30 mins of hypercapnia DT recovered to 64.66 +/- 4.05% of control, but no significant recovery in pHi was detected. Intracellular sodium concentration slowly rose to 61.05 +/- 23.79% over basal level 10 mins after the onset of hypercapnia and it remained elevated for 10 mins before gradually returning to control levels. When pHo was kept at 7.40 during hypercapnia by increasing sodium bicarbonate concentration, DT recovered to 79.11 +/- 6.94% of control after 30 mins of hypercapnia, while a significant recovery of pHi (0.12 +/- 0.02 pH units) was detected. Low extracellular sodium concentration diminished contractility recovery during hypercapnia without changing the initial decrease in DT. 5-[N-ethyl-N-isopropyl] amiloride (EIPA) (5 microM) increased the initial fall in DT to 34.33 +/- 8.68% of control and abolished the recovery. Sarcoplasmic reticulum (SR) inhibition by ryanodine (0.5 microM) markedly reduced the recovery of contractility without altering the recovery in pHi.(ABSTRACT TRUNCATED AT 250 WORDS)