RESUMO
We examined whether coupling of a ventricular myocyte to a non-myocyte cell expressing HCN2 could create a two-cell syncytium capable of generating sustained pacing. Three non-myocyte cell types were transfected with the mHCN2 gene and used as sources of mHCN2-induced currents. They were human mesenchymal stem cells and HEK293 cells, both of which express connexin43 (Cx43), and HeLa cells transfected with Cx43. Cell-cell coupling between heterologous pairs increased with time in co-culture, and hyperpolarization of the myocyte induced HCN2 currents, indicating current transfer from the mHCN2-expressing cell to the myocyte via gap junctions. The magnitude of the HCN2 currents recorded in myocytes increased with increasing junctional conductance. Once a critical level of electrical cell-cell coupling between myocytes and mHCN2 transfected cells was exceeded spontaneous action potentials were generated at frequencies of approximately 0.6 to 1.7 Hz (1.09 +/- 0.05 Hz). Addition of carbenoxolone (200 microM), a gap junction channel blocker, to the media stopped spontaneous activity in heterologous cell pairs. Carbenoxolone washout restored activity. Blockade of HCN2 currents by 100 microM 9-amino-1,2,3,4-tetrahydroacridine (THA) stopped spontaneous activity and subsequent washout restored it. Neither THA nor carbenoxolone affected electrically stimulated action potentials in isolated single myocytes. In summary, the inward current evoked in the genetically engineered (HCN2-expressing) cell was delivered to the cardiac myocyte via gap junctions and generated action potentials such that the cell pair could function as a pacemaker unit. This finding lays the groundwork for understanding cell-based biological pacemakers in vivo once an understanding of delivery and target cell geometry is defined.
Assuntos
Potenciais de Ação/fisiologia , Relógios Biológicos/fisiologia , Comunicação Celular/fisiologia , Canais Iônicos/metabolismo , Rim/fisiologia , Miócitos Cardíacos/fisiologia , Animais , Células Cultivadas , Cães , Retroalimentação Fisiológica/fisiologia , Células HeLa , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Canais de PotássioRESUMO
The M-current regulates the subthreshold electrical excitability of many neurons, determining their firing properties and responsiveness to synaptic input. To date, however, the genes that encode subunits of this important channel have not been identified. The biophysical properties, sensitivity to pharmacological blockade, and expression pattern of the KCNQ2 and KCNQ3 potassium channels were determined. It is concluded that both these subunits contribute to the native M-current.
Assuntos
Canais de Potássio/metabolismo , Potássio/metabolismo , Adulto , Animais , Antracenos/farmacologia , Encéfalo/metabolismo , Gânglios Simpáticos/metabolismo , Expressão Gênica , Humanos , Indóis/farmacologia , Canal de Potássio KCNQ2 , Canal de Potássio KCNQ3 , Cinética , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Oócitos , Técnicas de Patch-Clamp , Canais de Potássio/química , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Piridinas/farmacologia , Ratos , Sistema Nervoso Simpático/efeitos dos fármacos , Sistema Nervoso Simpático/fisiologia , Tetraetilamônio/farmacologia , XenopusRESUMO
Ventricular pacemaker current (I(f)) shows distinct voltage dependence as a function of age, activating outside the physiological range in normal adult ventricle, but less negatively in neonatal ventricle. However, heterologously expressed HCN2 and HCN4, the putative molecular correlates of ventricular I(f), exhibit only a modest difference in activation voltage. We therefore prepared an adenoviral construct (AdHCN2) of HCN2, the dominant ventricular isoform at either age, and used it to infect neonatal and adult rat ventricular myocytes to investigate the role of maturation on current gating. The expressed current exhibited an 18-mV difference in activation (V(1/2) -95.9+/-1.9 in adult; -77.6+/-1.6 mV in neonate), comparable to the 22-mV difference between native I(f) in adult and neonatal cultures (V(1/2) -98.7 versus -77.0 mV). This did not result from developmental differences in basal cAMP, because saturating cAMP in the pipette caused an equivalent positive shift in both preparations. In the neonate, AdHCN2 caused a significant increase in spontaneous rate compared with control (88+/-5 versus 48+/-4 bpm). In adult, where HCN2 activates more negatively, the effect was evident only during anodal excitation, requiring significantly less stimulus energy than control (2149+/-266 versus 3140+/-279 mV. ms). Thus, ventricular maturational state influences the voltage dependence of expressed HCN2, resulting in distinct physiological impact of expressed channels in neonate and adult myocytes. The full text of this article is available at http://www.circresaha.org.
Assuntos
Ventrículos do Coração/crescimento & desenvolvimento , Canais Iônicos/fisiologia , Proteínas Musculares , Função Ventricular , Animais , Animais Recém-Nascidos , Células Cultivadas , AMP Cíclico/farmacologia , Condutividade Elétrica , Ventrículos do Coração/efeitos dos fármacos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Ativação do Canal Iônico , Canais Iônicos/genética , Canais de Potássio , RatosRESUMO
The Ca(2+)-independent portion of transient outward K(+) current (I(to)) exhibits a transmural gradient in ventricle. To investigate control mechanisms for this gradient, we studied canine epicardial and endocardial ventricular myocytes with use of the whole-cell patch-clamp technique. I(to) was larger in amplitude, had a more negative voltage threshold for activation, and had a more negative midpoint of inactivation in epicardium. Recovery from inactivation was >10-fold slower in endocardium. Incubation of epicardial myocytes with angiotensin II for 2 to 52 hours altered I(to) to resemble unincubated endocardium and reduced the amplitude of the phase 1 notch of the action potential. In contrast, incubation of endocardial myocytes with losartan for 2 to 52 hours altered I(to) to resemble unincubated epicardium and induced a phase 1 notch in the action potential. With RNase protection assays, we determined that incubations with angiotensin II or losartan did not alter mRNA levels for either Kv4.3 or Kv1.4; thus, a change in the alpha subunit for I(to) is unlikely to be responsible. To test whether posttranslational modification produced the effects of angiotensin II, we coexpressed Kv4.3 and the angiotensin II type 1a receptor in Xenopus oocytes. Incubation with angiotensin II increased the time constant for recovery from inactivation of the expressed current by 2-fold with an incubation time constant of 3.7 hours. No effect on activation or inactivation voltage dependence was observed. These results demonstrate that the properties of I(to) in endocardium and epicardium are plastic and likely under the tonic-differing influence of the renin-angiotensin system.
Assuntos
Endocárdio/fisiologia , Pericárdio/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/fisiologia , Sistema Renina-Angiotensina/fisiologia , Função Ventricular , Potenciais de Ação , Angiotensina II/farmacologia , Animais , Cães , Condutividade Elétrica , Endocárdio/efeitos dos fármacos , Feminino , Ativação do Canal Iônico , Canal de Potássio Kv1.4 , Masculino , Miocárdio/citologia , Oócitos/metabolismo , Pericárdio/efeitos dos fármacos , Canais de Potássio/genética , RNA Mensageiro/metabolismo , Receptor Tipo 1 de Angiotensina , Receptor Tipo 2 de Angiotensina , Receptores de Angiotensina/metabolismo , Canais de Potássio Shal , Xenopus laevisRESUMO
HCN cation channel mRNA expression was determined in the rabbit heart and neonatal and adult rat ventricle using RNase protection assays. In the rabbit SA node, the dominant HCN transcript is HCN4, representing >81% of the total HCN message. HCN1 is also expressed, representing >18% of the total HCN mRNA. Rabbit Purkinje fibers contained almost equal amounts of HCN1 and HCN4 transcripts with low levels of HCN2, whereas rabbit ventricle contained predominantly HCN2. The SA node contained 25 times the total HCN message of Purkinje fibers and 140 times the total HCN message of ventricle. No reports of hyperpolarization-activated current (If) exist in rabbit Purkinje fibers, and we could not record If in rabbit ventricular myocytes. To investigate the possible role of isoform switching in determining the voltage dependence of If, we determined the prevalence of HCN isoforms in neonatal and adult rat ventricle. We had previously determined the threshold for activation of If to be approximately -70 mV in neonatal rat ventricle and -113 mV in adult rat ventricle. In both neonatal and adult rat ventricle, only HCN2 and HCN4 transcripts are present. The ratio of HCN2 to HCN4 is approximately 5:1 in the neonate and 13:1 in the adult. Taken together, these results suggest that different cardiac regions express different isoforms of the HCN family. The HCN1 and HCN4 isoforms are most closely associated with a depolarized threshold for If activation, whereas the HCN2 isoform is associated with a more negative activation curve.
Assuntos
Ventrículos do Coração/metabolismo , Canais Iônicos/metabolismo , Nó Sinoatrial/metabolismo , Animais , Animais Recém-Nascidos , Eletrofisiologia , Canais Iônicos/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , Coelhos , Ratos , Nó Sinoatrial/fisiologia , Função VentricularRESUMO
The HCN family of ion channel subunits underlies the currents I(f) in heart and I(h) and I(q) in the nervous system. In the present study, we demonstrate that minK-related peptide 1 (MiRP1) is a beta subunit for the HCN family. As such, it enhances protein and current expression as well as accelerating the kinetics of activation. Because MiRP1 also functions as a beta subunit for the cardiac delayed rectifier I(Kr), these results suggest that this peptide may have the unique role of regulating both the inward and outward channels that underlie cardiac pacemaker activity. The full text of this article is available at http://www.circresaha.org.
Assuntos
Canais Iônicos/metabolismo , Proteínas Musculares , Proteínas do Tecido Nervoso , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/metabolismo , Subunidades Proteicas , Animais , Northern Blotting , Células Cultivadas , Canais de Cátion Regulados por Nucleotídeos Cíclicos , Expressão Gênica , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Ativação do Canal Iônico/fisiologia , Canais Iônicos/genética , Potenciais da Membrana/fisiologia , Camundongos , Dados de Sequência Molecular , Família Multigênica , Oócitos/citologia , Oócitos/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio/genética , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Coelhos , Ratos , Transfecção , Xenopus laevisRESUMO
The ionic basis of automaticity in the sinoatrial node and His-Purkinje system, the primary and secondary cardiac pacemaking regions, is discussed. Consideration is given to potential targets for pharmacologic or genetic therapies of rhythm disorders. An ideal target would be an ion channel that functions only during diastole, so that action potential repolarization is not affected, and one that exhibits regional differences in expression and/or function so that the primary and secondary pacemakers can be selectively targeted. The so-called pacemaker current, If, generated by the HCN gene family, best fits these criteria. The biophysical and molecular characteristics of this current are reviewed, and progress to date in developing selective pharmacologic agents targeting If and in using gene and cell-based therapies to modulate the current are reviewed.
Assuntos
Sistema de Condução Cardíaco/fisiologia , Canais Iônicos/fisiologia , Potenciais de Ação , Animais , Fascículo Atrioventricular/fisiologia , Canais de Cálcio/fisiologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Modelos Animais , Proteínas Musculares/fisiologia , Canais de Potássio , Ramos Subendocárdicos/fisiologia , Nó Sinoatrial/fisiologia , Canais de Sódio/fisiologiaRESUMO
The effects of the potassium channel openers (KCOs) Cromakalim and Lemakalim on rabbit sinoatrial and atrial myocytes were examined by means of the whole-cell patch-clamp technique. Lemakalin (up to 100 microM) had no effect on potassium current in sinoatrial cells. Both Lemakalim and Cromakalim (100 microM) displayed a two-fold action on atrial myocytes: (1) they increased an outwardly rectifying conductance at potentials positive to EK and, (2) they markedly decreased a TTX-sensitive Na+ current active in the voltage range -50/-30 mV. This novel action on TTX-sensitive currents is of particular interest since these two benzopyrans have been thought to specifically target potassium channels.
Assuntos
Átrios do Coração/metabolismo , Canais de Potássio/efeitos dos fármacos , Potássio/metabolismo , Nó Sinoatrial/metabolismo , Sódio/metabolismo , Animais , Benzopiranos/farmacologia , Células Cultivadas , Cromakalim , Pirróis/farmacologia , Coelhos , TetrodotoxinaRESUMO
Our previous results have demonstrated that tyrosine kinase inhibition reduces i(f) in rabbit SA node myocytes, suggesting that tyrosine kinases regulate i(f). One receptor tyrosine kinase the EGF receptor kinase is known to increase heart rate. To determine if this action is mediated through changes in i(f), we examined the effect of epidermal growth factor (EGF) on i(f) with the permeabilized patch-clamp technique. 0.1 microM EGF increased i(f) amplitude in response to single-step hyperpolarizations in the diastolic range of potentials. This increase was 20+/-3%, n=11 at -75 mV. This effect is caused by activating a tyrosine kinase because 50 microM genistein, a tyrosine kinase inhibitor, eliminated this EGF action. A two-step pulse protocol showed that maximal i(f) conductance was increased by EGF. We further examined this conductance change by constructing the activation curve. The maximal i(f) conductance was increased by 23% with no change in midpoint, V(1/2), control=-74+/-2 mV, V(1/2) EGF=-74+/-1 mV. Thus EGF acts via a tyrosine kinase to increase maximal i(f) conductance with no change in the voltage dependence of activation. These results suggest that EGF effects on i(f) contribute to the positive chronotropic effect of EGF on SA node.
Assuntos
Fator de Crescimento Epidérmico/farmacologia , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/metabolismo , Proteínas Tirosina Quinases/metabolismo , Nó Sinoatrial/efeitos dos fármacos , Nó Sinoatrial/metabolismo , Animais , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Genisteína/farmacologia , Frequência Cardíaca/efeitos dos fármacos , Frequência Cardíaca/fisiologia , Técnicas In Vitro , Potenciais da Membrana , Técnicas de Patch-Clamp , Proteínas Tirosina Quinases/antagonistas & inibidores , Coelhos , Nó Sinoatrial/citologiaRESUMO
BACKGROUND: ACE inhibitors may not adequately suppress deleterious levels of angiotensin II in patients with heart failure. An angiotensin receptor blocker added to an ACE inhibitor may exert additional beneficial effects. METHODS AND RESULTS: Eighty-three symptomatic stable patients with chronic heart failure receiving long-term ACE inhibitor therapy were randomly assigned to double-blind treatment with valsartan 80 mg BID, valsartan 160 mg BID, or placebo while receiving their usual ACE inhibitor therapy. Studies were performed before and after the first dose of the test drug and again after 4 weeks of therapy. A single dose of lisinopril was administered during study days to ensure sustained ACE inhibition. Compared with placebo, the first dose of valsartan 160 mg resulted in a significantly greater reduction in pulmonary capillary wedge pressure at 3, 4, and 8 hours and during the prespecified 4- to 8-hour interval after the dose and in systolic blood pressure at 2, 3, 6, 8, and 12 hours and 4 to 8 hours after the dose. A pressure reduction from valsartan 80 mg did not achieve statistical significance. After 4 weeks of therapy, net reductions in 0-hour trough pulmonary capillary wedge pressure (-4.3 mm Hg; P=0. 16), pulmonary artery diastolic pressure (-4.7 mm Hg; P=0.013), and systolic blood pressure (-6.8 mm Hg; P=0.013) were observed in the valsartan 160 mg group compared with placebo. After 4 weeks of therapy, plasma aldosterone was reduced by valsartan 80 mg BID (-52. 1 pg/mL; P=0.001) and 160 mg BID (-47.8 pg/mL; P<0.001) compared with placebo, and there was a trend for a reduction in plasma norepinephrine (-97 pg/mL; P=0.10). Seventy-four of the 83 patients completed the trial. CONCLUSIONS: Physiologically active levels of angiotensin II persist during standard long-term ACE inhibitor therapy.
Assuntos
Antagonistas de Receptores de Angiotensina , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Insuficiência Cardíaca/tratamento farmacológico , Hemodinâmica/efeitos dos fármacos , Hormônios/sangue , Lisinopril/uso terapêutico , Tetrazóis/uso terapêutico , Valina/análogos & derivados , Idoso , Relação Dose-Resposta a Droga , Método Duplo-Cego , Quimioterapia Combinada , Humanos , Pessoa de Meia-Idade , Método Simples-Cego , Tetrazóis/efeitos adversos , Fatores de Tempo , Valina/efeitos adversos , Valina/uso terapêutico , ValsartanaRESUMO
BACKGROUND: Cardiac memory refers to an altered T-wave morphology induced by ventricular pacing or arrhythmias that persist for variable intervals after resumption of sinus rhythm. METHODS AND RESULTS: We induced long-term cardiac memory (LTM) in conscious dogs by pacing the ventricles at 120 bpm for 3 weeks. ECGs were recorded daily for 1 hour, during which time pacing was discontinued. At terminal study, the heart was removed and the electrophysiology of left ventricular epicardial myocytes was investigated. Control (C) and LTM ECG did not differ, except for T-wave amplitude, which decreased from 0.12+/-0.18 to -0.34+/-0.21 mV (+/-SEM, P<0.05), and T-wave vector, which shifted from -37+/-12 degrees to -143+/-4 degrees (P<0.05). Epicardial action potentials revealed loss of the notch and lengthening of duration at 20 days (both P<0.05). Calcium-insensitive transient outward current (Ito) was investigated by whole-cell patch clamp. No difference in capacitance was seen in C and LTM myocytes. Ito activated on membrane depolarization to -25+/-1 mV in C and -7+/-1 mV (P<0.05) in LTM myocytes, indicating a positive voltage shift of activation. Ito density was reduced in LTM myocytes, and a decreased mRNA level for Kv4.3 was observed. Recovery of Ito from inactivation was significantly prolonged: it was 531+/-80 ms (n=10) in LTM and 27+/-6 ms (n=9) in C (P<0.05) at -65 mV. CONCLUSIONS: Ito changes are associated with and can provide at least a partial explanation for action-potential and T-wave changes occurring with LTM.
Assuntos
Estimulação Cardíaca Artificial , Coração/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana , 4-Aminopiridina/farmacologia , Potenciais de Ação/fisiologia , Animais , Cães , Condutividade Elétrica , Eletrocardiografia , Coração/efeitos dos fármacos , Técnicas In Vitro , Miocárdio/citologia , Pericárdio/fisiologia , Canais de Potássio/genética , RNA Mensageiro/metabolismo , Canais de Potássio Shal , Fatores de TempoRESUMO
We have examined slow inactivation of L-type calcium current in canine Purkinje myocytes with the whole cell patch clamp technique. Slow inactivation is voltage dependent. It is negligible at -50 mV but can inactivate more than half of available iCaL at -10 mV. There are two major consequences of this slow inactivation. First, standard protocols for the measurement of T-type current can dramatically overestimate its contribution to total calcium current, and second, the position and steepness of the inactivation versus voltage curve for iCaL will depend on the method of measurement. Given the widespread attempts to identify calcium current components and characterize them biophysically, an important first step should be to determine the extent of slow inactivation of calcium current in each preparation.
Assuntos
Canais de Cálcio/fisiologia , Miocárdio/citologia , Células de Purkinje/citologia , Células de Purkinje/fisiologia , Animais , Canais de Cálcio/análise , Cães , Estimulação Elétrica , Coração/fisiologia , Potenciais da Membrana/fisiologia , Miocárdio/química , Miocárdio/ultraestrutura , Células de Purkinje/químicaRESUMO
Membrane current following prolonged periods of rapid stimulation was examined in short (less than 1.5 mm) canine cardiac Purkinje fibers of radius less than 0.15 mm. The Purkinje fibers were repetitively stimulated by delivering trains of depolarizing voltage clamp pulses at rapid frequencies. The slowly decaying outward current following repetitive stimulation ("post-drive" current) is eliminated by the addition of 10(-5) M dihydro-ouabain. The post-drive current is attributed to enhanced Na/K exchange caused by Na loading during the overdrive. Depolarizing voltage clamp pulses initiated from negative (-80 mV) or depolarized (-50 mV) holding potentials can give rise to post-drive current because of activation of tetrodotoxin-sensitive or D600-sensitive channels. The magnitude of the post-drive current depends on the frequency of voltage clamp pulses, the duration of each pulse, and the duration of the repetitive stimulation. The time constant of decay of the post-drive current depends on extracellular [K] in accordance with Michaelis-Menten kinetics. The Km is 1.2 mM bulk [K], [K]B. The mean time constant in 4 mM [K]B is 83 s. Epinephrine (10(-5) M) decreases the time constant by 20%. The time constant is increased by lowering [Ca]o between 4 and 1 mM. Lowering [Ca]o further, to 0.1 mM, eliminates post-drive current following repetitive stimulation initiated from depolarized potentials. The latter result suggests that slow inward Ca2+ current may increase [Na]i via Na/Ca exchange.
Assuntos
Sistema de Condução Cardíaco/fisiologia , Canais Iônicos/fisiologia , Ramos Subendocárdicos/fisiologia , Animais , Cálcio/farmacologia , Cães , Condutividade Elétrica , Estimulação Elétrica , Epinefrina/farmacologia , Galopamil/farmacologia , Líquido Intracelular/metabolismo , Modelos Biológicos , Ouabaína/análogos & derivados , Ouabaína/farmacologia , Sódio/metabolismo , Temperatura , Tetrodotoxina/farmacologia , Fatores de TempoRESUMO
The role of the Na/Ca exchanger in the control of cellular excitability and tension development is a subject of current interest in cardiac physiology. It has been suggested that this coupled transporter is responsible for rapid changes in intracellular calcium activity during single beats, generation of plateau currents, which control action potential duration, and control of intracellular sodium during Na/K pump suppression, which may occur during terminal states of ischemia. The actual behavior of this exchanger is likely to be complex for several reasons. First, the exchanger transports two ionic species and thus its instantaneous flux rate depends on both intracellular sodium and calcium activity. Secondly, the alteration in intracellular calcium activity, which is caused by a given transmembrane calcium flux, and which controls the subsequent exchanger rate, is a complex function of available intracellular calcium buffering. The buffers convert the ongoing transmembrane calcium fluxes into changes in activity that are a small and variable fraction of the change in total calcium concentration. Using a number of simple assumptions, we model changes in intracellular calcium and sodium concentration under the influence of Na/Ca exchange, Na/K ATPase and Ca-ATPase pumps, and passive sodium and calcium currents during periods of suppression and reactivation of the Na/K ATPase pump. The goal is to see whether and to what extent general notions of the role of the Na/Ca exchanger used in planning and interpreting experimental studies are consistent with its function as derived from current mechanistic assumptions about the exchanger. We find, for example, that based on even very high estimates of intracellular calcium buffering, it is unlikely that Na/Ca exchange alone can control intracellular sodium during prolonged Na/K pump blockade. It is also shown that Na/Ca exchange can contaminate measurements of Na/K pump currents under a variety of experimental conditions. The way in which these and other functions are affected by the dissociation constants and total capacity of the intracellular calcium buffers are also explored in detail.
Assuntos
Membranas Intracelulares/metabolismo , Íons , Modelos Biológicos , Transporte Biológico , Soluções Tampão , Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Concentração Osmolar , Canais de Potássio/metabolismo , Sódio/metabolismo , Canais de Sódio/metabolismo , Trocador de Sódio e CálcioRESUMO
We have characterized the inward rectifying background potassium current, iK1, of canine cardiac Purkinje myocytes in terms of its reversal potential, voltage activation curve, and "steady-state" current-voltage relation. The latter parameter was defined from the difference current between holding currents in the presence and absence of 20 mM cesium. Our data suggest that iK1 rectification does not arise exclusively from voltage-dependent gating or exclusively from voltage-dependent blockade by internal magnesium ions. The voltage activation curve constructed from tail currents fit to a Boltzmann two-state model predicts less outward current than is actually observed. The magnesium-dependent rectification due to channel blockade is too fast to account for the time-dependent gating of iK1 that gives rise to the tail currents. We propose a new model of rectification that assumes that magnesium blockade of the channel occurs simultaneously with voltage-dependent gating. The new model incorporates the kinetic schema elaborated by Matsuda, H. (1988. J. Physiol. 397:237-258) to explain the appearance of subconducting states of the iK1 channel in the presence of blocking ions. That schema suggested that iK1 channels were composed of three parallel pores, each of which could be blocked independently. In our model we considered the consequences of partial blockade of the channel. If the channels are partially blocked at potentials where normally they are mostly gated closed, and if the partially blocked channels cannot close, then blockade will have the paradoxical result of enhancing the current carried by iK1.
Assuntos
Condutividade Elétrica/fisiologia , Potenciais da Membrana/fisiologia , Ramos Subendocárdicos/citologia , Trifosfato de Adenosina/farmacologia , Animais , Césio/farmacologia , Cães , Relação Dose-Resposta a Droga , Condutividade Elétrica/efeitos dos fármacos , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/fisiologia , Canais Iônicos/ultraestrutura , Magnésio/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Modelos Biológicos , Ramos Subendocárdicos/fisiologia , Ramos Subendocárdicos/ultraestruturaRESUMO
It is generally assumed that in cardiac Purkinje fibers the hyperpolarization activated inward current i(f) underlies the pacemaker potential. Because some findings are at odds with this interpretation, we used the whole cell patch clamp method to study the currents in the voltage range of diastolic depolarization in single canine Purkinje myocytes, a preparation where many confounding limitations can be avoided. In Tyrode solution ([K+]o = 5.4 mM), hyperpolarizing steps from Vh = -50 mV resulted in a time-dependent inwardly increasing current in the voltage range of diastolic depolarization. This time-dependent current (iKdd) appeared around -60 mV and reversed near EK. Small superimposed hyperpolarizing steps (5 mV) applied during the voltage clamp step showed that the slope conductance decreases during the development of this time-dependent current. Decreasing [K+]o from 5.4 to 2.7 mM shifted the reversal potential to a more negative value, near the corresponding EK. Increasing [K+]o to 10.8 mM almost abolished iKdd. Cs+ (2 mM) markedly reduced or blocked the time-dependent current at potentials positive and negative to EK. Ba2+ (4 mM) abolished the time-dependent current in its usual range of potentials and unmasked another time-dependent current (presumably i(f)) with a threshold of approximately -90 mV (> 20 mV negative to that of the time-dependent current in Tyrode solution). During more negative steps, i(f) increased in size and did not reverse. During i(f) the slope conductance measured with small (8-10 mV) superimposed clamp steps increased. High [K+]o (10.8 mM) markedly increased and Cs+ (2 mM) blocked i(f). We conclude that: (a) in the absence of Ba2+, a time-dependent current does reverse near EK and its reversal is unrelated to K+ depletion; (b) the slope conductance of that time-dependent current decreases in the absence of K+ depletion at potentials positive to EK where inactivation of iK1 is unlikely to occur. (c) Ba2+ blocks this time-dependent current and unmasks another time-dependent current (i(f)) with a more negative (> 20 mV) threshold and no reversal at more negative values; (d) Cs+ blocks both time-dependent currents recorded in the absence and presence of Ba2+. The data suggest that in the diastolic range of potentials in Purkinje myocytes there is a voltage- and time-dependent K+ current (iKdd) that can be separated from the hyperpolarization-activated inward current i(f).
Assuntos
Coração/efeitos dos fármacos , Coração/fisiologia , Potenciais da Membrana/fisiologia , Potássio/farmacologia , Células de Purkinje/fisiologia , Animais , Cães , Feminino , Masculino , Técnicas de Patch-Clamp , Canais de Potássio/fisiologia , Fatores de TempoRESUMO
IsK is a K+ channel of the delayed rectifier type widely distributed throughout both excitable and nonexcitable cells. Its structure is different from other cloned K+ channels and molecular details of its gating remain obscure. Here we show that the activation kinetics of IsK expressed in Xenopus oocytes depend upon the amount of its mRNA injected, with larger amounts resulting in slower activation kinetics with a longer initial delay during activation. Similar changes in activation kinetics occur with time after a single injection of IsK mRNA. We present two kinetic schemes which illustrate how our experimental results could arise. Both imply an interaction among individual channel proteins during IsK activation. The dependence of channel gating on mRNA concentration provides a novel mechanism for long term regulation of ion current kinetics.
Assuntos
Ativação do Canal Iônico , Oócitos/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/metabolismo , RNA Mensageiro/metabolismo , Animais , Computadores , Feminino , Técnicas In Vitro , Potenciais da Membrana , Microeletrodos , Canais de Potássio/genética , RNA Mensageiro/genética , Xenopus laevisRESUMO
The whole-cell patch-clamp technique was used to voltage clamp acutely isolated myocytes at -60 mV and study effects of ionic environment on Na/K pump activity. In quiescent guinea pig myocytes, normal intracellular Na+ is approximately 6 mM, which gives a total pump current of 0.25 +/- 0.09 pA/pF, and an inward background sodium current of 0.75 +/- 0.26 pA/pF. The average capacitance of a cell is 189 +/- 61 pF. Our main conclusion is the total Na/K pump current comprises currents from two different types of pumps, whose functional responses to the extracellular environment are different. Pump current was reversibly blocked with two affinities by extracellular dihydro-ouabain (DHO). We determined dissociation constants of 72 microM for low affinity (type-1) pumps and 0.75 microM for high affinity (type-h) pumps. These dissociation constants did not detectably change with two intracellular Na+ concentrations, one saturating and one near half-saturating, and with two extracellular K+ concentrations of 4.6 and 1.0 mM. Ion effects on type-h pumps were therefore measured using 5 microM DHO and on total pump current using 1 mM DHO. Extracellular K+ half-maximally activated the type-h pumps at 0.4 mM and the type-1 at 3.7 mM. Extracellular H+ blocked the type-1 pumps with half-maximal blockade at a pH of 7.71 whereas the type-h pumps were insensitive to extracellular pH. Both types of pumps responded similarly to changes in intracellular-Na+, with 9.6 mM causing half-maximal activation. Neither changes in intracellular pH between 6.0 and 7.2, nor concentrations of intracellular K+ of 140 mM or below, had any effect on either type of pump. The lack of any effect of intracellular K+ suggests the dissociation constants are in the molar range so this step in the pump cycle is not rate limiting under normal physiological conditions. Changes in intracellular-Na+ did not affect the half-maximal activation by extracellular K+, and vice versa. We found DHO-blockade of Na/K pump current in canine ventricular myocytes also occurred with two affinities, which are very similar to those from guinea pig myocytes or rat ventricular myocytes. In contrast, isolated canine Purkinje myocytes have predominantly the type-h pumps, insofar as DHO-blockade and extracellular K+ activation are much closer to our type-h results than type-1. These observations suggest for mammalian ventricular myocytes: (a) the presence of two types of Na/K pumps may be a general property. (b) Normal physiological variations in extracellular pH and K+ are important determinants of Na/K pump current. (c) Normal physiological variations in the intracellular environment affect Na/K pump current primarily via the Na+ concentration. Lastly, Na/K pump current appears to be specifically tailored for a tissue by expression of a mix of functionally different types of pumps.
Assuntos
Miocárdio/enzimologia , ATPase Trocadora de Sódio-Potássio/fisiologia , Animais , Cobaias , Ventrículos do Coração/química , Concentração de Íons de Hidrogênio , Masculino , Matemática , Miocárdio/química , Miocárdio/citologia , Ouabaína/análogos & derivados , Ouabaína/farmacologia , Técnicas de Patch-Clamp , Potássio/metabolismo , Potássio/farmacologia , Sódio/metabolismo , Sódio/farmacologia , ATPase Trocadora de Sódio-Potássio/análise , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidoresRESUMO
Percutaneous balloon valvuloplasty was performed on a patient with pulmonary stenosis. Right to left shunting through a patent foramen ovale during balloon inflation was documented by contrast two-dimensional echocardiography. Right and left ventricular pressures recorded during balloon inflation showed a decrease in left ventricular end-diastolic pressure and equilibration with right ventricular end-diastolic pressure. Systemic hypotension was minimal during balloon inflation, possibly due to persistent filling of the left ventricle via the patent foramen ovale. Persistent right ventricular systolic hypertension immediately after valvuloplasty may have been due to infundibular narrowing and resolved on restudy 2 weeks later.
Assuntos
Cateterismo/métodos , Comunicação Interatrial/complicações , Estenose da Valva Pulmonar/terapia , Adulto , Pressão Sanguínea , Cateterismo Cardíaco , Dilatação , Ecocardiografia , Comunicação Interatrial/fisiopatologia , Ventrículos do Coração/fisiopatologia , Humanos , Hipotensão/etiologia , Hipotensão/prevenção & controle , Masculino , Contração Miocárdica , Valva Pulmonar/fisiopatologia , Estenose da Valva Pulmonar/complicações , Estenose da Valva Pulmonar/fisiopatologiaRESUMO
OBJECTIVE: The aim was to determine the extent to which endogenous release of vasoactive intestinal polypeptide (VIP) might be implicated in the modulation of sinoatrial rate in the presence and absence of muscarinic blockade or beta blockade. METHODS: Langendorff perfused rat hearts were studied with the right vagus intact. The hearts were maintained in sinus rhythm and subjected to right vagal stimuli of 5, 10, 20, and 30 Hz. RESULTS: Administration of exogenous VIP, 10(-8) M, increased sinus rate by 20% (p < 0.05). This increase in heart rate was reduced significantly to 8% by the VIP antagonist [D-p-Cl-Phe6, Leu17]VIP, 10(-7) M, which alone had no effect on sinus rate. Vagal stimulation reduced sinus rate from a control of 254(SEM 2) to 164(17) beats.min-1 (p < 0.05) at 20 Hz. VIP, 10(-8) M, increased these rates to 284(6) and 220(21) beats.min-1 (p < 0.05). In another eight vagally stimulated hearts, frequencies of 5-20 Hz reduced sinus rate. At 30 Hz heart rate increased in five, and the resultant rate was significantly faster in these [154(10) beats.min-1] than in the remainder [98(12) beats.min-1, p < 0.05]. Vagal stimulation also increased sinus rate (p < 0.05) in four of seven additional hearts perfused with atropine, 2 x 10(-6) M. This increase was completely abolished by [D-p-Cl-Phe6, Leu17]VIP. That the effect was not beta adrenergic was demonstrated in eight experiments using atropine plus propranolol, 1 x 10(-7) M. A vagally induced increment in rate still occurred (p < 0.05) and was abolished by [D-p-CL-Phe6, Leu17]VIP. The ability to ascribe a rate change to VIP release was maximal in the presence of propranolol and atropine, intermediate in the presence of atropine alone, and minimal in the absence of muscarinic or beta blockade. CONCLUSIONS: Vagally released VIP is capable of limiting the decrement in sinus rate that occurs at high frequencies of vagal stimulation, and in some circumstances can actually increment sinus rate. Its role as an endogenous modulator of vagal effects on heart rate and as a possible cause of vagal and postvagal tachycardias should be further explored.