RESUMO
Despite the important roles played by ventricular fibroblasts and myofibroblasts in the formation and maintenance of the extracellular matrix, neither the ionic basis for membrane potential nor the effect of modulating membrane potential on function has been analyzed in detail. In this study, whole cell patch-clamp experiments were done using ventricular fibroblasts and myofibroblasts. Time- and voltage-dependent outward K(+) currents were recorded at depolarized potentials, and an inwardly rectifying K(+) (Kir) current was recorded near the resting membrane potential (RMP) and at more hyperpolarized potentials. The apparent reversal potential of Kir currents shifted to more positive potentials as the external K(+) concentration ([K(+)](o)) was raised, and this Kir current was blocked by 100-300 muM Ba(2+). RT-PCR measurements showed that mRNA for Kir2.1 was expressed. Accordingly, we conclude that Kir current is a primary determinant of RMP in both fibroblasts and myofibroblasts. Changes in [K(+)](o) influenced fibroblast membrane potential as well as proliferation and contractile functions. Recordings made with a voltage-sensitive dye, DiBAC(3)(4), showed that 1.5 mM [K(+)](o) resulted in a hyperpolarization, whereas 20 mM [K(+)](o) produced a depolarization. Low [K(+)](o) (1.5 mM) enhanced myofibroblast number relative to control (5.4 mM [K(+)](o)). In contrast, 20 mM [K(+)](o) resulted in a significant reduction in myofibroblast number. In separate assays, 20 mM [K(+)](o) significantly enhanced contraction of collagen I gels seeded with myofibroblasts compared with control mechanical activity in 5.4 mM [K(+)](o). In combination, these results show that ventricular fibroblasts and myofibroblasts express a variety of K(+) channel alpha-subunits and demonstrate that Kir current can modulate RMP and alter essential physiological functions.
Assuntos
Fibroblastos/fisiologia , Potenciais da Membrana/fisiologia , Contração Miocárdica/fisiologia , Canais de Potássio/fisiologia , Função Ventricular , Animais , Divisão Celular , Técnicas Eletrofisiológicas Cardíacas/métodos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Células Musculares/fisiologia , Contração Miocárdica/efeitos dos fármacos , Técnicas de Patch-Clamp , Potássio/farmacologia , Canais de Potássio/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
An imaging system suitable for recordings from Langendorff-perfused rat hearts using the voltage-sensitive dye 4-[beta-[2-(di-n-butylamino)-6-naphthyl]vinyl]pyridinium (di-4-ANEPPS) has been developed. Conduction velocity was measured under hyper- and hypokalemic conditions, as well as at physiological and reduced temperature. Elevation of extracellular [K(+)] to 9 mM from 5.9 mM caused a slowing of conduction velocity from 0.66 +/- 0.08 to 0.43 +/- 0.07 mm/ms (35%), and reduction of the temperature to 32 degrees C from 37 degrees C caused a slowing from 0.64 +/- 0.07 to 0.46 +/- 0.05 mm/ms (28%). Ventricular activation patterns in sinus rhythm showed areas of early activation (breakthrough) in both the right and left ventricle, with breakthrough at a site near the apex of the right ventricle usually occurring first. The effects of mechanically immobilizing the preparation to reduce motion artifact were also characterized. Activation patterns in epicardially paced rhythm were insensitive to this procedure over the range of applied force tested. In sinus rhythm, however, a relatively large immobilizing force caused prolonged PQ intervals as well as altered ventricular activation patterns. The time-dependent effects of the dye on the rat heart were characterized and include 1) a transient vasodilation at the onset of dye perfusion and 2) a long-lasting prolongation of the PQ interval of the electrocardiogram, frequently resulting in brief episodes of atrioventricular block.
Assuntos
Mapeamento Potencial de Superfície Corporal , Corantes/farmacologia , Sistema de Condução Cardíaco/fisiologia , Coração/fisiologia , Compostos de Piridínio/farmacologia , Animais , Estimulação Cardíaca Artificial , Corantes/farmacocinética , Eletrocardiografia/efeitos dos fármacos , Técnicas Eletrofisiológicas Cardíacas/instrumentação , Técnicas Eletrofisiológicas Cardíacas/métodos , Coração/efeitos dos fármacos , Sistema de Condução Cardíaco/efeitos dos fármacos , Frequência Cardíaca/efeitos dos fármacos , Frequência Cardíaca/fisiologia , Técnicas In Vitro , Masculino , Perfusão , Potássio/metabolismo , Potássio/farmacologia , Pressão , Compostos de Piridínio/farmacocinética , Ratos , Ratos Sprague-Dawley , Nó Sinoatrial/fisiologia , TemperaturaRESUMO
The recently cloned retinal cone Na(+)-Ca(2+)-K(+) exchanger (NCKX) was expressed in cultured insect cells, and whole-cell patch clamp was used to measure transmembrane currents generated by this transcript and compare them with currents generated by retinal rod NCKX or by a deletion mutant rod NCKX from which the two large hydrophilic loops were removed. We have characterized the ionic currents generated by both the forward (Ca(2+) extrusion) and reverse (Ca(2+) influx) modes of all three NCKX proteins. Reverse NCKX exchange generated outward current that required the simultaneous presence of both external Ca(2+) and external K(+). Forward NCKX exchange carried inward current with Na(+), but not with Li(+) in the bath solution. The cation dependencies of the three NCKX tested (external K(+), external Na(+), internal Ca(2+)) were very similar to each other and to those reported previously for the in situ rod NCKX. These findings provide the first electrophysiological characterization of cone NCKX and the first electrophysiological characterization of potassium-dependent Na(+)-Ca(+) exchangers in heterologous systems. Our results demonstrate the feasibility of combining heterologous expression and biophysical measurements for detailed NCKX structure/function studies.