RESUMEN
Angiotensin II (Ang II) has powerful modulatory actions on cardiovascular function that are mediated by specific receptors located on neurons within the hypothalamus and brain stem. Incubation of neuronal cocultures of rat hypothalamus and brain stem with Ang II elicits an Ang II type 1 (AT1) receptor-mediated inhibition of total outward K+ current that contributes to an increase in neuronal firing rate. However, the exact K+ conductance(s) that is inhibited by Ang II are not established. Pharmacological manipulation of total neuronal outward K+ current revealed a component of K+ current sensitive to quinine, tetraethylammonium, and 4-aminopyridine, with IC50 values of 21.7 micromol/L, 1.49 mmol/L, and 890 micromol/L, respectively, and insensitive to alpha-dendrotoxin (100 to 500 nmol/L), charybdotoxin (100 to 500 nmol/L), and mast cell degranulating peptide (1 micromol/L). Collectively, these data suggest the presence of Kv2.2 and Kv3.1b. Biophysical examination of the quinine-sensitive neuronal K+ current demonstrated a macroscopic conductance with similar biophysical properties to those of Kv2.2 and Kv3.1b. Ang II (100 nmol/L), in the presence of the AT2 receptor blocker PD123,319, elicited an inhibition of neuronal K+ current that was abolished by quinine (50 micromol/L). Reverse transcriptase-polymerase chain reaction analysis confirmed the presence of Kv2.2 and Kv3.1b mRNA in these neurons. However, Western blot analyses demonstrated that only Kv2.2 protein was present. Coexpression of Kv2.2 and the AT1 receptor in Xenopus oocytes demonstrated an Ang II-induced inhibition of Kv2.2 current. Therefore, these data suggest that inhibition of Kv2.2 contributes to the AT1 receptor-mediated reduction of neuronal K+ current and subsequently to the modulation of cardiovascular function.
Asunto(s)
Tronco Encefálico/fisiología , Hipotálamo/fisiología , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/fisiología , Receptores de Angiotensina/fisiología , Angiotensina II/farmacología , Animales , Canales de Potasio de Tipo Rectificador Tardío , Femenino , Canales de Potasio/genética , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Canales de Potasio Shab , Xenopus laevisRESUMEN
Cardiac Cl- channels may play an important role in both the physiological as well as the pathophysiological regulation of the heart. Due to the present controversy regarding the role that Na+ may play in the regulation of the adenosine 3',5'-cyclic monophosphate (cAMP)-dependent Cl- channels in the heart, we have re-examined the effects of intracellular and extracellular Na+ on ICl,cAMP in isolated guinea-pig ventricular myocytes. In conventional, whole-cell patch-clamp experiments, exchanging of extracellular Na+ for N-methyl-d-glucamine (NMG) did not have a significant effect on the magnitude of the ICl,cAMP activated by external forskolin (FSK) in the presence of beta-adrenergic and muscarinic receptor blockade compared with normal rundown of the current with time. Utilization of the amphotericin B-perforated-patch technique prevented rundown of FSK-stimulated ICl, cAMP, and failed to reveal alterations in this conductance when NMG was substituted for extracellular Na+. Finally, intracellular dialysis of cells during whole-cell experiments with both 0 and 10 mM Na+-containing solutions failed to show any significant effect of intracellular Na+ on the magnitude of ICl,cAMP. We therefore conclude that intracellular or extracellular Na+ plays little or no direct regulatory role in modulation of cAMP-dependent Cl- channels in heart.
Asunto(s)
Canales de Cloruro/metabolismo , AMP Cíclico/metabolismo , Miocardio/metabolismo , Sodio/metabolismo , Animales , Calcio/metabolismo , Colforsina/farmacología , Cobayas , Masculino , Canales de Potasio/metabolismoRESUMEN
cAMP-dependent chloride channels in heart contribute to autonomic regulation of action potential duration and membrane potential and have been inferred to be due to cardiac expression of the epithelial cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. In this report, a cDNA from rabbit ventricle was isolated and sequenced, which encodes an exon 5 splice variant (exon 5-) of CFTR, with >90% identity to human CFTR cDNA present in epithelial cells. Expression of this cDNA in Xenopus oocytes gave rise to robust cAMP-activated chloride currents that were absent in control water-injected oocytes. Antisense oligodeoxynucleotides directed against CFTR significantly reduced the density of cAMP-dependent chloride currents in acutely cultured myocytes, thereby establishing a direct functional link between cardiac expression of CFTR protein and an endogenous chloride channel in native cardiac myocytes.
Asunto(s)
Canales de Cloruro/genética , AMP Cíclico/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/genética , Ventrículos Cardíacos/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Canales de Cloruro/fisiología , Clonación Molecular , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , ADN Complementario , Exones , Cobayas , Humanos , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional , Conejos , Función Ventricular , XenopusRESUMEN
OBJECTIVES: The cAMP-dependent Cl- conductance in heart is believed to be due to cardiac expression of the cystic fibrosis transmembrane conductance regulator (CFTR). While CFTR expressed in rabbit and guinea-pig heart (CFTRcardiac) is an alternatively spliced isoform of the epithelial gene product, little information is known regarding possible expression of CFTR in primate heart. In this study, we examined molecular expression of CFTR in human and simian atrium and ventricle and functional expression of cAMP-dependent Cl- currents in isolated human atrial and simian ventricular cells. METHODS: The reverse transcription polymerase chain reaction (RT-PCR) was performed on human and simian atrial and ventricular mRNA using primers designed to border regions of the CFTR gene product corresponding to transmembrane segments I-VI (TSI-VI), the first nucleotide binding domain (NBD1), transmembrane segments VII-XII (TSVII-XII), and the large cytoplasmic domain which includes the regulatory (R) domain and NBD1. Functional expression of CFTR Cl- channels in human atrial and simian ventricular myocytes was determined using whole-cell and giant inside-out patch-clamp techniques. RESULTS: Southern blot analysis of these RT-PCR products demonstrated expression of CFTR transcripts in human and simian atrial and ventricular tissue and revealed a novel pattern of expression compared to most animal species studies: both the exon 5 plus (unspliced) and exon 5 minus (spliced) CFTR transcripts are co-expressed in human and simian atrium and ventricle. Whole-cell experiments demonstrated a Cl- sensitive time-independent background conductance in both human atrial and simian ventricular myocytes that was activated by forskolin (FSK) and insensitive to 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). In inside-out patches utilizing the giant patch technique on human atrial myocytes, unitary Cl- sensitive channels resembling CFTR Cl- channels (approximately 14 pS conductance) were activated by the catalytic subunit of protein kinase A (PKA) in 3/12 patches examined. CONCLUSIONS: These results clearly demonstrate the molecular expression of CFTR Cl- channels and provide electrophysiological evidence consistent with functional expression of these channels in human atrial and simian ventricular myocardium.