RESUMO
BACKGROUND: The late sodium current (INa-L) contributes importantly to rate-dependent change in action potential duration (APD) and transmural dispersion of repolarization (TDR). However, little is known about the mechanisms of increased APD rate-dependence and amplified TDR in left ventricular hypertrophy (LVH) and failure. The purpose of this study was to investigate the role of INa-L in rate-adaptation of transmural APD heterogeneity. METHODS: APD, its rate-dependence and INa-L current were examined in myocytes isolated from the endocardium and epicardium of the control and LVH rabbits. AP was recorded using the standard microelectrode technique, and INa-L was recorded using the whole-cell patch clamp technique. RESULTS: Early afterdepolarizations (EADs) were frequently recorded in the isolated myocytes of the LVH rabbits but not in those of controls. LVH prolonged APD more significantly in the endocardial myocytes than in the epicardium (31.7±3.4 vs. 21.6±1.5% n=6, p<0.05), leading to a marked increase in TDR. LVH endocardial myocytes exhibited a greater rate-dependent change in APD compared to the epicardial myocytes. INa-L densities were significantly increased in both LVH endocardium and epicardium. However, LVH increased the INa-L density preferentially in the endocardial myocytes compared to the epicardial myocytes (54.5±4.8% vs. 39.2±3.3%, n=6, p<0.05). CONCLUSIONS: Our results demonstrate that LVH increased the INa-L preferentially in the endocardium over the epicardium, which contributes importantly to the stronger rate-dependent change in repolarization and longer APD in the endocardium. This results in an amplified TDR capable of initiating EAD and ventricular arrhythmias.
Assuntos
Potenciais de Ação/fisiologia , Endocárdio/citologia , Hipertrofia Ventricular Esquerda/metabolismo , Hipertrofia Ventricular Esquerda/fisiopatologia , Células Musculares/metabolismo , Canais de Sódio/metabolismo , Animais , Endocárdio/fisiopatologia , Masculino , Técnicas de Patch-Clamp , CoelhosRESUMO
Alternative splicing of the voltage-gated Ca(2+) (CaV) α1-subunit adds to the functional diversity of Ca(2+) channels. A variant with a 73-nt deletion in exon 15 of the Cav1.2 α1-subunit (Cav1.2Δ73) produced by alternative splicing that predicts a truncated protein has been described, but its function, if any, is unknown. We sought to determine if, by analogy to other truncated CaV α1-subunits, Cav1.2Δ73 acts as an inhibitor of wild-type Cav1.2 currents. HEK-293 cells were transfected with Cav1.2Δ73 in a pIRES vector with CD8 or in pcDNA3.1 with a V5/his COOH-terminal tag plus ß2 and α2δ1 accessory subunits and pEGFP. Production of Cav1.2Δ73 protein was confirmed by Western blotting and immunofluorescence. Voltage-clamp studies revealed the absence of functional channels in transfected cells. In contrast, cells transfected with full-length Cav1.2 plus accessory subunits and pEGFP exhibited robust Ca(2+) currents. A7r5 cells exhibited endogenous Cav1.2-based currents that were greatly reduced (>80%) without a change in voltage-dependent activation when transfected with Cav1.2Δ73-IRES-CD8 compared with empty vector or pIRES-CD8 controls. Transfection of A7r5 cells with an analogous Cav2.3Δ73-IRES-CD8 had no effect on Ca(2+) currents. Immunofluorescence showed intracellular, but not plasma membrane, localization of Cav1.2Δ73-V5/his, as well as colocalization with an endoplasmic reticulum marker, ER Organelle Lights. Expression of Cav1.2Δ73 α1-subunits in A7r5 cells inhibits endogenous Cav1.2 currents. The fact that this variant arises naturally by alternative splicing raises the possibility that it may represent a physiological mechanism to modulate Cav1.2 functional activity.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Miócitos de Músculo Liso/metabolismo , Processamento Alternativo , Animais , Sequência de Bases , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/genética , Linhagem Celular , Células HEK293 , Humanos , Masculino , Técnicas de Patch-Clamp , Estrutura Terciária de Proteína , Ratos , TransfecçãoRESUMO
A gain of function mutation N588K in the KCNH2 gene that encodes HERG channels has been shown to underlie the SQT1 form of short QT syndrome (SQTS). We describe a different mutation in the KCNH2 gene in a Chinese family with clinical evidence of SQTS. A Chinese family with a markedly short QT interval (QTc=316 ± 9 ms, n=4) and a strong family history of sudden death was investigated. Analysis of candidate genes contributing to ventricular repolarization identified a C1853T mutation in the KCNH2 gene coding for the HERG channel, resulting in an amino acid change (T618I) that was found to 100% co-segregate with the SQTS phenotype (n=4). Whole cell voltage clamp studies of the T618I mutation in HEK-cells demonstrated a 6-fold increase in maximum steady state current (146.1 ± 16.7 vs 23.8 ± 5.5 pA/pF) that occurred at a 20 mV more positive potential compared to the wild type channels. The voltage dependence of inactivation was significantly shifted in the positive voltage direction (WT -78.6 ± 6.8 vs T618I -29.3 ± 1.7 mV). Kinetic analysis revealed slower inactivation rates of T618I but faster rates of recovery from inactivation. Quinidine (5 µM) and sotalol (500 µM) had similar inhibitory effects on steady currents measured at +20 mV in WT and T618I but were less effective in inhibiting tail currents of mutant channels. The altered function of T618I-HERG channels suggests that this mutation in the KCNH2 gene is responsible for the SQTS phenotype in this family. Both quinidine and sotalol may be therapeutic options for patients with the T618I HERG mutation.
Assuntos
Arritmias Cardíacas/genética , Canais de Potássio Éter-A-Go-Go/genética , Mutação , Adolescente , Adulto , Animais , Antiarrítmicos/farmacologia , Arritmias Cardíacas/tratamento farmacológico , Arritmias Cardíacas/metabolismo , Criança , Canal de Potássio ERG1 , Eletrocardiografia/métodos , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Canais de Potássio Éter-A-Go-Go/metabolismo , Feminino , Testes Genéticos/métodos , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Linhagem , Fenótipo , Quinidina/farmacologia , Análise de Sequência de DNA , Sotalol/farmacologia , Adulto JovemRESUMO
The Smith-Lemli-Opitz syndrome (SLOS) is an inherited disorder of cholesterol synthesis caused by mutations in DHCR7 which encodes the final enzyme in the cholesterol synthesis pathway. The immediate precursor to cholesterol synthesis, 7-dehydrocholesterol (7-DHC) accumulates in the plasma and cells of SLOS patients which has led to the idea that the accumulation of abnormal sterols and/or reduction in cholesterol underlies the phenotypic abnormalities of SLOS. We tested the hypothesis that 7-DHC accumulates in membrane caveolae where it disturbs caveolar bilayer structure-function. Membrane caveolae from skin fibroblasts obtained from SLOS patients were isolated and found to accumulate 7-DHC. In caveolar-like model membranes containing 7-DHC, subtle, but complex alterations in intermolecular packing, lipid order and membrane width were observed. In addition, the BK(Ca) K(+) channel, which co-migrates with caveolin-1 in a membrane fraction enriched with cholesterol, was impaired in SLOS cells as reflected by reduced single channel conductance and a 50 mV rightward shift in the channel activation voltage. In addition, a marked decrease in BK(Ca) protein but not mRNA expression levels was seen suggesting post-translational alterations. Accompanying these changes was a reduction in caveolin-1 protein and mRNA levels, but membrane caveolar structure was not altered. These results are consistent with the hypothesis that 7-DHC accumulation in the caveolar membrane results in defective caveolar signaling. However, additional cellular alterations beyond mere changes associated with abnormal sterols in the membrane likely contribute to the pathogenesis of SLOS.
Assuntos
Cavéolas/metabolismo , Desidrocolesteróis/metabolismo , Fibroblastos/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Síndrome de Smith-Lemli-Opitz/metabolismo , Caveolina 1/metabolismo , Células Cultivadas , Desidrocolesteróis/química , Genótipo , Humanos , Immunoblotting , Membranas Artificiais , Microscopia Eletrônica , Estrutura Molecular , Pele/citologia , Esteróis/metabolismo , Difração de Raios XRESUMO
BACKGROUND: This study was designed to test the hypothesis that differences exist in the inactivation properties of voltage-gated Ca(2+) channels (Ca(V)) in hypertensive arterial smooth muscle cells (ASMCs), and that these differences contribute to enhanced Ca(V) activity. METHODS: The properties of Ca(V) were studied in freshly isolated myocytes from small mesenteric arteries (SMAs) of Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHRs) using whole-cell patch-clamp methods. RESULTS: Peak currents (I(Ca)) were larger in SHR with either 2 mmol/l Ca(2+) or Ba(2+) as the charge carrier. In WKY and SHR, the peak current was larger with Ba(2+) than with Ca(2+) with no difference in their ratio. The voltage dependence of Ca(V) activation was shifted to the left in SHR as compared to WKY for Ca(2+) but not for Ba(2+), while availability was not different. The time course of inactivation of current could be represented by two time constants, both of which were larger in SHR than in WKY and also larger for Ba(2+) than for Ca(2+), with a greater fraction of inactivation being associated with the process slower in SHR and with Ba(2+). The time courses of availability, inactivation, and recovery from inactivation were faster in SHR than in WKY in the case of Ca(2+), but there was no difference in the case of Ba(2+). CONCLUSIONS: These results demonstrate that there are differences between WKY and SHR in the inactivation properties of SMA Ca(V), and that these differences could contribute to larger steady-state currents. The differences cannot be explained merely by the presence of a larger number of identical Ca(V) complexes, and it appears likely that differences in intrinsic compositions, primary structures, and/or regulation are involved.
Assuntos
Canais de Cálcio/metabolismo , Hipertensão/metabolismo , Artérias Mesentéricas/metabolismo , Células Musculares/metabolismo , Contração Muscular/fisiologia , Músculo Liso Vascular/metabolismo , Animais , Modelos Animais de Doenças , Hipertensão/patologia , Hipertensão/fisiopatologia , Masculino , Potenciais da Membrana/fisiologia , Artérias Mesentéricas/patologia , Artérias Mesentéricas/fisiopatologia , Músculo Liso Vascular/patologia , Músculo Liso Vascular/fisiopatologia , Técnicas de Patch-Clamp , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKYRESUMO
BACKGROUND: We have previously demonstrated differences in the gene expression of voltage-gated K v1.X channel alpha-subunits in arteries from Wistar-Kyoto rats (WKYs) and spontaneously hypertensive rats (SHRs). The purpose of this study was to test the hypothesis that these differences are also present at the protein level. METHODS: Proteins were isolated from the aorta, mesenteric (MAs) and tail arteries (TAs) of 12- to 15-week-old male WKY and SHR, and analyzed by immunoblotting. K(v) currents were recorded from MA myocytes by patch clamp methods. RESULTS: Expression of Kv1.2, Kv1.5, and Kv2.1 was higher in MAs but was not different in aortas of SHRs as compared to WKYs. In the TA, expression of Kv1.2 and Kv1.5 was higher while that of Kv2.1 was lower in SHR compared to WKY. In the MA, the larger expression of an 80 kDa species of Kv1.2 in SHRs was associated with a lower expression of a 60 kDa species. Kv2.1 gene expression was larger in MAs from SHRs but not different in TAs. K(v) currents associated with Kv1.X and Kv2.1 channels were both larger in MA myocytes from SHRs but less than expected based upon differences in K(v) alpha-subunit protein expression. CONCLUSIONS: For the MA, K(v) protein expression and current components between WKYs and SHRs were qualitatively consistent, but differences in gene and protein expression were not closely correlated. The higher expression of K(v) subunits in small mesenteric arteries (SMAs) of SHR would tend to maintain normal myogenic activity and vasoconstrictor reserve, and could be viewed as a form of homeostatic remodeling.
Assuntos
Hipertensão/genética , Hipertensão/fisiopatologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Animais , Especificidade de Anticorpos , Células CHO , Cricetinae , Cricetulus , Expressão Gênica/fisiologia , Humanos , Rim/citologia , Canal de Potássio Kv1.2/genética , Canal de Potássio Kv1.2/imunologia , Canal de Potássio Kv1.2/fisiologia , Canal de Potássio Kv1.5/genética , Canal de Potássio Kv1.5/imunologia , Canal de Potássio Kv1.5/fisiologia , Masculino , Artérias Mesentéricas/fisiologia , Monócitos/fisiologia , Técnicas de Patch-Clamp , Canais de Potássio de Abertura Dependente da Tensão da Membrana/imunologia , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Canais de Potássio Shab/genética , Canais de Potássio Shab/imunologia , Canais de Potássio Shab/fisiologia , Cauda/irrigação sanguínea , Artérias Torácicas/fisiologiaRESUMO
OBJECTIVE: Anti-NMDA receptor encephalitis (ANRE) is a potentially lethal encephalitis attributed to autoantibodies against the N-methyl-D-aspartate receptor (NMDAR). We sought to clone and characterize monoclonal antibodies (mAbs) from an ANRE patient. METHODS: We used a hybridoma method to clone two IgG mAbs from a female patient with ANRE without teratoma, and characterized their binding activities on NMDAR-transfected cell lines, cultured primary rat neurons, and mouse hippocampus. We also assessed their effects on voluntary locomotor activity in mice and binding to NMDAR in vivo. RESULTS: The mAbs are structurally distinct and arose from distinct B-cell lineages. They recognize different epitopes on the GluN1 amino terminal domain (ATD), yet both require amino acids important for post-translational modification. Both mAbs bind subsets of GluN1 on cultured rat hippocampal neurons. The 5F5 mAb binds mouse brain hippocampal tissues, and the GluN1 recognized on cultured rat neurons was substantially extra-synaptic. Antibody binding to primary hippocampal neurons induced receptor internalization. The NMDAR inhibitor MK-801 inhibited internalization without preventing mAb binding; AP5 inhibited both mAb binding and internalization. Exposure of mice to the mAbs following permeabilization of the blood brain barrier increased voluntary wheel running activity, similar to low doses of the NMDAR inhibitor, MK-801. INTERPRETATION: These mAbs recapitulate features demonstrated in previous studies of ANRE patient CSF, and exert effects on NMDAR in vitro and in vivo consistent with modulation of NMDAR activity.
RESUMO
Many of the structural domains involved in Ca2+ channel (CACN) inactivation are also involved in determining their sensitivity to antagonist inhibition. We hypothesize that differences in inactivation properties and their structural determinants may suggest candidate domains as targets for the development of novel, selective antagonists. The characteristics of Ca2+ current (ICa) inactivation, steady-state inactivation (SSIN), and recovery from inactivation were studied in freshly dispersed smooth muscle cells from rabbit portal vein (RPV) using whole-cell, voltage-clamp methods. The time course of inactivation could be represented by two time constants. Increasing ICa by increasing [Ca2+]o or with more negative holding potentials decreased both time constants. With Sr2+, Ba2+, or Na+ as the charge carrier, ICa inactivation was also represented by two time constants, both of which were larger than those found with Ca2+. With Ca2+, Sr2+, or Ba2+ as the charge carrier, both time constants had minimum values near the voltage associated with maximum current. When Na+ (140 mM) was the charge carrier, voltages for Imax (-20 mV) or taumin (0 mV) did not correspond. SSIN of ICa had a half-maximum voltage of -32 +/- 4 mV for Ca2+, -43 mV +/- 5 mV for Sr2+, -41 +/- 5 mV for Ba2+, and -68 +/- 6 mV for Na+. The slope factor for SSIN per e-fold voltage change was 6.5 +/- 0.2 mV for Ca2+, 6.8 +/- 0.3 for Sr2+, and 6.6 +/- 0.2 for Ba2+, representing four equivalent charges. When Na+ or Li+ was the charge carrier, the slope factor was 13.5 +/- 0.7 mV, representing two equivalent charges. For ICa in rat left ventricular (rLV) myocytes, there was no difference in the slope factor of SSIN for Ca2+ and Na+. The rate of recovery of ICa from inactivation varied inversely with recovery voltage and was independent of the charge carrier. These results suggest that inactivation of ICa in PV myocytes possess an intrinsic voltage dependence that is modified by Ca2+. For RPV but not rLV ICa, the charge of the permeating ion confers the voltage-dependency of SSIN.
Assuntos
Canais de Cálcio/fisiologia , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Ativação do Canal Iônico/fisiologia , Modelos Biológicos , Fibras Musculares Esqueléticas/fisiologia , Músculo Liso Vascular/fisiologia , Animais , Células Cultivadas , Masculino , Potenciais da Membrana/fisiologia , CoelhosRESUMO
Multiple K v channel complexes contribute to total K v current in numerous cell types and usually subserve different physiological functions. Identifying the complete compliment of functional K v channel subunits in cells is a prerequisite to understanding regulatory function. It was the goal of this work to determine the complete K v subunit compliment that contribute to functional K v currents in rat small mesenteric artery (SMA) myocytes as a prelude to studying channel regulation. Using RNA prepared from freshly dispersed myocytes, high levels of K v 1.2, 1.5, and 2.1 and lower levels of K v 7.4 α-subunit expressions were demonstrated by quantitative PCR and confirmed by Western blotting. Selective inhibitors correolide (K v 1; COR), stromatoxin (K v 2.1; ScTx), and linopirdine (K v 7.4; LINO) decreased K v current at +40 mV in SMA by 46 ± 4, 48 ± 4, and 6.5 ± 2 %, respectively, and K v current in SMA was insensitive to α-dendrotoxin. Contractions of SMA segments pretreated with 100 nmol/L phenylephrine were enhanced by 27 ± 3, 30 ± 8, and 7 ± 3 % of the response to 120 mmol/L KCl by COR, ScTX, and LINO, respectively. The presence of K v 6.1, 9.3, ß1.1, and ß1.2 was demonstrated by RT-PCR using myocyte RNA with expressions of K vß1.2 and K v 9.3 about tenfold higher than K vß1.1 and K v 6.1, respectively. Selective inhibitors of K v 1.3, 3.4, 4.1, and 4.3 channels also found at the RNA and/or protein level had no significant effect on K v current or contraction. These results suggest that K v current in rat SMA myocytes are dominated equally by two major components consisting of K v 1.2-1.5-ß1.2 and K v 2.1-9.3 channels along with a smaller contribution from K v 7.4 channels but differences in voltage dependence of activation allows all three to provide significant contributions to SMA function at physiological voltages.
Assuntos
Regulação da Expressão Gênica , Artérias Mesentéricas/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Subunidades Proteicas/genética , Animais , Células HEK293 , Humanos , Masculino , Artérias Mesentéricas/anatomia & histologia , Tamanho do Órgão , Especificidade de Órgãos , Ratos , Ratos WistarRESUMO
We have shown that three components contribute to functional voltage gated K+ (K v) currents in rat small mesenteric artery myocytes: (1) Kv1.2 plus Kv1.5 with Kvß1.2 subunits, (2) Kv2.1 probably associated with Kv9.3 subunits, and (3) Kv7.4 subunits. To confirm and address subunit stoichiometry of the first two, we have compared the biophysical properties of K v currents in small mesenteric artery myocytes with those of Kv subunits heterologously expressed in HEK293 cells using whole cell voltage clamp methods. Selective inhibitors of Kv1 (correolide, COR) and Kv2 (stromatoxin, ScTx) channels were used to separate these K v current components. Conductance-voltage and steady state inactivation data along with time constants of activation, inactivation, and deactivation of native K v components were generally well represented by those of Kv1.2-1.5-ß1.2 and Kv2.1-9.3 channels. The slope of the steady state inactivation-voltage curve (availability slope) proved to be the most sensitive measure of accessory subunit presence. The availability slope curves exhibited a single peak for both native K v components. Availability slope curves for Kv1.2-1.5-ß1.2 and Kv2.1-9.3 channels expressed in human embryonic kidney cells also exhibited a single peak that shifted to more depolarized voltages with increasing accessory to α subunit transfection ratio. Availability slope curves for SxTc-insensitive currents were similar to those of Kv1.2-1.5 expressed with Kvß1.2 at a 1:5 molar ratio while curves for COR-insensitive currents closely resembled those of Kv2.1 expressed with Kv9.3 at a 1:1 molar ratio. These results support the suggested Kv subunit combinations in small mesenteric artery, and further suggest that Kv1 α and Kvß1.2 but not Kv2.1 and Kv9.3 subunits are present in a saturated (4:4) stoichiometry.
Assuntos
Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Animais , Células Cultivadas , Células HEK293 , Humanos , Masculino , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Técnicas de Patch-Clamp , Plasmídeos/genética , Plasmídeos/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidores , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ratos , Ratos WistarRESUMO
Voltage-gated K+ channels (Kv) play an important role in regulating contraction of vascular smooth muscle cells (VSMC) through their effects on membrane potential and on voltage-gated Ca2+ channel activity. Kv channels are tetrameric structures consisting of four identical or closely related pore-forming alpha subunits that may be associated with accessory subunits. More than 30 different gene products that contribute to Kv channel complexes have been identified to date, some of which are subject to alternative splicing. Consequently, there is an enormous potential diversity in the molecular composition and properties of possible Kv channel complexes. Electrophysiologic measurements of K+ currents in VSMC suggest the presence of multiple Kv channel assemblies including: (1) rapidly inactivating, 4-aminopyridine-sensitive, (2) slowly inactivating, tetraethylammonium-insensitive, and (3) noninactivating, tetraethylammonium-sensitive components. Based on electro physiological and expression studies, it is likely that the latter two components are represented by a heteromultimeric complex of Kv1.2 with either Kv1.4 or Kv1.5 and a Kvbeta1 subunit, and by at least Kv2.1, respectively. The identity of the first A-type current component, however, is not clear at this time. The relative abundance of these current components appears to vary in VSMC from different anatomical sites, from animals of different ages, and perhaps in VSMC within specific vascular segments. Expression of numerous Kv alpha and beta subunits has been demonstrated in VSMC at both the gene and protein level. However, the number of expressed subunits appears to be much larger than the number of apparent Kv current components. It remains unclear if all of these transcripts are expressed in VSMC or in other cell types in the tissue, or if expression patterns are homogenous or heterogeneous in VSMC at a given site.
Assuntos
Ativação do Canal Iônico/fisiologia , Potenciais da Membrana/fisiologia , Proteínas Musculares/metabolismo , Músculo Liso Vascular/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Animais , Humanos , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Enhanced function of dihydropyridine-sensitive Ca2+ channels (CaV) in hypertensive arterial myocytes (HAM) is well accepted. Increased protein expression of pore forming α1-subunits contributes to this effect, but cannot explain all of the differences in CaV properties in HAM. We hypothesized that differences in expression of CaV subunits and/or their splice variants also contribute. METHODS: RNA, protein, and myocytes were isolated from small mesenteric arteries (SMA) of 20-week-old male WKY and SHR and analyzed by polymerase chain reaction (PCR), sequencing, immunoblotting, and patch clamp methods. RESULTS: Cav1.2 α1, ß2c, and α2δ1d were the dominant subunits expressed in both WKY and SHR with a smaller amount of ß3a. Real-time PCR indicated that the mRNA abundance of ß3a and α2δ1 but not total Cav1.2 α1 or ß2c were significantly larger in SHR. Analysis of alternative splicing of Cav1.2 α1 showed no differences in abundance of mutually exclusive exons1b, 8, 21 and 32 or alternative exons33 and 45. However, inclusion of exon9* was higher and a 73 nucleotide (nt) deletion in exon15 (exon15Δ73) was lower in SHR. Immunoblot analysis showed higher protein levels of Cav1.2 α1 (1.61±0.05), ß3 (1.80±0.32), and α2δ1 (1.80±0.24) but not ß2 in SHR. CONCLUSIONS: The lower abundance of exon15Δ73 transcripts in SHR results in a larger fraction of total Cav1.2 mRNA coding for full-length CaV protein, and the higher abundance of exon9* transcripts and CaVß3a protein likely contribute to differences in gating and kinetics of CaV currents in SHR. Functional studies of Ca2+ currents in native SMA myocytes and HEK cells transiently transfected with CaV subunits support these conclusions.
Assuntos
Canais de Cálcio/metabolismo , Hipertensão/metabolismo , Artérias Mesentéricas/metabolismo , Miócitos de Músculo Liso/metabolismo , Animais , Perfilação da Expressão Gênica , Masculino , Isoformas de Proteínas/metabolismo , Subunidades Proteicas/metabolismo , Ratos Endogâmicos SHR , Ratos Endogâmicos WKYRESUMO
Hypertension is associated with a remodeling of arterial smooth muscle K(+) channels with Ca(2+)-gated K(+) channel (BK(Ca)) activity being enhanced and voltage-gated K(+) channel (K(v)) activity depressed. Because both of these channel types are modulated by intracellular Ca(2+), we tested the hypothesis that Ca(2+) had a larger effect on both BK(Ca) and K(v) channels in arterial myocytes from hypertensive animals. Myocytes were enzymatically dispersed from small mesenteric arteries (SMA) of 12-week-old Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Using whole cell patch clamp methods, BK(Ca) and K(v) current components were determined as iberiotoxin-sensitive and -insensitive currents, respectively. The effects of Ca(2+) on these K(+) current components were determined from measurements made with 0.2 and 2 mmol/L external Ca(2+). Increasing external Ca(2+) from 0.2 to 2 mmol/L Ca(2+) increased BK(Ca) currents recorded using myocytes from both WKY rats and SHR with a larger effect in SHR. Increasing external Ca(2+) decreased K(v) currents recorded using myocytes from both WKY and SHR also with a larger effect in SHR. In other experiments, currents through voltage-gated Ca(2+) channels (Ca(v)) measured at 0.2 mmol/L external Ca(2+) were 12 +/- 2% (n = 12) of those recorded at 2 mmol/L Ca(2+) with no differences in percent effect between WKY and SHR. In isolated SMA segments, isometric force development in response to 140 mmol/L KCl at 0.2 mmol/L external Ca(2+) was about 23 +/- 6% (n = 8) of that measured at 2 mmol/L external Ca(2+). These results suggest that an increase in Ca(2+) influx through Ca(v) or in intracellular Ca(2+) secondary to an increase in external Ca(2+) augments BK(Ca) currents and inhibits K(v) currents in SMA myocytes with a larger effect in SHR compared to WKY. This mechanism may contribute to the functional remodeling of K(+) currents of arterial myocytes in hypertensive animals.
Assuntos
Cálcio/metabolismo , Hipertensão/metabolismo , Artérias Mesentéricas/metabolismo , Miócitos de Músculo Liso/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Animais , Canais de Cálcio/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta , Potenciais da Membrana/fisiologia , Artérias Mesentéricas/citologia , Técnicas de Patch-Clamp , Potássio/metabolismo , Canais de Potássio Cálcio-Ativados/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Especificidade da EspécieRESUMO
Numerous studies have emphasized the important role of altered Ca(2+) channel function in hypertension. We previously showed that Ca(2+) currents measured in myocytes isolated from both Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) small mesenteric arteries closely correlated with systolic blood pressure (BP) during normal development. The purpose of the present experiments was to determine whether antihypertensive therapy with an angiotensin converting enzyme inhibitor normalizes Ca(2+) channel function in SHR myocytes along with BP. Ramipril (3.5 mg/kg/day) was added to the drinking water of 12-week-old male WKY and SHR for 8 weeks. Segments of small mesenteric arteries were used for isometric contraction studies, and for isolation of myocytes for measurement of Ca(2+) and K(+) currents (I(Ca) and I(K)) by patch clamp methods. Ramipril treatment decreased systolic pressure in WKY and SHR, decreased heart weight and heart weight-to-body weight ratio in SHR, and decreased body weight in WKY. Maximum contractile responses to Bay k 8644 in SMA from ramipril-treated SHR were smaller compared to untreated SHR (10% +/- 2% v 55% +/- 7% of the response to 120 mmol/L KCl). The smaller responses in WKY were not affected by ramipril treatment (11% +/- 4% v 8% +/- 3%). Contractile responses to 10 mmol/L tetraethylammonium (TEA) were not different in untreated versus ramipril-treated SHR (65% +/- 6% v 82% +/- 8%) but were increased in treated WKY (4% +/- 1% v 35% +/- 9%). Ramipril treatment decreased peak I(Ca) and equalized the voltage-dependence of I(Ca) activation between SHR and WKY. The I(K) measured from holding potentials of -60 and -20 mV were significantly smaller in treated SHR and WKY compared to their untreated counterparts, as was the component of I(K) measured in the presence of 100 nmol/L iberiotoxin. These results show that ramipril treatment decreases arterial pressure and Ca(2+) channel function in SHR as expected but unexpectedly also decreases I(K) in both WKY and SHR. These results suggest that angiotensin may have a BP independent effect on ion channel function in arterial smooth muscle.
Assuntos
Anti-Hipertensivos/farmacologia , Canais de Cálcio Tipo L/metabolismo , Artérias Mesentéricas/efeitos dos fármacos , Canais de Potássio/metabolismo , Ramipril/farmacologia , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Animais , Agonistas dos Canais de Cálcio/farmacologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Artérias Mesentéricas/fisiologia , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , Bloqueadores dos Canais de Potássio/farmacologia , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Tetraetilamônio/farmacologiaRESUMO
Altered function of K+ channels associated with hypertension has been inferred from the effects of K+ channel blockers on contraction of arterial smooth muscle cells (SMCs) and from K+ efflux measurements. Of the classes of K+ channels known to exist in the smooth muscle, the contribution of voltage-gated (KV) and high-conductance, Ca2+ gated K+ (BKCa) channels to the regulation of arterial SMC contractile function has been the most studied in hypertension. The effects of selective and nonselective K+ channel blockers on tonic contraction suggest that these two K+ channel gene families contribute differently to total K+ conductance in arterial SMCs from normal and hypertensive subjects. Direct measurements of K+ channel properties by electrophysiological methods generally support this conclusion. Studies have demonstrated larger BKCa currents in SMCs from several arteries of hypertensive rats, which have been reported to result from a greater Ca2+ sensitivity of BKCa channels and/or from greater protein expression. Some, but not all, studies have shown decreased KV currents in arterial SMCs from hypertensive animals measured under Ca(2+)-replete conditions. However, when external Ca2+ is removed or when Ca2+ influx is inhibited, KV currents are larger in SMCs exposed to chronic hypertension. Gene expression studies of Shaker KV1 transcripts have shown that of the dominant species present in arterial SMCs, KV1.2 expression is higher, whereas KV1.5 is the same in SMCs from hypertensive compared to normal animals. This finding is consistent with the larger KV currents in vascular SMCs from hypertensive animals under low Ca2+ conditions and suggests that Ca2+ influx and/or intracellular Ca2+ per se exerts a greater inhibitory effect on KV currents in the myocytes from these animals. The pathways by which these K+ channel differences are produced during hypertension remain to be elucidated, as does the potential for these channel proteins to be targeted by novel antihypertensive therapies.
Assuntos
Hipertensão/metabolismo , Canais de Potássio Cálcio-Ativados/biossíntese , Canais de Potássio de Abertura Dependente da Tensão da Membrana/biossíntese , Vasoconstrição/efeitos dos fármacos , Animais , Artérias/metabolismo , Artérias/fisiologia , Hipertensão/fisiopatologia , Canais de Potássio Ativados por Cálcio de Condutância Alta , Potenciais da Membrana/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiologia , Bloqueadores dos Canais de Potássio/farmacologiaRESUMO
The diameters of small arteries and arterioles are tightly regulated by the dynamic interaction between Ca(2+) and K(+) channels in the vascular smooth muscle cells. Calcium influx through voltage-gated Ca(2+) channels induces vasoconstriction, whereas the opening of K(+) channels mediates hyperpolarization, inactivation of voltage-gated Ca(2+) channels, and vasodilation. Three types of voltage-sensitive ion channels have been highly implicated in the regulation of resting vascular tone. These include the L-type Ca(2+) (Ca(L)) channels, voltage-gated K(+) (K(V)) channels, and high-conductance voltage- and Ca(2+)-sensitive K(+) (BK(Ca)) channels. Recently, abnormal expression profiles of these ion channels have been identified as part of the pathogenesis of arterial hypertension and other vasospastic diseases. An increasing number of studies suggest that high blood pressure may trigger cellular signaling cascades that dynamically alter the expression profile of arterial ion channels to further modify vascular tone. This article will briefly review the properties of Ca(L), K(V), and BK(Ca) channels, present evidence that their expression profile is altered during systemic hypertension, and suggest potential mechanisms by which the signal of elevated blood pressure may result in altered ion channel expression. A final section will discuss emerging concepts and opportunities for the development of new vasoactive drugs, which may rely on targeting disease-specific changes in ion channel expression as a mechanism to lower vascular tone during hypertensive diseases.