RESUMO
We wanted to elucidate whether extracellular calcium may regulate the expression of the cardiac gap-junction proteins connexin 40 and connexin43. In the free wall of the left atria of 126 cardiac surgery patients with either sinus rhythm (SR) or chronic atrial fibrillation (AF), we determined the expression of the cardiac gap-junction proteins Cx43 and Cx40 by Western blot and immunohistology. For deeper investigation, we incubated cultured neonatal rat cardiomyocytes at 2 or 4 mM Ca(++) for 24 h and determined intercellular coupling, Cx40, Cx43 protein and mRNA expression, protein trafficking and sensitivity to verapamil (10-100 nM), cyclosporin A (1 microM),and BMS605401 (100 nM), a specific inhibitor of Ca(2+)-sensing receptor (CaSR). We found in patients that both Cx are up-regulated in AF in the left atrium (by 100-200%). Interestingly, Cx40 was mainly up-regulated, if total serum calcium was >or=2.2 mM, while Cx43 was independent from extracellular [Ca(++)]. In cultured cells, 4 mM Ca(++)-exposure lead to up-regulation of Cx40, but not Cx43. We found enhanced Cx40 in the plasma membrane and reduced Cx40 in the Golgi apparatus. The membrane Cx40 up-regulation resulted in enhanced gap-junction intercellular coupling with a shift in the Boltzmann fit of voltage-dependent inactivation indicating a higher contribution of Cx40 as revealed by dual whole cell voltage clamp experiments. BMS605401 could prevent all Ca(2+)-induced changes. Moreover, cyclosporin A completely abolished the Ca(2+)-induced changes, while verapamil was ineffective. We conclude that extracellular calcium (24 h exposure) seems to up-regulate Cx40 but not Cx43.
Assuntos
Cálcio/fisiologia , Junções Comunicantes/fisiologia , Animais , Fibrilação Atrial/metabolismo , Células Cultivadas , Conexina 43/análise , Conexina 43/fisiologia , Conexinas/análise , Conexinas/fisiologia , Ciclosporina/farmacologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ratos , Ratos Wistar , Transdução de Sinais , Verapamil/farmacologia , Proteína alfa-5 de Junções ComunicantesRESUMO
BACKGROUND: Oxidatively modified LDL (oxLDL) plays an important role in the development of atherosclerosis. OxLDL effects, eg, foam cell formation, are mediated in part by the classic scavenger receptor, whereas other effects may involve the recently cloned endothelial oxLDL receptor, LOX-1 (lectinlike oxLDL receptor-1), which is distinct from macrophage scavenger receptors. Because the regulation of LOX-1 must still be defined, we investigated whether LOX-1 is regulated by the potentially proatherosclerotic stimulant angiotensin II (Ang II). METHODS AND RESULTS: Using competitive reverse transcription-polymerase chain reaction (RT-PCR), we quantified mRNA expression of LOX-1 in primary cultures of human umbilical vein endothelial cells (HUVECs). After treatment with Ang II for 3 hours (1 nmol/L to 1 micromol/L), LOX-1 mRNA was concentration-dependently induced (from 6.9+/-1.4 to 23.1+/-5.5 relative units [RU] by 1 micromol/L Ang II; P<0.05). The angiotensin II type 1 (AT(1)) receptor antagonist losartan prevented this induction. Incubation of HUVECs with Ang II (100 nmol/L, 3 hours) induced LOX-1 protein expression (212+/-21% of control level; P<0. 01) and uptake of 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled oxLDL (209+/-17% of control level; P<0.05) by an AT(1)-dependent pathway, reaching its maximum after 24 hours (680+/-89%; P<0.05). In internal mammary artery biopsy samples from patients with or without ACE inhibitor treatment before coronary artery bypass surgery, LOX-1 mRNA was downregulated by ACE inhibition (6.4+/-2.0 versus 19.3+/-5. 9 RU; n=12 each; P<0.05). CONCLUSIONS: We conclude that LOX-1 is regulated by Ang II in vitro and in vivo, that induction of LOX-1 is mediated by the AT(1) receptor, and that repression of LOX-1 by long-term ACE inhibitor treatment may contribute to the antiatherosclerotic potential of this therapy.
Assuntos
Angiotensina II/metabolismo , Doença da Artéria Coronariana/metabolismo , Receptores de LDL/metabolismo , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Antiarrítmicos/farmacologia , Anti-Hipertensivos/farmacologia , Células Cultivadas , Ponte de Artéria Coronária , Doença da Artéria Coronariana/tratamento farmacológico , Doença da Artéria Coronariana/cirurgia , Regulação para Baixo/efeitos dos fármacos , Endotélio Vascular/citologia , Humanos , Losartan/farmacologia , Artéria Torácica Interna , RNA Mensageiro/análise , Receptores de LDL/genética , Receptores de LDL Oxidado , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptores Depuradores Classe E , Veias UmbilicaisRESUMO
In this study, we analyzed the effect of endothelin-1 (ET-1) on expression of the lectin-like oxidized low-density lipoprotein (oxLDL) receptor-1 LOX-1 and on oxLDL uptake in primary cultures of human umbilical vein endothelial cells (HUVEC). LOX-1 mRNA was quantified by standard-calibrated competitive RT-PCR, LOX-1 protein expression by Western analysis and endothelial oxLDL uptake using DiI-labeled oxLDL. ET-1 induces LOX-1 mRNA expression, reaching its maximum after 1 h (160 +/- 14% of control, 100 nM ET-1, P < 0.05). This increased ET-1-mediated LOX-1 mRNA expression could be inhibited by endothelin receptor B antagonist BQ-788. In addition, ET-1 stimulates LOX-1 protein expression and oxLDL uptake in HUVEC. The augmented oxLDL uptake by ET-1 is mediated by endothelin receptor B, but not by protein kinases. These data support a new pathophysiological mechanism how locally and systemically increased ET-1 levels could promote LOX-1-mediated oxLDL uptake in human endothelial cells and the development and progression of endothelial dysfunction and atherosclerosis.
Assuntos
Endotelina-1/farmacologia , Endotélio Vascular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Receptores de LDL/genética , Transcrição Gênica/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Western Blotting , Células Cultivadas , Antagonistas dos Receptores de Endotelina , Humanos , Cinética , Lipoproteínas LDL/metabolismo , Oligopeptídeos/farmacologia , Piperidinas/farmacologia , Proteínas Quinases/metabolismo , RNA Mensageiro/genética , Receptor de Endotelina B , Receptores de LDL/análise , Receptores de LDL/biossíntese , Receptores de LDL Oxidado , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptores Depuradores Classe E , Veias Umbilicais , Regulação para Cima/efeitos dos fármacosRESUMO
Superoxide anions (O(*-)(2)) induce oxidative stress and reduce endothelial NO availability by peroxynitrite formation. In human endothelial cells gp91(phox) was identified as the limiting subunit of the forming NAD(P)H oxidase. Because endothelin-1 (ET-1) is considered as a pro-atherosclerotic stimulus, we analyzed the effect of ET-1 on gp91(phox) expression and O(*-)(2) generation in primary cultures of human umbilical vein endothelial cells (HUVECs). The gp91(phox) mRNA expression was quantified by standard calibrated competitive reverse transcriptase-polymerase chain reaction. ET-1 induces gp91(phox) mRNA expression in HUVEC (max. after 1 h). The induction of gp91(phox) expression was dose-dependent, reaching its maximum at 10 nmol/L ET-1. The increased gp91(phox) expression is mediated by endothelial receptor type B (ET(B)). Furthermore, ET-1 augments O(*-)(2) generation in human endothelial cells as measured by coelenterazine chemiluminescence. These data support a new mechanism: how ET-1 increases oxidative stress in the vessel wall leading to endothelial dysfunction and enhanced susceptibility to atherosclerosis.