| ObjectiveCigarette smoking exposure is a well-known risk factor for hypertension, atherosclerosis (AS), coronary heart disease (CHD), stroke, myocardial infarction, aortic aneurysm, peripheral vascular disease and other cardiovascular diseases. There is plenty of evidences show that smoking may induce damage of vessel wall and disorder of blood vessel endothelium, also enhance the form of atheromatous plaque. There is different receptor subtypes located on or inside vascular endothelial cell and smooth muscle cells, among them, G-protein coupled receptors (GPCR) which located in the surface of the cell are most capital. Mountains of experimental and clinical studies have demonstrated that the GPCR in vascular smooth muscle (VSMC) play an important role on blood vessel contraction. The activation of GPCR can regulate blood flow and the level of blood pressure. The dysfunction of contraction and proliferation mediated by GPCR in vascular smooth muscle is an important cause for the development of vascular diseases. However, the underlying molecular mechanisms that cigarette smoke leads to higher morbidity of cardiovascular disease are not fully understood.The present study was designed to examine if organ culture or DSP regulate TP, ETA and ETB receptors in renal arteries, and which intracellular signal pathways are involved in the organ culture or DSP's effects. Understanding the intracellular signal mechanisms behind cigarette smoke-induce renal arterial dysfunction and damage may provide a new target for developing therapies for hypertension and cardiovascular disease.MethodsRat renal arteries were dissected and after removal of endothelium, they were cut into about 1~1.5mm long cylindrical segments and subjected to organ culture. Specific intracellular signal pathway inhibitors were added to the culture medium to test the involvement of different signal pathways. A sensitive myograph system was used to record isometric tension of the segments, and selective agonists were added to tissue baths in turn to evaluate the TP, ETA and ETB receptor-mediated vessel contractility. The receptor mRNA expression was quantified by real-time reverse transcription PCR, while the receptor protein expression was examined using a laser scanning confocal microscope after immunohistochemistry staining.Based on the result of organ culture, the ring segments of renal arteries from rats were exposed to DSP (0.1~0.4 ml﹒L-1), nicotine or LPS, in the organ culture for up to 24 h. The alteration of GPCR caused by DSP was studied by three levels: functional evaluation, mRNA analysis and protein localization.ResultsWe observed down-regulation of the TP in rat renal artery after organ culture for 24h. The down-regulation of the TP was partly abolished by general transcriptional inhibitor ACD, but not abolished by general translational inhibitor CHX. The results of RT-PCR and immunohistochemistry showed that the mRNA and protein expression of TP decreased after organ culture. This suggests that the down-regulation of TP involves a transcriptional mechanism. Furthermore, the decreased contraction caused by organ culture was partly abolished by U0126 (a ERK1/2 pathway inhibitor), but not by SP600125 (a JNK pathway inhibitor) and SB203580 (a p38 pathway inhibitor). This suggests that ERK1/2 signal pathway involves the down-regulation of TP. The NF-κB pathway inhibitor BMS345541, co-incubated with the segments during the organ culture, revealed that organ culture-induced down-regulation of the TP significantly was attenuated. This means the down-regulation of TP receptor occurs through the NF-κB pathway. Additionally, we observed that the down-regulation of the TP was abolished by MG-132. This suggests that the down-regulation of TP receptor was activated through the IκB pathway; both IκBαand IκBβwere involved.ETA and ETB receptors were up-regulated after organ culture. The enhanced contraction mediated by ETB receptor was inhibited by U0126 and SB203580, while the enhanced contraction mediated by ETA receptor was not affected by U0126,SP600125 and SB203580. Suggesting that the up-regulated of ETB receptor maybe caused by the activation of ERK1/2and P38 pathway, while MAPK do not involve in the up-regulation of ETA receptor. Furthermore, MG-132 abolished the up- up-regulated of ETB and ETA receptor, but the inhibitors of PDE had no effects. After the segments organ culture for 24h, the mRNA expression of Il-6 and Il-1βincreased.The contractions mediated by TP, ETB and ETA receptors were significantly increased after the arterial segments exposed to DSP in a concentration- dependent (0.1~0.4 ml﹒L-1) manner and ETB receptor also presented in a time-dependent (0～24h) manner, compared to control (DMSO). This was in parallel with enhanced mRNA expression for ETB receptor, but not ETA and TP receptors. The results of immunohistochemistry showed that the protein expression of TP, ETA and ETB receptor increased after the segments cultured with DSP for 24h. BMS345541 totally abolished ETB receptor up-regulation, but not ETA and TP receptor up-regulations. Our results suggest that DSP transcriptionally up-regulated ETB receptor expression in rat renal artery via NF-кB signal pathways, whereas up-regulation of ETA and TP receptor-mediated contraction may involve post-transcriptional mechanisms. Both nicotine and LPS were unlikely involved in DSP effects, although LPS at the higher concentrations had such effects.ConclusionsTP was down-regulated after organ culture, while ETA and ETB receptors were up-regulated. The down-regulation of TP receptor occurs through the ERK1/2 and the downstream NF-κB pathway. The up-regulated of ETB receptor maybe caused by the activation of ERK1/2, P38 and NF-κB pathway, while MAPK and NF-κB pathway do not involve in the up-regulation of ETA receptor.DSP transcriptionally up-regulated ETB receptor expression in rat renal artery via NF-кB signals pathways, whereas up-regulation of ETA and TP receptor-mediated contraction may involve post-transcriptional mechanisms. Both nicotine and LPS were unlikely involved in DSP effects.
|
PDF Full Text Request