RESUMO
Ht30/=Ht5). Cells with reduced mitochondrial activity also showed abnormal responses to the stimulation of NGF output. Thrombin and phorbol ester elevated NGF production from Ht100, Ht30 and Ht10 cells, but not from Ht5 cells. Ht30 cells, despite secreting less NGF basally than Ht100 cells, reached a similar or greater NGF output upon stimulation. Mitogens increased NGF output and NGF mRNA levels with the largest effect on NGF protein in Ht30 cells. Free radical production and the ability of cells to respond to NGF-inducing agents were related. These data suggest that chronic impairment of mitochondrial function associates with disturbances in cellular production of a signaling protein.
Assuntos
Mitocôndrias/fisiologia , Músculo Liso Vascular/metabolismo , Fator de Crescimento Neural/biossíntese , Animais , Linhagem Celular , Respiração Celular , Etídio/farmacologia , Radicais Livres/análise , Fator de Crescimento Neural/genética , RNA Mensageiro/biossíntese , Ratos , Transdução de Sinais/fisiologia , Acetato de Tetradecanoilforbol/farmacologia , Trombina/farmacologia , Fatores de TempoRESUMO
Altered nerve growth factor (NGF) regulation has been linked to the pathophysiology of hypertension. Vascular smooth muscle cells from an inbred hypertensive, but normoactive rat strain (WKHT) secreted NGF at a greater rate than from a hyperactive, normotensive strain (WKHA). Exposure to phorbol ester increased NGF secretion rates from WKHT by 400-800% but not from WKHA vascular muscle. NGF secretion rates from both WKHT and WKHA vascular cells were elevated by co-application of platelet-derived growth factor (PDGF) and transforming growth factor-beta1 (TGF-beta1) by 300-1000%. This response was partially attenuated by actinomycin D, an inhibitor of RNA transcription. These results suggest that regulation of NGF production does not occur solely at the level of transcription and post-transcriptional mechanisms operate. Analysis of NGF mRNA stability in the two strains following PDGF and TGF-beta1 treatment showed that NGF mRNA in WKHT had a half-life of 126.2+/-11.68 min while in WKHA vascular smooth muscle cells, the half-life was 47. 33+/-11.98 min. In addition to increased NGF mRNA stability in WKHT vascular muscle, these cells have an increased translational efficiency of NGF protein; elevated synthesis of NGF protein per unit NGF mRNA. Differences in signaling pathways may result in increased NGF mRNA stability and translational efficiency that may account for the elevated NGF protein in WKHT vascular smooth muscle cells.
Assuntos
Hipertensão/metabolismo , Músculo Liso Vascular/metabolismo , Fatores de Crescimento Neural/genética , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Animais , Aorta Torácica/citologia , Células Cultivadas , Cruzamentos Genéticos , Hipercinese/genética , Hipertensão/genética , Músculo Liso Vascular/inervação , Fatores de Crescimento Neural/metabolismo , Ratos , Ratos Endogâmicos SHR/genética , Ratos Endogâmicos WKY/genética , Ratos Mutantes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido Nucleico , Transdução de SinaisRESUMO
The spontaneously hypertensive rat (SHR) was developed as a genetic model of essential hypertension. In vivo and in vitro evidence demonstrates that vascular smooth muscle cells (VSMCs) from the SHR produce more nerve growth factor (NGF) than the normotensive Wistar-Kyoto (WKY) control strain. This increased NGF production is accompanied by excessive innervation of target tissues in the SHR. In the present study, a sensitive, competitive, quantitative, reverse-transcriptase polymerase chain reaction (C Q RT-PCR) assay is characterized and used to analyze levels of NGF mRNA in cultured VSMCs derived from the SHR and WKY strains as well as bladder tissue. Differences in NGF secretion rates between SHR and WKY VSMCs were partially due to an increased stability of NGF mRNA in SHR VSMCs. Following treatment with platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta 1) to elevate NGF production, the half-life of the NGF mRNA was 104.5 +/- 18.0 min in SHR VSMCs, compared to only 36.5 +/- 11.6 min in WKY VSMCs. Sequence analysis of the 3' untranslated region (UTR) revealed no strain differences in cis-acting sequences potentially involved in determining mRNA stability. Thus, it seems unlikely to be a 3'UTR mutation that prolongs mRNA lifetime. Rather, differential regulation of an RNA-binding protein may play a role in the abnormal NGF mRNA stability in SHR VSMCs. SHR VSMCs also demonstrate an increased translational efficiency of NGF protein; more NGF protein is synthesized per unit of NGF mRNA. The use of a C Q RT-PCR assay has allowed the determination that abnormal NGF mRNA stabilization as well as altered translational efficiency may contribute to excess NGF synthesis and progressive hypertension in the SHR.
