RESUMO
Hydrogen sulfide has been shown to have a sympathoinhibitory effect in the rostral ventrolateral medulla (RVLM). The present study examined the function of cystathionine ß-synthase (CBS)/hydrogen sulfide system in the RVLM, which plays a crucial role in the control of blood pressure and sympathetic nerve activity. Adenovirus vectors encoding CBS (AdCBS) or enhanced green fluorescent protein (AdEGFP) were transfected into the RVLM in normotensive rats. Identical microinjection of AdCBS into the RVLM had no effect on systolic blood pressure and heart rate (HR) in conscious rats. Acute experiments were performed at day 7 after gene transfer in anesthetized rats. Microinjection of the CBS inhibitors hydroxylamine (HA) or amino-oxyacetate into the RVLM produced an increase in the renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP), and HR. There was a potentiation of the increases in RSNA, MAP, and HR because of the CBS inhibitors in AdCBS-injected rats compared with AdEGFP-injected rats. Pretreatment with pinacidil, a ATP-sensitive potassium (KATP) channel activator, abolished the effects of HA in two groups. Microinjection of glibenclamide, a KATP channel blocker, produced increases in RSNA, MAP, and HR in AdCBS-injected rats. No changes in behavior were observed in AdEGFP-injected rats. Furthermore, Western blot analysis indicated an increase in the expression of sulfonylurea receptor 2 and inward rectifier K(+) 6.1 in AdCBS-injected rats. These results suggest that the increase in KATP channels in the RVLM may be responsible for the greater sympathetic outflow and pressor effect of HA in AdCBS-injected rats compared with AdEGFP-injected rats.
Assuntos
Sistema Cardiovascular/inervação , Cistationina beta-Sintase/biossíntese , Técnicas de Transferência de Genes , Sulfeto de Hidrogênio/metabolismo , Canais KATP/metabolismo , Rim/inervação , Bulbo/enzimologia , Inibição Neural , Sistema Nervoso Simpático/metabolismo , Adenoviridae/genética , Animais , Pressão Arterial , Cistationina beta-Sintase/antagonistas & inibidores , Cistationina beta-Sintase/genética , Inibidores Enzimáticos/farmacologia , Vetores Genéticos , Frequência Cardíaca , Canais KATP/antagonistas & inibidores , Masculino , Bulbo/efeitos dos fármacos , Inibição Neural/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Ratos Sprague-Dawley , Transdução de Sinais , Receptores de Sulfonilureias/metabolismo , Sistema Nervoso Simpático/efeitos dos fármacos , Sistema Nervoso Simpático/fisiopatologia , Fatores de Tempo , Regulação para CimaRESUMO
AIM: to study the molecular mechanisms underlying α-tocopheryl succinate (α-TOS)-induced apoptosis in erbB2-positive breast cancer cells and to determine whether α-TOS and the human recombinant TNF-related apoptosis-inducing ligand (hrTRAIL) act synergically to induce cell death of erbB2-expressing breast cancer cells. METHODS: the annexin V binding method was used to measure apoptosis induced by α-TOS and/or hrTRAIL. RT-PCR and Western blotting were performed to detect gene and protein expression. A colorimetric assay was performed to detect caspase activity. The TransAM(TM) NF-κB p65 kit was used to assess NF-κB activation. RESULTS: α-TOS (100 µmol/L) significantly inhibited NF-κB nuclear translocation in erbB2-expressing breast cancer cells; this inhibition is expected to result in the inactivation of NF-κB. α-TOS (50 and 100 µmol/L) inhibited the expression of Flice-like inhibitory protein (FLIP) and cellular inhibitor of apoptosis protein 1 (c-IAP1) in erbB2-positive cells. α-TOS (100 µmol/L) inhibited Akt activation and augmented the activity of caspase 3 and caspase 8 in breast cancer cells expressing erbB2. α-TOS (50 µmol/L) and hrTRAIL (30 mg/mL) acted synergically to induce apoptosis in breast cancer cells. α-TOS also decreased the hrTRAIL-induced transient activation of NF-κB . CONCLUSION: our results suggest that α-TOS mediates the apoptosis of erbB2-positive breast cancer cells and acts synergically with hrTRAIL via the NF-κB pathway.
Assuntos
Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , NF-kappa B/fisiologia , Receptor ErbB-2/metabolismo , alfa-Tocoferol/farmacologia , Neoplasias da Mama , Caspases/genética , Caspases/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
An intact L1 define element is 6 kb in length in human genome. The majority of the L1s is truncated and has direction difference, implying that it is interesting to study the effects of different length and directions of L1s on gene. In this work, 7 different segments were obtained from L1-open reading frame 2 (ORF2), each of which was 280 bp in length. Each segment was connected into 8 repeats in head and tail tandem manner and was inserted to downstream of GFP gene in different directions in pEGFP-C1. The inserted ORF2 segments in the sense orientation caused much stronger inhibition on gene transcription and protein expression than antisense sequences did. Among all segments, the first and ninth 280 bp segments of ORF2 in both orientations induced weaker inhibition on gene transcription than other segments in the same orientations and did not induce transcriptional elongation. The distribution of Alu in most regions of genome was inverse ratio with L1. The inserted Alus in both orientations inhibited GFP gene expression, but the inhibition in antisense orientation was stronger than that in sense orientation and the sense Alu was the sequence of inducing transcription elongation. A-rich of ORF2 was probably the molecular basis of its sense orientation with stronger inhibition on gene expression.
Assuntos
Regulação da Expressão Gênica , Genes Reporter/genética , Fases de Leitura Aberta/genética , Transcrição Gênica/genética , Northern Blotting , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Microscopia de Fluorescência , Reação em Cadeia da Polimerase , TransfecçãoRESUMO
BACKGROUND: Hydrogen sulfide (H2S) is an important endogenous gaseous transmitter in many physiological functions. Plasma H2S decreased, and angiotensin II (Ang II) type 1 receptor (AT1R) increased in the myocardial tissues in 2-kidney 1-clip (2K1C) rats than in normotensive rats. Accumulating evidences suggest that H2S inhibited Ang II/AT1R pathway to regulate cardiovascular function. Therefore, we hypothesized that H2S may exert beneficial effects on myocardial remodeling in 2K1C rat models of renovascular hypertension. METHODS AND RESULTS: Sodium hydrosulfide (NaHS, 56 µmol/kg/day) was administered intraperitoneally to the rats from the 7th day after 2K1C operation. Systolic blood pressure was significantly increased from the first week after the operation and was lowered after NaHS treatment for 4 weeks. H2S could also inhibit the ratio of left ventricle and septum weight to body weight, improve cross-sectional area, and ameliorate ventricular dysfunction. Additionally, the protein expression of AT1R and Ang II serum content were downregulated, whereas superoxide dismutase (SOD) protein was upregulated in 2K1C rats by NaHS treatment for 4 weeks. Furthermore, the reactive oxygen species level and AT1R protein were increased, whereas SOD protein was decreased in cardiomyocytes treated with Ang II compared with the control group. NaHS could reverse these changes. Losartan and N-acetylcysteine could also reverse Ang II-induced changes. CONCLUSIONS: The protective effect of H2S is attributable to the suppression of oxidative stress. This process involves the inhibition of the Ang II/AT1R pathway and upregulation of antioxidant enzymes in 2K1C rats.
Assuntos
Cardiomegalia/prevenção & controle , Gasotransmissores/uso terapêutico , Sulfeto de Hidrogênio/uso terapêutico , Hipertensão Renovascular/complicações , Remodelação Ventricular/efeitos dos fármacos , Angiotensina II/sangue , Animais , Pressão Sanguínea/efeitos dos fármacos , Cardiomegalia/etiologia , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Gasotransmissores/farmacologia , Sulfeto de Hidrogênio/sangue , Sulfeto de Hidrogênio/farmacologia , Hipertensão Renovascular/metabolismo , Masculino , Estresse Oxidativo/efeitos dos fármacos , Ratos Sprague-Dawley , Receptor Tipo 1 de Angiotensina/metabolismo , Superóxido Dismutase-1/metabolismoRESUMO
BACKGROUND: Rostral ventrolateral medulla (RVLM) plays a crucial role in the central regulation of cardiovascular functions. Cystathionine-ß-synthase (CBS) is a major hydrogen sulfide (H2S)-generating enzyme that has been identified mainly in the brain. The present study was designed to examine CBS expression and determine its roles and mechanisms of regulating sympathetic outflow and blood pressure (BP) in the RVLM in spontaneously hypertensive rats (SHR). METHODS AND RESULTS: CBS expression was decreased in the RVLM in SHR compared to Wistar-Kyoto (WKY) rats. Accumulating evidences suggest that H2S interacts with nitric oxide (NO) to regulate cardiovascular function. Therefore, we hypothesize that the decrease in CBS expression in the RVLM may be involved in the disorder of l-arginine/NO pathway, which subsequently affects BP in SHR. Overexpression of CBS in the RVLM caused significant increases in BP, heart rate, and urinary norepinephrine excretion in SHR but not in WKY. Acute experiments were carried out at day 7 after gene transfer. NO metabolite levels, neuronal NO synthase, and γ-amino butyric acid were decreased in SHR after CBS gene transfer. Furthermore, pressor responses to microinjection of NG-monomethyl-l-arginine into RVLM were blunt in SHR transfected with AdCBS compared to SHR transfected with AdEGFP. CONCLUSIONS: Overexpression of CBS in the RVLM elicits enhanced pressor responses in SHR, but not in WKY, and the NO system is involved in these effects. The results suggest that alterations of H2S signaling in the brain may be associated with the development of hypertension.