Asunto(s)
Humanos , Femenino , Persona de Mediana Edad , Neutropenia/complicaciones , Hipertiroidismo/complicaciones , Lupus Eritematoso Sistémico/complicaciones , Peritonitis/complicaciones , Neutropenia/patología , Hipertiroidismo/patología , Lupus Eritematoso Sistémico/patología , Peritonitis/patología , Resultado FatalRESUMEN
Thyrotropin-releasing hormone (TRH) plays an important role in central cardiovascular regulation. Recently, we described that the TRH precursor gene overexpression induces hypertension in the normal rat. In addition, we published that spontaneously hypertensive rats (SHR) have central extrahypothalamic TRH hyperactivity with increased TRH synthesis and release and an elevated TRH receptor number. In the present study, we report that intracerebroventricular antisense (AS) treatment with a phosphorothioate oligonucleotide against the TRH precursor gene significantly diminished up to 72 hours and in a dose-dependent manner the increased diencephalic TRH content, whereas normalized systolic blood pressure (SABP) was present in the SHR compared with Wistar-Kyoto (WKY) rats. Although basal thyrotropin was higher in SHR compared with WKY rats and this difference disappeared after antisense treatment, no differences were observed in plasma T4 or T3 between strains with or without AS treatment, indicating that the effect of the AS on SABP was independent of the thyroid status. Because the encephalic renin-angiotensin system seems to be crucial in the development and/or maintenance of hypertension in SHR, we investigated the effect of antisense inhibition of TRH on that system and found that TRH antisense treatment significantly diminished the elevated diencephalic angiotensin II (Ang II) content in the SHR without any effect in control animals, suggesting that the Ang II system is involved in the TRH cardiovascular effects. To summarize, the central TRH system seems to be involved in the etiopathogenesis of hypertension in this model of essential hypertension.
Asunto(s)
ADN sin Sentido/farmacología , Hipertensión/etiología , Hormona Liberadora de Tirotropina/fisiología , Angiotensina II/metabolismo , Animales , Presión Sanguínea , Ventrículos Cerebrales/metabolismo , ADN sin Sentido/administración & dosificación , Modelos Animales de Enfermedad , Hipertensión/genética , Hipertensión/metabolismo , Masculino , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/farmacología , Radioinmunoensayo , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Sistema Renina-Angiotensina/fisiología , Hormonas Tiroideas/sangre , Hormona Liberadora de Tirotropina/análisis , Hormona Liberadora de Tirotropina/biosíntesis , Hormona Liberadora de Tirotropina/genéticaRESUMEN
The human glioblastoma-astrocytoma cell line U-373-MG shows morphological features typical of its neuroectodermal origin. Cells showed positive immunostaining for the glial fibrillary acidic protein. We used this cell culture for studying the putative production of TRH and TRH-related peptides. In a cell extract and conditioned medium, cation and anion exchange chromatography and HPLC revealed the presence of TRH and acidic TRH-like peptides which were identified, at least in part, as pGlu-Glu-ProNH(2). These findings demonstrated that U-373-MG cells are able to produce and release these peptides. Further evidence of TRH synthesis was obtained by amplification using RT-PCR of a 396 bp fragment that corresponds to the TRH precursor mRNA. Our results therefore suggest that the U-373-MG cell line may be a useful model for studying the regulation of TRH and TRH-related peptide production and the interaction of these peptides with other classical neurotransmitter systems. In fact, pilocarpine (a muscarinic cholinergic agonist) enhanced and nicotine (a nicotinic cholinergic agonist) decreased TRH and TRH-related compound production by this cell line. These data also point out that glia may produce substances with neuromodulatory action.
Asunto(s)
Astrocitoma/química , Neoplasias Encefálicas/química , Glioblastoma/química , Hormona Liberadora de Tirotropina/aislamiento & purificación , Análisis de Varianza , Astrocitoma/metabolismo , Neoplasias Encefálicas/metabolismo , Sistema Nervioso Central/metabolismo , Cromatografía DEAE-Celulosa , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Glioblastoma/metabolismo , Humanos , Modelos Biológicos , Ácido Pirrolidona Carboxílico/análogos & derivados , ARN Mensajero/análisis , Radioinmunoensayo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Hormona Liberadora de Tirotropina/análogos & derivados , Hormona Liberadora de Tirotropina/genética , Hormona Liberadora de Tirotropina/metabolismo , Células Tumorales Cultivadas/química , Células Tumorales Cultivadas/metabolismoRESUMEN
OBJECTIVE: We studied the expression of parathyroid hormone (PTH)-related protein in vascular smooth muscle cells of spontaneously hypertensive rats (SHR) using Wistar-Kyoto (WKY) and Sprague-Dawley rats as normotensive controls. METHODS: Aortae from 4- and 18-week-old SHR versus age-matched WKY and Sprague-Dawley rats were excised to obtain total RNA or smooth muscle cells. The cells were subcultured in Dulbecco's Modified Eagle's Medium containing 10% fetal calf serum, then serum-deprived for 72 h and stimulated with 0.1 micromol/I angiotensin II. PTH-related protein, c-myc and angiotensin II type qa receptor (AT1aR) messenger (m)RNA levels were measured by Northern blot, using total RNA extracted by phenol/chloroform. The effects of PTH-related protein(1-34)NH2 intravenous injections on arterial blood pressure and the heart rate were studied in anesthetized SHR and WKY rats. RESULTS: The Northern blots showed a significantly higher abundance of PTH-related protein mRNA in aortae of SHR versus WKY rats in the prehypertensive state but no significant difference in adult animals. In cultured aortic smooth muscle cells, angiotensin II induced a four- to sixfold increase in PTH-related protein mRNA levels in smooth muscle cells from normotensive animals, but failed to elicit a significant response in smooth muscle cells derived from SHR in either the prehypertensive or the hypertensive state. This lack of response to angiotensin II in SHR smooth muscle cells was not due to decreased expression or responsiveness of the AT1aR, since SHR smooth muscle cells had more AT1aR mRNA than Sprague-Dawley smooth muscle cells, and angiotensin II-induced activation of c-myc was faster and greater in smooth muscle cells derived from 4- or 18-week-old SHR than in Sprague-Dawley smooth muscle cells. In contrast, PTH-related protein(1-34)NH2 induced a long-lasting dose-dependent hypotensive and tachycardic response in both SHR and WKY rats, indicating that SHR retained responsiveness to the vasodilator. CONCLUSIONS: PTH-related protein gene expression in response to angiotensin II is impaired in SHR arteries. A deficiency in this potent local vasodilator may contribute to the development and/or maintenance of arterial hypertension in this model.
Asunto(s)
Angiotensina II/farmacología , Hipertensión/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Proteínas/metabolismo , Vasoconstrictores/farmacología , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/metabolismo , Northern Blotting , Hemodinámica/efectos de los fármacos , Inyecciones Intravenosas , Masculino , Músculo Liso Vascular/metabolismo , Hormona Paratiroidea/metabolismo , Proteína Relacionada con la Hormona Paratiroidea , Proteínas/genética , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Ratas Sprague-Dawley , Receptor de Angiotensina Tipo 1 , Receptor de Angiotensina Tipo 2 , Receptores de Angiotensina/genética , Receptores de Angiotensina/metabolismoRESUMEN
Extrahypothalamic TRH participates in cardiovascular regulation and spontaneous hypertension of the rat. To investigate whether an increase in central TRH activity produces hypertension we studied the effect of the preTRH overproduction induced by I.C.V. transfection with a naked eukaryotic expression plasmid vector which encodes preTRH (pCMV-TRH). Northern blot analysis and RT-PCR showed that pCMV-TRH was transcribed in vitro and in vivo. At 24, 48, and 72 hours, pCMV-TRH (100 microg) in a significant and dose-dependent manner increased 37%, 84%, and 49%, respectively, the diencephalic TRH content and SABP (42+/-3, 50+/-2, and 22+/-2 mm Hg, respectively) with respect to the vector without the preTRH cDNA insert (V[TRH(-)]) as measured by RIA and the plethysmographic method, respectively, in awake animals. In addition, using immunohistochemistry we found that the increase of TRH was produced in circumventricular areas where the tripeptide is normally located. To further analyze the specificity of these effects we studied the actions of 23-mer sense (S), antisense (AS), and 3'self-stabilized sense (Ss) and antisense (ASs) phosphorothioate oligonucleotides against the initiation codon region. Only ASs inhibited the increase of TRH content and SABP induced by pCMV-TRH treatment. In addition, pCMV-TRH-induced hypertension seems not to be mediated by central Ang II or serum TSH. To summarize, central TRH overproduction in periventricular areas induced by I.C.V. transfection produces hypertension in rats which is reversed by specific antisense treatment. This model may help in testing effective antisense oligodeoxynucleotides against other candidate genes.
Asunto(s)
Encéfalo/metabolismo , Regulación de la Expresión Génica , Hipertensión/etiología , Oligonucleótidos Antisentido/farmacología , Precursores de Proteínas/genética , Hormona Liberadora de Tirotropina/genética , Animales , Humanos , Masculino , Ratas , Ratas Wistar , Hormona Liberadora de Tirotropina/fisiología , TransfecciónRESUMEN
The interleukin-1 (IL-1) system is constituted by IL-1 alpha and IL-1 beta and IL-1 receptor antagonist (IL-1ra) that bind the same IL-1 receptors. Hypothalamic-pituitary-adrenal axis hormones are major mediators of the neuroendocrine control over immune function. Corticotropin-releasing hormone (CRH) is produced in peripheral inflammatory sites; its direct effects on inflammatory cytokine synthesis, however, remain unclear. We have studied the effects of CRH (0.1-100 nM) on IL-1 beta and IL-1ra expression by human peripheral monocytes in culture activated with different doses of lipopolysaccharide (LPS). In the absence of LPS, CRH up-regulated IL-1ra and IL-1 beta messenger RNA expression as well as protein synthesis. No significant changes were observed with low doses of LPS (1 ng/ml). In contrast, in combination with high doses of LPS (1 microgram/ml), CRH caused inhibition of IL-1ra and IL-1 beta transcription and secretion. The CRH effects were blocked by its antagonist alpha-helical CRH and mediated by intracellular cAMP. These data indicate that CRH modulates the IL-1 system; depending on the state of activation of the monocyte, CRH exerts an inhibitory control on the activated cell and a stimulatory action on the resting monocyte.
Asunto(s)
Hormona Liberadora de Corticotropina/farmacología , Interleucina-1/genética , Lipopolisacáridos/farmacología , Monocitos/efectos de los fármacos , Sialoglicoproteínas/genética , Células Cultivadas , AMP Cíclico/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Proteína Antagonista del Receptor de Interleucina 1 , Interleucina-1/antagonistas & inhibidores , Interleucina-1/metabolismo , Monocitos/metabolismo , ARN Mensajero/análisis , Sialoglicoproteínas/metabolismoRESUMEN
Thyrotropin-releasing hormone (TRH) plays an important role in central cardiovascular regulation through the activation of different neurotransmitter systems at distinct extrahypothalamic sites. To study possible alterations in the TRH system in the hypertensive state, we measured TRH concentration in cerebrospinal fluid and TRH content of the preoptic area in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) by radioimmunoassay. In addition, we also measured the density of the TRH receptor in this area by a rapid filtration technique using [3H]methyl-TRH. We found a significant increase in both the TRH content (634 +/- 61 versus 350 +/- 26 pg/mg protein, SHR versus WKY; P < .01, n = 5) and density of TRH receptors without changes in affinity (Bmax, 5.0 +/- 0.1 versus 3.3 +/- 0.1 fmol/mg protein, P < .01, n = 4). An increase in TRH concentration was also found in the cerebrospinal fluid of SHR (30 +/- 3 versus 21 +/- 2 pg/mL, P < .01, n = 5), suggesting increased TRH release in the central nervous system. Northern blot analysis indicated a threefold augmented abundance of TRH precursor mRNA in the preoptic area of SHR. A polyclonal antibody raised against TRH injected peripherally or intracerebroventricularly lowered arterial blood pressure in SHR but not in WKY. In addition, long-term treatment with enalapril (5 mg/kg twice daily), which was effective in inhibiting serum angiotensin-converting enzyme activity by more than 50%, decreased arterial blood pressure and preoptic area TRH content of SHR, whereas another vasodilator, diltiazem (10 mg/kg every 8 hours), failed to produce a similar change.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Hipertensión/fisiopatología , Área Preóptica/química , Hormona Liberadora de Tirotropina/fisiología , Análisis de Varianza , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Antihipertensivos/farmacología , Presión Sanguínea/efectos de los fármacos , Northern Blotting , Bloqueadores de los Canales de Calcio/farmacología , Diltiazem/administración & dosificación , Diltiazem/farmacología , Enalapril/administración & dosificación , Enalapril/farmacología , Hipertensión/tratamiento farmacológico , Hipertensión/etiología , Hipertensión/genética , Masculino , ARN Mensajero/análisis , Radioinmunoensayo , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Receptores de Hormona Liberadora de Tirotropina/análisis , Receptores de Hormona Liberadora de Tirotropina/genética , Hormona Liberadora de Tirotropina/análisis , Hormona Liberadora de Tirotropina/líquido cefalorraquídeo , Factores de Tiempo , Regulación hacia ArribaRESUMEN
Synenkephalin (proenkephalin 1-70) is produced and secreted as an intact molecule or as a part of precursors in the adult brain and adrenal medulla, respectively. However, it is cleaved to low molecular weight peptides in proliferating immune cells. Considering that the pre-proenkephalin gene is expressed in the embryonic rat brain during the cell proliferation stage, we studied the processing of synenkephalin in embryonic rat brains (E18) and compared it with the processing in adult rat brains. IR-synenkephalin was measured by RIA using a C-terminally directed antiserum. Adult rat brains contained higher concentrations of immunoreactive (IR)-synenkephalin (2,612 + 264) than embryonic rat brain (1,361 + 100) (results in fmol/mg proteins, n = 5). Gel filtration chromatography (Sephadex G-50) showed that in the extracts of adult rat brain, 50% of the IR-synenkephalin eluted in the position of the authentic peptide (8 kDa) and the rest of the immunoreactivity corresponded to partially processed peptides of 4.0 and 2.5 kDa. In embryonic rat brains synenkephalin was processed to intermediate peptides of 2.5, 1.7 and mainly to a low molecular weight peptide of 1.0 kDa. The concentration of this last peptide, which was further characterized by affinity column and HPLC, represented 45% of the total immunoreactivity. IR-met-enkephalin in embryonic rat brains (analyzed before and after enzymatic digestion with trypsin and carboxypeptidase B) corresponded principally to non-processed or partially processed products. However, these were cleaved to free met-enkephalin in adult rat brains.(ABSTRACT TRUNCATED AT 250 WORDS)