RESUMEN
In DM (differentiation medium), Sol 8 myoblasts spontaneously form myotubes and express the betaMHC (beta-myosin heavy chain), their main marker of terminal differentiation. This marker is detectable at 24 h, and increases up to 72 h. Our aim was to define temporal effects of TGFbeta (transforming growth factor beta) on betaMHC expression in Sol 8 cells. TGFbeta1 (1 ng/ml) added at time zero to DM decreased MyoD expression and completely inhibited betaMHC expression in Sol 8 cells. This inhibition of betaMHC expression was progressively lost when TGFbeta1 was added from 8 to 34 h. After 34 h, the cells were irreversibly differentiated, and TGFbeta1 did not inhibit betaMHC accumulation any longer. Two independent approaches showed that a TGFbeta autocrine regulatory loop retarded and partially impaired Sol 8 cell terminal differentiation. First, permanent immunoneutralization of the active TGFbetas released by the cells into DM increased betaMHC levels at 72 h compared with controls. Secondly, a dominant-negative mutant of the TGFbeta type II receptor was overexpressed in Sol 8 cells under the control of the betaMHC promoter. Both the dominant-negative receptor and the betaMHC gene were expressed after 24 h in DM. The delayed blocking of the TGFbeta signalling pathway by the dominant-negative receptor was as effective as permanent immunoneutralization to promote betaMHC expression. To conclude, TGFbeta inhibits Sol 8 cell terminal differentiation within a narrow time interval (24-34 h) that coincides with the onset of betaMHC expression.
Asunto(s)
Comunicación Autocrina/fisiología , Diferenciación Celular/fisiología , Mioblastos/fisiología , Factor de Crecimiento Transformador beta/fisiología , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/fisiología , Ratones , Ratones Endogámicos C3H , Mioblastos/efectos de los fármacos , Mioblastos/metabolismo , Cadenas Pesadas de Miosina/biosíntesis , ARN Mensajero/biosíntesis , Receptores de Factores de Crecimiento Transformadores beta/biosíntesis , Receptores de Factores de Crecimiento Transformadores beta/fisiología , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/fisiología , Homología de Secuencia de Aminoácido , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Factor de Crecimiento Transformador beta1RESUMEN
In Lyon hypertensive (LH) rats, a model of low-renin genetic hypertension, we investigated adrenal sensitivity to angiotensin II in terms of angiotensin II receptor (AT1 and AT2 receptors) regulation, morphological changes, and aldosterone and corticosterone secretion. Twelve-week-old LH rats, compared with normotensive LN and LL rats, were either untreated or treated for 4 weeks with AT1 receptor antagonist irbesartan (50 mg/kg/d), angiotensin-converting enzyme inhibitor perindopril (3 mg/kg/d), or perindopril (3 mg/kg/d) plus angiotensin II infusion (200 ng/kg/min). At 16 weeks, untreated LH rats had high systolic blood pressure (P<0.05), low aldosterone (P<0.05), and increased corticosterone (P<0.05) plasma levels. AT1-receptor binding density in the zona glomerulosa was similar in the three strains. In LH rats, angiotensin II infusion increased the relative adrenal weight from 10.5+/-0.3 to 16.7+/-0.7 mg/100g (P<0.05), whereas this change was very modest in normotensive rats. Zona glomerulosa enlarged and plasma aldosterone increased after angiotensin II infusion in the 3 strains, but more markedly in LH versus normotensive rats (2.4- versus 1.3- and 1.6-fold, respectively; 20- versus 10-fold in normotensive rats, P<0.05). Surprisingly, after angiotensin II infusion, despite the absence of angiotensin II receptors in the three strains, the zona fasciculata-reticularis enlarged 1.5-fold and plasma corticosterone increased 1.7-fold only in LH rats (P<0.05), suggesting an indirect control of this compartment by angiotensin II. The hypertrophy and hypersecretory activity of both zona glomerulosa and zona fasciculata-reticularis in LH rats in response to angiotensin II point to the adrenal cortex as a pivotal tissue in the pathophysiology of hypertension in LH rats.
Asunto(s)
Corteza Suprarrenal/metabolismo , Corteza Suprarrenal/patología , Angiotensina II/farmacología , Hipertensión/metabolismo , Hipertensión/patología , Corteza Suprarrenal/efectos de los fármacos , Glándulas Suprarrenales/efectos de los fármacos , Glándulas Suprarrenales/patología , Aldosterona/sangre , Animales , Presión Sanguínea/efectos de los fármacos , Corticosterona/sangre , Hipertensión/genética , Masculino , Tamaño de los Órganos , Ratas , Ratas Mutantes , Receptor de Angiotensina Tipo 1/metabolismo , Receptor de Angiotensina Tipo 2/metabolismoRESUMEN
The human corticotropin-releasing factor receptor type 1 (hCRF-R1) functional transcript is mainly expressed in the anterior pituitary corticotrophs, a tissue usually not available for clinical investigation. Splice variants translated into defective isoforms of the receptor have been described in few peripheral tissues. The aim of this work was to determine whether peripheral white blood cells from healthy individuals, an accessible tissue for clinical investigation, were suitable for the analysis of the hCRF-R1 transcript and gene. We report that: (i) specific amplification of the hCRF-R1 transcript from peripheral white blood cells mRNAs is feasible; (ii) this transcript is similar to the functional transcript; (iii) the draft sequence of chromosome 17 and unrelated sequences allow direct sequencing of all 14 exons of the gene, adjacent splice sites and related branch points. In conclusion, these approaches would be suitable for studies in patients having isolated secondary glucocorticoids deficiency to implicate the hCRH-R1 in the etiology of the disease.