RESUMEN
1. The present study investigates whether chemical sympathectomy compromises the relaxation of the rabbit femoral artery precontracted with serotonin. The vasodilating agents promoted cyclic adenosine monophosphate (cAMP) accumulation or opening of the potassium channels. The effect of denervation on the adenylyl cyclase transduction pathway was also studied. 2. 6-Hydroxydopamine treatment did not impair the relaxation to adenosine, 8-bromoadenosine-cAMP (a membrane-permeable analog of cAMP) and 3-isobutyl-1-methylxanthine (a cAMP phosphodiesterase inhibitor). Moreover, denervation enhanced the relaxation to forskolin (a direct Gs-type protein activator) and pinacidil (a potassium channel opener). 3. Denervation modified neither adenosine diphosphate ribosylation of Gs- and Gi-proteins nor adenylyl cyclase activity.
Asunto(s)
Adenilil Ciclasas/metabolismo , Colforsina/farmacología , Arteria Femoral/inervación , Guanidinas/farmacología , Músculo Liso Vascular/inervación , Transducción de Señal/fisiología , Vasodilatadores/farmacología , 1-Metil-3-Isobutilxantina/farmacología , 3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Toxina de Adenilato Ciclasa , Adenilil Ciclasas/fisiología , Animales , Toxina del Cólera/farmacología , Electroforesis en Gel de Poliacrilamida , Arteria Femoral/efectos de los fármacos , Arteria Femoral/enzimología , Técnicas In Vitro , Masculino , Desnervación Muscular , Relajación Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/enzimología , Norepinefrina/metabolismo , Inhibidores de Fosfodiesterasa/farmacología , Pinacidilo , Canales de Potasio/efectos de los fármacos , Canales de Potasio/metabolismo , Conejos , Transducción de Señal/efectos de los fármacos , Simpatectomía , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
We show that the levels and activity of the alpha-subunits of Gs and Gi proteins in plasma membrane of GH4C1 cells are regulated by the availability of mevalonate (MVA), and not by changes in cholesterol cell content. Changes in the levels of MVA, induced by modulation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, determine the amount of both membrane-bound G alpha-subunits, which correlated with the activity of their effector adenylyl cyclase. Lipoprotein deficient serum (LPDS) decreases cholesterol content and increases both HMG-CoA reductase activity and G alpha-subunits in the membrane. Cholesterol and 25-hydroxycholesterol (25-HC) each repress HMG-CoA reductase and diminish G alpha-subunit levels. However, while cholesterol cell content is also decreased by 25-HC, exogenous cholesterol increases it. In addition, the decrease of both G alpha-subunits is reversed by the presence of MVA. This regulation appears to be mediated by nonsterol products generated from MVA. We assume that the first is the prenylation of the gamma-subunits, since the attachment of G alpha-subunits to the membrane is dependent on this modification. However, as neither of our treatments completely abolished protein prenylation, we conclude that another MVA derivative is required in addition to prenyl residues to the presence and activity of alpha-subunits in the membrane.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Ácido Mevalónico/metabolismo , Sangre , Línea Celular , Membrana Celular/metabolismo , Colesterol/farmacología , Hidroxicolesteroles/farmacología , Transducción de SeñalRESUMEN
It is generally accepted that cholesterol affects dynamic membrane properties and the function of membrane bound proteins involved in secretion processes. In the present study we employed GH4C1 cells treated with human lipoprotein-deficient serum (h-LPDS), exogenous cholesterol and high density lipoprotein (HDL3) to investigate the role of cholesterol cell content on PRL and GH basal release. Incubation of GH4C1 cells with h-LPDS decreased free cholesterol content and cholesterol added to the media increased it. HDL3 did not act as a cholesterol acceptor in either cholesterol-depleted or cholesterol-loaded cells; however, in depleted cells HDL3 was a net donor, significantly increasing cell cholesterol. Control or cholesterol loaded cells incubated in media with h-LPDS increased their secretion of PRL in parallel with the loss of cell cholesterol. Conversely, the addition of either cholesterol or HDL3 to cholesterol depleted cells inhibited PRL release. However, GH secretion was not modified by changes in free cholesterol in any of these situations. In the experiments in which HDL3 was present, a highly positive correlation was found between cholesterol cell content at the end of the experiment and PRL secretion, no effect could be related to the amount of added HDL3, suggesting that the HDL3 had no specific effect on the secretion of PRL or GH. Our results indicate that cholesterol cell content is an important factor in the release of PRL but not of GH, and emphasize the differences in the basal regulation of the secretion of both hormones.
Asunto(s)
Colesterol/metabolismo , Hormona del Crecimiento/metabolismo , Hipófisis/metabolismo , Prolactina/metabolismo , Línea Celular , Colesterol/farmacología , Humanos , Lipoproteínas/sangre , Lipoproteínas/deficiencia , Lipoproteínas HDL/farmacología , Hipófisis/citología , Factores de TiempoRESUMEN
In pituitary GH1 cells, a rat growth hormone-producing cell line, butyrate elicited a dose-dependent increase in cholera toxin receptors as measured by an increased binding of 125I-labeled cholera toxin to the intact cells. Butyrate did not alter the affinity of cholera toxin binding, the dissociation constant being 0.4 nM for both control and butyrate-treated cells. Despite the increased binding, the cAMP response to cholera toxin was strongly reduced after exposure to butyrate. This reduction was dose-dependent and with butyrate 1--5 mM, intracellular and extracellular (medium) cAMP levels were decreased by more than 70% in cells incubated for 24 h with 1 nM cholera toxin. Forskolin (30 microM) elicited a cAMP response similar to that found with the toxin, and a similar inhibition of cAMP was also found after incubation of GH1 cells with butyrate. Butyrate also affected basal cAMP levels which were reduced by 40--60% in cells cultured for 24--48 h with the fatty acid. In order to study whether butyrate influenced cAMP synthesis and/or cAMP degradation, adenylyl cyclase and phosphodiesterase activities were determined in control cells and in cells incubated for 24 h with cholera toxin or forskolin. Butyrate had a dual effect since, besides activating phosphodiesterase by more than twofold, it also inhibited the cyclase by 40--50% in all groups. The in vitro response of adenylyl cyclase to stimulatory (NaF) and inhibitory (carbachol and adenosine) effectors was also examined. The absolute activity of the cyclase was always 40--50% lower in the cells incubated with butyrate, but the percentage change of activity obtained in butyrate-treated and untreated cells was unaltered. In addition, ADP-ribosylation of the guanine nucleotide stimulatory component of the cyclase (Gs) was not affected in the cells incubated with butyrate. These results suggest that the catalytic (C) subunit of adenylyl cyclase and/or its interaction with the regulatory components might be altered in butyrate-treated GH1 cells. The inhibition of the cAMP response in GH1 cells was accompanied by an inhibition of a biological action of the nucleotide, namely growth hormone (somatotropin) production which is primarily controlled by thyroid hormones in these cells. Forskolin alone did not affect the somatotropin levels but potentiated the growth hormone response to triiodothyronine. Butyrate produced a dose-dependent inhibition of this response, which was totally abolished at concentrations of butyrate higher than 1 mM.