Thrombin, phorbol ester, and cAMP regulate thrombin receptor protein and mRNA expression by different pathways

Human mesangial cells have been used to study the regulation of thrombin receptor protein and mRNA expression during cross-talk between different signal transduction pathways. Persistent activation of thrombin receptor by thrombin led to homologous down-regulation of thrombin receptor protein. However, thrombin receptor mRNA expression was not affected, suggesting that increased receptor degradation is responsible for homologous down-regulation. Chronic activation of protein kinase C by phorbol 12-myristate 13-acetate (PMA) and of adenylylcyclase by prostaglandin E1 (PGE1) resulted in heterologous down-regulation of thrombin receptor protein. In contrast to thrombin, PMA and PGE1 reduced in parallel thrombin receptor mRNA levels to 51% and 24% of control, respectively, indicating that heterologous down-regulation of thrombin receptor protein is, at least in part, due to inhibition of receptor mRNA expression. The mechanisms of heterologous down-regulation of thrombin receptor protein have been studied in detail and compared to homologous down-regulation. PMA-induced down-regulation was completely blocked by GF 109 203 X, an inhibitor of protein kinase C. However, the loss of thrombin receptor induced by thrombin was not prevented by GF 109 203 X, indicating that homologous regulation is not dependent on protein kinase C activation. The heterologous effect of PGE1 was mimicked by 8-bromo-cAMP, isobutylmethylxanthine, and forskolin, suggesting that an increase in intracellular cAMP level is involved in heterologous regulation. Interestingly, heterologous down-regulation induced by PGE1 seems not to require previous internalization of thrombin receptor. These data indicate that thrombin receptor protein and mRNA expression can be regulated in homologous and heterologous ways by different mechanisms

Effect of calcium and hydrogen ion on the fibrinolytic activity of isolated renal glomeruli from rat

Effect of calcium and hydrogen ion on the fibrinolytic activity of isolated renal glomeruli from rat. The fibrinolytic activity of glomeruli isolated from rat kidney has been measured with125I-fibrin-coated tubes. This technique allows the study of the two successive steps of the reaction: the release of the activator of plasminogen from the cells in the presence of isolated glomeruli, and the enzymatic process itself in the presence of the supernatant obtained after a 30-min incubation period with isolated glomeruli and centrifugation. Calcium in the range of 0.01 to 1.0mM increased the rate of fibrinolysis and shortened the lag-time preceding this effect. The fibrinolytic activity was also measured in the presence of isolated glomeruli as a function of the plasminogen concentration. Calcium at 1mM clearly increased the maximum velocity but did not change the concentration of plasminogen corresponding to 50% of the maximum. Supernatants were obtained after preincubation of isolated glomeruli with increasing doses of calcium. Addition of these supernatants to the125I-fibrin-coated tubes produced a progressive increase in the rate of fibrinolysis, depending on the dose of calcium. When calcium was added to the supernatants only, after having discarded the glomeruli, there was no change or, above 0.5mM calcium, a slight inhibition of the enzymatic reaction itself. The fibrinolytic activity measured in the presence of isolated glomeruli was maximum at a pH of 8. There was a clear-cut interaction in this condition between pH and calcium concentration. This interaction was not observed when the isolated glomeruli had been discarded. These results clearly demonstrate the major role of calcium in the release from glomeruli of an activator of plasminogen.Effet du calcium et de l'ion hydrogène sur l'activité fibrinolytique de glomérulus isolés de reins de rat. L'activité fibrinolytique de glomérules isolés de rein de rat a été mesurée en utilisant des tubes sur lesquels de la125I fibrine a été fixée. Cette technique permet d'apprécier les 2 étapes de la réaction: sortie de l'activateur cellulaire du plasminogène lorsque les glomérules isolés sont présents dans le milieu d'incubation et réaction enzymatique ellemême lorsque le surnageant obtenu après 30min d'incubation et élimination des glomérules isolés par centrifugation est étudié. Le calcium entre 0.01 et 1.0mM augmente la quantité de fibrine lysée et raccourcit le temps de latence nécessaire à cet effet. L'activité fibrinolytique a aussi été mesurée en présence de glomérules isolés en fonction de la concentration de plasminogène. Le calcium à la concentration de 1mM augmente clairement la vitesse maximale mais ne change pas la concentration de plasminogène correspondant à 50% de l'effet maximum. Des surnageants ont été obtenus après pré incubation de glomérules isolés dans des milieux contenant des concentrations croissantes de calcium. L'adjonction de ceux-ci aux tubes sur lesquels de la125I fibrine a été fixée entraine une augmentation croissante de la quantité de fibrine lysée dépendante de la dose de calcium. Quand du calcium a été ajouté à un surnageant obtenu après avoir éliminé les glomérules isolés il n'y a pas eu de modification de la fibrinolyse ou une légère inhibition de la réaction enzymatique pour des concentrations de calcium supérieures à 0.5mM. L'activité fibrinolytique mesurée en présence de glomérules isolés a été maximum à pH 8. Il y a eu une interaction claire entre le pH et la concentration de calcium qui n'a pas été observée lorsque les glomérules ont été éliminés. Ces résultats démontrent clairement le rôle majeur du calcium dans la sortie des glomérules d'un activateur du plasminogène

Isolation and propagation of glomerular mesangial cells.

Cultures of glomerular mesangial cells (MC) of rodent or human origin have been extensively employed in renal research laboratories since the early 1980s. Cultured MC retain extensive analogies with the fairly undifferentiated in vivo phenotype of an intercapillary mesenchymal cell population, i.e., a myofibroblast. MC proliferating in response to mitogens and growth factors can be growth-arrested by withdrawal of serum or 3D culture in collagen gels. They synthesize an extracellular matrix that includes interstitial collagens and has analogies with the glomerular basement membrane; a prominent cytoskeleton acts as a functional contractile apparatus. Cultured MC have been extensively employed as a tool for studying pathophysiological events such as mesangial expansion, scarring, and glomerulosclerosis. Current technology for MC isolation and culture is reviewed, with emphasis on methodological issues relevant to characterization, propagation, and long-term maintenance of homogeneous clones