RESUMEN
ACE inhibitors improve endothelial dysfunction, possibly by blocking endothelial angiotensin production. Prorenin, through its binding and activation by endothelial mannose 6-phosphate (M6P) receptors, may contribute to this production. Here, we investigated this possibility as well as prorenin activation kinetics, the nature of the prorenin-activating enzyme, and M6P receptor-independent prorenin binding. Human umbilical vein endothelial cells (HUVECs) were incubated with wild-type prorenin, K/A-2 prorenin (in which Lys42 is mutated to Ala, thereby preventing cleavage by known proteases), M6P-free prorenin, and nonglycosylated prorenin, with or without M6P, protease inhibitors, or angiotensinogen. HUVECs bound only M6P-containing prorenin (K(d) 0.9+/-0.1 nmol/L, maximum number of binding sites [B(max)] 1010+/-50 receptors/cell). At 37 degrees C, because of M6P receptor recycling, the amount of prorenin internalized via M6P receptors was >25 times B(max). Inside the cells, wild-type and K/A-2 prorenin were proteolytically activated to renin. Renin was subsequently degraded. Protease inhibitors interfered with the latter but not with prorenin activation, thereby indicating that the activating enzyme is different from any of the known prorenin-activating enzymes. Incubation with angiotensinogen did not lead to endothelial angiotensin generation, inasmuch as HUVECs were unable to internalize angiotensinogen. Most likely, therefore, in the absence of angiotensinogen synthesis or endocytosis, M6P receptor-mediated prorenin internalization by endothelial cells represents prorenin clearance.
Asunto(s)
Endotelio Vascular/metabolismo , Precursores Enzimáticos/metabolismo , Receptor IGF Tipo 2/fisiología , Renina/metabolismo , Angiotensinas/biosíntesis , Células Cultivadas , Frío , Endocitosis , Precursores Enzimáticos/genética , Humanos , Cinética , Mutación , Renina/genéticaRESUMEN
Mannose-6-phosphate (man-6-P)/insulin-like growth factor-II (man-6-P/IgF-II) receptors are involved in the activation of recombinant human prorenin by cardiomyocytes. To investigate the kinetics of this process, the nature of activation, the existence of other prorenin receptors, and binding of native prorenin, neonatal rat cardiomyocytes were incubated with recombinant, renal, or amniotic fluid prorenin with or without man-6-P. Intact and activated prorenin were measured in cell lysates with prosegment- and renin-specific antibodies, respectively. The dissociation constant (K(d)) and maximum number of binding sites (B(max)) for prorenin binding to man-6-P/IGF-II receptors were 0.6 +/- 0.1 nM and 3,840 +/- 510 receptors/myocyte, respectively. The capacity for prorenin internalization was greater than 10 times B(max). Levels of internalized intact prorenin decreased rapidly (half-life = 5 +/- 3 min) indicating proteolytic prosegment removal. Prorenin subdivision into man-6-P-free and man-6-P-containing fractions revealed that only the latter was bound. Cells also bound and activated renal but not amniotic fluid prorenin. We concluded that cardiomyocytes display high-affinity binding of renal but not extrarenal prorenin exclusively via man-6-P/IGF-II receptors. Binding precedes internalization and proteolytic activation to renin thereby supporting the concept of cardiac angiotensin formation by renal prorenin.
Asunto(s)
Precursores Enzimáticos/metabolismo , Miocardio/metabolismo , Procesamiento Proteico-Postraduccional , Receptor IGF Tipo 2/metabolismo , Renina/metabolismo , Líquido Amniótico/enzimología , Animales , Animales Recién Nacidos , Células Cultivadas , Activación Enzimática , Fibroblastos/metabolismo , Humanos , Riñón/enzimología , Cinética , Inhibidores de Proteasas/farmacología , Unión Proteica , Ratas , Proteínas Recombinantes/metabolismoRESUMEN
Newly developed IRMAs to measure the plasma concentrations of renin and prorenin were validated for clinical use and compared with a classical enzyme kinetic assay. The IRMAs involve two monoclonal antibodies, one that reacts equally well with renin and prorenin and one that recognizes renin well but prorenin only minimally. Prorenin reactivity with the second antibody was enhanced by adding the renin inhibitor, Remikiren, to plasma. The complex of prorenin with this active-site ligand undergoes a conformational change, whereby prorenin is converted into a form that cannot be differentiated from renin by the IRMA. The linear working range of the assay was 4.0-3000 mU/L. The concentration of prorenin was calculated by subtracting the assay result obtained without Remikiren (i.e., renin) from the result obtained with Remikiren (i.e., renin plus prorenin). No more than 2% of prorenin present in plasma was detected as renin. The interassay CVs for renin quantification were 18%, 13%, and 8% at low, medium, and high concentrations, respectively. The interassay CV for calculated prorenin was 8% at both low and high concentrations. The IRMA results were highly correlated with those of an enzyme kinetic assay in healthy subjects; in patients with such conditions as primary hyperaldosteronism, renovascular hypertension, and low-, medium-, and high-renin essential hypertension; and in women undergoing gonadotropin stimulation.
Asunto(s)
Precursores Enzimáticos/sangre , Imidazoles , Ensayo Inmunorradiométrico/métodos , Ensayo Inmunorradiométrico/estadística & datos numéricos , Inhibidores de Proteasas , Renina/antagonistas & inhibidores , Renina/sangre , Adulto , Anciano , Anticuerpos Monoclonales , Anticoagulantes , Estabilidad de Enzimas , Femenino , Humanos , Indicadores y Reactivos , Masculino , Persona de Mediana Edad , Proteínas Recombinantes , Valores de Referencia , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadAsunto(s)
Precursores Enzimáticos/sangre , Ensayo Inmunorradiométrico/métodos , Renina/antagonistas & inhibidores , Renina/sangre , Adulto , Análisis Químico de la Sangre/métodos , Anticonceptivos Orales/farmacología , Diabetes Mellitus Tipo 1/sangre , Estudios de Evaluación como Asunto , Femenino , Gonadotropinas/farmacología , Humanos , Imidazoles , Masculino , Persona de Mediana Edad , Preeclampsia/sangre , EmbarazoRESUMEN
Plasma atrial natriuretic factor (ANF) concentrations were measured, and relationships to intracardiac pressures and atrial dimensions were assessed in a series of 17 heart transplant recipients undergoing cardiac catheterization during their annual evaluation. Despite excellent cardiac function (normal filling pressures, adequate cardiac output, ejection fraction above 60%), plasma levels of ANF were elevated. Step-up levels across the heart were consistent with increased cardiac secretion, whereas both the metabolic clearance rate and plasma half-life were normal. Correlations between plasma concentrations of ANF at different cardiac sites and atrial filling pressures were low. However, right and left atrial dimensions in our patients were greatly enlarged. These findings suggest that increased atrial size (by virtue of the atrial anastomoses) and augmented wall stress (law of Laplace) rather than pressure per se are responsible for the increased ANF production after heart transplantation.