RESUMEN
Valepotriates are iridoids found in variable amounts in Valerianaceae and might be among the bioactive compounds which confer anxiolytic properties to the Valeriana species. On the other hand, unspecific cytotoxicity has also been described. Presently, however, no particular molecular target has been defined for these compounds. Here we studied the effect of valtrate, acevaltrate, and 1- ß-acevaltrate isolated from Valeriana glechomifolia on the enzymatic activity of rat P-type ATPases. Valepotriates did not affect rat skeletal muscle sarco/endoplasmic reticulum Ca²âº-ATPase (SERCA) activity at the highest concentration used (100 µM). In contrast, the same concentration inhibited roughly half of the total Hâº/Kâº-ATPase activity from rat gastric epithelium (valtrate 54.6 ± 3.2â%, acevaltrate 60.7 ± 7.3â%, 1- ß-acevaltrate 50.2 ± 3.1â%; mean ± SEM, n = 3-5). Finally, these substances showed the highest inhibitory potency toward Naâº/Kâº-ATPase, and the inhibition curves obtained provided a similar IC50 (in µM) for rat kidney α1 isoform (valtrate 21.2, acevaltrate 22.8, 1- ß-acevaltrate 24.4) and brain hemispheres α2/ α3 isoforms (valtrate 19.4, acevaltrate 42.3, 1- ß-acevaltrate 38.3). Our results suggest that P-type ATPases are differentially inhibited by valepotriates and that Naâº/Kâº-ATPase might be one of their molecular targets in vivo.
Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Iridoides/farmacología , Valeriana/química , Adenosina Trifosfatasas/efectos de los fármacos , Adenosina Trifosfatasas/metabolismo , Animales , Encéfalo/enzimología , Epitelio/enzimología , ATPasa Intercambiadora de Hidrógeno-Potásio/efectos de los fármacos , ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , Concentración 50 Inhibidora , Iridoides/química , Iridoides/aislamiento & purificación , Riñón/enzimología , Masculino , Ratas , Ratas Wistar , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/efectos de los fármacos , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/efectos de los fármacos , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Estómago/enzimologíaRESUMEN
The kinetic properties of a microsomal gill (Na+,K+)-ATPase from the freshwater shrimp, Macrobrachium olfersii, acclimated to 21 per thousand salinity for 10 days were investigated using the substrate p-nitrophenylphosphate. The enzyme hydrolyzed this substrate obeying cooperative kinetics at a rate of 123.6+/-4.9 U mg-1 and K0.5=1.31+/-0.05 mmol L-1. Stimulation of K+-phosphatase activity by magnesium (Vmax=125.3+/-7.5 U mg-1; K0.5=2.09+/-0.06 mmol L-1), potassium (Vmax=134.2+/-6.7 U mg-1; K0.5=1.33+/-0.06 mmol L-1) and ammonium ions (Vmax=130.1+/-5.9 U mg-1; K0.5=11.4+/-0.5 mmol L-1) was also cooperative. While orthovanadate abolished p-nitrophenylphosphatase activity, ouabain inhibition reached 80% (KI=304.9+/-18.3 micromol L-1). The kinetic parameters estimated differ significantly from those for freshwater-acclimated shrimps, suggesting expression of different isoenzymes during salinity adaptation. Despite the approximately 2-fold reduction in K+-phosphatase specific activity, Western blotting analysis revealed similar alpha-subunit expression in gill tissue from shrimps acclimated to 21 per thousand salinity or fresh water, although expression of phosphate-hydrolyzing enzymes other than (Na+,K+)-ATPase was stimulated by high salinity acclimation.