RESUMO
BACKGROUND AND PURPOSE: AMP-activated protein kinase (AMPK) is activated by metformin, phenformin, and the AMP mimetic, 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR). We have completed an extensive study of the pharmacological effects of these drugs on AMPK activation, adenine nucleotide concentration, transepithelial amiloride-sensitive (I(amiloride)) and ouabain-sensitive basolateral (I(ouabain)) short circuit current in H441 lung epithelial cells. EXPERIMENTAL APPROACH: H441 cells were grown on permeable filters at air interface. I(amiloride), I(ouabain) and transepithelial resistance were measured in Ussing chambers. AMPK activity was measured as the amount of radiolabelled phosphate transferred to the SAMS peptide. Adenine nucleotide concentration was analysed by reverse phase HPLC and NAD(P)H autofluorescence was measured using confocal microscopy. KEY RESULTS: Phenformin, AICAR and metformin increased AMPK (alpha1) activity and decreased I(amiloride). The AMPK inhibitor Compound C prevented the action of metformin and AICAR but not phenformin. Phenformin and AICAR decreased I(ouabain) across H441 monolayers and decreased monolayer resistance. The decrease in I(amiloride) was closely related to I(ouabain) with phenformin, but not in AICAR treated monolayers. Metformin and phenformin increased the cellular AMP:ATP ratio but only phenformin and AICAR decreased cellular ATP. CONCLUSIONS AND IMPLICATIONS: Activation of alpha1-AMPK is associated with inhibition of apical amiloride-sensitive Na(+) channels (ENaC), which has important implications for the clinical use of metformin. Additional pharmacological effects evoked by AICAR and phenformin on I(ouabain), with potential secondary effects on apical Na+ conductance, ENaC activity and monolayer resistance, have important consequences for their use as pharmacological activators of AMPK in cell systems where Na+K+ATPase is an important component.
Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Hipoglicemiantes/farmacologia , Metformina/farmacologia , Complexos Multienzimáticos/efeitos dos fármacos , Fenformin/farmacologia , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Ribonucleotídeos/farmacologia , Sódio/metabolismo , Proteínas Quinases Ativadas por AMP , Nucleotídeos de Adenina/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Amilorida , Aminoimidazol Carboxamida/farmacologia , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Células Epiteliais , Canais Epiteliais de Sódio/efeitos dos fármacos , Fluorescência , Humanos , Pulmão , Microscopia Confocal , Complexos Multienzimáticos/metabolismo , Ouabaína , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Hypothalamic 5HT concentrations are transiently lower in male compared to female Wistar rats in the second week post partum (pp) and our previous findings have shown that pharmacologically potentiating 5HT activity over this period feminizes certain aspects of sexually differentiated behaviours in adult males and androgenized females. In order to investigate whether neonatal testosterone and 5HT interact to influence physiological and morphological brain sexual differences, females, androgenized females and males were treated with the 5HT2 agonist (-) [2,5 dimethoxy-4-iodophenyl]-2-amino propane HCl [(-) DOI], over days 8-16 pp. In androgenized females (250 microg testosterone proprionate, day 2 pp) (-) DOI prevented the delay in vaginal opening, but did not prevent the androgen-induced constant oestrus in females treated with 100 microg TP, day 2 pp. (-) DOI overcame the neonatal androgen effect in suppressing the positive feedback of ovarian steroids in a few males and androgenized females. (-) DOI had a feminizing effect on the volume of the anteroventral periventricular nucleus (normally smaller in males), by significantly increasing its volume in male and androgenized females. It also had a significant antagonistic effect on the testosterone-induced increase in the volume of the sexually dimorphic nucleus of the preoptic area in males and androgenized females. These findings support the view that raised 5HT activity in the second week of life antagonizes the masculinizing effect of neonatal testosterone.