RESUMO
We have identified an agent (SP-303) that shows efficacy against in vivo cholera toxin-induced fluid secretion and in vitro cAMP-mediated Cl- secretion. Administration of cholera toxin to adult mice results in an increase in fluid accumulation (FA) in the small intestine (FA ratio = 0.63 vs. 1.86 in control vs. cholera toxin-treated animals, respectively). This elevation in FA induced by cholera toxin was significantly reduced (FA ratio = 0.70) in animals treated with a 100 mg/kg dose of SP-303 at the same time as the cholera treatment. Moreover, when SP-303 was administered 3 h after cholera toxin, a dose-dependent inhibition of FA levels was observed with a half-maximal inhibitory dose of 10 mg/kg. In Ussing chamber studies of Caco-2 or T84 monolayer preparations, SP-303 had a significant effect on both basal current and forskolin-stimulated Cl- current. SP-303 also induced an increase in resistance that paralleled the observed decrease in current. These data suggest that SP-303 has an inhibitory effect on cAMP-mediated Cl- and fluid secretion. Thus SP-303 may prove to be a useful broad-spectrum antidiarrheal agent.
Assuntos
Biopolímeros/farmacologia , Líquidos Corporais/metabolismo , Catequina/análogos & derivados , Cloretos/antagonistas & inibidores , AMP Cíclico/fisiologia , Animais , Líquidos Corporais/efeitos dos fármacos , Células CACO-2 , Catequina/farmacologia , Linhagem Celular , Toxina da Cólera/administração & dosagem , Toxina da Cólera/farmacologia , Colforsina/farmacologia , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Esquema de Medicação , Condutividade Elétrica , Impedância Elétrica , Feminino , Humanos , Intestino Delgado/citologia , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/metabolismo , Intestino Delgado/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
We evaluated the role of the activated Ras and Src/PyMT (Polyoma Middle T) signaling pathways on the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) in human colonic Caco-2 cell lines. Control vector-transfected Caco-2 cell monolayer preparations (Caco-2-H) responded to forskolin with an increase in short circuit current (Isc) mediated by CFTR. Furthermore, Caco-2-H cells responded to ATP, a reported stimulator of intracellular Ca2+ (Cai2+), and a potential source of adenosine-mediated elevation of cAMP. In contrast, Caco-2 cells transfected with PyMT (Caco-2-MT), expressing high levels of PKC, showed no sustained Isc response to forskolin or ATP. Pretreatment of Caco-2-MT cells with 2.5 microM phorbol 12-myristate 13-acetate (PMA) for 24 hr. effectively down-regulated PKC activity and restored expression of CFTR mRNA but failed to re-establish functional CFTR. These data suggest that, stable up-regulation of PKC alpha, consequent to activation of the Ras or Src/PyMT pathways, leads to an absence of CFTR expression and Cl- secretion mediated by either cAMP or Cai2+. Moreover, Cl- secretion in the colonic Caco-2 epithelial cell line is mediated primarily by CFTR and an alternate Cai(2+)-activated Cl- channel is not functional in these cells.