RESUMEN
Mammary epithelial 31EG4 cells (MEC) were grown as monolayers on filters to analyze the apical membrane mechanisms that help mediate ion and fluid transport across the epithelium. RT-PCR showed the presence of cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial Na(+) channel (ENaC) message, and immunomicroscopy showed apical membrane staining for both proteins. CFTR was also localized to the apical membrane of native human mammary duct epithelium. In control conditions, mean values of transepithelial potential (apical-side negative) and resistance (R(T)) are -5.9 mV and 829 Omega x cm(2), respectively. The apical membrane potential (V(A)) is -40.7 mV, and the mean ratio of apical to basolateral membrane resistance (R(A)/R(B)) is 2.8. Apical amiloride hyperpolarized V(A) by 19.7 mV and tripled R(A)/R(B). A cAMP-elevating cocktail depolarized V(A) by 17.6 mV, decreased R(A)/R(B) by 60%, increased short-circuit current by 6 microA/cm(2), decreased R(T) by 155 Omega x cm(2), and largely eliminated responses to amiloride. Whole cell patch-clamp measurements demonstrated amiloride-inhibited Na(+) currents [linear current-voltage (I-V) relation] and forskolin-stimulated Cl(-) currents (linear I-V relation). A capacitance probe method showed that in the control state, MEC monolayers either absorbed or secreted fluid (2--4 microl x cm(-2) x h(-1)). Fluid secretion was stimulated either by activating CFTR (cAMP) or blocking ENaC (amiloride). These data plus equivalent circuit analysis showed that 1) fluid absorption across MEC is mediated by Na(+) transport via apical membrane ENaC, and fluid secretion is mediated, in part, by Cl(-) transport via apical CFTR; 2) in both cases, appropriate counterions move through tight junctions to maintain electroneutrality; and 3) interactions among CFTR, ENaC, and tight junctions allow MEC to either absorb or secrete fluid and, in situ, may help control luminal [Na(+)] and [Cl(-)].
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Glándulas Mamarias Animales/metabolismo , Canales de Sodio/fisiología , Adenosina Trifosfato/farmacología , Amilorida/farmacología , Animales , Transporte Biológico/fisiología , Western Blotting , Línea Celular , Membrana Celular/fisiología , AMP Cíclico/farmacología , Impedancia Eléctrica , Células Epiteliales/metabolismo , Células Epiteliales/fisiología , Canales Epiteliales de Sodio , Inmunohistoquímica , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/efectos de los fármacos , Glándulas Mamarias Animales/fisiología , Ratones , Modelos Biológicos , Técnicas de Placa-Clamp , Reacción en Cadena de la Polimerasa , Valores de ReferenciaRESUMEN
Low levels of digoxin were noted in a patient receiving digoxin and sulfasalazine (SSA). Discontinuation of SSA resulted in a significant increase in serum digoxin levels. To determine whether or not SSA consistently interfered with the therapeutic effect of digoxin, both drugs were administered to 10 normal subjects in a crossover study. Each received 2 doses of digoxin (0.5 mg, elixir): one dose given alone, and a second dose after 6 days of treatment with SSA. When digoxin was given with SSA, the average area under the serum digoxin curve fell from the control value of 8.79 ng-hr-ml(-1) to 6.66 ng-hr-ml(-1) (p less than 0.05), fell and total urinary excretion decreased from 278 mcg/10 days to 228 mcg/10 days (p less than 0.025). These changes suggest interference with the bioavailability of digoxin by SSA. Studies were conducted to determine whether SSA inhibited digoxin absorption by physically absorbing the glycoside from solution. In vitro tests failed to reveal any significant adsorptive properties for SSA.
