Bumetanide inhibits (Na + K + 2Cl) co-transport at a chloride site

258

239

The mechanisms underlying mucus-associated pathologies in cystic fibrosis (CF) remain obscure. However, recent studies indicate that CF transmembrane conductance regulator (CFTR) is required for bicarbonate (HCO 3 -) transport and that HCO 3 -is critical for normal mucus formation. We therefore investigated the role of HCO 3 -in mucus secretion using mouse small intestine segments ex vivo. Basal rates of mucus release in the presence or absence of HCO 3 -were similar. However, in the absence of HCO 3 -, mucus release stimulated by either PGE 2 or 5-hydroxytryptamine (5-HT) was approximately half that stimulated by these molecules in the presence of HCO 3 -. Inhibition of HCO 3 -and fluid transport markedly reduced stimulated mucus release. However, neither absence of HCO 3 -nor inhibition of HCO 3 -transport affected fluid secretion rates, indicating that the effect of HCO 3 -removal on mucus release was not due to decreased fluid secretion. In a mouse model of CF (mice homozygous for the most common human CFTR mutation), intestinal mucus release was minimal when stimulated with either PGE 2 or 5-HT in the presence or absence of HCO 3 -. These data suggest that normal mucus release requires concurrent HCO 3 -secretion and that the characteristically aggregated mucus observed in mucin-secreting organs in individuals with CF may be a consequence of defective HCO 3 -transport.

Section: Figurementioning

confidence: 99%

Normal mouse intestinal mucus release requires cystic fibrosis transmembrane regulator–dependent bicarbonate secretion

García

Yang²,

Quinton³

2009

258

239

“…The dose of bumetanide which resulted in 50% inhibition of 86Rb+ uptake was 2 X 10-6 M, as compared with 7 X 10-6 M for inhibition of VIP-induced I,,. The discrepancy may be explained by the large experimental error in the Ussing chamber experiments and by changes in the affinity of bumetanide for the cotransporter by temperature or small differences in Na', K+, or Clconcentrations for the two experimental conditions (37,38).…”

Section: Resultsmentioning

confidence: 99%

Vasoactive intestinal polypeptide-induced chloride secretion by a colonic epithelial cell line. Direct participation of a basolaterally localized Na+,K+,Cl- cotransport system.

Dharmsathaphorn¹,

Mandel²,

Masui³

et al. 1985

224

100

We have used a well-differentiated human colonic cell line, the T4 cell line, as a model system to study the pathways of cellular ion transport involved in vasoactive intestinal polypeptide (VIP)-induced chloride secretion. A modified Ussing chamber was used to study transepithelial Na' and Cl-fluxes across confluent monolayer cultures of the T84 cells grown on permeable supports. In a manner analogous to isolated intestine, the Addition of VIP caused an increase of net Cl-secretion which accounted for the increase in short circuit current (1). The effect of VIP on I,, was dose dependent with a threshold stimulation at 10-10 M VIP, and a maximal effect at 10-8 M.Bumetanide prevented or reversed the response to VIP. Inhibition by bumetanide occurred promptly when it was added to the serosal, but not to the mucosal bathing media. Ion replacement studies demonstrated that the response to VIP required the simultaneous presence of Na', K+, and CF-in the serosal media. Utilizing cellular ion uptake techniques, we describe an interdependence of bumetanide-sensitive 22Na', "Rb+, and MCl-uptake, which is indicative of a Na+,K+iCl-cotransport system in this cell line. This transport pathway was localized to the basolateral membrane. Extrapolated initial velocities of uptake for each of the three ions was consistent with the electroneutral cotransport of 1 Na+:1 K+ (Rb+):2 Cl-. Our findings indicate that VIP-induced Cl-secretion intimately involves a bumetanide-sensitive Na+,K+,CF cotransport system which is functionally localized to the basolateral membrane.

“…The results also suggest that this transport pathway serves as the Cl-uptake step in the CF-secretory process, and is required to sustain the action ofprostaglandin El. Na',K+,Clcotransport has been observed in a variety of cells and is inhibitable by bumetanide (30)(31)(32)(33)(34)(35)(36)(37). In a recent study, the existence of a bumetanide-sensitive Na',K+,Cl-cotransport pathway has been demonstrated in the T84 cells and localized to the basolateral membrane (10).…”

Section: Discussionmentioning

confidence: 99%

Chloride secretory mechanism induced by prostaglandin E1 in a colonic epithelial cell line.

Weymer¹,

Huott²,

Liu³

et al. 1985

137

Confluent T8 monolayers grown on permeable supports and mounted in a modified Ussing chamber secrete chloride (Cl-) in response to prostaglandin El. The threshold stimulation was observed at 10-' M and a maximal effect at 10' M. Unidirectional flux studies showed an increase in both serosal to mucosal and mucosal to serosal CI-fluxes with 10' M prostaglandin El; the increase in serosal to mucosal Cl-flux exceeded the increase in mucosal to serosal flux, resulting in net Cl-secretion.Na' transport was not affected in either direction and the changes in net Cla flux correlated well with the changes in short circuit current. To identify the electrolyte transport pathways involved in the Cla secretory process, the effect of prostaglandin El on ion fluxes was tested in the presence of putative inhibitors. Bumetanide was used as an inhibitor for the basolaterally localized Na',K+,Ca-cotransport system whose existence and bumetanide sensitivity have been verified in earlier studies (Dharmsathaphorn et al. 1984. J. Clin. Invest. 75:462-471). Barium was used as an inhibitor for the K+ efflux pathway on the basolateral membrane whose existence and barium sensitivity were demonstrated in this study by preloading the monolayers with "Rb+ (as a tracer for K+) and simultaneously measuring MRb+ efflux into both serosal and mucosal reservoirs. Both bumetanide and barium inhibited the net chloride secretion induced by prostaglandin El suggesting the involvement of the Na+,K+,Ca-cotransport and a K+ efflux pathways on the basolateral membrane in the Clsecretory process. The activation of another Cl-transport pathway on the apical membrane by prostaglandin El was suggested by Cl-uptake studies. Our findings indicate that the prostaglandin El-stimulated Cl-secretion, which is associated with an increase in cyclic AMP level, intimately involves (a) a bumetanide-sensitive Na+,K+,Cl-cotransport pathway that serves as a C1-uptake step across the basolateral membrane, (b) the stimulation of a barium-sensitive K+ efflux mechanism on the basolateral membrane that most likely acts to recycle K+, and (c) the activation of a Cl-transport pathway on the apical membrane that serves as a Cl-exit pathway.