Rabit distal colon epithelium: II. Characterization of (Na+, K+, Cl−)-cotransport and [3H]-bumetanide binding

Abstract

Summary Loop diuretic-sensitive (Na⁺,K⁺,Cl⁻)-cotransport activity was found to be present in basolateral membrane vesicles of surface and crypt cells of rabbit distal colon epithelium. The presence of grandients of all three ions was essential for optimal transport activity (Na⁺,K⁺) gradien-driven³⁶Cl fluxes weree half-maximally inhibited by 0.14 μm bumetanide and 44 μm furosimide. While⁸⁶Rb uptake rates showed hyperbolic dependencies on Na⁺ and K⁺ concentrations with Hill coefficients of 0.8 and 0.9, respectively, uptakes were sigmoidally related to the Cl⁻ concentration, Hill coefficient 1.8, indicating a 1 Na⁺: 1 K⁺:2 Cl⁻ stoichiometry of ion transport. The interaction of putative (Na⁺, K⁺, Cl⁻)-cotransport proteins with loop diuretics was studied from equilibrium-binding experiments using [³H]-bumetanide. The requirement for the simulataneous presence of Na⁺,K⁺, and Cl⁻, saturability, reversibility, and specificity for diuretics suggest specific binding to the (Na⁺, K⁺, Cl⁻)-cotransporter. [³H]-bumetanide recognizes a minimum of two classes of diuretic receptors sites. high-affinity (K _D1=0.13 μm;B _max1 =6.4 pmol/mg of protein) and low-affinity (K _D2=34 μm;B _max2=153 pmol/mg of protein) sites. The specific binding to the high-affinity receptor was found to be linearly competitive with Cl⁻ (K ₁=60mm), whereas low-affinity sites seem to be unaffected by Cl⁻. We have shown that only high-affinity [³H]-bumetanide binding correlates with transport inhibition raising questions on the physiological significance of diuretic receptor site heterogeneity observed in rabbit distal colon epithelium.