Abstract
Uptake of guanidine, an endogenous organic cation, into brush-border membrane vesicles isolated from human term placentas was investigated. Initial uptake rates were manyfold greater in the presence of an outward-directed H+ gradient ([pH]o greater than [pH]i) than in the absence of a H+ gradient ([pH]o = [pH]i). Guanidine was transiently accumulated inside the vesicles against a concentration gradient in the presence of the H+ gradient. The H+ gradient-dependent stimulation of guanidine uptake was not due to a H+-diffusion potential because an ionophore (valinomycin or carbonylcyanide p-trifluoromethoxyphenylhydrazone)-induced inside-negative membrane potential failed to stimulate the uptake. In addition, uphill transport of guanidine could be demonstrated even in voltage-clamped membrane vesicles. The H+ gradient-dependent uptake of guanidine was inhibited by many exogenous as well as endogenous organic cations (cis-inhibition) but not by cationic amino acids. The presence of unlabeled guanidine inside the vesicles stimulated the uptake of labeled guanidine (trans-stimulation). These data provide evidence for the presence of an organic cation-proton antiporter in human placental brush-border membranes. Kinetic analysis of guanidine uptake demonstrated that the uptake occurred via two saturable, carrier-mediated transport systems, one being a high affinity, low capacity type and the other a low affinity, high capacity type. Studies on the effects of various cations on the organic cation-proton antiporter and the Na+-H+ exchanger revealed that these two transport systems are distinct.
Highlights
From the Departments of $Cell and Molecular Biology and Physiology and Endocrinology, MedicalCollege of Georgia, Augusta, Georgia 30912
An endogenous organic cation, the active transport of organic cations into the brush-border into brush-border membranveesicles isolated from hu- membrane vesicles is energized by an outward-directedproton man term placentas was investigated
The well known excretory functions of the kidney and theapparent role of the organic cation-proton antiporter in the renal excretion of various organic cations are the probable reasons for the relative lack of attention on the handling of organic cations by organs other than the Theterm “organic cation-protonantiporter” refers to a specific transport system originally described in the brushborder membrane of renal proximal tubular cells [1,2,3,4]
Summary
Effects of a H + Gradient and a Membrane Potential on Guanidine Uptake-We investigated the effect of an outwarddirected H' gradient ([H'], > [H'],) on the uptake of guanidine in humanplacental brush-border membrane vesicles. MM Hepes, 10 mM Tris buffer, pH 7.2, containing 150 mM KCl. In the case of the membrane vesicles preloaded with the pH 5.5 buffer, guanidine uptake was measured using the uptake buffer whose composition was 18 mM Hepes, 12 mM Tris, 150mM KCl, pH 7.5. In the case of the membrane vesicles preloaded with thepH 7.2 buffer, guanidine uptake was measured using the uptake buffer whose composition was the same as that of the preloading buffer. The equilibrium value howeverremained the same in the presence and absence of a H' gradient, showing that thegreater uptake observed in the presence of a H' gradient was not due to changes in the intravesicular volume These data show that an outward-directed H' gradient energizes the uphill transport of guanidine into theplacental brush-border membrane vesicles.
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