Calcium uptake in isolated brush-border vesicles from rat small intestine

Abstract

Ca2+ uptake in brush-border vesicles isolated from rat duodena was studied by a rapid-filtration technique. Ca2+ uptake showed saturation kinetics, was dependent on the pH and ionic strength of the medium and was independent of metabolic energy. Uptake activity was readily inhibited by Ruthenium Red, La3+, tetracaine, EGTA, choline chloride and Na+ or K+. The effect of variations in medium osmolarity on Ca2+ uptake and the ionophore A23 187-induced efflux of the cation from preloaded vesicles indicated that the Ca2+-uptake process involved binding to membrane components, as well as transport into an osmotically active space. Scatchard-plot analyses of the binding data suggested at least two classes of Ca2+-binding sites. The high-affinity sites, Ka = (2.7 + 1.1) x 104M-1 (mean + S.D.) bound 3.2 + 0.8 nmol of Ca2+/mg of protein, whereas the low-affinity sites (Ka = 60 ± 6 mF1) bound 1 10 + 17 nmol of Ca2+/mg of protein. In the presence of l10mM-NaCl, 1.7 and 53nmol of Ca2+/mg of protein were bound to the high-and low-affinity sites respectively. Decreased Ca2+-uptake activity was observed in vesicles isolated from vitamin D-deficient as compared with vitamin D-replete animals and intraperitoneal administration of 1,25-dihydroxycholecalciferol to vitamin D-deficient rats 16h before membrane isolation stimulated the initial rate of Ca2+ uptake significantly. The data indicated that Ca2+ entry and/or binding was passive and may involve a carrier-mediated Ca2+-uptake component that is associated with the brush-border membrane. Altering the electrochemical potential difference across the membrane by using anions of various permeability and selected ionophores appeared to increase primarily binding to the membrane rather than transport into the intravesicular space. Since there is considerable binding of Ca2+ to the vesicle interior, a comprehensive analysis of the transport remains difficult at present.

Evidence for multiple effects of vitamin D3 on calcium absorption: response of rachitic chicks, with or without partial vitamin D3 repletion, to 1,25-dihydroxyvitamin D3.

“…This is supported by the observations of others showing that (a) isolated brush border membrane vesicles from animals treated with D3 (or a D3 metabolite) accumulate calcium more rapidly than do those from vitamin D-deficient animals (9,(33)(34)(35), and (b) cortisol inhibits 1,25(OH)2D3-mediated calcium absorption by chick intestine (18,35) but has no effect on the 1,25(OH)2D3-dependent uptake of calcium by isolated brush border vesicles (35), indicating that the rate-determining transfer step was not at the brush border but elsewhere in the transport path.…”

Calcium uptake by intestinal brush border membrane vesicles. Comparison with in vivo calcium transport.

“…i.e., in the range reported by other investigators (12,(22)(23)(24)(25). Our space measurements were made at equilibrium in the medium we used for calcium uptake studies (100 mM KCl, 5 mM MgC12, and 20 mM Hepes-Tris, pH 7.…”

“…They prepared duodenal vesicles from male rats fed a low calcium (0.06%) and low phosphorus (0.2%) semisynthetic diet for several days. Miller and Bronner (22) found that calcium uptake at 0.5 min showed saturation kinetics. Scatchard-plot analysis was used to define high (K. 2.7 X 104 M-l) and low (K. 60 M-1) affinity testine.…”

“…Nonsaturable calcium transport was greater in the distal than the proximal segment, unlike the rat, where both segments showed the same Kd. Concentration range 0-6.8 mM for rat (10); 0-27 mM for hamster (9 (22) studied vesicles from rats stimulated by low calcium and low phosphorous diet, rather than from animals maintained under usual laboratory conditions. Rasmussen (26) and Bikle (27) studied the chick, which appears to behave differently from the rat.…”

1,25-Dihydroxyvitamin D increases calmodulin binding to specific proteins in the chick duodenal brush border membrane.

“…This process does not require ATP. Administration of 1,25(OH)2D in vivo to vitamin D-deficient chicks results in an enhanced ability of the subsequently isolated BBMV to accumulate calcium (5,10,11). Hypotheses explaining this effect include the synthesis of new proteins such as IMcal (12) or the vitamin D-dependent calciumbinding protein (13), and changes in the lipid composition of the BBM (14,15).…”