Characterization of calcium transport by basolateral membrane vesicles of human small intestine
Kikuchi, K.; Kikuchi, T.; Ghishan, F.K.
American Journal of Physiology 255(4 Pt 1): G482-G489
1988
ISSN/ISBN: 0002-9513 PMID: 3140674 Document Number: 323948
The present studies investigated the mechanism of Ca2+ transport across basolateral membrane vesicles (BLMVs) prepared from human small intestine. Ca2+ uptake represented transport into the intravesicular space as evident by osmolality study and by the demonstration of Ca2+ efflux from the intravesicular space by Ca2+ ionophore A23187. Ca2+ uptake was stimulated by Mg2+-ATP. Kinetic parameters for ATP-dependent Ca2+ uptake revealed a Michaelis constant (Km) of 0.02 .+-. 0.01 .mu.M and a maximum rate of uptake (Vmax) of 1.00 .+-. 0.03 nmol .cntdot. mg protein-1 .cntdot. min-1. Ca2+ uptake in the absence of Mg2+ was inhibited by 75%. The Km of ATP concentration required for half-maximal Ca2+ uptake was 0.50 .+-. 0.1 mM. Calmodulin (10 .mu.g/mL) increased Vmax to 1.62 .+-. 0.02 nmol .cntdot. mg protein-1 .cntdot. min-1 (P < 0.001). Km values were 0.017 .+-. 0.001 .mu.M, which was not significantly different from control values. Basolateral membranes depleted of calmodulin by EDTA osmotic shock decreased ATP-dependent Ca2+ uptake by 65%. Trifluoperazine, an anticalmodulin drug, inhibied ATP-dependent Ca2+ uptake by 50%, while no inhibition was noted in calmodulin-depleted membranes. Efflux of Ca2+ in the BLMVs was stimulated by trans-Na+-dependent Ca2+ uptake was saturable with respect to Ca2+ concentration and exhibited a Km of 0.09 .+-. 0.03 .mu.M and a Vmax of 1.08 .+-. 0.01 nmol .cntdot. mg protein-1 .cntdot. min-1. These results are consistent with the existence of a Na+-Ca2+ exchange system and ATP and Mg2+-dependent, calmodulin-regulated Ca2+. transport mechanism in BLMVs of human enterocytes.