Mechanisms of calcium transport in human colonic basolateral membrane vesicles

Human colon has been suggested to play an important role in calcium absorption especially after extensive disease or resection of the small intestine. We have previously demonstrated the presence of a carrier-mediated calcium uptake mechanism in the human colonic luminal membrane vesicles. Current studies were, therefore, undertaken to investigate the mechanism(s) of calcium exit across the basolateral membrane domain of the human colon. Human colonic basolateral membrane vesicles (BLMVs) were isolated and purified from mucosal scrapings of organ donor colons, utilizing a technique developed in our laboratory. ⁴⁵Ca uptake was measured by a rapid filtration technique. ⁴⁵Ca uptake represented transport into the intravesicular space as evidenced by an osmolarity study and by the demonstration of Ca²⁺ efflux from calcium preloaded vesicles by Ca²⁺ ionophore A23187. Calcium uptake was stimulated by Mg²⁺ ATP. The kinetic parameters for ATP-dependent Ca²⁺ uptake revealed saturation kinetics with Michaelis constant (K_m) of 0.22 ± 0.04 μM and a maximum rate of uptake (V_max) of 0.38 ± 0.12 nmol/mg protein/min. The K_m of ATP concentration required for half maximal Ca²⁺ uptake was 0.39 ± 0.04 mM. ATP-stimulated calcium uptake into these vesicles was further stimulated in the presence of calmodulin and was inhibited by calmodulin antagonist, trifluoperazine. Uptake of ⁴⁵Ca into BLMVs was markedly inhibited by cis-Na⁺ but was significantly stimulated by trans-Na⁺ (40-50% stimulation). Our results demonstrate the presence of a Mg²⁺/ATP-dependent calmodulin-regulated Ca²⁺ transport system and a Na⁺-Ca²⁺ exchange process in the human colonic basolateral membranes.