In rabbit ileum, Ca2+/calmodulin (CaM) appears to be involved in physiologically inhibiting the linked NaCl absorptive process, since inhibitors of Ca2+/CaM stimulate linked Na+ and Cl- absorption. The role of Ca2+/CaM-dependent phosphorylation in regulation of the brush-border Na+/H+ antiporter, which is believed to be part of the neutral linked NaCl absorptive process, was studied using purified brush-border membrane vesicles, which contain both the Na+/H+ antiporter and Ca2+/CaM-dependent protein kinase(s) and its phosphorprotein substrates. Rabbit ileal villus cell brush-border membrane vesicles were prepared by Mg precipitation and depleted of ATP. Using a freezethaw technique, the ATP-depleted vesicles were loaded with Ca2+, CaM, ATP and an ATP-regenerating system consisting of creatine kinase and creatine phosphate. The combination of Ca2+/CaM and ATP inhibited Na+/H+ exchange by 45 +/- 13%. This effect was specific since Ca2+/CaM and ATP did not alter diffusive Na+ uptake, Na+-dependent glucose entry, or Na+ or glucose equilibrium volumes. The inhibition of the Na+/H+ exchanger by Ca2+/CaM/ATP was due to an effect on the Vmax and not on the Km for Na+. In the presence of CaM and ATP, Ca2+ caused a concentration-dependent inhibition of Na+ uptake, with an effect 50% of maximum occurring at 120 nM. This Ca2+ concentration dependence was similar to the Ca2+ concentration dependence of Ca2+/CaM-dependent phosphorylation of specific proteins in the vesicles. The Ca2+/CaM/ATP-inhibition of Na+/H+ exchange was reversed by W13, a Ca2+/CaM antagonist, but not by a hydrophobic control, W12, or by H-7, a protein kinase C antagonist. We conclude that Ca2+, acting through CaM, regulates ileal brush-border Na+/H+ exchange, and that this may be involved in the regulation of neutral linked NaCl absorption.

Brush-border vesicles purified from rabbit ileal villus cells were used to evaluate how Ca++/calmodulin (CaM) regulates the neutral linked NaCl absorptive process, part of which is a Na+/H+ exchanger. After freezing and thawing to allow incorporation of macromolecules into the vesicles, the effect of Ca++/CaM on brush-border Na+ uptake with an acid inside pH gradient, and on Na+/H+ exchange was determined.