Summary
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 phosphoprotein 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 theV max and not on theK m 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 120nm. 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.
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Bartfai, T. 1979. Preparation of metal-chelate complexes and the design of steady-state kinetic experiments involving metal nucleotide complexes.Adv. Cyclic Nucleotide Res. 10:219–242
Carboni, J.M., Howe, C.L., West, A.B., Barwick, K.W., Mooseker, M.S., Morrow, J.S. 1987. Characterization of intestinal brush border cytoskeletal proteins of normal and neoplastic human epithelial cells. A comparison with the avian brush border.Am. J. Pathol. 129:589–600
Cassel, D., Katz, M., Rotman, M. 1986. Depletion of cellular ATP inhibits Na+/H+ antiport in cultured human cells.J. Biol. Chem. 261:5460–5466
Cohen, M.E., Sharp, G.W.G., Donwitz, M. 1986. Suggestion of a role for calmodulin and phosphorylation in regulation of rabbit ileal electrolyte transport: Effects of promethazine.Am. J. Physiol. 251:G710-G717
Costa-Casnellie, M.R., Segel, G.B., Cragoe, E.J. Jr., Lichtman, M.A. 1987. Characterization of the Na+/H+ exchanger during maturation of HL-60 cells induced by dimethyl sulfoxide.J. Biol. Chem. 262:9093–9097
Donowitz, M., Cohen, M., Gudewich, R., Taylor, D., Sharp, G.W.G. 1984. Ca++/calmodulin, cyclic AMP and cyclic GMP-induced phosphorylation of proteins in purified microvillus membrane of rabbit ileum.Biochem. J. 219:573–581
Donowitz, M., Emmer, E., McCullen, J., Reinlib, L., Cohen, M.E., Rood, R.P., et al. 1987. Freeze-thaw and highvoltage discharge allow macromolecule uptake into ileal brush-border vesicles.Am. J. Physiol. 252:G723-G735
Donowitz, M., Cohen, M.E., Gould, M., Sharp, G.W.G. 1989. Elevated intracellular Ca2+ acts through protein kinase C to regulate rabbit ileal NaCl absorption: Evidence for sequential control by Ca2+/calmodulin and protein kinase C.J. Clin. Invest. (in press)
Donowitz, M., Welsh, M.J. 1986. Ca++ and cyclic AMP in regulation of intestinal Na, K, and Cl transport.Annu. Rev. Physiol. 48:135–150
Donowitz, M., Wicks, J., Madara, J., Sharp, G.W.G. 1985. Studies on role of calmodulin in Ca++ regulation of rabbit ileal Na and Cl absorption.Am. J. Physiol. 248:G726-G740
Fan, C.C., Powell, D.W. 1983. Calcium inhibition of NaCl uptake in rabbit ileal brush border membrane vesicles.Proc. Natl. Acad. Sci. USA 80:5248–5252
Grinstein, S., Cohen, S., Goetz, J.D., Rothstein, A. 1985. Osmotic and phorbol ester-induced activation of Na+/H+ exchange: Possible role of protein phosphorylation in lymphocyte volume regulation.J. Cell Biol. 101:269–276
Grinstein, S., Rothstein, A. 1986. Mechanisms of regulation of the Na+/H+ exchanger.J. Membrane Biol. 90:1–12
Huganir, R.L., Delcour, A.H., Greengard, P., Hess, G.P. 1986. Phosphorylation of the nicotinic acethylcholine receptor regulates its rate of desensitization.Nature (London) 321:774–776
Kinsella, J., Cujdik, T., Sacktor, B. 1984. Na+/H+ exchange in isolated renal brush border membrane vesicles in response to metabolic acidosis. Kinetic effects.J. Biol. Chem. 259:13224–13227
Knickelbein, R., Aronson, P.S., Atherton, W., Dobbins, J.W. 1983. Na and Cl transport across rabbit ileal brush border. I. Evidence for Na+/H+ exchange.Am. J. Physiol. 245:G504-G510
Knickelbein, R., Aronson, P.S., Schron, C.M., Seifter, J., Dobbins, J.W. 1985. Na and Cl transport across rabbit ileal brush border. II. Demonstration of Cl−/HCO −3 exchange and mechanism for coupling.Am. J. Physiol. 249:G236-G245
Levitan, I.B. 1985. Phosphorylation of ion channels.J. Membrane Biol. 87:177–190
Levitan, I.B., Lemor, J.R., Novar-Hoffer, I.L. 1983. Protein phosphorylation and the regulation of ion channels.Trends Neurol. Sci. 6:496–499
Liedtke, C.M., Hopfer, U. 1982. Mechanism of Cl translocation across small intestinal brush-border membrane. I. Absence of NaCl cotransport.Am. J. Physiol. 242:G263-G271
Liedtke, C.M., Hopfer, U. 1982. Mechanism of Cl translocation across small intestinal brush-border membrane. II. Demonstration of Cl−/OH− exchange and mechanism of coupling.Am. J. Physiol. 242:G272-G280
Miller, R.T., Pollock, A.S. 1987. Modification of the internal pH sensitivity of the Na+/H+ antiporter by parathyroid hormone in a cultured renal cell line.J. Biol. Chem. 262:9115–9120
Rood, R.P., Emmer, E., Wesolek, J., McCullen, J., Hussain, Z., Cohen, M.E., et al. 1988. Regulation of the rabbit ileal brush border Na+/H+ exchanger by an ATP requiring Ca++, calmodulin-mediated process.J. Clin. Invest. 82:1091–1097
Segel, I.H. 1976. Biochemical Calculations. pp. 208–323. John Wiley & Sons, New York
Van Dommelen, F.S., Hamer, C.M., DeJonge, H.R. 1986. Efficient entrapment of large and small compounds during vesiculation of intestinal microvilli.Biochem. J. 236:771–778
Weinman, E.J., Dubinsky, W.P., Shenolikar, S. 1988. Reconstitution of cAMP-dependent protein kinase regulated renal Na+−H+ exchanger.J. Membrane Biol. 101:11–18
Weinman, E.J., Shenolikar, S., Cragoe, E.J., Jr., Dubinsky, W.P. 1988. Solubilization and reconstruction of renal brush border Na+−H+ exchanger.J. Membrane Biol. 101:1–9
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Emmer, E., Rood, R.P., Wesolek, J.H. et al. Role of calcium and calmodulin in the regulation of the rabbit ileal brush-border membrane Na+/H+ antiporter. J. Membrain Biol. 108, 207–215 (1989). https://doi.org/10.1007/BF01871735
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DOI: https://doi.org/10.1007/BF01871735