Skip to main content

Some NLM-NCBI services and products are experiencing heavy traffic, which may affect performance and availability. We apologize for the inconvenience and appreciate your patience. For assistance, please contact our Help Desk at info@ncbi.nlm.nih.gov.

The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1988 Sep;82(3):1091–1097. doi: 10.1172/JCI113665

Regulation of the rabbit ileal brush-border Na+/H+ exchanger by an ATP-requiring Ca++/calmodulin-mediated process.

R P Rood 1, E Emmer 1, J Wesolek 1, J McCullen 1, Z Husain 1, M E Cohen 1, R S Braithwaite 1, H Murer 1, G W Sharp 1, M Donowitz 1
PMCID: PMC303623  PMID: 2843567

Abstract

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. Freezing and thawing vesicles with 0.85 microM free Ca++ plus 5 microM exogenous CaM failed to alter Na+/H+ exchange as did the addition of exogenous ATP plus an ATP regenerating system, which was sufficient to elevate intravesicular ATP to 47 microM from a basal level of 0.4 microM. However, the combination of Ca++/CaM plus ATP inhibited Na+ uptake in the presence of an acid inside pH gradient and inhibited Na+/H+ exchange, while Na+ uptake in the absence of a pH gradient was not altered. This effect required a hydrolyzable form of ATP, and did not occur when the nonhydrolyzable ATP analogue, AMP-PNP, replaced ATP. Under the identical intravesicular conditions used for the transport studies, Ca++ (0.85 microM) plus exogenous CaM (5 microM), in the presence of magnesium plus ATP, increased phosphorylation of five brush-border peptides. These data are consistent with Ca++/CaM acting via phosphorylation to regulate the ileal brush-border Na+/H+ exchanger.

Full text

PDF
1091

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aronson P. S., Suhm M. A., Nee J. Interaction of external H+ with the Na+-H+ exchanger in renal microvillus membrane vesicles. J Biol Chem. 1983 Jun 10;258(11):6767–6771. [PubMed] [Google Scholar]
  2. BEUTLER E., BALUDA M. C. SIMPLIFIED DETERMINATION OF BLOOD ADENOSINE TRIPHOSPHATE USING THE FIREFLY SYSTEM. Blood. 1964 May;23:688–698. [PubMed] [Google Scholar]
  3. Besterman J. M., May W. S., Jr, LeVine H., 3rd, Cragoe E. J., Jr, Cuatrecasas P. Amiloride inhibits phorbol ester-stimulated Na+/H+ exchange and protein kinase C. An amiloride analog selectively inhibits Na+/H+ exchange. J Biol Chem. 1985 Jan 25;260(2):1155–1159. [PubMed] [Google Scholar]
  4. Cassano G., Stieger B., Murer H. Na/H- and Cl/OH-exchange in rat jejunal and rat proximal tubular brush border membrane vesicles. Studies with acridine orange. Pflugers Arch. 1984 Mar;400(3):309–317. doi: 10.1007/BF00581565. [DOI] [PubMed] [Google Scholar]
  5. Donowitz M., Cohen M. E., Gudewich R., Taylor L., Sharp G. W. Ca2+-calmodulin-, cyclic AMP- and cyclic GMP-induced phosphorylation of proteins in purified microvillus membranes of rabbit ileum. Biochem J. 1984 Apr 15;219(2):573–581. doi: 10.1042/bj2190573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Donowitz M., Emmer E., McCullen J., Reinlib L., Cohen M. E., Rood R. P., Madara J., Sharp G. W., Murer H., Malmstrom K. Freeze-thaw and high-voltage discharge allow macromolecule uptake into ileal brush-border vesicles. Am J Physiol. 1987 Jun;252(6 Pt 1):G723–G735. doi: 10.1152/ajpgi.1987.252.6.G723. [DOI] [PubMed] [Google Scholar]
  7. Donowitz M., Welsh M. J. Ca2+ and cyclic AMP in regulation of intestinal Na, K, and Cl transport. Annu Rev Physiol. 1986;48:135–150. doi: 10.1146/annurev.ph.48.030186.001031. [DOI] [PubMed] [Google Scholar]
  8. Donowitz M., Wicks J., Madara J. L., Sharp G. W. Studies on role of calmodulin in Ca2+ regulation of rabbit ileal Na and Cl transport. Am J Physiol. 1985 Jun;248(6 Pt 1):G726–G740. doi: 10.1152/ajpgi.1985.248.6.G726. [DOI] [PubMed] [Google Scholar]
  9. Fan C. C., Powell D. W. Calcium/calmodulin inhibition of coupled NaCl transport in membrane vesicles from rabbit ileal brush border. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5248–5252. doi: 10.1073/pnas.80.17.5248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Huganir R. L., Delcour A. H., Greengard P., Hess G. P. Phosphorylation of the nicotinic acetylcholine receptor regulates its rate of desensitization. Nature. 1986 Jun 19;321(6072):774–776. doi: 10.1038/321774a0. [DOI] [PubMed] [Google Scholar]
  11. Knickelbein R., Aronson P. S., Atherton W., Dobbins J. W. Sodium and chloride transport across rabbit ileal brush border. I. Evidence for Na-H exchange. Am J Physiol. 1983 Oct;245(4):G504–G510. doi: 10.1152/ajpgi.1983.245.4.G504. [DOI] [PubMed] [Google Scholar]
  12. Knickelbein R., Aronson P. S., Schron C. M., Seifter J., Dobbins J. W. Sodium and chloride transport across rabbit ileal brush border. II. Evidence for Cl-HCO3 exchange and mechanism of coupling. Am J Physiol. 1985 Aug;249(2 Pt 1):G236–G245. doi: 10.1152/ajpgi.1985.249.2.G236. [DOI] [PubMed] [Google Scholar]
  13. Lai Y., Nairn A. C., Greengard P. Autophosphorylation reversibly regulates the Ca2+/calmodulin-dependence of Ca2+/calmodulin-dependent protein kinase II. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4253–4257. doi: 10.1073/pnas.83.12.4253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Levitan I. B. Phosphorylation of ion channels. J Membr Biol. 1985;87(3):177–190. doi: 10.1007/BF01871217. [DOI] [PubMed] [Google Scholar]
  15. Liedtke C. M., Hopfer U. Mechanism of Cl- translocation across small intestinal brush-border membrane. I. Absence of Na+-Cl- cotransport. Am J Physiol. 1982 Mar;242(3):G263–G271. doi: 10.1152/ajpgi.1982.242.3.G263. [DOI] [PubMed] [Google Scholar]
  16. Liedtke C. M., Hopfer U. Mechanism of Cl- translocation across small intestinal brush-border membrane. II. Demonstration of Cl--OH- exchange and Cl- conductance. Am J Physiol. 1982 Mar;242(3):G272–G280. doi: 10.1152/ajpgi.1982.242.3.G272. [DOI] [PubMed] [Google Scholar]
  17. Lou L. L., Lloyd S. J., Schulman H. Activation of the multifunctional Ca2+/calmodulin-dependent protein kinase by autophosphorylation: ATP modulates production of an autonomous enzyme. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9497–9501. doi: 10.1073/pnas.83.24.9497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Velasco G., Iglesias C. F., Domínguez P., Barros F., Gascón S., Lazo P. S. Protein kinase C from small intestine epithelial cells. Biochem Biophys Res Commun. 1986 Sep 30;139(3):875–882. doi: 10.1016/s0006-291x(86)80259-5. [DOI] [PubMed] [Google Scholar]
  19. Whitehouse S., Feramisco J. R., Casnellie J. E., Krebs E. G., Walsh D. A. Studies on the kinetic mechanism of the catalytic subunit of the cAMP-dependent protein kinase. J Biol Chem. 1983 Mar 25;258(6):3693–3701. [PubMed] [Google Scholar]
  20. van Dommelen F. S., Hamer C. M., De Jonge H. R. Efficient entrapment of large and small compounds during vesiculation of intestinal microvilli. Biochem J. 1986 Jun 15;236(3):771–778. doi: 10.1042/bj2360771. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES