Skip to main content
PMC home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1986 May;77(5):1682–1688. doi: 10.1172/JCI112486

Intracellular pH regulation in rabbit renal medullary collecting duct cells. Role of chloride-bicarbonate exchange.

M L Zeidel, P Silva, J L Seifter
PMCID: PMC424574  PMID: 2871045

Abstract

The renal medullary collecting duct (MCD) secretes protons into its lumen and HCO3 into its basolateral space. Basolateral HCO3 transport is thought to occur via Cl/HCO3 exchange. To further characterize this Cl/HCO3 exchange process, intracellular pH (pHi) regulation was monitored in freshly prepared rabbit outer MCD cells. Cells were separated by protease digestion and purified by Ficoll gradient centrifugation. pHi was estimated fluorometrically using the entrapped intracytoplasmic pH indicator, 6-carboxyfluorescein. Cells were preincubated in bicarbonate-containing solutions and then abruptly diluted into bicarbonate-free media. The MCD cell pHi response to abrupt removal of CO2/HCO3 included an initial alkalinization due to rapid CO2 efflux, followed by an acidification due to HCO3 efflux and a gradual recovery to the resting pHi of 7.24 +/- 0.06 partly due to the action of a plasma membrane H+-ATPase. The initial alkalinization required a CO2/HCO3 gradient and did not occur in the presence of acetazolamide. The acidification phase required intracellular HCO3 and extracellular Cl, which was consistent with a Cl/HCO3 exchange. MCD HCO3 efflux exhibited saturable kinetics for extracellular Cl, with a Michaelis constant (Km) of 29.9 +/- 7.7 mM. HCO3 efflux also exhibited preference for halides over NO3, SCN, and gluconate, and striking sensitivity to disulfonic stilbene and acetazolamide inhibition, with an apparent K1 of 5 X 10(-7) M for DIDS. The final pHi recovery required intracellular ATP, which indicated that Cl/HCO3 and H+-ATPase activities are present in the same cells in these suspensions. The results provide direct evidence for MCD Cl/HCO3 exchange and describe some of the properties of this transport process.

Full text

PDF
1688

Selected References

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

  1. Beck J. C., Sacktor B. Membrane potential-sensitive fluorescence changes during Na+-dependent D-glucose transport in renal brush border membrane vesicles. J Biol Chem. 1978 Oct 25;253(20):7158–7162. [PubMed] [Google Scholar]
  2. Bowen J. W., Levinson C. H+ transport and the regulation of intracellular pH in Ehrlich ascites tumor cells. J Membr Biol. 1984;79(1):7–18. doi: 10.1007/BF01868522. [DOI] [PubMed] [Google Scholar]
  3. Brazy P. C., Gunn R. B. Furosemide inhibition of chloride transport in human red blood cells. J Gen Physiol. 1976 Dec;68(6):583–599. doi: 10.1085/jgp.68.6.583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burckhardt B. C., Cassola A. C., Frömter E. Electrophysiological analysis of bicarbonate permeation across the peritubular cell membrane of rat kidney proximal tubule. II. Exclusion of HCO3(-)-effects on other ion permeabilities and of coupled electroneutral HCO3(-)-transport. Pflugers Arch. 1984 May;401(1):43–51. doi: 10.1007/BF00581531. [DOI] [PubMed] [Google Scholar]
  5. Burg M., Stoner L., Cardinal J., Green N. Furosemide effect on isolated perfused tubules. Am J Physiol. 1973 Jul;225(1):119–124. doi: 10.1152/ajplegacy.1973.225.1.119. [DOI] [PubMed] [Google Scholar]
  6. Cabantchik Z. I., Knauf P. A., Rothstein A. The anion transport system of the red blood cell. The role of membrane protein evaluated by the use of 'probes'. Biochim Biophys Acta. 1978 Sep 29;515(3):239–302. doi: 10.1016/0304-4157(78)90016-3. [DOI] [PubMed] [Google Scholar]
  7. Dalmark M. Effects of halides and bicarbonate on chloride transport in human red blood cells. J Gen Physiol. 1976 Feb;67(2):223–234. doi: 10.1085/jgp.67.2.223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Davis G. R., Morawski S. G., Santa Ana C. A., Fordtran J. S. Evaluation of chloride/bicarbonate. Exchange in the human colon in vivo. J Clin Invest. 1983 Feb;71(2):201–207. doi: 10.1172/JCI110760. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dobyan D. C., Magill L. S., Friedman P. A., Hebert S. C., Bulger R. E. Carbonic anhydrase histochemistry in rabbit and mouse kidneys. Anat Rec. 1982 Nov;204(3):185–197. doi: 10.1002/ar.1092040303. [DOI] [PubMed] [Google Scholar]
  10. Ericson A. C., Spring K. R. Volume regulation by Necturus gallbladder: apical Na+-H+ and Cl(-)-HCO-3 exchange. Am J Physiol. 1982 Sep;243(3):C146–C150. doi: 10.1152/ajpcell.1982.243.3.C146. [DOI] [PubMed] [Google Scholar]
  11. Eveloff J., Haase W., Kinne R. Separation of renal medullary cells: isolation of cells from the thick ascending limb of Henle's loop. J Cell Biol. 1980 Dec;87(3 Pt 1):672–681. doi: 10.1083/jcb.87.3.672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fischer J. L., Husted R. F., Steinmetz P. R. Chloride dependence of the HCO3 exit step in urinary acidification by the turtle bladder. Am J Physiol. 1983 Nov;245(5 Pt 1):F564–F568. doi: 10.1152/ajprenal.1983.245.5.F564. [DOI] [PubMed] [Google Scholar]
  13. Friedman P. A., Andreoli T. E. CO2-stimulated NaCl absorption in the mouse renal cortical thick ascending limb of Henle. Evidence for synchronous Na +/H+ and Cl-/HCO3- exchange in apical plasma membranes. J Gen Physiol. 1982 Nov;80(5):683–711. doi: 10.1085/jgp.80.5.683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gluck S., Al-Awqati Q. An electrogenic proton-translocating adenosine triphosphatase from bovine kidney medulla. J Clin Invest. 1984 Jun;73(6):1704–1710. doi: 10.1172/JCI111378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Grantham J. J. Studies of organic anion and cation transport in isolated segments of proximal tubules. Kidney Int. 1982 Nov;22(5):519–525. doi: 10.1038/ki.1982.205. [DOI] [PubMed] [Google Scholar]
  16. Guder W. G., Ross B. D. Enzyme distribution along the nephron. Kidney Int. 1984 Aug;26(2):101–111. doi: 10.1038/ki.1984.143. [DOI] [PubMed] [Google Scholar]
  17. Gutknecht J., Bisson M. A., Tosteson F. C. Diffusion of carbon dioxide through lipid bilayer membranes: effects of carbonic anhydrase, bicarbonate, and unstirred layers. J Gen Physiol. 1977 Jun;69(6):779–794. doi: 10.1085/jgp.69.6.779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kaissling B., Kriz W. Structural analysis of the rabbit kidney. Adv Anat Embryol Cell Biol. 1979;56:1–123. doi: 10.1007/978-3-642-67147-0. [DOI] [PubMed] [Google Scholar]
  19. Koeppen B. M. Conductive properties of the rabbit outer medullary collecting duct: inner stripe. Am J Physiol. 1985 Apr;248(4 Pt 2):F500–F506. doi: 10.1152/ajprenal.1985.248.4.F500. [DOI] [PubMed] [Google Scholar]
  20. Lombard W. E., Kokko J. P., Jacobson H. R. Bicarbonate transport in cortical and outer medullary collecting tubules. Am J Physiol. 1983 Mar;244(3):F289–F296. doi: 10.1152/ajprenal.1983.244.3.F289. [DOI] [PubMed] [Google Scholar]
  21. Murer H., Hopfer U. Demonstration of electrogenic Na+-dependent D-glucose transport in intestinal brush border membranes. Proc Natl Acad Sci U S A. 1974 Feb;71(2):484–488. doi: 10.1073/pnas.71.2.484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Palfrey H. C., Feit P. W., Greengard P. cAMP-stimulated cation cotransport in avian erythrocytes: inhibition by "loop" diuretics. Am J Physiol. 1980 Mar;238(3):C139–C148. doi: 10.1152/ajpcell.1980.238.3.C139. [DOI] [PubMed] [Google Scholar]
  23. Rehm W. S., Sanders S. S. Implications of the neutral carrier Cl-HCO3- exchange mechanism in gastric mucosa. Ann N Y Acad Sci. 1975 Dec 30;264:442–455. doi: 10.1111/j.1749-6632.1975.tb31502.x. [DOI] [PubMed] [Google Scholar]
  24. Reuss L., Costantin J. L. Cl-/HCO3- exchange at the apical membrane of Necturus gallbladder. J Gen Physiol. 1984 Jun;83(6):801–818. doi: 10.1085/jgp.83.6.801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Roos A., Boron W. F. Intracellular pH. Physiol Rev. 1981 Apr;61(2):296–434. doi: 10.1152/physrev.1981.61.2.296. [DOI] [PubMed] [Google Scholar]
  26. Seifter J. L., Aronson P. S. Cl- transport via anion exchange in Necturus renal microvillus membranes. Am J Physiol. 1984 Dec;247(6 Pt 2):F888–F895. doi: 10.1152/ajprenal.1984.247.6.F888. [DOI] [PubMed] [Google Scholar]
  27. Simchowitz L., Roos A. Regulation of intracellular pH in human neutrophils. J Gen Physiol. 1985 Mar;85(3):443–470. doi: 10.1085/jgp.85.3.443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Steinmetz P. R. Cellular mechanisms of urinary acidification. Physiol Rev. 1974 Oct;54(4):890–956. doi: 10.1152/physrev.1974.54.4.890. [DOI] [PubMed] [Google Scholar]
  29. Stone D. K., Seldin D. W., Kokko J. P., Jacobson H. R. Anion dependence of rabbit medullary collecting duct acidification. J Clin Invest. 1983 May;71(5):1505–1508. doi: 10.1172/JCI110905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Thomas J. A., Buchsbaum R. N., Zimniak A., Racker E. Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry. 1979 May 29;18(11):2210–2218. doi: 10.1021/bi00578a012. [DOI] [PubMed] [Google Scholar]
  31. Warnock D. G., Greger R., Dunham P. B., Benjamin M. A., Frizzell R. A., Field M., Spring K. R., Ives H. E., Aronson P. S., Seifter J. Ion transport processes in apical membranes of epithelia. Fed Proc. 1984 Jul;43(10):2473–2487. [PubMed] [Google Scholar]
  32. Wieth J. O. Bicarbonate exchange through the human red cell membrane determined with [14C] bicarbonate. J Physiol. 1979 Sep;294:521–539. doi: 10.1113/jphysiol.1979.sp012944. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wright E. M., Diamond J. M. Anion selectivity in biological systems. Physiol Rev. 1977 Jan;57(1):109–156. doi: 10.1152/physrev.1977.57.1.109. [DOI] [PubMed] [Google Scholar]
  34. Zeidel M. L., Silva P., Seifter J. L. Intracellular pH regulation and proton transport by rabbit renal medullary collecting duct cells. Role of plasma membrane proton adenosine triphosphatase. J Clin Invest. 1986 Jan;77(1):113–120. doi: 10.1172/JCI112264. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES