Skip to main content
PMC home page
The Journal of General Physiology logoLink to The Journal of General Physiology
. 1994 May 1;103(5):917–936. doi: 10.1085/jgp.103.5.917

Effects of ammonium on intracellular pH in rat medullary thick ascending limb: mechanisms of apical membrane NH4+ transport

PMCID: PMC2219218  PMID: 8035168

Abstract

The renal medullary thick ascending limb (MTAL) actively reabsorbs ammonium ions. To examine the effects of NH4+ transport on intracellular pH (pHi) and the mechanisms of apical membrane NH4+ transport, MTALs from rats were isolated and perfused in vitro with 25 mM HCO3(-)-buffered solutions (pH 7.4). pHi was monitored using the fluorescent dye BCECF. In the absence of NH4+, the mean pHi was 7.16. Luminal addition of 20 mM NH4+ caused a rapid intracellular acidification (dpHi/dt = 11.1 U/min) and reduced the steady state pHi to 6.67 (delta pHi = 0.5 U), indicating that apical NH4+ entry was more rapid than entry of NH3. Luminal furosemide (10(-4) M) reduced the initial rate of cell acidification by 70% and the fall in steady state pHi by 35%. The residual acidification observed with furosemide was inhibited by luminal barium (12 mM), indicating that apical NH4+ entry occurred via both furosemide (Na(+)-NH4(+)-2Cl- cotransport) and barium- sensitive pathways. The role of these pathways in NH4+ absorption was assessed under symmetric ammonium conditions. With 4 mM NH4+ in perfusate and bath, mean steady state pHi was 6.61 and net ammonium absorption was 12 pmol/min/mm. Addition of furosemide to the lumen abolished net ammonium absorption and caused pHi to increase abruptly (dpHi/dt = 0.8 U/min) to 7.0. Increasing luminal [K+] from 4 to 25 mM caused a similar, rapid cell alkalinization. The pronounced cell alkalinization observed with furosemide or increasing [K+] was not observed in the absence of NH4+. In symmetric 4 mM NH4+ solutions, addition of barium to the lumen caused a slow intracellular alkalinization and reduced net ammonium absorption only by 14%. Conclusions: (a) ammonium transport is a critical determinant of pHi in the MTAL, with NH4+ absorption markedly acidifying the cells and maneuvers that inhibit apical NH4+ uptake (furosemide or elevation of luminal [K+]) causing intracellular alkalinization; (b) most or all of transcellular ammonium absorption is mediated by apical membrane Na(+)- NH4(+)-2Cl- cotransport; (c) NH4+ also permeates a barium-sensitive apical membrane transport pathway (presumably apical membrane K+ channels) but this pathway does not contribute significantly to ammonium absorption under physiologic (symmetric ammonium) conditions.

Full Text

The Full Text of this article is available as a PDF (1.1 MB).

Selected References

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

  1. Bleich M., Schlatter E., Greger R. The luminal K+ channel of the thick ascending limb of Henle's loop. Pflugers Arch. 1990 Jan;415(4):449–460. doi: 10.1007/BF00373623. [DOI] [PubMed] [Google Scholar]
  2. Chaillet J. R., Boron W. F. Intracellular calibration of a pH-sensitive dye in isolated, perfused salamander proximal tubules. J Gen Physiol. 1985 Dec;86(6):765–794. doi: 10.1085/jgp.86.6.765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. DuBose T. D., Jr, Lucci M. S., Hogg R. J., Pucacco L. R., Kokko J. P., Carter N. W. Comparison of acidification parameters in superficial and deep nephrons of the rat. Am J Physiol. 1983 May;244(5):F497–F503. doi: 10.1152/ajprenal.1983.244.5.F497. [DOI] [PubMed] [Google Scholar]
  4. Garvin J. L., Burg M. B., Knepper M. A. Active NH4+ absorption by the thick ascending limb. Am J Physiol. 1988 Jul;255(1 Pt 2):F57–F65. doi: 10.1152/ajprenal.1988.255.1.F57. [DOI] [PubMed] [Google Scholar]
  5. Good D. W. Active absorption of NH4+ by rat medullary thick ascending limb: inhibition by potassium. Am J Physiol. 1988 Jul;255(1 Pt 2):F78–F87. doi: 10.1152/ajprenal.1988.255.1.F78. [DOI] [PubMed] [Google Scholar]
  6. Good D. W. Adaptation of HCO-3 and NH+4 transport in rat MTAL: effects of chronic metabolic acidosis and Na+ intake. Am J Physiol. 1990 May;258(5 Pt 2):F1345–F1353. doi: 10.1152/ajprenal.1990.258.5.F1345. [DOI] [PubMed] [Google Scholar]
  7. Good D. W. Effects of osmolality on bicarbonate absorption by medullary thick ascending limb of the rat. J Clin Invest. 1992 Jan;89(1):184–190. doi: 10.1172/JCI115560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Good D. W. Effects of potassium on ammonia transport by medullary thick ascending limb of the rat. J Clin Invest. 1987 Nov;80(5):1358–1365. doi: 10.1172/JCI113213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Good D. W. Inhibition of bicarbonate absorption by peptide hormones and cyclic adenosine monophosphate in rat medullary thick ascending limb. J Clin Invest. 1990 Apr;85(4):1006–1013. doi: 10.1172/JCI114530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Good D. W., Knepper M. A., Burg M. B. Ammonia and bicarbonate transport by thick ascending limb of rat kidney. Am J Physiol. 1984 Jul;247(1 Pt 2):F35–F44. doi: 10.1152/ajprenal.1984.247.1.F35. [DOI] [PubMed] [Google Scholar]
  11. Good D. W., Knepper M. A. Mechanisms of ammonium excretion: role of the renal medulla. Semin Nephrol. 1990 Mar;10(2):166–173. [PubMed] [Google Scholar]
  12. Good D. W. The thick ascending limb as a site of renal bicarbonate reabsorption. Semin Nephrol. 1993 Mar;13(2):225–235. [PubMed] [Google Scholar]
  13. Good D. W., Vurek G. G. Picomole quantitation of ammonia by flow-through fluorometry. Anal Biochem. 1983 Apr 1;130(1):199–202. doi: 10.1016/0003-2697(83)90670-x. [DOI] [PubMed] [Google Scholar]
  14. Greger R. Ion transport mechanisms in thick ascending limb of Henle's loop of mammalian nephron. Physiol Rev. 1985 Jul;65(3):760–797. doi: 10.1152/physrev.1985.65.3.760. [DOI] [PubMed] [Google Scholar]
  15. Greger R., Schlatter E., Lang F. Evidence for electroneutral sodium chloride cotransport in the cortical thick ascending limb of Henle's loop of rabbit kidney. Pflugers Arch. 1983 Mar;396(4):308–314. doi: 10.1007/BF01063936. [DOI] [PubMed] [Google Scholar]
  16. Greger R., Schlatter E. Properties of the lumen membrane of the cortical thick ascending limb of Henle's loop of rabbit kidney. Pflugers Arch. 1983 Mar;396(4):315–324. doi: 10.1007/BF01063937. [DOI] [PubMed] [Google Scholar]
  17. Guggino W. B., Oberleithner H., Giebisch G. The amphibian diluting segment. Am J Physiol. 1988 May;254(5 Pt 2):F615–F627. doi: 10.1152/ajprenal.1988.254.5.F615. [DOI] [PubMed] [Google Scholar]
  18. Hebert S. C., Andreoli T. E. Effects of antidiuretic hormone on cellular conductive pathways in mouse medullary thick ascending limbs of Henle: II. determinants of the ADH-mediated increases in transepithelial voltage and in net Cl-absorption. J Membr Biol. 1984;80(3):221–233. doi: 10.1007/BF01868440. [DOI] [PubMed] [Google Scholar]
  19. Hebert S. C., Andreoli T. E. Ionic conductance pathways in the mouse medullary thick ascending limb of Henle. The paracellular pathway and electrogenic Cl- absorption. J Gen Physiol. 1986 Apr;87(4):567–590. doi: 10.1085/jgp.87.4.567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hebert S. C. Hypertonic cell volume regulation in mouse thick limbs. I. ADH dependency and nephron heterogeneity. Am J Physiol. 1986 Jun;250(6 Pt 1):C907–C919. doi: 10.1152/ajpcell.1986.250.6.C907. [DOI] [PubMed] [Google Scholar]
  21. Hebert S. C. Hypertonic cell volume regulation in mouse thick limbs. II. Na+-H+ and Cl(-)-HCO3- exchange in basolateral membranes. Am J Physiol. 1986 Jun;250(6 Pt 1):C920–C931. doi: 10.1152/ajpcell.1986.250.6.C920. [DOI] [PubMed] [Google Scholar]
  22. Hebert S. C., Reeves W. B., Molony D. A., Andreoli T. E. The medullary thick limb: function and modulation of the single-effect multiplier. Kidney Int. 1987 Feb;31(2):580–589. doi: 10.1038/ki.1987.38. [DOI] [PubMed] [Google Scholar]
  23. Hurst A. M., Hunter M. Apical membrane potassium channels in frog diluting segment: stimulation by furosemide. Am J Physiol. 1992 Apr;262(4 Pt 2):F606–F614. doi: 10.1152/ajprenal.1992.262.4.F606. [DOI] [PubMed] [Google Scholar]
  24. Kikeri D., Sun A., Zeidel M. L., Hebert S. C. Cell membranes impermeable to NH3. Nature. 1989 Jun 8;339(6224):478–480. doi: 10.1038/339478a0. [DOI] [PubMed] [Google Scholar]
  25. Kikeri D., Sun A., Zeidel M. L., Hebert S. C. Cellular NH4+/K+ transport pathways in mouse medullary thick limb of Henle. Regulation by intracellular pH. J Gen Physiol. 1992 Mar;99(3):435–461. doi: 10.1085/jgp.99.3.435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kinne R., Kinne-Saffran E., Schütz H., Schölermann B. Ammonium transport in medullary thick ascending limb of rabbit kidney: involvement of the Na+,K+,Cl(-)-cotransporter. J Membr Biol. 1986;94(3):279–284. doi: 10.1007/BF01869723. [DOI] [PubMed] [Google Scholar]
  27. Knepper M. A., Packer R., Good D. W. Ammonium transport in the kidney. Physiol Rev. 1989 Jan;69(1):179–249. doi: 10.1152/physrev.1989.69.1.179. [DOI] [PubMed] [Google Scholar]
  28. Kone B. C., Madsen K. M., Tisher C. C. Ultrastructure of the thick ascending limb of Henle in the rat kidney. Am J Anat. 1984 Oct;171(2):217–226. doi: 10.1002/aja.1001710207. [DOI] [PubMed] [Google Scholar]
  29. Krapf R. Basolateral membrane H/OH/HCO3 transport in the rat cortical thick ascending limb. Evidence for an electrogenic Na/HCO3 cotransporter in parallel with a Na/H antiporter. J Clin Invest. 1988 Jul;82(1):234–241. doi: 10.1172/JCI113576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Oberleithner H., Kersting U., Hunter M. Cytoplasmic pH determines K+ conductance in fused renal epithelial cells. Proc Natl Acad Sci U S A. 1988 Nov;85(21):8345–8349. doi: 10.1073/pnas.85.21.8345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rink T. J., Tsien R. Y., Pozzan T. Cytoplasmic pH and free Mg2+ in lymphocytes. J Cell Biol. 1982 Oct;95(1):189–196. doi: 10.1083/jcb.95.1.189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. Wingo C. S. Effect of acidosis on chloride transport in the cortical thick ascending limb of Henle perfused in vitro. J Clin Invest. 1986 Nov;78(5):1324–1330. doi: 10.1172/JCI112718. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of General Physiology are provided here courtesy of The Rockefeller University Press

RESOURCES