Skip to main content
PMC home page
The Journal of General Physiology logoLink to The Journal of General Physiology
. 1985 Oct 1;86(4):527–564. doi: 10.1085/jgp.86.4.527

Volume-regulatory responses of Amphiuma red cells in anisotonic media. The effect of amiloride

PMCID: PMC2228807  PMID: 4056735

Abstract

Amphiuma red cells were incubated for several hours in hypotonic or hypertonic media. They regulate their volume in both media by using ouabain-insensitive salt transport mechanisms. After initially enlarging osmotically, cells in hypotonic media return toward their original size by losing K, Cl, and H2O. During this volume-regulatory decrease (VRD) response, K loss results from a greater than 10-fold increase in K efflux. Cells in hypertonic media initially shrink osmotically, but then return toward their original volume by gaining Na, Cl, and H2O. The volume-regulatory increase (VRI) response involves a large (greater than 100-fold) increase in Na uptake that is entirely blocked by the diuretic amiloride (10(-3) M). Na transport in the VRI response shares many of the characteristics of amiloride-sensitive transport in epithelia: (a) amiloride inhibition is reversible; (b) removal of amiloride from cells pretreated with amiloride enhances Na uptake relative to untreated controls; (c) amiloride appears to act as a competitive inhibitor (Ki = 1-3 microM) of Na uptake; (d) Na uptake is a saturable function of external Na (Km approximately 29 mM); (e) Li can substitute for Na but K cannot. Anomalous Na/K pump behavior is observed in both the VRD and the VRI responses. In the VRD response, pump activity increases 3-fold despite a decrease in intracellular Na concentration, while in the VRI response, a 10-fold increase in pump activity is observed when only a doubling is predicted from increases in intracellular Na.

Full Text

The Full Text of this article is available as a PDF (2.3 MB).

Selected References

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

  1. Aceves J., Cereijido M. The effect of amiloride on sodium and potassium fluxes in red cells. J Physiol. 1973 Mar;229(3):709–718. doi: 10.1113/jphysiol.1973.sp010162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Benos D. J. Amiloride: a molecular probe of sodium transport in tissues and cells. Am J Physiol. 1982 Mar;242(3):C131–C145. doi: 10.1152/ajpcell.1982.242.3.C131. [DOI] [PubMed] [Google Scholar]
  3. Benos D. J., Mandel L. J., Balaban R. S. On the mechanism of the amiloride-sodium entry site interaction in anuran skin epithelia. J Gen Physiol. 1979 Mar;73(3):307–326. doi: 10.1085/jgp.73.3.307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bentley P. J. Amiloride: a potent inhibitor of sodium transport across the toad bladder. J Physiol. 1968 Mar;195(2):317–330. doi: 10.1113/jphysiol.1968.sp008460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Blaustein M. P. The interrelationship between sodium and calcium fluxes across cell membranes. Rev Physiol Biochem Pharmacol. 1974;70:33–82. doi: 10.1007/BFb0034293. [DOI] [PubMed] [Google Scholar]
  6. Boron W. F., Boulpaep E. L. Intracellular pH regulation in the renal proximal tubule of the salamander. Na-H exchange. J Gen Physiol. 1983 Jan;81(1):29–52. doi: 10.1085/jgp.81.1.29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cala P. M. Volume regulation by Amphiuma red blood cells. The membrane potential and its implications regarding the nature of the ion-flux pathways. J Gen Physiol. 1980 Dec;76(6):683–708. doi: 10.1085/jgp.76.6.683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cala P. M. Volume regulation by flounder red blood cells in anisotonic media. J Gen Physiol. 1977 May;69(5):537–552. doi: 10.1085/jgp.69.5.537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cuthbert A. W., Shum W. K. Does intracellular sodium modify membrane permeability to sodium ions? Nature. 1977 Mar 31;266(5601):468–469. doi: 10.1038/266468a0. [DOI] [PubMed] [Google Scholar]
  10. DICK D. A., LOWENSTEIN L. M. Osmotic equilibria in human erythrocytes studied by immersion refractometry. Proc R Soc Lond B Biol Sci. 1958 Feb 18;148(931):241–256. doi: 10.1098/rspb.1958.0016. [DOI] [PubMed] [Google Scholar]
  11. Dalmark M. Chloride and water distribution in human red cells. J Physiol. 1975 Aug;250(1):65–84. doi: 10.1113/jphysiol.1975.sp011043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ehrlich E. N., Crabbé J. The mechanism of action of amipramizide. Pflugers Arch. 1968;302(1):79–96. doi: 10.1007/BF00586783. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Erlij D., Smith M. W. Sodium uptake by frog skin and its modification by inhibitors of transepithelial sodium transport. J Physiol. 1973 Jan;228(1):221–239. doi: 10.1113/jphysiol.1973.sp010083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. FLYNN F., MAIZELS M. Cation control in human erythrocytes. J Physiol. 1949 Dec;110(3-4):301–318. doi: 10.1113/jphysiol.1949.sp004440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. FRAZIER H. S., DEMPSEY E. F., LEAF A. Movement of sodium across the mucosal surface of the isolated toad bladder and its modification by vasopressin. J Gen Physiol. 1962 Jan;45:529–543. doi: 10.1085/jgp.45.3.529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Freedman J. C., Hoffman J. F. Ionic and osmotic equilibria of human red blood cells treated with nystatin. J Gen Physiol. 1979 Aug;74(2):157–185. doi: 10.1085/jgp.74.2.157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fuchs W., Larsen E. H., Lindemann B. Current-voltage curve of sodium channels and concentration dependence of sodium permeability in frog skin. J Physiol. 1977 May;267(1):137–166. doi: 10.1113/jphysiol.1977.sp011805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Fugelli K. Regulation of cell volume in flounder (Pleuronectes flesus) erythrocytes accompanying a decrease in plasma osmolarity. Comp Biochem Physiol. 1967 Jul;22(1):253–260. doi: 10.1016/0010-406x(67)90185-5. [DOI] [PubMed] [Google Scholar]
  20. Garay R. P., Garrahan P. J. The interaction of sodium and potassium with the sodium pump in red cells. J Physiol. 1973 Jun;231(2):297–325. doi: 10.1113/jphysiol.1973.sp010234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Greenwald L. E., Kirschner L. B. The effect of poly-L-lysine, amiloride and methyl-L-lysine on gill ion transport and permeability in the rainbow trout. J Membr Biol. 1976 May;26(4):371–383. doi: 10.1007/BF01868884. [DOI] [PubMed] [Google Scholar]
  22. Grinstein S., Clarke C. A., Rothstein A. Activation of Na+/H+ exchange in lymphocytes by osmotically induced volume changes and by cytoplasmic acidification. J Gen Physiol. 1983 Nov;82(5):619–638. doi: 10.1085/jgp.82.5.619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Grinstein S., Rothstein A., Sarkadi B., Gelfand E. W. Responses of lymphocytes to anisotonic media: volume-regulating behavior. Am J Physiol. 1984 Mar;246(3 Pt 1):C204–C215. doi: 10.1152/ajpcell.1984.246.3.C204. [DOI] [PubMed] [Google Scholar]
  24. Haas M., Schmidt W. F., 3rd, McManus T. J. Catecholamine-stimulated ion transport in duck red cells. Gradient effects in electrically neutral [Na + K + 2Cl] Co-transport. J Gen Physiol. 1982 Jul;80(1):125–147. doi: 10.1085/jgp.80.1.125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Helman S. I., Fisher R. S. Microelectrode studies of the active Na transport pathway of frog skin. J Gen Physiol. 1977 May;69(5):571–604. doi: 10.1085/jgp.69.5.571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Herrera F. C. Inhibition of lithium transport across toad bladder by amiloride. Am J Physiol. 1972 Feb;222(2):499–502. doi: 10.1152/ajplegacy.1972.222.2.499. [DOI] [PubMed] [Google Scholar]
  27. Hoffman J. F., Kaplan J. H., Callahan T. J. The Na:K pump in red cells is electrogenic. Fed Proc. 1979 Oct;38(11):2440–2441. [PubMed] [Google Scholar]
  28. Kinsella J. L., Aronson P. S. Amiloride inhibition of the Na+-H+ exchanger in renal microvillus membrane vesicles. Am J Physiol. 1981 Oct;241(4):F374–F379. doi: 10.1152/ajprenal.1981.241.4.F374. [DOI] [PubMed] [Google Scholar]
  29. Knott G. D. Mlab--a mathematical modeling tool. Comput Programs Biomed. 1979 Dec;10(3):271–280. doi: 10.1016/0010-468x(79)90075-8. [DOI] [PubMed] [Google Scholar]
  30. Kregenow F. M., Caryk T., Siebens A. W. Further studies of the volume-regulatory response of Amphiuma red cells in hypertonic media. Evidence for amiloride-sensitive Na/H exchange. J Gen Physiol. 1985 Oct;86(4):565–584. doi: 10.1085/jgp.86.4.565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kregenow F. M. Osmoregulatory salt transporting mechanisms: control of cell volume in anisotonic media. Annu Rev Physiol. 1981;43:493–505. doi: 10.1146/annurev.ph.43.030181.002425. [DOI] [PubMed] [Google Scholar]
  32. Kregenow F. M., Robbie D. E., Orloff J. Effect of norepinephrine and hypertonicity on K influx and cyclic AMP in duck erythrocytes. Am J Physiol. 1976 Aug;231(2):306–311. doi: 10.1152/ajplegacy.1976.231.2.306. [DOI] [PubMed] [Google Scholar]
  33. Kregenow F. M. The response of duck erythrocytes to hypertonic media. Further evidence for a volume-controlling mechanism. J Gen Physiol. 1971 Oct;58(4):396–412. doi: 10.1085/jgp.58.4.396. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kregenow F. M. The response of duck erythrocytes to nonhemolytic hypotonic media. Evidence for a volume-controlling mechanism. J Gen Physiol. 1971 Oct;58(4):372–395. doi: 10.1085/jgp.58.4.372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Kregenow F. M. The response of duck erythrocytes to norepinephrine and an elevated extracellular potassium. Volume regulation in isotonic media. J Gen Physiol. 1973 Apr;61(4):509–527. doi: 10.1085/jgp.61.4.509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. LINDLEY B. D., HOSHIKO T. THE EFFECTS OF ALKALI METAL CATIONS AND COMMON ANIONS ON THE FROG SKIN POTENTIAL. J Gen Physiol. 1964 Mar;47:749–771. doi: 10.1085/jgp.47.4.749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Lewis S. A., Eaton D. C., Diamond J. M. The mechanism of Na+ transport by rabbit urinary bladder. J Membr Biol. 1976 Aug 27;28(1):41–70. doi: 10.1007/BF01869690. [DOI] [PubMed] [Google Scholar]
  38. Lipton P. Effect of changes in osmolarity on sodium transport across isolated toad bladder. Am J Physiol. 1972 Apr;222(4):821–828. doi: 10.1152/ajplegacy.1972.222.4.821. [DOI] [PubMed] [Google Scholar]
  39. MCCONAGHEY P. D., MAIZELS M. The osmotic coefficients of haemoglobin in red cells under varying conditions. J Physiol. 1961 Jan;155:28–45. doi: 10.1113/jphysiol.1961.sp006611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. MULLINS L. J., FRUMENTO A. S. The concentration dependence of sodium efflux from muscle. J Gen Physiol. 1963 Mar;46:629–654. doi: 10.1085/jgp.46.4.629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Nelson M. T., Blaustein M. P. Properties of sodium pumps in internally perfused barnacle muscle fibers. J Gen Physiol. 1980 Feb;75(2):183–206. doi: 10.1085/jgp.75.2.183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. ORSKOV S. L. Experiments on the influence of adrenaline and noradrenaline on the potassium absorption of red blood cells from pigeons and frogs. Acta Physiol Scand. 1956 Nov 5;37(4):299–306. doi: 10.1111/j.1748-1716.1956.tb01365.x. [DOI] [PubMed] [Google Scholar]
  43. ORSKOV S. L. Experiments on the potassium absorption of the erythrocytes of Rana esculenta and Rana temporaria after bleeding and in hypertonic plasma. Acta Physiol Scand. 1956 Nov 5;37(4):295–298. doi: 10.1111/j.1748-1716.1956.tb01364.x. [DOI] [PubMed] [Google Scholar]
  44. POST R. L., JOLLY P. C. The linkage of sodium, potassium, and ammonium active transport across the human erythrocyte membrane. Biochim Biophys Acta. 1957 Jul;25(1):118–128. doi: 10.1016/0006-3002(57)90426-2. [DOI] [PubMed] [Google Scholar]
  45. Palfrey H. C., Greengard P. Hormone-sensitive ion transport systems in erythrocytes as models for epithelial ion pathways. Ann N Y Acad Sci. 1981;372:291–308. doi: 10.1111/j.1749-6632.1981.tb15482.x. [DOI] [PubMed] [Google Scholar]
  46. Parker J. C., Castranova V. Volume-responsive sodium and proton movements in dog red blood cells. J Gen Physiol. 1984 Sep;84(3):379–401. doi: 10.1085/jgp.84.3.379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Parker J. C. Dog red blood cells. Adjustment of salt and water content in vitro. J Gen Physiol. 1973 Aug;62(2):147–156. doi: 10.1085/jgp.62.2.147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Parker J. C., Gitelman H. J., Glosson P. S., Leonard D. L. Role of calcium in volume regulation by dog red blood cells. J Gen Physiol. 1975 Jan;65(1):84–96. doi: 10.1085/jgp.65.1.84. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Parker J. C. Volume-responsive sodium movements in dog red blood cells. Am J Physiol. 1983 May;244(5):C324–C330. doi: 10.1152/ajpcell.1983.244.5.C324. [DOI] [PubMed] [Google Scholar]
  50. Pollack L. R., Tate E. H., Cook J. S. Turnover and regulation of Na-K-ATPase in HeLa cells. Am J Physiol. 1981 Nov;241(5):C173–C183. doi: 10.1152/ajpcell.1981.241.5.C173. [DOI] [PubMed] [Google Scholar]
  51. Riddick D. H., Kregenow F. M., Orloff J. The effect of norepinephrine and dibutyryl cyclic adenosine monophosphate on cation transport in duck erythrocytes. J Gen Physiol. 1971 Jun;57(6):752–766. doi: 10.1085/jgp.57.6.752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Rindler M. J., Taub M., Saier M. H., Jr Uptake of 22Na+ by cultured dog kidney cells (MDCK). J Biol Chem. 1979 Nov 25;254(22):11431–11439. [PubMed] [Google Scholar]
  53. SCHATZMANN H. J. Herzglykoside als Hemmstoffe für den aktiven Kalium- und Natriumtransport durch die Erythrocytenmembran. Helv Physiol Pharmacol Acta. 1953;11(4):346–354. [PubMed] [Google Scholar]
  54. Schmidt W. F., 3rd, McManus T. J. Ouabain-insensitive salt and water movements in duck red cells. III. The role of chloride in the volume response. J Gen Physiol. 1977 Jul;70(1):99–121. doi: 10.1085/jgp.70.1.99. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Schultz S. G., Frizzell R. A., Nellans H. N. Active sodium transport and the electrophysiology of rabbit colon. J Membr Biol. 1977 May 12;33(3-4):351–384. doi: 10.1007/BF01869524. [DOI] [PubMed] [Google Scholar]
  56. Spring K. R., Ericson A. C. Epithelial cell volume modulation and regulation. J Membr Biol. 1982;69(3):167–176. doi: 10.1007/BF01870396. [DOI] [PubMed] [Google Scholar]
  57. Stoner L. C., Kregenow F. M. A single-cell technique for the measurement of membrane potential, membrane conductance, and the efflux of rapidly penetrating solutes in Amphiuma erythrocytes. J Gen Physiol. 1980 Oct;76(4):455–478. doi: 10.1085/jgp.76.4.455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Sudou K., Hoshi T. Mode of action of amiloride in toad urinary bladder. An electrophysiological study of the drug action on sodium permeability of the mucosal border. J Membr Biol. 1977 Apr 7;32(1-2):115–132. doi: 10.1007/BF01905212. [DOI] [PubMed] [Google Scholar]
  59. TOSTESON D. C., CARLSEN E., DUNHAM E. T. The effects of sickling on ion transport. I. Effect of sickling on potassium transport. J Gen Physiol. 1955 Sep 20;39(1):31–53. doi: 10.1085/jgp.39.1.31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Taylor A., Windhager E. E. Possible role of cytosolic calcium and Na-Ca exchange in regulation of transepithelial sodium transport. Am J Physiol. 1979 Jun;236(6):F505–F512. doi: 10.1152/ajprenal.1979.236.6.F505. [DOI] [PubMed] [Google Scholar]
  61. ØRSKOV S. L. The potassium absorption by pigeon blood cells; a considerable potassium absorption by pigeon- and hen blood cells in observed when a hypertonic sodium chloride solution is added. Acta Physiol Scand. 1954 Jul 18;31(2-3):221–229. doi: 10.1111/j.1748-1716.1954.tb01133.x. [DOI] [PubMed] [Google Scholar]

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

RESOURCES