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. 1988 Dec;406:225–246. doi: 10.1113/jphysiol.1988.sp017378

Intracellular chloride regulation in amphibian dorsal root ganglion neurones studied with ion-selective microelectrodes.

F J Alvarez-Leefmans 1, S M Gamiño 1, F Giraldez 1, I Noguerón 1
PMCID: PMC1191097  PMID: 3254412

Abstract

1. Intracellular Cl- activity (aiCl) and membrane potential (Em) were measured in frog dorsal root ganglion neurones (DRG neurones) using double-barrelled Cl- -selective microelectrodes. In standard Ringer solution buffered with HEPES (5 mM), equilibrated with air or 100% O2, the resting membrane potential was -57.7 +/- 1.0 mV and aiCl was 23.6 +/- 1.0 mM (n = 53). The value of aiCl was 2.6 times the activity expected for an equilibrium distribution and the difference between Em and ECl was 25 mV. 2. Removal of external Cl- led to a reversible fall in aiCl. Initial rates of decay and recovery of aiCl were 4.1 and 3.3 mM min-1, respectively. During the recovery of aiCl following return to standard Ringer solution, most of the movement of Cl- occurred against the driving force for a passive distribution. Changes in aiCl were not associated with changes in Em. Chloride fluxes estimated from initial rates of change in aiCl when external Cl- was removed were too high to be accounted for by electrodiffusion. 3. The intracellular accumulation of Cl- was dependent on the extracellular Cl- activity (aoCl). The relationship between aiCl and aoCl had a sigmoidal shape with a half-maximal activation of about 50 mM-external Cl-. 4. The steady-state aiCl depended on the simultaneous presence of extracellular Na+ and K+. Similarly, the active reaccumulation of Cl- after intracellular Cl- depletion was abolished in the absence of either Na+ or K+ in the bathing solution. 5. The reaccumulation of Cl- was inhibited by furosemide (0.5-1 x 10(-3) M) or bumetanide (10(-5) M). The decrease in aiCl observed in Cl- -free solutions was also inhibited by bumetanide. 6. Cell volume changes were calculated from the observed changes in aiCl. Cells were estimated to shrink in Cl- -free solutions to about 75% their initial volume, at an initial rate of 6% min-1. 7. The present results provide direct evidence for the active accumulation of Cl- in DRG neurones. The mechanism of Cl- transport is electrically silent, dependent on the simultaneous presence of external Cl-, Na+ and K+ and inhibited by loop diuretics. It is suggested that a Na+:K+:Cl- co-transport system mediates the active transport of Cl- across the cell membrane of DRG neurones.

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Selected References

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  1. Aickin C. C., Brading A. F. Advances in the understanding of transmembrane ionic gradients and permeabilities in smooth muscle obtained by using ion-selective micro-electrodes. Experientia. 1985 Jul 15;41(7):879–887. doi: 10.1007/BF01970005. [DOI] [PubMed] [Google Scholar]
  2. Alvarez-Leefmans F. J., Gamiño S. M., Giraldez F., González-Serratos H. Intracellular free magnesium in frog skeletal muscle fibres measured with ion-selective micro-electrodes. J Physiol. 1986 Sep;378:461–483. doi: 10.1113/jphysiol.1986.sp016230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bolton T. B., Vaughan-Jones R. D. Continuous direct measurement of intracellular chloride and pH in frog skeletal muscle. J Physiol. 1977 Sep;270(3):801–833. doi: 10.1113/jphysiol.1977.sp011983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Deschenes M., Feltz P., Lamour Y. A model for an estimate in vivo of the ionic basis of presynaptic inhibition: an intracellular analysis of the GABA-induced depolarization in rat dorsal root ganglia. Brain Res. 1976 Dec 24;118(3):486–493. doi: 10.1016/0006-8993(76)90318-8. [DOI] [PubMed] [Google Scholar]
  5. Ellory J. C., Stewart G. W. The human erythrocyte Cl-dependent Na-K cotransport system as a possible model for studying the action of loop diuretics. Br J Pharmacol. 1982 Jan;75(1):183–188. doi: 10.1111/j.1476-5381.1982.tb08771.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gallagher J. P., Higashi H., Nishi S. Characterization and ionic basis of GABA-induced depolarizations recorded in vitro from cat primary afferent neurones. J Physiol. 1978 Feb;275:263–282. doi: 10.1113/jphysiol.1978.sp012189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gallagher J. P., Nakamura J., Shinnick-Gallagher P. The effects of temperature, pH and Cl-pump inhibitors on GABA responses recorded from cat dorsal root ganglia. Brain Res. 1983 May 16;267(2):249–259. doi: 10.1016/0006-8993(83)90877-6. [DOI] [PubMed] [Google Scholar]
  8. Geck P., Heinz E. The Na-K-2Cl cotransport system. J Membr Biol. 1986;91(2):97–105. doi: 10.1007/BF01925787. [DOI] [PubMed] [Google Scholar]
  9. Giraldez F. Active sodium transport and fluid secretion in the gall-bladder epithelium of Necturus. J Physiol. 1984 Mar;348:431–455. doi: 10.1113/jphysiol.1984.sp015118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. HODGKIN A. L., HOROWICZ P. The influence of potassium and chloride ions on the membrane potential of single muscle fibres. J Physiol. 1959 Oct;148:127–160. doi: 10.1113/jphysiol.1959.sp006278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. HODGKIN A. L., KATZ B. The effect of sodium ions on the electrical activity of giant axon of the squid. J Physiol. 1949 Mar 1;108(1):37–77. doi: 10.1113/jphysiol.1949.sp004310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hoffmann E. K. Anion transport systems in the plasma membrane of vertebrate cells. Biochim Biophys Acta. 1986 Jun 12;864(1):1–31. doi: 10.1016/0304-4157(86)90014-6. [DOI] [PubMed] [Google Scholar]
  13. Kenyon J. L., Gibbons W. R. Effects of low-chloride solutions on action potentials of sheep cardiac Purkinje fibers. J Gen Physiol. 1977 Nov;70(5):635–660. doi: 10.1085/jgp.70.5.635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lederer W. J., Spindler A. J., Eisner D. A. Thick slurry bevelling: a new technique for bevelling extremely fine microelectrodes and micropipettes. Pflugers Arch. 1979 Sep;381(3):287–288. doi: 10.1007/BF00583261. [DOI] [PubMed] [Google Scholar]
  15. Levy R. A. The role of GABA in primary afferent depolarization. Prog Neurobiol. 1977;9(4):211–267. doi: 10.1016/0301-0082(77)90002-8. [DOI] [PubMed] [Google Scholar]
  16. Mayer M. L., Westbrook G. L. A voltage-clamp analysis of inward (anomalous) rectification in mouse spinal sensory ganglion neurones. J Physiol. 1983 Jul;340:19–45. doi: 10.1113/jphysiol.1983.sp014747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Nicoll R. A., Alger B. E. Presynaptic inhibition: transmitter and ionic mechanisms. Int Rev Neurobiol. 1979;21:217–258. doi: 10.1016/s0074-7742(08)60639-x. [DOI] [PubMed] [Google Scholar]
  18. Nicoll R. A. The blockade of GABA mediated responses in the frog spinal cord by ammonium ions and furosemide. J Physiol. 1978 Oct;283:121–132. doi: 10.1113/jphysiol.1978.sp012491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Nishi S., Minota S., Karczmar A. G. Primary afferent neurones: the ionic mechanism of GABA-mediated depolarization. Neuropharmacology. 1974 Mar;13(3):215–219. doi: 10.1016/0028-3908(74)90110-5. [DOI] [PubMed] [Google Scholar]
  20. O'Grady S. M., Palfrey H. C., Field M. Na-K-2Cl cotransport in winter flounder intestine and bovine kidney outer medulla: [3H] bumetanide binding and effects of furosemide analogues. J Membr Biol. 1987;96(1):11–18. doi: 10.1007/BF01869330. [DOI] [PubMed] [Google Scholar]
  21. Russell J. M. Cation-coupled chloride influx in squid axon. Role of potassium and stoichiometry of the transport process. J Gen Physiol. 1983 Jun;81(6):909–925. doi: 10.1085/jgp.81.6.909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Thomas R. C., Cohen C. J. A liquid ion-exchanger alternative to KCl for filling intracellular reference microelectrodes. Pflugers Arch. 1981 Apr;390(1):96–98. doi: 10.1007/BF00582719. [DOI] [PubMed] [Google Scholar]
  23. Vaughan-Jones R. D. Chloride activity and its control in skeletal and cardiac muscle. Philos Trans R Soc Lond B Biol Sci. 1982 Dec 1;299(1097):537–548. doi: 10.1098/rstb.1982.0150. [DOI] [PubMed] [Google Scholar]
  24. Wojtowicz J. M., Nicoll R. A. Selective action of piretanide on primary afferent GABA responses in the frog spinal cord. Brain Res. 1982 Mar 18;236(1):173–181. doi: 10.1016/0006-8993(82)90043-9. [DOI] [PubMed] [Google Scholar]

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