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Intracellular pH regulation in cultured rat astrocytes in CO2/HCO −3 -containing media

Experimental Brain Research volume 95, pages 371–380 (1993)Cite this article

Abstract

We studied the regulation of intracellular pH (pHi) and the mechanisms of pHi regulation in cultured rat astrocytes using microspectrofluorometry and the pH-sensitive fluorophore 2′,7′-bis(carboxyethyl-)-5,6-carboxyfluorescein. Control pHi was 7.00±0.02 in HCO -3 containing solutions at an extracellular pH of 7.35. Addition of 4, 4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS) or amiloride decreased pHi, as did removal of extracellular Na+, while removal of extracellular Cl- was followed by an increase in pHi. Following exposure to an acid transient induced by increasing the CO2 content from 5 to 15%, pHi rapidly returned to base line, with an average initial rate of recovery of 0.10 pH units min-1 (corresponding to a mean acid extrusion rate of 6.3±0.36 mmolo 1-1 min-1). Regulation of pHi was impaired when either amiloride or DIDS was added or Cl- was removed. This inhibition was enhanced when both DIDS and amiloride were present, and pHi regulation was completely blocked in the absence of extracellular Na+. The rapid regulation of pHi normally seen following a transient alkalinisation was not inhibited by amiloride or removal of Na+, but was partially inhibited by DIDS and by the absence of extracellular Cl-. The results are compatible with the presence of at least three different pHi-regulating mechanisms: a Na+/H+ antiporter, a Na+-dependent HCO -3 /Cl- exchanger (both regulating pHi during a transient acidification), and a passive Cl-/HCO -3 exchanger (regulating pHi during transient alkalinisation). The results fail to provide firm evidence of the presence of an electrogenic Na+/HCO -3 symporter.

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References

  • Astion ML, Orkand RK (1988) Electrogenic Na+/HCO -3 cotransport in neuroglia. Glia 1:355–357

    Google Scholar 

  • Boron WF (1983) Transport of H+ and of ionic weak acids and basis. J Membr Biol 72:1–16

    Google Scholar 

  • Boron W (1985) Intracellular pH-regulating mechanism of the squid axon: relationship between external Na+ and HCO3-dependencies. J Gen Physiol 85:325–345

    Google Scholar 

  • Boron W, Boulpaep E (1983) Intracellular pH regulation in the renal proximal tubule of the salamander: Na-H exchange. J Gen Physiol 81:29–52

    Google Scholar 

  • Boron W, De Weer P (1976) Intracellular pH transients in squid giant axons caused by CO2, NH3 and metabolic inhibitors. J Gen Physiol 67:91–112

    Google Scholar 

  • Boron W, Knakal R (1989) Intracellular pH-regulating mechanism of the squid axon: interaction between DNDS and extracellular Na+ and HCO -3 . J Gen Physiol 93:123–150

    Google Scholar 

  • Boyarsky G, Ganz M, Sterzel R, Boron W (1988) pH regulation in single glomerular mesangial cells I. Acid extrusion in absence and presence of HCO -3 . Am J Physiol 255: C844-C856

    Google Scholar 

  • Cabantchik ZI, Knauf OA, Rothstein A (1978) The anion transport system of the red blood cell: the role of membrane protein evaluated by the use of “probes”. Biochim Biophys Acta 515:239–302

    Google Scholar 

  • Caldwell PC (1958) Studies on the internal pH of large muscle and nerve fibers. J Physiol (Lond) 142:22–62

    Google Scholar 

  • Chesler M (1990) The regulation and modulation of pH in the nervous system. Prog Neurobiol 34:401–427

    Google Scholar 

  • Chesler M, Kaila K (1992) Modulation of pH by neuronal activity. Trends Neurosci 15:396–402

    Google Scholar 

  • Chesler M, Kraig RP (1987) Intracellular pH of astrocytes increases rapidly with cortical stimulation. Am J Physiol 253: R666-R670

    Google Scholar 

  • Chow S, Yen-Chow Y, White H, Woodbury D (1991) pH regulation after acid load in primary cultures of mouse astrocytes. Brain Res Dev Brain Res 60:69–78

    Google Scholar 

  • Deitmer J (1991) Electrogenic sodium-dependent bicarbonate secretion by glial cells of the leech central nervous system. J Gen Physiol 98:637–655

    Google Scholar 

  • Deitmer J, Schlue W-R (1987) The regulation of intracellular pH by identified glia and neurons in the central nervous system of the leech. J Physiol (Lond) 388:261–283

    Google Scholar 

  • Deitmer J, Schlue W-R (1989) An inwardly directed electrogenic sodium-bicarbonate co-transport in leech glial cells. J Physiol (Lond) 411:179–194

    Google Scholar 

  • Deitmer J, Szatkowski M (1990) Membrane potential dependence of intracellular pH regulation by identified glial cells in the leech central nervous system. J Physiol (Lond) 421:617–631

    Google Scholar 

  • Dixon S, Wilson J (1988) Plasma membrane proton transport in rat astroglia. J Gen Physiol 92:26a-27a

    Google Scholar 

  • Frelin C, Vigne P, Ladoux A, Lazdunski M (1988) The regulation of the intracellular pH in cells from vertebrates. Eur J Biochem 174:3–14

    Google Scholar 

  • Grinstein S, Rothstein R (1986) Mechanisms of regulation of the Na+/H+ exchanger. J Membr Biol 90:1–12

    Google Scholar 

  • Hansson E, Rönnbäck L (1989) Cultures of Astroglia and Neurons from Different Brain Regions. In: Shahar A, De Vellis J, Vernadakis A, Haber B (eds) Dissection and tissue culture. A manual of the nervous system. Alan Liss Inc, pp 92–104

  • Katsura K, Asplund B, Ekholm A, Siesjö B (1992) Extraand intracellular pH in the brain during ischemia, related to tissue lactate content in normaand hypercapnic rats. Eur J Neurosci 4:166–176

    Google Scholar 

  • Katsura K, Mellergård P, Theander S, Ouyang Y, Siesjö B (1993) Buffer capacity of rat cortical tissue, cultured neurons and astrocytes as evaluated by different methods. Brain Res (in press)

  • Kettenmann H, Schlue W-R (1988) Intracellular pH regulation in cultured mouse oligodendrocytes. J Physiol (Lond) 406:147–162

    Google Scholar 

  • Khadouri C, Cheval L, Marsy S, Barlet-Bas C, Doucet A (1991) Characterization and control of proton-ATPase along the nephron. Kidney Int [Suppl 33] 40: S71-S78

    Google Scholar 

  • Kimelberg HK (1983) Primary astrocyte culture a key to astrocyte function. Cell Mol Neurobiol 3:1–16

    Google Scholar 

  • Kimelberg H, Biddlecome S, Bourke R (1979a) SITS-inhibitable Cl- transport and Na+-dependent H+ production in primary astroglial culture. Brain Res 173:111–124

    Google Scholar 

  • Kimelberg H, Bowman C, Biddlecome S, Bourke R (1979b) Cation transport and membrane potential properties of primary astroglial cultures from neonatal rat brains. Brain Res 177:533–550

    Google Scholar 

  • Kraig R, Chesler M (1990) Astrocytic acidosis in hyperglycemic and complete ischemia. J Cereb Blood Flow Metab 10:104–114

    Google Scholar 

  • Kraig RP, Pulsinelli WA, Plum F (1986) Carbonic acid buffer changes during complete brain ischemia. Am J Physiol 250: R348-R357

    Google Scholar 

  • MacVicar B (1984) Voltage-dependent calcium channels in glial cells. Science 226:1345–1347

    Google Scholar 

  • Mellergård P (1993) pH regulation in brain cells. Doctoral thesis, University of Lund, Sweden

    Google Scholar 

  • Mellergård P, Siesjö BK (1991) Astrocytes fail to regulate intracellular pH at moderately reduced extracellular pH. Neuroreport 2:695–698

    Google Scholar 

  • Mellergård P, Ou-Yang Y, Siesjö B (1992) Regulation of intracellular pH in cultured astrocytes and neuroblastoma cells: dependence on extracellular pH. Can J Physiol Pharmacol 70:S293-S300

    Google Scholar 

  • Messeter K, Siesjö BK (1971) The intracellular pH in the brain in acute and sustained hypercapnia. Acta Physiol Scand 83:210–219

    Google Scholar 

  • Moody W (1981) The ionic mechanism of intracellular pH regulation in crayfish neurones. J Physiol (Lond) 316:433–351

    Google Scholar 

  • Mürer A, Hopfer U, Kinne R (1976) Sodium/proton antiport in brush-border membrane vesicles isolated from small intestine and kidney. Biochem J 154:597–605

    Google Scholar 

  • Raley-Susman KM, Cragoe EJ, Sapolsky RM, Kopito RR (1991) Regulation of intracellular pH in cultured hippocampal neurons by an amiloride-insensitive Na+/H+ exchanger. J Biol Chem 266:2739–2745

    Google Scholar 

  • Rink TJ, Tsien RY, Pozzan T (1982) Cytoplasmatic pH and free Mg++ in lymphocytes. J Cell Biol 95:189–196

    Google Scholar 

  • Roos A, Boron WF (1981) Intracellular pH. Physiol Rev 61:296–434

    CAS  PubMed  Google Scholar 

  • Siesjö B (1972) The regulation of cerebrospinal fluid pH. Kidney Int 1:360–374

    Google Scholar 

  • Siesjö B (1973) Metabolic control of intracellular pH. Scand J Clin Lab Invest 32:97–104

    Google Scholar 

  • Siesjö B, Folbergova J, MacMillan V (1972) The effect of hypercapnia upon intracellular pH in the brain evaluated by the bicarbonate-cabonic acid method and from the creatinine phosphokinase equilibrium. J Neurochem 19:2483–2495

    Google Scholar 

  • Siesjö B, Katsura K, Mellergård P, Ekholm A, Lundgren J, Smith M-L (1993) Acidosis-related brain damage. Prog Brain Res 96:23–48

    Google Scholar 

  • Thomas R (1976) The effect of carbon dioxide on the intracellular pH and buffering power of snail neurones. J Physiol (Lond) 255:715–735

    Google Scholar 

  • Thomas RC (1977) The role of bicarbonate, chloride and sodium ions in the regulation of intracellular pH in snail neurons. J Physiol (Lond) 273:317–338

    Google Scholar 

  • Tønnessen T, Ludt J, Sandvig K, Olsnes S (1987) Bicarbonate/chloride antiport in Vero cells. I. Evidence for both sodium-linked and sodium-independent exchange. J Cell Physiol 132:183–191

    Google Scholar 

  • Walz W, Wuttke W, Hertz L (1984) Astrocytes in primary cultures: membrane potential characteristics reveal exclusive potassium conductance and potassium accumulator properties. Brain Res 292:367–374

    Google Scholar 

  • Wuttke W, Walz W (1990) Sodiumand bicarbonate-independent regulation of intracellular pH in cultured mouse astrocytes. Neurosci lett 117:105–110

    Google Scholar 

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Affiliations

  1. Laboratory for Experimental Brain Research, Lund University Hospital, S-221 85, Lund, Sweden

    Pekka Mellergård, Yi-bing Ouyang & Bo K Siesjö

  2. Department of Neurosurgery, Lund University Hospital, S-221 85, Lund, Sweden

    Pekka Mellergård

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  1. Pekka Mellergård
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  2. Yi-bing Ouyang
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  3. Bo K Siesjö
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Mellergård, P., Ouyang, Yb. & Siesjö, B.K. Intracellular pH regulation in cultured rat astrocytes in CO2/HCO −3 -containing media. Exp Brain Res 95, 371–380 (1993). https://doi.org/10.1007/BF00227129

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  • DOI: https://doi.org/10.1007/BF00227129

Key words

  • Astrocytes
  • Na+/H+ exchanger
  • HCO -3 /Cl-exchanger
  • pH regulation
  • Rat