Acid/base transport across the leech giant glial cell membrane at low external bicarbonate concentration.
1. We have studied acid/base transport across the cell membrane of the giant neuropile glial cell in the leech (Hirudo medicinalis) central nervous system induced by changing the external pH (pHo), using double-barrelled, pH-sensitive microelectrodes. In the presence of 5 % CO2 and 24 mM HCO3-, the intracellular pH (pHi) rapidly changes due to a potent, reversible Na+-HCO3- cotransport across the glial membrane. We have now investigated the transport mechanism which leads to pHi changes in the nominal absence of CO2/HCO3-, where the HCO3- concentration is expected to be below 1 mM. 2. The intracellular pH increased and then decreased when pHo was altered from 7.4 to 7.8 and then 7.0 with a rate of increase of +0.026 +/- 0.008 and a rate of decrease of -0.028 +/- 0.009 pH units min-1 (+/- s.d., n = 49), indicating an acid/base flux rate of 0.64 and 0.71 mM min-1 across the glial membrane, respectively. 3. In the absence of external sodium (Na+replaced by N-methyl-D-glucamine), pHi slowly decreased, and the rate of alkali and acid loading was reduced to 19 and 28 %, respectively, (n = 12). Amiloride (2 mM), which inhibits Na+-H+ exchange, had no effect on the alkali/acid loading (n = 6). 4. The alkali and acid loading were not impaired after the removal of external chloride (Cl-o, replaced by gluconate; n = 11), but were significantly reduced by the anion transport inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS, 0.5 mM) to 23 and 16 %, respectively, of the control (P < 0.001; n = 5). 5. Alkali and acid loading were affected differently by manipulating the availability of residual HCO3-. After adding the membrane-permeable carbonic anhydrase inhibitor ethoxyzolamide (EZA, 2 microM) to the saline, the acid loading, but not the alkali loading, was significantly reduced (by 25 %, P < 0.01), while lowering the residual CO2/HCO3- concentration in the saline by O2 bubbling significantly reduced the alkali loading (by 59 %, P < 0. 02), but not the acid loading. 6. Changing the membrane holding potential in voltage-clamped glial cells or raising the external K+ concentration to 30 mM had no significant effect on acid/base loading. 7. It is concluded that a residual HCO3- concentration of less than 1 mM in nominally CO2/HCO3--free salines and HCO3- produced endogenously in the glial cells support alkali and acid loading across the glial cell membrane, presumably by activation of the reversible Na+-HCO3- cotransporter. The results suggest a very high selectivity and affinity of this cotransporter for HCO3-; they imply that HCO3--dependent processes may not be negligible even in the nominal absence of CO2/HCO3-, when the HCO3- concentration is expected to be in the submillimolar range.
Deitmer
J W
JW
Abteilung fur Allgemeine Zoologie, FB Biologie, Universitat Kaiserslautern, Postfach 3049, D-67653 Kaiserslautern, Germany. deitmer@rhrk.uni-kl.de
Schneider
H P
HP
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000136:Acid-Base Equilibrium / Q000502:physiology*; D000818:Animals; D001639:Bicarbonates / Q000378:metabolism*; D001693:Biological Transport, Active / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism; D004558:Electric Stimulation; D004594:Electrophysiology; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007865:Leeches / Q000378:metabolism*; D008564:Membrane Potentials / Q000502:physiology; D009457:Neuroglia / Q000378:metabolism*; D018408:Patch-Clamp Techniques
D001639:Bicarbonates; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid