The regulation of intracellular pH by identified glial cells and neurones in the central nervous system of the leech.

Abstract

SUMMARY1. Double-barrelled, neutral-carrier pH-sensitive micro-electrodes were used to measure the intracellular pH (pHi) and the pHi regulation of neuropile glial (n.g.) cells and of identified neurones of the leech Hirudo medicinalis.2. The distribution of H+ in the n.g. cells and in the neurones was found not to be in electrical equilibrium. The mean pHi ofthe n.g. cells was 6-87 + 0 13 (mean + S.D.of mean here and for all following data n = 27) in HEPES-buffered leech saline (pHo = 7 4) and 7418 + 0-19 (n = 13) in 2 % C02-11 mM-HC03--saline. The mean pHi was 7'28+0-1 (n = 20) in Retzius neurones and 7-32+0415 (n = 12) in noxious neurones in HEPES-buffered leech saline, and 7-20+0415 (n = 10) and 7-27 +016 (n = 6) in 2 % C02-1 1 mM-HC03--buffered saline in these two types of neurones, respectively.3. The cytoplasmic buffering power, as calculated by the change in pHi following the change from 2 % C02-11 mM-HC03-to 5 % C02-22 mM-HCO.-in the leech saline, was 20-30 mM/pH unit in the n.g. cells and between 12 and 33 mM/pH unit in the neurones.4. The recovery of pHi in n.g. cells from an experimentally induced acidification (addition and removal of 20 mm-NH4Cl) was dependent on the presence of external Na+. Independent of the buffer system used, pHi recovery was inhibited when external Na+ was exchanged by N-methyl-D-glucamine. Amiloride (2-3 mM) reduced the rate of pHi recovery by about 50 % in these n.g. cells.5. In CO2-HCO3--free saline, or in the presence of the anion exchange blocker 4-acetamido-4'-isothiocyanostilbene-2, 2'-disulphonic acid (SITS, 0 5 mM), pHi recovery from an acid load was often slowed by up to 50 % in n.g. cells. This suggests that there is a significant contribution of a HC03--dependent membrane transport to pHi regulation in n.g. cells.6. When a HEPES-buffered saline was exchanged by a 2 % C02-11 mM-HCO3-buffered saline, the pHi of n.g. cells increased by 0-31 pH units. This alkaline shift was reversible upon removal of the CO2-HC03-and was absent in the Na+-free saline. It was not inhibited by 1 mM-furosemide or by 0 5 mM-SITS. Successive addition of first Na+ and then CO2-HCO3-to an Na+-free HEPES-buffered saline produced two separate phases of intracellular alkalization, suggesting two different pHi regulation transport systems.J. W. DEITMER AND W.-R. SCHLUE 7. In sensory and Retzius neurones pHi recovery from acidification was also dependent on external Na+, both in the presence and in the absence of CO2-HC03-.The rate of pHi recovery was reversibly reduced by 2 mM-amiloride by up to 90 % in these neurones.8. In conclusion, bicarbonate buffer affects n.g. cell pHi differently from the way in which it affects neuronal pHi. The regulation of pHi depends on the presence of external Na+ in both n.g. cells and neurones. The experiments suggest the presence of an Na+-H+ exchange and a SITS-sensitive HCO3--and Na+-dependent acid extrusion mechanism across n.g. (and nerve cell) membranes and another, SITSinsensitive HC03-and Na+-dependent transport system only in n.g. cells.

Regulation of Intracellular pH in Single Rat Cortical Neurons in vitro: A Microspectrofluorometric Study

“…A possible can didate would be an (electrogenic) Na + -HC03 -cotransporter. Such a cotransporter was first de scribed in salamander kidney (Boron and Boulpaep, 1983) and has since been reported in several types of glial cells, including glia of leech CNS (Deitmer and Schlue, 1987) and amphibian optic nerve (As tion and Orkand, 1988). To our knowledge, this transporter has never been identified in cells of neu ronal origin.…”

“…In most vertebrate cells studied, pHj regulation during acid transients is achieved by an amiloride-sensitive N a + IH + anti porter , often working in conjunction with Na + -dependent or Na + -independent HC03 -1 Cl-exchangers (Mahnensmith and Aronsson, 1985;Boyarsky et aI., 1988b;Frelin et aI., 1988;Grinstein et aI., 1989). Recent studies have identified addi tional mechanisms that influence pHi' e.g., an elec trogenic N a + IHC03 -symporter, which carries HC03 -inward at the expense of the Na + gradient (Boron and Boulpaep, 1983;Deitmer and Schlue, 1987; for reviews see Madshus, 1988;Chesler and Kaila, 1992).…”

Molecular Cloning, Chromosomal Localization, Tissue Distribution, and Functional Expression of the Human Pancreatic Sodium Bicarbonate Cotransporter

“…Of interest, cAMP stimulates transepithelial bicarbonate secretion and basolateral Na(HCO 3 ) n cotransport in pancreatic ducts (7,45), whereas in the renal proximal tubule, cAMP inhibits basolateral Na(HCO 3 ) n cotransport (46). The unique N terminus of pNBC not shared by kNBC could have an important regulatory role in functioning as a target for phosphorylation by protein kinase A. Na(HCO 3 ) n cotransport has been functionally demonstrated in pancreas (3)(4)(5)(6)(7)(8), kidney (1,2,21,22,(25)(26)(27)31), leech glial cells (17,18), retinal Mü ller cells (15,16), type II alveolar cells (20), hepatocytes (10 -12), colon (9), gastric parietal cells (19), cardiac Purkinje fibers (13) and ventricular myocytes (14), although the stoichiometry of the transported species appears to be tissue-dependent. The lack of detectable transcripts in heart and lung with any of the three probes used in this study suggests the possibility that cardiac and lung Na(HCO 3 ) n cotransport is mediated by an alternative protein(s).…”

“…Functional Na(HCO 3 ) n cotransport was first identified in the salamander Ambystoma tigritum kidney (2) and has since been documented functionally in numerous other cell types including pancreas (3)(4)(5)(6)(7)(8), colon (9), liver (10 -12), heart (13,14), retinal Mü eller cells (15,16), glial cells (17,18), parietal cells (19),and type II alveolar cells (20). Depending on the cell type, the stoichiometry of Na ϩ to HCO 3 Ϫ flux is 3:1, 2:1, or 1:1.…”

Neutral-Carrier-Based Ion-Selective Microelectrodes Design and Application

“…In certain cases, the pH microelectrodes were claimed to respond to C02. [46][47][48][49] As mentioned in ref. 1 (p. 211), the interference can Table 2 The electrode properties of the two types of neutralcarrier-based sensors mentioned above are quite similar, the membrane resistance corresponding to ca.…”