Activation of group I metabotropic glutamate receptors elicits pH changes in cultured rat cortical glia and neurons.

Activation of metabotropic glutamate receptors is known to elicit a rise in intracellular Ca2+ and the present study was undertaken to see whether they also modulate the intracellular pH (pHi) of neurons and glia. Measurements of the pHi of neurons and astrocytes were made with the ratiometric fluorescent dye 2',7'-biscarboxyethyl-5,6-carboxyfluorescein. In the absence of bicarbonate, stimulation with the specific metabotropic glutamate receptor agonist 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid caused a fall in pHi in both astrocytes and neurons. In the presence of bicarbonate, stimulation with 25 microM 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid elicited a rise in pHi in the astrocytes, while the neurons responded with a small acidification. The astrocytic alkalinization could also be elicited by the specific group I metabotropic glutamate receptor agonist (S)-3-hydroxyphenylglycine but not by the group II agonist (2S,1'S,2'S)-(2-carboxycyclopropyl)glycine or by the group III agonist L(+)-2-amino-4-phosphonobutyric acid. The alkalinization of glial cells could be reduced by preloading the cells with BAPTA, but not by removal of extracellular Ca2+. Depolarization of the astrocytes with potassium elicited a small alkalinization, but stimulation with 100 microM 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid in high potassium medium elicited a further alkalinization. It is concluded that activation of group I metabotropic glutamate receptors leads to an alkalinization of astrocytes by a process that involves an elevation of intracellular Ca2+. The pHi changes that follow activation of the metabotropic glutamate receptors may play a role in initiation of glial proliferation following cerebral injury.

Characterization of an intracellular alkaline shift in rat astrocytes triggered by metabotropic glutamate receptors.

The modulation of intracellular pH by activation of metabotropic glutamate receptors was investigated in cultured and acutely dissociated rat astrocytes. One minute superfusion of 100 microM (1S,3R)-1-aminocyclopentane-1, 3-dicarboxcylic acid (ACPD) evoked an alkaline shift of 0.13 +/- 0. 013 (mean +/- SE) and 0.16 +/- 0.03 pH units in cultured (cortical or cerebellar) and acutely dissociated cortical astrocytes, respectively. Alkalinizations were elicited by concentrations of ACPD as low as 1 muM. The ACPD response was mimicked by S-3-hydroxyphenylglycine (3-HPG) and by (s)-4-carboxy-3-hydroxyphenylglycine (4C-3HPG) but was not blocked by alpha-methyl-4-carboxyphenylglycine (MCPG) or (RS)-1-aminoindan-1, 5-dicarboxcylic acid (AIDA), features consistent with an mGluR5 receptor-mediated mechanism. The ACPD-evoked alkaline shift was insensitive to amiloride, 4,4'-diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS), and the v-type ATPase inhibitors 7-chloro-4-nitrobenz-2-oxa-1,3-diazol (NBD-Cl), bafilomycin, and concanamycin. The alkaline response persisted in Na+- or Cl--free saline, but was reversibly blocked in bicarbonate-free, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solutions. A bicarbonate-dependent and Na+-independent alkaline shift could also be elicited by either 3 mM caffeine or 1 muM ionomycin. These data suggest that a rise in cytosolic Ca2+ activity is instrumental in triggering the alkalinizing mechanism and that this response is independent of the classic depolarization-induced alkalinization mediated by electrogenic sodium-bicarbonate cotransport.

On the role of bicarbonate as a hydrogen ion buffer in rat CNS neurones.

The role of bicarbonate as a hydrogen ion buffer has been investigated using the fluorescent dye BCECF in individual rat cerebellar, hippocampal and neocortical neurones maintained in culture. The steady-state intracellular pH (pHi) was estimated to be 7.07 +/- 0.05 (n = 22) in CO2-HCO3(-)-buffered media. Buffering power (beta) estimated from the addition and removal of weak bases was ca 10 mM (pH unit)-1 and was found to be similar in both CO2-HCO3(-)- and Hepes-buffered media. The membrane-permeant carbonic anhydrase inhibitor, acetazolamide (10-20 microM), did not affect estimates of beta. The results indicate that CO2-HCO3- does not act as an open buffer system in these neurones.

Intrinsic hydrogen ion buffering in rat CNS neurones maintained in culture.

The intrinsic proton buffering power (beta 1) of individual rat hippocampal and neocortical neurones maintained in culture has been investigated using the fluorescent dye 2', 7'-bis(carboxymethyl)-5, 6-(carboxyfluorescein) (BCECF). The steady-state intracellular pH (pH1) was estimated to be 7.03 +/- 0.04 (n = 22) in Hepes-buffered media and beta 1 estimated from the addition and removal of weak bases was ca 10 mM (pH unit)-1 at pH1 values near to 7. Estimates of beta 1 made from butyric acid challenges were inconsistent with estimates made at the same pH1, using NH4Cl withdrawal. However, estimating beta 1 with butyrate in the presence of the monocarboxylate ion transport inhibitor alpha-cyano-hydroxy-cinnamate (CHC) yielded beta 1 values commensurate with those measured using NH4Cl. Application of CHC alone lead to a rapid fall in pH1, suggesting a significant contribution of the monocarboxylate transporter to pH1 regulation. beta 1 was also estimated from a step increase in extracellular P(CO2). This yielded a value of 11 mM at an average pH1 of 7.1, which is similar to that of the other estimates reported here. beta 1 was found to increase with decreasing pH1: each unit drop in pH1 increased buffering power by about 60%. Blockade of pH1 regulation did not significantly affect estimates of beta 1. The change in buffering power with pH could be closely modelled from the known concentrations of free amino acids and organic phosphates.

Projet completion report : Groundwater exploration in southeast Zimbabwe using remote sensing and ground geophysical techniques

Regional and local scale investigations, involving remote sensing, ground geophysics and drilling, have carried out in southeast Zimbabwe aimed at increasing the understanding of crytalline, hard-rock aquifers and improving borehole siting procedures. New products derived from satellite imagery that simplify interpretation have been developed and are described. Detailed field studies at a small number of locations suggest that, whilst photolineaments, thought to represent rock fractures for the most part, are an important element in borehole targeting, they are not by themselves a wholly reliable. The role of ground geophysical surveys in site selection is described and evaluated. A hypothesis to describe the nature and role of fracturing in this region is presented, based on the evidence of the field test undertaken. A strategy for medium-term groundwater development in low-rainfall areas underlain by crystalline basement, such as southeast Zimbabwe, is proposed. It is suggested that more effort is put into establishing a network of high-yielding wells at strategic locations to supplement individual village supplies during periods of drought. The work summarised in this report was carried out under the ODA/BGS Research & Development Programme of technical assistance to the devloping countries. A full list of reports arising from the study is provided (AU)