Endogenous Na(+)-K+ (or NH4+)-2Cl- cotransport in Rana oocytes; anomalous effect of external NH4+ on pHi.

1. In Rana oocytes, measurements with chloride-sensitive microelectrodes show that the mean intracellular chloride activity (34.8 +/- 6.3 mM, n = 79) is three times higher than that expected for the passive distribution of chloride ions across the outer membrane (12.4 mM, mean membrane potential -43 +/- 8.8 mV, n = 79). 2. Reuptake of chloride into oocytes depleted by prolonged exposure to chloride-free saline takes place against the electrochemical gradient. 3. Chloride reuptake does not take place in sodium-free solution or in a sodium-substituted potassium-free solution. It is inhibited by bumetanide (10(-5) M) in the bathing medium. 4. The overall stoichiometry of the transport mechanism deduced from simultaneous measurements of intracellular sodium and chloride using ion-selective electrodes is 1Na+:1K+:2Cl-. 5. Ammonium ions substitute for potassium on the cotransporter. 6. In oocytes smaller than 0.9 mm in diameter, exposure to external ammonium causes an alkaline shift in intracellular pH as the NH3 enters and takes up H+ to form NH4+. We propose that chloride-dependent NH4+ transport contributes to the accumulation of NH4+ and causes the 'postexposure' acidification as the intracellular NH4+ releases H+ to form NH3 which is then lost from the cell. 7. In larger oocytes ammonium exposure produces a rapid reduction in pHi which may be explained in part by cotransport-mediated uptake of NH4+. Evidence is also provided for a second chloride-dependent NH4+ transport mechanism and a chloride-independent process.

The abundance of Aplysia gliagrana depends on Ca2+ and/or Na+ concentrations in sea water.

The glial cells surrounding the identified giant nerve cell bodies R2 or LP1 of Aplysia punctata were studied by quantitative electron microscopy. They contain specific, electron-dense but non-osmiophilic membrane-bound granules, approximately 0.3 microns in diameter, called gliagrana. Similar glial granules are more often found in marine than in freshwater molluscs, possibly because they represent a calcium store used to compensate excess Na+ in the extracellular milieu of marine species and to regulate perineuronal calcium concentration. In agreement with this hypothesis, the abundance of gliagrana (= number of glial granules per microns 2) is found to be higher in animals adapted to low Ca2+ artificial sea water than in animals kept in high Ca2+ (or low Na+) conditions. This finding is not observed after 1 week but after 2 weeks of adaptation.

The calcium loading of secretory granules. A possible key event in stimulus-secretion coupling.

The review focuses on calcium accumulation by secretory organelles. The observation that secretory granules contain variable and often important quantities of calcium (1-200 mM of total calcium) can be interpreted as a maturation index. A progressive loading with calcium would be permitted by a Ca2(+)-transport mechanism on the granular membrane and calcium-binding molecules in the granular core. The saturation of this store by the stimulus-induced calcium transient would permit in mature (calcium-loaded) granules the ionic crisis leading to exocytosis. The inside of secretory organelles being acidic, calcium influx into the granule can be driven by calcium-proton exchange. The calcium-proton exchanger could be a Ca2(+)-ATPase.

Ca(2+)-ATPase and Mg(2+)-ATPase in Aplysia glial and interstitial cells: an EM cytochemical study.

The localization of Ca(2+)- and Mg(2+)-ATPases was determined in Aplysia central and peripheral nervous system, using an electron microscopic cytochemical method. The enzyme activity appeared localized to the membrane of glial granules (gliagrana), particularly in the peripheral nervous system of the esophagus, and on the plasma membrane of central glial cells adjacent to neuronal cell bodies. No calcium- and/or magnesium-ATPase activity was detectable on the plasma membrane of glial cells surrounding nerve axons in the pleuro-visceral connectives. These findings are discussed along two main lines: (a) the calcium-ATPase of the gliagrana coincides with a high intragranular calcium and/or proton concentration; and (b) the presence of a calcium-ATPase activity at the glio-neuronal interface around the neuronal cell bodies coincides with the use of calcium ions as charge carriers of the action potential, and its absence at the level of the axon with the concurrent functional use of sodium ions.

The Desheathed Periphery of Aplysia Giant Neuron. Fine Structure and Measurement of [Ca2+]o Fluctuations with Calcium-selective Microelectrodes.

The visceral ganglion of Aplysia was mechanically desheathed after protease softening of the connective tissue to permit the positioning of ion-selective electrodes in the vicinity of the neuronal membrane. The effects of this treatment on satellite glia and neuronal cytology were observed by electron microscopy. The intracellular alterations were not suggestive of serious membrane damage but the cohesion between glial and neuronal membranes was affected-the glial processes appeared to retract from the trophospongium and in some cases the neuronal membrane was completely naked. The external calcium activity [Ca2+]o at the surface of identified giant neuron, R2, was measured using double-barrelled calcium-selective microelectrodes. A decrease of approximately 1 mM in [Ca2+]o could be recorded only during trains of action potentials induced by intracellular depolarizing current injection, and when the electrode was pushed firmly against the neuron surface. A recovery from this decrease in [Ca2+]o could sometimes, but not always, be observed during the phase of induced neuronal activity.

The lacunar glial zone at the periphery of Aplysia giant neuron: volume of extracellular space and total calcium content of gliagrana.

The relative volume of perineuronal extracellular space, the number of gliagrana and their total calcium content have been measured in Aplysia punctata and A. californica, at the periphery of giant neurons R2 and LP1. After chemical fixation, the extracellular space amounts to 26% of the periganglionic glial zone, but this increases to 36% after quick freezing and freeze-substitution. The glial cytoplasm contains gliagrana, membrane-bound granules approximately 0.3 micron in diameter. The number of gliagrana per micron 2 of section, defined as "abundance", was counted in electron micrographs of chemically fixed tissues. The abundance of gliagrana appears to be directly proportional to the volume of the extracellular space when the values are averaged per individual Aplysia. The total calcium concentration of the gliagrana is measured by X-ray microanalysis on sections of ganglia processed by rapid freezing and freeze-substitution in the presence of oxalic acid: it was found to be very high. An individual granule may contain 100 mM Ca in A. californica and 50 mM in A. punctata but in both species the calcium concentration varies along a wide range as if there were different functional states of the granules with respect to this concentration. The total calcium stored in the specific granules of the glial zone was estimated. It was calculated that should the glial calcium store be entirely diluted in the extracellular space of the glial zone, it would raise the calcium concentration of this space by approximately 1 mM (0.1-2.7 mM). These findings are discussed with regard to the hypothesis of glial cells regulating the perineuronal calcium concentration.

X-ray microanalysis of calcium containing organelles in resin embedded tissue.

The localization of calcium in cell organelles at the electron microscope level is often achieved through cytochemical techniques, and verified by X-ray microanalysis. Various methods have been used to cytochemically detect calcium or calcium-binding sites: calcium loading, calcium substitution by strontium, barium, or even lead, and calcium precipitation by oxalate, phosphate, fluoride, or pyroantimonate. Their results may have heuristic value, particularly in preliminary studies of poorly known cell types. A complementary and more physiological approach is offered by quantitative measurement of the total calcium content of organelles after cryofixation. Resin embedding is less demanding than cryomicrotomy and gives better images: it can be used after cryosubstitution in the presence of oxalic acid. This technique was tested, and applied to several cell types.