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G Nicaise

Publications and source records attributed to G Nicaise.

At least 19 recordsLinked to original sources
PubMed1999

Stimulus-secretion coupling in neurohypophysial nerve endings: a role for intravesicular sodium?

It is generally accepted that Ca is essentially involved in regulated secretion, but the role of this cation, as well as others such as Na, is not well understood. An illustrative example occurs in neurohypophysial secretion, where an experimentally induced increase in the cytosolic concentration of Na+ can induce continuous neuropeptide release. In contrast, an increase in cytosolic Ca2+ will have only a transient stimulatory effect. The secretion-promoting targets for Ca2+ are not known; they may be cytosolic, as is usually assumed, but they may also be intravesicular, especially in view of evidence that Ca-rich secretory vesicles are preferentially secreted. In the present work, we have investigated the movements of these cations into and out of secretory vesicles during stimulus-secretion coupling. Isolated rat neurohypophysial nerve endings were stimulated by potassium (55 mM) depolarization, and at 6 min (peak secretion) and 20 min after the onset of stimulation, the elemental content of individual secretory vesicles was measured by quantitative x-ray microanalysis. A depolarization-induced transient increase in intravesicular Na+ concentration was found to coincide with the onset of secretion. Moreover, only a predicted small fraction of peripheral vesicles-presumably the docked ones-were Na+-loaded. The low sulfur concentration of Na+-rich vesicles most likely resulted from vesicle swelling. The results suggest that high intravesicular Na+ concentrations in docked vesicles, occurring by Na+/Ca2+ exchange or by transient fusion pore opening, is a proximal event in exocytosis.

Animals↗
PubMed1998

ATP-evoked increases in [Ca2+]i and peptide release from rat isolated neurohypophysial terminals via a P2X2 purinoceptor.

1. The effect of externally applied ATP on cytosolic free Ca2+ concentration ([Ca2+]i) was tested in single isolated rat neurohypophysial nerve terminals by fura-2 imaging. The release of vasopressin (AVP) and oxytocin (OT) upon ATP stimulation was also studied from a population of terminals using specific radioimmunoassays. 2. ATP evoked a sustained [Ca2+]i increase, which was dose dependent in the 1-100 microM range (EC50 = 4.8 microM). This effect was observed in only approximately 40 % of the terminals. 3. Interestingly, ATP, in the same range (EC50 = 8.6 microM), evoked AVP, but no significant OT, release from these terminals. 4. Both the [Ca2+]i increase and AVP release induced by ATP were highly and reversibly inhibited by suramin, suggesting the involvement of a P2 purinergic receptor in the ATP-induced responses. Pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), another P2 purinergic receptor antagonist, strongly reduced the ATP-induced [Ca2+]i response. 5. To further characterize the receptor, different agonists were tested, with the following efficacy: ATP = 2-methylthio-ATP > ATP-gamma-S > alpha, beta-methylene-ATP > ADP. The compounds adenosine, AMP, beta, gamma-methylene-ATP and UTP were ineffective. 6. The ATP-dependent [Ca2+]i increase was dependent on extracellular Ca2+ concentration ([Ca2+]o). Fluorescence-quenching experiments with Mn2+ showed that externally applied ATP triggered a Mn2+ influx. The ATP-induced [Ca2+]i increase and AVP release were independent of and additive to a K+-induced response, in addition to being insensitive to Cd2+. The ATP-induced [Ca2+]i increase was strongly reduced in the presence of Gd3+. These results suggest that the observed [Ca2+]i increases were elicited by Ca2+ entry through a P2X channel receptor rather than via a voltage-dependent Ca2+ channel. 7. We propose that ATP, co-released with neuropeptides, could act as a paracrine-autocrine messenger, stimulating, via Ca2+ entry through a P2X2 receptor, the secretion of AVP, in particular, from neurohypophysial nerve terminals.

Adenosine Triphosphate↗
PubMed1998

Calcium-induced calcium increase in secretory vesicles of permeabilized rat neurohypophysial nerve terminals.

Digitonin-permeabilized isolated neurohypophysial nerve terminals are known to release their secretory vesicle content under calcium challenge. On this preparation, we monitored intra-organelle Ca2+ concentration using digital fluorescence microscopy of Fura-2. The superfusion of artificial intracellular solution containing 10 to 50 microM Ca2+ induced an intra-organelle [Ca2+] increase. Two major organelles are candidates for this increase: secretory vesicles and mitochondria. In an attempt to detect calcium changes in the vesicles, ruthenium red was used to impair mitochondrial calcium uptake. Part of the ruthenium red-insensitive intra-organelle [Ca2+] increase was abolished by raising sodium in the solution. Removing sodium boosted the intra-organelle [Ca2+] increase. These results taken together suggest the participation of Na/Ca exchange, known to exist in the membrane of these secretory vesicles. In addition to Na/Ca exchange, there would be at least another mechanism of vesicular calcium intake, as suggested by the partial inhibition of intra-organelle [Ca2+] increase obtained under acidic compartments: neutralization with NH4Cl. This mechanism remains to be defined. The main conclusion presented here, that an intravesicular [Ca2+] increase takes place at the rate of secretion, was predicted by the hypothesis that intravesicular Ca2+ changes would be involved in stimulus-secretion coupling.

Animals↗
PubMed1997

Calcium in secretory vesicles of neurohypophysial nerve endings: quantitative comparison by X-ray microanalysis of cryosectioned and freeze-substituted specimens.

The calcium content of individual secretory vesicles in rat neurohypophysial nerve endings was measured by quantitative electron probe X-ray microanalysis. Directly frozen control and potassium-depolarized isolated endings were analysed using two presumably equivalent preparative techniques: (1) freeze-substitution in presence of oxalic acid followed by sectioning of resin-embedded pellets; or (2) direct cryosectioning of the frozen pellets followed by freeze-drying in the column of the microscope. In the pellets of stimulated endings, both approaches revealed an increase in the calcium content of neurosecretory vesicles. This increase was statistically more significant in the specimens prepared by cryosectioning, probably because in this case the contribution of 'dead' nerve endings could be eliminated on the basis of excessive cytoplasmic sodium and chloride. The results demonstrate that an increase in cytosolic calcium can lead to an increase in intravesicular calcium, and that when this occurs, it occurs within a subpopulation of vesicles in a given nerve ending. In addition, measured intravesicular calcium was dispersed over a wide range of concentrations, as predicted by the hypothesis of intravesicular calcium priming. When the vesicular calcium content was averaged per nerve ending, a relatively wide distribution of concentrations was again observed, indicating that some nerve endings respond more strongly to the stimulation than others.

Animals↗
PubMed1996

Cytochemical localization of Ca(2+)-ATPases and demonstration of ATP-dependent calcium sequestration in giant smooth muscle fibres of Beroe.

A cytochemical analysis of the mechanisms underlying cytosolic calcium regulation was undertaken in the giant smooth muscle fibres of the marine invertebrate Beroe. The ability of the sarcoplasmic reticulum to accumulate Ca2+ was demonstrated on living skinned single cells. In the presence of oxalate, and physiological concentrations of Ca2+, calcium oxalate crystals were formed in the lumen of tubules and cisternae of the sarcoplasmic reticulum. The subcellular distribution of Ca(2+)-ATPase was studied with a cytochemical technique; a dense precipitate resulting from Ca(2+)-ATPase activity was found on the plasma membrane, on the membranes of tubules and cisternae of the sarcoplasmic reticulum, and in mitochondria.

Adenosine Triphosphate↗
PubMed1996

Cytochemical localization of ecto-ATPases in rat neurohypophysis.

We studied the distribution of Ca(2+)- or Mg(2+)-dependent ATPase activity in rat neurohypophysis using the lead cytochemical method of Ando et al. In electron microscopy, precipitates were found lining the outer surface of the plasma membrane surrounding nerve endings and pituicytes. These precipitates were believed to represent the activity of ecto-ATPases (as opposed to Ca pump ATPases) for the following reasons: there was equal activation by Ca2+ in the absence of Mg2+ or Mg2+ in the absence of Ca2+; the effects of the two ions were not additive; there was activation by ATP or GTP; and there was resistance to glutaraldehyde fixation, to high (10 mM) Ca2+ concentrations, and to various inhibitors such as NEM, vanadate, oligomycin, quercetin, p-chloromercuribenzoate, ouabain, and levamisole. Cytosolic activity observed in certain nerve endings in the same conditions of incubation but more sensitive to NEM is also described and discussed.

Adenosine Triphosphatases↗
PubMed1995

Calcium loading of secretory granules in stimulated neurohypophysial nerve endings.

The total calcium content of secretory granules, Cag, was evaluated in isolated neurohypophysial nerve endings. The Cag in the resting state, as measured by X-ray microanalysis, is relatively high with an average of 7.4 +/- 0.6 mmol/kg wet weight. Following a depolarizing potassium challenge, a subpopulation of granules with even higher Cag could be detected, dispersed over a wider range of concentrations (up to 70 mmol/kg wet weight). After subsequent rinsing in physiological saline, Cag decreased to control values. This could have resulted from Ca2+ extrusion, or from preferential secretion of calcium-enriched granules. Our data can be interpreted in favor of the second explanation since no decrease in Cag was observed when secretion was blocked by a hyperosmotic saline. The effect of hyperosmotic conditions on isolated nerve endings was further studied by monitoring free cytoplasmic Ca2+ with the calcium-sensitive dye Fura-2 and by conventional electron microscopy. It was demonstrated that hyperosmotic treatment alone did not increase basal cytosolic Ca2+ concentrations but did significantly reduce the potassium-induced cytosolic rise in Ca2+. Electron microscopy of nerve endings in hyperosmotic conditions showed numerous exocytotic figures at various stages. The observed changes in Cag are in accord with a published hypothesis which proposes that intragranular calcium is a significant variable in regulated secretion.

Animals↗
PubMed1992

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.

Animals↗
PubMed1992

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.

Animals↗
PubMed1991

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.

Animals↗
PubMed1991

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.

Animals↗
PubMed1989

Quantitative X-ray microanalysis of calcium in sea urchin eggs after quick-freezing and freeze-substitution. Validity of the method.

Freeze-substitution was used to study the distribution of calcium in sea urchin eggs, and the validity of the technique was assessed. We followed the fate of both total and exchangeable calcium of sea urchin eggs in two species (Paracentrotus lividus and Arbacia lixula) after the various treatments needed for freeze-substitution and embedding. We compared the calcium content either by X-ray microanalysis of Epon-embedded sections of freeze-substituted eggs (6.2 +/- 0.71 mmoles/kg of Epon-embedded tissue) or by flame spectrometry analysis of living eggs (32.3 +/- 1.30 nmoles/mg protein). After standardization of units, both values lead to similar total calcium content. We also measured the movements of 45Ca from prelabelled eggs. Exchangeable 45Ca as well as total calcium appeared unaffected by the preparative treatment for X-ray microanalysis. In conclusion, our preparative technique for X-ray microanalysis can be considered appropriate for our material and allows us to undertake a subcellular quantification of calcium in various organelles.

Animals↗
PubMed1989

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.

Acrylic Resins↗
PubMed1988

Increase in the calcium content of cardiac tissue after postfixation with osmium tetroxide.

The concentration of osmium has been measured by destructive chemical analysis in glutaraldehyde fixed heart tissue postfixed with osmium tetroxide and embedded in epoxy resin. After such treatment, the mean atomic number of the specimen (Z) is close to 10, which permits a quantitative analysis of calcium (Ca) by the continuum method, using Z2/A as a correcting factor (A: atomic weight). Wavelength-dispersive X-ray microanalysis has been used to determine the Ca concentration of frog cardiac tissue fixed in glutaraldehyde and embedded in resin. These measurements have been repeated on tissue postfixed in osmium tetroxide; contrary to expectations, the apparent Ca concentration is much higher in osmium treated than in nontreated tissue. However, this result is observed with OsO4 solutions prepared in glass, not with solutions prepared in plastic. It is shown by energy dispersive X-ray analysis of droplets that OsO4 solutions prepared in glass contain large amounts of calcium, potassium and silicon. Care must be taken in preparing OsO4 fixatives when the fixed tissues are to be subjected to X-ray microanalysis of such elements as Ca or Si.

Animals↗
PubMed1987

Quantitative X-ray microanalysis of calcium with the Camebax-TEM system in frozen, freeze-substituted and resin-embedded tissue sections. Application to molluscan glio-interstitial granules.

The relevance of the continuum method for a quantitative X-ray microanalysis of epon embedded tissue sections in the particular conditions offered by the Camebax-TEM system was tested and an improved model of specimen holder is proposed. The absolute calcium concentration [Ca] of membrane-bound intracellular glio-interstitial granules was determined by X-ray microanalysis in transmission electron microscopy of Mytilus retractor muscle. The Ca peak and background values were measured by the wavelength-dispersive spectrometer of the Camebax; the mass thickness of the section was recorded simultaneously with an added energy-dispersive detector. The tissue was frozen at approximately equal to 77 K in a mixture of liquid propane and butane, freeze-substituted in the presence of oxalic acid and embedded in epoxy resin. The calcium concentration of glio-interstitial granules can be as high as 180 mmol.kg-1 of epoxy-embedded tissue, with an average of 40 mmol.kg-1. The sampling of the data through repeated experiments is discussed and it is proposed that the cell would be the main level of variation. The Ca content of glio-interstitial granules is significantly lower in the tissues of animals submitted to high-potassium artificial sea-water for 10 min. This finding was predicted by the hypothesis that glio-interstitial tissue is a regulator of calcium concentration in extracellular spaces.

Animals↗
PubMed1984

Calcium pathway through a mineralizing epithelium in the crustacean Orchestia in pre-molt: ultrastructural cytochemistry and X-ray microanalysis.

During the pre-exuvial period of the terrestrial crustacean Orchestia, the calcium of the old cuticle is almost entirely reabsorbed and stored as calcareous concretions in the lumen of the midgut posterior caeca. The elaboration of these concretions is due to transport by the caecal epithelium. With ultrastructural cytochemistry controlled by X-ray microanalysis, it can be demonstrated that the main sites of ionized or ionizable calcium are the apical microvilli and an extracellular (lateral and basal) network of channels. Direct precipitating cytochemical methods, using potassium pyroantimonate or pyrophosphate, potassium oxalate or oxalic acid, sodium fluoride, sodium tungstate, and indirect substitution methods, using lead acetate or nitrate and cobalt nitrate were comparatively used. The results are interpreted in favour of the hypothesis of an extracellular transport pathway for calcium through the lateral smooth septate junctions, in conjunction with a more classical apical transport through the microvilli.

Animals↗