Ghislain Nicaise

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 μM range (EC50= 4·8 μM). This effect was observed in only ≈40 % of the terminals. 3 Interestingly, ATP, in the same range (EC50= 8·6 μM), 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‐γ‐S > α,β‐methylene‐ATP > ADP. The compounds adenosine, AMP, β,γ‐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.

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 (calciumloaded) 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.

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.

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.

GIANT SMOOTH MUSCLE FIBERS OF THE CTENOPHORE MNEMIOPSIS LEYDII: ULTRASTRUCTURAL STUDY OF IN SITU AND ISOLATED CELLS

The lobate ctenophore Mnemiopsis leydii possesses giant smooth muscle fibers grouped in two sagittal bundles. Functional isolated cells were obtained by an enzymatic digestion of mesoglea and epithelia.Each bundle is made of 30 to 50 multinucleated cylindrical cells which may reach 35 µm in diameter and 4 cm in length. The nuclei and non-contractile organelles (mitochondria, golgi, rough endoplasmic reticulum) are contained in a discontinuous axial core, surrounded by a thick sheath of myofilaments. Thin (actin) filaments, 5.9 nm in diameter, form irregular rosettes around the thick (myosin) filaments, 16.1 nm in diameter. An actin:myosin filament ratio of 7:2 and a myosin density of 249 filaments per µm2 were found in cross-sections of relaxed in situ cells. No dense bodies nor attachment plates were observed. From the coiled shape of contracted single cells and from the rearrangement of organelles in such coiled cells, we propose that myofilaments are organized in thin long myofibrils attached to the ce...

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.

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 ∼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.

Immunocytochemical analysis of rat vagus nerve by antibodies against glycogen phosphorylase isozymes

Glycogen is an endogenous store of glucose equivalents for energy metabolism in many tissues. The brain contains a significant amount of glycogen the role of which as an energy reserve is currently under debate. Apparently little is known concerning a possible role of glycogen in peripheral nerves. We have demonstrated immunocytochemically the presence of glycogen phosphorylase (GP), a key enzyme in glycogen metabolism, in large and small axons of the rat vagus nerve, but not in Schwann cells. Furthermore, the isozyme-specific antibodies applied detected only the presence of the brain isoform BB of GP, but not the muscle isoform MM. This is in agreement with the occurrence of solely the BB isoform in the few brain and spinal cord neurons that contain GP. In contrast, astroglial cells in brain and spinal cord have previously been shown to contain both isoforms. Since GP isozymes are regulated differentially, the expression of isoform BB may provide hints to possible functions of glycogen in the vagus nerve.

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

SummaryFreeze-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.

Intercellular Junctions in Ctenophore Integument

The ctenophoran integument consists basically of a single-layered epidermis which covers the entire body, including appendages such as the tentacular apparatus, the lobes and auricles, and Unes the stomodeal cavity (generally referred to as the gastric cavity). This integument is always devoid of any cuticle or hard secretion, but is permanently covered by a film of mucus. It rests on a gelatinous mesoglea, which is an unusual connective tissue devoid of collagen and elastin fibers (Franc et al., 1976), and harboring mesenchymal cells and numerous true muscle cells. The mesoglea may be considered as the internal milieu of the ctenophore; it is fed and oxygenated by a system of gastrovascular channels, which may be very elaborate in large species. Franc (1972) demonstrated that specialized ciliated cells, located in the canal walls and grouped in ciliated rosettes, regulated the relative ionic and osmotic composition of the mesoglea.