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. 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. Interestingly, ATP, in the same range (EC50= 8·6 μM), evoked AVP, but no significant OT, release from these terminals. 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. 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. 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. 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.

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.

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.

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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 ^m. 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 ftm^ 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 cell membrane at both ends, and that the attachment sites follow two (sets of) enantiomorphic helices. The sarcoplasmic reticulum is a longitudinally oriented 3-dimensional network of narrow tubules among the myofilaments. Its relative volume, estimated from cross sections, amounts to 0.9% of the contractile cytoplasm. No peripheral couplings have been observed, nor any tubular or vesicular invagination of the sarcolemma.

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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 Ca 2؉ will have only a transient stimulatory effect. The secretion-promoting targets for Ca 2؉ 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 ؉ ͞Ca 2؉ exchange or by transient fusion pore opening, is a proximal event in exocytosis. Ca 2ϩplays an essential role in several stimulatory, and perhaps also inhibitory, steps of regulated exocytosis but, despite much recent progress, its precise targets are still largely a matter of debate (1-3). In neurohypophysial secretion particularly, it is clear that Ca 2ϩ cannot be the ultimate and sufficient trigger for neurosecretion, because the obligatory depolarizationinduced increase in cytosolic Ca 2ϩ concentration stimulates release for only a few minutes (4, 5), even when depolarization is maintained. This behavior contrasts with the known but mechanistically obscure secretagogue effect of Na ϩ (6-8), wherein an artificially induced increase in cytosolic Na ϩ concentration can induce a sustained and continuous secretion (8). In previous work on neurohypophysial nerve endings, one of our laboratories reported that the Ca content of the secretory vesicles increased upon secretory stimulation, and that Ca-rich vesicles accumulated when secretion was blocked (9), suggesting a role of intravesicular ion concentration in the secretory mechanism. This hypothesis is further supported by observation that in insulin-secreting cells, Ca 2ϩ depletion from secretory granules inhibits exocytosis (10). Because the swelling of some granule matrices is inhibited by Ca 2ϩ and promoted by Na ϩ (11, 12), we have investigated in isolated and stimulated rat neurohypophysial nerve endings possible changes in the concentra...