Novel role of calcium in exocytosis: mechanism of nematocyst discharge as shown by x-ray microanalysis.

Abstract

Mature nematocysts of the sea anemones Rhodactis rhodostoma and Anthopleura elegantissima contain a fluid that has a high concentration of solutes and is extraordinarily rich in calcium (ca. 500-0 mmol/kg wet weight); this contrasts with the surrounding cytoplasm which is rich in potassium but poor in calcium. The undischarged capsule is surrounded by a membrane that probably acts as a selective permeability barrier between the cytoplasm and the nematocyst fluid. During discharge the nematocyst moves to the surface of the nematocyte and comes into contact with the external sea water medium. Calcium, which may be bound to proteins in the undischarged state, is rapidly lost from the fluid; at the same time, sea water enters the capsule. In vitro experiments have already shown that calcium loss increases the osmotic pressure of the capsular fluid, causing an influx of water from the external medium; this influx appears to increase the hydrostatic pressure inside the capsule to the point that the thread everts explosively.Nematocysts are stinging organelles found in cnidarians; each one consists ofa closed capsule which contains an inverted harpoon-like thread that can be discharged explosively. Although the structural events during eversion of the thread have been described in detail (1), little is known about the mechanism of discharge (2-5). Recently, Lubbock and Amos (6) found that discharge could be initiated in vitro by the removal of bound calcium from the fluid inside the nematocyst; calcium removal increased the osmotic pressure of the nematocyst fluid, thus producing an influx of water from the external medium and an increase in the internal hydrostatic pressure of the capsule. It was notable that alterations in the permeability of the capsule wall to water did not seem to be involved in controlling discharge in vitro. At that time, the relevance of the in vitro experiments to nematocyst discharge in vivo was not clearly established because data were not available on the ionic changes occurring under natural conditions.In the present study, we examined, by x-ray microanalysis of frozen sections, the elemental composition of both excited and unexcited nematocytes in the sea anemones Rhodactis rhodostoma and Anthopleura elegantissima. Our results enable us to present a generalized model of the events that take place during excitation. MATERIALS AND METHODSMesenterial filaments from R. rhodostoma were placed in synthetic seawater (Tropic Marin Neu; specific gravity, 1.021) containing 16.8% (wt/wt) dextran (250,000 daltors; Sigma, clinical grade). To quench freeze, filaments with droplets of dextran/ sea water were placed on the truncated conical tips of copper pins and rapidly immersed in a well-stirred slush of (monochlorodifluoromethane, m.p. -181'C) (7-9). In certain cases, filaments mounted on copper pins were electrically stimulated <3 sec before instant quenching in order to induce nematocyst discharge. Acrorhagial tissue was obtained from A. elegantissima by removing inflated acrorhagi with ...

Electron and ion microprobe analysis of calcium distribution and transport in coral tissues

“…Furthermore, calcium concentration in the mesogloea of the oral epithelium approaches 18·mmol·kg -1 . These values are similar to those observed in settled larvae of Pocillopora damicornis been recorded at less than 5·mmol·kg -1 wet mass (reviewed by Gupta, 1993), although a concentration of 9·mmol·kg -1 wet mass has been reported for the cytoplasm of nematoblast cells in an anemone (Lubbock et al, 1981). Our values are considerably higher than the estimate of 2.8·mmol·l -1 derived from 45 Ca compartment analysis in Stylophora pistillata .…”

Occurrence, distribution, and localisation of metals in cnidarians

“…The latter theory received considerable support from the investigation of Lubbock et al (1981). This work was carried out by X-ray microanalysis of 1-2 m cryosections of tentacles and acrorhagi in both the hydrated and freeze-dried states.…”

Cloning and functional expression of voltage-gated ion channel subunits from cnidocytes of the Portuguese Man O'War Physalia physalis

“…One consequence of the fact that multiple cell types are involved in the regulation of discharge has been the challenge of localizing the site of action of some experimental manipulations. For example, several studies have concluded that voltage-gated Ca 2+ channels are involved in discharge (Lubbock et al, 1981;Gitter and Thurm, 1993;Gitter et al, 1994), and while it has been assumed that Ca 2+ channels play a direct role on the exocytotic event underlying discharge, the possibility that those channels are present at synapses within the nerve nets that innervate cnidocytes cannot be excluded. Distinguishing between these possibilities is compounded by the small size and diffuse nature of many of the cells in question, particularly the neurons.…”

“…Because of the complexity of the cell, and the fact that a cnidocyte can be used only once, their discharge is very tightly regulated to minimize what is likely to be the considerable energetic cost of replacement. Studies of the regulation of cnidocyte discharge have employed a variety of physiological (Gitter et al, 1994;Brinkmann et al, 1995;Purcell and Anderson, 1995;Salleo et al, 1996), structural (Lubbock et al, 1981;Westfall and Grimmelikhuijzen, 1993;Westfall, 2004) and histochemical approaches (Anderson et al, 2004;Kass-Simon and Scappaticci, Jr, 2004), using representatives of all cnidarian classes. Given that cnidocyte discharge is thought to be an exocytotic event, special attention (Lubbock et al, 1981;Gitter and Thurm, 1993;Gitter et al, 1994) has been given to the potential role of voltage-gated ionic currents, particularly Ca 2+ currents of the type that trigger exocytosis at synapses and exocrine cells (Sudhof, 2004).…”