Effect of cobalt ions on the metabolism of some volatile and polar compounds in the marine invertebrates Mytilus galloprovincialis and Actinia equina

The compositions of the volatile and polar fractions from two coexisting Black Sea invertebrates, the mussel Mytilus galloprovincialis and the beadlet anemone Actinia equina, were established. The main metabolites in the volatile fraction from the investigated animals appeared to be methyl esters of fatty acids and fatty aldehydes. In the polar fraction from both animals low concentrations of free acids and nitrogen-containing compounds were obtained. Free carbohydrates were in much higher concentrations in M. galloprovincialis than in A. equina. Some sterols, probably as polar conjugates, were identified mainly in A. equina. Significant changes among all compounds appeared after treatment of both invertebrates with two different concentrations of cobalt ions. The variety of changes in each invertebrate could be due to their different evolutionary status. The effect of cobalt ions was often stronger at medium cobalt-ion concentrations.     

海葵毒素多肽的分离和初步表征

经冻融法提取,丙酮沉淀,SP Sephadex C 25强离子交换和HPLC ODS C₁₈反相色谱层析,从海葵Actinia cari中提纯到两个新的毒素多肽,命名为RSAPⅠ和RSAPⅡ,经SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）和基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）鉴定RSAPⅠ和RSAPⅡ均为单一成分,相对分子质量分别为5 008和4 992.初步钠通道药理学实验表明,RSAPⅠ和RSAPⅡ对豚鼠心室肌细胞钠通道表现出活性,RSAPⅠ使钠离子内流增加,起开放作用;RSAPⅡ使钠离子内流减少,起阻滞作用.二者的圆二色谱均表现为100% β转角.     

Experimental habituation of aggression in the sea anemone Actinia equina

Behavioural plasticity in Actinia equina (L.) was examined in experimental contests using a range of pedal disc colour phenotypes, which characterize 3 known, ecologically distinct morphs. With repeated pairing of individuals in auto-phenotypic encounters, habituation was easily induced in the 2 mid-shore morphs, but was not obvious in the less aggressive, low-shore form. Subsequent pairing with a different partner revealed that anemones remained aggressive towards a novel opponent. Following novel contact, repairing of the dark red pedal phenotype with the original partner provided some evidence of retention of habituation to a previous opponent, and thus of a specific inducible memory.     

Etude histologique et ultrastructurale de la granulosa à certains stades de développement du follicule ovarien chez un Lézard: Lacerta vivipara Jacquin

Résumé La granulosa s'épaissit, sans apport extérieur de cellules, par division mitotique de petites cellules folliculaires. Les cellules piriformes proviennent de la transformation de certaines petites cellules, mais elles présentent de nombreuses ressemblances morphologiques et cytologiques avec de jeunes ovocytes. A l'exception de ces similitudes aucun autre argument ne permet actuellement d'attribuer une nature germinale initiale aux cellules piriformes. Le nucléole de ces cellules montre quelques particularités et une séparation nette entre ses constituants fibrillaire et granulaire. Le rôle physiologique des cellules piriformes reste à préciser, mais elles ne possèdent pas de canal et leur appareil de Golgi très développé n'a pas de rapport avec la production des granules incorporés par l'ovocyte. La dégénérescence de nombreuses cellules piriformes, dont certains aspects ultrastructuraux sont décrits, est une des causes de la réduction de la granulosa.

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Histological and ultrastructural study of the granulosa during certain developmental stages of the ovarian follicle of a lizard, Lacerta vivipara Jacquin

Summary The granulosa thickens by mitotic division of the small follicle cells, without any external contribution. The pyriform cells arise from the transformation of certain small cells and show many morphological and cytological similarities with young oocytes. In spite of this resemblance, there is no proof for the germinal nature of the pyriform cells. The nucleolus of these cells shows certain peculiarities, and a clear separation of fibrillar and granular components. The physiological significance of the pyriform cells remains to be determined, but they have no duct and their large Golgi apparatus has no relationship with the granules incorporated by the oocyte. The degeneration of many pyriform cells is one of the reasons for the reduction of the granulosa. Some ultrastructural features of this degenerative process are described.

Avec la collaboration technique de Mme M. Hubert.     

Etude ultrastructurale de l'innervation des glandes salivaires postérieures chez Octopus vulgaris

Résumé Le canal salivaire contient dans sa paroi deux types de nerfs, moteurs et sécréteurs. Le diamètre des fibres nerveuses est en moyenne plus faible dans les nerfs moteurs, où il diminue encore en direction des glandes, tandis qu'au contraire celui des fibres sécrétrices augmente, accentuant ainsi les différences morphologiques entre les deux ners. Ces différences de calibre entre les fibres sont en rapport avec le nombre plus ou moins élevé des neurites qu'elles comportent, et dont les mésaxones ont des formes variées. Les caractères cytologiques des cellules satellites et des axones des deux types de nerfs sont comparables. Les axones contiennent des vésicules de types morphologiques très divers dont la signification est discutée.

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Ultrastructural study of the innervation of the posterior salivary glands in Octopus vulgaris I. The nervous trunks of the salivary duct

Summary Two types of nerves, motor and secretory, are found in the wall of the salivary duct. The average diameter of the motor fibers is smaller and decreases still more towards the glands, while on the contrary it increases for the secretory fibers. The disparity of diameter between the two types of fibers is due to the different number of neurites they contain, embedded in satellite cells with mesaxons of various forms. Axons and satellite cells have similar cytological features in the two types of nerves. Vesicles of several morphological types occur in the axons; their significance is discussed.

     

The fine structure of granular amoebocytes from the gonads of the sea anemoneActinia fragacea (Cnidaria: Anthozoa)

Summary The structure of granular amoebocytes of the intertidal sea anemoneActinia fragacea (Cnidaria: Anthozoa) has been investigated using the electron microscope. Cells from the gonads of large, intact individuals were studied in most detail, but other regions of the anemone were also examined. The amoebocytes are cells of variable appearance which are widely distributed both in the mesogloea and in the epithelial cell layers. They contain numbers of characteristic dense granules, which may enclose spherical cores of greater or lesser electron density. They also contain rough endoplasmic reticulum, Golgi apparatus and a range of inclusions, some of which may have lysosomal origins. They may contain extensive deposits of glycogen, and usually smaller quantities of lipid droplets. They may take on a variety of forms, depending partly on their location within the various types of mesogloea and epithelia. The amoebocytes appear to be motile and phagocytic, and may also be involved in the storage and transport of glycogen. They are involved with gametogenesis, both during the development of the oocytes and spermatogenic cysts and during the resorption of degenerating gametes. Their possible role in the secretion or maintenance of the mesogloea remains uncertain. No evidence of amoebocytes differentiating into other cell types was obtained.     

Action de pesticides chez des insectes en fonction de leur etat physiologique: Etude de 2 insecticides et d'un melange d'herbicides chez les larves aquatiques de cloeon. (Ephemeropteres).

For the larvae of the ephemeron Cloeon, our studies of acute toxicity show that the degradation products of fenthion are more toxic than the insecticide itself.Doses of fenthion and lindane, responsible of a hight mortality, lead to a decrease in the moulting frequency of these larvae. A mixture of herbicides and degradation products of fenthion gave different results.

     

Ultrastructure de l'iléon de Blatella germanica L. (Dictyoptère)

Résumé L'iléon de Blatella germanica est un important segment d'accumulation minérale. Les nombreuses concrétions d'origine ergastoplasmique, contiennent du phosphore, du chlore, du calcium, du magnesium, du potassium et du fer dans un stroma glycoprotéique. La paroi de ce segment protodéal est constituée d'un type cellulaire unique caractérisé par la présence de feuillets apicaux et d'invaginations basales, différenciations membranaires décrites dans d'autres organes de transit, mais dont la coexistence constitue l'originalité de l'iléon. La signification physiologique de ce segment digestif est discutée.

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Ultrastructure of the ileum in Blatella germanica L. (Dictyoptera)Localisation, origin and composition of intracellular mineralized concretions

Summary The ileum of Blatella germanica is an important proctodeal segment of mineral accumulation. The numerous concretions, elaborated by the ergastoplasm, contain P, Cl, Ca, Mg, K and Fe in a glycoproteic matrix. The epithelium of this segment is composed by only one type of cells which are characterized by apical leaflets and basal infoldings. These membraneous differenciations have been already described in other transit organs, but their coexistence is typical of the ileum. The physiological significance of this digestive segment is discussed.

Avec la collaboration technique de Melle M. Oricco. Recherches effectuées dans le cadre de la RCP no 162 du C.N.R.S.     

Aspects ultrastructuraux des relations entre les couches folliculaires et l'ovocyte depuis la formation du follicule jusqu'au début de la vitellogenèse chez le Lézard Lacerta vivipara Jacquin

Résumé Des contacts étroits s'établissent d'abord entre la granulosa et la membrane plasmique de l'ovocyte. L'aire pellucide les sépare ensuite. L'ovocyte émet de nombreuses microvillosités digitiformes et des processus intenses de micropinocytose se produisent au niveau des invaginations ovocytaires. Des granules provenant de la thèque externe, passent dans les espaces intercellulaires de la granulosa, se répandent dans l'aire pellucide et envahissent les invaginations de l'ovocyte. Des vésicules tapissées, différenciées à partir de la membrane plasmique de ces invaginations, captent des granules et s'isolent dans le cytoplasme; elles perdent leur revêtement externe et donnent naissance à des sphères vitellines corticales. Le rôle attribué jusqu'alors aux cellules piriformes n'est pas confirmé. Le grand développement de l'appareil de Golgi dans ces cellules peut indiquer une fonction sécrétoire qui serait à l'origine du matériel amorphe périovocytaire.

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Ultrastructural aspects of the relationships between follicular layers and oocyte from the formation of the follicle to the beginning of vitellogenesis in the lizard Lacerta vivipara Jacquin

Summary First the granulosa cells establish close contact with the plasma membrane of the oocyte. Then, the zona pellucida separates them. The oocyte acquires numerous microvillosities, and an intensely active micropinocytosis is observed in the ovocytic invaginations. Granules originating in the theca externa pass through the intercellular spaces of the granulosa and invade the zona pellucida and the invaginations of the oocyte. At the level of these invaginations, coated vesicles incorporate granules and form granular vesicles without coating. These granular vesicles are transformed into cortical vitelline spheres. The nutritive function, generally assigned to the pyriform cells, is not confirmed in this study. The great development of the Golgi apparatus in these cells suggests a secretory function. Perhaps the pyriform cells secrete the amorphous component of the periovocytic space.

Avec la collaboration technique de Madame M. Hubert.     

Kunitz-type protease inhibitors from acrorhagi of three species of sea anemones

Sea anemones are rich in biologically active polypeptides such as toxins and protease inhibitors. These polypeptides have so far been isolated from whole bodies, tentacles or secreted mucus. Recently, two novel peptide toxins with crab lethality have been isolated from acrorhagi (specialized aggressive organs elaborated by only certain species of sea anemones belonging to the family Actiniidae) of Actinia equina. This prompted us to survey biologically active polypeptides in the acrorhagi of two species of sea anemones, Anthopleura aff. xanthogrammica and Anthopleura fuscoviridis. No potent crab lethality was displayed by the acrorhagial extracts of both species. However, significantly high protease inhibitory activity was instead detected in the acrorhagial extracts of the two species and also in that of A. equina. From the acrorhagi of A. equina, A. aff. xanthogrammica and A. fuscoviridis, one (AEAPI), one (AXAPI) and two (AFAPI-I and AFAPI-III) protease inhibitors were isolated, respectively. The complete amino acid sequences of the four inhibitors were elucidated by N-terminal sequencing and sequencing of the C-terminal peptide fragment produced upon asparaginylendopeptidase digestion. The determined amino acid sequences revealed that all the four inhibitors are new members of the Kunitz-type protease inhibitor family.     

Early embryogenesis in Tropaeolum majus L.: Diversification of plastids

Embryogeny in the nasturtium is characterized by the development of a large, tripartite suspensor and storing cotyledons. A light and electron microscopic study revealed an early diversification of the plastids in the various regions of the suspensor and the embryo proper. Amyloplasts are found in the developing cotyledons of the heart-like embryo, while chloroplasts occur within the meristematic part of the embryo and the adjacent portion of the suspensor. The cells between the meristem and the storing cotyledons display undifferentiated leukoplasts, whereas leukoplasts with an electron-dense matrix occur in the basal cell mass of the embryo-suspensor. Etioplasts develop in several cells of the placental haustorium of the suspensor. The carpel haustorium shows rather undifferentiated leukoplasts, which are transformed into electron-dense plastids during autolysis of the suspensor. This early plastidal differentiation in discussed with respect to its control and functional significance.     

Etude ultrastructurale des spermatogonies et de la croissance spermatocytaire chez Lithobius forficatus L. (Myriapode Chilopode)

Résumé L'étude ultrastructurale des spermatogonies et des spermatocytes a été envisagée chez Lithobius forficatus L. (Myriapode Chilopode). Les spermatogonies présentent un noyau à chromatine dispersée dont le nucléole est condensé. Leur cytoplasme renferme des ribosomes, libres ou associés en polysomes; les mitochondries et les dictyosomes sont peu abondants.Au cours de la croissance spermatocytaire, le noyau et le cytoplasme augmentent considérablement de volume. La dispersion de la chromatine est plus importante et le nucléole présente de nombreuses figures de bourgeonnement. Les ribosomes, les mitochondries, les dictyosomes et le reticulum endoplasmique sont très abondants. De nouvelles formations sont observables: 1) des amas de saccules réticulaires concentriques, délimitant une zone cytoplasmique; 2) des empilements lamellaires ayant leur origine soit dans le reticulum banal, soit dans les amas concentriques.L'activité synthétique se manifeste lors de la croissance spermatocytaire par: 1) d'abondants échanges nucléo-cytoplasmiques; 2) une augmentation du nombre de mitochondries et des ribosomes; 3) une intense activité golgienne. Elle est vraisemblablement en rapport avec le gigantisme spermatocytaire observé chez les Myriapodes Chilopodes.

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Ultrastructural study of spermatogonia and spermatocyte growth of Lithobius forficatus L. (Myriapoda, Chilopoda)

Summary The ultrastructure of spermatogonia and spermatocytes of Lithobius was studied. The spermatogonia show a nucleus with dispersed chromatin and a compact nucleolus. Their cytoplasm contains free ribosomes and polysomes; mitochondria and dictyosomes are not abundant.During spermatocyte growth, nucleus and cytoplasm increase markedly in volume. The dispersion of the chromatin is more pronounced; the nucleolus shows frequent budding. Ribosomes, mitochondria, dictyosomes and endoplasmic reticulum are very numerous. New structures are (1) accumulations of concentric reticular saccules with a cytoplasmic center, (2) piles of narrow cisternae originating from the regular endoplasmic reticulum or from concentric saccules.The synthetic activity during spermatocyte growth manifests itself by (1) numerous signs of nucleo-cytoplasmic exchanges, (2) an increase in the number of mitochondria and ribosomes, (3) a high activity of the Golgi apparatus. This is probably related to the gigantism of spermatocytes in Chilopoda.

     

Pore formation by the sea anemone cytolysin equinatoxin II in red blood cells and model lipid membranes

Summary The interaction ofActinia equina equinatoxin II (EqT-II) with human red blood cells (HRBC) and with model lipid membranes was studied. It was found that HRBC hemolysis by EqT-II is the result of a colloid-osmotic shock caused by the opening of toxin-induced ionic pores. In fact, hemolysis can be prevented by osmotic protectants of adequate size. The functional radius of the lesion was estimated to be about 1.1 nm. EqT-II increased also the permeability of calcein-loaded lipid vesicles comprised of different phospholipids. The rate of permeabilization rised when sphingomyelin was introduced into the vesicles, but it was also a function of the pH of the medium, optimum activity being between pH 8 and 9; at pH 10 the toxin became markedly less potent. From the dose-dependence of the permeabilization it was inferred that EqT-II increases membrane permeability by forming oligomeric channels comprising several copies of the cytolysin monomer. The existence of such oligomers was directly demonstrated by chemical cross-linking. Addition of EqT-II to one side of a planar lipid membrane (PLM) increases the conductivity of the film in discrete steps of defined amplitude indicating the formation of cation-selective channels. The conductance of the channel is consistent with the estimated size of the lesion formed in HRBC. High pH and sphingomyelin promoted the interaction even in this system. Chemical modification of lysine residues or carboxyl groups of this protein changed the conductance, the ion selectivity and the current-voltage characteristic of the pore, suggesting that both these groups were present in its lumen.     

Etude au microscope électronique de la neurohypophyse de Rana esculenta L.

Résumé L'éminence médiane et la pars nervosa de Rana esculenta diffèrent du point de vue de leur structure.L'éminence médiane se compose de 2 zones différentes: la zone externe placée près du lobe distal et la zone interne située sous l'épendyme. Dans la zone externe, on distingue, d'après la taille et la forme des grains de sécrétion, 5 types de terminaisons.1. des terminaisons avec de fins granules sphériques denses de 800 à 1000 Å de diamètre; 2. des terminaisons avec des granules de 1000 à 1200 Å de diamètre; 3. des terminaisons avec des grains de forme irrégulière de diamètre supérieur aux précédents (1200 à 1600 Å); 4. des terminaisons avec de volumineux grains denses sphériques d'environ 1200 à 1800 Å de diamètre; 5. un petit nombre de terminaisons ne contenant que des vésicules. Les terminaisons des catégories 3 et 4 sont probablement du type neurosécrétoire.La zone interne contient de nombreuses fibres neurosécrétrices. Elles sont de 2 types, l'une avec de gros granules (1600–2400 Å), l'autre avec des granules moins volumineux (1300–2000 Å). Des fibres non neurosécrétrices ont également été observées.Dans la pars nervosa, on rencontre deux types principaux de fibres neurosécrétrices, l'une avec des grains denses de 1600 à 2400 Å de diamètre, l'autre avec des grains moins denses d'environ 1300 à 2000 Å de diamètre. Dans la zone externe bordant la pars intermedia des fibres aminergiques avec de fines granulations ont été observées.

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Electron microscopic study of the neurohypophysis of Rana esculenta L.

Summary The median eminence and the pars nervosa of Rana esculenta have a different structure.The median eminence has 2 different zones: the outer zone situated near the pars distalis and the inner zone under the ependyme. In the outer zone there are, according to the size and the shape of the granules, 5 types of nerve terminals.1. Endings containing spherical fine dense granules of 800 to 1000 Å in diameter; 2. Endings with spherical granules from 1000 to 1200 Å in diameter; 3. Endings with granules of irregular shape which are bigger than the former (1200 to 1600 Å); 4. Endings with spherical dense granules of about 1200 to 1800 Å in diameter; 5. A few endings containing only clear vesicles. Type 3 and type 4 endings are probably neurosecretory.The inner zone contains numerous neurosecretory fibres. They are of two types: one with big granules (1600–2400 Å), the second with smaller granules (1300–2000 Å). Non-neurosecretory fibres have also been observed.The pars nervosa contains two principal types of neurosecretory fibres: one with dense granules of 1600 to 2400 Å in diameter, the other with lighter granules of about 1300 to 2000 Å. In the external zone lining the pars intermedia, aminergic fibres with fine granules have been observed.

Je tiens à exprimer mes vifs remerciements, à Monsieur le Professeur E. Follenius pour l'intérêt constant qu'il prend à ce travail. Je remercie également Madame R. O. Clauss, collaboratrice technique et Madame Schwoerer, photographe, pour leur aide précieuse.     

Secretory tapetum of Brassica oleracea L.: polarity and ultrastructural features

Summary The ultrastructure of the secretory, binucleate tapetum of Brassica oleracea in the micro spore mother cell (MMC) stage through to the mature pollen stage is reported. The tapetal cells differentiate as highly specialized cells whose development is involved in lipid accumulation in their final stage. They start breaking down just before anther dehiscence. Nuclei with dispersed chromatin, large nucleoli and many ribosomes in the cytoplasm characterize the tapetal cells. The wall-bearing tapetum phase ends at the tetrade stage. The dissolution of tapetal walls begins from the inner tangential wall oriented towards the loculus and proceeds gradually along the radial walls to the outer tangential one. The plasmodesmata transversing the radial walls between tapetal cells persist until the mature microspore, long after loss of the inner tangential wall. After wall dissolution, the tapetal protoplasts retain their integrity and position within the anther locule. The tapetal cell membrane is in direct contact with the exine of the microspores/pollen grains and forms tubular evaginations that increase its surface area and appear to be involved in the translocation of solutes from the tapetal cells to the microspores/ pollen grains. The tapetal cells exhibit a polarity expressed by spatial differentiation in the radial direction.     

The ultrastructure of the swimbladder of the toadfish,Opsanus tau L.

Summary The anterior chamber of the swimbladder of the toadfish Opsanus tau L. is lined by a single layer of columnar gas gland cells, cuboidal cells that resemble gas gland cells but are located outside of the gas gland region, and squamous cells. Multilamellar bodies are numerous in the gas gland cells and the cuboidal cells and are present in smaller numbers in the squamous cells. Capillaries lie in the lamina propria directly below the epithelial lining. A thick continuous muscularis mucosae and a submucosa consisting of tightly packed cells, cell processes, and connective tissue may contribute to the impermeability to gases of the wall of the anterior chamber.The posterior chamber of the swimbladder is lined by a single type of squamous epithelial cell. Multilamellar bodies were occasionally observed in these cells also. Other types of cells frequently form a partial second layer between the epithelial lining and the basement lamina. A thin muscularis mucosae lies directly below the basement lamina and the capillaries of the posterior chamber are located in the submucosa. The tunica externa is a layer of dense connective tissue that surrounds both the anterior and posterior chambers. Collagen fibrils in the form of tactoids are present in this layer.Part of this work was submitted by S.M.M. in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Biology Department, Boston University. S.M.M. is grateful for a National Science Foundation Traineeship.     

Cycle biologique de l'hirudinée rhynchobdelle Piscicola geometra L.

Observations carried out in nature and in laboratory conditions show that the life cycle of P. geometra includes three or four generations per year. The winter population is composed of much bigger leeches than the summer ones.

     

Observations on the ultrastructure ofFrankia sp. in root nodules ofDatisca cannabina L.

Summary The fine structures of the microsymbiont inside the root nodules ofDatisca cannabina have been studied by light, by transmission- and by scanning-electron microscopy. The endophyte is prokaryotic and actinomycetal in nature. The hyphae are septate and branched, diameter 0.3–0.5 m. The tips of hyphae are swollen to form electron-dense, clubshaped to filamentous vesicles, ranging in diameter: 0.4–1.4 m. The endophyte penetrates through walls of the cortial cells. The infected zone is kidney shaped and confined to one side of the acentric stele. The orientation of infection is reversed from other actinorhizae exceptCoriaria. The hyphae are near the host cell wall and vesicles are directed towards the central vacuole. Vesicles are aseptate and no collapsing of the vesicle cell wall (void area) has been observed. Vesicle clusters structures are globular with an opening at one side of the cluster. The host cell is multinucleate or contains a lobed nucleus. Groups of mitochondria are located in between the hyphae, suggesting a strong association between the host and the endophyte for energy supply and amino acid production. The consequences of the inability to separate the mitochondria from the vesicle clusters in nodule homogenates in physiological studies have been discussed.Isolated vesicles clusters showed dehydrogenase activity, indicated by the presence of formazan crystals, after incubation with NADH and NBT. Strongest reducing activity was found within the vesicles. The possible role of filamentous vesicles in nitrogen fixation has been discussed.     

Cytotoxicity of equinatoxin II from the sea anemoneActinia equina involves ion channel formation and an increase in intracellular calcium activity

Summary Equinatoxin Il is a 20-kDa basic protein isolated from the sea anemoneActinia equina. The aim of our work was to investigate the primary molecular basis for the cytotoxic effects of equinatoxin II in two model systems: single bovine lactotrophs and planar lipid bilayers. Previous work has shown that equinatoxin II produces rapid changes in cell morphology, which are dependent on external calcium. It has also been reported that addition of equinatoxin II increases membrane electrical conductance, which suggests that the cytotoxic action of equinatoxin II involves an increase in the permeability of membranes to Ca²⁺. Extensive changes in cytosolic Ca²⁺ activity are thought to invoke irreversible changes in cell physiology and morphology. In this paper, we show that morphological changes brought about by equinatoxin II in bovine lactotrophs are associated with a rapid rise in cytosolic Ca²⁺ activity, monitored with a fura-2 video imaging apparatus. Moreover, incorporation of equinatoxin II into planar lipid bilayers produces Ca²⁺ permeable ion channels. This suggests that the mode of equinatoxin II cytotoxicity involves the formation of cation (Ca²⁺) permeable channels in cell membranes.