Secondary Messengers in the Locomotor-Sensory System of Early Multicellulars. Inositol-Containing Compartments in Receptor Cells of Ctenophores and Coelenterates according to the Data of Cytochemical Investigation

The electron microscopy cytochemical data have been obtained on compartmentalization of a secondary messenger, inositol (1,4,5)-triphosphate (InsP₃), in receptor cells of different modalities in representatives of early multicellular animals, ctenophores Bero cucumis and coelenterates, scyphomedusas Cyanea arctica. In the gravitation mechanoreceptor cells of the ctenophores and medusas, the inositol-containing compartments were revealed predominantly in cilia and stereocilia. The presence of uniformly distributed discrete granules of the precipitate of the enzymatic cytochemical reaction product in the light-sensitive lamellar structures of photoreceptor cells of ctenophores is discussed in the connection with the possible existence of peculiar rhodopsin–inositol complexes participating in photoreceptor processes. The peculiarities of compartmentalization of the precipitate in photo- and gravitation mechanoreceptor cells allow considering InsP₃ as one of the components that that are able to contribute to development of mechanisms of modality in the ciliary receptor cells of ancient multicellular animals.     

Morphology and dynamic behavior of anal pores during defecation in the ctenophore Pleurobrachia pileus

Defecation in the lobate ctenophore Mnemiopsis leidyi was recently shown to occur periodically with an ultradian rhythm through a single transient anal pore which suddenly appeared, expelled waste, and disappeared afterward. To discover whether this novel method of defecation occurs in other kinds of ctenophores, I examined individuals of Pleurobrachia pileus and Beroe cucumis. Both ctenophores were found to have two identical and permanent anal pores as described in the scientific literature and textbooks. In P. pileus, both anal pores commonly participated in a defecation, but they did so asynchronously with independent and irregular opening and closing kinetics. Individuals of P. pileus defecated with an ultradian rhythm. Closed anal pores in P. pileus and B. cucumis consisted of a continuous ectodermal epithelium overlying a continuous endodermal epithelium with a cup-shaped group of thickened endodermal cells bearing a tuft of cilia which beats into the anal cavity. The rims of opening or closing pores were smooth and uniform without encircling muscles or fibers. This morphology and the continuity of the epithelial layers between defecations suggest that anal pores may not operate by a contractile sphincter, but by a reversible ring of tissue fusion between apposed ectodermal and endodermal epithelia to create an adjustable hole to expel waste.     

Ultrastructural identification of glial cells in the oral area of the comb-bearer <Emphasis Type="Italic">Berë cucumis</Emphasis>

In the electron microscopic study of oral epithelium and underlying areas of mesoglea of adult comb-bearers Berë cucumis (Ctenophora), there were described peculiar light cells with a characteristic localization, ultrastructure, ways of interaction with cells of other types including developing and mature chemoreceptors and neuronal processes. Comparison of the obtained results with the literature data allows identification of the light cells as glial cells, one of the first such cells in representatives of the first multicellular animals.Translated from Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, Vol. 40, No. 6, 2004, pp. 579–588.Original Russian Text Copyright © 2004 by Aronova, Alekseeva.     

Ökologische Untersuchungen anPleurobrachia pileus

Zusammenfassung 1. Die Populationsdynamik der tentaculaten CtenophorePleurobrachia pileus Fabr. 1780 wurde in den Jahren 1966 bis 1968 bei Helgoland untersucht.2. Die in der hydrographisch komplizierten Deutschen Bucht ablaufende Populationsdynamik konnte durch die punktförmige Probennahme und die sie ergänzenden Messungen nur annäherungsweise erfaßt werden.3. Der Jahresgang vonP. pileus zeichnet sich im Untersuchungsgebiet aus durch einen in allen Untersuchungsjahren übereinstimmenden Abundanzanstieg unter Zunahme junger Individuen von März bis Mai. Im Juni wurden die höchsten Abundanzwerte ermittelt; der Rückgang der Population erfolgt anschließend sehr schnell bis zum völligen Fehlen vonP. pileus in den Planktonfängen vor Helgoland.4. In den Jahren 1966 und 1968, alsPleurobrachia pileus eine Abundanz von etwa 10 Individuen pro m³ erreichte, fehlte sie im Spätsommer völlig. 1967, als ihre maximale Abundanz 1–2 Individuen pro m³ betrug, war sie im Spätsommer und Herbst regelmäßig bis häufig im Plankton vertreten.5. Der mittlere Körperdurchmesser der gefangenenP. pileus ist im Winter größer als im Sommer. Gegen Ende des Winters zeigen einzelne Individuen Reduktionserscheinungen an den Lokomotionsorganen.6. Die Tiefenverteilung vonP. pileus zeigt ganzjährig eine Präferenz der bodennahen Wasserschichten, die durch Seegangseinwirkungen anscheinend gefördert wird.7. Die Massenentwicklung vonP. pileus im Frühjahr folgt der Frühjahrsblüte des Phytoplanktons und dem daran gebundenen Auftreten von Copepoden und Evertebratenlarven.8. Der Populationsrückgang wird maßgeblich durchBeroe gracilis verursacht. Deren Population ist somit für die Populationsdynamik vonP. pileus der einflußreichste biotische Faktor.9. DaBeroe gracilis Nahrungsspezialist ist, bilden beide Arten ein Regelsystem, das bei hoher Abundanz vonP. pileus wirksam wird.10.Bolinopsis infundibulum undBeroe cucumis, die synchron im gleichen Gebiet als ökologisches Regelsystem verwandter Struktur vertreten sind, haben nur einen geringen direkten Einfluß auf die Populationsdynamik vonPleurobrachia pileus undBeroe gracilis.11. Die Konsequenzen der Abhängigkeit des Auftretens vonBeroe gracilis vonP. pileus werden diskutiert.

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Ecological investigtions onPleurobrachia pileus. 1. Field studies

The tentaculate ctenophorePleurobrachia pileus Fabr. belongs to the most abundant holoplanktonic zooplankters of the German Bight (North Sea). Its population dynamics have been studied from May 1966 to August 1968. Samples were taken mainly near the island Helgoland; hence the survey on population dynamics, which depend upon the complicated hydrographical conditions of the German Bight, is quite limited. Plankton samples were taken either as surface hauls in turbulent water, or as Hensen vertical hauls or horizontal hauls with the Knüppelnetz. In all three years the annual cycle ofP. pileus reveals a characteristic population increase from March to the end of May, followed by a steep population decrease. Maximum abundances varied from about 20 individuals per m³ to about 1 individual per m³. The population increase corresponds to the spring plankton bloom. The decrease is mainly due to the influence ofBeroe gracilis, whose population dynamics were also studied, as well as those ofBolinopsis infundibulum andBeroe cucumis. IfP. pileus andB. gracilis were abundant in spring,P. pileus could not be found during the subsequent summer, but reappeared in autumn and winter. In 1967, whenP. pileus andB. gracilis were less abundant, representatives could be caught throughout the following months. The population dynamics ofPleurobrachia pileus andBeroe gracilis, as well as ofBolinopsis infundibulum andBeroe cucumis, provide examples of ecological feedback systems.

     

Development of Chemoreceptor Cells in Oral Epithelium of Adult Jelly-Fish Beroë cucumis

In oral epithelium of adult jelly-fish Beroë cucumis, the main stages of differentiation of chemoreceptor cells replacing damaged degenerating chemoreceptors are described. The initial stages are connected with transformation of the so-called precursor cells that on transformation into juvenile cells, are inserted from mesoglea into basal epithelial areas. In the juvenile chemoreceptor cells, there continue ultrastructural transformations connected with differentiation of the apical sensory apparatus, central process, and innervation. Consecutive spatial translocations of these cells in epithelium are traced, up to their insertion into the superficial layer and replacement of degenerated chemoreceptors. The literature and the authors' own data about possible sources of origin and ways of utilization of damaged jelly-fish chemoreceptor cells are discussed.     

NADPH-Diaphorase in the Olfactory System of the Carp Cyprinus carpio

Ultrastructural distribution of NADPH-diaphorase (NADPH-d) in olfactory epithelium and bulb of the carp Cyprinus carpio L. was studied using light and electron microscopy. The diaphorase staining was revealed in the supranuclear area of the sensory and indifferent epithelium, in the olfactory nerve, as well as in the outer layers of the olfactory bulb—in fibers and glomeruli. NADPH-d-positive neurons were found in the interglomerular neuropil. Electron microscopy showed that NADPH-d in the olfactory lining epithelium was related only to receptor cells and ciliary supporting cells and was present in submembranous structures. Besides, in both parts of the olfactory system the main, cytosolic part of the enzyme is bound to cytoskeleton and is also present in membranes of endoplasmic reticulum and in mitochondria. In general, the NADPH-d of the carp olfactory system is characterized by predominantly intracellular localization and widespread contacts of the enzyme with cytosol.     

Renaissance of cytochemical localization of membrane ATPases in the myocardium

ATPases of cardiac cells are known to be among the most important enzymes to maintain the fluxes of vital cations by hydrolysis of the terminal high-energy phosphate of ATP. Biochemically the activities of Ca²⁺-pump ATPase, Ca²⁺/Mg²⁺-ecto ATPase, Na⁺,K⁺-ATPase and Mg²⁺-ATPase are determined in homogenates and isolated membranes as well as in myofibrillar and mitochondrial fractions of various purities. Such techniques permit estimation of enzyme activitiesin vitro under optimal conditions without precise enzyme topography. On the other hand, cytochemical methods demonstrate enzyme activityin situ, but not under optimal conditions. Until recently several cytochemical methods have been employed for each enzyme in order to protect its specific activity and precise localization but the results are difficult to interpret. To obtain more consistent data from biochemical and cytochemical point of view, we modified cytochemical methods in which unified conditions for each ATPase were used. The fixative solution (1% paraformaldehyde –0.2% glutaraldehyde in 0.1 M Tris Base buffer, pH 7.4), the same cationic concentrations of basic components in the incubation medium (0.1 M Tris Base, 2mM Pb(NO₂)₃, 5 mM MgSO₄, 5 mM ATP) and selective stimulators or inhibitors were employed. The results reveal improved localization of Ca²⁺-pump ATPase, Na⁺–K⁺ ATPase and Ca²⁺/Mg²⁺-ecto ATPase in the cardiac membrane.     

The cytochemical localization of nucleotide pyrophosphatase activity in plant tissues using naphthyl esters of thymidine-5''-phosphate.

Synopsis A cytochemical method for the localization of nucleotide pyrophosphatase activity in plants employing naphthol AS-BI thymidine 5-monophosphate and -naphthyl thymidine 5-monophosphate as specific substrates is reported. Biochemical evidence for the validity of this method is presented and the synthesis of the naphthol AS-BI ester is described.The application of this cytochemical technique to shoots ofTriticum sp. and roots ofVicia faba has shown nucleotide pyrophosphatase to be ubiquitous in its distribution in these organs and to occur in a structurally-bound form in the cytoplasm. The highest activity was detected in developing fibres adjacent to the leaf vascular bundles, in the coleoptile epidermal and hypodermal cells and in the coleoptile and leaf xylem.     

Localization of Phosphatase Responsible for Hydrolysis of Inositol 1,4,5-Triphosphate in the Olfactory Lining of True Sturgeons

The paper presents results of a cytochemical study of localization of phosphatase responsible for hydrolysis of inositol 1,4,5-triphosphate (ITP) in the olfactory lining of true sturgeons (the sturgeon, starred sturgeon, and sterlet). Reaction products as a dark discrete granules are localized in the apical parts of epithelium, practically in the same manner in all the species studied. The precipitate is found on the plasma membranes of cilia, microvilli, and clava of the olfactory cells. Occasionally, the precipitate is also found in the cilia, basal bodies, and rootlets of microvillar cells. The ITP-hydrolyzing phosphatase is supposed to restrict development of transduction process by removing excess messengers from the operating system. The data obtained indicate that in the true sturgeons, the phospholipase cascade of olfactory transduction is concentrated predominantly in the cilia and microvilli of olfactory cells.     

Primary inhibition: a mechanism for sudden stoppage of metachrony in ctenophores

The ctenophores Beroë cucumis and Pleurobrachia pileus exhibit rapid stoppage of comb plate beating, without accompanying muscular contraction, in response to an orally applied mechanical stimulus. This phenomenon, termed ‘primary inhibition’ by Göthlin, was re-examined by high-speed video microscopy and intracellular recording. The most remarkable features of this event were that (1) inhibition was associated with only one or two comb plates in the row, (2) inhibition occurred nearly anywhere in the ciliary beat cycle, (3) the inhibited plate acted as a mechanical blockade to the propagation of additional metachronal waves, and (4) a single depolarizing post-synaptic potential occurred nearly simultaneously with comb plate inhibition. B. cucumis and P. pileus have evolved a neurally controlled behavior to rapidly stop comb plate metachrony.

     

Ultrastructural localization of endogenous calcium in the teleost retina

Summary The ultrastructural localization of endogenous calcium in the retina of adult cichlid fishOreochromis mossambicus (Teleostei) was studied using the cytochemical osmiate-bichromate method of Probst (1986). The specificity of this method for calcium localization was proven by means of EGTA treatment of ultrathin sections and electronspectroscopic-imaging technique (ESI) with an energy-filtering transmission electron microscope (CEM 902, Zeiss). Large amounts of electron-dense calcium containing deposits were found in the outer segments of rods, in the synaptic vesicles of receptor terminals and bipolar cells, in the perinuclear space of photoreceptors and in the endoplasmic reticulum of different cell types, especially in the inner segment and fibres of photoreceptor cells. In the inner plexiform layer calcium was detected in the extracellular space with greater accumulations in the synaptic cleft. Principal differences in the localization of calcium between rods and cones and between several types of synapses and vesicles are shown. The possible role of calcium in the subcellular structures of retinal cells is discussed.     

Immunocytochemical identification and localization of lipase in cells of the mycelium of Penicillium cyclopium variety

The localization of lipase in cells of the fungus Penicillium cyclopium was investigated. It was shown by differential centrifugation of a homogenate of mycelial cells that the activity of the enzyme is associated with the cell wall. A study of ultrathin sections of mycelium fixed using the method of Zvyagintseva in an electron microscope showed that the final products of lipolytic activity of the enzyme is localized on the cell wall. Antibodies were raised against the purified A and B lipases from P. cyclopium and their specificity was assessed by enzyme-linked immunosorbent assay. The antibody preparation was used in cytochemical investigation by immunogold labelling. This study permits the localization of cell-bound lipase mainly in the cell wall and in the periplasmic space. The identity of the cell-bound lipase with one of the two extracellular lipases is also demonstrated.     

New methods for the demonstration of lysosomal hydrolases by the formation of osmium blacks

Summary Methods are described for the direct cytochemical demonstration of the enzymes nonspecific esterase and acid phosphatase based on synthetic substrates which initially deposit Hatchett's brown (cupric ferrocyanide, Cu₂Fe(CN)₆·7 H₂O) at their subcellular sites. The small amounts of Hatchett's brown deposited as a result of the enzyme's activity may be intensified by bridging to osmium through thiocarbohydrazide. Alternatively, even greater amplification of the sites of activity may be attained by utilizing the Hatchett's brown as a catalyst to effect the oxidative coupling of 3,3-diaminobenzidine resulting in the formation of an osmiophilic indamine-type polymer.One of the major advantages of this new approach is that it permits the study of acid hydrolase localization without lead in the incubation medium. Studies were performed with these methods having identical incubation media except for synthetic substrate in many different cell types and tissues. They verify a frequent nonlysosomal localization for acid phosphatase and the heterogeneity of lysosomes and lysosomal populations with respect to hydrolase content.These methods give information obtained by direct cytochemical observation an advantage not previously held, in comparison with information from cell-fractionation cytochemical or biochemical studies. Initial studies with these methods on many tissues reinforce previous suggestions of the involvement of acid hydrolases in extralysosomal sites in subcellulur anabolic processes.Supported by U.S.P.H.S. Grant DE-02668.Dr. Anderson's work was performed at the Department of Anatomy of the University of Chicago and was supported by Research Grant No. M 71-077C from the Population Council.     

The Olfactory System in the Hagfish Myxine glutinosa

The apical part of the olfactory epithelium in Myxine glutinosa was investigated by optical and electron microscopy. This part of the epithelium consists of supporting cells and two types of olfactory receptor cells, i.e., ciliated receptor cells and microvillous receptor cells. The olfactory cilia have a 9 + 0 pattern of the microtubules, occasionally with one pair of the doublets dislocated towards the center of the cilium. Giant cilia were observed. The supporting cells bear microvilli and are rich in tonofilaments. The supporting cells also have a secretory function, their secretion consisting mainly of acid mucopolysaccharides. An asymmetrical type of desmosome was found between the olfactory receptor cells and the supporting cells.     

A cytochemical and immunofluorescence study of endocrine cells in the gut of the ascidian Styela clava

Summary Strong secretin-like immunofluorescence has been demonstrated in endocrine-like cells from the gastric epithelium of Styela. These cells also stain with lead haematoxylin and exhibit a brilliant formaldehyde-induced fluorescence, but do not show any other cytochemical features characteristic of the mammalian APUD series. Tests with antisera to glucagon, gastrin and somatostatin all proved negative. In the oesophagus tests with all four antisera proved negative. The significance of these results is discussed in relation to the phylogeny of vertebrate gastro-intestinal hormones.     

Lithium Enhances Muscarinic Receptor–Stimulated CDP-Diacylglycerol Formation in Inositol-Depleted SK-N-SH Neuroblastoma Cells

Abstract: The psychotherapeutic action of Li⁺ in brain has been proposed to result from the depletion of cellular inositol secondary to its block of inositol monophosphatase. This action is thought to slow phosphoinositide resynthesis, thereby attenuating stimulated phosphoinositidase-mediated signal transduction in affected cells. In the present study, the effect of Li⁺ on muscarinic receptor–stimulated formation of the immediate precursor of phosphatidylinositol, CDP-diacylglycerol (CDP-DAG), has been examined in human SK-N-SH neuroblastoma cells that have been cultured under conditions that alter the cellular content of myo-inositol. Resting neuroblastoma cells, like brain cells in vivo, were found to concentrate inositol from the culture medium, achieving an intracellular level of 60.0 ± 4 nmol/mg of protein. The addition of carbachol to [³H]cytidine-prelabeled cells elicited a four- to fivefold increase in the accumulation of labeled CDP-DAG. This stimulated formation of [³H]CDP-DAG was completely blocked by the addition of 10 μM atropine, was not dependent on the presence of Li⁺, nor was it affected by co-incubation with myo-inositol. This result was in sharp contrast to findings in rat brain slices, in which carbachol-stimulated formation of [³H]CDP-DAG was potentiated ～ 10-fold by Li⁺ and substantially reduced by coincubation with inositol. The formation of [³H]CDP-DAG in labeled SK-N-SH cells by carbachol was both concentration and time dependent. The order of efficacy of muscarinic ligands in stimulating [³H]-CDP-DAG accumulation paralleled that established in these cells for inositol phosphate accumulation, i.e., carbachol ≥ oxotremorine-M > bethanecol ≥ arecoline > oxotremorine > pilocarpine. Extended culture of the SK-N-SH cells in an inositol-free chemically defined growth medium progressively reduced the intracellular inositol content to <5 nmol/mg of protein, a level comparable with that seen in cortical slices. In these inositol-depleted cells, Li⁺ potentiated carbachol-stimulated [³H]CDP-DAG formation, and this effect was completely reversed by coincubation with inositol (EC₅₀ 0.2 mM). The present study thus demonstrates, in the same cultured cell line, the effects of normal and reduced intracellular inositol levels on the ability of Li⁺ to attenuate phosphoinositide resynthesis, as inferred from [³H]CDP-DAG accumulation. The results indicate that Li⁺ can lead to a slowing of stimulated phosphoinositide turnover in neuroblastoma cells, provided that the intracellular inositol content has been significantly reduced.     

Substance P-Induced Reduction in the Initial Accumulation of Cytosolic myo-[3H]Inositol in Rat Parotid Acinar Cells Mediated by the NK1 Tachykinin Receptor

Abstract: Stimulation of rat parotid acinar cells by the tachykinin neurokinin (NK) 1 receptor agonist substance P (SP) resulted in a significant reduction in the initial accumulation of cytosolic myo-[³H]inositol. This effect was rapid, because a reduction of ～15% could be seen already at 30 s, with the maximal effect (～45%) being observed at 15 min. The response to SP stimulation Was temperature dependent, because at 4°C no reduction was found, jln addition, at 4°C, cytosolic myo-[³H]inositol represented only 10% of the labeled inositol accumulated at 37°C. The SP-induced reduct on in cytosolic ravo[³H]inositol accumulation was concentration dependent; the EC₅₀ obtained for SP was 5.8 ± 2.5 nM. Spantide [N Arg¹, D-Trp⁷⁹, Leu]SP), a SP antagonist, used at a concentration oif 10⁵ A/, gave a competitive shift of the dose-response curve to SP. Various tachykinins and their analogs were evaluated for their ability to reduce cytosolic mvo-[³H]inositol. [L-Pro⁹]SP and SP methyl ester, two highly selective agonists of NK1 receptors, reduced the initial accumulation of myo-H]inositol with EQo values of 2.3 and 67.0 nM, respectively. Long SP C-terminal fragments were more potent than shorter ones. SP N-terminal fragments and SP free acid were -without effect. [Pro⁷]NKB, a selective NKB analog, had no effect. The rank order of potency of mammalian tachykinins was SP > NKA > NKB. These findings and the close correlation between EC50 values and IC₅₀ values obtained in binding studies implicate the NK 1 receptor. In addition, stimulation of muscarinic receptors by carbachol alscp resulted in a reduction in level of cytosolic mjw-[³H]inositol, with this effect being reversed by atropine. Moreover, atropine was unable tjo alter the SP-induced reduction in cytosolic myo-[³H]inositol accumulation. Other neurotransmitters, such as glutamic acid, serotonin, chplecystokinin, neurotensin, bradykinin, and neuropeptide Y, were without effect on initial cytosolic myo-[³H]inositol accumulation. In conclusion, NK1 and muscarinic receptors seem to regulate the membrane transport of inositol in acinar cells of the rat parotid gland. Measurement of the initial accumulation of cytosolic myo-[³H]inositol in this tissue could profitably be adopted as a very simple, rapid, [sensitive, and specific biochemical procedure for screening the activity of potential agonists and antagonists at NK1 receptors.     

Light microscopic characterization of glycoconjugates in secretory cells of the carp (Cyprinus carpio) gill epithelium

Summary Secretory products of granular and mucous cells in the gill epithelium of the carp, Cyprinus carpio, were distinguished by their cytochemical reactions with peroxidase-labelled lectins and with the galactose oxidase (GO)-Schiff reagents. Secretory products of granular cells reacted with lectins from Triticum vulgaris (WGA), Arachis hypogaea (PNA), Dolichos biflorus (DBA), Glycine max (SAB), and Lotus tetragonolobus (LTA). They also reacted with GO-Schiff reagents. After sialic acid cleavage with HCl, new binding sites for DBA and SBA appeared, suggesting the terminal sequence sialic acid-N-acetylgalactosamine (SA-GalNAc) for the secretion of this cell type. In mucous cells, binding sites for WGA, DBA, and SBA and, after acid hydrolysis, binding sites for PNA and a positive GO-Schiff reaction were detected. The terminal trisaccharide sialic acid-galactose (1-3)-N-acetylgalactosamine (SA-Gal-GalNAc) is proposed for the secretion of mucuous cells. These cytochemical differences are discussed in light of the involvement of both cell types in fish mucus elaboration.     

Assessment of a strontium capture technique for cytochemical localization of potassium-dependent phosphatase in Malpighian tubules of Locusta

Abstract A strontium capture method, using p-nitrophenyl phosphate as substrate, was used to determine the subcellular localization of (Na⁺ + K⁺)-ATPase activity in Malpighian tubules of Locusta migratoria L. Ultrastructural studies revealed that (Na⁺ + K⁺)-ATPase activity was restricted to the basolateral plasma membranes with little evidence of activity associated with the apical microvilli. In contrast, alkaline phosphatase activity was specifically associated with the apical cell membrane. Biochemical assays of fixed and non-fixed tubule homogenates were used to evaluate the p-nitrophenyl phosphate-strontium procedure for localization of the phosphatase component of (Na⁺ + K⁺)-ATPase. No significant potassium-dependent, ouabain-sensitive p-nitrophenyl phosphatase activity was demonstrated in homogenates under conditions necessary for the cytochemical procedure, viz fixation, pH 9.0 and the presence of strontium. The significance of the biochemical results are discussed in relation to the validity of such cytochemical techniques for (Na⁺ + K⁺)-ATPase localization.