Ciliary/decarboxylase activities as indicators of PTZ,photoperiod and Ca-ionophore effect on Crassostrea virginica (gmelin)

• 1.1. Lateral ciliary activity and DOPA decarboxylase were measured in the ctenidium of Crassostrea virginica (Gmelin).
• 2.2. Activity of the lateral cilia is dependent upon branchial nerve (Paparo, 1985a,b) and on intracellular calcium homostasis (Baker, 1963; Rassmussen, 1970, 1971; Romero and Wittman, 1971; Blanstein et al., 1978).
• 3.3. PTZ induced lamella morphogenesis in eytosomes with subsequent release of calcium into the cytosol. This cilio-inhibition was enhanced in the presence of additional calcium in the perfusate.
• 4.4. Prolonged exposure to light also induces fully converted membranous eytosomes with subsequent production of a gradual lateral cilio-inhibition. Darkness produces the opposite effect, in that secondary membranous conversions of cytosomes are inhibited.
• 5.5. In the presence of A-23187 (a calcium releasing agent), inhibition of lateral activity is produced, independent of cytosomal conversion.
• 6.6. It is postulated that photic electrical and chemical stimulation of neuronal chromoproteins can lead to release of calcium from sequestered cytosomal stores which triggers a neuro-exocytosis of a neuroinhibitory transmitter, dopamine.

     

The relationship between lamellar-type cytosomes,DOPA decarboxylase,and lateral ciliary activity of the oyster Crassostrea virginica (Gmelin): Response to salinity change

Lamellar transformations of cytosomes, DOPA decarboxylase and lateral ciliary activity were measured in neuronal components and/or ctenidia of the oyster, Crassostrea virginica (Gmelin). Prolonged exposure to light, anoxic conditions, EGTA (a calcium chelator), and exogenous perfusion of dopamine (a cilioinhibitory neurotransmitter) increases the frequency of lamellar type cytosomes. This is accompanied by decreases in enzymatic activity and a lateral cilioinhibition. Prolonged periods of darkness, electrical stimulation of the branchial nerve and exogenous perfusion of serotonin (a cilioexcitatory neurotransmitter) reduces the conversion of lamellar type configurations, increases enzymatic activity and produces a lateral cilioexcitation. Salinity changes that produce an increase to higher acclimation values result in a gradual decrease in the number of lamellar configurations with a subsequent increase in enzymatic activity and lateral cilioexcitation. The magnitude of the lateral cilioexcitation-cilioinhibition and decarboxylase activity is directly related to the number of lamellar conversions.     

Transient elevations in intracellular calcium are sufficient to induce sustained responsiveness to the neurotrophic factor bFGF

The present study investigates how a neuron's past history of neural activity may alter its responsiveness to subsequent signals. We demonstrate that a depolarizing pulse of extracellular potassium can prime neurons to become responsive to basic fibroblast growth factor (bFGF), even when the pulse is brief and occurs prior to addition of bFGF. Specifically, we subjected cultured embryonic chick ciliary ganglion neurons (E7) to a short pulse of elevated extracellular potassium followed by addition of bFGF and tested the effect of such treatment on neuronal survival. Neurons treated in this manner produced high levels of survival, whereas neurons exposed to either the pulse alone or the continuous presence of bFGF alone failed to promote any significant levels of survival. This priming effect of depolarization on bFGF-induced survival was blocked by calcium channel antagonists. To test the time dependency of this effect, we increased the time interval between termination of the calcium pulse and addition of bFGF. Our results demonstrate that a brief elevation in intracellular calcium has long lasting effects, up to 8 h after cessation of the depolarizing pulse, on neuronal responsiveness to bFGF. These findings suggest how a developing neuron's history of activity can alter its subsequent ability to respond to neurotrophic factors and has significant implications on the mechanisms by which activity may influence neuronal survival. © 1996 John Wiley & Sons, Inc.     

Specific localization of scallop gill epithelial calmodulin in cilia

Calmodulin has been isolated and characterized from the gill of the bay scallop aequipecten irradians. Quantitative electrophoretic analysis of epithelial cell fractions show most of the calmodulin to be localized in the cilia, specifically in the detergent- solubilized membrane-matrix fraction. Calmodulin represents 2.2 +/- 0.3 percent of the membrane-matrix protein or 0.41 +/- 0.5 percent of the total ciliary protein. Its concentration is at least 10(-4) M if distributed uniformly within the matrix. Extraction in the presence of calcium suggests that the calmodulin is not bound to the axoneme proper. The ciliary protein is identified as a calmodulin on the basis of its calcium- dependent binding to a fluphenazine-sepharose affinity column and its comigration with bovine brain calmodulin on alkaline-urea and SDS polyacrylamide gels in both the presence and absence of calcium. Scallop ciliary calmodulin activates bovine brain phosphodiesterase to the same extent as bovine brain and chicken gizzard calmodulins. Containing trimethyllysine and lacking cysteine and tryptophan, the amino acid composition of gill calmodulin is typical of known calmodulins, except that it is relatively high in serine and low in methionine. Its composition is less acidic than other calmodulins, in agreement with an observed isoelectric point approximately 0.2 units higher than that of bovine brain. Comparative tryptic peptide mapping of scallop gill ciliary and bovine brain calmodulins indicates coincidence of over 75 percent of the major peptides, but at least two major peptides in each show no near-equivalency. Preliminary results using ATP-reactivated gill cell models show no effect of calcium at micromolar levels on ciliary beat or directionality of the lateral cilia, the cilia which constitute the vast majority of those isolated. However, ciliary arrest will occur at calcium levels more than 150 muM. Because calmodulin usually functions in the micromolar range, its role in this system is unclear. Scallop gill ciliary calmodulin may be involved in the direct regulation of dyneintubule sliding, or it may serve some coupled calcium transport function. At the concentration in which it is found, it must also at least act as a calcium buffer.     

Calcium signals and the in vitro migration of chick ciliary ganglion cells

We have studied calcium signals and their role in the migration of neuronal and nonneuronal cells of embryonic chick ciliary ganglion (CG). In vitro, neurons migrate in association with nonneuronal cells to form cellular aggregates. Changes in the modulus of the velocity of the neuron-nonneuronal cell complex were observed in response to treatments that increased or decreased intracellular calcium concentration. In addition, both cell types generated spontaneous calcium activity that was abolished by removal of extracellular calcium. Calcium signals in neurons could be characterized as either spikes or waves. Neuronal spikes were found to be related to action potential generation whereas neuronal waves were due to voltage-independent calcium influx. Nonneuronal cells generated calcium oscillations that were dependent on calcium release from intracellular stores and on voltage-independent calcium influx. Application of thimerosal, a compound that stimulates calcium mobilization from internal stores, increased: (1) the amplitude of spontaneous nonneuronal oscillations; (2) the area of migrating nonneuronal cells; and (3) the velocity of the neuronal-nonneuronal cell complex. We conclude that CG cell migration is a calcium dependent process and that nonneuronal cell calcium oscillations play a key role in the modulation of velocity.     

Apical sensory organ in larvae of the patellogastropod Tectura scutum

The apical sensory organ in veliger larvae of a patellogastropod, a basal clade of gastropod molluscs, was studied using ultrastructural and immunohistochemical techniques. Immediately before veligers of Tectura scutum undergo ontogenetic torsion, the apical sensory organ consists of three large cells that generate a very long apical ciliary tuft, two cells that generate a bilateral pair of shorter ciliary tufts, and a neural ganglion (apical ganglion). Putative sensory neurons forming the ganglion give rise to dendrites that extend to the apical surface of the larva and to basal neurites that contribute to a neuropil. The ganglion includes only one ampullary neuron, a distinctive neuronal type found in the apical ganglion of other gastropod veligers. Serotonin immunoreactivity is expressed by a medial and two lateral neurons, all having an apical dendrite, and also by neurites within the neuropil and by peripheral neurites that run beneath the ciliated prototrochal cells that power larval swimming. The three cells generating the long apical ciliary tuft are lost soon after ontogenetic torsion, and the medial serotonergic cell stops expressing serotonin antigenicity in late-stage veligers. The lateral ciliary tuft cells of T. scutum may be homologs of lateral ciliary tuft cells in planktotrophic opisthobranch veligers. A tripartite arrangement of sensory dendrites, as described previously for veligers of other gastropod clades, can be recognized in T. scutum after loss of the apical ciliary tuft cells.     

The calcium-dependent neuronal release of serotonin and its antagonism by lithium

The cilio-excitatory serotonergic innervation of lateral gill cilia of Mytilus edulis was studied in vivo. Peripheral serotonin release was dependent on the external calcium concentration. Serotonin release was inhibited by autodialyzing calcium from the tissue or by increasing the calcium concentration in the medium, as determined by measuring ciliary activity stroboscopically and by biochemical and radioassays of serotonin. Lithium also inhibited serotonin release when added to the external bathing medium. Concomitantly, altering calcium concentrations altered the degree of inhibition of serotonin release caused by lithium. The study demonstrates that the terminal release of the monoamine serotonin is a calcium-dependent mechanism. The pharmacological effects of lithium in this system appear to be interrelated with the calcium-dependent releasing mechanism.     

Cholinergic neuronotrophic factors: III. Developmental increase of trophic activity for chick embryo ciliary ganglion neurons in their intraocular target tissues

Between stages 34 and 40 in the chick embryo, the ciliary ganglion (CG) undergoes a 50% loss of neurons. Such neuronal death is a common feature in neural development and it has been proposed that neurons are dependent for survival on trophic support from their target tissues. Using an in vitro bioassay it was previously shown in this laboratory that trophic activity for CG neurons is highly concentrated in eye structures containing CG target tissues. In the present study we have found that trophic activity in the eye increases markedly between stages 37 and 39, the time when neuronal death in the ciliary ganglion is ending. Thus, a developmental increase in trophic activity within the eye may be involved in determining neuronal survival in the CG. Furthermore, this study provides the first indication that the trophic content of target tissue is itself developmentally regulated.     

Ciliary Neuronotrophic Factor Stimulates Choline Acetyltransferase Activity in Cultured Chicken Retina Neurons

It has been demonstrated that cultured cholinergic retinal neurons from 8-day-old chicken embryos respond to a polypeptide factor present in retinal cell-conditioned medium (RCM) and in retinal extracts. Compared with control cultures, the activity of acetyl-CoA:choline O-acetyltransferase (EC 2.3.1.6; ChAT) is enhanced more than twofold in neuronal retinal cultures grown for 7 days in the presence of RCM. The present study demonstrates that both ciliary neuronotrophic factor (CNTF), which is characterized by its trophic activity on parasympathetic ciliary neurons, and RCM exhibit identical stimulatory effects on ChAT activity in retinal monolayer cultures. Similarly, RCM supports the in vitro survival of ciliary neurons to the same extent as CNTF. The active species in RCM has a molecular weight (20,900 +/- 1,000) identical to that of CNTF, as determined by preparative sodium dodecyl sulfate gel electrophoresis. The results indicate that cholinergic retinal neurons represent a central neuronal target for CNTF or a closely related protein.     

The effects of photoperiod,A-23187 and PTZ on the habitat salinity adaptation of Crassostrea virginica (Gmelin)

• 1.1. Electrical stimulation and DOPA decarboxylase activities were studied in Crassostrea virginica maintained at 30‰ habitat salinity.
• 2.2. Exposure to light significantly reduced the effectiveness of the electrical stimulation and 30‰ habitat acclimation. However, periods of exposure to darkness had the opposite result. Recovery to the 30‰ habitat ctenidial ciliary rate was significantly faster (more than 2 × ) in animals dissected and then maintained in darkness during the acclimation period.
• 3.3. Acclimation time of dark-dissected ctenidial preparation was significantly increased in the presence of A-23187 (a calcium cell membrane pore facilitator) or PTZ (a cytosomal calcium releasor). The latter treatment exhibited only about a 65% recovery to the control basal rate of beating.
• 4.4. This study elucidates a cytosomal role in the acclimation process via a neuronal regulatory mechanism controlling ciliary activity on the ctenidium. Cytosomes could conceivably furnish an extension of the transport activity of the plasma membrane to bring about a sophisticated microscopic control of solute (i.e. calcium) homoeostasis in the cytosol.

     

Ciliary sieving and active ciliary response in capture of particles by suspension-feeding brachiopod larvae

Larvae of a brachiopod, Glottidia pyramidata, used at least two ciliary mechanisms to capture algal cells upstream from the lateral band of cilia that produces a feeding/swimming current. (1) Filtration: the larvae retained algal cells on the upstream (frontal) side of a sieve composed of a row of stationary laterofrontal cilia. Movement of the laterofrontal cilia could not be observed during capture or rejection of particles, but the laterofrontal cilia can bend toward the beating lateral cilia, a possible mechanism for releasing rejected particles from the ciliary sieve. (2) Localized changes of ciliary beat: the larvae may also concentrate particles by a local change in beat of lateral cilia in response to particles. The evidence is that the beat of lateral cilia changed coincident with captures of algal cells and that captured particles moved on paths consistent with a current redirected toward the frontal side of the tentacle by an induced local reversal of the lateral cilia. The change of beat of lateral cilia could have been an arrest rather than a reversal of ciliary beat, however. The similar ciliary bands in adult and larval lophophorates (brachiopods, phoronids, and bryozoans) suggest that these animals share a range of ciliary behaviours. The divergent accounts of ciliary feeding of lophophorates could be mostly the result of different authors observing different aspects of ciliary feeding.     

Ciliary beating in three dimensions: Steps of a quantitative description

We document a novel approach for quantitative assessment of ciliary activity, exemplified in rapid three-dimensional cyclic motion of the frontal cirri of Stylonychia. Cells held under voltage-clamp control are stimulated by step pulses to elicit reproducible hyperpolarization- or depolarization-induced ciliary motor responses. High-speed video recording at 200 fields per second is used for imaging ciliary organelles of the same cell in two perspectives: the axial view and, following cell rotation by 90 degrees, the lateral view. From video sequences of typically 1 s, the contours of the cirral images are determined and digitized. Computer programs are established to (1) reduce an observed image to a "ciliary axis", (2) sort series of axes by template to generate an averaged ciliary cycle in 2D-projection, and (3) to associate the generalized axial and lateral 2D-images for generation of a sequence of three-dimensional images, which quantitatively represent the cycle in space and time. The method allows us to produce predetermined perspectives of images selected from the ciliary cycle, and to generate stereo views for graphical representation of ciliary motion. The approach includes a potential for extraction of the complete microtubular sliding program of a cilium under reproducible electric stimulation of the ciliary membrane.     

Loading neurons with BAPTA-AM activates xbp1 processing indicative of induction of endoplasmic reticulum stress

Loading cells with the calcium chelator BAPTA-AM is an analytical tool which has been used to suppress a rise in cytoplasmic calcium activity under various experimental conditions and thus, to evaluate the role of elevated cytoplasmic calcium levels in the process under investigation. BAPTA-AM may, however, not only have an isolated effect on cytoplasmic processes but also on functions of other subcellular compartments such as the endoplasmic reticulum (ER). Under conditions associated with ER dysfunction, the unfolded protein response is activated which is characterized by suppression of translation and processing of xbp1 mRNA, resulting in activation of the expression of genes coding for ER stress proteins. To investigate whether BAPTA-AM causes ER stress, primary neuronal cell cultures were loaded with varying amounts of BAPTA-AM. Exposure of cells to BAPTA-AM induced a marked rise in processed xbp1 mRNA levels, correlating with exposure times and BAPTA-AM concentrations in the medium used for loading. The increase in processed xbp1 mRNA was associated with suppression of protein synthesis and induction of cell injury. The results of this study indicate that loading primary neuronal cell cultures with BAPTA-AM activates xbp1 processing, implying that this calcium chelator does not have an isolated effect on cytoplasmic calcium activity but also an affect on ER function.     

Na,K-ATPase alpha2 and Ncx4a regulate zebrafish left-right patterning

A conserved molecular cascade involving Nodal signaling that patterns the laterality of the lateral mesoderm in vertebrates has been extensively studied, but processes involved in the initial break of left-right (LR) symmetry are just beginning to be explored. Here we report that Na,K-ATPase alpha2 and Ncx4a function upstream of Nodal signaling to regulate LR patterning in zebrafish. Knocking down Na,K-ATPase alpha2 and Ncx4a activity in dorsal forerunner cells (DFCs), which are precursors of Kupffer's vesicle (KV), is sufficient to disrupt asymmetric gene expression in the lateral plate mesoderm and randomize the placement of internal organs, indicating that the activity of Na,K-ATPase alpha2 and Ncx4a in DFCs/KV is crucial for LR patterning. High-speed videomicroscopy and bead implantation experiments show that KV cilia are immobile and the directional fluid flow in KV is abolished in Na,K-ATPase alpha2 and Ncx4a morphants, suggesting their essential role in KV ciliary function. Furthermore, we found that intracellular Ca(2+) levels are elevated in Na,K-ATPase alpha2 and Ncx4a morphants and that the defects in ciliary motility, KV fluid flow and placement of internal organs induced by their knockdown could be suppressed by inhibiting the activity of Ca(2+)/calmodulin-dependent protein kinase II. Together, our data demonstrate that Na,K-ATPase alpha2 and Ncx4a regulate LR patterning by modulating intracellular calcium levels in KV and by influencing cilia function, revealing a previously unrecognized role for calcium signaling in LR patterning.     

Ciliary beat co-ordination by calcium

Motile cilia in the airway epithelium are the engine for mucociliary clearance, the mechanism responsible for cleaning the airways from inhaled particles. Human airway epithelial cilia appear to have a slow constitutive rate of beating, driven by inherent and spontaneous dynein ATPase activity. Additionally, cilia can increase their beating frequency by activation of several different control mechanisms. One of these controllers is calcium. Its intracellular concentration is regulated by purinergic and acetylcholine receptors. Besides the rate regulatory effect of calcium on ciliary beat, calcium is also involved in synchronizing the beat among cilia of one single cell as well as between cilia on different cells. This article gives an overview of the complex effects of calcium on the beating of motile cilia in the airways.     

Comparison of mechanical agitation and calcium shock methods for preparation of a membrane fraction enriched in olfactory cilia

Calcium plays an important regulatory role in olfactory signal transduction. Many investigations into the regulation of the olfactory signaling pathway have been performed using fractions enriched in ciliary membranes from olfactory sensory neurons. The traditional method of preparing ciliary fractions uses high calcium concentrations, thought to dislodge cilia from the dendritic knobs of the olfactory neurons in the nasal epithelium. However, calcium, an important second messenger in the odorant signaling cascade, modulates the activity of many enzymatic reactions in this cascade. Pre-exposure of cilia to high calcium concentrations may modify these signaling events. Therefore, we sought to develop a method of isolating cilia-enriched membranes that avoids exposing the cilia to high calcium concentrations. Our method of isolation, referred to as the mechanical agitation method, involves mechanical disruption and sonication of the olfactory epithelium to dislodge the cilia. To evaluate this method of cilia preparation, basal adenylyl cyclase activity, as well as forskolin- and odorant-activated adenylyl cyclase, were analyzed. Specific activity of adenylyl cyclase and protein yield were compared for the mechanical agitation and the high calcium preparations. Immunoblots were analyzed for the presence of transduction components enriched in olfactory cilia: adenylyl cyclase type III (ACIII), heterotrimeric G-protein subunit Galphaolf and the 1 C2 isoform of phosphodiesterase (PDE 1 C2). Based on these analyses, the ciliary fraction prepared by the mechanical agitation method appears to be very similar to that prepared by the high calcium method, with a higher yield.     

Control of ciliary beat by calcium: the effects of lindane, a potent insecticide

The ciliary beat and cell motility of Dunaliella, a biflagellate unicellular green alga, have been studied by means of computer analysis of high-speed microcinematography and laser doppler velocimetry. Lindane was found rapidly to inhibit cell velocity in less than 5 min. and in a dose-related manner. After the initial 5-min. period, inhibition remained nearly stable for at least 3 hr. The waveform of the ciliary beat was not uniformly affected by lindane and only the effective stroke was greatly slowed down. The recovery stroke was not significantly modified, and the general form of the wave did not seem to be altered. Bending parameters automatically measured from high-speed movies showed that lindane induced a considerable lengthening of the initiation phase of wave propagation. Since lindane interacts specifically with calcium transport and can induce an increase in cytoplasmic calcium, the strong effect of lindane on ciliary beat is probably correlated with a modification of the calcium balance of the cell. These results support the hypothesis of a control of bend initiation by calcium.     

The interaction of cadmium with calcium and cisplatin with chloride

Changes in the locomotor rate of the ciliateTetrahymena pyriformis were used to quantitatively evaluate chemical interactions produced by: cadmium in combination with varying amounts of calcium, andcis-dichlorodiammineplatinum (II) (cisplatin) with varying amounts of sodium chloride. Cadmium (as CdCl₂) produces a measurable decline in the locomotor rate of the cells. Cadmium's detrimental effect can be reduced by the addition of calcium (as CaCl₂) in combination with cadmium. At a ratio of 30∶1 (calcium: cadmium), cadmium's negative effect upon motility is essentially nullified. It is suggested that the “protective” action afforded by calcium stems from the chemical similarity of the two cations and their involvement/competition for molecular sites responsible for the energy release and/or delivery of ciliary activity. Cisplatin will also effect a reduction in ciliary activity. However, the interaction between cisplatin, sodium chloride, and the cell appears more complex than that found with cadmium-calcium. At the lower range of chloride (as NaCl) used in this study, increased chloride concentration produces an increase in cisplatin's action against ciliary activity. At the higher levels, the chloride reduced cisplatin's negative effects. It is suggested that the increases in cisplatin's effects are caused by mass chemical action of increased chloride, which increases the concentration of the nonpolar cisplatin. The reduced effects found with the higher concentrations of sodium chloride may be because of the presence and action of elevated NaCl in/on the cell. This study clearly demonstrates differences in biologically relevant chemical interactions occurring with the two sets: cadmium-calcium and cisplatin-chloride.     

A morphologic and cytochemical study on the great alveolar cell.

Lungs from marsupials, bats and rodents were studied by light and electron microscopy. In all three groups, the great alveolar cells exhibit similar morphologic and cytochemical characteristics. Cytoplasmic vacuoles seen in these cells by light microscopy correspond to cytosomes that are demonstrable in them by electron microscopy. Such cytosomes are osmiophilic, periodic acid-Schiff-positive and stainable with Sudan black after acetone extraction. After fixation in a mixture of aldehydes, followed by extraction in chloroform-methanol and postfixation in osmium tetroxide, cytosomes lose their osmiophilia. The cytoplasm of the great alveolar cell is notable for a loosely ordered granular endoplasmic reticulum, an extensive Golgi apparatus and numerous multivesicular bodies. Many forms transitional in appearance between multivesicular bodies and cytosomes are present. In these, osmiophilic matter occupies the intervesicular space. It is proposed that these bodies are the precursors of cytosomes. The cytosomes are interpreted to be products of the "lysosomal" system in this cell. Ultimately they are secreted onto the alveolar surface.