A comparative study of the epidermis of the common carp and the three Indian major carp

A comparative study has been made of the mucogenic epidermis of the common carp, Cyprinus carpio var. communis, and the three Indian major carps, Catla catla, Labeo rohita and Cirrhina mrigala: on the basis of epidermis structural organization, these species are easily differentiated. The epithelial cells in the superficial layer, as in most fishes, show secretory activity, evidenced by positive histochemical reactions, which is high in C. carpio var. communis, moderate in C. catla and low in L. rohita and C. mrigala. The epithelial cells in the underlying two or three layers also give positive reactions, though their intensity is relatively weak. The mucous cells in C. carpio var. communis are distributed in large numbers arranged in several superimposed layers in the outer regions of the epidermis, whereas in C. catla they are fewer in number and are widely separated in the surface layers as well as in the deeper layers of the epidermis; in both species the mucous cells appear rounded, large, and open on the surface by wide pores. In contrast, in L. rohita and C. mrigala the mucous cells are smaller, restricted mainly to the superficial layer, close together in a single row, and open on the surface by narrow pores. The overall density of mucous cells in L. rohita and C. mrigala, as in C. catla, is much lower than in C. carpio var. communis. In the epidermis of C. carpio var. communis there are a large number of mucous cells, and the few club cells are restricted to the deeper layers. In contrast, in the epidermis of the three Indian major carp the overall density of the mucous cells is much lower and the club cells are very numerous. It is suggested that the high density of club cells compensates an overall low density of mucous cells as an adaptation for an effective defence mechanism. Increased mucus production in the epidermis of C. carpio var. communis, as evidenced by a large number of mucous cells in outer regions and high secretory activity of superficial layer epithelial cells, is associated with increased precipitation of mud held in suspension, needed as an adaptation to the species’peculiar bottom-scooping habits. The varied density of the taste buds in the epidermis of the four carp is associated with their feeding habits.     

Ultrastructure of Phasmid Development in Meloidodera floridensis and M. charis (Heteroderinae)

Phylogenetic characters for Heteroderinae Luc. et al., 1988 are evaluated in Meloidodera which is believed to have primarily ancestral characters. Phasmid ultrastructure is observed in second-stage juveniles (J2), third-stage juvenile males, fourth-stage juvenile males, and fifth-stage males of Meloidodera floridensis and M. charis. Phasmid secretion occurs inside the egg before the J1-J2 molt. Before J2 hatch, concentric lamellar membranes occur within the sheath and socket cells. Some membranes become lamellae of the sheath cell plasma membrane; others become multilamellar bodies. During early molting, plasma membrane lamellae disappear and a distal dendrite segment appears in a rudimentary canal. After the molt, the distal dendrite is not present within the canal. The phylogenetic utility of phasmid features is discussed. In both species the ampulla shape and size between molts are stable features in juveniles and males. The posthatch J2 sheath cell receptor cavity may vary in a species specific manner, but comparative morphology requires precise timing after hatch.     

Voltage-gated chloride currents in cultured canine tracheal epithelial cells

Summary Chloride ions (Cl^–) are concentrated in airway epithelial cells and subsequently secreted into the tracheal lumen by downhill flux through apical Cl^– channels. We have studied Cl^– currents in cultured canine tracheal cells using the whole-cell voltage-clamp technique. Ultrastructural techniques demonstrated that the cells used in the electrophysiological experiments possessed apical membrane specializations known to be present in the intact, transporting cell type. Cultured cells 2–6 days old were characterized by an input resistance of 3.4±0.8 G (n=11) and a capacitance of 63.8±10.8 pF (n=26). A comparison of 3 and 4 day-old cells with 5 and 6 day-old cells showed that the input resistance decreased almost 50%, and the cell capacitance and the inward and outward currents increased concomitantly approximately 200%. Cultured cells 3–4 days old held at –40 mV produced currents of 196±22 pA at 50 mV and –246±27 pA at –90 mV (n=212) with pipette and bath solutions containing primarily 140 KCl and 140 NaCl, respectively. The chloride channel blocker diphenylamine-2-carboxylate (DPC, 100 m) suppressed whole-cell currents by 76.8% at 60 mV; however, currents were unaffected by the stilbenes SITS (1mm) and DNDS (1–30 m). Replacement of K⁺ with Cs⁺ in the pipette solution did not affect the outward current, the current reversal potential, or the input resistance of the cells, indicating that the current was not significantly K⁺ dependent when the intrapipette solution was buffered to a Ca²⁺ concentration of 20nm. The Cl^–/Na⁺ permeability ratio was estimated to be greater than 11 as calculated from reversal potential measurements in the presence of an internal to external NaCl concentration ratio of 12. Current equilibrium permeabilities, relative to Cl^– were: I^– (2.9)NO ₃ ^– (1.1)Br^– (1.1)Cl^– (1.0)F^– (0.93)MeSO ₄ ^– (0.19)gluconate (0.18)aspartate (0.14). Depolarizations to potentials greater than 20 mV elicited a time-dependent component in the outward current in 71% of the cells studied. Currents inactivated with a double exponential time course at the most depolarized voltages. Recovery from inactivation was fast, holding potential-dependent, and followed a double exponential time course. Current amplitude was increased via a cAMP-dependent pathway as has been demonstrated for single Cl-selective channels in cell-attached patches from cultured canine and human tracheal epithelial cells. Forskolin, an activator of adenylate cyclase, produced a 260% increase in the outward current at +50 mV. In summary, cultured canine tracheal cells have a single resting conductance that is Cl^– selective, voltage-dependent, and modulated by a cAMP-dependent mechanism. This preparation appears to be appropriate for analysis of cellular modulation of airway Cl^– channels and Cl^– secretion.     

Characterisation of chloride transport at the tonoplast of higher plants using a chloride-sensitive fluorescent probe

The characteristics of Cl^– transport in isolated tonoplast vesicles from red-beet (Beta vulgaris L.) storage tissue have been investigated using the Cl^–-sensitive fluorescent probe, 6-methoxy-1-(3-sulfonatopropyl)-quinolinium (SPQ). The imposition of (inside) positive diffusion potentials, generated with K⁺ and valinomycin, increased the initial rate of Cl^– transport, demonstrating that Cl^– could be electrically driven into the vesicles. Chloride influx was unaffected by SO ₄ ^2- , but was competitively blocked by NO ₃ ^– , indicating that both Cl^– and NO ₃ ^– may be transported by the same porter. In some preparations, increases in free-Ca²⁺ concentration from 10^–8 to 10^–5 mol·dm^–3 caused a significant decrease in Cl^– influx, which may indicate that cytosolic Ca²⁺ concentration has a role in controlling Cl^– fluxes at the tonoplast. However, this effect was only seen in about 50% of membrane preparations and some doubt remains over its physiological significance. A range of compounds known to block anion transport in other systems was tested, and some partially blocked Cl^– transport. However, many of these inhibitors interfered with SPQ fluorescence and so only irreversible effects could be tested. The results are discussed in the context of recent advances made using the patch-clamp technique on isolated vacuoles.Abbreviations and Symbols BTP 1,3-bis[tris(hydroxymethyl)-methylamino]propane - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - membrane potential - pH pH gradient - SPQ 6-methoxy-1-(3-sulfonatopropyl)quinolinium - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl] glycine     

Distribution and Pharmacological Properties of the GABAA/ Benzodiazepine/Chloride Ionophore Receptor Complex in the Brain of the Fish Anguilla anguilla

In the present study, we characterized the distribution and the pharmacological properties of the different components of the GABAA receptor complex in the brain of the eel (Anguilla anguilla). Benzodiazepine recognition sites labeled "in vitro" with [3H]flunitrazepam ([3H]FNT) were present in highest concentration in the optic lobe and in lowest concentration in the medulla oblongata and spinal cord. A similar distribution was observed in the density of gamma-[3H]aminobutyric acid ([3H]GABA) binding sites. GABA increased the binding of [3H]FNT in a concentration-dependent manner, with a maximal enhancement of 45% above the control value, and, vice versa, diazepam stimulated the binding of [3H]GABA to eel brain membrane preparations. The density of benzodiazepine and GABA recognition sites and their reciprocal regulation were similar to those observed in the rat brain. In contrast, the binding of the specific ligand for the Cl- ionophore, t-[35S]butylbicyclophosphorothionate ([35S]TBPS), to eel brain membranes was lower than that found in the rat brain. In addition, [35S]TBPS binding in eel brain was less sensitive to the inhibitory effects of GABA and muscimol and much more sensitive to the stimulatory effect of bicuculline, when compared with [35S]TBPS binding in the rat brain. Moreover, the uptake of 36Cl- into eel brain membrane vesicles was only marginally stimulated by concentrations of GABA or muscimol that significantly enhanced the 36Cl- uptake into rat brain membrane vesicles. Finally, intravenous administration of the beta-carboline inverse agonist 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylic acid methyl ester (20 mg/kg) and of the chloride channel blocker pentylenetetrazole (80 mg/kg) produced convulsions in eels that were antagonized by diazepam at doses five to 20 times higher than those required to produce similar effects in rats. The results may indicate a different functional activity of the GABA-coupled chloride ionophore in the fish brain as compared with the mammalian brain.     

Effect of Halide Ions on t-[35S]Butylbicyclophosphorothionate Binding

The binding of t-[35S]butylbicyclophosphorothionate [( 35S]TBPS) to a site on the GABAA receptor complex is ion dependent. This study was conducted to determine the effects of ion species and concentration on the time course, affinity, and number of sites of [35S]TBPS binding. At a concentration of 200 mM ion, the time to equilibrium for [35S]TBPS binding was shortest for I-, followed by Br- less than Cl- less than F-. A similar rank order was observed for the concentration of ion required to produce half-maximal [35S]TBPS binding. Saturation binding experiments were conducted to evaluate the effect of increasing ion concentration on the KD and Bmax of [35S]TBPS binding. The Bmax was independent of both ion species and concentration. The receptor affinity, however, increased with increasing concentration for each ion. Calculated maximal affinity values were not different between ions; however, the EC50 to produce those values was different among ions and ranked in the same order as that for time course and maximal binding data. Association and dissociation rates for [35S]TBPS binding were greater in I- than in Cl-. These data emphasize the importance of ion selection and incubation times on [35S]TBPS binding.     

Evaluation of the annexins as potential mediators of membrane fusion in exocytosis

Membrane fusion is a central event in the process of exocytosis. It occurs between secretory vesicle membranes and the plasma membrane and also among secretory vesicle membranes themselves during compound exocytosis. In many cells the fusion event is regulated by calcium. Since the relevant membranes do not undergo fusion in vitro when highly purified, much attention has been paid to possible protein mediators of these calcium-dependent fusion events. The annexins comprise a group of calcium-dependent membrane-aggregating proteins, of which synexin is the prototype, which can initiate contacts between secretory vesicle membranes which will then fuse if the membranes are further perturbed by the addition of exogenous free fatty acids. This review discusses the secretory pathway and the evidence obtained fromin vitro studies that suggests the annexins may be mediators or regulators of membrane fusion in exocytosis.     

SecA protein: Autoregulated initiator of secretory precursor protein translocation across theE. coli plasma membrane

Several classes ofsecA mutants have been isolated which reveal the essential role of this gene product forE. coli cell envelope protein secretion. SecA-dependent,in vitro protein translocation systems have been utilized to show that SecA is an essential, plasma membrane-associated, protein translocation factor, and that SecA's ATPase activity appears to play an essential but as yet undefined role in this process. Cell fractionation studies suggested that SecA protein is in a dynamic state within the cell, occurring in soluble, peripheral, and integral membraneous states. These data have been used to argue that SecA is likely to promote the initial insertion of secretory precursor proteins into the plasma membrane in a manner dependent on ATP hydrolysis. The protein secretion capability of the cell has been shown to translationally regulatesecA expression with SecA protein serving as an autogenous repressor, although the exact mechanism and purpose of this regulation need to be defined further.     

Protein translocationin vitro: Biochemical characterization of genetically defined translocation components

Recent years have seen the convergence of both genetic and biochemical approaches in the study of protein translocation inE. coli. The powerful combination of these approaches is exemplified in the use of anin vitro protein synthesis-protein translocaltion system to analyze the role of genetically defined components of the protein translocation machinery. We describe in this review recent results focusing on the function of thesecA, secB, andsecY gene products and the demonstration of their requirement forin vitro protein translocation. The SecA protein was recently shown to possess ATPase activity and was proposed to be a component of the translocation ATPase. We present a speculative working model whereby the translocator complex is composed of the integral membrane proteins SecY, SecD, SecE, and SecF, forming an aqueous channel in the cytoplasmic membrane, and the tightly associated peripheral membrane protein SecA functioning as the catalytic subunit of the translocator or protein-ATPase.     

Chloride-induced Ca2+ Release from the Sarcoplasmic Reticulum of Chemically Skinned Rabbit Psoas Fibers and Isolated Vesicles of Terminal Cisternae

There is increasing evidence that Ca²⁺ release from sarcoplasmic reticulum (SR) of mammalian skeletal muscle is regulated or modified by several factors including ionic composition of the myoplasm. We have studied the effect of Cl⁻ on the release of Ca²⁺ from the SR of rabbit skeletal muscle in both skinned psoas fibers and in isolated terminal cisternae vesicles. Ca²⁺ release from the SR in skinned fibers was inferred from increases in isometric tension and the amount of release was assessed by integrating the area under each tension transient. Ca²⁺ release from isolated SR was measured by rapid filtration of vesicles passively loaded with ⁴⁵Ca²⁺. Ca²⁺ release from SR was stimulated in both preparations by exposure to a solution containing 191 mm choline-Cl, following pre-equilibration in Ca²⁺-loading solution that had propionate as the major anion. Controls using saponin (50 μg/ml), indicated that the release of Ca²⁺ was due to direct action of Cl⁻ on the SR rather than via depolarization of T-tubules. Procaine (10 mm) totally blocked Cl⁻- and caffeine-elicited tension transients recorded using loading and release solutions having ([Na⁺] + [K⁺]) × [Cl⁻] product of 6487.69 mm ² and 12361.52 mm ², respectively, and blocked 60% of Ca²⁺ release in isolated SR vesicles. Surprisingly, procaine had only a minor effect on tension transients elicited by Cl⁻ and caffeine together. The data from both preparations suggests that Cl⁻ induces a relatively small amount of Ca²⁺ release from the SR by activating receptors other than RYR-1. In addition, Cl⁻ may increase the Ca²⁺ sensitivity of RYR-1, which would then allow the small initial release of Ca²⁺ to facilitate further release of Ca²⁺ from the SR by Ca²⁺-induced Ca²⁺ release. Received: 6 February 1996/Revised: 17 July 1996