J. E. G. Downing

Differential expression of voltage-activated Na+ currents in two prostatic tumour cell lines: contribution to invasiveness in vitro

The voltage‐gated ionic currents of two rodent prostatic cancer cell lines were investigated using the whole‐cell patch clamp technique. The highly metastatic Mat‐Ly‐Lu cells expressed a transient, inward Na+ current (blocked by 600 nM tetrodotoxin), which was not found in any of the weakly metastatic AT‐2 cells. Although both cell lines expressed a sustained, outward K+ current, this occurred at a significantly higher density in the AT‐2 than in the Mat‐Ly‐Lu cells. Incubation of the Mat‐Ly‐Lu cell line with 600 nM tetrodotoxin significantly reduced the invasive capacity of the cells in vitro. Under identical conditions, tetrodotoxin had no effect on the invasiveness of the AT‐2 cells.

Without nerves, immunology remains incomplete - In vivo veritas

Interest in the interactions between nervous and immune systems involved in both pathological and homeostatic mechanisms of host defence has prompted studies of neuroendocrine immune modulation and cytokine involvement in neuropathologies. In this review we concentrate on a distinct area of homeostatic control of both normal and abnormal host defence activity involving the network of peripheral c‐fibre nerve fibres. These nerve fibres have long been recognized by dermatologists and gastroenterologists as key players in abnormal inflammatory processes, such as dermatitis and eczema. However, the involvement of nerves can all too easily be regarded as that of isolated elements in a local phenomenon. On the contrary, it is becoming increasingly clear that neural monitoring of host defence activities takes place, and that involvement of central/spinal mechanisms are crucial in the co‐ordination of the adaptive response to host challenge. We describe studies demonstrating neural control of host defence and use the specific examples of bone marrow haemopoiesis and contact sensitivity to highlight the role of direct nerve fibre connections in these activities. We propose a host monitoring system that requires interaction between specialized immune cells and nerve fibres distributed throughout the body and that gives rise to both neural and immune memories of prior challenge. While immunological mechanisms alone may be sufficient for local responsiveness to subsequent challenge, data are discussed that implicate the neural memory in co‐ordination of host defence across the body, at distinct sites not served by the same nerve fibres, consistent with central nervous mediation.

Plasticity of cone horizontal cell functioning in cyprinid fish retina: effects of background illumination of moderate intensity

SummaryIn electrophysiological experiments involving intracellular recording from horizontal cells in the isolated retina of the roach, light adaptation of the retina has been shown to result in potentiation both of (1) the depolarizing component of biphasic chromaticity type S-potentials, and (2) the temporal frequency transfer functions of photopic luminosity type horizontal cells. Under identical light adaptation conditions, the number of spinules on horizontal cell dendrites positioned laterally at cone pedicle ribbon synapses, increase by some threefold. The latter effect occurs equally in pedicles of red- and green-sensitive cones. Thus, horizontal cells are ‘plastic’ in both structural and electrophysiological respects. Furthermore, since the two electrophysiological parameters studied depend on negative feedback from horizontal cells onto cones, the results suggest that it is the inhibitory synapses that are plastic and that spinules may be sites of the negative feedback interaction. Physiological and behavioural aspects of light-dependent horizontal cell plasticity are also discussed.

The cellular origin of an unusual type of S-potential: an intracellular horseradish peroxidase study in a cyprinid fish retina

SummaryL2-type S-potentials are mainly blue/green-sensitive hyperpolarizing responses with a red-sensitive depolarizing component which is either weak or absent. They were first described in the retina of the roach, a cyprinid fish, by Djamgoz (1978, 1984) and Djamgoz & Ruddock (1978, 1979a). The cellular origin of these responses has been determined and characterized by intracellular recording, horseradish peroxidase staining, and light and electron microscopy. They were found to arise in horizontal cells with H2-like morphologies on average (Stell & Lightfoot, 1975). The dendrites of thesg cells contacted green- and blue-sensitive cone pedicles within which both lateral and central contacts were made at ribbon synapses. The laterally-positioned dendrites had incompletely formed spinules associated with them. A number of similarities between these units and the biphasic, chromaticity (Cb)-type S-potentials have been outlined and it is suggested that L2 units are essentially Cb-units with a weak depolarizing component. In turn, it is suggested that the depolarizing component is reduced as a consequence of the relatively dark-adapted states of the retinae. It is concluded that the negative feed-back pathway that subserves the generation of depolarizing (Cb-type) S-potentials is weak or absent in dark-adapted retinae and that spinules may be the site of this feed-back interaction.

Neuropeptides Exert Direct Effects on Rat Thymic Epithelial Cells in Culture

To determine if major thymic neuropeptides and neurotransmitters can directly influence the functional activity of cultured rat thymic epithelium, neuropeptides and neurotransmitters were applied, and intercellular communication, proliferation, and thymulin secretion assessed. After injections of a mixture of lucifer yellow dextran (too large to pass gap junctions) and cascade blue (which does) into single cells, some neuropeptides decrease dye coupling: 0.1 mM GABA (P < 0.0001), 100 nM NPY (P < 0.0001), 100 nM VIP (P < 0.001), 100 nM CGRP (P < 0.001), 100 nM SP (P < 0.01), and 0.1 mM histamine (P < 0.01), whereas 0.1 mM 5-HT, mM acetylcholine, and 1 μM isoproterenol (β-adrenergic agonist) had no effect. Proliferation (incorporation of tritiated thymidine) was increased by CGRP (P = 0.004) and histamine (P < 0.02), but decreased by isoproterenol (P = 0.002), 5-HT (P = 0.003), and acetylcholine (P < 0.05). The percentage of multinucleate cells was decreased after isoproterenol (2.5%), and increased after 5-HT (21.3%), GABA (15%), and histamine (15.1%). Compared to controls, thymulin in the supernatant was decreased after challenge with acetylcholine (52%), isoproterenol (71%), 5-HT (73%), and histamine (84%). This study demonstrates direct effects of neuropeptides and neurotransmitters on functional aspects of cultured thymic epithelial cells.

Amacrine cells in the retina of a cyprinid fish: functional characterization and intracellular labelling with horseradish peroxidase

SummaryForty amacrine cells in retinae of a cyprinid fish, the roach, were intracellularly labelled with horseradish peroxidase following electrophysiological identification as sustained depolarizing, sustained hyperpolarizing or transient units. Labelled cells were analysed by light microscopy and compared with a catalogue of amacrine cells established in a previous Golgi study on the same species. About 30% of the cell types characterized by the Golgi method were encountered in the present study. When intracellularly labelled cells were differentiated on the basis of their dendritic organization in the plane of the retina, a given electrophysiological response pattern was found to be generated by different morphological types, and vice versa. However, examination of the ramification patterns of the dendrites within the inner plexiform layer (i.e. in the radial dimension of the retina), showed that this morphological parameter of a given amacrine cell could be correlated with its light-evoked response. Several amacrine cell types were found to possess special distal dendrites which arose from the main dendritic branches and extended well over a mm in the retina. Distal dendrites were oriented tangentially with respect to the optic nerve papilla, but did not appear to be involved in any synaptic connectivity. It is concluded that the Golgi-based classification is a valuable tool for identifying intracellularly labelled amacrine cells. However, although the correlation between layering of dendrites in the inner plexiform layer and electrophysiology was generally good, additional physiological parameters would be required to determine whether more extensive parallels exist between structural and functional characteristics of amacrine cells. Alternatively, the considerable morphological diversity of amacrine cells may be of limited physiological significance.

A Horizontal Cell Selectively Contacts Blue-Sensitive Cones in Cyprinid Fish Retina: Intracellular Staining with Horseradish Peroxidase

A horizontal cell selectively contacting blue-sensitive cones has been intracellularly stained with horseradish peroxidase in the retina of a cyprinid fish, the roach. The light microscopical morphology of the cell belonged to the H3 category of horizontal cells found in cyprinid fish retinae. In response to spectral stimuli, the cell generated chromaticity-type S-potentials that were hyperpolarizing to blue and depolarizing to yellow-orange. A red-sensitive hyperpolarizing component was absent possibly because of suppression of the negative feedback pathway between luminosity-type (H1) horizontal cells and green-sensitive cones.

Modulation of dye-coupling and proliferation in cultured rat thymic epithelium by factors involved in thymulin secretion

Cultures of rat thymic epithelium were used to measure the effect of thymulin secretagogues on dye‐coupling and proliferation. Dye‐coupling was assessed after the injection of lucifer yellow dextran which cannot permeate the connexin pore of gap junctions and the smaller, permeant cascade blue. In addition to gap junctional communication, larger intercellular bridges were demonstrated by the transfer of lucifer yellow dextran between cells. The extent of intercellular communication was found to be influenced by both cell density and the number of passages. In control cultures, intercellular communication was reduced in cell groups of low (<20 cells/group) or high cell densities (>100 cells/group) compared with groups of 20–60 cells. The highest coupling indices were found in subcultures 20–30. Taking these factors into account, significant decreases in coupling index were observed after pretreatment of test cultures with factors known to influence the secretion of thymulin (5 U/ml interleukin 1 (α and β), 1 μM progesterone, 1 μM oestrogen, 1 μM testosterone, 1 ng/ml adrenocorticotropic hormone, 100 nm rat growth hormone) but 7.5 ng/ml thymulin had no effect on dye‐coupling. The nonspecific gap junction uncoupler, octanol, abolished dye‐coupling. Cellular proliferation, as measured by the uptake of tritiated thymidine, showed that the same factors that reduced coupling also increased proliferation. None of these factors affected the number of multinucleate cells present, except interleukin‐1β which caused a significant reduction in the average number of nuclei per cell. Thus rat thymic epithelium in vitro provides a model for the study of the direct action of factors on cells of the thymic microenvironment.

Rapid Progesterone Actions on Thymulin-Secreting Epithelial Cells Cultured from Rat Thymus

Many soluble factors of neural, endocrine, paracrine and autocrine origin are present in the thymus and modulate its function. Long-term effects of sex steroids have been documented for thymocytes and cells of the thymic microenvironment. In this report we examine rapid actions of progesterone upon aspects of epithelial cell physiology. Progesterone (0.1–10 µM) was applied to cultured thymulin-secreting thymic epithelial cells (TS-TEC) and changes in transmembrane potential, transmembrane current, intracellular calcium levels and thymulin secretion were assessed. Rapid changes in electrophysiology and intracellular calcium provide evidence for a membrane-bound progesterone receptor in these cells, in addition to classical cytoplasmic receptors. Application of progesterone to TS-TEC caused electrophysiological changes in 56% of cells (n = 40), activating an inward current (–24 ± 9 pA at 1 µM, n = 7, p < 0.02) and dose-dependent depolarization (7.1 ± 1.8 mV at 1 µM, n = 19, p < 0.01). Intracellular calcium levels, monitored by the ratiometric fluorescent calcium indicator fura-2, increased within seconds of progesterone (1 µM) application. Progesterone (1 µM) increased thymulin levels in supernatant, as measured by ELISA, above the levels in the preapplication period (142 ± 16% of the preapplication period, n = 3, p < 0.02). This effect was reduced in the presence of cobalt chloride which blocks voltage-dependent calcium channels. In addition, TS-TEC in culture were immunoreactive to antibody AG7. This antibody was raised to a membrane-bound antigen involved in calcium influx subsequent to progesterone binding in sperm. Thus we suggest that progesterone acts upon many aspects of TS-TEC physiology through both cytoplasmic and membrane-bound receptors.

Cat retinal ganglion cells show transient responses to acetylcholine and sustained responses to L-glutamate.

The neuropharmacological basis for the different receptive field properties of cat retinal ganglion cells was investigated using whole cell voltage-clamp recordings from acutely dissociated adult tissue. Subclasses of physiologically characterised ganglion cells were determined on the basis of (i) their soma diameters and (ii) their projection to central visual nuclei (identified by microinjection of fluorescent dyes into the lateral geniculate and/or superior colliculus). The sensitivities of all categories of ganglion cells, prepared from peripheral retina were found to be similar for gamma-amino butyric acid, glycine, acetylcholine and glutamate. The kinetics of desensitisation differed among receptor subtypes, revealing possible physiologically significant molecular specialisations that could be involved in shaping synaptic transmission.