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 microM) 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 microM, n = 7, p < 0.02) and dose-dependent depolarization (7.1 +/- 1.8 mV at 1 microM, n = 19, p < 0.01). Intracellular calcium levels, monitored by the ratiometric fluorescent calcium indicator fura-2, increased within seconds of progesterone (1 microM) application. Progesterone (1 microM) 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.

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, 1 mM acetylcholine, and 1 microM isoproterenol (beta-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.

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 (alpha and beta), 1 microM progesterone, 1 microM oestrogen, 1 microM 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-1beta 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.

Electrophysiological signs of retinal dopamine deficiency in recently diagnosed Parkinson's disease and a follow up study.

Electrophysiological studies, including electrooculogram (EOG), and simultaneously recorded flash and pattern evoked electroretinograms (FERG and PERG) and visually evoked potentials (FVEP and PVEP) were made in 1988 on 10 newly diagnosed untreated Parkinson's patients at Stage 1 of the Hoehn and Yahr scale. Follow up studies were made on five out of the 10 patients when their disease had progressed to Stage 2 during 1993. The earliest and only sign of abnormality detected in the Stage 1 of Parkinson's patients in 1988 was a delay in the time to reach the peak light rise in the EOG. When the disease had progressed to Stage 2, not only a delay in the time to reach the peak light rise but also a reduction in the amplitude of the peak light rise in the EOG, together with changes in PERG, FERG and PVEPs were demonstrable. These changes observed in PERG, FERG and PVEPs were generally consistent with those reported by previous studies. It is suggested that the reason for the susceptibility of pigment epithelial function to dopamine deficiency in Parkinson's disease may be due to the pigment epithelium being at the extremity of the diffusion pathway from dopamine release sites at the inner plexiform layer.

The medium frequency (7.5 MHz) ultrasound image characteristics of cattle skin.

The medium frequency (7.5 MHz) ultrasound images of cattle skin, as a model for other domestic animals, are described and compared with the published information for human skin. Although some difficulties are apparent in identifying the skin surface and therefore measuring tissue thicknesses, the papillary and reticular layers of cattle skin, about 2 and 4 mm thick, respectively, can be differentiated using a water standoff with a vegetable oil coupling medium. The difference in echo intensities and patterns between these dermal components supports the concept that ultrasound tissue characterisation using higher frequencies may prove to be a useful non-invasive procedure in livestock production and veterinary science. However, a detailed protocol is required. Care is needed in the choice of ultrasound coupling medium for good resolution of cattle skin. Higher frequency ultrasound equipment would be necessary for imaging the skin of small animals. Potential applications of this new technology are discussed.