Mice immunised with partially purified surface molecules of photoreceptor outer segments or with a synthetic peptide corresponding to the N-terminus of a cone-specific rhodopsin-like protein, gave rise to the production of monoclonal antibodies with only general immunoreactivity against the retina. Immunisation with interphotoreceptor matrix or crude photoreceptor outer segment preparations resulted in the production of hybridomas which secreted monoclonal antibodies 1001.A1 and 1001.A3. 1001.A1 binds to the interstitial retinol-binding protein (IRBP), associated with the rod photoreceptor cells and the IPM, as demonstrated by immunohisto chemistry and by Western blotting and dot blotting of IPM and purified IRBP. 1001.A3 binds a soluble high molecular weight chondroitin sulphase proteoglycan. Immunohistochemistry indicated the antigen to be present in the form of distinct sheath-like structures surrounding the photoreceptor cells. Gel filtration chromatography both in native conditions and in the presence of guanidinium chloride showed the antigen to have an apparent molecular mass of greater than 2000kDaltons and indicated the antigen was not a loosely associated aggregate of smaller components. Binding of 1001.A3 to fixed tissue sections of the bovine retina was completely abolished by their prior treatment with either chondroitinase ABC, chondroitinase AC, hyaluronidase (testicular) or trypsin. Prior treatment of tissue sections with either heparinase or neuraminidase had no effect on binding. Treatment of tissue sections with hyaluronidase (<i>Streptomyces</i>) had no effect on the ability of 1001.A3 to bind, but the structure of the antigen was altered. The sheath-like structure surrounding the photoreceptors was broken down and immunoreactivity was seen in the same area of the IPM, adjacent to the photoreceptors but with no defined structure. In conclusion, the antigen is a chondroitin sulphate molecule which is associated with hyaluronic acid molecules and which together form a defined sheath-like structure surrounding the photoreceptor cells.

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A specific radioimmunoassay was developed and characterised for the measurement of arginine vasotocin (A VT) in teleost fish. Crossreactivity of the antibody with the other teleost neurohypophysial peptide, isotocin, was less than 1 % and the sensitivity of the assay 0.25 pglassay tube. Rapid blood sampling, within 1 minute of capture, by needle puncture of the caudal blood vessels, and collection of blood through chronic arterial cannula 1 - 4 days after surgery were identified as procedures which did not influence plasma ion levels, osmolality or hormone concentrations. These procedures were used in later experimental protocols examining the effects of chronic and acute osmotic stimuli on plasma A VT concentrations. Plasma A VT concentrations in a range of euryhaline and stenohaline teleost fish were measured at 10-12 to low 10-11 M (1-20 pglml). There was no consistent difference in circulating A VT levels in euryhaline fish (flounder, eel and trout) adapted to fresh water (FW) or seawater (SW) for at least 2 weeks. The flounder was studied in greater detail and seasonal influences were identified as having an effected on osmoregulation. In the winter/spring period plasma osmolality was significantly lower in FW adapted fish than at other times of the year. In SW adapted fish at this time plasma osmolality was elevated and this was accompanied by elevated plasma A VT concentrations. The winter/spring period was associated with the development of the gonads and this factor may affect osmoregulation in the flounder. In SW adapted flounder a positive linear relationship between plasma A VT and plasma osmolality and ion levels was observed, indicating a link between these two parameters but no such relationship was evident in FW adapted fish. Chronically cannulated SW adapted flounder were used to examine the effects of acute osmotic stimuli on plasma A VT concentrations. Intraperitoneal injection of hypertonic saline acutely increased plasma osmolality and was associated with a significant increase in plasma A VT concentration, indicating that A VT secretion was sensitive to increased plasma osmolality. The expansion of extracellar fluid volume via intravascular infusion of isotonic saline reduced plasma A VT concentrations independently of any change in plasma osmolality. This indicated that vascular volume also influences plasma A VT concentrations. Reducing blood volume by haemorrhage and lowering plasma osmolality by infusion of hypotonic saline did not result in any change in plasma A VT concentrations. Plasma A VT concentrations were measured in lamprey at a 10-12 M concentration, and the transfer of lamprey from FW to Baltic SW caused an initial rise in plasma A VT concentrations, suggesting that A VT is involved in osmoregulation in these fish. A VT immunoreactivity was also demonstrated in two species of elasmobranch fish in a 10-12 to low 10-11 M range.

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No description available.