The purpose of this study was to examine nitric oxide synthase (NOS) expression in the retinal vasculature in vivo and to study nitric oxide (NO) synthesis in vitro in retinal microvascular endothelial cells and pericytes. Immunoreactivity was examined using a polyclonal antibody raised against porcine cerebellar nitric oxide synthase on frozen sections cut from postmortem human retina and trypsin digests of rat retinal vasculature. The synthesis of nitrite, a stable end product from the interaction of NO with molecular oxygen, was measured in culture supernatants of retinal microvascular cells under basal and stimulated conditions. Expression of constitutive NOS (cNOS) in these cells was examined using the polymerase chain reaction (PCR). Strong NOS immunoreactivity was seen in the endothelium of choroidal and retinal vessels. Nitrite synthesis was documented in supernatants from cultured microvascular endothelial cells which increased significantly following exposure to A23187 and cytokines. Nitrite synthesis by pericytes was not detectable under basal conditions or following stimulation with A23187. Bacterial lipopolysaccharide (LPS), a potent inducer of NOS, caused an increase in nitrite concentrations in pericyte supernatants 24 h after stimulation suggesting the presence of inducible NOS (iNOS). PCR amplification confirmed the presence of the cNOS gene in endothelial cells but not in pericytes. Retinal vascular endothelial cells express significant amounts of NOS constitutively in vivo and in vitro which is activated by Ca++. Also, endothelial cells can be stimulated to synthesize iNOS by cytokines. Retinal pericytes too show iNOS activity following exposure to bacterial LPS. These results suggest that the nitric oxide synthase/nitric oxide pathway may be involved in the regulation of microcirculatory haemodynamics in the retina.

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The synthesis of two biotinylated affinity labels for chymotrypsin and trypsin-like serine proteinases is described, along with their kinetic characterization and application to the detection of these proteinases after PAGE and Western blotting. Thus the chloromethane analogues biotinylphenylalanylchloromethane (Bio-Phe-CH2Cl; reagent 1) and biotinylarginylchloromethane (Bio-Arg-CH2Cl, reagent 2), have been shown to be potent active-site-directed inactivators of chymotrypsin and trypsin respectively. The apparent overall second-order rate constants (kobs./[I]) for the inactivation of chymotrypsin and trypsin by reagent 1 (-4.9 x 103 M-1 min-') and reagent 2 (-1.0 x 105 M-1 min-') respectively are comparable with those obtained by other workers with simple urethane-protected analogues and demonstrates that the presence of the bulky biotinyl moiety is compatable with inhibitor effectiveness. Samples of chymotrypsin and trypsin that have been inactivated by reagents 1 and 2 respectively and which have been subjected to SDS/PAGE and Western blotting can be revealed with a streptavidin/alkaline phosphatase label. We can presently detect down to 20 ng of inactivated proteinase by using this system. The utility of the arginine derivative for the detection of the plasma trypsin-like proteinases plasmin and thrombin has also been demonstrated, thus holding out the possibility that this reagent may find general application as an active-site-directed label for this class of proteinase.

Epidermal growth factor (EGF) and its homologue, transforming growth factor alpha (TGF alpha), are mitogenic, angiogenic and tumour-promoting polypeptides. Much effort has therefore been directed towards the development of EGF/TGF alpha antagonists as a potential cancer therapy. Initial reports that some EGF/TGF alpha synthetic fragments possess EGF-receptor binding activity have not been confirmed in subsequent studies. We have found, however, that the murine EGF B-loop sequence: Ac-[(S-acetamidomethyl)-Cys20,31]-EGF-(20-31)-NH2 [(mEGF-(20-31)] produces biological effects consistent with the parent molecule in bovine, murine, chick and human, but not rat, model systems. In parallel experiments, both mEGF and mEGF-(20-31) elicit migratory, cytoprotective, growth-stimulatory, growth-inhibitory and angiogenic responses. The reverse B-loop sequence, mEGF-(31-20), is also mitogenic and angiogenic. The C-loop sequence, mEGF-(33-42), has no mitogenic or angiogenic activity when applied alone, does not block the mitogenic effect of mEGF, but does block the angiogenic effect of mEGF. It has not been established that the EGF receptor is the target for these fragments, but the results suggest that the residual biological activities of EGF fragments merit further investigation.

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