Janice R. Bailie

Nitric oxide synthase activity and expression in retinal capillary endothelial cells and pericytes

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.

Molecular characterization of endothelin receptors and the effect of insulin on their expression in retinal microvascular pericytes.

Summary: Microvascular pericytes contain predominantly endothelin A (ETA)-like binding sites and also a smaller number of ETB binding sites. In this study we verified the expression of both receptors in these cells. We then examined the effect of insulin, a potent mediator of vasodilatation, on the expression of these receptors in pericytes by Northern hybridization. Northern hybridization studies showed that ETA mRNA levels were not influenced by exposure to insulin (1 x 10-7 M).By contrast, ETB receptor mRNA, which was minimal under basal conditions, was significantly increased by 4 h after exposure to insulin.

Growth-inhibitory and growth-stimulatory effects of epidermal growth factor on human breast cancer cell line, MDA.MB.436: Dependence on culture conditions

Epidermal growth factor (EGF) is growth inhibitory for some cell lines, especially those having over-expressed EGF receptors. We have examined the effects of murine EGF on the growth of the human breast cancer cell line, MDA.MB.436, which has low numbers of EGF receptors. In the presence or absence of serum a 6 day exposure to 0.1 ng/ml EGF causes inhibition of growth if the culture medium is left unchanged during the course of the experiment but the same concentration of EGF causes stimulation above control if the EGF-containing medium is replaced daily. A 1 day exposure to 0.1 ng/ml followed by return to control medium has no effect on subsequent growth. The cells do not synthesize EGF receptor binding activity and added EGF is degraded within 2 days, suggesting that the inhibitory effects of EGF persist in its absence.

An Investigation of Receptor-Mediated Endocytosis and Endosomal Sorting of Albumin and Transferrin in Retinal Vascular Endothelial Cells

Confluent monolayers of cultured retinal microvascular endothelial cells (RVECs) were exposed simultaneously to two different protein-gold conjugates to determine if partitioning or sorting of protein ligands occurs during receptor mediated endocytosis in CNS microvascular cells. A mixture of bovine serum albumin (BSA), conjugated with 5nm colloidal gold (BSA-gold) and transferrin (TO, conjugated with 15nm gold (Tf-gold) were added to RVECs and the monolayers processed, in situ, for TEM. At 0 mins the gold conjugates bound diffusely to the apical plasma membrane, but after 2 mins, both ligands were observed to cluster in clathrin-coated pits prior to internalisation in coated vesicles. At 5 mins the gold-ligands were co-localised in coated vesicles and early endosomes. After 10 mins the probes were observed in larger late endosomes where segregation was first noted and by 30mins, the mixture of gold particles appeared partitioned within late endosomes, with BSA-gold localised at the periphery and Tf-gol...