Sean F. Hackett

Human retinal pigment epithelial cells possess muscarinic receptors coupled to calcium mobilization

Human retinal pigment epithelial (RPE) cells in culture demonstrated saturable specific binding of [3H]quinuclidinyl benzilate (QNB). Specific binding represents about 75% of total binding. Scatchard analysis yields a Kd of 0.178 nM and Bmax of 42 fmol/mg protein. Atropine and carbachol show typical displacement curves, and a Hill plot has a slope of 0.96, suggesting a homogeneous population of receptors. Muscarinic agonists have no effect on intracellular cyclic adenosine monophosphate levels in RPE cells measured by radioimmunoassay, nor do they alter the isoproterenol-induced stimulation of adenylate cyclase. However, both acetylcholine and carbachol cause a rapid increase in intracellular calcium concentration measured by the fluorescent indicator quin 2. Atropine reverses the calcium rise when added after agonist and prevents the rise when added prior to agonist. These data suggest that human RPE cells possess muscarinic receptors coupled to calcium mobilization.

Characterization of adenylate cyclase in human retinal pigment epithelial cells in vitro.

Human retinal pigment epithelial cells in culture demonstrate adenylate cyclase activity. It is membrane-bound and modulated by GTP regulatory proteins. It is effectively activated only by beta-adrenergic agonists (L-isoproterenol greater than or equal to L-epinephrine greater than L-norepinephrine) and some prostaglandins (PGE1 and PGE2, but not PGF1 alpha). The adrenergic response appears to be mediated by beta-2 receptors. No inhibitory ligands could be demonstrated. Its characteristics, which are similar to functional adenylate cyclase complexes in other mammalian cells, and its selective and sensitive agonist responsiveness, suggest a possible physiologic role in the regulation of human retinal pigment epithelial-cell function.

Human retinal pigment epithelial cells in culture possess A2-adenosine receptors

Adenosine agonists cause a marked stimulation in cyclic AMP accumulation in whole human retinal pigment epithelial (RPE) cells in the presence of adenosine deaminase and papaverine, a phosphodiesterase inhibitor. N-Ethylcarboxamidoadenosine (NECA) stimulates cyclic AMP accumulation 16.1-fold above basal with an EC50 of 2.5 x 10(-7) M. It is also an effective (1.9-fold) stimulator of adenylate cyclase activity in RPE membrane preparations and a modest (1.22-fold) stimulator in the presence of forskolin in RPE cell membranes prepared from freshly isolated porcine RPE. N6-Cyclopentyladenosine (CPA) and N6-phenylisopropyladenosine (PIA) also increase cyclic AMP levels with EC50s of 4.9 x 10(6) M (8.9-fold above basal) and 3.5 x 10(-6) M (8.0-fold above basal) respectively. This potency order (NECA greater than PIA greater than CPA) is typical of A2-adenosine receptors. The relatively A1-selective agonists 10(-7) M indicating that RPE cells do not have A1-receptors which inhibit adenylate cyclase. Three adenosine receptor antagonists, BW-A1433U, 8-cyclopentyltheophylline and 8-sulfophenyltheophylline, blocked the NECA-induced stimulation of cyclic AMP accumulation with IC50s of 0.36 microM, 1.5 microM, and 75 microM respectively. Since alteration of cAMP levels has been demonstrated to affect several RPE functions, including cell migration, resorption of subretinal fluid, and phagocytosis, adenosine may play a significant regulatory role in RPE.

A morphological and immunohistochemical study of human retinal pigment epthelial cells, retinal glia, and fibroblasts grown on Gelfoam matrix in an organ culture system - A comparison of structural and nonstructural proteins and their application to cell type identification

Retinal pigment epithelial (RPE) cells, retinal glia, and fibroblasts undergo marked phenotypical change when outside their usual microenvironment, as occurs in epiretinal membrane formation. To explore their phenotypic potential without the influence of other cell types, each was cultured on Gelfoam matrix and assessed immunohistochemically and ultrastructurally. All cell types demonstrated vimentin and a universalβ-tubulin epitope, TU27. RPE and retinal glial cells were positive for cytokeratin, Len 7, and neuron-specific (γγ) enolase, as were glia and ibroblasts for S-100 protein and RPE cells and fibroblasts for glutamine synthetase. RPE cells alone showed positivity for class III β-tubulin and retinal S-antigen (monolayer cultures only) ; occasional retinal glia, which immunohistochemical findings suggest are Willer cell derived, demonstrated GFA protein. Therefore, class III β-tubulin may be useful in distinguishing RPE cells from retinal glia and ibroblasts, and Leu-7 may help to identify RPE cells and fibroblasts; these cell types are difficult to distinguish in clinical material using more traditional morphological criteria.

Thrombin is a stimulator of retinal pigment epithelial cell proliferation

Two preparations of bovine thrombin were found to stimulate DNA synthesis in cultured human retinal pigment epithelial cells. DNA synthesis was assessed by both [3H]thymidine incorporation into TCA precipitable material and nuclear labeling with [3H]thymidine. Cultures grown in the presence of thrombin for 48 hr showed a significant increase in cell number. When the concentrations of the two thrombin preparations were normalized for clotting activity, they had almost identical dose-response curves and both caused a tenfold maximal stimulation of [3H]thymidine incorporation. The EC50 for the preparation with higher specific activity was 20 ng ml(-1). Hirudin, a specific high affinity inhibitor of thrombin, completely blocked the mitogenic effect. When a maximally effective concentration of thrombin was used in combination with maximally effective concentrations of other growth factors (insulin, acidic fibroblast growth factor, platelet-derived growth factor, epidermal growth factor), they were found to be strongly synergistic in stimulating DNA synthesis. These data suggest that thrombin may act as an endocrine mediator of retinal pigment epithelial cell proliferation and participate in normal and exaggerated ocular wound healing.

A Method for Measuring Polymorphonuclear Leukocyte Concentrations in Nasal Mucus

A method is described for measuring polymorphonuclear leukocyte (PMN) concentrations in expelled nasal mucus. Nasal mucus specimens (0.05-0.15 ml) were diluted 1:60 in Hanks balanced salt solution. Diluted mucus was vigorously agitated to disperse contained cells, and cells were counted in a hemocytometer. The mean PMN counts in nasal mucus from healthy young adults was 7.1 X 10(6) PMNs/ml mucus, but large daily fluctuations were observed in PMN counts in the same individual. This method may be useful to investigate inflammatory conditions of the nasal cavity.

Implication of Protein Carboxymethylation in Retinal Pigment Epithelial Cell Chemotaxis

Two chemoattractants for retinal pigment epithelial (RPE) cells, fibronectin (FN) and platelet-derived growth factor (PDGF) were found to enhance protein carboxymethylation mediated by S-adenosyl-L-methionine in RPE cells measured by [3H]methanol hydrolyzed from TCA precipitable protein methyl esters labelled with [3H]methionine. The effect was rapid, peaking at 2 min when [3H]methanol production was enhanced 120% by FN and 150% by PDGF. Erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA; 10 microM), adenosine (100 microM), and L-homocysteine thiolactone (100 microM) inhibited FN-induced carboxymethylation by 60%, and PDGF-induced carboxymethylation by 59%. In modified Boyden chamber assays, the combination of EHNA (10 microM), adenosine (100 microM), and L-homocysteine thiolactone (100 microM) markedly inhibited FN-induced chemotaxis by 88% and PDGF-induced chemotaxis by 93%). Migration of RPE cells has been implicated in the pathogenesis of proliferative vitreoretinopathy (PVR). Inhibition of protein carboxymethylation may provide a new target in the development of pharmacologic therapy for PVR.

Cyclic 3′,5′-adenosine monophosphate modulates vascular endothelial cell migration in vitro

Using a modified Boyden chamber assay, we have examined the effect of cyclic nucleotides on bovine aortic endothelial cell migration in vitro. Dibutyrl cyclic 3,5-adenosine monophosphate (5 mM) inhibited endothelial cell random migration by 67% and inhibited fibronectin-induced chemotaxis by 75%. Agents which significantly stimulated adenylate cyclase activity in endothelial cell membranes were also effective inhibitors of endothelial cell migration. Timolol blocked both the isoproterenol-induced stimulation of adenylate cyclase and the ability of isoproterenol to inhibit endothelial cell migration. Caffeine and isoproterenol together had a greater inhibitory effect on endothelial cell motility than either alone. These data suggest that cAMP may modulate vascular endothelial cell migration in an inhibitory fashion.