Richard B. Crook

Culture and characterization of epithelial cells from bovine choroid plexus.

Abstract Epithelial cells were isolated from choroid plexus, which plays a major role in cerebrospinal fluid production and regulation. Incubation of bovine choroid plexuses with pronase released cells which attached to plastic dishes with a plating efficiency of 5%. The cells were predominantly polygonal as judged by phase‐contrast microscopy. These polygonal cells undergo limited cell division and survive for 1 – 2 weeks in culture before being overgrown by fibroblasts. The fibroblastic cells could be selectively removed from the cultures by the addition of 100 μg/ml cis‐hydroxyproline to the medium for several days. The specific activities of three membrane‐bound enzymes, γ‐glutamyl transpeptidase, alkaline phosphatase, and leucine aminopeptidase were compared in selective cultures of polygonal cells and fibroblasts. Polygonal cells were found to have 4–5 times the γ‐glutamyl transpeptidase of fibroblasts, whereas fibroblasts have 2–3 times the alkaline phosphatase of polygonal cells. Leucine aminopeptidase levels in the two cultures were roughly equivalent. The polygonal cells rapidly lost γ‐glutamyl transpeptidase activity over a 4‐day period in culture but acquired increased levels of leucine aminopeptidase. Alkaline phosphatase remained roughly constant. Under similar conditions fibroblasts showed a 3‐ to 4‐fold increase in the specific activities of all three enzymes; these changes coincided with a substantial increase in cell density. Based on morphology, resistance to cis‐hydroxyproline, absence of antihemophilic factor antigen, and enzymatic characteristics, we believe the polygonal cells to be of epithelial origin.

Hormones and neurotransmitters control cyclic AMP metabolism in choroid plexus epithelial cells.

Abstract: The choroid plexus is a major site of CSF production. When primary cultures of bovine choroid plexus epithelial cells were exposed to 1 μg/ml cholera toxin, a 50‐fold increase of intracellular cyclic AMP was found 1 h later. Exposure of cells to 10−5M isoproterenol, 10−4M prostaglandin E1, 10−5M histamine, and 10−5M serotonin caused increases of intracellular cyclic concentrations of 100‐, 50‐, 20‐, and 4‐fold, respectively. From 5 to 15 min were required for these maximal responses to occur. Many other molecules including prolactin, vasopressin, and corticotropin did not alter cellular cyclic AMP levels. The accumulation of cyclic AMP could be inhibited by specific antagonists: propranolol inhibited the isoproterenol‐mediated stimulation while diphenhydramine and metiamide inhibited the histamine response. In addition, diphenhydramine inhibited serotonin‐dependent cyclic AMP accumulation. Combinations of isoproterenol, prostaglandin E1, histamine, and serotonin elicited additive responses as measured by cyclic AMP accumulation with one exception, i.e., serotonin inhibited the histamine response. Our findings suggest that distinct receptor sites on choroid plexus epithelia exist for isoproterenol, prostaglandin E1, and histamine. Efflux of cyclic AMP into the extracellular medium was found to be a function of the intracellular cyclic AMP levels over a wide range of concentrations. Our studies provide direct evidence for hormonal regulation of cyclic AMP metabolism in epithelial cells of the choroid plexus.

H2 Histamine Receptors on the Epithelial Cells of Choroid Plexus

Abstract: A major site of cerebrospinal fluid production in vertebrates is the choroid plexus. The epithelial cells of the choroid plexus accumulate intracellular cyclic AMP in response to several effectors, including histamine. Since histamine is known to regulate fluid secretion in the stomach via H2 histamine receptors, we asked whether H2 receptors might also be present on epithelial cells of bovine choroid plexus. Using agonists and antagonists of histamine, we show that an agonist and antagonist pair specific for the H2 subtype were clearly more effective than an H1 agonist and antagonist pair in mimicking or inhibiting histamine stimulation of cellular cyclic AMP. Analysis by Schild plot allowed assignment of an apparent dissociation constant to the H2 antagonist metiamide which was 34‐fold lower than that of its H1 counterpart, diphenhydramine. These results indicate that epithelial cells of the choroid plexus possess H2 histamine receptors.

Calcitonin gene-related peptide stimulates intracellular camp via a protein kinase C- controlled mechanism in human ocular ciliary epithelial cells

Calcitonin gene-related peptides I and II (CGRP I and II) were found to stimulate cAMP levels by approximately 4-6 fold in human nonpigmented ciliary epithelial cells with half-maximal effective concentrations of 20 x 10(-10) and 3 x 10(-10) M, respectively. Prior exposure of cells to 6 x 10(-7) M phorbol 12-myristate, 13-acetate for 15 min resulted in a 40-50% inhibition of CGRP II-dependent cAMP stimulation. Phorbol didecanoate and dioctanoylglycerol also effectively inhibited, whereas 4 alpha phorbol didecanoate, an ineffective activator of protein kinase C, had no effect. Staurosporine, a protein kinase C inhibitor, blocked the inhibition of cAMP formation by phorbol esters. cAMP stimulation by forskolin or cholera toxin was not inhibited by phorbol esters, suggesting that neither a Gs protein nor adenylyl cyclase is the site of inhibition by protein kinase C. These data therefore suggest that CGRP receptors are required for inhibition of adenylate cyclase by protein kinase C.

Vasoactive intestinal peptide stimulates cyclic AMP metabolism in choroid plexus epithelial cells.

Some peptides of the glucagon-secretin family were found to stimulate intracellular cyclic AMP accumulation in cultured bovine choroid plexus epithelial cells. Vasointestinal peptide and porcine intestinal peptide at concentrations of 30 and 300 nM, respectively, evoked 50-fold elevations of cyclic AMP; half-maximal responses were obtained with concentrations of 15 and 102 nM for the two peptides, respectively. Secretin and glucagon each produced 25- to 50-fold elevations of cyclic AMP at 330 microM, but showed no effect below 3 microM. Gastric inhibitory peptide and prealbumin had little or no response at any concentration tested. Experiments measuring the cellular cyclic AMP accumulation in response to pairs of peptides suggested that vasointestinal peptide, porcine intestinal peptide and secretin act through a common receptor. Studies with antagonists to isoproterenol and histamine indicated that this receptor is distinct from the beta-adrenergic and H2-histamine receptors known to exist on choroidal cells.

Histamine stimulation of inositol phosphate metabolism in cultured human non-pigmented ciliary epithelial cells

Treatment of cultured human non-pigmented ciliary epithelial cells with 100 microM histamine for 30 minutes resulted in a 3-5 fold increase in intracellular inositol phosphates. The stimulation by histamine was dose-dependent, with a half-maximal concentration of 3 microM and a maximal concentration of 100 microM. In response to histamine, inositol monophosphate increased approximately linearly for 30 min in the presence of 10 mM LiCl2, while inositol bisphosphate and inositol trisphosphate showed rapid rises complete within a few minutes. Treatment of cells with the H1 antagonist diphenhydramine resulted in a complete inhibition of the histamine effect at 1 microM, with a half-maximal inhibition at 56 nM, whereas cimetidine, an H2 antagonist, had little effect at any concentration tested. Schild analysis of the diphenhydramine/histamine receptor interaction gave an apparent dissociation constant of 7.1 nM. The data suggest that human non-pigmented ciliary epithelial cells possess H1 histamine receptors.

High affinity vasoactive intestinal peptide receptors on fetal human nonpigmented ciliary epithelial cells

The effect of vasoactive intestinal peptide (VIP) on stimulation of adenylyl cyclase in fetal human nonpigmented ciliary epithelial (NPE) and pigmented ciliary epithelial (CPE) cells was studied. 1 microM VIP elicited a 5-10 fold increase in intracellular cAMP in NPE cells from three fetal donors, but caused little or no response in CPE from two fetal donors and other ocular cell types employed as controls. Appearance of cAMP in the extracellular medium was stimulated in NPE but not in CPE in response to VIP. Both NPE and CPE gave similar cAMP responses (8-13 fold) to the beta-adrenergic agonist, isoproterenol. Binding studies of [125I]VIP to intact NPE and CPE revealed that VIP bound to NPE cells at a high affinity site (KD = .33 nM and a low affinity site (KD = 16 nM), whereas VIP bound to CPE cells only at the low affinity site (KD = 18 nM). In NPE cells, VIP stimulated cAMP formation with an EC50 of approximately 0.6-1 nM, similar to the high affinity binding site KD, with maximal stimulation at 10 nM. Four peptides with various degrees of sequence homology to VIP were also studied. Of these, PHM and PHI stimulated cAMP with EC50s of 50 and 300 nM, respectively, while secretin and glucagon stimulated only at concentrations above 0.1 microM. These results suggest that in fetal human ciliary epithelium, as in rabbit ciliary epithelium (Mittag et al., J Pharm Exp Ther 241: 230, [1987]), VIP stimulation of adenylyl cyclase is a characteristic of NPE but not CPE cells.

Stimulation of inositol phosphate formation in cultured human retinal pigment epithelium.

Several hormones, neurotransmitters, and neuropeptides were screened for the ability to stimulate inositol phosphate formation in cultured human retinal epithelial (RPE) cells. Carbachol, vasopressin and thrombin were found to be effective. Treatment of RPE cells with all three agents produced increases in inositol monophosphate, inositol bisphosphate and inositol trisphosphate in the presence of 10 mM LiCl. Carbachol stimulated a 4-fold increase in the total of inositol phosphates at 1 mM. Studies with cholinergic antagonists showed a rank order of 4 DAMP greater than QNX greater than pirenzepine greater than methoctramine, suggesting the presence of M3 muscarinic receptors. Vasopressin gave a 2.5-fold stimulation at 10 microM. Agonists of vasopressin were also tested and gave differential responses. Studies using a V1 agonist (PIOVP) and a V2 agonist (DAVP) showed DAVP matching the level of stimulation elicited by vasopressin whereas treatment with PIOVP only reached 50% of the vasopressin response. These data suggested the presence of V2 receptors in the RPE cells. Several proteases were tested for their ability to stimulate RPE inositol phosphates. Thrombin caused a 7-fold increase in inositol phosphate formation at 1 U/ml, whereas trypsin and plasmin elicited smaller responses (approximately 2-fold). The thrombin effect was blocked by the thrombin-specific inhibitor, hirudin, but not by other protease inhibitors. Several mediators of inflammation such as bradykinin, histamine and serotonin were also tested, and they were ineffective in stimulating inositol phosphate turnover in the RPE cells.

Natriuretic peptide receptors on human trabecular meshwork cells

The effects of natriuretic peptides on cGMP formation and [125I]ANP binding in human trabecular meshwork cells were investigated. CNP at 1 microM stimulated cGMP formation approximately 18-25 fold, with a half maximal effective concentration approximately 20-30nM. BNP at 1 microM stimulated approximately 7 fold, while ANP stimulated cGMP formation 2-fold at 1 microM but had little or no effect at concentrations below 1 microM. Displacement binding of [125I]ANP to intact TM cells in the presence of unlabeled ANP indicated a single binding site with a dissociation constant approximately 0.15nM.c-ANP, which binds specifically to natriuretic peptide C receptors, displaced > 95% [125I]ANP binding to surface receptor sites with a half-maximal effective concentration comparable to that of ANP or BNP. c-ANP had no inhibitory effect on CNP stimulation of cGMP formation. The data suggest that human TM cells possess natriuretic peptide B receptors as the primary guanylyl cyclase-containing subtype and C receptors as the numerically predominant subtype of natriuretic peptide receptors.

Histamine H1 receptor occupancy triggers inositol phosphates and intracellular calcium mobilization in human non-pigmented ciliary epithelial cells.

Agonists and antagonists of histamine were used to characterize the stimulation of inositol phosphates formation and elevation of intracellular Ca2+ by histamine in cultured non-pigmented epithelial (NPE) cells from human ciliary body. Agonists specific for the H1 histamine receptor subtype were 20- to 200-fold more potent than the H2-specific agonists tested, and 5-16% as potent as histamine in inositol phosphates stimulation. An H1 antagonist was 10,000-fold more potent than an H2 antagonist in blocking histamine stimulation of inositol phosphates. H1 agonists also mimicked and H1 antagonists inhibited the elevation of intracellular Ca2+ by histamine. The first phase of the Ca2+ response to histamine was largely independent of extracellular Ca2+ while the second phase required extracellular Ca2+. Dose-response curves for histamine elevation of intracellular Ca2+ (EC50 = 10 microM, maximum at 100 microM) and inositol phosphates (EC50 = 2 microM, maximum at 100 microM) were similar. These data support the characterization of the NPE histamine receptor as an H1 receptor linked to elevation of inositol phosphates and intracellular Ca2+.