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A F Clark

Publications and source records attributed to A F Clark.

At least 55 records · Page 3Linked to original sources
PubMed1996

Effects of muscarinic agents on cultured human trabecular meshwork cells.

Intracellular calcium measurements were performed in cultured human trabecular meshwork cells preloaded with the cell permeant dye fura 2-AM. Fluctuations in calcium levels were then monitored with microscope-based ratio fluorometry. Carbachol increased intracellular calcium in a dose-dependent manner; as did oxotremorine-M, aceclidine, and pilocarpine. Carbachol's effect was blocked by the non-selective muscarinic antagonist atropine, as well as by muscarinic receptor subtype-selective antagonists such as pirenzepine (M1-selective), p-fHHSiD (M3-selective), and 4-DAMP (M1, M3 subtypes). Rank order of potencies for the antagonists' effects was atropine = 4-DAMP > p-fHHSiD > pirenzepine, a profile suggesting that the M3 receptor subtype is essential in the carbachol effect. Phospholipase C activity was estimated via measurement of total production of inositol phosphates in cultured human trabecular meshwork cells pre-exposed to 3H-myoinositol. In these cells, carbachol also stimulated phosphoinositide production in a dose-dependent manner, and an antagonist profile similar to that seen for calcium response was obtained when carbachol was used as the effector. The data indicate that muscarinic effects on cultured human trabecular meshwork calcium mobilization and phospholipase C activity are mediated by an M3-like receptor subtype. Therefore, the muscarinic M3 receptor may play a role in trabecular meshwork cell function(s).

Adolescent↗
PubMed1996

Influence of testosterone on chondroitin sulphate proteoglycan in the rat prostate.

There are recognized interactions between prostatic stromal and epithelial cells. These interactions may be influenced by the composition of the extracellular matrix, which is composed of proteins such as collagen, laminin, fibronectin, and proteoglycans (PGs) such as chondroitin sulphate proteoglycan (CSPG). In our continuing studies on prostate biology, we examined the three lobes of the normal adult rat prostate, i.e., ventral, dorsal, and lateral, for CSPG by indirect immunofluorescence, using an immunospecific monoclonal antibody (CS-56) for the chondroitin sulphate (CS) moiety of the PG. Staining of the prostate sections with CS-56 antibody followed by labelling with IgG fluorescein isothiocyanate conjugate indicated strong fluorescent signals associated with the ventral lobe basement membrane. The signal was stronger and more continuous in the distal acini than in the proximal acini. The staining of the dorsal and lateral lobes was less intense than that of the ventral lobe. Following castration of the rats, the basement membrane staining became discontinuous. Androgen replacement by administration of testosterone propionate (TP) reversed the effects of castration. Quantification of the total CS content showed decreases of about 60% in the ventral and lateral lobes after castration. TP administration for 14 days increased the total CS content several fold above the values for castrated rats in all lobes. The results demonstrated that CS content was significantly higher for TP-treated animals, suggesting that the expression of prostate CSPG is regulated by androgens. This approach should be useful in the study of the extracellular matrix in prostate biology.

Animals↗
PubMed1996

Glycosaminoglycans in the three lobes of the rat prostate following castration and testosterone treatment.

Androgen dependence of glycosaminoglycans (GAGs) in the prostate was studied using tissue from intact (sham control), castrated, and androgen-treated castrated rats. GAGs from the ventral, dorsal, and lateral lobes of the prostate were isolated and characterized by cellulose electrophoresis using appropriate GAG standards and enzymatic digestion or nitrous acid hydrolysis. Androgen deprivation was initiated by castration and rats were sacrificed at various time intervals after 7 days castration. After castration, the total GAG content decreased in three prostate lobes. At day 7 after castration, the total hyaluronic acid (HA) content decreased by 74% (ventral lobe) and 34% (lateral lobe) compared with the sham control. No effect was observed for HA content in the dorsal lobe. Castration decreased the total heparan sulphate (HS), dermatan sulphate (DS), and chondroitin sulphate (CS) contents in the three prostate lobes at 0 days of treatment, except for the CS content in the dorsal and lateral lobes. Androgen replacement increased the total GAG contents in the three prostate lobes. At 14 days of testosterone propionate treatment, there were 9-, 6.8-, 4.1- and 3.7-fold increases in HA, HS, DS, and CS, respectively, in the ventral lobe. These increases were more rapid and profound in the ventral than in the dorsal and lateral lobes. These findings indicate that all GAGs are regulated by androgen and there may be lobe-specific differences in their regulation. This could be a function of the heterogeneous populations of cells in each lobe.

Animals↗
PubMed1996

Targeted inactivation of the Ren-2 gene in mice.

Several recent studies have demonstrated that ablation of genes of the renin-angiotensin system can have wide-ranging and sometimes unexpected effects. Renin is directly involved in blood pressure regulation and is encoded by a single gene in most mammals. Wild mouse strains and some inbred laboratory strains have a duplicated renin gene (Ren-2), the physiological significance of which is unclear. Significant differences exist in the structure and expression of these renin genes, but as yet, no distinct biological function that distinguishes these genes has been defined. We have used gene targeting to discover the effects of inactivating the duplicated (Ren-2) gene in strain 129 mice, and we show that mice lacking the Ren-2 gene are viable and healthy. There appear to be no histopathological differences in renin-expressing tissues between Ren-2-null mice and their controls. Studies of our Ren-2-null mice allow, for the first time, a direct evaluation of the ability of the Ren-1d gene to regulate blood pressure in the absence of expression of the Ren-2 enzyme. We observed no alteration to blood pressure in adult mice homozygous for the mutated Ren-2 gene, even though the concentration of active renin is increased and of prorenin is decreased in plasma of these mice. Ren-1d is therefore capable of regulating normal blood pressure and despite a different tissue expression profile, is functionally equivalent to Ren-1c.

Animals↗
PubMed1996

Inhibition of dexamethasone-induced cytoskeletal changes in cultured human trabecular meshwork cells by tetrahydrocortisol.

PURPOSE: To determine the cellular mechanism of action of the intraocular pressure (IOP) lowering steroid tetrahydrocortisol (THF). METHODS: Tetrahydrocortisol was evaluated for glucocorticoid antagonist activity using in vitro and in vivo assays. Systemically administered THF was evaluated for its ability to inhibit dexamethasone-induced body weight loss and systemic hypertension in rats. In vitro receptor antagonism was tested using the supernatant fraction of IM9 cells as the source of soluble glucocorticoid receptor in 3H-dexamethasone displacement binding assays. In addition, six different primary human trabecular meshwork (TM) cell lines were cultured for 0 to 14 days in the absence or presence of dexamethasone (10(-7) M) and/or THF (10(-6) to 10(-8) M). The effects of these steroids on the TM cytoskeleton were determined by epifluorescent microscopy and by transmission electron microscopy. RESULTS: Tetrahydrocortisol was unable to inhibit the dexamethasone (DEX)-induced systemic hypertension and decrease in body mass in rats and was unable to displace 3H-DEX from the soluble human glucocorticoid receptor. However, THF inhibited the DEX-induced formation of cross-linked actin networks in cultured human TM cells in a progressive and dose-dependent manner (IC50 = 5.7 x 10(-7) M). Dexamethasone caused changes in the TM cell microtubules that were reversed partially by concomitant treatment with THF. Tetrahydrocortisol alone appeared to increase microfilament bundling in TM cells. CONCLUSIONS: Tetrahydrocortisol was not a glucocorticoid antagonist at the level of the classical glucocorticoid receptor and did not appear to antagonize systemically mediated glucocorticoid activity in the rat. Tetrahydrocortisol inhibited DEX-induced changes in the TM microfilaments and microtubules. These results may explain partially the IOP lowering activity of THF because glucocorticoid-mediated changes in the TM cytoskeleton have been proposed to be involved in the generation of ocular hypertension.

Actins↗
PubMed1995

Dexamethasone-induced ocular hypertension in perfusion-cultured human eyes.

PURPOSE: Glucocorticoid administration can lead to the development of ocular hypertension and corticosteroid glaucoma in a subset of the population through a decrease in the aqueous humor outflow facility. The purpose of this study was to determine whether glucocorticoid treatment can directly affect the outflow facility of isolated, perfusion-cultured human eyes. METHODS: The anterior segments of human donor eyes from regional eye banks were placed in a constant flow, variable pressure perfusion culture system. Paired eyes were perfused in serum-free media with or without 10(-7) M dexamethasone for 12 days. Intraocular pressure was monitored daily. After incubation, the eyes were morphologically characterized by light microscopy, transmission and scanning electron microscopy, and scanning laser confocal microscopy. RESULTS: A significant increase in intraocular pressure developed in 13 of the 44 pairs of eyes perfused with dexamethasone with an average pressure rise of 17.5 +/- 3.8 mm Hg after 12 days of dexamethasone exposure. The contralateral control eyes, which did not receive dexamethasone, maintained a stable intraocular pressure during the same period. The outflow pathway of the untreated eyes appeared morphologically normal. In contrast, the dexamethasone-treated hypertensive eyes had thickened trabecular beams, decreased intertrabecular spaces, thickened juxtacanalicular tissue, activated trabecular meshwork cells, and increased amounts of amorphogranular extracellular material, especially in the juxtacanalicular tissue and beneath the endothelial lining of the canal of Schlemm. The dexamethasone-treated nonresponder eyes appeared to be morphologically similar to the untreated eyes, although several subtle dexamethasone-induced morphologic changes were evident. CONCLUSION: Dexamethasone treatment of isolated, perfusion-cultured human eyes led to the generation of ocular hypertension in approximately 30% of the dexamethasone-treated eyes. Steroid treatment resulted in morphologic changes in the trabecular meshwork similar to those reported for corticosteroid glaucoma and open angle glaucoma. This system may provide an acute model in which to study the pathogenic mechanisms involved in steroid glaucoma and primary open angle glaucoma.

Aged↗
PubMed1994

Electrophysiological properties of cultured human trabecular meshwork cells.

Previous studies using cultured bovine trabecular meshwork cells demonstrated at least two different cell types which were distinguishable morphologically and electrophysiologically. The purpose of the present study was to evaluate the electrical membrane properties of cultured human trabecular meshwork cells. Seven different human trabecular meshwork cell lines were grown from four different donors. One trabecular meshwork cell line was transformed by microinjection with SV40 DNA. The electrochemical properties of these TM cells were determined by micropuncture with glass microelectrodes. The mean membrane voltage at resting conditions differed between the cell lines (-33.3 to -58.7 mV). Application of 10-mmol l-1 Ba2+ induced repetitive voltage spikes in all cell lines. The voltage transients similar to action potentials were inhibited by nifedipine, but insensitive to tetrodotoxin. Acetylcholine evoked depolarizations in three cell lines which were blocked by atropine. In one cell line isoproterenol caused sustained depolarizations sensitive to metipranolol. All three of the cell lines tested depolarized upon application of the vasoactive peptide endothelin-1. All untransformed cell lines showed voltage spikes typical for smooth muscle cells and functional receptors for endothelin-1 and cholinergic agonists. One out of three cell lines tested possessed beta-adrenergic receptors influencing the membrane voltage.

Acetylcholine↗
PubMed1994

Preliminary characterization of a transformed cell strain derived from human trabecular meshwork.

Cells isolated from the trabecular meshwork (TM) of a male glaucoma patient were transformed by transfection with an origin defective mutant of SV40 virus. Transformation dramatically increased the growth rate of these cells (designated HTM-3 cells), allowing biochemical and pharmacological characterization. The HTM-3 cells had cytoskeletal components that were reported to be present in TM tissue and non-transformed TM cells. Vimentin, tubulin and smooth muscle specific alpha-actin, but not desmin, were localized in these cells by immunocytochemistry. The extracellular matrix components collagen types I, III and IV, fibronectin and laminin were found in HTM-3 cells as well as their non-transformed parental cells. As predicted, the protein profile of the HTM-3 cells revealed by two-dimensional gel electrophoresis was different from that of the non-transformed cells, probably due to the enhanced growth characteristics of these cells. Furthermore, HTM-3 cells had various intracellular second messenger systems that responded to pharmacological agents. Forskolin, prostaglandin E2, beta-adrenergic and adenosine A2 agonists stimulated the adenylyl cyclase in these cells, whereas muscarinic, serotonergic, dopaminergic and other agonists were ineffective. Sodium nitroprusside increased the intracellular concentration of cGMP, demonstrating the presence of a functional guanylyl cyclase. Phospholipase C activity in these cells was also detected. Muscarinic agonists, histamine and bradykinin, but not adrenergic, serotonergic agonists or prostaglandins, increased phosphoinositide turnover. These drug responses of HTM-3 cells agree with published data on primary TM cells and TM tissues, suggesting that the transformed cells may be a valid substitute for certain pharmacological studies of TM.

Aged↗
PubMed1994

Glucocorticoid-induced formation of cross-linked actin networks in cultured human trabecular meshwork cells.

PURPOSE: To determine the effects of glucocorticoid treatment on the microfilament structure of cultured human trabecular meshwork cells. Topical or systemic administration of glucocorticoids can lead to the development of ocular hypertension and to the development of vision loss, which is clinically similar to primary open angle glaucoma. However, the mechanism(s) by which glucocorticoids cause ocular hypertension is not well defined. Alterations in the trabecular meshwork, the site of drainage of aqueous humor from the eye, have been linked to the development of ocular hypertension. METHODS: Human trabecular meshwork cells were cultured in the presence and absence of glucocorticoids for 0 to 21 days. The microfilament organization of the cultured trabecular meshwork cells was examined by epifluorescent and transmission electron microscopy. RESULTS: Glucocorticoids caused a progressive change in the organization of microfilaments in the trabecular meshwork cells, but not in other cultured ocular cells. By fluorescence microscopic analysis, the actin stress fibers found in control trabecular meshwork cells were reorganized on treatment with glucocorticoids into cross-linked actin networks that resembled geodesic-dome-like polygonal lattices. The cross-linked actin networks were reversible on withdrawal of the glucocorticoid treatment. Dose-response data for dexamethasone, relative ranking of activity with glucocorticoid potency, and partial inhibition with glucocorticoid antagonists all suggest the involvement of the trabecular meshwork glucocorticoid receptor in cross-linked actin network formation. The reorganization of the trabecular meshwork cytoskeleton alters cell function because glucocorticoid treatment of cultured trabecular meshwork cells also inhibited trabecular meshwork cell migration and proliferation. CONCLUSION: The steroid-induced alteration in trabecular meshwork cytoskeleton may be an important factor in the development of steroid-induced ocular hypertension and may play a role in the ocular hypertension associated with primary open angle glaucoma.

Actin Cytoskeleton↗
PubMed1993

Dexamethasone induced ultrastructural changes in cultured human trabecular meshwork cells.

Glucocorticoid-induced ocular hypertension has been demonstrated in both animals and humans. It is possible that glucocorticoid-induced changes in trabecular meshwork (TM) cells are responsible for this hypertension. In order to elaborate further the effect of glucocorticoids on the trabecular meshwork, the ultrastructural consequences of dexamethasone (DEX) treatment were examined in three different human TM cell lines. Confluent TM cells were treated with 0.1 microM of DEX for 14 days, and then processed for light, epifluorescent microscopy or transmission electron microscopy (TEM). The effect of DEX treatment on TM cell and nuclear size was quantified using computer assisted morphometrics. Morphometric analysis showed a significant increase in both TM cell and nuclear size after 14 days of DEX treatment. Epifluorescent microscopy of rhodamine-phalloidin stained, control TM cells showed the normal arrangement of stress fibers. In contrast, DEX-treated TM cells showed unusual geodesic dome-like cross-linked actin networks. Control TM cells had the normal complement and arrangement of organelles as well as electron dense inclusions and large vacuoles. DEX-treated TM cells showed stacked arrangements of smooth and rough endoplasmic reticulum, proliferation of the Golgi apparatus, pleomorphic nuclei and increased amounts of extracellular matrix material. The DEX-induced alterations observed in the present study may be an indication of the processes that are occurring in the in vivo disease process.

Actins↗
PubMed1992

Angiostatic activity and metabolism of cortisol in the chorioallantoic membrane (CAM) of the chick embryo.

There is considerable interest in the discovery of compounds which inhibit angiogenesis dependent (neovascular) diseases. The chick embryo, due to the rapid development of an extensive vascular capillary network in the chorioallantoic membrane (CAM), has been used extensively as a model for studying angiogenesis. Angiostatic steroids are a new class of compounds which inhibit the growth of new capillaries in the chick CAM and in other models of neovascularization. Despite the potential therapeutic importance of these compounds, little is known about the ability of the CAM to metabolize these steroids. We have evaluated the ability of the chick CAM to metabolize cortisol which is both an angiostatic steroid as well as a glucocorticoid. When CAM homogenate was incubated with [3H]cortisol and NADPH at 37 degrees C and pH 7.4, and the reaction products analyzed by reverse phase HPLC, [3H]cortisol was converted exclusively to 20 beta-dihydrocortisol (4-pregnen-11 beta,17 alpha,20 beta,21-tetrol-3-one). The cortisol metabolite, 20 beta-dihydrocortisol, has very little glucocorticoid activity, but shows significant angiostatic activity in the CAM comparable to cortisol. The apparent Km determined for cortisol metabolism was 12 microM and the observed Vmax was 1.4 mumol cortisol/mg protein/min. The majority of the 20 beta-reductase activity was found in the soluble (242,000 g) fraction of CAM homogenate. 20 beta-Reductase activity in chick embryo CAM has not been previously reported.

Allantois↗
PubMed1992

The effects of dexamethasone on fibronectin expression in cultured human trabecular meshwork cells.

Topical administration of glucocorticoids to the eye can lead to the development of ocular hypertension. This increase in intraocular pressure is caused by the heightened resistance to flow of aqueous humor from the eye, presumably at the trabecular meshwork (TM). This study reports the effects of dexamethasone (DEX) on the expression of the extracellular matrix protein fibronectin (FN) in cultured human TM cells (HTM). The expression of FN was evaluated in four HTM cell strains by epifluorescence microscopy and immunoblotting and autofluorography of electrophoretically separated cell proteins. There was a heterogeneous response of the four cell strains tested. Treatment of cell strain HTM4 with DEX (10(-7) mol/l) for 17 d caused an approximate doubling of cell-associated and secreted FN. This DEX-induced increase in FN expression was progressive after the first 7 d of treatment and was blocked partially with a glucocorticoid antagonist, cortexolone. By contrast, DEX treatment induced an intermediate 50-60% increase in FN expression in cell strains HTM10 and HTM2; in HTM6, FN was unchanged after exposure to the glucocorticoid. This model system may be useful to examine molecular changes associated with corticosteroid-induced ocular hypertension and evaluate glaucomatous changes in the TM because increased FN deposition occurs in the aqueous humor outflow pathway of patients with open-angle glaucoma.

Adolescent↗
PubMed1991

Epithelial-stromal interactions in the regulation of rat ventral prostate function: identification and characterization of pathways for androgen metabolism in isolated cell types.

Androgen metabolism plays a significant role in the androgen regulation of prostate cell function. In this report the various pathways for androgen metabolism in primary cultures of rat ventral prostate epithelial and stromal cells were identified and characterized by in vitro whole cell assays, using HPLC. Confluent cultures of both cell types were incubated with supraphysiological concentrations (50 nM) of tritiated androgens (testosterone, 5 alpha-dihydrotestosterone, 5 alpha-androstane-3 alpha(and 3 beta), 17 beta-diols, and delta 4-androstene-3,17-dione), and the metabolites were analyzed at several time points over a 24-h period. The metabolism studies indicated that 5 alpha-reductase activity, the oxidative reactions of 3 alpha-, 3 beta-, and 17 beta-hydroxysteroid oxidoreductases, and the reductive reaction of 3 beta-hydroxysteroid oxidoreductase were expressed at significantly higher levels in epithelial cells compared to stromal cells. The reductive reactions of 3 alpha- and 17 beta-hydroxysteroid oxidoreductases were similar in both cell types. In contrast, stromal cells exhibited substantially higher levels of 6 alpha/7 alpha-hydroxylase activity. In addition, stromal cells were capable of metabolizing 5 alpha-dihydrotestosterone directly to a new unidentified polar androgen metabolite (HO5 alpha-DHT). Overall, epithelial cells were approximately 29 times more capable than stromal cells of forming the biologically active androgen 5 alpha-dihydrotestosterone. Conversely, stromal cells were more capable of forming biologically inactive polar androgen metabolites.

17-Hydroxysteroid Dehydrogenases↗
PubMed1990

Nocturnal faecal soiling and anal masturbation.

Two cases of late onset faecal soiling as a result of anal masturbation in children who were neither mentally handicapped nor psychotic were studied. The role of soiling in aiding the young person and his family to avoid separating and maturing is highlighted. We suggest that the association of anal masturbation and resistant nocturnal soiling may be unrecognised.

Adolescent↗
PubMed1990

Differential androgen modulation of acid phosphatase isozymes in primary cultures of rat ventral prostate epithelial and stromal cells.

The influence of androgen on prostate differentiated cell function was investigated using primary cultures of rat ventral prostate epithelial and stromal cells developed from sexually immature animals (21 days of age). As a biochemical marker of androgen action, total acid phosphatase activity, which comprises both the secretory and lysosomal isoforms, was measured. Testosterone increased total acid phosphatase activity approximately 2-fold in epithelial cell cultures. This increase occurred only after the cessation of cell proliferation (i.e. upon reaching a confluent monolayer). In contrast, stromal cells showed no significant change in total acid phosphatase activity in response to androgen. Polyacrylamide gel isoelectric focusing of total acid phosphatase activity from epithelial and stromal cell extracts revealed that secretory acid phosphatase activity was localized exclusively in the epithelial cells while lysosomal acid phosphatase activity was present in both cell types. Furthermore, the androgen-induced increases in epithelial total acid phosphatase activity were found to result from increases in the secretory isoform.

Acid Phosphatase↗
PubMed1989

Histopathologic analysis of experimental autoimmune uveitis attenuated by intracameral injection of S-antigen.

Pretreatment of rats and mice with intracameral injections of soluble protein antigens induces Anterior Chamber Associated Immune Deviation, an unusual spectrum of immune reactivities that is dominated by suppression of the efferent limb of the immune response. Pretreatment of Lewis rats with an intracameral injection of S antigen (S Ag) mitigates against the development of clinically detectable experimental autoimmune uveitis if these rats are subsequently injected with a uveitogenic dose of S Ag. In this report, we describe a series of histopathologic studies designed to determine the extent and severity of changes that occur in the eyes of rats pretreated with intracameral S Ag. The results indicate that pretreatment with intracameral injection of S Ag alone or mixed with Complete Freund's Adjuvant significantly inhibited the development of severe uveoretinitis. In the eyes of the majority of rats, the histologic appearance was completely normal, without any evidence of pathologic changes. A minority of rats displayed clinical evidence of uveitis, and their eyes contained histopathologic changes. These changes suggested that a mild form of uveoretinitis had taken place. It is proposed that S Ag-specific suppression of delayed hypersensitivity is induced by the anterior chamber injection of this antigen, and that this suppressor activity accounts for the reduced severity of disease in the eyes of treated rats. The potential physiologic significance of suppression in response to exposure to unique ocular antigens is discussed.

Animals↗