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J R Polansky

Publications and source records attributed to J R Polansky.

At least 37 records · Page 2Linked to original sources
PubMed1997

Cellular pharmacology and molecular biology of the trabecular meshwork inducible glucocorticoid response gene product.

Studies of the effects of glucocorticoid (GC) and oxidative stress stimuli in differentiated cultures of human trabecular meshwork (HTM) cells have provided the rationale for our studies of a major new gene termed TIGR (trabecular meshwork inducible GC response). The TIGR clone was isolated by differential library screening using selection criteria based on the induction pattern of a new protein/glycoprotein found in HTM cultures after prolonged but not brief exposure to GCs. This GC induction pattern matched the time course and dose response required for intraocular pressure elevation in patients receiving corticosteroids. The very large, progressive induction of TIGR combined with specific structural features of its cDNA suggested that TIGR should be considered a candidate gene for outflow obstruction in glaucoma. Among the properties of TIGR cDNA were a signal sequence for secretion, several structural features for interactions with glycosaminoglycans and other glycoproteins and putative sites for cell surface interactions. In addition, the leucine zippers in the structure were related to TIGR-TIGR oligomerization that was shown to occur with native and recombinant TIGR protein. The verification that TIGR was a major stress response protein in HTM cells following hydrogen peroxide (or phorbol esters) exposure provided a potential link between GC and oxidative mechanisms thought to be involved in glaucoma pathogenesis. Pharmacological evaluation showed that basic fibroblast growth factory and transforming growth factor beta decreased the GC induction of TIGR, and certain nonsteroidal anti-inflammatory drugs protected against both GC- and oxidation-induced stress responses in HTM cells. Our recent studies of TIGR's genomic structure have shown motifs in the promoter region that suggest a basis by which multiple hormonal/environmental stimuli can regulate TIGR production and by which putative genetic alterations could lead to an overexpression of the protein. Further application of cell biology/biochemistry, molecular biology, genetic and histological approaches will be helpful in understanding the role of TIGR in different glaucoma syndromes.

Aged↗
PubMed1997

Hyaluronan in the human trabecular meshwork.

PURPOSE: Hyaluronan (HA) is a high-molecular weight glycosaminoglycan that can affect water and solute fluxes in the extracellular matrix. The distribution of HA in the human trabecular meshwork of nonglaucomatous eyes was examined to help understand the potential role of HA in the regulation of aqueous outflow resistance. METHODS: Histolocalization of HA was established in situ in the trabecular meshwork of human eyes with no known diseases of the anterior segment. A specific biotinylated HA-binding peptide was used as a probe for this study, with enhanced sensitivity of HA detection achieved by modifications of the fixation and staining procedures. RESULTS: Evaluation of HA staining in the aqueous outflow pathway in comparison to that in other ocular structures (e.g., the vitreous) showed pronounced staining in the trabecular meshwork. The staining intensity was similar between various layers of the meshwork. Both the filtering and the anterior nonfiltering portions of the trabecular meshwork showed pronounced HA staining. The staining was localized primarily to the trabecular meshwork endothelial cells. CONCLUSIONS: Pronounced HA staining observed in the various layers of the trabecular meshwork suggests that substantial amount of HA is present in the nonglaucomatous outflow pathway. The staining pattern suggests that HA is associated with the endothelial cells lining the trabecular beams. This finding supports potential roles for this glycosaminoglycan in the regulation of the physiological aqueous outflow resistance or in the maintenance of the outflow channels or both. Histochemical localization of HA in the various layers of the non-glaucomatous meshwork provides a useful basis for future comparative studies of HA distribution and relative amounts in the trabecular meshworks of eyes affected by various types of glaucoma.

Aged↗
PubMed1996

Halogen lamp phototoxicity.

BACKGROUND: The radiation from quartz halogen light bulbs has been reported to cause erythema in humans and skin tumors in experimental animals. OBJECTIVE: The objective of this study was to determine the relative risk of injury to humans due to the radiation from these lamps. METHODS: We studied a 12-volt 50-watt quartz halogen lamp. We measured its ultraviolet output spectrum and the biological toxicity of its radiation by means of its pyrimidine dimer-inducing capability. RESULTS: At a 1-cm distance, the 254-nm light output of the lamp is 3 x 10(-7) W/cm2/nm. Solar radiation at this wavelength is undetectable on the earth's surface, i.e., less than 10(-12) W/cm2/nm. At 290 nm, the lamp output is approximately 3 x 10(-6) W/cm2/nm, 5,000 times greater than the summertime sun at 1 p.m. at sea level in Kuwait. The UVB and UVA output of the lamp is roughly similar to the solar output under these conditions. We also measured the pyrimidine dimer-forming potential of the lamps relative to the sun for the purpose of estimating the DNA toxicity of the lamps. At a 7-cm distance the lamp induces approximately 3.9 pyrimidine dimers/100,000 bp/min. This is approximately 4 times more rapid than that of the noontime summer sun in Michigan. CONCLUSIONS: We conclude that direct radiation from these lamps can cause damage to human skin, due to their UVC and UVB output. We estimate that the relative risk to keratinocyte DNA in human skin, in vivo, from exposure to the 50-watt halogen lamp at a distance of 7 cm is between 27 and 400% of the noontime summer sun in Michigan.

DNA↗
PubMed1996

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.

Adult↗
PubMed1994

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.

Adenylyl Cyclases↗
PubMed1994

Stimulation of Na+,K+,Cl- cotransport by forskolin-activated adenylyl cyclase in fetal human nonpigmented epithelial cells.

PURPOSE: To determine the stoichiometry of Na+,K+,Cl- cotransport in fetal human nonpigmented ciliary epithelial cells and the effect of forskolin, an adenylyl cyclase activator, on Na+,K+,Cl- cotransport. METHODS: 86Rb+ as a marker for K+ was used to study ouabain-insensitive, bumetanide-sensitive 86Rb+ uptake in cultured human nonpigmented epithelial (NPE) monolayers. RESULTS: The dependence of ouabain-insensitive, bumetanide-sensitive 86Rb+ uptake upon Na+, K+, and Cl- concentrations was determined. Maximal uptake was observed at about 12 mM, 20 mM, and 120 mM of these ions, respectively. Analysis by Hill plot suggested that the stoichiometry of Na+,K+,Cl- cotransport is 1:1:2, making this an electroneutral process. Na+,K+,Cl- cotransport was found to be stimulated approximately 1.5- to 2-fold after incubation of cells for 15 minutes with 1 microM forskolin. neither ouabain-sensitive 86Rb+ uptake nor bumetanide-insensitive, ouabain-insensitive uptake was affected. 8-Bromoadenosine cAMP and 8-chlorophenylthio cAMP at 1 mM stimulated Na+,K+,Cl- cotransport approximately 30% to 40%, whereas 1,9 dideoxyforskolin, a non-adenylyl cyclase-activating analogue of forskolin, had little effect. Stimulation of Na+,K+,Cl- cotransport by forskolin was blocked by prior exposure of cells to 10 microM H-89, a protein kinase A inhibitor. Stimulation by forskolin was also observed in the presence of either 1 mM DIDS, 30 microM NPPB, 3 mM DPC, or 5 mM BaCl2, although all four channel blockers inhibited Na+,K+,Cl- cotransport to various degrees. CONCLUSIONS: The data suggest that the human NPE Na+,K+,Cl- cotransporter transports Na+, K+, and Cl- in the ratio of 1:1:2. Activation of adenylyl cyclase stimulates Na+,K+,Cl(-)-cotransport via a mechanism involving protein kinase A. Reduction of Na+,K+,Cl- cotransport by chloride channel blockers raises the possibility that activities of some ion channels can influence the rate of ion influx via Na+,K+,Cl- cotransport.

Adenylyl Cyclases↗
PubMed1992

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.

Angiotensin II↗
PubMed1992

Potassium transport in nonpigmented epithelial cells of ocular ciliary body: inhibition of a Na+, K+, Cl- cotransporter by protein kinase C.

The mechanisms by which 86Rb+ (used as a tracer for K+) enters human nonpigmented ciliary epithelial cells were investigated. Ouabain-inhibitable bumetanide-insensitive 86Rb+ transport accounted for approximately 70-80% of total, whereas bumetanide-inhibitable ouabain-insensitive uptake accounted for 15-25% of total. K+ channel blockers such as BaCl2 reduced uptake by approximately 5%. Bumetanide inhibited 86Rb+ uptake with an IC50 of 0.5 microM, while furosemide inhibited with an IC50 of about 20 microM. Bumetanide-inhibitable 86Rb+ uptake was reduced in Na(+)-free or Cl(-)-free media, suggesting that Na+ and Cl- were required for optimal uptake via this mechanism. These characteristics are consistent with a Na+, K+, Cl- cotransporter in NPE cells. Treatment of NPE cells for 15 min with phorbol 12-myristate, 13-acetate (PMA), an activator of protein kinase C, caused a 50-70% decrease in 86Rb+ uptake via the Na+, K+, Cl- cotransporter. Other 86Rb+ uptake mechanisms were not affected. 86Rb+ uptake via the Na+, K+, Cl- cotransporter could be inhibited by other phorbol esters and by dioctanoylglycerol, an analog of diacylglycerol, but not by 4 alpha phorbol didecanoate, an ineffective activator of protein kinase C. Staurosporine, a protein kinase C inhibitor, blocked phorbol ester inhibition of 86Rb+ uptake. These data suggest that a Na+, K+, Cl- cotransporter in NPE cells is inhibited by activation of protein kinase C.

Carrier Proteins↗
PubMed1992

Thrombin stimulates inositol phosphate formation, intracellular calcium fluxes and DNA synthesis in cultured fetal human non-pigmented ciliary epithelial cells.

Thrombin at concentrations as low as 20 pM (0.002 U ml-1) was found to stimulate inositol phosphate levels in cultured human non-pigmented ciliary epithelial cells. Several other proteases, including trypsin and plasmin, had little or no effect, of several protease inhibitors tested, only those with specificity for thrombin blocked the effect. Studies with active site-blocked thrombin suggested that the esterolytic active site of thrombin is required for inositol phosphate stimulation, while gamma-thrombin, which has reduced binding affinity to fibrinogen also showed reduced effectiveness in stimulating inositol phosphates. In the presence of 10 mM LiCl, thrombin stimulated inositol monophosphate, inositol bisphosphate and inositol trisphosphate formation, with a prolonged rise of the first and transient early rises in the latter two species. Thrombin also elevated intracellular Ca2+ levels as measured with the fluorescent calcium probe, indo-1-AM. This elevation could be blocked by prior addition to cells of the thrombin inhibitor, hirudin, and was dependent upon extracellular Ca2+ for the maintenance of an elevated level in the presence of thrombin. Incorporation of thymidine into DNA in confluent cultures was also stimulated by thrombin, with a four-fold increase in incorporation at 35 hr in thrombin-treated cells compared to controls. The half-maximal concentration for this process was 0.25 U ml-1. Pretreatment with 100 ng ml-1 pertussis toxin greatly reduced the thrombin effect, which is consistent with a role for a G-protein in stimulation of DNA synthesis by thrombin.

Binding Sites↗
PubMed1992

Neurotransmitters and neuropeptides stimulate inositol phosphates and intracellular calcium in cultured human nonpigmented ciliary epithelium.

The effects of several neurotransmitters and neuropeptides on the inositol phosphate/diacylglycerol pathway were examined in human nonpigmented ciliary epithelial cells. Maximal stimulation of inositol phosphate formation by vasopressin (approximately 3-fold), carbachol (approximately 2-fold) and histamine (approximately 5-fold) was observed only after cells had been confluent for at least six days. In contrast, a response to bombesin (approximately 3-fold) declined with extended time in confluent culture. Inositol monophosphate, inositol bisphosphate, and inositol trisphosphate all were stimulated by these agonists. Dose-response studies showed a close correlation between the EC50s of the different agonists when elevation of inositol phosphates was compared to stimulation of intracellular Ca2+, with the exception of bombesin. Preliminary pharmacologic characterization of the receptors for vasopressin, carbachol, and bombesin provided rank order of potencies for selective agonists and antagonists. The data suggest that the muscarinic receptor on human NPE cells is the M3 subtype, whereas the vasopressin receptor, as defined by its linkage to the inositol phosphate/diacylglycerol pathway, is the V1 subtype.

Analgesics↗
PubMed1991

Parasympathetic denervation of the ciliary muscle following panretinal photocoagulation.

Cynomolgus monkeys underwent unilateral panretinal scatter photocoagulation (PRP) and/or nasal and temporal horizontal retinal meridional photocoagulation (HRMP) with xenon arc or argon or krypton laser light. Shortly thereafter, in the PRP-treated eyes, accommodative responsiveness to topical eserine and electrical stimulation of the Edinger-Westphal nucleus (EWN) was diminished, accommodative responsiveness to intramuscular (i.m.) pilocarpine was enhanced, and the number of muscarinic receptors in the ciliary muscle was reduced compared to the contralateral controls. In most instances, these parameters returned to normal over 6-12 wks and the abnormalities could be induced again by another round of PRP. However, in some PRP-treated eyes, accommodative responsiveness to EWN stimulation and topical eserine remained subnormal permanently (greater than 1 yr). Shortly after HRMP alone, accommodative responses to i.m. pilocarpine, topical eserine, and central stimulation did not differ markedly in the treated and control eyes. Morphologic studies 1 to 78 wk following PRP revealed that myelinated and unmyelinated nerves within the entire circumference of the choroid and ciliary muscle were severely damaged early on. The number of unmyelinated nerves between the individual ciliary muscle fibers was drastically reduced, those which remained were swollen or deteriorated, and agranular synaptic vesicles were rarely seen. Thereafter, the nerves in the choroid and ciliary muscle gradually regenerated. Following HRMP, only the choroidal nerves which passed through the photocoagulated areas and the ciliary muscle nerves in the corresponding meridians showed signs of deterioration, and there was minimal effect on the physiologic responses examined. These findings collectively indicate that intraocular parasympathetic denervation of the ciliary muscle is produced by PRP, although all nerve types are likely damaged.

Accommodation, Ocular↗
PubMed1991

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+.

Calcium↗
PubMed1991

Accommodation and ciliary muscle muscarinic receptors after echothiophate.

Twice daily topical administration of echothiophate for 2 weeks to the eyes of living cynomolgus monkeys produced profound subsensitivity of the accommodative response to pilocarpine and an approximately 50% decrease in the number of specific binding sites for 3H-quinuclidinyl benzilate (3H-QNB) in the ciliary muscle without a change in their affinity. When echothiophate treatment was discontinued, functional cholinergic sensitivity and the number of QNB binding sites both returned to normal over a similar 4-8 week period. Most animals had a modest overshoot of both functional sensitivity and number of binding sites for at least several weeks thereafter. The treated to control eye ratios for the number of binding sites and accommodative response to pilocarpine were correlated and the plot of log treated to control binding site ratio versus treated to control accommodation ratio resembled a dose-response curve. Similarly, the treated versus control eye differences for the two parameters were correlated, with the regression line passing through the 0-0 axis intercept. Collectively, these findings suggest that agonist-induced modulation of functional cholinergic sensitivity in the parasympathetically innervated (as opposed to denervated) ciliary muscle occurs by a muscarinic receptor-mediated mechanism. This system appears to provide a useful model to study the regulation of ciliary muscle cholinergic sensitivity.

Accommodation, Ocular↗
PubMed1990

Ciliary muscle muscarinic binding sites, choline acetyltransferase, and acetylcholinesterase in aging rhesus monkeys.

Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activity, and the affinity and number of muscarinic binding sites (as reflected by specific 3H-quinuclidinyl benzilate binding) were determined in the ciliary muscle of rhesus monkeys ranging in age from 1-34 years. No age dependence was evident for any of these parameters. Within the limits of their specificity and precision, the data indicate that biochemical alterations in ciliary neuromuscular mechanisms do not account for the age-related loss of ciliary muscle configurational responses to topical pilocarpine and electrical stimulation of the Edinger-Westphal nucleus in the rhesus monkey.

Accommodation, Ocular↗
PubMed1989

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.

Cells, Cultured↗
PubMed1989

Eicosanoid production and glucocorticoid regulatory mechanisms in cultured human trabecular meshwork cells.

The techniques we developed to propagate HTM cells in serial cell culture have provided an opportunity to investigate the spectrum of endogenous PGs and other eicosanoids that are produced by these cells. PGE2 and PGF2 alpha were the major cyclooxygenase products detected by both radioimmunoassay and thin-layer chromatography. A small amount of 6-keto PGF1 alpha was also detected, indicating that these cells are able to produce prostacyclin. The observation of a substantial increase in the proportion of PGF2 alpha relative to PGE2 at later time periods after a media change suggests a metabolic conversion of PGE2 to PGF2 alpha by these cells. Bradykinin, thrombin, platelet activating factor, and serum were found to be effective stimulators of PG production by HTM cells, whereas calcium ionophore produced only a minor effect. Using high pressure liquid chromatography, elution profiles of radiolabeled metabolites of AA suggested the presence of certain lipoxygenase products, including LTB4, 12-HETE, 15-HETE, and a small amount of 5-HETE in HTM cells. The formation of these products was inhibited by both DEX and BW 755c, reinforcing the view that metabolic conversions of AA through the lipoxygenase pathway were possible in the trabecular meshwork. We also examined the effects of glucocorticoids on specific protein synthesis in the HTM cells, using 35S-methionine labeling and SDS-PAGE techniques. Short-term (1 day) DEX treatment revealed a major induction of a protein band at approximately 30 kDa. Longer treatments (1 to 3 weeks) resulted in major inductions at approximately 55 kDa inside the cells, with the presence of secreted forms (probably glycoproteins) between 55 and 72 kDa. The short-term DEX effect on protein synthesis a phospholipase inhibitor regulating eicosanoid production within the HTM. The longer-term induction may, on the other hand, be related more directly to the development of steroid glaucoma, based on our findings that the inductions of these proteins correlate with the observed time course and dose-dependence topical glucocorticoid effects on IOP. Continued in vitro and in vivo evaluations of the eicosanoid pathways in cultured HTM cells obtained from normal and glaucomatous human eyes may help to delineate their relationship to IOP regulation and the pathogenesis and treatment of glaucoma. Glucocorticoid-induced proteins may be key participants in the regulation of phospholipase activity and hence may represent a major control mechanism of the AA cascade.(ABSTRACT TRUNCATED AT 400 WORDS)

Cells, Cultured↗
PubMed1989

Kinetics of phagocytosis in trabecular meshwork cells. Flow cytometry and morphometry.

Confluent human trabecular meshwork (HTM) cells from three different donors and at various stages of serial passage were fed fluorescein-labeled polystyrene beads. Phagocytosis was monitored for up to 6 days using flow cytometry, fluorescence microscopy, and morphometric calculations from comprehensive electron microscopic observations at key time points. During the first 4 hr after initiation of phagocytosis, the confluent endothelial monolayer lost its cohesiveness and became segregated into separate cells. During the first 3 days the cells underwent marked and progressive changes in shape and size. After 4 days, some cells detached from the dish, as necrotic debris and degenerative changes appeared. The kinetics of phagocytosis in this stable, confluent monolayer showed that recruitment (the percentage of cells which had ingested at least one bead) proceeded semilogarithmically, with 50% of the cells recruited by 8 hr and 97% by 96 hr. The time course of phagocytosis (ie, the average number of beads phagocytosed per cell) is described by a sigmoidlike curve, reaching half-maximum at 40 hr and maximum (about 500 beads per cell) at 96 hr. The rate of uptake (ie, the first derivative of the average number of beads per cell) reached a peak (nine beads per cell per hr) at 24 hr and then decelerated slowly over the next 5 days. Cytochalasin B treatment, as a control, reduced phagocytosis by approximately 70%. Flow cytometry, when combined with electron microscopy, should provide a useful tool to examine phagocytosis in HTM cells exposed to steroids and other hormones and drugs.

Adult↗