摘要:

Taurine uptake into cultures of human retinal pigment epithelial (HRPE) cells was monitored for 7 days after seeding. A culture medium containing 16% fetal bovine serum (FBS) was used for 2 days and switched to one with 8% FBS. Uptake of taurine (25 nM) was approximately 1.5 pmol/mg protein/15 min for 3 days, then decreased by 45% and was maintained at a decreased level till the 7th day. When the 16% FBS medium was used for the entire culture period, a similar profile of taurine uptake was observed but decrease of the uptake started on the 3rd day. Treatment of cells with 100 ng/ml cholera toxin (CT) for 24 h between the 6th and 7th days returned taurine uptake to its high level observed at the beginning of the cell culture. A similar CT treatment of cells between the 2nd and 3rd days enhanced taurine uptake significantly but this enhancement was much smaller. CT increased taurine uptake in treatment-time and dose dependent manners. Forskolin (FSK) (10 mM) and 8-Bromocyclic adenosine 3′,5′-monophosphate (1 mM) also increased taurine uptake. KT5720 at 1μM, a selective inhibitor of cAMP-dependent protein kinase (PKA), partially blocked CT-induced enhancement of taurine uptake. The level of cAMP was higher on the 3rd day than the 7th day but its response to 3-isobutyl-1-methyl-xanthine, FSK and CT was similar on both days. A kinetic analysis revealed that CT treatment decreases the apparent Michaelis-Menten constant of the taurine transporter while the drastic reduction of taurine uptake during the cell culture period is due to a decrease in the maximal velocity. The results show that cAMP elevated by CT treatment enhances taurine uptake via an increase in the affinity of the transporter. The decrease of taurine uptake during the culture period seems to be related to a decrease in the amount of the transporter.

ISSN:0271-3683    

DOI:10.3109/02713689609007616

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Calcium-activated potassium current was studied in cultured rabbit retinal pigment epithelial (RPE) cells using whole-cell and single channel patch-clamp recording techniques. When K+was the principal cation in the electrode, depolarizing voltage steps from a holding potential of–60 mV activated outwardly rectifying current. Outward K+current was increased by the Ca2+ionophore ionomycin and reduced when the extracellular Ca2+concentration was decreased from 2.5 mM to 100 nM in the presence of ionomycin. Outward K+current recorded in the presence of ionomycin was blocked by iberiotoxin and by charybdotoxin. Single channel recording from cell-attached and excised membrane patches revealed a large conductance Ca2+-activated K+(K(Ca)) channel. Identification of K(Ca)channels was based on: 1) the voltage-dependence of channel opening; 2) the large unitary conductance (>200 pS with symmetrical 130 mM K+); 3) the dependence of the reversal potential on the K+gradient; and 4) increased channel opening after exposure of the cytosolic surface of excised membrane patches to elevated Ca2+. These results demonstrate that Ca2+-activated K+channels are present in rabbit RPE cells and may play an essential role in the regulation of membrane potential and ion transport.

ISSN:0271-3683    

DOI:10.3109/02713689609007617

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Antimetabolites such as 5-fluorouracil and mitomycin C are known to delay wound healing. Epidermal growth factor (EGF) has been shown to accelerate corneal epithelial wound healing. This study was designed to investigate the effects of EGF on corneal epithelial healing that has been modified by antimetabolite treatment.New Zealand White rabbits were pretreated with either saline (controls) or 5-fluorouracil (5FU) injected subconjunctivally, or mitomycin C (MMC) applied topically. Circular anterior stromal wounds were created, followed by a 6-hour perfusion of normal saline or 50μg/ml of EGF. Subconjunctival saline or 5FU, or topical MMC treatments were continued after wounding for a total of 6 days. Corneas were photographed and quantitative morphometry of the wound site was performed.Compared with saline controls, MMC significantly delayed wound healing (P0.05). Compared with 5FU, MMC significantly delayed wound healing with either normal saline or EGF perfusion (P0.05).Corneal wound healing is not affected by subconjunctivally injected 5FU while it is delayed by topically applied MMC. EGF treatment does not overcome the inhibitory effects of MMC. EGF therapy may not be useful in the treatment of complications related to antimetabolite therapy.

ISSN:0271-3683    

DOI:10.3109/02713689609007618

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Proliferative vitreoretinopathy (PVR) is characterized by the proliferation and migration of retinal pigment epithelial (RPE) cells in the vitreous cavity. The drug hypericin, which is already in clinical use as an antidepressant, has shown promise as an antiviral and antineoplastic agent. To investigate the therapeutic potential of hypericin in PVR, we incubated RPE cells in standard medium with various serum concentrations containing 0.5 to 5μM hypericin. In some experiments we studied the effects of hypericin in conjunction with the RPE growth stimulating cytokine tumor necrosis factor alpha (TNF-α). Dose-dependent inhibition of RPE cell proliferation with IC50values of 0.7μM and 3.3μM in 1% and 5% serum respectively, was found. Even in conjunction with TNF-α, hypericin inhibited RPE proliferation with an IC50value of 1.5μM. The drug inhibited PKC activity in cells treated with a 2.5μM dose by 72% after 30 min and by 100% after 180 min. Finally, hypericin induced RPE cells to undergo apoptotic cell death, as shown by the presence of DNA laddering. These results suggest that hypericin may have potential as a therapeutic drug for PVR and that its antiproliferative and apoptotic effects on RPE cellsin vitroare in part mediated by PKC.

ISSN:0271-3683    

DOI:10.3109/02713689609007619

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

This study describes the effects of three cryopreservation media on the specific activity of corneal endothelial plasma membrane Na+,K+ATPase activity, a transporter required for the fluid pump in the cornea. Bovine corneal endothelial cell cultures were used as a model system for these studies. Cryopreserved primary cells were thawed and passaged once to increase cell number. The specific activity plasma membrane Na+,K+ATPase activity was subsequently measured on 4–6 replicate cultures. One freeze/thaw cycle depleted the Na+,K+ATPase specific activity of corneal endothelial cell cultures by approximately 90%, as compared to cells of equivalent passage which had not been cryopreserved. Cell morphology of the cryopreserved cultures was indistinguishable from that of control cultures. In other experiments, first passage cultures which had not been subjected to cryopreservation were incubated with a dimethyl sulfoxide-, glycerol-, or propane diol-based freezing medium and Na+,K+ATPase was measured on plasma membranes subsequently isolated from the cultures. Incubation of cells with cryopreservation media in the absence of the freezing process also depleted Na+,K+ATPase by approximately 90%. Radiolabeled ouabain was used to measure Na+,K+ATPase sites on cell cultures pretreated with dimethyl sulfoxide-based freezing media. A 4 h treatment with DMSO-based freezing medium had no effect on ouabain binding; treatment for 18 h reduced binding by only 50%. Thus, the method used to assess pump function (determination of Na+,K+ATPase specific activity versus ouabain binding) may provide conflicting data concerning the level of pump function cultured cells. The cryoprotectants present in many common media used to freeze tissue culture cells appear to inhibit corneal endothelial Na+,K+ATPase. Since the fluid pump of corneal endothelial cells is coupled to Na+,K+ATPase activity, care must be taken to insure that pump function is not impaired during cryopreservation of cell cultures.

ISSN:0271-3683    

DOI:10.3109/02713689609007620

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

The purpose of this research was to evaluate the effect of age on protective antioxidant enzyme activity of normal fresh cadaver human retina of the macula and periphery. Antioxidant enzymes were assayed in tissue extracts generated from 5 mm trephined punches of retina obtained centered over the macula and the superior midperiphery of normal fresh human cadaver retina. Cadaver tissue was obtained from donors of a wide age range (age 7 to 85 years). The assays were performed within 6 h of enucleation and within 24 h of donor death. Antioxidant enzymes assayed included superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase. Hexokinase and glucose-6-phosphate dehydrogenase, enzymes not directly involved in protection against oxidative damage, were assayed for comparison. Enzyme specific activities were calculated for the macula and periphery using protein concentration of the extract as the denominator. Using linear regression analysis, over the age range of 25 to 75 years, superoxide dismutase activity of the periphery but not the macula tended to decline with age (p = 0.04, R2= 0.21). Interindividual variability was high, and variability increased with age. The difference between the macular and peripheral enzyme activities for glutathione peroxidase tended to decline with increasing donor age (p = 0.025, R2= 0.33). There was no effect of age on the specific activities of catalase, glucose-6-phosphate dehydrogenase, and glutathione reductase. The specific activity of hexokinase from the macula declined with increasing donor age (p = 0.022, R2= 0.43). Time from death to enucleation or beginning of experiment was not a significant factor.In summary, age does not have an effect on the activity of major antioxidant enzymes of the macula in normal human retina. There is a tendency for an effect of age on peripheral superoxide dismutase activity and the difference between macular and peripheral glutathione peroxidase activity. High interindividual variability of antioxidant enzyme activity exists in humans.

ISSN:0271-3683    

DOI:10.3109/02713689609007621

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Previous research has indicated that the lacrimal gland may be a target organ for sex steroids and that androgen effects on this tissue may be inhibited by pituitary deficiency or diabetes. To extend these findings, the objectives of the current investigation were 3-fold: [a] to determine whether specific and high-affinity binding sites for androgens and estrogens exist in rat lacrimal tissue; [b]to assess whether the number and affinity of androgen binding sites in the lacrimal gland may be influenced by hypophysectomy or acute diabetes; and [c] to examine whether androgen receptor mRNA may be detected in lacrimal tissues of a variety of species. Following the collection of lacrimal gland samples, tissues were processed for the conduct of equilibrium binding methods or molecular biological techniques. Our results demonstrated that a single class of saturable, high-affinity and stereochemically selective binding sites for androgens exist in lacrimal tissues of male and female rats. These sites possessed a dissociation constant of approximately 1 nM and were also present in isolated acinar epithelial cells. In contrast, we were unable to find any evidence for the presence of specific or high-affinity receptors for estrogens in the rat lacrimal gland. With regard to changes in the endocrine environment, hypophysectomy led to an increase in the number and affinity of androgen binding sites in rat lacrimal tissue cytosol, whereas diabetes reduced the total quantity of these sites. Of interest, androgen receptor mRNA was detected in lacrimal glands of mice, rats, hamsters, guinea pigs, rabbits and humans. Overall, our findings show that the lacrimal gland is a target organ for androgens and that androgen action in this tissue may be mediated through an interaction with specific and high-affinity binding sites.

ISSN:0271-3683    

DOI:10.3109/02713689609007622

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

We evaluated the ability of Lensmeter 701 (LOM) to detect changes in the transparency of the lens graded with the Lens Opacities Classification System II (LOCS II). In this prospective study 410 middle-aged Eastern Finnish men participating in the Kuopio Atherosclerosis Prevention Study were examined three times at eighteen month intervals, and lens opacities were graded with both LOM and LOCS II, the latter serving as the standard.Majority of the change in the LOM reading during the follow-up fell within the 95% tolerance interval of the apparatus (3.08 units). Only four eyes showing progression by LOCS II were detected by LOM. The association between LOM change and the change observed by LOCS II was not statistically significant, and the correspondence of the two methods was weak. It seems that the sensitivity of the LOM is not sufficient to detect small changes in the transparency of the lens over time.

ISSN:0271-3683    

DOI:10.3109/02713689609007623

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

To investigate the role of lymphocytes in the pathogenesis of uveitis, we analyzed the expression of memory markers, CD29 and CD45RO antigens, and apoptosis-related Fas antigen on T lymphocytes in the aqueous humor (AH) and peripheral blood (PB) from patients with uveitis. Using three-color flow cytometry, we assessed the number of T lymphocyte subsets that stained with fluorescence-conjugated anti-CD3, CD4, CD8, CD29, CD45RA, CD45RO, HLA-DR, and Fas monoclonal antibodies in the AH and PB from 19 patients with active uveitis who were diagnosed as having sarcoidosis, Vogt-Koyanagi-Harada disease, HLA-B27+uveitis, or idiopathic uveitis. Cells from AH and PB were evaluated by light and electron microscopy before and after 6 h of incubation. The majority of lymphocytes in AH but not in PB, were CD3+HLA-DR+(activated) T cells. The percentage of CD4+lymphocytes was significantly higher in uveitic AH than in PBL (P<0.01). While the percentage of CD4+CD45RA+(naive) cells within T cells was much lower in uveitic AH than in PB, the percentage of CD4+CD29+or CD4+CD45RO+(memory) cells was significantly higher in uveitic AH than in PBL (P<0.01). Fas antigen was expressed preferentially on memory cells in uveitic AH. Apoptosis of cells in the AH was observed by microscopically following after incubation with no stimulation. Lymphocytes from the AH of patients with uveitis were more activated than those from PB. The majority of T lymphocytes from uveitic AH expressed memory markers and Fas antigen. Results suggest that an increase in the number of Fas+memory T lymphocytes in AH is involved in the pathogenesis of uveitis.

ISSN:0271-3683    

DOI:10.3109/02713689609007624

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

The nucleotide sequence of the sheep homologue of the lens-specific mouse connexin50, chicken connexin45.6, and human connexin50 has been obtained following screening of a sheep genomic library. This connexin comprises 1323 nucleotides, coding for a protein of 440 amino acid residues and a predicted molecular weight of 49,160 daltons, so by convention is termed sheep connexin49. A connexin49 cDNA probe detected a single major band with a mobility of 6.8 kb in sheep lens RNA, but not in RNA isolated from five other sheep organs. The N-terminal amino acid sequence of sheep connexin49 is identical to that of mouse connexin50 and closely matches that of MP70, indicating the identity of sheep connexin49 with MP70. The nucleotide and translated amino acid sequences of connexin49 have 69–87% and 76%–87% identity respectively with chicken connexin45.6, human connexin50 and mouse connexin50. Like other members of this lens connexin family, sheep connexin49 coding region is completely contained within one exon, and the sequence of the N-terminal region, the four transmembrane domains and the two extracellular loops are highly conserved.

ISSN:0271-3683    

DOI:10.3109/02713689609007625

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor