Keith G. Duncan

Cathepsin B Is a Prorenin Processing Enzyme

Conversion of prorenin to renin results from proteolytic cleavage of a 43-amino-acid prorenin prosegment in renal juxtaglomerular cells. The enzyme that performs this processing is not known. Of several enzymes proposed, cathepsin B is a candidate because it colocalizes with renin in juxtaglomerular cell secretory granules and accurately cleaves the prosegment of human prorenin in vitro. It is not known whether cathepsin B can perform this function in the cell. We examined this using secretory granule-containing rat GH4C1 cells transfected with a human preprorenin expression vector. When treated with secretagogue (KCl 50 mmol/L + forskolin 10 micromol/L), these cells secrete 95% prorenin and 5% active renin into the medium, indicating little prorenin processing activity. In contrast, when the cells are cotransfected with a vector that expresses human preprocathepsin B or mouse prohormone convertase 1, secretagogue-induced secretion of active renin increased to 12% and 16.5%, respectively. With antisera that recognize the prosegment and renin, prorenin and renin were identified as proteins of 47 and 43 kD, respectively, and an antibody specific to the prosegment precipitated only the 47-kD species. These results do not address whether cathepsin B is the authentic renal prorenin processing enzyme. However, the results do demonstrate that cathepsin B can localize to the appropriate subcellular compartment and process prorenin to renin in GH4C1 cells and are consistent with a role for this enzyme in prorenin processing.

Expression of reverse cholesterol transport proteins ATP-binding cassette A1 (ABCA1) and scavenger receptor BI (SR-BI) in the retina and retinal pigment epithelium

Aims: Excessive lipid accumulation in Bruch’s membrane (BrM) is a hallmark of ageing, the major risk factor for age-related macular degeneration (AMD). Retinal pigment epithelial (RPE) cells may utilise reverse cholesterol transport (RCT) activity to move lipid into BrM, mediated through ATP-binding cassette A1 (ABCA1) and scavenger receptor BI (SR-BI). Methods: ABCA1 expression was assessed by reverse transcription polymerase chain reaction (RT-PCR) and western blotting of human RPE cell extracts. Lipid transport assays were performed using radiolabelled photoreceptor outer segments (POS). ABCA1 and SR-BI expression was examined in normal mouse eyes by immunofluorescence staining. BrMs of ABCA1 and SR-BI heterozygous mice were examined microscopically. Results: Human RPE cells expressed ABCA1 mRNA and protein. The ABCA1 and SR-BI inhibitor glyburide (also known as glibenclamide) abolished basal transport of POS-derived lipids in RPE cells in the presence of high-density lipoprotein. Mouse retina and RPE expressed ABCA1 and SR-BI. SR-BI was highly expressed in RPE. BrMs were significantly thickened in SR-BI heterozygous mice, but not in ABCA1 heterozygous mice. Conclusion: RPE cells express ABCA1 and SR-BI. This implies a significant role for SR-BI and ABCA1 in lipid transport and RCT in the retina and RPE.

High density lipoprotein mediated lipid efflux from retinal pigment epithelial cells in culture

Backgound/aim: The transport of radiolabelled photoreceptor outer segments (POS) lipids was investigated by cultured retinal pigment epithelial cells (RPE). Phagocytosis of POS by the RPE is essential to maintain the health and function of the photoreceptors in vivo. POS are phagocytised at the apical cell surface of RPE cells. Phagocytised POS lipids may be either recycled to the photoreceptors for reincorporation into new POS or they may be transported to the basolateral surface for efflux into the circulation. Results: The authors have demonstrated that high density lipoprotein (HDL) stimulates efflux of radiolabelled lipids, of POS origin, from the basal surface of RPE cells in culture. Effluxed lipids bind preferentially to HDL species of low and high molecular weight. Effluxed radiolabelled phosphotidyl choline was the major phospholipid bound to HDL, with lesser amounts of phosphatidyl ethanolamine, phosphatidyl inosotol. Effluxed radiolabelled triglycerides, cholesterol, and cholesterol esters also bound to HDL. Lipid free apolipoprotein A-I (apoA-I) and apoA-I containing vesicles also stimulate lipid efflux. Conclusion: The findings suggest a role for HDL and apoA-I in regulating lipid and cholesterol transport from RPE cells that may influence the pathological lipid accumulation associated with age related macular degeneration.

Experimental study of tetrodotoxin, a long-acting topical anesthetic

PURPOSE To determine the effectiveness and toxicity of tetrodotoxin for use as a long-acting topical anesthetic. METHODS Four groups of six rabbits each received a 40-microl aliquot of either tetrodotoxin in one of three concentrations (10 mM, 1 mM, or 0.1 mM) or proparacaine 0.5% into the inferior conjunctival cul-de-sac of one eye, with the fellow eye of each rabbit receiving 40 microl of a 60-mM, pH 4.3 sodium citrate vehicle as a control. Corneal sensation was tested for up to 8 hours after administration of drugs, and response was noted by no blink, partial blink without full eyelid closure, and full blink. Slit-lamp examination at 12 and 24 hours after administration and pachymetry before and 24 hours after administration were performed to detect corneal toxicity. RESULTS Rabbits receiving all three concentrations of tetrodotoxin did not demonstrate any ocular irritation, corneal thickening, or signs of systemic toxicity. At a dose of 10 mM, tetrodotoxin produced an anesthetic effect lasting up to 8 hours. At 1 mM, tetrodotoxin was an effective but shorter-acting anesthetic. At 0.1 mM, tetrodotoxin had no significant anesthetic effect. Proparacaine-treated rabbits initially were anesthetic, but this effect was largely gone by 1 hour and completely gone by 3 hours. CONCLUSIONS Tetrodotoxin is a long-acting topical anesthetic in the rabbit cornea. Although additional toxicity studies are required, tetrodotoxin may provide an effective, long-lasting topical anesthetic for use in pain control after corneal procedures such as photorefractive keratectomy.

Molecular biology of human renin and its gene.

The molecular biology of renin, prorenin, and the renin gene have been studied. A tissue-specific pattern of expression was found in rat and human tissues. In the human placenta, the transfected and endogenous renin promoters are active, and renin mRNA levels and transfected promoter activity are increased by a calcium ionophore plus cAMP. Cultured pituitary AtT-20 cells transfected with a preprorenin expression vector mimick renal renin release by converting prorenin to renin and releasing renin in response to 8Br-cAMP. Studies with mutant renin genes suggest that the body of renin directs renin to the regulated secretory pathway, and renin glycosylation affects its trafficking. Chinese hamster ovary cells were used to produce recombinant prorenin. Infused prorenin was not converted to renin in monkeys. Renin crystals were used to determine its three-dimensional structure. Renin resembles other aspartyl proteases in the active site and core, but it differs in other regions that probably explain renins unique substrate specificity. Based on structural and mutational analysis, a model for human prorenin was built that suggests lysine -2 of the prosegment interacts with active site aspartate residues, and that the prosegment inactivation of renin is stabilized by binding of an amino terminal beta strand into a groove on renin.

Hypothermia reduces secretion of vascular endothelial growth factor by cultured retinal pigment epithelial cells.

Aim Visual loss in age-related macular degeneration usually develops secondary to choroidal neovascularisation. Vascular endothelial growth factor (VEGF) is a critical regulator of retinal angiogenesis and vascular permeability, especially in hypoxic conditions. We hypothesise that hypothermia may reduce the retinal pigment epithelium (RPE) metabolism and, consequently, the levels of VEGF secretion by cultured RPE cells under hypoxic conditions. The effects of hypothermia were compared with the metabolic inhibiting effects of thiopental and nicotinamide. Methods ARPE-19 cells were grown in culture for up to 5 days under normoxic (20% O2) and hypoxic (1% O2) conditions at temperatures ranging from 27°C to 40°C. For experiments with pharmacological agents, thiopental and nicotinamide were added to the media. VEGF levels in the media were measured by ELISA and cell metabolic activity was measured by a fluorescent cell metabolic assay. Results We found that hypothermia reduced ARPE-19 cell metabolism in a temperature-dependent fashion. Hypothermia also reduced ARPE-19 cell VEGF secretion in a temperature-dependent fashion. ARPE-19 cell VEGF secretion was reduced by 38% at 34°C compared with cells grown at 37°C. Conversely, ARPE-19 cell VEGF secretion was increased by 32% at 40°C compared with cells grown at 37°C. Hypoxia increased ARPE-19 cell VEGF secretion by 84% at 37°C. However, hypothermia decreased the hypoxia-induced increase of ARPE-19 cell VEGF secretion by 30%. The effect of hypothermia on ARPE-19 cell VEGF secretion was reversible after 4 days. In contrast to hypothermia, thiopental and nicotinamide were able to reduce RPE cell metabolism but not VEGF secretion. Conclusion Hypothermia decreases both VEGF secretion and cellular metabolism in ARPE-19 cells. Hypothermia also mitigates the hypoxia-induced increase in ARPE-19 cell VEGF secretion. These effects of hypothermia are potentially unique and cannot be obtained by other pharmacological agents that slow cellular metabolism.

Corneal Toxicity of Intraocular Hyaluronidase

The purpose of this study was to examine the corneal toxicity of different preparations of intraocular hyaluronidase. SDS-PAGE analysis of bovine testicular hyaluronidase (Wydase) and chromatographically purified hyaluronidase (Sigma) was performed. These two preparations were injected into the anterior chamber of rabbits in amounts ranging from 1.5-150 IU (Wydase) and 1.5-300 IU (Sigma). A third set of rabbit eyes received Wydase vehicle alone or in combination with Sigma hyaluronidase. Treated control eyes were injected with saline. Slit lamp examination and indirect ophthalmoscopy were performed preoperatively and on postoperative days 1 and 7. Light microscopy of the corneas was performed. SDS-PAGE of Wydase revealed numerous protein impurities, while Sigma demonstrated one protein band consistent with mammalian hyaluronidase. Persistent corneal edema, severe anterior chamber fibrin, and endothelial necrosis, were seen in the majority of eyes injected with Wydase in amounts of 50 IU and greater (n = 11). Thirty percent (30%) of the eyes injected with the Sigma preparation (n = 11) had localized corneal opacity similar to 50% of eyes injected with saline (n = 2). Of the rabbit eyes injected with the Wydase vehicle (n = 19), 68% had toxic changes. Intracameral injection of Wydase is toxic to the rabbit cornea in amounts of 50 IU and greater. A chromatographically purified preparation showed only transient local toxicity. Toxicity of Wydase may be due to protein impurities and the thimerosal-containing vehicle.

Human trabecular meshwork cells as a thyroid hormone target tissue: presence of functional thyroid hormone receptors

Abstract. · Purpose: To deter- mine whether human trabecular meshwork cells (HTM) are a po- tential target tissue for thyroid hormone (3,3′,5-triiodothyronine, T3). · Methods: Cultured HTM were assayed for the presence of thyroid hormone receptors (TRs) and retinoid X receptors (RXRs) by reverse-transcriptase polymerase chain reaction (RT-PCR) to detected TR and RXR mRNA, and by immunohistochemistry to detect nuclear TR and RXR proteins. Functionality of the TR was determined by analysis of 125I-T3 binding affinity and capacity in HTM nuclear extracts. Effects of T3 on modulation of hyaluronic acid (HA) levels in HTM were measured as a function of dose and duration of T3 administration. · Results: Analysis of RT-PCR and immunohistochemistry demonstrated that cultured HTM expressed TRα1, TRα2, and TRβ1 but not TRβ2; and RXRα but not RXRβ and RXRγ isoforms. Saturation binding and analysis of 125I-T3 to HTM nuclear extracts revealed Kd of 57 pM. The number of T3 binding sites extrapolated from a Scatchard plot was 7.3×1010/μg of HTM nuclear protein extract. T3 supplementation reduced the concentration of HA in the cell medium by 32–43% compared to cells grown in the absence of T3. · Conclusions: Cultured HTM express three TR isoforms and one RXR isoform, bind T3 with an affinity similar to that of TR in responsive cells, and modulate their HA production in response to T3. These findings indicate that the human trabecular meshwork tissue has the capacity to respond to thyroid hormones.

Tetrodotoxin: anesthetic activity in the de-epithelialized cornea.

Abstract · Background: Tetrodotoxin (TTX) binds with high affinity to sodium channels and produces local anesthesia. We investigated whether TTX is an effective, long-acting corneal anesthetic in rabbits. · Methods: After mechanical debridement of the central corneal epithelium, topical TTX (1 mM, 0.1 mM, or 0.01 mM) was applied to one eye each of 18 New Zealand White rabbits. The fellow eye of each rabbit was treated with control vehicle. Blink response to a mechanical stimulus was assessed. Blink response was also assessed every 3 h for 30 h in 6 rabbits treated with 1 mM TTX administered every 6 h. In a separate group of 12 rabbits with central epithelial debridement, the rate of epithelial healing was compared between animals treated with topical 1.0 mM TTX and animals receiving no treatment. · Results: After 4 h, eyes treated with 1.0 mM and 0.1 mM TTX were anesthetic. At 6 h, five of six rabbit eyes treated with 1.0 mM TTX were still partially anesthetic. By 8 h, the mean anesthesia score for 1.0 mM TTX was approaching normal. With multiple dosing, all six rabbit eyes remained anesthetic for the duration of the experiment. There was no significant difference in the rate of re-epithelialization between eyes treated with TTX and untreated controls. There was no evidence of systemic or local toxicity from topical TTX. · Conclusion: In a rabbit model, TTX is a long-acting topical anesthetic that retains its effectiveness when administered repeatedly over 24 h and does not inhibit epithelial healing. It may have application in management of pain after photorefractive keratectomy.

Experimental use of tetrodotoxin for corneal pain after excimer laser keratectomy.

PURPOSE To determine the duration of anesthesia, effect on corneal reepithelialization, and systemic toxicity of topical tetrodotoxin (TTX) administered after excimer laser keratectomy. METHODS Two groups of six rabbits each underwent excimer laser keratectomy in the right eye to create a 5-mm-diameter wound, 75 mm in depth. One group then received a 40-microl aliquot of topical 1 mM TTX into the injured eye, whereas the other group received 40 microl of the sodium citrate vehicle as a control. The rabbits were treated with TTX or vehicle again at 6, 12, 18, and 24 h. Corneal sensation was tested at 3, 6, 9, 12, 15, 18, 21, 24, 30, 32, and 40 h. To determine whether TTX inhibited corneal reepithelialization, compared with vehicle-treated control eyes, the healing rate of the epithelial defect was measured. RESULTS Administration of TTX every 6 h for 24 h produced nearly complete anesthesia for > or = 30 h. At 32 h, 8 h after the final application of TTX, there was still significant anesthesia of the TTX-treated corneas (p = 0.0325, Wilcoxon test). Normal corneal sensation in all TTX-treated animals returned at 40 h, or 16 h after the final dose. In contrast, vehicle-treated eyes all had normal sensation for nearly the entire duration of the experiment. At 40 h, the TTX-treated eyes had slightly larger defects than vehicle-treated eyes, 7.85+/-1.74 versus 4.54+/-1.31 mm2 (p < 0.025, t test). However, at 49 h and thereafter, both groups were equally healed (p > 0.05, t test). No systemic toxicity was observed in any of the rabbits. CONCLUSION Topical TTX is a long-acting and nontoxic local anesthetic in a rabbit model of excimer laser keratectomy.