Evaluation of occupation hot exposure in industrial workplaces in a subtropical country.

OBJECTIVES: The objective of this study has been to evaluate the occupational heat exposure of 12 workers at 5 plants in a subtropical country. MATERIAL AND METHODS: The heat stresses and strain on workers in 5 plants were assessed by the International Organization for Standardization (ISO) 7243 index (wet bulb globe temperature - WBGT) and the ISO 7933 index (maximum allowable exposure time - Dlim). RESULTS: Results indicated that 42% of the subjects (5 workers) surpassed the WBGT limits. According to the Dlim, 42% of the subjects could not continue working in the hot environments. The relationships between the various heat stress indices and the WBGT index were also correlated. However, further studies from different heat environments and more subjects should be performed. CONCLUSIONS: The sensitive dependence of skin temperature on meteorological and physiological indices for each subject was clearly observed. Obviously, the heart rate response to metabolic rate was much greater than that caused by environmental heat alone. The exponential relationship between workers' duration-limited exposure time, predicted by various estimated criteria, and WBGT were also found. Int J Occup Med Environ Health 2017;30(3):379-395.

5-Fluorouracil-Induced Apoptosis Changes in Cultured Corneal Epithelial Cells.

PURPOSE: Repeated subconjunctival injections with 5-fluorouracil (5-FU) after trabeculectomy are used in glaucoma patients for the inhibition of overproliferation in wound site. Thus, a certain amount of the drug may penetrate into epithelial layer, where it causes toxicity to corneal epithelial cells. The aim of this study was to evaluate the toxic effects of 5-FU and mechanisms of drug-induced apoptosis in cultured corneal epithelial cells. METHODS: Cellular damage and the caspase pathway were estimated with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The apoptotic characteristics were detected by flow cytometry, a TUNEL test, and western blotting in cultured corneal epithelial cells. RESULTS: The results indicated that 5-FU was toxic to corneal epithelial cells in a time- and dose-dependent manner. Pretreatment with a general caspase inhibitor (Z-VAD-FMK), a caspase-8 inhibitor (Z-IETD-FMK), and a caspase-9 inhibitor (Z-LEHD-FMK) reversed 5-FU-induced cellular damage. Following exposure to 5-FU, a flow cytometric assay with MitoLight dye demonstrated the significant loss of mitochondrial membrane potential. A positive TUNEL test revealed that cellular DNA apoptosis occurred following exposure to 0.5, 1, and 5 mg/mL 5-FU for 15 h. Positive annexin V-FITC and negative propidium iodide (PI) staining indicated that the cell membrane exhibited apoptosis upon exposure to 1 and 5 mg/mL 5-FU for 15 h. The western blot assay demonstrated upregulation of the p21 protein but downregulation of the Bcl-2 proteins induced by 5-FU. CONCLUSION: These data reveal that 5-FU-induced cellular apoptosis in corneal epithelial cells may be mediated through caspase-8, caspase-9, and mitochondria-regulated pathways, as well as by upregulation of p21 and downregulation of Bcl-2-dependent signal transduction pathways.

Determination of Oleanolic and Ursolic Acids in Hedyotis diffusa Using Hyphenated Ultrasound-Assisted Supercritical Carbon Dioxide Extraction and Chromatography.

Oleanolic acid (OA) and ursolic acid (UA) were extracted from Hedyotis diffusa using a hyphenated procedure of ultrasound-assisted and supercritical carbon dioxide (HSC-CO2) extraction at different temperatures, pressures, cosolvent percentages, and SC-CO2 flow rates. The results indicated that these parameters significantly affected the extraction yield. The maximal yields of OA (0.917 mg/g of dry plant) and UA (3.540 mg/g of dry plant) were obtained at a dynamic extraction time of 110 min, a static extraction time of 15 min, 28.2 MPa, and 56°C with a 12.5% (v/v) cosolvent (ethanol/water = 82/18, v/v) and SC-CO2 flowing at 2.3 mL/min (STP). The extracted yields were then analyzed by high performance liquid chromatography (HPLC) to quantify the OA and UA. The present findings revealed that H. diffusa is a potential source of OA and UA. In addition, using the hyphenated procedure for extraction is a promising and alternative process for recovering OA and UA from H. diffusa at high concentrations.

Ultrasound-assisted extraction and quantitation of oils from Syzygium aromaticum flower bud (clove) with supercritical carbon dioxide.

This study evaluated ultrasound-assisted supercritical carbon dioxide (USC-CO2) extraction for determining the extraction yields of oils and the contents of eugenol, ß-caryophyllene, eugenyl acetate and α-humulene from clove buds. Compared to traditional SC-CO2 extraction, USC-CO2 extraction might provide a 13.5% increase in the extraction yield for the oil while utilizing less severe operating parameters, such as temperature, pressure, CO2 flow rate and the time consumed by the process. Our results were comparable to those obtained using the heat reflux extraction method, though the yield was improved by 20.8% using USC-CO2. In kinetic studies, the USC-CO2 extraction of clove oil followed second-order kinetics. The activation energy for the oil extraction was 76.56kJ/mol. The USC-CO2 procedure facilitated the use of mild extraction conditions, improved extraction efficiency and the quality of products and is a potential method for industry.

Development of a hyphenated procedure of heat-reflux and ultrasound-assisted extraction followed by RP-HPLC separation for the determination of three flavonoids content in Scutellaria barbata D. Don.

A hyphenated procedure of heat-reflux and ultrasound-assisted extraction (HUAE), and an accurate high-performance liquid chromatographic (HPLC) method were developed for the determination of apigenin, baicalin and luteolin content in Scutellaria barbara D. Don. The suitable HUAE conditions for the extraction of target compounds from the herb were identified as an ultrasonic frequency of 40kHz, power of 185W, duty cycle of 75% (intermittent sonication), mean particle size of 0.355mm, extraction temperature of 50°C, ratio of solvent to raw material of 12:1 (mL/g), ethanol concentration of 60% (v/v), extraction time of 30min and three cycles. Compared with a traditional heat-reflux extraction method, the proposed method reduced the extraction time, extraction temperature and solvent consumption. Also, this HUAE method achieved superior apigenin, baicalin and luteolin yields. Furthermore, the developed HUAE-HPLC method was applied successfully for the simultaneous evaluation of three bioactive compounds in five samples of S. barbara D. Don obtained from different geographical regions. These results clearly demonstrated that the combined HUAE-HPLC process is feasible in the future commercialized manufacture of this highly valuable Chinese herbal medicine.

Novel usage of intraocular pressure-lowering drugs as wound-healing inhibitors after trabeculectomy with cell culture and animal models.

Inhibiting the healing of wounds to ensure that the aqueous humor can drain into the scleral space unimpeded and form a filtering bleb plays a crucial role in determining the success rate of glaucoma surgery. The aim of this study was to investigate the novel use, with cell culture and animal models, of some commercial intraocular pressure (IOP)-lowering drugs in inhibiting the healing of fibroblast wounds. The Tenon's fibroblasts of rabbits were cultured to evaluate 13 IOP-lowering drugs for cellular proliferation, collagen formation, and migration. These were measured using [(3)H]thymidine and [(3)H]proline uptake, and Transwell chambers. A preservative of benzalkonium chloride (BAK) was initially used, with 0.02% as a maximal original concentration. All of the drugs and the BAK were diluted from original commercial concentrations to 1/10, 1/100, and 1/1000. The more inhibitive drugs screened from the cell cultures were then selected for further short-term application during and after trabeculectomy surgeries had been performed on the rabbits. Expression of the proliferative cell nuclear antigen was immunohistochemically examined 3 and 7 days after surgery. The results revealed that the inhibitive effects of BAK in cellular [(3)H]thymidine and [(3)H]proline uptake, and cellular migration were only evident at 0.002% concentrations. Based on the results of the cell cultures, timolol, latanoprost, and unoprostone exhibited a greater inhibitory effect than the other drugs. Moreover, the animal studies showed that latanoprost and unoprostone significantly suppressed the positive expression of proliferative cell nuclear antigen around the operative excision area 7 days after the trabeculectomy surgeries. The results indicate that short-term use of some IOP-lowering drugs, such as latanoprost and unoprostone, may inhibit postoperative wound healing after glaucoma surgery.

Mechanism of carteolol-induced cytosolic Ca2+ mobilization in cultured vascular smooth muscle cells.

An increase in cytosolic calcium concentration triggers intracellular signal transduction in vascular cells, which then regulates the vascular contraction. In the present study, the regulatory mechanism of carteolol on the intracellular free Ca(2+) ([Ca(2+)](i)) mobilization was investigated in cultured A7r5 vascular smooth muscle cells. The A7r5 cells were cultured and loaded with fura-2-AM, which was used as a Ca(2+) sensitive fluorescent probe. In both the presence and absence of external Ca(2+), carteolol increased [Ca(2+)](i) with a dose-dependent manner in A7r5 cells at concentrations between 608 microM and 6.08 microM. In a Ca(2+)-containing buffer, carteolol-induced [Ca(2+)](i) showed an initial peak followed by a secondary and persistent plateau. Pretreatment of the cells with La(3+), the plasma membrane Ca(2+) pump inhibitor, and nifedipine, a L-type Ca(2+) channel inhibitor, both partially restrained the carteolol-induced initial peak in [Ca(2+)](i) by 92% and 86%, respectively. Pretreatment of the cells with adrenoceptor antagonists, prazosin inhibited the [Ca(2+)](i) response by 80%, and propranolol enhanced the response by 61%. In the Ca(2+-)-free buffer, pretreatment of the cells with carteolol inhibited the endoplasmic reticulum Ca(2+) pump inhibitor of thapsigargin-induced [Ca(2+)](i) increase by 97%. Pretreatment of the cells with thapsigargin also inhibited the carteolol-induced [Ca(2+)](i) rise by 98%. The internal Ca(2+) release induced by the carteolol was partially inhibited by U73122 (phospholipase C inhibitor) and aristolochic acid, quinacrine (phospholipase A(2) inhibitors). After incubation of carteolol in the Ca(2+)-free buffer, the addition of CaCl(2) increased the Ca(2+) influx, implying that the release of Ca(2+) from internal stores further induced capacitative Ca(2+) entry. These results suggest that carteolol-induced [Ca(2+)](i) increase is mediated by the initial influx via the alpha(1)-adrenoceptor, L-type Ca(2+) channel, nonselective calcium entry channels and release of Ca(2+) from an intracellular store, which is mainly in the endoplasmic reticulum followed by capacitative Ca(2+) entry but decrease via the beta(2)-adrenoceptor. The intracellular Ca(2+) release was also modulated by phospholipase A(2), C-coupled events.

Mechanism of mitomycin-induced apoptosis in cultured corneal endothelial cells.

PURPOSE: Previous studies have indicated that improper use of mitomycin C (MMC) caused cytotoxicity in corneal endothelial cells. The aim of the present study was to investigate whether MMC induces cellular apoptosis in corneal endothelial cells and to determine the mechanism by which this may occur. METHODS: Porcine corneal endothelial cells were acquired from primary culture. Cellular damage and caspase pathway were estimated with a MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay. The apoptotic characteristics were detected by means of flow cytometry, the TUNEL (terminal deoxyribonucleotidyl-transferase-(TdT)-mediated deoxyuridine-5'-triphosphate-digoxigenin (dUTP) nick-end labeling) test, immunofluorescent staining, and western blotting. RESULTS: The results indicated that MMC was toxic to corneal endothelial cells in a time-dependent and dose-dependent manner. Pretreatment with a general caspase inhibitor (Z-VAD-FMK), a caspase-8 inhibitor (Z-IETD-FMK), and a caspase-9 inhibitor (Z-LEHD-FMK) reversed MMC-induced cellular damage. Following exposure to MMC, a change in the mitochondrial membrane potential was positively detected by flow cytometric assay with MitoLight dye while cellular cytochrome c that was released from the mitochondria to the cytoplasm was detected by immunofluorescent staining. A positive TUNEL test revealed that cellular DNA apoptosis had occurred following exposure to 0.001 and 0.01 mg/ml MMC for 24 h. Positive annexin V-FITC, and negative propidium iodide (PI) staining indicated that the cellular plasma membrane underwent apoptosis following 0.001 mg/ml MMC exposure for 24 h. Western blot assay demonstrated down-regulation of the Bcl-2 protein and upregulation of the p53 and p21 proteins, which were all involved in apoptosis induced by MMC. CONCLUSIONS: These results indicate that mitomycin-induced cellular apoptosis in corneal endothelial cells may be mediated through caspase-8, caspase-9, and the mitochondrial regulated pathways as well as through upregulation of p53-dependent and p21-dependent signal transduction pathways.

Endothelin-1 enhances corneal fibronectin deposition and promotes corneal epithelial wound healing after photorefractive keratectomy in rabbits.

The objective was to study the effects of endothelin-1 (ET1) on corneal wound healing after photorefractive keratectomy (PRK) in rabbit corneas. Following PRK, 18 New Zealand white rabbits were treated with ET1 in the right eyes and with phosphate-buffered salt solution (PBS) in the left eyes. Corneal epithelial wound size, corneal haze and corneal thickness were recorded. Corneal extracellular matrixes, including collagen types 3, 4 and 7, chondroitin sulfate and fibronectin, were investigated using immunohistochemistry study. ET1 increased the rate of healing of corneal epithelial wounds in rabbits. Anti-fibronectin fluorescence was present at week 12 and week 24 in ET1-treated eyes but not in the control eyes. There were no significant differences in corneal haze, corneal thickness and changes in other extracellular matrixes between ET1- and PBS-treated eyes. ET1 can enhance the deposition of fibronectin in corneal stroma and promote corneal epithelial wound healing after PRK. The increase in fibronectin probably explains the increased healing rate of corneal epithelial wounds.

Mechanism of unoprostone-induced cytosolic Ca2+ mobility in cultured porcine corneal endothelial cells.

The effect of unoprostone isopropyl on the intracellular free Ca(2+) signaling ([Ca(2+)](i)) in cultured porcine corneal endothelial cells was evaluated by using fura-2-AM as a Ca(2+) probe. In Ca(2+)-containing buffer and Ca(2+)-free buffer, unoprostone increased [Ca(2+)](i) concentration dependently from 28.2 to 0.282microM, diluted from original concentration to 1/100, 1/1000 and 1/10,000. The response was inhibited on extracellular Ca(2+) removal. In Ca(2+)-free buffer, pretreatment of the cells with unoprostone inhibited the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin-induced [Ca(2+)](i) increase and the mitochondria uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP)-induced Ca(2+) release by 96 and 95%, respectively. Pretreatment of the cells with thapsigargin or CCCP also inhibited unoprostone-1-induced [Ca(2+)](i) rise by 84 and 57%, respectively. The intracellular calcium release caused by unoprostone was partially inhibited by phospholipase C inhibitor (U73122) and by phospholipase A(2) inhibitor aristolochic acid. After incubation of the cells with unoprostone in Ca(2+)-free buffer, the addition of 5mM CaCl(2) increased Ca(2+) influx, implying that release of Ca(2+) from internal stores caused by unoprostone further induced capacitative Ca(2+) entry. These results suggest that unoprostone-induced [Ca(2+)](i) increase in corneal endothelial cells results from Ca(2+) influx from external buffer and release of Ca(2+) from intracellular stores from the endoplasmic reticulum and mitochondria Ca(2+) stores followed by capacitative Ca(2+) entry. Unoprostone-induced intracellular Ca(2+) release was also modulated by phospholipase A(2) and C-coupled events.

Cellular cytotoxicity of antiglaucoma drugs in cultured corneal endothelial cells.

In this study, the various antiglaucoma drugs including betaxolol, timolol, levobunolol, carteolol, brimonidine, dipivefrin, dorzolamide, brinzolamide, latanoprost, unoprostone, and pilocarpine were used to investigate the effects of cellular cytotoxicity in cultured bovine corneal endothelial cells. After exposure to the drugs in three dilutions, 1/100, 1/1,000, and 1/10,000, for 100 minutes, cells were estimated based on the release assay of lactate dehydrogenase (LDH) enzyme. It was found that cellular LDH was significantly released in the medium only at 1/100th dilution of betaxolol, brimonidine, dorzolamide, dipivefrin, latanoprost and unoprostone to 130%, 123%, 145%, 157%, 128% and 237%, respectively, compared with controls upon exposure to drugs for 100 minutes. Moreover, benzalkonium chloride preservative at the concentrations ranging from 0.001 to 0.00001 mg/mL did not affect cellular LDH release in bovine corneal endothelial cells. These results indicate that high concentrations of antiglaucoma drugs may induce cytotoxicity in corneal endothelial cells.

Effects of antibiotics and corticosteroid eyedrops on cellular proliferation in cultured human corneal keratocytes.

The purpose of this study is to investigate the effects of antibiotics and glucocorticoid eyedrops, including gentamicin, sulfisomezole, fluorometholone, dexamethasone, and betamethasone, on cellular proliferation in cultured human corneal keratocytes. Human corneal keratocytes were cultured in RPMI-1640 containing 10% fetal bovine serum. Drugs were prepared from original concentrations to 1/10, 1/100, and 1/1,000 dilutions. After exposure to drugs for 100 minutes, cellular proliferation was estimated by [3H]-thymidine uptake. It was found that cellular proliferation in corneal keratocytes was not affected by any of the three dilutions of gentamicin but was inhibited by 1/10 and 1/100 dilutions of sulfisomezole to 82% and 90% of control. [3H]-thymidine uptake values were inhibited to 75% by 1/10 dilution of fluorometholone and by 1/10and 1/100 dilutions of betamethasone to 84% and 86% of control. Meanwhile, cellular proliferation was significantly inhibited by 1/10, 1/100, and 1/1,000 dilutions of dexamethasone to 82%,86%, and 90%, respectively, in comparison with control values. It was demonstrated that commercial eyedrops of glucocorticoids inhibit cellular proliferation in corneal keratocytes, which may modulate the wound healing of corneal stroma.

Effects of antiglaucoma drugs on cellular proliferation in cultured human corneal keratocytes.

The purpose of this study was to investigate the effects of various antiglaucoma drugs, including timolol, betaxolol, carteolol, levobunolol, dipivefrin, and pilocarpine, on cellular proliferation in cultured human corneal keratocytes. Human corneal keratocytes were cultured with RPMI-1640 medium containing 10% fetal bovine serum. Antiglaucoma drugs were prepared from original concentrations to dilutions of 1/10, 1/100, and 1/1,000. After exposure to drugs for 100 minutes, cellular proliferation was estimated by [3H]thymidine uptake methodology. It was found that cellular proliferation in corneal keratocytes was inhibited by only a 1/10 dilution of various drugs including timolol, betaxolol, carteolol, levobunolol, dipivefrin, and pilocarpine. The [3H]thymidine uptake values were significantly inhibited to 63%, 18%, 87%, 68%, 55%, and 67% by a 1/10 dilution of the above drugs. However, the cellular proliferation was also significantly suppressed by 0.01 mg/mL of benzalkonium chloride preservative. It is shown that the inhibition of cellular proliferation by high concentrations of antiglaucoma drugs may result from the benzalkonium chloride preservative contained in these drugs.

Effects of antiglaucoma drugs on calcium mobility in cultured corneal endothelial cells.

The aim of this study was to estimate the effects of various antiglaucoma drugs including betaxolol, timolol, levobunolol, brimonidine, carteolol, dipivefrin, dorzolamide, brinzolamide, latanoprost, unoprostone, and pilocarpine on intracellular free Ca2+ ([Ca2+]i) mobility in cultured bovine corneal endothelial cells. Various antiglaucoma drugs were diluted from original concentrations to 1/ 100, 1/ 1,000, and 1/ 10,000. The [Ca2+] mobility was studied by spectrofluorophotometry after loading with the ester of fura-2 (fura-2/AM). It was found that timolol (58 microM and 5.8 microM), levobunolol (171 microM, 17.1 microM, and 1.71 microM), betaxolol (162 microM, 16.2 microM, and 1.62 microM), carteolol (680 microM and 68 microM), dipivefrin (28 microM and 2.8 microM), dorzolamide (616 microM and 61.6 microM), brinzolamide (260 microM), latanoprost (1.1 microM), unoprostone (28.2 microM, 2.82 microM, and 0.282 microM), and pilocarpine (408 micro and 40.8 microM) induced a significant increase in [Ca2+]i. Nevertheless, only brimonidine (68 microM and 6.8 microM) decreased [Ca2+]i concentration significantly. Benzalkonium chloride preservative did not affect [Ca2+]i after addition of 0.001, 0.0001 and 0.00001 mg/mL to cells. These results indicate that all antiglaucoma drugs may affect the physiologic function of corneal endothelial cells through change of [Ca2+]i mobility.

Effect of latanoprost on cultured porcine corneal stromal cells.

PURPOSE: Latanoprost reduces intraocular pressure mainly by enhancing uveoscleral outflow that may be involved in the decreased of extracellular matrixes such as collagens. However, the effect of latanoprost on corneal stromal cells is not well understood. In the current study, we investigated the changes of cultured porcine corneal stromal cells upon exposure to latanoprost. METHODS: Porcine corneal stromal cells were acquired from primary culture and maintained in fetal bovine serum-containing medium. Cells were estimated on 3H-thymidine, 3H-leucine, 3H-uridine, 3H-proline uptakes and migration. Dead and living cells were estimated with MTT assay. The changes of type 1 collagen and fibronectin proteins were detected by means of immunofluorescent staining and Western blot assay. Intracellular free Ca2+ ([Ca2+]i) mobility was studied by spectrofluorophotometer after loading with fura-2-AM. RESULTS: Latanoprost has remarkable effects inhibiting cultured corneal stromal cells on 3H-thymidine, 3H-leucine, 3H-uridine, 3H-proline uptakes and cellular migration. The inhibitory effects are in a dose-dependent manner at concentrations ranging from 10(- 5), 10(- 6), 10(- 7) to 10(- 8) M. The 50% inhibitory dosages (ID50) for latanoprost to corneal stromal cells, as measured by 3H-thymidine uptake, 3H-uridine uptake, 3H-leucine uptake, 3H-proline uptakes and cellular migration were 5.01 x 10(- 6) M, 2.81 x 10(- 6) M, 2.09 x 10(- 6) M, 3.89 x 10(- 7) M and 2.2 x 10(- 6) M, respectively. In the presence of latanoprost, the cellular MTT values were also decreased significantly. Immunofluorescent staining displayed that latanoprost changed type 1 collagen distribution in cultured corneal stromal cells. Western blot assay revealed that latanoprost caused cells to decrease in fibronectin protein. In Ca2+-containing buffer, latanoprost induced a significant rise in [Ca2+]i at 10(- 5) and 10(- 6) M. CONCLUSIONS: These results indicate that latanoprost may induce the morphological and biochemical changes in cultured corneal stromal cells. Long-term use of latanoprost needs to be carefully monitored for change in corneal stroma.

Inhibition of endothelin-1 and KCL-induced increase of [CA2+]i by antiglaucoma drugs in cultured A7r5 vascular smooth-muscle cells.

Over contraction of vascular smooth muscle may result in ischemia to ocular neuronal cells and deteriorate the glaucoma. The purpose of this study was to investigate the inhibitory effects of various commercial antiglaucoma drugs including brimonidine, dipivefrin, betaxolol, timolol, levobunolol, carteolol, brinzolamide, dorzolamide, unoprostone, latanoprost, pilocarpine, and preservative benzalkonium chloride on endothelin-1(ET-1) and KCl-induced increase of intracellular free Ca2+ ([Ca2+]i) in cultured rat A7r5 vascular smooth muscle cells. These drugs were diluted from original concentrations to 1/100, 1/1000, and 1/10000. [Ca2+]i mobility was analyzed by spectrofluorometry after loading with fura-2-AM. Betaxolol, timolol, levobunolol, and carteolol were found to inhibit KCl-induced release of [Ca2+]i in a dose-dependent manner. High concentrations of betaxolol, timolol, levobunolol, carteolol, and unoprostone also inhibited ET-1-induced increase of [Ca2+]i in A7r5 cells. However, ET-1- and KCl-induced increase of [Ca2+]i was not diminished by other drugs including brimonidine, dipivefrin, brinzolamide, dorzolamide, latanoprost, pilocarpine, and benzalkonium chloride. These results indicate that high concentrations of unoprostone and beta-adrenergic blocking agents including betaxolol, timolol, levobunolol, and carteolol may inhibit ET-1-induced increase of [Ca2+]i. The mechanism may be mediated by inhibition of extracellular calcium influx via blocking of L-type voltage-dependent Ca2+ channel in A7r5 cells.

Differential effects of endothelin-1 on calcium mobility in cultured porcine pigment epithelial cells of iris and retina.

BACKGROUND AND PURPOSE: Photoreceptor function depends on an intact retina pigment epithelial (RPE) cell layer. Transplantation of iris pigment epithelial (IPE) cells to the subretinal space offers a potential alternative to RPE transplantation in patients with severe retinal diseases such as macular degeneration. Whether the functions of IPE and RPE are similar or different is not well established. The purpose of this study was to identify the characteristics of IPE cells and RPE cells by detecting the effects of endothelin-1 (ET-1) on intracellular free Ca(2+) ([Ca(2+)](i)) mobility in these cells. METHODS: Iris and retina pigment epithelial cells were cultured from porcine eyeballs. After the cells were loaded with fura-2/AM, fluorescence was monitored with a fluorescent spectrophotometer by continuously recording excitation signals at 340 nm and 380 nm and emission signals at 510 nm. RESULTS: IPE and RPE cells induced a significant increase in [Ca(2+)](i) under exposure to 10(-7), 10(-8), and 10(-9) mol/L ET-1 in Ca(2+)-containing buffer. In the presence of Ca(2+)-free buffer, ET-1 alone induced an increase in [Ca(2+)](i) in iris but not in retina pigment epithelial cells. In Ca(2+)-free buffer, pretreatment of the IPE cells with 10(-7) mol/L ET-1 partially inhibited thapsigargin-induced [Ca(2+)](i) increase and carbonylcyanide m-chlorophenylhydrazone (CCCP)-induced Ca(2+) release. Similarly, pretreatment of the IPE cells with thapsigargin or CCCP also partially inhibited ET-1-induced [Ca(2+)](i) increase. The [Ca(2+)](i) release induced by 10(-7) mol/L ET-1 was not inhibited by the phospholipase C inhibitor U73122. In Ca(2+)-containing buffer, the ET(A) receptor antagonist BQ123 and ET(B) receptor antagonist BQ788 partially inhibited 10(-7) mol/L ET-1-induced [Ca(2+)](i) increase in IPE cells. However, BQ123 partially inhibited the 10(-7) mol/L ET-1-induced [Ca(2+)](i) increase in RPE cells. Nifedipine partially inhibited 10(-7) mol/L ET-1-induced [Ca(2+)](i) increase in both types of pigment cells. CONCLUSIONS: These results suggest that iris and retina pigment epithelial cells possess distinguishing characteristics because the ET-1-induced [Ca(2+)](i) increases in these 2 types of pigment epithelial cells are regulated by distinct mechanisms.

Effects of Toona sinensis leaf extract on lipolysis in differentiated 3T3-L1 adipocytes.

The effect of substances extracted from Toona sinensis leaves with 50% alcohol solution on lipolysis was investigated in cultured 3T3-L1 differentiated adipocytes. The amount of glycerol released from cells into culture medium was used to measure lipolysis activity. Glycerol release was increased by Toona sinensis leaf extract in a dose-dependent and time-dependent manner. Following treatment of 3T3-L1 adipocyte cells with various concentrations of Toona sinensis leaf extract (0.001, 0.01, and 0.1 mg/mL) for 6 hours, the amounts of glycerol released from 3T3-L1 cells increased from a control value of 99 nmol/mg protein to 127, 144, and 154 nmol/mg protein, respectively. The lipolytic effect of Toona sinensis leaf extract was not inhibited by pretreatment of cells with cycloheximide, econazole, baicalein, or indomethacin. However, the lipolytic activity induced by Toona sinensis leaf extract was diminished by dibutyryl cyclic adenosine-5'-monophosphate (dibutyryl cAMP) and the protein kinase C inhibitor calphostin C. These results indicate that the lipolytic effect induced by Toona sinensis leaf substances may be involved in the protein kinase C pathway and may be down-regulated by cAMP.

Enhancement of glucose uptake in 3T3-L1 adipocytes by Toona sinensis leaf extract.

The effects of substances extracted from Toona sinensis leaves, using 50% alcohol/water, on cellular [3H]-2-deoxyglucose uptake in differentiated cultured 3T3-L1 adipocytes were investigated. Following treatment of cells with 0.001, 0.01, or 0.1 mg/mL extracts for 60 minutes, [3H]-2-deoxyglucose uptake increased from a basal value of 0.23 nmol/min/mg protein to 0.30, 0.33, and 0.38 nmol/min/mg protein, respectively. In insulin-stimulated cells, cellular [3H]-2-deoxyglucose uptake was enhanced by Toona sinensis leaf extract from a basal value of 0.35 nmol/min/mg protein to 0.41, 0.46, and 0.52 nmol/min/mg protein, respectively. Cellular glucose uptake was also enhanced by Toona sinensis leaf extract after incubation of cells with 20 mM glucose for 48 hours. Cellular glucose uptake with a combination of Toona sinensis leaf extract and insulin was significantly inhibited by pretreatment of cells with the protein synthesis inhibitor cycloheximide and the protein kinase C inhibitor calphostin C in normal-, medium- and high-glucose media. However, the glucose uptake-enhancing effect of Toona sinensis leaf extract was not diminished by cycloheximide and calphostin C in the absence of insulin. These results indicate that enhancement of cellular glucose uptake by Toona sinensis leaf extract in basal and insulin-stimulated 3T3-L1 adipocytes may be mediated by distinct mechanisms.

Effects of commercial antiglaucoma drugs to glutamate-induced [Ca2+)]i increase in cultured neuroblastoma cells.

Over releasing of glutamate and cellular calcium influx always results in neuronal death. In the present study, we investigated various commercial antiglaucoma drugs including timolol (0.58 microM to 58 microM), betaxolol (1.62 microM to 162 microM), carteolol (6.8 microM to 680 microM), pilocarpine (4.08 microM to 408 microM), latanoprost (0.01 microM to 1.1 microM), dorzolamide (6.16 microM to 616 microM), brinzolamide (2.6 microM to 260 microM), brimonidine (0.68 microM to 68 microM), dipivefrin (0.28 microM to 28 microM) and preservative benzalkonium chloride on their effects to inhibit glutamate-induced intracellular free Ca(2+) ([Ca(2+)](i)) increase in cultured N1E-115 neuroblastoma cells. These drugs were diluted from original concentrations to 1/100, 1/1000 and 1/10000. The [Ca(2+)](i) mobility was studied after loading with fura-2-AM and analyzed by spectrofluorometry. It was found that betaxolol, dipivefrin and brimonidine have remarkable effects not only to inhibit the glutamate-induced [Ca(2+)](i) increase but also to decrease the basal [Ca(2+)](i). In the case of other drugs, only high concentration of timolol (58 microM) exhibited significant effect to completely prevent glutamate-induced [Ca(2+)](i) increase. Moreover, benzalkonium chloride did not exhibit any inhibitive effect. These results indicate that betaxolol, dipivefrin and brimonidine may have neuroprotective effects to inhibit the glutamate-induced over Ca(2+) influx damage.