Show-Jen Hong

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.

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 0.00001mg/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.

Toxic Effects of Mitomycin-C on Cultured Ciliary Process Cells and Trabecular Meshwork Cells

Mitomycin-C has recently become an adjunct medication for inhibition of fibroblast proliferation in glaucoma filtering procedures. Prolonged postoperative ocular hypotony has been a frequent complication of trabeculectomy with mitomycin-C. In order to characterize the hypotony mechanism, we compared the toxic effects of mitomycin-C on cultured rabbit ciliary process cells and trabecular meshwork cells. The results indicate that mitomycin-C has a more marked effect on ciliary process cells on 3H-thymidine uptake than on trabecular meshwork cells at concentrations ranging from 10(-1) to 10(-5) mg/ml after 3-, 5- and 60-min treatment, respectively. The living cells after mitomycin-C treatment were estimated with MTT assay that was converted tetrazolium dye of living cells only into insoluble purple formazan crystals within mitochondria. In the presence of mitomycin-C for 3, 5, and 60 min, the cellular MTT values in ciliary process cells were more decreased than in trabecular meshwork cells. Depolarization of the trabecular meshwork cells with 50 mM KCl led to an increase in intracellular calcium concentration, whereas application of mitomycin-C at 10(-3) mg/ml resulted in decrease of KCl-induced intracellular calcium increase. Mitomycin-C (10(-3) mg/ml) decreased cAMP concentration in ciliary process cells following 3- and 5-min treatment; however, it did not significantly affect the cellular cAMP concentration after only a 1-min exposure. Mitomycin-induced marked ladder pattern of DNA fragmentation was observed in ciliary process tissues after treatment with 10(-1) mg/ml of mitomycin-C for 3 and 5 min. However, the DNA pattern in trabecular meshwork tissues was not obviously affected by mitomycin-C. These findings from our results indicate that mitomycin-induced ocular hypotony may result from damage to both ciliary process and trabecular meshwork tissues.

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+]i mobility was studied by spectrofluorophotometry after loading with the ester of fura‐2 (fura‐2/AM). It was found that timolol (58 μM and 5.8 μM), levobunolol (171 μM, 17.1 μM, and 1.71 μM), betaxolol (162 μM, 16.2 μM, and 1.62 μM), carteolol (680 μM and 68 μM), dipivefrin (28 μM and 2.8 μM), dorzolamide (616 μM and 61.6 μM), brinzolamide (260 μM), latanoprost (1.1 μM), unoprostone (28.2 μM, 2.82 μM, and 0.282 μM), and pilocarpine (408 μM and 40.8 μM) induced a significant increase in [Ca2+]i. Nevertheless, only brimonidine (68 μM and 6.8 μM) 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.

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.

Effects of crude drugs on glucose uptake in 3T3-L1 adipocytes.

In this study, various water-extracted crude drugs from Radix Asparagi, Radix Ginseng, Radix Scutellariae, Cortex Lycii Radicis, Cortex Phellodendri and Radix Ophiopogonis were investigated in their effects on [3H]-2-deoxyglucose uptake in 3T3-L1 adipocytes. Following treatment of cells with various crude drugs for 60 mim, the basal [3H]-2-deoxyglucose uptake in cultured 3T3-L1 cells was changed by Radix Asparagi from 140 pmole/min/mg protein of control to 513 (0.1 mg/ml), 201 (1 mg/ml) and 97 (10 mg/ml). Glucose uptake was changed to 324 (0.1 mg/ml), 146 (1 mg/ml) and 46 (10 mg/ml) with Radix Ginseng. In the presence of Radix Scutellariae, glucose uptake was changed to 215 (0.1 mg/ml), 213 (1 mg/ml) and 34 (10 mg/ml). In the presence of Cortex Lycii Radicis, glucose uptake was 230 (0.1 mg/ml), 188 (1 mg/ml) and 38 (10 mg/ml). In the case of Cortex Phellodendri and Radix Ophiopogonis, uptake was changed to 142 (0.1 mg/ml), 132 (1 mg/ml), 24 (10 mg/ml) and 489 (0.1 mg/ml), 374 (1 mg/ml), 344 (10 mg/ml), respectively. In insulin-stimulated cells, the [3H]-2-deoxyglucose uptake was changed by Radix Asparagi from 570 pmole/min/mg protein of the control to 816 (0.1 mg/ml), 674 (1 mg/ml) and 532 (10 mg/ml). After incubation with Radix Ginseng, the glucose uptake was changed to 254 (0.1 mg/mi), 123 (1 mg/mi) to 76 (10 mg/mi). In the presence of Radix Scutellariae, the glucose uptake was changed to 315 (0.1 mg/ml), 265 (1 mg/ml) and 33 (10 mg/ml). After incubation of Cortex Lycii Radicis, the uptake activity was changed to 281 (0.1 mg/ml), 248 (1 mg/ml) and 37 (10 mg/ml). In the case of Cortex Phellodendri and Radix Ophiopogonis, the activity of glucose uptake was measured as 747 (0.1 mg/ml), 523 (1 mg/ml), 33 (10 mg/ml) and 753 (0.1 mg/ml), 740 (1 mg/ml), and 421 (10 mg/ml), respectively. These results indicate that the water-extracted materials of Radix Asparagi and Radix Ophiopogonis increase the glucose uptake in basal and insulin-stimulated 3T3-L1 adipocytes.

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.

Change of cytosolic Ca2+ mobility in cultured bovine corneal endothelial cells by endothelin-1

The effect of endothelin-1 (ET-1) on the intracellular free Ca(2+) ([Ca(2+)](i)) in cultured bovine corneal endothelial cells was studied after loading with fura-2-AM. In Ca(2+)-containing buffer and Ca(2+)-free buffer, ET-1 induced a significant rise in [Ca(2+)](i) at concentrations from 10(-9) to 10(-7) M. In Ca(2+)-free buffer, pretreatment of the cells with ET-1 inhibited thapsigargin-induced [Ca(2+)](i) increase and carbonylcyanide m-chlorophenylhydrazone (CCCP)-induced Ca(2+) release by 99% and 62%, respectively. Pretreatment of the cells with thapsigargin or CCCP also inhibited ET-1-induced [Ca(2+)](i) rise by 36% and 92%, respectively. In Ca(2+)-containing buffer, the ET(A) receptor antagonist (BQ123) and ET(B) receptor antagonist (BQ788) partially inhibited ET-1-induced [Ca(2+)](i) by 92% and 98%, respectively. Nifedipine and La(3+) also inhibited ET-1-induced [Ca(2+)](i) increase by 26% and 91%, respectively. The intracellular calcium release caused by ET-1 was partially inhibited by phospholipase C inhibitor (U73122). After incubation of the cells with ET-1 in Ca(2+)-free buffer, the addition of 5 mM CaCl(2) increased Ca(2+) influx, implying that release of Ca(2+) from internal stores caused by ET-1 further induced capacitative Ca(2+) entry. These data suggest that ET-1-induced [Ca(2+)](i) rise in bovine corneal endothelial cells are mediated by ET(A) receptor, ET(B) receptor, La(3+)-sensitive Ca(2+) pump and L-type voltage-operated Ca(2+) channel, leading to Ca(2+) influx. ET-1 also increased the internal Ca(2+) release from endoplasmic reticulum and mitochondria Ca(2+) stores followed by capacitative Ca(2+) entry. ET-1-induced intracellular Ca(2+) release was modulated by phospholipase C-coupled events.

Endothelin-Induced Changes of Secondary Messengers in Cultured Corneal Endothelial Cells

The effect of endothelins on corneal endothelial cells is not well understood. We have investigated the effects of endothelin-1 (ET-1), endothelin-2 (ET-2) and endothelin-3 (ET-3) on bovine corneal endothelial cellular proliferation and the secondary messenger changes in cells in the presence of ET-1. It was found that the 3H-thymidine uptake was enhanced by ET-1 significantly, whereas ET-2 and ET-3 had no effect. ET-1 remarkably affects the increase of corneal endothelial cells on 3H-thymidine, 3H-leucine, and 3H-uridine uptakes in a dose-dependent manner. The 50% effective concentrations (EC50) for ET-1, as measured by 3H-thymidine uptake, 3H-uridine uptake, and 3H-leucine uptake were 10(-8.78) M, 10(-8.53) M and 10(-8.04) M, respectively. It was found that endothelin-1 increased intracellular calcium concentration by using the method of preloading with Fura-2-AM and assaying with spectrophotometry. The cellular IP1, IP2, and IP3 were also stimulated in the presence of ET-1. Moreover, ET-1 enhanced the basal cellular cAMP and cGMP concentrations in corneal endothelial cells in a dose-dependent manner. Immunofluorescent staining revealed that ET-1 increased the fibronectin protein concentration and changed protein distribution in corneal endothelial cells. These findings indicate that endothelin-1 increases in cell proliferation and biological changes may be involved in changing intracellular calcium mobility, increasing intracellular phosphoinositides, enhancing intracellular cGMP and cAMP accumulation, and fibronectin protein synthesis.

Ocular hypotensive and vasodilative effects of two β-adrenergic blockers with intrinsic sympathomimetic activity

PURPOSE The ocular hypotensive and vasodilative effects of vaninolol and eugenolol, two beta-adrenergic blockers with intrinsic sympathomimetic activity, were tested in rabbits and their pharmacologic mechanisms were also studied in vitro. METHODS Intraocular pressure was measured in ocular hypertonic rabbits which were induced by infusing 20% NaCl or 5% glucose solution. The rabbits ocular blood flow was determined using the colored microsphere technique. The concentrations of cAMP were evaluated in porcine ciliary body and cultured A7r5 smooth muscle cells by radioimmunoassay. Ca+2 concentration was measured in A7r5 cells by spectrofluorometry after loading cells with Fura-2-AM. RESULTS It was found that 0.5% eugenolol and vaninolol could suppress the intraocular pressure in glucose-induced ocular hypertensive rabbits and delay the intraocular pressure recovery in NaCl-induced ocular hypertensive rabbits. In addition, both agents improved the ocular blood flow in the iris, ciliary body, retina and choroid. Vaninolol and eugenolol of 10 microM inhibited the basal cAMP accumulation from 23.9 +/- 2.0 of control to 8.7 +/- 0.4 and 2.4 +/- 0.1 respectively and inhibited the isoproterenol-induced cAMP accumulation from 154.3 +/- 13.6 to 120.6 +/- 8.3 and 74.2 +/- 6.1 respectively in the porcine ciliary body. The cellular cAMP concentration was significantly increased from 10 +/- 1 of control to 96 +/- 5 (vaninolol) and 38 +/- 3 (eugenolol) in cultured A7r5 smooth muscle cells. Both agents also increased the intracellular calcium concentration in A7r5 cells. CONCLUSIONS These results indicate that the lowering of intraocular pressure by vaninolol and eugenolol may be due to cAMP suppression in the ciliary body by beta-antagonist and/or alpha2-agonist activities. Both agents cause vasodilation via beta2-agonist action that increase the smooth muscle cellular cAMP level more than vasoconstriction via alpha-agonist activity by increasing an influx of extracellular Ca+2.