探討抗青光眼藥對Glutamate引起視網膜傷害的保護作用及血管平滑肌的影響

Effects of antiglaucoma drugs to glutamate-induced damage in retina and to vascular smooth muscle cells

李佳芬(Chia-Fen Li)

指導教授 ： 洪秀貞 博士

高雄醫學大學/醫學院/醫學研究所/碩士(2003年)

摘要

1. 本實驗中主要探討不同種類降眼壓藥物，例如timolol ( 58 μM - 0.58 μM ), betaxolol ( 162 μM - 1.62 μM ), levobunolol ( 171 μM - 1.71 μM ), dipivefrin ( 28 μM - 0.28 μM ), brimonidine ( 68 μM - 0.68 μM ), latanoprost ( 1.1 μM - 0.01 μM ), carteolol ( 680 μM - 6.8 μM ), pilocarpine ( 408 μM - 4.08 μM ), dorzolamide ( 616 μM - 6.16 μM ), brinzolamide ( 260 μM - 2.6 μM )及unoprostone ( 31 μM - 0.31 μM ) 對視網膜神經的保護作用及對血管平滑肌的影響。
2. 結果發現兔子視網膜經10-5 M Glutamate處理4小時後，視網膜中H2O2引起自由基的量沒有增加，但是neuronal nitric oxide synthase (nNOS) 和inducible nitric oxide synthase (iNOS) 活性卻明顯增加，同時Propyl - L - arginine (nNOS 抑制劑) 會降低nNOS活性，而Methylisothiourea sulfate (iNOS抑制劑) 也會使iNOS活性減少。
3. 利用western blotting分析視網膜神經節細胞之表面抗原Thy-1 及nNOS protein在經過4小時的glutamate反應後，於視網膜的表現並未有顯著的差異。而且也沒有iNOS蛋白質的出現。
4. betaxolol (162 mM, 16.2 mM及1.62 mM) 都能抑制nNOS activity，然而高濃度的carteolol (680 mM), pilocarpine (408 μM)和unoprostone (31 μM) 也有抑制nNOS activity的效果。
5. 所有濃度的Brimonidine, dipivefrin, latanoprost, dorzolamide,timolol, brinzolamide和benzalkonium chloride對於nNOS activity則完全無抑制效果。
6. betaxolol (162 mM, 16.2 mM及1.62 mM) 都能抑制iNOS活性，然而高濃度的timolol (58 mM) , carteolol (680 mM), pilocarpine (408 μM) 和levobunolol (171 μM) 也有抑制iNOS活性的效果。
7. 所有濃度的Brimonidine, dipivefrin, latanoprost, dorzolamide, unoprostone, brinzolamide和防腐劑 (benzalkonium chloride) 對於iNOS activity則完全無抑制效果。
8. 兔子視網膜經Glutamate處理4小時後，經切片染色觀察視網膜並沒有明顯的改變。
9. 內皮素-1 (ET - 1)會引起A7r5血管平滑肌細胞內游離鈣離子濃度([Ca2+]i)的增加。
10. betaxolol (162 mM，16.2 mM及1.62 mM ) 都能抑制ET-1誘發A7r5細胞[Ca2+]i增加，而高濃度的timolol (58 mM), carteolol (680 mM), levobunolol (171mM) 及unoprostone (31 μM) 也有抑制效果。

關鍵字

青光眼

並列摘要

In the present study, we investigated the neuroproteictive effects of commercial antiglaucoma drugs including timolol, betaxolol, levobunolol, dipivefrin, brimonidine, latanoprost, carteolol, pilocarpine, dorzolamide, brinzolamide and unoprostone in rabbits’ retinas.
2. The results indicated that neuronal nitric oxide synthase ( nNOS ) and inducible nitric oxide synthase ( iNOS ) activities were significantly increased after in the treatment with 10-5 M Glutamate for 4 hours in retinas of rabbits but H2O2 - induced free radical were not changed. Meanwhile, propyl - L — arginine ( nNOS inhibitor ) and methylisothiourea sulfate ( iNOS inhibitor ) also inhibited the nNOS and iNOS activities in retinas respectively.
3. By western blotting assay, the nNOS protein and retina ganglial cell marker, thy-1 proteins did not obviously change of the content after glutamate treatment. The iNOS protein also did not appear after in the treatment with glutamate.
4. The nNOS activities were inhibited by all concentrations of betaxolo including 162 mM, 16.2 mM and 1.62 mM and by high concentrations of carteolol (680 mM), pilocarpine (408 μM) and unoprostone (31 μM).
5. All concentrations of Pilocarpine, brimonidine, dorzolamide, latanoprost, dipivefrin, timolol, brinzolamide and benzalkonium chloride did not exhibit the inhibitory effects on glutamate - induced nNOS increase in rabbits’ retina.
6. The iNOS activities were inhibited by all concentrations of betaxolol including 162 mM, 16.2 mMand 1.62 mM and by high concentrations of timolol (58mM), carteolol (680 mM), pilocarpine (408 μM) and levobunool (171 μM).
7. All concentrations of Pilocarpine, brimonidine, dorzolamide, latanoprost, dipivefrin, unoprostone, brinzolamide and benzalkonium chloride did not exhibit the inhibitory effects on glutamate - induced iNOS increase in rabbits’ retina.
8. By histological study, the morphology of retina was not significantly changed in the presence of glutamate for 4 hours.
9. In this study, the endothelin-1 (ET-1) was found to induce intracellular free calcium [Ca2+]i increase in cultured A7r5 aortic smooth muscle cells.
10. The ET-1-induced [Ca2+]i increase were inhibited by all concentrations of betaxolol (162 mM, 16.2 mM and 1.62 mM) and by high concentrations of timolol (58 mM), carteolol (680 mM), unoprostone (31 μM) and levobunool (171 μM).

並列關鍵字

glaucoma

參考文獻

參考文獻

Akins PT, Atkinson RP: Glutamate AMPA receptor antagonist treatment for ischemia stroke. Curr Med Res Opin 18:s9-13, 2002.

Anderson DR: Introductory comments on blood flow autoregulation in the optic nerve head and vascular risk factors in glaucoma. Surv Ophthalmol 43:5-9, 1999.

Archer SL, Nelson DP, Weir EK: Detection of activated O2 species in vitro and in rat lungs by chemiluminescence. J Appl Physiol 67:1912-21, 1989.

Bessho H, Suzuki J, Tobe A: Vascular effects of betaxolol, a cardioselective beta-adrenoceptor antagonist, in isolated rat arteries. Jpn J Pharmacol 55:351-58, 1991.