L-Arginine與Taicatoxin對外毛細胞運動性之影響

中文摘要 本研究在探討 L—arginine 與 taicatoxin對天竺鼠之耳蝸外毛細胞的作用。L—arginine 能增加天竺鼠外毛細胞的長度，而 D—arginine 或其他類似胺基酸 (如 L—aspartate 或 L-glutamate) 的作用卻不會引起相同的反應。同樣的，一種具有穿透細胞膜能力的環鳥糞嘌呤核苷單磷酸(cGMP)類似物，8-(-4 chlorophenylthio) guanosine 3’，5’-cyclic monophosphate (pCPT—cGMP) 或 8—bromo-guanosine 3’，5-cyclic mono- phosphate (8-Br-cGMP) 也能延長天竺鼠外毛細胞。L—arginine 延長外毛細胞之作用以一氧化氮合成酶抑制劑(NOS)，NG—nitro—L—arginine methyl ester hydrochloride (L-NAME) 或 7—nitroindazole (7-NINA)，前處理30 分鐘可減弱。比較L—arginine和 ionomycin 延長外毛細胞的作用後發現，L—arginine 不會改變細胞內鈣離子的螢光表現強度。用 EGTA (3 mM) 預處理外毛細胞 40 分鐘來減少細胞外鈣離子濃度，並不能改變 L—arginine 的作用。這些結果指示，一氧化氮(NO)/環鳥糞嘌呤核苷單磷酸(cGMP)路徑可能參與哺乳類動物外毛細胞的運動性。此外，L—arginine 的作用並不依賴細胞外的鈣離子濃度。 Taicatoxin萃取自澳洲大斑蛇(Oxyuranus scutellatus)，為一種高閾值鈣離子管道阻斷劑以及低傳導鈣離子依賴性鉀離子管道阻斷劑 (small conductanced Ca2+-activated K+ channels)。以taicatoxin (0.19 mM) 前處理外毛細胞能明顯阻斷高鉀溶液 (50 mM) 縮短外毛細胞之作用。同樣的，cobaltous chloride，一種非選擇性鈣離子管道阻斷劑，亦能明顯阻斷高鉀溶液縮短毛細胞之作用，但ionomycin 延長毛細胞之作用不受其影響。其他的鈣離子管道阻斷劑nifedipine (L-型阻斷劑)、ω— conotoxin GVIA (N-型阻斷劑)、ω — agatoxin IVA (P-型阻斷劑)，皆無法影響高鉀溶液所誘發之細胞長度的改變。比較 taicatoxin 和 cobaltous chloride 影響外毛細胞之長度與細胞內鈣離子濃度的變化，taicatoxin 與 cobaltous chloride 二者都能阻斷高鉀溶液縮短毛細胞，但taicatoxin不能抑制高鈣溶液 (5 mM) 增加細胞內鈣離子濃度。總而言之，taicatoxin 的作用與細胞膜鈣離子管道或細胞內鈣離子濃度之間並無直接關係。 不論L-arginine或taicatoxin的作用皆與鈣離子濃度的變化或鈣離子管道無關，這也支持了之前其它實驗室所發表的非鈣依賴性的毛細胞運動假說。

Abstract The effect of L-arginine on the mammalian cochlear outer hair cell was studied in this experiment. L-arginine but not D-arginine or other amino acids (L-aspartate or L-glutamine) induced length increases of guinea pig outer hair cell. Similarity, a membrane permeable cyclic GMP analog, 8-(-4- chloro- phenylthio) guanosine 3’，5’-cyclic monophosphate (pCPT -cGMP) or 8-bromo-guanosine 3’，5’-cyclic monopho- sphate induced length increases of guinea pig outer hair cells. These length increases induced by L-arginine can be attenuated by a 30 min preincubation of the cells with the nitric oxide synthase inhibitors, NG-nitro-L-arginine methyl ester hydrochloride (L-NAME) or 7-nitroindazole (7-NINA). Comparison on the elongate effect of cell length of L-arginine and ionomycin, L-arginine did not change the fluorescence intensity of fluo-3. Preincubation of the cells with EGTA (3 mM) for 40 min to reduce the extracellular calcium concentration did not influence the effect of L-arginine. The results demonstrated that nitric oxide/cGMP pathway involved in regulating the slow motility of mammalian outer hair cell can not be ruled out.In addition, the effect of L-arginine is independent of extracellular calcium concentration. Taicatoxin isolated from the venom of the Australian taipan snake (Oxyuranus scutellatus), has been previously regarded as a blockers of high threshold Ca2+ channels and small conductance Ca2+-activated K+ channels. Taicatoxin (0.19 mM) significantly prevented the cell shortening induced by high potassium (50 mM) medium. Similarly, cobaltous chloride, a nonselective calcium channel blocker, was to significantly reduced the effect of cell shortening induced by high potassium but not the effect of cell elongation induced by ionomycin. Other calcium channel blockers, nifedipine, w-conotoxin GVIA, w -agatoxin IVA did no such effect in prevented the cell length change induced by high potassium. Comparing the effects of taicatoxin and cobaltous chloride on the cell length and intracellular calcium change in outer hair cell, both taicatoxin and cobaltous chloride were able to prevent the cell shortening induced by high potassium but taicatoxin did not prevent the intracellular calcium increase induced by high calcium medium. The effect of taicatoxin was no relative with the membrane calcium channels or intracellular calcium concentration. In summary, the effects of L-arginine and taicatoxin were not relative with the calcium channels or cellular calcium concentration. Neither the action of L-arginine, nor taicatoxin were relative with the changes of calcium concentration or calcium channels. These findings support the hypothesis of calcium-independent slow motility of outer hair cells as described previously.