Wang Qi, Steyger Peter S
Oregon Hearing Research Center, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA.
J Assoc Res Otolaryngol. 2009 Jun;10(2):205-19. doi: 10.1007/s10162-009-0160-4. Epub 2009 Mar 3.
Aminoglycosides enter inner ear hair cells across their apical membranes via endocytosis, or through the mechanoelectrical transduction channels in vitro, suggesting that these drugs enter cochlear hair cells from endolymph to exert their cytotoxic effect. We used zebrafish to determine if fluorescently tagged gentamicin (GTTR) also enters hair cells via apically located calcium-sensitive cation channels and the cytotoxicity of GTTR to hair cells. We then examined the serum kinetics of GTTR following systemic injection in mice and which murine cochlear sites preferentially loaded with systemically administered GTTR over time by confocal microscopy. GTTR is taken up by, and is toxic to, wild-type zebrafish neuromast hair cells. Neuromast hair cell uptake of GTTR is attenuated by high concentrations of extracellular calcium or unconjugated gentamicin and is blocked in mariner mutant zebrafish, suggestive of entry via the apical mechanotransduction channel. In murine cochleae, GTTR is preferentially taken up by the stria vascularis compared to the spiral ligament, peaking 3 h after intra-peritoneal injection, following GTTR kinetics in serum. Strial marginal cells display greater intensity of GTTR fluorescence compared to intermediate and basal cells. Immunofluorescent detection of gentamicin in the cochlea also revealed widespread cellular labeling throughout the cochlea, with preferential labeling of marginal cells. Only GTTR fluorescence displayed increasing cytoplasmic intensity with increasing concentration, unlike the cytoplasmic intensity of fluorescence from immunolabeled gentamicin. These data suggest that systemically administered aminoglycosides are trafficked from strial capillaries into marginal cells and clear into endolymph. If so, this will facilitate electrophoretically driven aminoglycoside entry into hair cells from endolymph. Trans-strial trafficking of aminoglycosides from strial capillaries to marginal cells will be dependent on as-yet-unidentified mechanisms that convey these drugs across the intra-strial electrical barrier and into marginal cells.
氨基糖苷类药物通过内吞作用穿过内耳毛细胞的顶膜进入细胞，或在体外通过机械电转导通道进入细胞，这表明这些药物从内淋巴进入耳蜗毛细胞以发挥其细胞毒性作用。我们使用斑马鱼来确定荧光标记的庆大霉素（GTTR）是否也通过位于顶端的钙敏感阳离子通道进入毛细胞以及GTTR对毛细胞的细胞毒性。然后，我们通过共聚焦显微镜检查了小鼠全身注射GTTR后的血清动力学，以及随着时间的推移，哪些小鼠耳蜗部位优先摄取全身给药的GTTR。GTTR被野生型斑马鱼的神经丘毛细胞摄取并对其有毒性。高浓度的细胞外钙或未结合的庆大霉素会减弱神经丘毛细胞对GTTR的摄取，并且在水手突变斑马鱼中摄取被阻断，这表明GTTR是通过顶端机械转导通道进入细胞的。在小鼠耳蜗中，与螺旋韧带相比，血管纹优先摄取GTTR，在腹腔注射后3小时达到峰值，这与血清中的GTTR动力学一致。血管纹边缘细胞显示出比中间细胞和基底细胞更高强度的GTTR荧光。耳蜗中庆大霉素的免疫荧光检测还显示整个耳蜗存在广泛的细胞标记，边缘细胞有优先标记。与免疫标记的庆大霉素的细胞质荧光强度不同，只有GTTR荧光的细胞质强度随着浓度的增加而增加。这些数据表明，全身给药的氨基糖苷类药物从血管纹毛细血管运输到边缘细胞并清除到内淋巴中。如果是这样，这将有助于氨基糖苷类药物通过电泳从内淋巴进入毛细胞。氨基糖苷类药物从血管纹毛细血管到边缘细胞的跨血管纹运输将取决于尚未确定的机制，这些机制将这些药物穿过血管纹内的电屏障并进入边缘细胞。
