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Gentamicin-induced production of reactive oxygen species (ROS) and of nitric oxide (NO) in the vestibular end organs of the guinea pig was investigated by applying two new fluorescence indicators, 4,5-diaminofluorescein diacetate for direct detection of NO and dihydrotetramethylrosamine for ROS. The vestibular sensory cells produced both NO and ROS after exposure to gentamicin. A nonspecific inhibitor of NO synthase, L-NAME, inhibited the production of NO but did not appear to affect the production of ROS following exposure to gentamicin. In contrast, a radical scavenger, D-methionine, or the neurotrophin BDNF suppressed the production of ROS, in turn stimulating NO production. These findings could indicate that both NO and ROS play an important role in aminoglycoside ototoxicity., (Copyright 2002 S. Karger AG, Basel)

The distribution of rat alpha-atrial natriuretic polypeptide (ANP) was studied in the adult inner ear of the Sprague-Dawley rat using immunohistochemical methods. Immunostaining, in particular of the subepithelial stroma cells of the spiral ligament, the spiral prominence and below neuroepithelial areas of all five vestibular organs, was identified. We hypothesize that alpha-ANP is involved in perilymphatic dynamics rather than generating endolymph. The strong staining for alpha-ANP of outer hair cells indicates specific mechanisms for volume control as compared with inner hair cells and vestibular hair cells which all lack this immunostaining. The functional significance of the specific staining of nerve calyces in only the three cristae ampullaris but not in the two maculae remains to be clarified. The positivity of epithelial cells in the endolymphatic sac supports a previous hypothesis which looks upon this organ as a pressure regulation system in the inner ear.

The distribution of glycoconjugates in the endolymphatic sac (ES) and vestibular end organs from the 12th gestational day (GD) in the developing mouse to the 6th day after birth was analyzed using six biotinylated lectins: wheat germ agglutinin (WGA), Abrus precatorius agglutinin (APA), Ulex europaeus agglutinin I (UEA-I), Ricinus communis agglutinin 120 (RCA120), Helix pomatia agglutinin (HPA), concanavalin A (conA). In the 13th and 15th GD sections, the luminal contents in the fetal ES were strongly labelled with lectins, while only a small amount of substance in the ES was labelled with lectins after the 17th GD. In the 13th GD sections, before the cupula and otoconia were formed, the fetal ES started to produce glycoconjugates labelled with WGA, APA, RCA120 and ConA. In the 17th GD sections, the immature cupula and otoconia were strongly labelled with WGA, APA and RCA120. Sugar residues stained by lectins detected in the substance of the fetal ES were the same as those found in the immature cupula and otoconia. HPA only stained the ES epithelium around the 13th and 15th GD. The fetal ES may interact with the formation of the cupula and otoconia, especially in the early stage during evolution and that HPA-reactive glycoconjugates may be related to the intracellular elements of the ES epithelium only during a well-defined phase of development.

Vestibular hair cells were isolated from the macula utriculi, macula sacculi and crista ampullaris of the guinea pig, using enzymatic and mechanical techniques. The cells could be classified into two types: flask-shaped ones with a neck and rod- or round-shaped ones without a neck. The flask-shaped cells were thought to be type I cells, the rod-shaped cells type II cells, while the round-shaped cells could have been type I or type II cells. The intracellular free calcium ion concentrations [( Ca2+]i) of these cells were determined using the Ca2(+)-sensitive dye Fura-2 and digital imaging miroscopy. In the resting state, [Ca2+]i in the nucleus and cuticular plate was variable in both types of cells. The 150 mM KCl stimulation, which might induce a depolarization, resulted in a temporary increase in [Ca2+]i in both types of cells. In the presence of 1 micron ionomycin, there was an irreversible increase in [Ca2+]i in both types of cells. These observations aid in elucidating vestibular function.

The presence of carbohydrates in the glycocalyx of the vestibular end organs of the guinea pig was investigated at the ultrastructural level using lectin-gold solutions. The glycocalyx of both the sensory and the supporting cells has variable sugar components. The glycocalyx of the sensory cells including the ciliary interconnections contains N-acetyl-glucosamine, galactose and mannose. In contrast, the glycocalyx of the supporting cells had a lower amount of N-acetylglucosamine. The ciliary interconnections, which have been considered to be a part of the glycocalyx, have larger amounts of galactose and mannose than the surface glycocalyx. These findings indicate that the sugar components may be closely related to the functional significance of the inner-ear glycocalyx and that the functional properties of the glycocalyx may differ between the sensory and the supporting cells of the vestibular end organs.