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International audience; Ototoxins inhibit the muscarinic receptor-activated inositol phosphate synthesis in the rat cochlea. In order to study this inhibitory mechanism, we investigated the effects of the ototoxins ethacrynate, cisplatin, HgCI2 and neomycin on [3H]quinuclidinyl benzylate binding to muscarinic receptors in adult and 12-day-old rat cochleas. The results are similar whatever the age: at concentrations that inhibit the inositol phosphate synthesis, ethacrynate is without effect. Neomycin only reduces [3H]quinuclidinyl benzylate binding at concentrations in the millimolar range. Cisplatin and HgCI2 block the binding in a dose-dependent way. These results suggest that the block of the transduction system by cisplatin and HgCI2 is due to direct interactions with muscarinic binding sites. Moreover, considering these data together with previous results, ethacrynate and neomycin may affect the phosphoinositide signalling pathway at targets including phosphoinositides and G proteins.

International audience; In the rat cochlea, the activation of muscarinic receptors stimulates the hydrolysis of phosphoinositides but the importance of this muscarinic effect is still unknown. In order to find out about the role of the muscarinic receptors in the cochlea, we examined their functional distribution within this organ. This was achieved by measuring the formation of [3H]inositol phosphates induced by carbachol (1 mM) in two regions of the cochlea: the modiolus and the organ of Corti. At both sites, carbachol enhanced the accumulation of inositol phosphates in an atropine-sensitive way. These stimulations were completely antagonised by 4-diphenylacetoxy-N-methyl piperidine methiodide (1 ~zM) but unchanged by pirenzepine (1 p,M). In cochleas depleted of outer hair cells by a treatment with amikacin, the carbachol-induced formation of inositol phosphates is not altered with respect to control, undamaged cochleas. Conversely, when the medial cholinergic axons which form synapses with the outer hair cells are destroyed by the section of the crossed olivocochlear bundle the carbachol-stimulated inositol phosphates response is reduced by 35% in the organ of Corti. This section has no effect in the modiolus, despite the degeneration of some modiolar fibers. Our results show that functional muscarinic receptors are distributed both in the organ of Corti and in the modiolus. These two structures contain presumably the same class of cholinoceptor. The effects of selective destruction clearly demonstrate that a population of muscarinic receptors is located on presynaptic membranes at the level of the medial axon-outer hair cell contacts. They also point to spiral ganglion neurons and/or the Schwann cells as sites for the functional cholinoceptors in the modiolus.

International audience; The ability of amikacin, neomycin, ethacrynate, mercuric chloride and cisplatin to alter the inositol phosphate (IP) signalling pathway was assessed in the 12-day-old rat cochlea, where the turnover of IPs is coupled to muscarinic receptors. This study was motivated by: (1) the demonstration of neomycin binding to phosphatidylinositol 4,Sbiphosphate, the precursor of IPs, and (2) the fact that ototoxic drugs induce some common symptoms in outer hair cells. At concentrations below 1 mM, none of the compounds changed the control 'H-IP formation. Mercuric chloride, cisplatin and ethacrynate inhibited the carbachol-induced formation of IPs in a dose-dependent manner with IC?,, values of 74, 340 and 430 /LM. respectively. The aminoglycosides were less efficient in reducing the carbachol-stimulated accumulation of IPs, since neither amikacin nor neomycin. both at 1 mM, had any significant effect. However. neomycin applied at 15 and 30 PM induced 29% and 43% of inhibition of the stimulated IP response. Finally, additive effects are obtained between some of the toxic drugs. The results suggest that a block of the IP transduction system, associated with the cholinergic efferent innervation of the organ of Corti, is a feature that may he involved in some types of ototoxicity. The inefficiency of aminoglycosides and the putative targets of the ototoxic agents are discussed.

International audience; The maturation of the cholinergic innervation of the rat cochlea is associated with a transient increase in the muscarinic-receptor activated inositol phosphate synthesis. In order to investigate the mechanisms involved in this transient enhancement of the inositol phosphate response, the binding properties of the cochlear muscarinic receptors were studied during rat cochlear development. Incubating the membranes from 4-day-old, 12-day-old and adult cochleas with [3H]quinuclidinyl benzylate indicates that their respective, mean concentrations of cholinoceptors are 454 +/- 51 (+/- SEM), 39 +/- 2 and 42 +/- 3 fmol/mg of protein. The dissociation constants at equilibrium are 207 +/- 80, 42 +/- 7 and 28 +/- 3 pM for the binding sites of the 4-day-old, 12-day-old and adult cochleas, respectively. Pharmacological characterization of the binding, using selective antagonists, shows that M3 cholinoceptors are expressed in developing and adult cochleas. The data demonstrate that changes in muscarinic receptor affinity and number do not correlate with the previously observed peak of the inositol phosphate metabolism. The transient enhanced inositol phosphate response is therefore not due to changes in cholinoceptors, but probably due to alterations involving the intrinsic activity of the phospholipase C and/or the efficacy of coupling of the transduction system.