Identifying tinnitus-related genes based on a side-effect network analysis.

Tinnitus, phantom sound perception, is a worldwide highly prevalent disorder for which no clear underlying pathology has been established and for which no approved drug is on the market. Thus, there is an urgent need for new approaches to understand this condition. We used a network pharmacology side-effect analysis to search for genes that are involved in tinnitus generation. We analyzed a network of 1,313 drug-target pairs, based on 275 compounds that elicit tinnitus as side effect and their targets reported in databases, and used a quantitative score to identify emergent significant targets that were more common than expected at random. Cyclooxigenase 1 and 2 were significant, which validates our approach, since salicylate is a known tinnitus generator. More importantly, we predict previously unknown tinnitus-related targets. The present results have important implications toward understanding tinnitus pathophysiology and might pave the way toward the design of novel pharmacotherapies.CPT Pharmacometrics Syst. Pharmacol. (2014) 3, e97; doi:10.1038/psp.2013.75; published online 29 January 2014.

Positive modulation of the α9α10 nicotinic cholinergic receptor by ascorbic acid.

BACKGROUND AND PURPOSE: The activation of α9α10 nicotinic cholinergic receptors (nAChRs) present at the synapse between efferent olivocochlear fibres and cochlear hair cells can prevent acoustic trauma. Hence, pharmacological potentiators of these receptors could be useful therapeutically. In this work, we characterize ascorbic acid as a positive modulator of recombinant α9α10 nAChRs. EXPERIMENTAL APPROACH: ACh-evoked responses were analysed under two-electrode voltage-clamp recordings in Xenopus laevis oocytes injected with α9 and α10 cRNAs. KEY RESULTS: Ascorbic acid potentiated ACh responses in X. laevis oocytes expressing α9α10 (but not α4ß2 or α7) nAChRs, in a concentration-dependent manner, with an effective concentration range of 1-30 mM. The compound did not affect the receptor's current-voltage profile nor its apparent affinity for ACh, but it significantly enhanced the maximal evoked currents (percentage of ACh maximal response, 240 ± 20%). This effect was specific for the L form of reduced ascorbic acid. Substitution of the extracellular cysteine residues present in loop C of the ACh binding site did not affect the potentiation. Ascorbic acid turned into a partial agonist of α9α10 nAChRs bearing a point mutation at the pore domain of the channel (TM2 V13'T mutant). A positive allosteric mechanism of action rather than an antioxidant effect of ascorbic acid is proposed. CONCLUSIONS AND IMPLICATIONS: The present work describes one of the few agents that activates or potentiates α9α10 nAChRs and leads to new avenues for designing drugs with potential therapeutic use in inner ear disorders.

The alpha9/alpha10-containing nicotinic ACh receptor is directly modulated by opioid peptides, endomorphin-1, and dynorphin B, proposed efferent cotransmitters in the inner ear.

UNLABELLED: Opioid peptides have been detected in the auditory and vestibular efferent neurons where they colocalize with the major neurotransmitter, acetylcholine. We investigated the function of opioids to modulate neurotransmission mediated by hair cell's alpha9/alpha10-containing nicotinic acetylcholine receptors (alpha9/alpha10nAChRs). The endogenous opioid peptides, endomorphin-1 (mu agonist) and dynorphin B (kappa agonist), but not a delta agonist [D-Pen2,D-Pen-5]enkephalin, inhibited the acetylcholine-evoked currents in frog saccular hair cells and rat inner hair cells. This inhibition was noncompetitive, voltage-independent, and was accompanied by an acceleration of the rate of current decay. Selective mu- and kappa-opioid receptor antagonists did not block the inhibition, although partial reduction by naloxone was observed. All opioid antagonists tested also reduced the acetylcholine response. Endomorphin-1 and dynorphin B inhibited the acetylcholine-evoked currents in alpha9/alpha10-expressing Xenopus oocytes. Because oocytes lack opioid receptors, it provides strong evidence for the direct interaction of opioid peptides with alpha9/alpha10nAChR. CONCLUSION: alpha9/alpha10nAChR is a target for modulation by endomorphin-1 and dynorphin B, efferent cotransmitters in the inner ear.

alpha10: a determinant of nicotinic cholinergic receptor function in mammalian vestibular and cochlear mechanosensory hair cells.

We report the cloning and characterization of rat alpha10, a previously unidentified member of the nicotinic acetylcholine receptor (nAChR) subunit gene family. The protein encoded by the alpha10 nAChR subunit gene is most similar to the rat alpha9 nAChR, and both alpha9 and alpha10 subunit genes are transcribed in adult rat mechanosensory hair cells. Injection of Xenopus laevis oocytes with alpha10 cRNA alone or in pairwise combinations with either alpha2-alpha6 or beta2-beta4 subunit cRNAs yielded no detectable ACh-gated currents. However, coinjection of alpha9 and alpha10 cRNAs resulted in the appearance of an unusual nAChR subtype. Compared with homomeric alpha9 channels, the alpha9alpha10 nAChR subtype displays faster and more extensive agonist-mediated desensitization, a distinct current-voltage relationship, and a biphasic response to changes in extracellular Ca(2+) ions. The pharmacological profiles of homomeric alpha9 and heteromeric alpha9alpha10 nAChRs are essentially indistinguishable and closely resemble those reported for endogenous cholinergic eceptors found in vertebrate hair cells. Our data suggest that efferent modulation of hair cell function occurs, at least in part, through heteromeric nAChRs assembled from both alpha9 and alpha10 subunits.

Morphine inhibits an alpha9-acetylcholine nicotinic receptor-mediated response by a mechanism which does not involve opioid receptors.

Nicotinic acetylcholine (nACh) receptors are known to be targets for modulation by a number of substances, including the opiates. It is known that acetylcholine (ACh) coexists with opioid peptides in cochlear efferent neurons, and such a colocalization has been proposed for the vestibular system. In the present study we test the hypothesis that morphine, an opioid receptor agonist with a broad spectrum of selectivity, modulates alpha9nACh receptor-mediated responses in frog vestibular hair cells. Morphine dose-dependently and reversibly inhibited ACh-induced currents as recorded by the perforated patch-clamp method. In the presence of morphine the ACh dose-response curve was shifted to the right in a parallel fashion, suggesting a competitive interaction. However, naloxone did not antagonize the inhibition produced by morphine. To test the hypothesis that morphine could interact with the alpha9nACh receptor without the involvement of opioid receptors, experiments were performed using Xenopus laevis oocytes injected with the alpha9nACh receptor cRNA. The currents activated by ACh in Xenopus oocytes, a system that lacks opioid receptors, were also dose-dependently inhibited by morphine. We conclude that morphine inhibits the alpha9nACh receptor-mediated response in hair cells and Xenopus oocytes through a mechanism which does not involve opioid receptors but may be a direct block of the alpha9nACh receptor.

Mixed nicotinic-muscarinic properties of the alpha9 nicotinic cholinergic receptor.

The rat alpha9 nicotinic acetylcholine receptor (nAChR) was expressed in Xenopus laevis oocytes and tested for its sensitivity to a wide variety of cholinergic compounds. Acetylcholine (ACh), carbachol, choline and methylcarbachol elicited agonist-evoked currents, giving maximal or near maximal responses. Both the nicotinic agonist suberyldicholine as well as the muscarinic agonists McN-A-343 and methylfurtrethonium behaved as weak partial agonists of the receptor. Most classical cholinergic compounds tested, being either nicotinic (nicotine, epibatidine, cytisine, methyllycaconitine, mecamylamine, dihydro-beta-erythroidine), or muscarinic (muscarine, atropine, gallamine, pilocarpine, bethanechol) agonists and antagonists, blocked the recombinant alpha9 receptor. Block by nicotine, epibatidine, cytisine, methyllycaconitine and atropine was overcome at high ACh concentrations, suggesting a competitive type of block. The present results indicate that alpha9 displays mixed nicotinic-muscarinic features that resemble the ones described for the cholinergic receptor of cochlear outer hair cells (OHCs). We suggest that alpha9 contains the structural determinants responsible for the pharmacological properties of the native receptor.

Block of the alpha9 nicotinic receptor by ototoxic aminoglycosides.

In the present study, we report that the alpha9 nicotinic acetylcholine receptor (nAChR) expressed in Xenopus laevis oocytes is reversibly blocked by aminoglycoside antibiotics. The aminoglycosides tested blocked the alpha9 nAChR in a concentration-dependent manner with the following rank order of potency: neomycin>gentamicin>streptomycin>amikacin>kanamycin. The antagonistic effect of gentamicin was not overcome by increasing the concentration of acetylcholine (ACh), indicative of a non-competitive type of block. Blockage of ACh-evoked currents by gentamicin was found to be voltage-dependent, being more potent at hyperpolarized than at depolarized holding potentials. Furthermore, gentamicin blockage was dependent upon the extracellular Ca(2+) concentration, shown by the fact that increments in extracellular Ca(2+) significantly reduced the potency of this aminoglycoside to block the alpha9 nAChR. Possible mechanisms of blockage by the aminoglycosides are discussed. The present results suggest that the initial reversible actions of aminoglycosides at the organ of Corti, such as the elimination of the olivocochlear efferent function, are due in part to the interaction with the native alpha9-containing cholinergic receptor of the outer hair cells.

High calcium permeability and calcium block of the alpha9 nicotinic acetylcholine receptor.

At the synapse between olivocochlear efferent fibers and outer hair cells (OHCs) of the cochlea, a non-classical ionotropic cholinergic receptor allows Ca(2+) entry into the hair cell, thus activating a Ca(2+)-sensitive K(+) current which hyperpolarizes the cell's membrane. In the mammalian ear, this leads to a reduction in basilar membrane motion, altering auditory nerve fiber activity and reducing the dynamic range of hearing. The alpha9 nicotinic acetylcholine receptor (nAChR) subunit mediates synaptic transmission between cholinergic olivocochlear fibers and OHCs. Given that Ca(2+) is a key player at this inhibitory synapse, we evaluated the permeability to Ca(2+) of the recombinant alpha9 receptor expressed in Xenopus laevis oocytes and the modulation of its activity by extracellular Ca(2+). Our results show that the alpha9 receptor is highly permeable to Ca(2+) and that this cation potently blocks monovalent currents through this channel (IC(50)=100 microM, at -70 mV) in a voltage-dependent manner. At a Ca(2+) concentration similar to that found in the perilymph bathing the base of the OHCs, approximately 90% of the Na(+) current through the alpha9 receptor is blocked, suggesting that one of the main functions of this channel could be to provide a pathway for Ca(2+) influx.

Role of alpha9 nicotinic ACh receptor subunits in the development and function of cochlear efferent innervation.

Cochlear outer hair cells (OHCs) express alpha9 nACh receptors and are contacted by descending, predominately cholinergic, efferent fibers originating in the CNS. Mice carrying a null mutation for the nACh alpha9 gene were produced to investigate its role(s) in auditory processing and development of hair cell innervation. In alpha9 knockout mice, most OHCs were innervated by one large terminal instead of multiple smaller terminals as in wild types, suggesting a role for the nACh alpha9 subunit in development of mature synaptic connections. Alpha9 knockout mice also failed to show suppression of cochlear responses (compound action potentials, distortion product otoacoustic emissions) during efferent fiber activation, demonstrating the key role alpha9 receptors play in mediating the only known effects of the olivocochlear system.

The alpha9 nicotinic acetylcholine receptor shares pharmacological properties with type A gamma-aminobutyric acid, glycine, and type 3 serotonin receptors.

In the present study, we provide evidence that the alpha9 nicotinic acetylcholine receptor (nAChR) shares pharmacological properties with members of the Cys-loop family of receptors. Thus, the type A gamma-aminobutyric acid receptor antagonist bicuculline, the glycinergic antagonist strychnine, and the type 3 serotonin receptor antagonist ICS-205,930 block ACh-evoked currents in alpha9-injected Xenopus laevis oocytes with the following rank order of potency: strychnine > ICS-205,930 > bicuculline. Block by antagonists was reflected in an increase in the acetylcholine (ACh) EC50 value, with no changes in agonist maximal response or Hill coefficient, which suggests a competitive type of block. Moreover, whereas neither gamma-aminobutyric acid nor glycine modified ACh-evoked currents, serotonin blocked responses to ACh in a concentration-dependent manner. The present results suggest that the alpha9 nAChR must conserve in its primary structure some residues responsible for ligand binding common to other Cys-loop receptors. In addition, it adds further evidence that the alpha9 nAChR and the cholinergic receptor present at the base of cochlear outer hair cells have similar pharmacological properties.

Identification of the subunits of the nicotinic cholinergic receptors in the rat cochlea using RT-PCR and in situ hybridization.

There are two tissues in the adult mammalian cochlea that are post-synaptic to cholinergic efferent fibers: The outer hair cells (OHCs) and the dendrites of the afferent fibers of the type I spiral ganglion cells. The unusual nicotinic-like pharmacology of cochlear cholinergic responses and the unique embryonic development of cochlear tissues suggest that the inner-ear nicotinic cholinergic receptor (nAChR) may be different from nAChRs described previously at synapses in the mammalian brain, autonomic ganglia, or skeletal muscle. In this study, we determined the mRNA expression of the alpha2-7, alpha9, and beta2-4 subunits of the nicotinic acetylcholine receptor (nAChR) family in the rat cochlea. In micro-dissected tissue from the organ of Corti, spiral ganglion, and the membranous lateral wall, we found mRNA expression of the alpha7 and alpha9 subunits in the organ of Corti and alpha5-7, and beta2 and beta3 in the spiral ganglion using RT-PCR. Employing in situ hybridization with 35S-riboprobes, we localized alpha9 in hair cells regions and alpha6, alpha7 and beta2 in the type I cells of the spiral ganglion. No evidence of nAChR subunit mRNA expression was found in supporting cells, but beta2 was expressed in type II spiral ganglion cells, which are neither cholinergic nor cholinoceptive.

Expression of nicotinic acetylcholine receptor mRNA in the adult rat peripheral vestibular system.

The mRNA expression of the neuronal nicotinic acetylcholine receptor subunits was determined in adult rat vestibular end-organs and in Scarpa's ganglion (SCG) by in situ hybridization with [35S] riboprobes. Neurons in the SCG expressed the alpha 4-7 and beta 2-3 mRNAs, but not alpha 3 or beta 4 mRNAs. Not all SCG neurons expressed every mRNA found in SCG. The alpha 6 and beta 2-3 riboprobes labeled all neurons, but alpha 4, alpha 5, and alpha 7 mRNAs were selectively expressed in one or more subpopulations of SCG neurons. Vestibular sensory hair cells, in contrast, expressed only alpha 9 mRNA.

alpha-Conotoxin ImI exhibits subtype-specific nicotinic acetylcholine receptor blockade: preferential inhibition of homomeric alpha 7 and alpha 9 receptors.

Through a study of cloned nicotinic receptors expressed in Xenopus oocytes, we provide evidence that alpha-conotoxin ImI, a peptide marine snail toxin that induces seizures in rodents, selectively blocks subtypes of nicotinic acetylcholine receptors. alpha-Conotoxin ImI blocks homomeric alpha 7 nicotinic receptors with the highest apparent affinity and homomeric alpha 9 receptors with 8-fold lower affinity. This toxin has no effect on receptors composed of alpha 2 beta 2, alpha 3 beta 2, alpha 4 beta 2, alpha 2 beta 4, alpha 3 beta 4, or alpha 4 beta 4 subunit combinations. In contrast to alpha-bungarotoxin, which has high affinity for alpha 7, alpha 9, and alpha 1 beta 1 gamma delta receptors, alpha-conotoxin ImI has low affinity for the muscle nAChR. Related Conus peptides, alpha-conotoxins MI and GI, exhibit a distinct specificity, strictly targeting the muscle subtype receptor but not alpha 7 or alpha 9 receptors. alpha-Conotoxins thus represent selective tools for the study of neuronal nicotinic acetylcholine receptors.

Alpha 9: an acetylcholine receptor with novel pharmacological properties expressed in rat cochlear hair cells.

We report the isolation and functional characterization of a member of the nicotinic acetylcholine receptor subunit gene family, alpha 9. Xenopus oocytes injected with alpha 9 cRNA express a homomeric receptor-channel complex that is activated by acetylcholine. The alpha 9 receptor displays an unusual mixed nicotinic-muscarinic pharmacological profile. The unique properties of the alpha 9 receptor-channel complex closely match those described for the cholinergic receptor present in vertebrate cochlear hair cells. In situ hybridization studies reveal a restricted pattern of alpha 9 gene expression that includes the outer hair cells of the rat cochlea. Our results suggest that the alpha 9 receptor is involved in the cholinergic efferent innervation of cochlear hair cells and thus may modulate the encoding of auditory stimuli.

Molecular cloning and chromosomal localization of the human alpha 7-nicotinic receptor subunit gene (CHRNA7).

We have isolated cDNA and genomic clones coding for the human alpha 7 neuronal nicotinic receptor subunit, the major component of brain nicotinic receptors that are blocked by alpha-bungarotoxin. The human alpha 7 neuronal nicotinic cDNA encodes a mature protein of 479 amino acids that is highly homologous to the rat alpha 7 neuronal nicotinic subunit (90%). We have mapped the human alpha 7-nicotinic receptor subunit gene to chromosome 15, band q14, a region frequently rearranged in patients carrying a bisatellite 15 chromosome, large inv dup (15), whose clinical features include mental retardation and seizures.

Relaxant effect of benzodiazepines on uterine rings isolated from estrogen-treated rats.

K(+)-contracted rat uterine rings were relaxed in a concentration-dependent manner by the benzodiazepines Ro 5-4864, diazepam and clonazepam, as well as by the putative peripheral benzodiazepine antagonist PK 11195. The relaxation induced by diazepam was not counteracted by the central antagonist Ro 15-1788 (10 microM), and the relaxant effects of Ro 5-4864 and of diazepam were not prevented by either the GABAA antagonist bicuculline (10 microM) or the beta-adrenoceptor antagonist propranolol (1 microM). The mechanism underlying the relaxant effects of benzodiazepines on K(+)-contracted uterine rings is still under study.

Benzodiazepines decrease the release of [3H]noradrenaline and of [3H]acetylcholine in the cat superior cervical ganglion.

The effects of two benzodiazepines, diazepam and clonazepam, were studied on the release of [3H]noradrenaline and of [3H]acetylcholine elicited by preganglionic stimulation of the cat isolated superior cervical ganglion and on the contractile responses evoked by either nerve stimulation or exogenous noradrenaline in the cat isolated nictitating membrane. Both 10 microM diazepam and 10 microM clonazepam reduced by approximately 50% the release of [3H]noradrenaline and of [3H]acetylcholine in the cat ganglion whereas they did not modify the contractile responses in the nictitating membrane. It is concluded that benzodiazepines are also peripheral neuroactive agents and that they exhibit a tissue-selective action within the same animal species.

Different receptor subtypes mediate the dual presynaptic effects of 5-hydroxytryptamine on peripheral sympathetic neurones.

1. The aim of the present work was to characterize the presynaptic 5-HT receptors that mediate either the facilitation of the responses to nerve stimulation in the nictitating membrane of the cat or the inhibition of the responses to nerve stimulation in the guinea-pig atria. 2. In the nictitating membrane of the cat, the shift to the left in the frequency-response curves produced by 5-HT (0.1 microM) was prevented by the 5-HT3 receptor antagonists, metoclopramide (1 microM) and MDL 72222 (0.01 microM). 3. The facilitatory effect of 5-HT is also prevented by the 5-HT2 receptor antagonist, 0.01 microM ketanserin. Nevertheless, this drug reduced by itself the responses to both nerve stimulation and exogenous NA in the nictitating membrane. 4. In the guinea-pig isolated atria, the inhibitory effect of 5-HT on the chronotropic responses to cardioaccelerans nerve stimulation was mimicked by the mixed 5-HT1A + 5-HT1B + 5-HT1D receptor agonist 5-carboxamidotryptamine (5-CT 0.1 and 1 microM). The 5-HT1A receptor agonist 8-OH-DPAT (0.1 and 1 microM) did not modify the responses of the atria to the nerve stimulation. 5. The 5-HT2 receptor antagonists, ketanserin (0.01 and 0.1 microM) and cyproheptadine (1 microM), did not prevent the inhibitory effect of 5-HT in the guinea-pig atria. 6. The present results suggest that the facilitatory effects of 5-HT in the nictitating membrane of the cat are linked to the activation of 5-HT3 receptors whereas the inhibitory effects observed in the guinea-pig atria are mediated by 5-HT1-like receptors.

Serotonin uptake inhibitors and the prejunctional effects of serotonin on peripheral sympathetic nerves.

The experiments were designed to study whether the inhibitors of the uptake of serotonin (5-HT) potentiated the prejunctional effects of 5-HT on peripheral sympathetic nerves. The effect of two selective 5-HT uptake inhibitors, citalopram and fluoxetine, were studied on the presynaptic actions of 5-HT in the cat isolated nictitating membrane and in the guinea-pig isolated atria. Frequency-effect curves to nerve stimulation and concentration-response curves to noradrenaline (NA) were performed in both preparations. The facilitation that 0.1 microM 5-HT causes on the contractile responses to nerve stimulation of the nictitating membrane of the cat was not potentiated but entirely prevented by both 0.01 microM citalopram and 1.0 microM fluoxetine. On the other hand the diminution that 1.0 microM 5-HT evokes on the chronotropic responses to nerve stimulation of guinea-pig isolated atria was not modified at all by 0.1 and 1.0 microM fluoxetine and only partially prevented by 10.0 microM fluoxetine and by 0.001 microM, 0.01 microM and 0.1 microM citalopram. This latter effect of citalopram was unrelated to the concentration employed. The 5-HT uptake inhibitors did not modify per se either the responses to nerve stimulation or the sensitivity to exogenous NA in both tissues studied. In addition, the 5-HT uptake inhibitors did not interfere with the contractile responses caused by 5-HT in the cat isolated nictitating membrane. Taken together, these observations might indicate a pharmacological rather than a physiological role for the effects of 5-HT in guinea-pig isolated atria and cat nictitating membranes. It is concluded that the 5-HT uptake inhibitors do not potentiate but even antagonize the presynaptic effects of 5-HT. Our results also show that 5-HT uptake inhibitors are more effective to interfere with the facilitation rather than with the inhibition that 5-HT causes on sympathetic responses.

Effect of acute exposure to reduced atmospheric pressures on body weight, food intake and body composition of growing rats.

Growing male rats were exposed to simulated altitudes of 1850, 2900, 4100, 5450 or 7100 m in a hypobaric chamber to determine the effects of altitude on body weight gain and food intake as function of time of exposure. Female rats were exposed to a simulated altitude of 7100 m for 24 h to determine the effect of altitude on body composition. The results obtained indicate that in growing rats exposed to acute simulated altitude the initial body weight loss and the depressed growth rate, on one hand, and the reduced food intake, on the other hand, are related to the degree of the altitude; the parameters are not affected at altitudes below 1 850 m; the initial weight loss is not solely due to reduction in food intake, the additional loss being attributed to the added stress of hypoxia; the body weight loss occurs without marked alterations in body composition, although a tendency to dehydration exists; and the body compositional changes are the reflection of the altitude-induced hypophagia.