Receptor-mediated release of inositol phosphates in the cochlear and vestibular sensory epithelia of the rat

Various neurotransmitters, hormones and other modulators involved in intercellular communication exert their biological action at receptors coupled to phospholipase C (PLC). This enzyme catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) to inositol 1,4,5-trisphosphate (InsP3) and 1,2-diacylglycerol (DG) which act as second messengers. In the organ of Corti of the guinea pig, the InsP3 second messenger system is linked to muscarinic cholinergic and P2y purinergic receptors. However, nothing is known about the the InsP3 second messenger system in the vestibule. In this study, the receptor-mediated release of inositol phosphates (InsPs) in the vestibular sensory epithelia was compared to that in the cochlear sensory epithelia of Fischer-344 rats. After preincubation of the isolated intact tissues with myo-[3H] in-ositol, stimulation with the cholinergic agonist carbamylcholine or the P2 purinergic agonist ATP-[gamma]-S resulted in a concentration-dependent increase in the formation of [3H]InsPs in both epithelia. Similarly, the muscarinic cholinergic agonist muscarine enhanced InsPs release in both organs, while the nicotinic cholinergic agonist dimethylphenylpiperadinium (DMPP) was ineffective. The muscarinic cholinergic antagonist atropine completely suppressed the InsPs release induced by carbamylcholine, while the nicotinic cholinergic antagonist mecamylamine was ineffective. Potassium depolarization did not alter unstimulated or carbamylcholine-stimulated release of InsPs in either organ. In both tissues, the P2 purinergic agonist [alpha],[beta]-methylene ATP also increased InsPs release, but the P1 purinergic agonist adenosine did not. These results extend our previous observations in the organ of Corti of the guinea pig to the rat and suggest a similar control of the InsP3 second messenger system in the vestibular sensory epithelia. In contrast to the cochlear sensory epithelia, atropine also significantly suppressed unstimulated InsPs release in the vestibular sensory epithelia. This suggests that the physiological mechanisms of the efferent nervous systems involving InsP3 second messenger system might be different in vestibular versus cochlear sensory epithelia.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30596/1/0000233.pd

Nitric oxide/Cyclic GMP pathway attenuates ATP-evoked intracellular calcium increase in supporting cells of the guinea pig cochlea

We demonstrate here that nitric oxide (NO) attenuates ATP-evoked calcium transients in Deiters' and Hensen's cells, “supporting” (nonsensory) cells of the guinea pig cochlea, by means of activation of soluble guanylyl cyclase and protein kinase G. The enzymatic activities associated with the nitric oxide/cGMP/protein kinase G pathway had previously been demonstrated to be present in Deiters' and Hensen's cells. We now isolate these cells and measure changes in intracellular free calcium by using the calcium indicator fluo-3. In Deiters' cells, calcium increased rapidly in response to the application of ATP . The increase was attenuated when the pathway was stimulated by NO donors (diethylamine NONOate or sodium nitroprusside) or the cyclic GMP analog, 8-bromo-cyclic GMP. When the activation of the pathway was blocked by the additional presence of inhibitors of soluble guanylyl cyclase (LY83583) or protein kinase G (Rp-8-bromo-cyclic GMP or KT5823), the response to ATP was restored. The reactions also occurred in calcium-free media. Hensen's cells responded similarly. These results provide evidence that intracellular calcium is regulated by the NO/cGMP/protein kinase G pathway in the inner ear. J. Comp. Neurol. 423:452–461, 2000. © 2000 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34461/1/8_ftp.pd

GENE EXPRESSION KINETICS AND PROTEIN DISTRIBUTION OF NUCLEOTIDE EXCISION REPAIR FACTORS IN THE INNER EAR AS A FUNCTION OF cis-DIAMMINEDICHLOROPLATINUM-II DNA DAMAGE

The kinetics of the rate-limiting genes of the molecular DNA repair pathways of nucleotide excision repair (NER) were quantified from the inner ear as a function of cis-diamminedichloroplatinum-II (cisplatin) treatment. The distribution of the post-translational products of these genes was evaluated among neurons and sensory hair cells of the inner ear following cisplatin treatment. These NER factors (genes & post-translational products) are only potentiated by DNA damage and are particularly sensitive to cisplatin induced DNA damage. A 2 x 3 x 2 factorial design, consisting of two treatment conditions (saline and cisplatin treated Fischer344 rats), three survival times and two molecular analysis methods (polymerase chain reaction and immunohistochemistry) was employed in this dissertation. The results revealed at least five important findings. First, it revealed for the first time that complex DNA repair molecular pathways such as NER exist in the inner ear. Second, it revealed for the first time that molecules used by advanced tumor cells to detect and repair damaged DNA from cisplatin genotoxicity also generalize to the inner ear and are stimulated by even small sub-toxic doses of cisplatin. Third, it revealed for the first time that NER proteins reside in the cytoplasm of neurons under normal conditions and translocate to the nucleus under conditions of genomic stress. Fourth, it revealed for the first time that the basal coil of the mammalian cochlea differs from the apical coil in the magnitude and latency in which NER molecules translocate from the cytoplasm to the nucleus under conditions of genomic stress. Fifth, the current work provides the bases for a new line of hearing research focused on molecular mechanisms of inner ear DNA repair

Der Einfluß von Amilorid und Diazoxid auf die Innenohrfunktion der Taube (Columba livia)

1. Die Arbeit soll den Beitrag von Ionenleitfähigkeiten an der Funktionsweise der Vogelhaarzelle weiter aufklären. 2. Dazu werden zwei Ionenkanalmodulatoren sowohl in die Scala media, wie auch in Scala tympani appliziert. Amilorid ist ein Blocker v.a. von Natrium­abhängigen Ionleitfähigkeiten; Diazoxid ist ein Öffner ATP­abhängiger Kaliumleitfähigkeiten. 3. Amilorid hat bei Applikation in die Scala media keinen Effekt auf das endocochleäre Potential. Diazoxid senkt das endocochleäre Potential nach Applikation in die Scala media signifikant um 2,42mV ± 2,31. Da Diazoxid auf die Aktivität auditorischer Neurone keinerlei Einfluß hat, muß davon ausgegangen werden, daß Diazoxid das EP durch Beeinflussung anderer Ionkanäle unabhängig von der Haarzelle absenkt. Mögliche Kandidaten sind Ionkanäle im Bereich des Tegmentum vasculosum, das für die Generation des EP mit verantwortlich ist. 4. Eine endolymphatische Amilorid­Applikation erhöht frequenzabhängig und dosisabhängig die CAP­Schwelle, wobei die Schwellenanhebung mit Anstieg der Frequenz steigt (gemessener Bereich 125­2000Hz). Bis zu einer Frequenz von 400Hz hat Amilorid kaum einen Effekt auf das CAP, oberhalb 400Hz steigt die Schwelle mit einem Gradienten von 11 dB/Okt an. 5. Diazoxid hat bei Applikation in die Scala media keinen Einfluß auf das CAP. 6. Die endolymphatische Applikation von Amilorid erniedrigt die akustisch evozierte Entladungsrate und erhöht die spontane Entladungsrate afferenter Neurone aus dem Ganglion cochleare. Diese Veränderungen sind abhängig von der charakteristischen Frequenz und der applizierten Menge, wobei der Frequenzbereich der charakteristischen Frequenz der nicht reagierenden Einzelfaserableitungen. zwischen 126­632 Hz lag, der der reagierenden zwischen 704 und 1200 Hz. 7. Bei den afferenten Neuronen, bei der die akustisch evozierte Aktivität nach endolymphatischer Applikation von Amilorid ansteigt, kommt es auch zu einer Veränderung der von der evozierten Aktivität abhängigen Parameter Q10dB, Tief­ und Hochfrequenzflanke und charakteristischer Frequenz. Die endolymphatische Amilorid­Konzentration bei diesen Einzelfaserableitungen lag zwischen 91µM und 269 µM. 8. Die charakteristische Frequenz wird durch Amilorid erniedrigt, allerdings kommt dies durch eine stärkere Abnahme der evozierten Rate oberhalb der charakteristischen Frequenz zustande. 9. Die Gruppeneinteilung ist bis auf ein Neuron bei Veränderungen spontaner und akustisch evozierter Entladungsrate gleich. Bei einem kam es zu einem Anstieg der spontanen Entladungsrate, aber nicht zu einer Abnahme der akustisch evozierten Entladungsrate. Dies legt nahe, daß 1. die Veränderungen auf Beeinflussung unterschiedlicher Ionleitfähigkeiten beruht , und 2. die Ionleitfähigkeit, welche für die Veränderung der spontanen Entladungsrate verantwortlich ist, etwas sensibler für Amilorid ist, als jene, welche für die Veränderung der evozierten Entladungsrate verantwortlich ist. 10. Der Anstieg der evozierten Rate steht in guten Einklang mit den Ergebnissen von Jørgensen und Ohmori (1988), die zeigen konnten, daß Amilorid den mechano­elektrischen Transduktionskanal von Vogelhaarzellen mit einem IC 50 von 50µM blockiert. Der Anstieg der spontanen Entladungsrate kann mit einem Block des Transduktionskanals nicht erklärt werden. Es muß also noch eine andere Leitfähigkeit in dem Innenohr der Taube durch Amilorid blockiert werden. 11. Zusammen mit den Ergebnissen anderer Studien legen die Ergebnisse nahe, daß es sich hierbei um eine Ionleitfähigkeit im Bereich der apikalen Membran handeln muß. Mögliche Kandidaten wären Ca 2 ­Kanäle, welche an Ca 2 ­abhängigen Prozessen zur Regulierung der Ciliensteifigkeit bzw. Cilienmotilität beteiligt sind. 12. Diazoxid hat bei Applikation in die Scala media keinen Einfluß auf die Aktivität auditorischer Neurone. 13. Bei Applikation in die Scala tympani hat weder Amilorid noch Diazoxid Einfluß auf das CAP

Pharmacokinetics of caroverine and its protective and therapeutic roles in noise-induced hearing loss following round window administration in the guinea pig

Ph.DDOCTOR OF PHILOSOPH

A study of in situ outer hair cells from the adult mammalian cochlea

This thesis investigates three characteristics of outer hair cells (OHCs) of the adult mammalian cochlea. The first investigation compared the basolateral membrane K+ channel expression of turn 4 (T4) and turn 1 (T1) OHCs, cells that respond to low and high frequency sound respectively. The second and third studies investigated two further aspects of T4 OHCs, the equivalent concentration of endogenous Ca2+ buffer and the characteristics of the mechanoelectric transduction (MET) current. OHCs in situ were recorded from using the whole-cell patch-clamp technique. For the first study, the kinetics, pharmacology and Ca2+ sensitivity of T4 and T1 OHC K+ channels were investigated. This work demonstrated that T4 OHCs express at least three types of K+ channels, termed Ikss, IkCa and lkT4. In contrast, T1 OHCs express different ion channels that exhibit faster onset kinetics, different pharmacology and an insensitivity to raised intracellular Ca2+ concentrations compared to those channels expressed in T4. These T1 channels have been termed IkT1 and Ik,n. The characteristics of these ion channels are discussed in relation to the particular sound frequency to which the OHC best responds. The equivalent concentration of endogenous Ca2+ buffer in T4 OHCs was investigated by using the time constant of current onset as a tool to compare the effects of various concentrations of BAPTA, introduced into the cell via the patch pipette, with those of the endogenous buffer, assayed using the perforated-patch technique. The concentration of endogenous Ca2+ buffer was found to be equivalent to the Ca2+ binding capacity of 2.1 mM BAPTA. This value converts to a Ca2+ binding ratio of 10,500. These results indicate that OHCs posess an enormous Ca2+ buffering capacity, have a low free [Ca2+]i and a huge pool of bound Ca2+ within their cytosol. Finally, the biophysical properties of the MET current of T4 OHCs were investigated. The few recordings obtained were variable but indicated that MET currents in situ are small (60 pA), limited in their onset kinetics only by the kinetics of the fluid-jet stimulus and run-down over a period of 20 minutes in the whole-cell recording configuration. These currents were found to be physiologically effective in activating the motile response of the OHC

ATP receptors in hypothalamic neurons and pituitary cells : a novel mediator in the neuroendocrine system.

SIGLEAvailable from British Library Document Supply Centre-DSC:DX189424 / BLDSC - British Library Document Supply CentreGBUnited Kingdo