© 1991 S. Karger AG, Basel 0301-1569/91 /0532-0078$2.75/0
ORL 1991;53:78-81
Effect of Neuroregulators on the Intracellular Calcium Level in the Outer Hair Cell Isolated from the Guinea Pig Katsuhisa Ikedaa, Yoshitaka Saitob, Akinori Nishiyamab, Tomonori T a k a sa k a 1 Departments o f aOtolaryngology and bPhysiology, Tohoku University School of Medicine, Sendai, Japan
Key Words. Outer hair cell • Intracellular Ca2+ concentration • Adenosine triphosphate • Acetylcholine • Efferent synapse Abstract. The cytosolic calcium concentration ([Ca2+]i) of the isolated outer hair cell of the guinea pig was mea sured using fluorescence imaging microscopy and the effects of efferent neuroregulators such as acetylcholine, ATP, GABA, substance P, enkephalin, calcitonin gene-related peptide, serotonin, dopamine, norepinephrine, and gluta mate were investigated. Among the drugs tested only ATP induced an elevation of the [Ca2+]i of the outer hair cell. In the resting condition, [Ca2+]i averaged 104.5 ± 31.1 nM (n = 27), while 100 pAf ATP significantly increased [Ca2+]i to 146.3 ± 43.5 nM (n = 19). Superfusion with Ca2+-free solution (pCa = 7.5) abolished the increase in [Ca2+]i induced by ATP, suggesting that ATP causes an entry of external Ca2+. The relevance of [Ca2+]i to the inhibitory actions of efferent neuroregulators is discussed.
The outer hair cell (OHC) of the mammalian cochlea is extensively innervated by efferent fibers. The physio logical role of the efferent innervation to the OHC is believed to be inhibitory on the cochlear potential [1], however, the cellular and biological mechanism of action have not yet been elucidated. Two possibilities closely related to Ca2+ ions for explanation of the functional mechanism of the efferent actions in the cochlea have been considered. One is that efferent neurotransmitters may generate an OHC motility that may attenuate fre quency selectivity. Slow motility is initiated by an in crease in the concentration of intracellular free Ca2+ ([Ca2+]i) and interacts with the cytoskeletal proteins and calmodulin [2]. Another explanation for the inhibitory effects of the cochlear efferent system is the hyperpolar ization of the membrane potential in the OHC resulting from the efferent stimulation [3]. In light of the proposed 1 We thank Ms. Michiko Yokoyama for preparing the manu script.
model of mechanoelectrical signal transduction in the hair cell [4], it is proposed that an increase in [Ca2+]i activates the K+conductance and hyperpolarizes the hair cells. Therefore, it is of interest to explore the changes in [Ca2+]i of the OHC in response to efferent transmitters. In the present study we tested a number of candidate neurotransmitter substances to identify that physiologi cally involved in the increase in OHC [Ca2+]i.
Materials and Methods Albino guinea pigs (200-350 g) with a Preyer reflex were anes thetized by inhalation of ethyl ether and the temporal bones were rapidly removed after decapitation. After the tympanic bulla was opened, the bony capsules of the cochlea were picked away under a stereomicroscope to expose the spiral ligament. The lateral walls of the cochlea were peeled away from the basilar membrane and the organ of Corti in the apical to third turns was dissected and col lected. Microdissection was performed in a Petri dish filled with an oxygenated artificial perilymph composed of 150 mM NaCl, 3.5 mA/KCl, 1.0m 3/C aC l2, 1.0m 4/M gCl2, 2.75 nuV/HEPES, and 2.25 m M Tris. The pH and osmolality of the solution were adjusted to 7.4 with HC1 and 300 mosm/kg H 20 with D-glucose, respectively.
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Introduction
[Ca2’]i and Neuroregulators in the Outer Hair Cell
79
Fig. 1. Absence of effect of ACh ( 10-6, 10_s, 10“J M ) on [Ca2+]i. Fig. 2. Effects of 100 \iM ATP on [Ca2*]i. Fig. 3. Decrease in [Ca2^]i induced by 100 \iM ATP in the absence of external Ca2* ions.
Results ACh is one of the putative neurotransmitters of the cochlear efferent system on the basis of physiological [6], biochemical [7] and histochemical [8] studies. Addition of 1-100 \iM ACh to the superfused artificial perilymph, however, did not alter the OHC [Ca2+]i (fig. 1). On the other hand, an application of 100 pM ATP abruptly increased the OHC [Ca2+]i to a plateau within 3 min (fig. 2). The basal value of [Ca2+]i, 104.5 ± 31.1 nM(n = 27), was significantly increased to 119.8 ± 16.5 nM (n = 8) by 10 pM ATP and to 146.3 ± 43.5 nM (n = 19) by 100 pM ATP. When the external Ca2+ concentration was reduced to pCa 7.5 by adding EGTA, the application of 100 pA/ ATP decreased [Ca2+]i to 58.3 ± 19.2 nM (n = 4; fig. 3), which was not significantly different from the value (65.8 ± 21.5 nM, n = 3) obtained under the super fusion with Ca-free solution without ATP. The effect of ACh on the ATP-induced increase in [Ca2+]i was examined. After an elevation of [Ca2+]i with 100 pM ATP, addition of 100 pM ACh to ATP-contain-
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 11:05:13 AM
The OHCs were mechanically isolated by gentle reflux through a micropipette and were incubated in the artificial perilymph supple mented with 3 pAf fura-2 acetoxymethyl ester (Dojindo Lab.) at 37 °C for 60 min. An approximately 30-jj.I droplet containing fura2-loaded OHCs was plated on a glass coverslip coated with CellTak® (Collaborative Research Inc.) and was placed in a superfusion chamber which was mounted on the stage of an inverted microscope (TMD-EF, Nikon). During the experiments, OHCs were contin uously superfused with the oxygenated solutions at 37 °C at a flow rate of 2.4 ml/min, ensuring the exchange of the bathing solution within several seconds. Digital images of fura-2 in OHCs were focused on an intensified silicon intensifier target camera (C-2400-8, Hamamatsu Photonics) and analyzed by an image processor (ARGUS-100, Hamamatsu Photonics). An in vitro calibration experiment as described by Grynkiewicz et al. [5] showed that the ratio of epifluorescence at 510 nm emitted with 340 and 380 nm excitation lights was propor tional to the logarithm of the Ca2‘ concentration. The drugs applied in the present study were acetylcholine (ACh), adenosine triphosphate (ATP), substance P, [D-ala2, met5]-enkephalin, calcitonin gene-related peptide (CGRP), y-aminobutyric acid (GABA), serotonin, dopamine, and norepinephrine. The experimental data were expressed as means ± SD. Statisti cal difference was analyzed by Student’s t test; p < 0.05 was accepted as significant.
Ikeda/Saito/Nishiyama/Takasaka
80
Fig. 4. Lack of effect of 100 \iM ACh on the [Ca2t]i elevation induced by 100 \iM ATP.
Fig. 5. Lack of effect of 10 \iM nifedipine on the [Ca2+]i eleva tion induced by 100 \iM ATP.
ing superfusate had little effect on the level of [Ca2+]i (fig. 4). Nifedipine (10 pM), a potent inhibitor of Ca2+ channels, did not inhibit the [Ca2+]i increase induced by 100 pM ATP (fig. 5). Other neuroregulators such as substance P (10 pM), enkephalin (lOOpM), CGRP (0.1 pM), GABA (lOmM), serotonin (100 pM), dopamine (lOmAf), and norepi nephrine (10 pM), either alone or in combination with ACh, failed to induce a change in OHC [Ca2+]i.
in the cochlear OHC of the guinea pig. The discrepancy as to the release of Ca2+ from the intracellular stores between two studies may be due to experimental proto col and preparation method. It is possible that with the external Ca2+ concentration in the nominal 0 Ca solution used in the experiment by Ashmore and Ohmori [15], there may be an inwardly directed electrochemical gra dient for Ca2+, and a Ca2+ influx from the external site on addition of ATP cannot be ruled out. It is known that ATP coexists with ACh in presynaptic nerve terminals [16], The present study did not reveal a sign of interac tion between ATP and ACh to the [Ca2+]i; the addition of ACh failed to alter the ATP-induced elevation of [Ca2+]i. On the other hand, it is reported that the simul taneous application of high-dose ACh inhibited the ele vation of [Ca2+]i by ATP [15], The discrepancy between our results and those of Ashmore and Ohmori [15] can be explained by the dose ratio of ATP vs. ACh and if the affinity of the receptor site to ATP is much higher than to ACh. A stimulation of the efferent fibers may induce a release of ATP together with ACh from the synaptic end ings and an increase in [Ca2+]i. The increase in [Ca2+]i induced by ATP may contribute to the slow motility mediated by the Ca2+-dependent action-myosin system and to hyperpolarization of the membrane potential induced by the Ca2+-activated K+ channel. Further stud ies regarding motile responses, ionic fluxes and mem brane currents of OHCs induced by neuroregulators are needed. In conclusion, ATP induced an increase in the [Ca2+]i via the entry of Ca2+ ions across the membrane. Other
Discussion ACh is the most likely neurotransmitter of the coch lear efferent system. Cholinergic action on the efferent synapse of the cochlea has been thought to involve the muscarinic mechanisms [9, 10]. In numerous cells, mus carinic agonists require a mobilization of cytosolic Ca2+ and an influx of extracellular Ca2+, resulting in a variety of cellular responses [11, 12]. The present finding that the application of ACh did not increase [Ca2+]i is consis tent with that of Dulon et al. [13], In contrast, an appli cation of ATP induced an increase in [Ca2+]i, which required the presence of extracellular Ca2+ ions. Unlike voltage-gated Ca2+ channels, the increase in [Ca2+]i was not inhibited by nifedipine. This finding implies the entry of Ca2+ across the cell membrane, via the ATPinduced nonspecific cationic channels found in the basolateral membrane of the OHC [14], Recently, Ashmore and Ohmori [15] reported that ATP also induced an increase in [Ca2+]i due to both transmembranous Ca2+ influx and mobilization from the intracellular Ca2+ store
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 11:05:13 AM
Tim e (min)
[Ca-*]i and Neuroregulators in the Outer Hair Cell
References 1 Bledsoe SC Jr, Bobbin RP, Puel J-L: Neurotransmission in the inner ear; in Jahn AH, Santos-Sacchi J (eds): Physiology of the Ear. New York, Raven Press, 1988, pp 385-406. 2 Flock A, Flock B, Ulfendahl M: Mechanisms of movement in OHCs and a possible structure basis. Arch Otorhinolaryngol 1986;243:83-90. 3 Art JJ, Fettiplace R, Fuchs PA: Synaptic hyperpolarization and inhibition of turtle cochlear hair cells. J Physiol 1984;356:525— 550. 4 Hudspeth AJ: The cellular basis of hearing: The biophysics of hair cells. Science 1985;230:745-752. 5 Grynkiewicz G, Poenie M, Tsien RY: A new generation of Ca2t indicators with greatly improved fluorescence properties. J Biol Chem 1985;260:3440-3450. 6 Robertson D, Johnstone BM: Efferent transmitter substance in the mammalian cochlea: Single neuron support for acetylcho line. Hear Res 1978;1:31-35. 7 Norris CH, Guth PS: The release of acetylcholine (ACh) by the crossed olivo-cochlear bundle (COCB). Acta Otolaryngol 1974; 77:318-326. 8 Altschuler RA, Kachar B, Rubio JA, et al: Immunocytochemical localization of choline acetyltransferase-like immunoreactivity in the guinea pig cochlea. Brain Res 1985;338:1-11.
9 van Megen YJB, KJassen ABM, Rodrigues de Miranda, et al: Cholinergic muscarinic receptors in rat cochlea. Brain Res 1988; 474:185-188. 10 Guiramand J, Mayat E, Bartolami S, et al: A M 3 muscarinic receptor coupled to inositol phosphate formation in the rat coch lea. Biochem Pharmacol 1990;39:1913-1919. 11 Williamson JR, Monck JR: Hormone effects on cellular Ca2* fluxes. Annu Rev Physiol 1989;51:107-124. 12 Berridge MJ, Irvine RF: Inositol phosphates and cell signalling. Nature 1989;341:197-205. 13 Dulon D, Zajic G, Schacht J: Increasing intracellular free cal cium induces circumferential contractions in isolated cochlear outer hair cells. J Neurosci 1990;10:1388-1397. 14 Nakagawa T, Akaike N, Kimitsuki T, et al: ATP-induced current in isolated outer hair cells of guinea pig cochlea. J Neurophysiol 1990;63:1068-1074. 15 Ashmore JF, Ohmori H: Control of intracellular calcium by ATP in isolated outer hair cells of the guinea pig cochlea. J Physiol 1990;428:109-131. 16 Bumstock G: Neurotransmitters and trophic factors in the auto nomic nervous system. J Physiol 1981;313:1-35.
Received: October 15, 1990 Accepted: October 24, 1990 Katsuhisa Ikdea, MD Department of Otolaryngology Tohoku University School of Medicine 1-1 Seiryo-machi, Aoba-ku Sendai 980 (Japan)
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 11:05:13 AM
neuroregulator substances such as ACh, GABA, enkeph alin, CGRP, substance P, serotonin, dopamine, and nor epinephrine failed to increase the [Ca2+]i.
81
ORL 1991;53:78-81
Effect of Neuroregulators on the Intracellular Calcium Level in the Outer Hair Cell Isolated from the Guinea Pig Katsuhisa Ikedaa, Yoshitaka Saitob, Akinori Nishiyamab, Tomonori T a k a sa k a 1 Departments o f aOtolaryngology and bPhysiology, Tohoku University School of Medicine, Sendai, Japan
Key Words. Outer hair cell • Intracellular Ca2+ concentration • Adenosine triphosphate • Acetylcholine • Efferent synapse Abstract. The cytosolic calcium concentration ([Ca2+]i) of the isolated outer hair cell of the guinea pig was mea sured using fluorescence imaging microscopy and the effects of efferent neuroregulators such as acetylcholine, ATP, GABA, substance P, enkephalin, calcitonin gene-related peptide, serotonin, dopamine, norepinephrine, and gluta mate were investigated. Among the drugs tested only ATP induced an elevation of the [Ca2+]i of the outer hair cell. In the resting condition, [Ca2+]i averaged 104.5 ± 31.1 nM (n = 27), while 100 pAf ATP significantly increased [Ca2+]i to 146.3 ± 43.5 nM (n = 19). Superfusion with Ca2+-free solution (pCa = 7.5) abolished the increase in [Ca2+]i induced by ATP, suggesting that ATP causes an entry of external Ca2+. The relevance of [Ca2+]i to the inhibitory actions of efferent neuroregulators is discussed.
The outer hair cell (OHC) of the mammalian cochlea is extensively innervated by efferent fibers. The physio logical role of the efferent innervation to the OHC is believed to be inhibitory on the cochlear potential [1], however, the cellular and biological mechanism of action have not yet been elucidated. Two possibilities closely related to Ca2+ ions for explanation of the functional mechanism of the efferent actions in the cochlea have been considered. One is that efferent neurotransmitters may generate an OHC motility that may attenuate fre quency selectivity. Slow motility is initiated by an in crease in the concentration of intracellular free Ca2+ ([Ca2+]i) and interacts with the cytoskeletal proteins and calmodulin [2]. Another explanation for the inhibitory effects of the cochlear efferent system is the hyperpolar ization of the membrane potential in the OHC resulting from the efferent stimulation [3]. In light of the proposed 1 We thank Ms. Michiko Yokoyama for preparing the manu script.
model of mechanoelectrical signal transduction in the hair cell [4], it is proposed that an increase in [Ca2+]i activates the K+conductance and hyperpolarizes the hair cells. Therefore, it is of interest to explore the changes in [Ca2+]i of the OHC in response to efferent transmitters. In the present study we tested a number of candidate neurotransmitter substances to identify that physiologi cally involved in the increase in OHC [Ca2+]i.
Materials and Methods Albino guinea pigs (200-350 g) with a Preyer reflex were anes thetized by inhalation of ethyl ether and the temporal bones were rapidly removed after decapitation. After the tympanic bulla was opened, the bony capsules of the cochlea were picked away under a stereomicroscope to expose the spiral ligament. The lateral walls of the cochlea were peeled away from the basilar membrane and the organ of Corti in the apical to third turns was dissected and col lected. Microdissection was performed in a Petri dish filled with an oxygenated artificial perilymph composed of 150 mM NaCl, 3.5 mA/KCl, 1.0m 3/C aC l2, 1.0m 4/M gCl2, 2.75 nuV/HEPES, and 2.25 m M Tris. The pH and osmolality of the solution were adjusted to 7.4 with HC1 and 300 mosm/kg H 20 with D-glucose, respectively.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 11:05:13 AM
Introduction
[Ca2’]i and Neuroregulators in the Outer Hair Cell
79
Fig. 1. Absence of effect of ACh ( 10-6, 10_s, 10“J M ) on [Ca2+]i. Fig. 2. Effects of 100 \iM ATP on [Ca2*]i. Fig. 3. Decrease in [Ca2^]i induced by 100 \iM ATP in the absence of external Ca2* ions.
Results ACh is one of the putative neurotransmitters of the cochlear efferent system on the basis of physiological [6], biochemical [7] and histochemical [8] studies. Addition of 1-100 \iM ACh to the superfused artificial perilymph, however, did not alter the OHC [Ca2+]i (fig. 1). On the other hand, an application of 100 pM ATP abruptly increased the OHC [Ca2+]i to a plateau within 3 min (fig. 2). The basal value of [Ca2+]i, 104.5 ± 31.1 nM(n = 27), was significantly increased to 119.8 ± 16.5 nM (n = 8) by 10 pM ATP and to 146.3 ± 43.5 nM (n = 19) by 100 pM ATP. When the external Ca2+ concentration was reduced to pCa 7.5 by adding EGTA, the application of 100 pA/ ATP decreased [Ca2+]i to 58.3 ± 19.2 nM (n = 4; fig. 3), which was not significantly different from the value (65.8 ± 21.5 nM, n = 3) obtained under the super fusion with Ca-free solution without ATP. The effect of ACh on the ATP-induced increase in [Ca2+]i was examined. After an elevation of [Ca2+]i with 100 pM ATP, addition of 100 pM ACh to ATP-contain-
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 11:05:13 AM
The OHCs were mechanically isolated by gentle reflux through a micropipette and were incubated in the artificial perilymph supple mented with 3 pAf fura-2 acetoxymethyl ester (Dojindo Lab.) at 37 °C for 60 min. An approximately 30-jj.I droplet containing fura2-loaded OHCs was plated on a glass coverslip coated with CellTak® (Collaborative Research Inc.) and was placed in a superfusion chamber which was mounted on the stage of an inverted microscope (TMD-EF, Nikon). During the experiments, OHCs were contin uously superfused with the oxygenated solutions at 37 °C at a flow rate of 2.4 ml/min, ensuring the exchange of the bathing solution within several seconds. Digital images of fura-2 in OHCs were focused on an intensified silicon intensifier target camera (C-2400-8, Hamamatsu Photonics) and analyzed by an image processor (ARGUS-100, Hamamatsu Photonics). An in vitro calibration experiment as described by Grynkiewicz et al. [5] showed that the ratio of epifluorescence at 510 nm emitted with 340 and 380 nm excitation lights was propor tional to the logarithm of the Ca2‘ concentration. The drugs applied in the present study were acetylcholine (ACh), adenosine triphosphate (ATP), substance P, [D-ala2, met5]-enkephalin, calcitonin gene-related peptide (CGRP), y-aminobutyric acid (GABA), serotonin, dopamine, and norepinephrine. The experimental data were expressed as means ± SD. Statisti cal difference was analyzed by Student’s t test; p < 0.05 was accepted as significant.
Ikeda/Saito/Nishiyama/Takasaka
80
Fig. 4. Lack of effect of 100 \iM ACh on the [Ca2t]i elevation induced by 100 \iM ATP.
Fig. 5. Lack of effect of 10 \iM nifedipine on the [Ca2+]i eleva tion induced by 100 \iM ATP.
ing superfusate had little effect on the level of [Ca2+]i (fig. 4). Nifedipine (10 pM), a potent inhibitor of Ca2+ channels, did not inhibit the [Ca2+]i increase induced by 100 pM ATP (fig. 5). Other neuroregulators such as substance P (10 pM), enkephalin (lOOpM), CGRP (0.1 pM), GABA (lOmM), serotonin (100 pM), dopamine (lOmAf), and norepi nephrine (10 pM), either alone or in combination with ACh, failed to induce a change in OHC [Ca2+]i.
in the cochlear OHC of the guinea pig. The discrepancy as to the release of Ca2+ from the intracellular stores between two studies may be due to experimental proto col and preparation method. It is possible that with the external Ca2+ concentration in the nominal 0 Ca solution used in the experiment by Ashmore and Ohmori [15], there may be an inwardly directed electrochemical gra dient for Ca2+, and a Ca2+ influx from the external site on addition of ATP cannot be ruled out. It is known that ATP coexists with ACh in presynaptic nerve terminals [16], The present study did not reveal a sign of interac tion between ATP and ACh to the [Ca2+]i; the addition of ACh failed to alter the ATP-induced elevation of [Ca2+]i. On the other hand, it is reported that the simul taneous application of high-dose ACh inhibited the ele vation of [Ca2+]i by ATP [15], The discrepancy between our results and those of Ashmore and Ohmori [15] can be explained by the dose ratio of ATP vs. ACh and if the affinity of the receptor site to ATP is much higher than to ACh. A stimulation of the efferent fibers may induce a release of ATP together with ACh from the synaptic end ings and an increase in [Ca2+]i. The increase in [Ca2+]i induced by ATP may contribute to the slow motility mediated by the Ca2+-dependent action-myosin system and to hyperpolarization of the membrane potential induced by the Ca2+-activated K+ channel. Further stud ies regarding motile responses, ionic fluxes and mem brane currents of OHCs induced by neuroregulators are needed. In conclusion, ATP induced an increase in the [Ca2+]i via the entry of Ca2+ ions across the membrane. Other
Discussion ACh is the most likely neurotransmitter of the coch lear efferent system. Cholinergic action on the efferent synapse of the cochlea has been thought to involve the muscarinic mechanisms [9, 10]. In numerous cells, mus carinic agonists require a mobilization of cytosolic Ca2+ and an influx of extracellular Ca2+, resulting in a variety of cellular responses [11, 12]. The present finding that the application of ACh did not increase [Ca2+]i is consis tent with that of Dulon et al. [13], In contrast, an appli cation of ATP induced an increase in [Ca2+]i, which required the presence of extracellular Ca2+ ions. Unlike voltage-gated Ca2+ channels, the increase in [Ca2+]i was not inhibited by nifedipine. This finding implies the entry of Ca2+ across the cell membrane, via the ATPinduced nonspecific cationic channels found in the basolateral membrane of the OHC [14], Recently, Ashmore and Ohmori [15] reported that ATP also induced an increase in [Ca2+]i due to both transmembranous Ca2+ influx and mobilization from the intracellular Ca2+ store
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 11:05:13 AM
Tim e (min)
[Ca-*]i and Neuroregulators in the Outer Hair Cell
References 1 Bledsoe SC Jr, Bobbin RP, Puel J-L: Neurotransmission in the inner ear; in Jahn AH, Santos-Sacchi J (eds): Physiology of the Ear. New York, Raven Press, 1988, pp 385-406. 2 Flock A, Flock B, Ulfendahl M: Mechanisms of movement in OHCs and a possible structure basis. Arch Otorhinolaryngol 1986;243:83-90. 3 Art JJ, Fettiplace R, Fuchs PA: Synaptic hyperpolarization and inhibition of turtle cochlear hair cells. J Physiol 1984;356:525— 550. 4 Hudspeth AJ: The cellular basis of hearing: The biophysics of hair cells. Science 1985;230:745-752. 5 Grynkiewicz G, Poenie M, Tsien RY: A new generation of Ca2t indicators with greatly improved fluorescence properties. J Biol Chem 1985;260:3440-3450. 6 Robertson D, Johnstone BM: Efferent transmitter substance in the mammalian cochlea: Single neuron support for acetylcho line. Hear Res 1978;1:31-35. 7 Norris CH, Guth PS: The release of acetylcholine (ACh) by the crossed olivo-cochlear bundle (COCB). Acta Otolaryngol 1974; 77:318-326. 8 Altschuler RA, Kachar B, Rubio JA, et al: Immunocytochemical localization of choline acetyltransferase-like immunoreactivity in the guinea pig cochlea. Brain Res 1985;338:1-11.
9 van Megen YJB, KJassen ABM, Rodrigues de Miranda, et al: Cholinergic muscarinic receptors in rat cochlea. Brain Res 1988; 474:185-188. 10 Guiramand J, Mayat E, Bartolami S, et al: A M 3 muscarinic receptor coupled to inositol phosphate formation in the rat coch lea. Biochem Pharmacol 1990;39:1913-1919. 11 Williamson JR, Monck JR: Hormone effects on cellular Ca2* fluxes. Annu Rev Physiol 1989;51:107-124. 12 Berridge MJ, Irvine RF: Inositol phosphates and cell signalling. Nature 1989;341:197-205. 13 Dulon D, Zajic G, Schacht J: Increasing intracellular free cal cium induces circumferential contractions in isolated cochlear outer hair cells. J Neurosci 1990;10:1388-1397. 14 Nakagawa T, Akaike N, Kimitsuki T, et al: ATP-induced current in isolated outer hair cells of guinea pig cochlea. J Neurophysiol 1990;63:1068-1074. 15 Ashmore JF, Ohmori H: Control of intracellular calcium by ATP in isolated outer hair cells of the guinea pig cochlea. J Physiol 1990;428:109-131. 16 Bumstock G: Neurotransmitters and trophic factors in the auto nomic nervous system. J Physiol 1981;313:1-35.
Received: October 15, 1990 Accepted: October 24, 1990 Katsuhisa Ikdea, MD Department of Otolaryngology Tohoku University School of Medicine 1-1 Seiryo-machi, Aoba-ku Sendai 980 (Japan)
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 11:05:13 AM
neuroregulator substances such as ACh, GABA, enkeph alin, CGRP, substance P, serotonin, dopamine, and nor epinephrine failed to increase the [Ca2+]i.
81
