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Nitric oxide inhibits adenosine 5′-triphosphate-induced Ca2+ response in inner hair cells of the guinea pig cochlea
Neuroscience Letters 337 (2003) 135–138 www.elsevier.com/locate/neulet
Nitric oxide inhibits adenosine 5 0 -triphosphate-induced Ca 21 response in inner hair cells of the guinea pig cochlea Jing Shen a,b, Narinobu Harada a,*, Toshio Yamashita a a
Hearing Research Laboratory, Department of Otolaryngology, Kansai Medical University, Fumizonocho 10–15, Moriguchi, Osaka 570-8507, Japan b Hearing Research Laboratory, China Medical University, Shenyang 110001, P.R.China Received 2 October 2002; received in revised form 6 November 2002; accepted 9 November 2002
Abstract To investigate the interaction between Ca 21 and nitric oxide (NO) in inner hair cells of the guinea pig cochlea (IHCs), the extracellular adenosine 5 0 -triphosphate (ATP)-induced NO production and the effects of NO on ATP-induced increase of intracellular Ca 21 concentrations ([Ca 21]i) were investigated in IHCs using the NO-sensitive dye DAF-2 and the Ca 21sensitive dye Fura-2. Extracellular ATP induced an increase in DAF-2 fluorescence, which thus indicates NO production in IHCs. The ATP-induced NO production was mainly due to Ca 21 influx through the activation of P2 receptor. l-N G-nitroarginine methyl ester, a NO synthesis inhibitor, enhanced the ATP-induced [Ca 21]i increase in IHCs while S-nitroso-Nacetylpenicillamine, a NO donor, inhibited it. We conclude that NO inhibits the ATP-induced [Ca 21]i increase in IHCs by a negative-feedback mechanism. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Inner hair cell; P2 receptor; Nitric oxide; Negative-feedback; Ca 21 homeostasis; Cochlea
Cochlear inner hair cells (IHCs), as mechano-electrial transducers, encode acoustic information and transmit it to the central nervous system. The release of neurotransmitters is triggered by the entry of Ca 21 into IHCs [3]. As a result, Ca 21 homeostasis in IHCs plays a very important role in auditory signal transduction. Extracellular adenosine 5 0 triphosphate (ATP) has been suggested to play an important role as a neurotransmitter or neuromodulator in the inner ear [8]. In the cochlea, extracellular ATP has been reported to elicit an elevation of [Ca 21]i in IHCs and spiral ganglion cells [1,14]. These previous studies also suggest that ATP may act as an afferent neurotransmitter or neuromodulator in the cochlea [1,14]. There is increasing evidence that NO contributes to the cellular Ca 21 homeostasis in several cell types by either a positive or negative effect on Ca 21 transients [2]. The expression of the neuronal isoform (nNOS) and the endothelial isoform (eNOS) in the enzyme NOS requires an increase in the intracellular Ca 21 concentrations ([Ca 21]i) for them to obtain their maximal activity. Therefore, a rise in [Ca 21]i
* Corresponding author. Tel.: 181-6-6992-1001; fax: 181-66992-1055. E-mail address: [email protected] (N. Harada).
may serve to amplify the NO production as previously reported [12]. Both nNOS and eNOS, which are Ca 21dependent, have been recently detected in the guinea pig cochlea [5,13]. However the role of NO and the interaction between NO and Ca 21 in the auditory signal transduction pathway still remains to be elucidated. The present study was designed to investigate the effects of NO on ATPinduced Ca 21 mobilization and a possibility of interaction between NO and Ca 21 in IHCs using the NO-sensitive dye DAF-2 and the Ca 21-sensitive dye Fura-2. The isolation procedure of the IHCs was similar to that previously described [16]. In brief, the organ of Corti from the guinea pig cochlea was dissected in physiological standard solution (PSS) containing (mM): NaCl, 150; KCl, 5; MgCl2, 1; D-Glucose, 5; HEPES, 10; CaCl2, 2; adjusted to a pH 7.4 and 300 mOsm. The pieces of Corti organ were further treated with trypsin (0.5 mg/ml, Sigma) for 10 min followed by rinsing with bovine serum albumin (0.5 mg/ml, Sigma). Finally, the isolation procedure was completed by mechanical dissociation and pipetting in fresh PSS. The isolated IHCs settled and adhered to the bottom of the chamber coated in advance with Cell-Tak (Cosmo-Bio, Japan). The morphological criteria used in this experiment to identify the IHCs were compatible with those previously
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S03 04 -3 94 0( 02 ) 01 320 - 4
136
J. Shen et al. / Neuroscience Letters 337 (2003) 135–138
Fig. 1. Production of nitric oxide induced by extracellular ATP in IHCs for a variety of treatments. (a) Normalized changes in DAF-2 fluorescence induced by 100 mM ATP alone (up trace), pretreatment cells with 100 mM suramin (middle trace) and neither ATP nor suramin is added, as a control (bottom trace). (b) Normalized changes in DAF-2 fluorescence induced by 100 mM ATP in presence of 100 mM d-NAME (up trace), pretreatment cells with 200 mM l-NAME (middle trace), in Ca 21 -free medium (bottom trace), The traces in (a) and (b) represent the averaged response with error bars showing SD. (c) A histogram summarized the normalized maximum average for the above treatments. *P , 0:05 compared to the ATP-evoked NO production.
described [7,16]. In brief, the structured cytoplasm of IHCs was different from OHCs, the cuticular plate in IHCs was thick and in a curved surface and angled to cell body in contract to the flat-shape in the OHCs. Measurements of NO production were performed using the single excitation wavelength method described by Kojima et al. [9]. The cells were loaded with 5 mM DAF2DA (Daiichi pure chemicals CO., LTD. Tokyo Japan) for 40 min. The DAF-2 fluorescence in IHCs was imaged using an inverted fluorescent microscope (TMD-2, Nikon, Japan) equipped with a xenon lamp and excitation filter (485 , 495 nm) and an emission filter (510 , 540 nm). A fluorescent image was obtained via a low-light, silicon-intensified target camera, and analyzed with Argus-100 imaging-analysis system (Hamamatsu Photonics, Japan). The data were presented as relative changes in fluorescence intensity normalized to the baseline fluorescence (DF/F). In some experiments, l-N G-nitroarginine methyl ester (l-NAME)
and d-NAME were added 15 min before loading with DAF-2 DA. For measurements of NO and [Ca 21]i in the same IHCs, DAF-2 loaded IHCs were subsequently loaded with 2 mM Fura-2/AM after DAF-2 measurements. [Ca 21]i was measured as described below using the same equipment as for DAF-2 measurements. The measurements of [Ca 21]i were made following a protocol similar to that we reported elsewhere [1]. In brief, the isolated IHCs were loaded with 2 mM fura-2/AM (Dojindo, Japan) for 30 min. A fluorescence image was obtained by an excitation of a wavelength pair of 340 nm and 380 nm through a 500 nm long-pass filter. Each image was analyzed by a cooled-CCD camera system (HisCA/Argus system, Hamamatsu Photonics, Japan). The fluorescence ratio (340/380 nm) was calculated and transferred to the absolute values of [Ca 21]i based on the formula described by Grynkiewicz et al. [6] using (ethylenebis(oxononitrilo))tetraacetate (EGTA)-Ca 21 buffer solution. The fluorescence ratio obtained at 340 and 380nm was used as an index of [Ca 21]i in the present study. All of experiments were done at room temperature. The drugs were applied by perfusion using a microperpex pump (LKB2132, Pharmacia LKB Biotechnology). All the drugs were purchased from Sigma, unless otherwise specified. Ca 21-free solution was prepared by omitting CaCl2 from PSS but by adding 2 mM EGTA. All values are given as the means ^ standard deviation of the mean, and n denotes the number of the cells. Statistical significance was evaluated by the two-tailed unpaired Student’s t-test. A value of P of less than 0.05 was considered to be significant. The application of extracellular ATP (100 mM) caused a gradual saturable increase in DAF-2 fluorescence, indicative of NO production in IHCs (Fig. 1a). Suramin (100 mM), an antagonist for P2 receptor, inhibited the ATP-induced increase of DAF-2 fluorescence in IHCs (n ¼ 15. Fig. 1a). Furthermore, ATP-induced NO production in IHCs was significantly suppressed by the pretreatment with 200 mM l-NAME, a non-specific NO synthesis inhibitor (n ¼ 6, Fig. 1b) while 200 mM d-NAME, inactive isomer of l-NAME, did not inhibit the ATP-induced NO production (n ¼ 3, Fig. 1b). In the absence of extracellular Ca 21, ATP-induced NO production was abolished (n ¼ 14, Fig. 1b). The fluorescent imaging of both intracellular NO and [Ca 21]i was performed in the same IHC (Fig. 2). The DAF-2 loaded IHCs did not have any fluorescence under the excitation of both 340 nm and 380 nm at a resting state, during the control measurements with PSS and ATP stimulation (Fig. 2a). However, DAF-2 loaded IHCs demonstrated fluorescence under excitation at both 340 nm and 380 nm after loading with fura-2 (Fig. 2b). As a result, DAF-2 itself did not affect the Fura-2 measurements. Both NO production and [Ca 21]i increase induced by ATP were observed in the same IHCs (Fig. 2c–j). Continually, the experiments were designed to determine whether NO affects [Ca 21]i in the IHCs. The application of extracellular ATP (100 mM) caused a transient increase of [Ca 21]i in IHCs. The response to a subsequent application of
J. Shen et al. / Neuroscience Letters 337 (2003) 135–138
137
Fig. 2. Fluorescence images of nitric oxide production and [Ca 21]i changes induced by ATP in the same IHC. (a) The DAF-2 loaded IHCs did not have any fluorescence under the excitation of both 340 nm and 380 nm before loading with fura-2. (b) The fluorescence was identified under 340 nm and 380 nm after additional loading the same IHC with fura-2/AM. (c) Phase-contrast view of IHC, scale bar ¼ 10 mm. (d–f) Pseudo-color displays of the ATP-evoked NO production in DAF-2 loaded and fura-2 unloaded IHC. The fluorescence image of the IHC at pre-stimulation (d), 5 min (e) and 10 min (f) after stimulation with 100 mM ATP. (g–j) Pseudo-color displays of the ATP-induced [Ca 21]i changes in the same IHC loading with fura-2 after DAF-2 measurements. The fluorescence image of the IHC at pre-stimulation (g), 1 min (h), 1.5 min (i) and 3 min (f) after stimulation with 100 mM ATP. The color bar represents relative DAF-2 fluorescence intensity and the 340 nm/380 nm ratio level, respectively.
ATP at 15 min intervals was similar with the amplitude of the first response (Fig. 3a). Therefore, two applications of ATP at intervals of longer than 15 min were used in the present study. Thus, the effects of ATP were reversible and repeatable in our experimental arrangements. 200 mM l-NAME enhanced ATP-induced [Ca 21]i increases to 152 ^ 59% of the control (n ¼ 27, P , 0:05, Fig. 3b) while 100 mM S-nitroso-N-acetylpenicillamine (SNAP), NO donor, inhibited it to 78 ^ 23% of the control (n ¼ 9, P , 0:05, Fig. 3c). Neither 200 mM l-NAME nor 100 mM SNAP alone elevated the [Ca 21]i. The present study demonstrates that extracellular ATP can induce NO production in isolated cochlear IHCs using NOspecific fluorescent dye DAF-2. To our knowledge, this is the first direct evidence of NO production induced by extracellular ATP in IHCs. Our results also showed that ATP stimulation did not induce an increase in DAF-2 fluorescence in the absence of extracellular Ca 21 while ATP can induce a [Ca 21]i increase by Ca 21 release from intracellular stores in IHCs [14]. These results suggest that the ATP-induced NO production may be caused by the ATP-induced [Ca 21]i increase, which is mainly due to Ca 21 influx through the activation
of P2 receptor. Recent study by simultaneous measurements of DAF-2 and fura-2 showed that the increase of DAF-2 fluorescence induced by ATP lagged behind the rapid [Ca 21]i transient by Ca 21 release from internal stores and was observed during the late phase of the [Ca 21]i increase by Ca 21 influx in vascular endothelial cells [4]. They suggested that the [Ca 21]i increase induced by Ca 21 influx not release from internal stores was essential for NO production although both Ca 21 influx and Ca 21 release from internal stores could induce an increase of [Ca 21]i. Our findings that the ATP-induced NO production was mainly due to Ca 21 influx are line with the previous study. Several studies using fura-2 techniques suggested that the [Ca 21]i increase induced by ATP may activate NO production, which thus affects Ca 21 signaling by a positive feedback mechanism [11,15]. Our results also provided clear evidence that NO attenuated the ATP-induced calcium signaling in IHCs. These results are consistent with recent findings showing that NO attenuated an ATP-evoked [Ca 21]i increase in cochlear supporting cells [10]. Together with the abovementioned results, we presume that NO regulates the ATPinduced [Ca 21]i increase in IHCs by a negative-feedback
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J. Shen et al. / Neuroscience Letters 337 (2003) 135–138
Fig. 3. Effect of NO on the ATP-evoked [Ca 21]i increase in IHCs. (a) A representative recording of increase in [Ca 21]i induced by 100 mM ATP in different intervals. The amplitude was almost same as the first response when repetitive stimulation with ATP was performed at 15 min intervals. (b) Effect of l-NAME, a NO synthase inhibitor, on ATP-induced [Ca 21]i increase. The [Ca 21]i response to ATP was significantly enhanced in the presence of 200 mM l-NAME. (c) Effect of SNAP, a NO donor, on ATP-induced [Ca 21]i increase. 100 mM SNAP markedly decreased the response to ATP (n ¼ 9). (d) A summary of statistics results on the changes of ATP-induced [Ca 21]i increase on IHCs with the treatments as described above. The values obtained with the second response to ATP were normalized after referencing them to those obtained with the first response to ATP. Error bar showed SD. *P , 0:05 compared to the [Ca 21]i response to ATP as a control (n ¼ 14).
mechanism. Since both ATP and NO act as neuromodulators in the cochlea, we are also able to deduce the possibility that interactions occur between ATP and NO, which might thus represent a regulation loop of IHC physiology that critically modulates Ca 21 homeostasis in IHCs.
[1] Cho, H., Harada, N. and Yamashita, T., Extracellular ATPinduced Ca 21 mobilization of type I spiral ganglion cells from the guinea pig cochlea, Acta. Otolaryngol., 117 (1997) 545–552. [2] Clementi, E. and Meldolesi, J., The cross-talk between nitric oxide and Ca 21: a story with a complex past and a promising future, Trends Pharmacol. Sci., 18 (1997) 266–269. [3] Dallos, P., The active cochlea, J. Neurosci., 12 (1992) 4575– 4585. [4] Dedkova, E.N. and Blatter, L.A., Nitric oxide inhibits capacitative Ca 21 entry and enhances endoplasmic reticulum Ca 21 uptake in bovine vascular endothelial cells, J. Physiol., 539 (2002) 77–91. [5] Gosepath, K., Gath, I., Maurer, J., Pollock, J.S., Amedee, R., Forstermann, U. and Mann, W., Characterization of nitric oxide synthase isoforms expressed in different structures of the guinea pig cochlea, Brain Res., 747 (1997) 26– 33. [6] Grynkiewicz, G., Poenie, M. and Tsien, R.Y., A new generation of Ca 21 indicators with greatly improved fluorescence properties, J. Biol. Chem., 260 (1985) 3440–3450. [7] He, D.Z., Zheng, J., Edge, R. and Dallos, P., Isolation of cochlear inner hair cells, Hear. Res., 145 (2000) 156–160. [8] Housley, G.D., Extracellular nucleotide signaling in the inner ear, Mol. Neurobiol., 16 (1998) 21–48. [9] Kojima, H., Nakatsubo, N., Kikuchi, K., Urano, Y., Higuchi, T., Tanaka, J., Kudo, Y. and Nagano, T., Direct evidence of NO production in rat hippocampus and cortex using a new fluorescent indicator: DAF-2 DA, NeuroReport, 9 (1998) 3345–3348. [10] Matsunobu, T. and Schacht, J., Nitric oxide/cyclic GMP pathway attenuates ATP-evoked intracellular calcium increase in supporting cells of the guinea pig cochlea, J. Comp. Neurol., 423 (2000) 452–461. [11] Nobles, M. and Abbott, N.J., Modulation of the effects of extracellular ATP on [Ca 21]i in rat brain microvacular endothelial cells, Eur. J. Pharmacol., 361 (1998) 119–127. [12] Publicover, N.G., Hammond, E.M. and Sanders, K.M., Free in PMC Amplification of nitric oxide signaling by interstitial cells isolated from canine colon, Proc. Natl. Acad. Sci. USA, 90 (1993) 2087–2091. [13] Riemann, R. and Reuss, S., Nitric oxide synthase in identified olivocochlear projection neurons in rat and guinea pig, Hear. Res., 135 (1999) 181–189. [14] Sugasawa, M., Erostegui, C., Blanchet, C. and Dulon, D., ATP activates non-selective cation channels and calcium release in inner hair cells of the guinea-pig cochlea, J. Physiol., 491 (1996) 707–718. [15] Uzlaner, N. and Priel, Z., Interplay between the NO pathway and elevated [Ca 21]i enhances ciliary activity in rabbit trachea, J. Physiol., 516 (1999) 179–190. [16] Yang, S.H., Shen, J., Doi, T. and Yamashita, T., Isolation of guinea pig inner hair cells using manual microsurgical dissection, ORL., 64 (2002) 1–5.
Nitric oxide inhibits adenosine 5 0 -triphosphate-induced Ca 21 response in inner hair cells of the guinea pig cochlea Jing Shen a,b, Narinobu Harada a,*, Toshio Yamashita a a
Hearing Research Laboratory, Department of Otolaryngology, Kansai Medical University, Fumizonocho 10–15, Moriguchi, Osaka 570-8507, Japan b Hearing Research Laboratory, China Medical University, Shenyang 110001, P.R.China Received 2 October 2002; received in revised form 6 November 2002; accepted 9 November 2002
Abstract To investigate the interaction between Ca 21 and nitric oxide (NO) in inner hair cells of the guinea pig cochlea (IHCs), the extracellular adenosine 5 0 -triphosphate (ATP)-induced NO production and the effects of NO on ATP-induced increase of intracellular Ca 21 concentrations ([Ca 21]i) were investigated in IHCs using the NO-sensitive dye DAF-2 and the Ca 21sensitive dye Fura-2. Extracellular ATP induced an increase in DAF-2 fluorescence, which thus indicates NO production in IHCs. The ATP-induced NO production was mainly due to Ca 21 influx through the activation of P2 receptor. l-N G-nitroarginine methyl ester, a NO synthesis inhibitor, enhanced the ATP-induced [Ca 21]i increase in IHCs while S-nitroso-Nacetylpenicillamine, a NO donor, inhibited it. We conclude that NO inhibits the ATP-induced [Ca 21]i increase in IHCs by a negative-feedback mechanism. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Inner hair cell; P2 receptor; Nitric oxide; Negative-feedback; Ca 21 homeostasis; Cochlea
Cochlear inner hair cells (IHCs), as mechano-electrial transducers, encode acoustic information and transmit it to the central nervous system. The release of neurotransmitters is triggered by the entry of Ca 21 into IHCs [3]. As a result, Ca 21 homeostasis in IHCs plays a very important role in auditory signal transduction. Extracellular adenosine 5 0 triphosphate (ATP) has been suggested to play an important role as a neurotransmitter or neuromodulator in the inner ear [8]. In the cochlea, extracellular ATP has been reported to elicit an elevation of [Ca 21]i in IHCs and spiral ganglion cells [1,14]. These previous studies also suggest that ATP may act as an afferent neurotransmitter or neuromodulator in the cochlea [1,14]. There is increasing evidence that NO contributes to the cellular Ca 21 homeostasis in several cell types by either a positive or negative effect on Ca 21 transients [2]. The expression of the neuronal isoform (nNOS) and the endothelial isoform (eNOS) in the enzyme NOS requires an increase in the intracellular Ca 21 concentrations ([Ca 21]i) for them to obtain their maximal activity. Therefore, a rise in [Ca 21]i
* Corresponding author. Tel.: 181-6-6992-1001; fax: 181-66992-1055. E-mail address: [email protected] (N. Harada).
may serve to amplify the NO production as previously reported [12]. Both nNOS and eNOS, which are Ca 21dependent, have been recently detected in the guinea pig cochlea [5,13]. However the role of NO and the interaction between NO and Ca 21 in the auditory signal transduction pathway still remains to be elucidated. The present study was designed to investigate the effects of NO on ATPinduced Ca 21 mobilization and a possibility of interaction between NO and Ca 21 in IHCs using the NO-sensitive dye DAF-2 and the Ca 21-sensitive dye Fura-2. The isolation procedure of the IHCs was similar to that previously described [16]. In brief, the organ of Corti from the guinea pig cochlea was dissected in physiological standard solution (PSS) containing (mM): NaCl, 150; KCl, 5; MgCl2, 1; D-Glucose, 5; HEPES, 10; CaCl2, 2; adjusted to a pH 7.4 and 300 mOsm. The pieces of Corti organ were further treated with trypsin (0.5 mg/ml, Sigma) for 10 min followed by rinsing with bovine serum albumin (0.5 mg/ml, Sigma). Finally, the isolation procedure was completed by mechanical dissociation and pipetting in fresh PSS. The isolated IHCs settled and adhered to the bottom of the chamber coated in advance with Cell-Tak (Cosmo-Bio, Japan). The morphological criteria used in this experiment to identify the IHCs were compatible with those previously
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S03 04 -3 94 0( 02 ) 01 320 - 4
136
J. Shen et al. / Neuroscience Letters 337 (2003) 135–138
Fig. 1. Production of nitric oxide induced by extracellular ATP in IHCs for a variety of treatments. (a) Normalized changes in DAF-2 fluorescence induced by 100 mM ATP alone (up trace), pretreatment cells with 100 mM suramin (middle trace) and neither ATP nor suramin is added, as a control (bottom trace). (b) Normalized changes in DAF-2 fluorescence induced by 100 mM ATP in presence of 100 mM d-NAME (up trace), pretreatment cells with 200 mM l-NAME (middle trace), in Ca 21 -free medium (bottom trace), The traces in (a) and (b) represent the averaged response with error bars showing SD. (c) A histogram summarized the normalized maximum average for the above treatments. *P , 0:05 compared to the ATP-evoked NO production.
described [7,16]. In brief, the structured cytoplasm of IHCs was different from OHCs, the cuticular plate in IHCs was thick and in a curved surface and angled to cell body in contract to the flat-shape in the OHCs. Measurements of NO production were performed using the single excitation wavelength method described by Kojima et al. [9]. The cells were loaded with 5 mM DAF2DA (Daiichi pure chemicals CO., LTD. Tokyo Japan) for 40 min. The DAF-2 fluorescence in IHCs was imaged using an inverted fluorescent microscope (TMD-2, Nikon, Japan) equipped with a xenon lamp and excitation filter (485 , 495 nm) and an emission filter (510 , 540 nm). A fluorescent image was obtained via a low-light, silicon-intensified target camera, and analyzed with Argus-100 imaging-analysis system (Hamamatsu Photonics, Japan). The data were presented as relative changes in fluorescence intensity normalized to the baseline fluorescence (DF/F). In some experiments, l-N G-nitroarginine methyl ester (l-NAME)
and d-NAME were added 15 min before loading with DAF-2 DA. For measurements of NO and [Ca 21]i in the same IHCs, DAF-2 loaded IHCs were subsequently loaded with 2 mM Fura-2/AM after DAF-2 measurements. [Ca 21]i was measured as described below using the same equipment as for DAF-2 measurements. The measurements of [Ca 21]i were made following a protocol similar to that we reported elsewhere [1]. In brief, the isolated IHCs were loaded with 2 mM fura-2/AM (Dojindo, Japan) for 30 min. A fluorescence image was obtained by an excitation of a wavelength pair of 340 nm and 380 nm through a 500 nm long-pass filter. Each image was analyzed by a cooled-CCD camera system (HisCA/Argus system, Hamamatsu Photonics, Japan). The fluorescence ratio (340/380 nm) was calculated and transferred to the absolute values of [Ca 21]i based on the formula described by Grynkiewicz et al. [6] using (ethylenebis(oxononitrilo))tetraacetate (EGTA)-Ca 21 buffer solution. The fluorescence ratio obtained at 340 and 380nm was used as an index of [Ca 21]i in the present study. All of experiments were done at room temperature. The drugs were applied by perfusion using a microperpex pump (LKB2132, Pharmacia LKB Biotechnology). All the drugs were purchased from Sigma, unless otherwise specified. Ca 21-free solution was prepared by omitting CaCl2 from PSS but by adding 2 mM EGTA. All values are given as the means ^ standard deviation of the mean, and n denotes the number of the cells. Statistical significance was evaluated by the two-tailed unpaired Student’s t-test. A value of P of less than 0.05 was considered to be significant. The application of extracellular ATP (100 mM) caused a gradual saturable increase in DAF-2 fluorescence, indicative of NO production in IHCs (Fig. 1a). Suramin (100 mM), an antagonist for P2 receptor, inhibited the ATP-induced increase of DAF-2 fluorescence in IHCs (n ¼ 15. Fig. 1a). Furthermore, ATP-induced NO production in IHCs was significantly suppressed by the pretreatment with 200 mM l-NAME, a non-specific NO synthesis inhibitor (n ¼ 6, Fig. 1b) while 200 mM d-NAME, inactive isomer of l-NAME, did not inhibit the ATP-induced NO production (n ¼ 3, Fig. 1b). In the absence of extracellular Ca 21, ATP-induced NO production was abolished (n ¼ 14, Fig. 1b). The fluorescent imaging of both intracellular NO and [Ca 21]i was performed in the same IHC (Fig. 2). The DAF-2 loaded IHCs did not have any fluorescence under the excitation of both 340 nm and 380 nm at a resting state, during the control measurements with PSS and ATP stimulation (Fig. 2a). However, DAF-2 loaded IHCs demonstrated fluorescence under excitation at both 340 nm and 380 nm after loading with fura-2 (Fig. 2b). As a result, DAF-2 itself did not affect the Fura-2 measurements. Both NO production and [Ca 21]i increase induced by ATP were observed in the same IHCs (Fig. 2c–j). Continually, the experiments were designed to determine whether NO affects [Ca 21]i in the IHCs. The application of extracellular ATP (100 mM) caused a transient increase of [Ca 21]i in IHCs. The response to a subsequent application of
J. Shen et al. / Neuroscience Letters 337 (2003) 135–138
137
Fig. 2. Fluorescence images of nitric oxide production and [Ca 21]i changes induced by ATP in the same IHC. (a) The DAF-2 loaded IHCs did not have any fluorescence under the excitation of both 340 nm and 380 nm before loading with fura-2. (b) The fluorescence was identified under 340 nm and 380 nm after additional loading the same IHC with fura-2/AM. (c) Phase-contrast view of IHC, scale bar ¼ 10 mm. (d–f) Pseudo-color displays of the ATP-evoked NO production in DAF-2 loaded and fura-2 unloaded IHC. The fluorescence image of the IHC at pre-stimulation (d), 5 min (e) and 10 min (f) after stimulation with 100 mM ATP. (g–j) Pseudo-color displays of the ATP-induced [Ca 21]i changes in the same IHC loading with fura-2 after DAF-2 measurements. The fluorescence image of the IHC at pre-stimulation (g), 1 min (h), 1.5 min (i) and 3 min (f) after stimulation with 100 mM ATP. The color bar represents relative DAF-2 fluorescence intensity and the 340 nm/380 nm ratio level, respectively.
ATP at 15 min intervals was similar with the amplitude of the first response (Fig. 3a). Therefore, two applications of ATP at intervals of longer than 15 min were used in the present study. Thus, the effects of ATP were reversible and repeatable in our experimental arrangements. 200 mM l-NAME enhanced ATP-induced [Ca 21]i increases to 152 ^ 59% of the control (n ¼ 27, P , 0:05, Fig. 3b) while 100 mM S-nitroso-N-acetylpenicillamine (SNAP), NO donor, inhibited it to 78 ^ 23% of the control (n ¼ 9, P , 0:05, Fig. 3c). Neither 200 mM l-NAME nor 100 mM SNAP alone elevated the [Ca 21]i. The present study demonstrates that extracellular ATP can induce NO production in isolated cochlear IHCs using NOspecific fluorescent dye DAF-2. To our knowledge, this is the first direct evidence of NO production induced by extracellular ATP in IHCs. Our results also showed that ATP stimulation did not induce an increase in DAF-2 fluorescence in the absence of extracellular Ca 21 while ATP can induce a [Ca 21]i increase by Ca 21 release from intracellular stores in IHCs [14]. These results suggest that the ATP-induced NO production may be caused by the ATP-induced [Ca 21]i increase, which is mainly due to Ca 21 influx through the activation
of P2 receptor. Recent study by simultaneous measurements of DAF-2 and fura-2 showed that the increase of DAF-2 fluorescence induced by ATP lagged behind the rapid [Ca 21]i transient by Ca 21 release from internal stores and was observed during the late phase of the [Ca 21]i increase by Ca 21 influx in vascular endothelial cells [4]. They suggested that the [Ca 21]i increase induced by Ca 21 influx not release from internal stores was essential for NO production although both Ca 21 influx and Ca 21 release from internal stores could induce an increase of [Ca 21]i. Our findings that the ATP-induced NO production was mainly due to Ca 21 influx are line with the previous study. Several studies using fura-2 techniques suggested that the [Ca 21]i increase induced by ATP may activate NO production, which thus affects Ca 21 signaling by a positive feedback mechanism [11,15]. Our results also provided clear evidence that NO attenuated the ATP-induced calcium signaling in IHCs. These results are consistent with recent findings showing that NO attenuated an ATP-evoked [Ca 21]i increase in cochlear supporting cells [10]. Together with the abovementioned results, we presume that NO regulates the ATPinduced [Ca 21]i increase in IHCs by a negative-feedback
138
J. Shen et al. / Neuroscience Letters 337 (2003) 135–138
Fig. 3. Effect of NO on the ATP-evoked [Ca 21]i increase in IHCs. (a) A representative recording of increase in [Ca 21]i induced by 100 mM ATP in different intervals. The amplitude was almost same as the first response when repetitive stimulation with ATP was performed at 15 min intervals. (b) Effect of l-NAME, a NO synthase inhibitor, on ATP-induced [Ca 21]i increase. The [Ca 21]i response to ATP was significantly enhanced in the presence of 200 mM l-NAME. (c) Effect of SNAP, a NO donor, on ATP-induced [Ca 21]i increase. 100 mM SNAP markedly decreased the response to ATP (n ¼ 9). (d) A summary of statistics results on the changes of ATP-induced [Ca 21]i increase on IHCs with the treatments as described above. The values obtained with the second response to ATP were normalized after referencing them to those obtained with the first response to ATP. Error bar showed SD. *P , 0:05 compared to the [Ca 21]i response to ATP as a control (n ¼ 14).
mechanism. Since both ATP and NO act as neuromodulators in the cochlea, we are also able to deduce the possibility that interactions occur between ATP and NO, which might thus represent a regulation loop of IHC physiology that critically modulates Ca 21 homeostasis in IHCs.
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