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The production of free oxygen radicals and nitric oxide in the rat cochlea
NEUROCHEMISTRY International ELSEVIER
Neurochem. Int. 33 (1998) 55-59
The production of free oxygen radicals and nitric oxide in the rat cochlea M. A. L d p e z - G o n z f i l e z , a M. Lucas, b* F. D e l g a d o , a P. D i a z b aUnit of Pediatric Otorhinolaringology, Virgen del Rocio University Hospital bDepartment of Medical Biochemistry and Molecular Biology, Molecular Biology Service, Virgen Macarena University Hospital, Facultad de Medic&a, Sevilla, Spain
Received 25 August 1997; accepted 28 November 1997
Abstract
Rat cochleas were analysed for free oxygen radicals (FOR) and nitric oxide (NO) production by the chemiluminescent oxidation of luminol. 4/~-Phorbol-12fl-myristate-13~-acetate (PMA), a well-known agonist of protein kinase C, induced the release of FOR after a time lag close to 30 s and reverted to basal values in approximately 10 rain. Sphingosine inhibited by nearly 50% the response to PMA, whereas staurosporine caused an inhibition of 100%. The incubation of rat cochleas with 0.5 mM arginine potentiated the chemiluminescent reaction induced by PMA causing an additional oxidation of luminol that was inhibited by the NO synthase inhibitor N-methyl-arginine (NMA). Our results show for the first time the presence in the cochlea of cell populations producing FOR and NO and the real time production following cell activation. This procedure may help to explain the mechanisms involved in ototoxicity, as in the case of streptomycin and gentamicin that enhanced PMA-dependent production of FOR and NO. © 1998 Elsevier Science Ltd. All rights reserved.
1. Introduction
Superoxide anion 0 2 , hydroxyl radical OH. and hydrogen peroxide H202, known as reactive oxygen metabolites, are produced in the reduction of molecular oxygen to water during oxidative phosphorylation. Oxygenderived free radicals (FOR) are a highly reactive chemical species involved in a variety of clinical disorders (Halliwell and Gutteridge, 1995; Knight, 1995). Furthermore, different drugs and xenobiotics are themselves either converted to, or stimulate the formation of FOR. On the other hand, arginine is a precursor of nitric oxide (NO) (Knowles and Moncada, 1992), an intercellular signaling molecule synthesized in diverse m a m m a l i a n tissues by N O synthase. Arginine is produced by satellite cells surrounding neurons of the spiral ganglion, supporting or controlling the neural activity by providing L-arginine (Aoki et al., 1991). Besides, N O synthase has been described by histochemistry in the spiral ganglion cells of the rat cochlea and a role as neurotransmitter in neurotoxicity has been proposed (Zdanski et al., 1994). Indirect
data suggest that cochlear blood vessels produce N O and contributes to the regulation of cochlear blood flow (Brechtelsbauer, 1994). N O synthase activity has been described and characterized in extracts of the mammalian cochlea in the neural elements of the organ of Corti (Fessensen et al., 1994). We present here real time measurement of luminolchemiluminescence in intact rat cochlea. Its dependence on a protein kinase C (PKC) agonist and on arginine, suggests the presence in rat cochlea of the enzymatic machinery producing F O R and NO. As far as we know this is the first time that F O R and N O production by the rat cochlea has been shown. The sensitivity of the chemiluminescence method and the features of the continuous assay of the rat cochlea response could be very useful in the assay of the ototoxicity of chemical or physical agents.
2. Materials and methods 2.1. Chemicals
* To whom all correspondence should be addressed: Departamento de Bioqulmica M6dica y Biologia Molecular, Laboratorio de Gen6tica Molecular, Facultad de Medicina, Avda. Sfinchez Pizjufin 4, 41009Sevilla, Spain. Tel.: 345.455.7352; fax: 345,455.7481. 0197-0186/98 $19.00 © 1998 Elsevier Science Ltd. All rights reserved PII: SO 197-0 186(97)00 1 1 5-0
Streptomycin, gentamicin, ciprofloxacin, 4fl-phorbol12/%myristate- 13~-acetate (PMA), luminol (5-amino-2,3dihydro- 1,4-phthalazinedione), N-methyl-L-arginine
56
M.A. Ldpez-Gonzdlezet al./Neurochem. Int. 33 (1998) 55-59
(NMA), arginine, staurosporine, sphingosine and horseradish peroxidase (POD) were purchased from Sigma. Dimethyl-sulfoxide (DMSO) was from Merck. Superoxide dismutase and catalase were from Boehringer Mannheim. 2.2. Preparation of the rat cochlea
Wistar rats of 150-200 g weight, 6-8 weeks old, were anaesthetized under ether atmosphere, decapitated and the temporal bone immediately excised. Each cochlea was dissected and placed in 100 #1 of Hepes-buffered PBS (Hepes-PBS) consisting of: 20 mM Hepes, 137 raM NaC1, 1 mM KH2PO4 10 mM Na2HPO4, 2.7 mM KC1, 1.2 mM CaC12, 1,2 mM MgC12, 10 mM glucose, pH 7.4. The bony lateral wall of the cochlea was thoroughly opened and removed by microsurgery. The tissue remaining in the preparation included the modiolus and the membranous cochlea. The whole preparation of each cochlea was incubated in each separate experiment. Extensions of the preparation were stained with Giemsa and examined with an optic microscope. 2.3. Measurement of luminol chemiluminescence
FOR production was monitored by measuring luminoldependent chemiluminescence using a Berthold LB 9500 C luminometer attached to a chart-recorder as previously described (Lucas et al., 1995). Samples for chemiluminescence determination were prepared by adding the rat cochleas to Hepes-PBS supplemented with 20 #M luminol and 2 units/ml horseradish-peroxidase, which provided the catalytic component for luminol oxidation. Experiments were carried out at 37°C after 4 min preincubation at the same temperature. Reagents were injected via a microsyringe through a light-tight septum. Experimental protocols were designed to compare, in paired cochleas, the activity under control conditions vs the response to a given agonist. The degree of activation varied significantly among cochleas of different rats but we observed minor variations in paired cochleas from the same rat. In the latter case, the coefficient of variation of the chemiluminescence response to PMA was less than 3%. The variability was higher when the results of cochleas from different rats were compared. Therefore, experiments were designed to analyse, in a cochlear preparation, the response to a given agent in comparison to its paired cochlea of the same rat. In some experiments, figures are computer-drawn composites of the actual chart records. Further details are described in the legends to figures. It was ascertained that the solvent of the compounds, at the concentrations used in the present work, did not modify the chemiluminescent reaction. 2.4. Measurement of nitric oxide (NO) production
NO was detected by the chemiluminescence reaction of luminol with peroxynitrite, a strong oxidizing molecule,
according to procedures previously published (Kikuchi et al., 1993; Wang et al., 1993). The experimental approach relies in the reaction of O~- with NO to form peroxynitrite. The incubation mixture is the same as the above described for the measurement of FOR production. Peroxidase was omitted in some of these experiments as described in the Results section.
3. Results
Rat cochleas were isolated following mechanical disruption of bony structure (Fig. 1) and analysed for FOR production by a sensitive chemiluminescence procedure. Preliminary experiments were designed to find the intrarat (paired cochleas) and inter-rats variability of the experimental approach. Therefore, the time course and maximal chemiluminescence rate following PMA stimulation, calculated from the slope of the chemiluminescence traces in cm/min, was measured in a set of 60 cochleas obtained from 30 rats. The results are summarized in Figs 2 and 3 and show a minor variation in paired cochleas obtained from the same rat (variation coefficient of 2.2%), whereas the variability (variation coefficient of 60%) was higher among cochleas of different rats (Fig. 2). The low variability in paired cochleas, obtained from the same rat, is further shown in Fig. 3. In fact, a plot of the chemiluminescence rate of paired cochleas fitted a linear regression with a slope of 0.97 and a correlation coefficient of 0.99. Activation of the rat cochlea by PMA produced, after a time lag of 30-60 s, a slow increase in the chemiluminescence reaction caused by lumino[ oxidation. It reached maximal response in 3-5 min and reverted to basal rates in 10-15 rain. The chemiluminescence reaction induced by PMA required the presence of exogenously added horseradish peroxidase (POD) that provides the catalytic component of luminol oxidation. Figure 4 panel A shows a typical response to PMA that is also very illustrative of the degree of inhibition induced by PKC inhibitors. Sphingosine 5 ,aM inhibited PMA-induced chemiluminescence to nearly 50% whereas 0.1 #M staurosporine inhibited totally the response to PMA (Table 1). These results suggest that PKC-dependent mechanisms are involved in FOR production in the PMA-stimulated rat cochlea. We next sought the possible production of NO in the cochlea, since NO synthase has been described in cochlear extracts. The experimental approach relies on the reaction of O2 with NO to form peroxynitrite, a strong oxidizing species that triggers a chemiluminescence reaction with luminol. An arginine-dependent luminol chemiluminescence should be expected. The results were quite demonstrative and took advantage of the continuous recording procedure to measure NO release (Fig. 4, panels B and C). The cochleas incubated with arginine, without
57
M.A. L@ez-Gonzdlez et al./Neurochem. Int. 33 (1998) 55-59
Fig. 1. Photograph of cells of the cochlear preparation. Cochleas were dissected and the cells of the membranous cochlea stained by Giemsa. Arrows indicate the following: (a), outer hair cell; (b), inner hair cell; (c), cells of the Reissner's membrane; (d), basilar membrane; (e), spiral ligament.
i
"~
5,0
1500
qO C::: O ¢n E:
1000
. m
E
P
E .E
Cb
.~
5OO
0
, I 0
I 5
110
2,5-
e--
115
Time (rain)
Fig. 2. Time course of FOR production in the rat cochlea. The chemiluminescent oxidation of luminol was measured under resting conditions and following the addition of 50 nM PMA (see arrow). Chemiluminescence values in the ordinate scale are given as the integral in counts per 10 s obtained from the digital output of the chemi~uminometer. Results are the mean_+ SEM obtained from 30 identical experiments with unpaired cochleas prepared from 30 rats.
P M A , d i d n o t p r o d u c e a significant c h e m i l u m i n e s c e n c e response, w h e r e a s the c h e m i l u m i n e s c e n c e response to P M A was clearly e n h a n c e d b y 0.5 m M arginine (Fig. 4, p a n e l B). The p o t e n t i a t i o n was quantified b y the increase o f the rate o f P M A - d e p e n d e n t o x i d a t i o n o f luminol. These e x p e r i m e n t s were c a r r i e d o u t b y either a d d i n g arginine 4 rain before P M A s t i m u l a t i o n (Fig. 4, p a n e l B a n d T a b l e 1) o r after P M A a c t i v a t i o n at its m a x i m a l c h e m i l u m i n e s c e n c e response (Fig. 4, p a n e l C). U n d e r these c o n d i t i o n s , 2 m M N - m e t h y l - a r g i n i n e ( N M A ) inhibited the a r g i n i n e - d e p e n d e n t p o t e n t i a t i o n o f chemi-
0,0-
o,0
2;
Chemiluminescence rate Fig. 3. Correlation of FOR production in paired cochieas. Paired values of chemiluminescence in PMA-activated cochleas of the same rat were fitted by linear regression and the results accordingiy plotted. The coordinate Y and X axis values refer to the left and right cochlea respectively. The correlation coefficient was 0.99, the slope 0.97 and the Y intercept 0.017. The degree of significance of the linear regression was very high (P < 0.0001).
luminescence (Fig. 4, p a n e l C). The d e p e n d e n c e on P M A o f the effect o f arginine can be e x p l a i n e d by the requirem e n t o f O j to react with N O p r o d u c i n g peroxynitrite. T h e p r o d u c t i o n o f F O R a n d N O was inhibited up to n e a r l y 70% by s u p e r o x i d e d i s m u t a s e a n d a l m o s t t o t a l l y a b o l i s h e d b y s u p e r o x i d e d i s m u t a s e plus catalase (Table
1).
M.A. L@ez-Gonzdlez et al./Neurochern. Int. 33 (1998) 55-59
58 150---
A
Table 1 Chemiluminescencerate in the rat cochlea: effect of arginine, antibiotics and inhibitors
200-
Additions
I
ff
I
10
15
5
200-
200-
_
10
15
POD ÷ A r ~ i ~ e
D
INI-
wa I
I 5
I
10
15
5
f
I0
f
IS
A B C D E F G H I J K L M N
None PMA (30) PMA+Arginine(5) PMA+Sphingosine(5) PMA + Staurosporine (5) Arginine (8) PMA+Streptomycin(10) PMA + Streptomycin+ Arginine (4) PMA+Gentamicin(4) PMA+Gentamicin+Arginine(4) PMA + Ciprofloxacin(4) PMA + Ciprofloxacin+ Arginine (4) PMA+SOD(3) PMA+SOD+Catalase(3)
Chemiluminescence (mean +_SEM) 0.0_0.0 2.6_+0.18 3.6_+0.17 1.1 +__0.09 0.0 +-0.0 1.8_+0.07 5.6+_0.2 7.6 __0.31 6.2_+0.3 8.7+_0.34 2.3 +-0.1 3.2 _+0.09 0.8+_0.05 0.1 +_0.001
P P P P P P P P P P P P
< < < < < < < < > < < <
0.03 0.001 0.001 0.03 0.001 0.001 0.001 0.001 0.05 0.01 0.001 0.001
Time (rain) Fig. 4. Response of FOR and NO in rat cochlea. Rat cochleas were isolated and the oxidation of luminol assayed in the chemiluminometer as described in the Material and methods section. Pane[ A shows the inhibition by PKC inhibitors The upper trace is the response obtained by 50 nM PMA added at the time indicated by the line. The other traces show the chemiluminescencerate after PMA stimulation of the rat cochlea incubated in the presence of 5 #M sphingosine and 0.1 #M staurosporine. Panel B shows the arginine-dependent chemiluminescence.Rat cochleas were incubated in the presence (lower and upper traces) and in the absence (middle trace) of 0.5 mM arginine and at the time indicated by the arrow, stimulated with 50 nM PMA (upper and middle traces) or the solvent DMSO (lower trace). Pane[ C shows the inhibition by NMA of arginine-dependent chemiluminescence.Rat cochleas were analysed followingthe sequential stimulation (see arrows) with: 50 nM PMA (trace a), 50 nM PMA and 0.5 mM arginine (trace b), 50 nM PMA and 0.5 mM arginine and 2mM NMA (trace c). The traces of separate experiments were superimposed to show the decline of the chemiluminescencewhen arginine was omitted and when NMA was added. Panel D shows the effect of peroxidase on arginine-dependent chemiluminescence.Experiments were carried out on rat cochleas incubated in the reaction mixture supplemented as follows: none (lower trace), 0.5 mM L-arginine(arginine), 2 units/ml horseradish peroxidase (POD) and arginine plus POD (POD + arginine). At the time pointed by the arrow 50 nM PMA was injected and the chemiluminescencerate recorded. Chemiluminescencerate values are givenin counts per second (c.p.s.). Results in each panel are representative of two to ten other experiments. Additional details of these results are given in Table 1.
A set of experiments sought the dependence of chemiluminescence o n exogenously added peroxidase that provides the catalytic c o m p o n e n t for l u m i n o l o x i d a t i o n by HaO2. The results in Fig. 4, p a n e l D show the d e p e n d e n c e o n peroxidase for chemiluminescence response i n d u c e d by P M A . It is w o r t h p o i n t i n g o u t that in some cochlear p r e p a r a t i o n s P M A - d e p e n d e n t chemiluminescence was triggered even w i t h o u t exogenously added peroxidase. This v a r i a t i o n is p r o b a b l y due to the extent of 0 2 prod u c t i o n following P M A activation. A r g i n i n e stimulated the P M A - d e p e n d e n t o x i d a t i o n of l u m i n o l by the rat cochlea even in the absence of peroxidase, a l t h o u g h the maxi-
Rat cochleas were preincubated at 37°C for 4 min in the presence of none (A, B, F), 0.5 mM arginine (C), 5/~M sphingosine (D), 0.1 #M staurosporine (E), 100 ng/ml streptomycin (G, H), gentamicin (I, J), ciprofloxacin (K, L), 20 #g/ml superoxide dismutase, SOD, (M, N) and 20 #/ml catalase (N). Afterwards, the tubes were supplemented with 50 nM PMA, except control tubes (A) that received the solvent. The chemiluminescence rate was registered in a chart recorder and the maximal rate was calculated in cm/min from the slope of the traces. File F refers to results obtained in PMA activated cells when 0.5 mM arginine was added at the point of maximal chemiluminescencerate (Fig. 4 panel C). Number in parentheses refer to the number of separate experiments. Statistical significancewas calculated by the ANOVA test by comparison of each group with B. The effect of arginine in the presence of antibiotics was analysed by comparison of files H, J and L versus G, I and K respectively. The effect of antibiotics in the presence of PMA and arginine was analysed by comparison of H, J and L vs C: streptomycin and gentamicin increased very significantly(P < 0.0001) the chemiluminescencerate whereas the effect of ciprofloxacinwas not significant.
m a l rate was achieved in the presence of b o t h peroxidase a n d arginine (Fig. 4, p a n e l D). The assay of a set of antibiotics showed that streptomycin, g e n t a m i c i n a n d ciprofloxacin failed to induce the p r o d u c t i o n of F O R a n d N O in the rat cochlea. I n P M A activated cochleas, b o t h s t r e p t o m y c i n a n d gent a m i c i n increased very significantly the chemiluminescence rate b o t h in the absence a n d the presence of arginine. By contrast, ciprofloxacin did n o t modify it, even in the presence of P M A a n d arginine (Table 1).
4. D i s c u s s i o n Several reports have dealt with the possible i n v o l v e m e n t of F O R , in the m e c h a n i s m causing ototoxicity by indirect evidence of antioxidants, having a protective role (Garetz et al., 1994). This was the case of a l l o p u r i n o l a n d superoxide dismutase (Seidman et al., 1993). Moreover, sig-
M.A. L6pez-Gonzdlez et al,/Neurochem. Int. 33 (1998) 55-59
nificant activities of two antioxidative enzymes, superoxide dismutase and glutathione peroxidase, have been described in the cochlea of guinea-pigs (Zelck et al., 1993). However, a direct evidence of FOR production has still not been properly described. The sensitive chemiluminescence procedure described in the this article, shows the existence in the rat cochlea of the machinery to produce free radicals. Concerning NO production, a role for NO in the regulation of cochlear blood flow has been suggested (Brechtelsbauer et al., 1994). Moreover, NMA, an inhibitor of NO synthase, protects the guinea-pig cochlea from the cytotoxic effect of the toxin pneumolysin (Amee et al., 1995). The presence of L-arginine in the satellite cells that surround the spiral ganglion (Aoki et al., 1991), suggests the in vivo induction of NO synthase expression in a cell subpopulation of the cochlea. This could support the activation of NO synthase in the rat cochlea exposed in vitro to L-arginine. Our experiments suggest a cell subpopulation expressing NO synthase and the triggering of NO synthesis following the challenging to L-arginine, an effect inhibited by NMA. These results agree with the findings of previous reports on NO synthase activity in extracts of the mammalian cochlea (Fessenden et al., 1994) and in the spiral ganglion cells of the rat (Zdanski et al., 1994). NO synthase in the mammalian cochlea is the neuronal type I isoform and has been localized in the lateral wall and the neuronal elements of the organ of Corti (Fessenden et al., 1994). PKC inhibitors block FOR production and therefore the formation of peroxynitrite that is detectable by the chemiluminescent oxidation of luminol (Kikuchi et al., 1993). Nonetheless, a direct inhibition of NO synthase cannot be excluded since NO is directly involved in the chemiluminescence of PMA activated rat peritoneal macrophages and Kupffer cells (Wang et al., 1993). Our results show the induction by PMA of FOR and the cooperation of PMA and arginine in NO production both in the same and different cell subpopulation of the rat cochlea. The reaction of superoxide anion and NO produces peroxynitrite that increases the chemiluminescent oxidation of luminol. The contribution of superoxide anion and H202 in the overall chemiluminescence produced by the oxidation of luminol may be deduced from the strong inhibition caused by superoxide dismutase and catalase. We provided a sensitive experimental approach that showed for the first time the production of FOR and NO by rat cochlea. The procedure described here is easy to do and may be of further interest to gain greater insight into the mechanism of cell injury by ototoxic agents.
59
This was the case of streptomycin and gentamicin that enhanced PMA-dependent production of FOR and NO.
Acknowledgements This study was supported by grants 94/528-112 from the Direccidn General de Coordinacidn, Docencia e Investigacidn de la Consejerla de Salud de la Junta de Andaluci and 96/0278 from the Fondo de Investigaciones Sanitarias.
References Amee, F.R., Cosmis, S.D., Osborne, M.P., 1995. N~-methyl-L-arginine protects the guinea pig cochlea from the cytotoxic effects of pneumolysin. Acta Otolaryngology (Stockh) 115, 386-39i. Aoki, E., Semba, R., Kashiwamata, S., 1991. Evidence for the presence of L-arginine in the glial components of the peripheral nervous system. Brain Research 559, 159--I62. Brechtetsbauer, P.B., Nuttall, A.L., Miller, J.M., 1994. Basal nitric oxide production in regulation of cochlear cells. Hear Research 77, 38-42. Fessenden, J.D., Coling, D.E., Schacht, J., 1994. Detection and characterization of nitric oxide synthase in the mammalian cochlea. Brain Research 668, 9-15. Garetz, S.L., Rhee, D.J., Schacht, J., 1994. Sulfhydryl compounds and antioxidants inhibit cytotoxicity to outer hair cells of gentamicin metabolite in vitro. Hear Research 77, 75-80. Halliwel, B., Gutteridge, J.M.C., 1995. Oxygen radicals and the nervous system. Trends Neuroscience 79, 22-26. Kikuchi, K., Nagano, T., Hayakawa, H. et al., 1993. Detection of nitric oxide production from a perfused organ by a luminol-H202 system. Analytical Chemistry 65, 1794-1799. Knip:~t, J.A., 1995. Diseases related to oxygen-derived free radicals. Annals of Clinical and Laboratory Science 25, 111-121. Knowles, R.G., Moncada, S., 1992. Nitric oxide as a signal in blood vessels. Trends Biochemistry Science 17, 399-402. Lucas, M., S/mchez-Margalet, V., Pedrera, C., et al., 1995. A chemiluminescence method to analyse phosphatidyl-choline- phospholipase activity in plasma membrane preparations and in intact cells. Analytical Biochemistry 231,277-281. Seidman, M.D., Shivapuja, B.G., Quirk, W.S., 1993. The protective effects of allopurinol and superoxide dismutase on noise-induced cochlear damage. Otolaryngology and Head Neck Surgery 109, 1052-1056. Wang, J.F., Komarov, P., Groot, H., 1993. Luminol chemiluminescence in rat macrophages and granulocytes: The role of NO, Of/H20;, and HOCL. Archives Biochemistry and Biophysics 304, 189-I96. Zdanski, C.J., Prazma, J., Petrusz, P. et al., 1994. Nitric oxide synthase is an active enzyme in the spiral ganglion cells of the rat cochlea. Hear Research 79, 39-47. Zelck, U., Nowak, R., Karnstedt, U. et al., 1993. Specific activities of antioxidative enzymes in the cochlea of guinea pigs at different stages of development. European Archives of Otorhinoiaryngology 250, 2t8-219.
Neurochem. Int. 33 (1998) 55-59
The production of free oxygen radicals and nitric oxide in the rat cochlea M. A. L d p e z - G o n z f i l e z , a M. Lucas, b* F. D e l g a d o , a P. D i a z b aUnit of Pediatric Otorhinolaringology, Virgen del Rocio University Hospital bDepartment of Medical Biochemistry and Molecular Biology, Molecular Biology Service, Virgen Macarena University Hospital, Facultad de Medic&a, Sevilla, Spain
Received 25 August 1997; accepted 28 November 1997
Abstract
Rat cochleas were analysed for free oxygen radicals (FOR) and nitric oxide (NO) production by the chemiluminescent oxidation of luminol. 4/~-Phorbol-12fl-myristate-13~-acetate (PMA), a well-known agonist of protein kinase C, induced the release of FOR after a time lag close to 30 s and reverted to basal values in approximately 10 rain. Sphingosine inhibited by nearly 50% the response to PMA, whereas staurosporine caused an inhibition of 100%. The incubation of rat cochleas with 0.5 mM arginine potentiated the chemiluminescent reaction induced by PMA causing an additional oxidation of luminol that was inhibited by the NO synthase inhibitor N-methyl-arginine (NMA). Our results show for the first time the presence in the cochlea of cell populations producing FOR and NO and the real time production following cell activation. This procedure may help to explain the mechanisms involved in ototoxicity, as in the case of streptomycin and gentamicin that enhanced PMA-dependent production of FOR and NO. © 1998 Elsevier Science Ltd. All rights reserved.
1. Introduction
Superoxide anion 0 2 , hydroxyl radical OH. and hydrogen peroxide H202, known as reactive oxygen metabolites, are produced in the reduction of molecular oxygen to water during oxidative phosphorylation. Oxygenderived free radicals (FOR) are a highly reactive chemical species involved in a variety of clinical disorders (Halliwell and Gutteridge, 1995; Knight, 1995). Furthermore, different drugs and xenobiotics are themselves either converted to, or stimulate the formation of FOR. On the other hand, arginine is a precursor of nitric oxide (NO) (Knowles and Moncada, 1992), an intercellular signaling molecule synthesized in diverse m a m m a l i a n tissues by N O synthase. Arginine is produced by satellite cells surrounding neurons of the spiral ganglion, supporting or controlling the neural activity by providing L-arginine (Aoki et al., 1991). Besides, N O synthase has been described by histochemistry in the spiral ganglion cells of the rat cochlea and a role as neurotransmitter in neurotoxicity has been proposed (Zdanski et al., 1994). Indirect
data suggest that cochlear blood vessels produce N O and contributes to the regulation of cochlear blood flow (Brechtelsbauer, 1994). N O synthase activity has been described and characterized in extracts of the mammalian cochlea in the neural elements of the organ of Corti (Fessensen et al., 1994). We present here real time measurement of luminolchemiluminescence in intact rat cochlea. Its dependence on a protein kinase C (PKC) agonist and on arginine, suggests the presence in rat cochlea of the enzymatic machinery producing F O R and NO. As far as we know this is the first time that F O R and N O production by the rat cochlea has been shown. The sensitivity of the chemiluminescence method and the features of the continuous assay of the rat cochlea response could be very useful in the assay of the ototoxicity of chemical or physical agents.
2. Materials and methods 2.1. Chemicals
* To whom all correspondence should be addressed: Departamento de Bioqulmica M6dica y Biologia Molecular, Laboratorio de Gen6tica Molecular, Facultad de Medicina, Avda. Sfinchez Pizjufin 4, 41009Sevilla, Spain. Tel.: 345.455.7352; fax: 345,455.7481. 0197-0186/98 $19.00 © 1998 Elsevier Science Ltd. All rights reserved PII: SO 197-0 186(97)00 1 1 5-0
Streptomycin, gentamicin, ciprofloxacin, 4fl-phorbol12/%myristate- 13~-acetate (PMA), luminol (5-amino-2,3dihydro- 1,4-phthalazinedione), N-methyl-L-arginine
56
M.A. Ldpez-Gonzdlezet al./Neurochem. Int. 33 (1998) 55-59
(NMA), arginine, staurosporine, sphingosine and horseradish peroxidase (POD) were purchased from Sigma. Dimethyl-sulfoxide (DMSO) was from Merck. Superoxide dismutase and catalase were from Boehringer Mannheim. 2.2. Preparation of the rat cochlea
Wistar rats of 150-200 g weight, 6-8 weeks old, were anaesthetized under ether atmosphere, decapitated and the temporal bone immediately excised. Each cochlea was dissected and placed in 100 #1 of Hepes-buffered PBS (Hepes-PBS) consisting of: 20 mM Hepes, 137 raM NaC1, 1 mM KH2PO4 10 mM Na2HPO4, 2.7 mM KC1, 1.2 mM CaC12, 1,2 mM MgC12, 10 mM glucose, pH 7.4. The bony lateral wall of the cochlea was thoroughly opened and removed by microsurgery. The tissue remaining in the preparation included the modiolus and the membranous cochlea. The whole preparation of each cochlea was incubated in each separate experiment. Extensions of the preparation were stained with Giemsa and examined with an optic microscope. 2.3. Measurement of luminol chemiluminescence
FOR production was monitored by measuring luminoldependent chemiluminescence using a Berthold LB 9500 C luminometer attached to a chart-recorder as previously described (Lucas et al., 1995). Samples for chemiluminescence determination were prepared by adding the rat cochleas to Hepes-PBS supplemented with 20 #M luminol and 2 units/ml horseradish-peroxidase, which provided the catalytic component for luminol oxidation. Experiments were carried out at 37°C after 4 min preincubation at the same temperature. Reagents were injected via a microsyringe through a light-tight septum. Experimental protocols were designed to compare, in paired cochleas, the activity under control conditions vs the response to a given agonist. The degree of activation varied significantly among cochleas of different rats but we observed minor variations in paired cochleas from the same rat. In the latter case, the coefficient of variation of the chemiluminescence response to PMA was less than 3%. The variability was higher when the results of cochleas from different rats were compared. Therefore, experiments were designed to analyse, in a cochlear preparation, the response to a given agent in comparison to its paired cochlea of the same rat. In some experiments, figures are computer-drawn composites of the actual chart records. Further details are described in the legends to figures. It was ascertained that the solvent of the compounds, at the concentrations used in the present work, did not modify the chemiluminescent reaction. 2.4. Measurement of nitric oxide (NO) production
NO was detected by the chemiluminescence reaction of luminol with peroxynitrite, a strong oxidizing molecule,
according to procedures previously published (Kikuchi et al., 1993; Wang et al., 1993). The experimental approach relies in the reaction of O~- with NO to form peroxynitrite. The incubation mixture is the same as the above described for the measurement of FOR production. Peroxidase was omitted in some of these experiments as described in the Results section.
3. Results
Rat cochleas were isolated following mechanical disruption of bony structure (Fig. 1) and analysed for FOR production by a sensitive chemiluminescence procedure. Preliminary experiments were designed to find the intrarat (paired cochleas) and inter-rats variability of the experimental approach. Therefore, the time course and maximal chemiluminescence rate following PMA stimulation, calculated from the slope of the chemiluminescence traces in cm/min, was measured in a set of 60 cochleas obtained from 30 rats. The results are summarized in Figs 2 and 3 and show a minor variation in paired cochleas obtained from the same rat (variation coefficient of 2.2%), whereas the variability (variation coefficient of 60%) was higher among cochleas of different rats (Fig. 2). The low variability in paired cochleas, obtained from the same rat, is further shown in Fig. 3. In fact, a plot of the chemiluminescence rate of paired cochleas fitted a linear regression with a slope of 0.97 and a correlation coefficient of 0.99. Activation of the rat cochlea by PMA produced, after a time lag of 30-60 s, a slow increase in the chemiluminescence reaction caused by lumino[ oxidation. It reached maximal response in 3-5 min and reverted to basal rates in 10-15 rain. The chemiluminescence reaction induced by PMA required the presence of exogenously added horseradish peroxidase (POD) that provides the catalytic component of luminol oxidation. Figure 4 panel A shows a typical response to PMA that is also very illustrative of the degree of inhibition induced by PKC inhibitors. Sphingosine 5 ,aM inhibited PMA-induced chemiluminescence to nearly 50% whereas 0.1 #M staurosporine inhibited totally the response to PMA (Table 1). These results suggest that PKC-dependent mechanisms are involved in FOR production in the PMA-stimulated rat cochlea. We next sought the possible production of NO in the cochlea, since NO synthase has been described in cochlear extracts. The experimental approach relies on the reaction of O2 with NO to form peroxynitrite, a strong oxidizing species that triggers a chemiluminescence reaction with luminol. An arginine-dependent luminol chemiluminescence should be expected. The results were quite demonstrative and took advantage of the continuous recording procedure to measure NO release (Fig. 4, panels B and C). The cochleas incubated with arginine, without
57
M.A. L@ez-Gonzdlez et al./Neurochem. Int. 33 (1998) 55-59
Fig. 1. Photograph of cells of the cochlear preparation. Cochleas were dissected and the cells of the membranous cochlea stained by Giemsa. Arrows indicate the following: (a), outer hair cell; (b), inner hair cell; (c), cells of the Reissner's membrane; (d), basilar membrane; (e), spiral ligament.
i
"~
5,0
1500
qO C::: O ¢n E:
1000
. m
E
P
E .E
Cb
.~
5OO
0
, I 0
I 5
110
2,5-
e--
115
Time (rain)
Fig. 2. Time course of FOR production in the rat cochlea. The chemiluminescent oxidation of luminol was measured under resting conditions and following the addition of 50 nM PMA (see arrow). Chemiluminescence values in the ordinate scale are given as the integral in counts per 10 s obtained from the digital output of the chemi~uminometer. Results are the mean_+ SEM obtained from 30 identical experiments with unpaired cochleas prepared from 30 rats.
P M A , d i d n o t p r o d u c e a significant c h e m i l u m i n e s c e n c e response, w h e r e a s the c h e m i l u m i n e s c e n c e response to P M A was clearly e n h a n c e d b y 0.5 m M arginine (Fig. 4, p a n e l B). The p o t e n t i a t i o n was quantified b y the increase o f the rate o f P M A - d e p e n d e n t o x i d a t i o n o f luminol. These e x p e r i m e n t s were c a r r i e d o u t b y either a d d i n g arginine 4 rain before P M A s t i m u l a t i o n (Fig. 4, p a n e l B a n d T a b l e 1) o r after P M A a c t i v a t i o n at its m a x i m a l c h e m i l u m i n e s c e n c e response (Fig. 4, p a n e l C). U n d e r these c o n d i t i o n s , 2 m M N - m e t h y l - a r g i n i n e ( N M A ) inhibited the a r g i n i n e - d e p e n d e n t p o t e n t i a t i o n o f chemi-
0,0-
o,0
2;
Chemiluminescence rate Fig. 3. Correlation of FOR production in paired cochieas. Paired values of chemiluminescence in PMA-activated cochleas of the same rat were fitted by linear regression and the results accordingiy plotted. The coordinate Y and X axis values refer to the left and right cochlea respectively. The correlation coefficient was 0.99, the slope 0.97 and the Y intercept 0.017. The degree of significance of the linear regression was very high (P < 0.0001).
luminescence (Fig. 4, p a n e l C). The d e p e n d e n c e on P M A o f the effect o f arginine can be e x p l a i n e d by the requirem e n t o f O j to react with N O p r o d u c i n g peroxynitrite. T h e p r o d u c t i o n o f F O R a n d N O was inhibited up to n e a r l y 70% by s u p e r o x i d e d i s m u t a s e a n d a l m o s t t o t a l l y a b o l i s h e d b y s u p e r o x i d e d i s m u t a s e plus catalase (Table
1).
M.A. L@ez-Gonzdlez et al./Neurochern. Int. 33 (1998) 55-59
58 150---
A
Table 1 Chemiluminescencerate in the rat cochlea: effect of arginine, antibiotics and inhibitors
200-
Additions
I
ff
I
10
15
5
200-
200-
_
10
15
POD ÷ A r ~ i ~ e
D
INI-
wa I
I 5
I
10
15
5
f
I0
f
IS
A B C D E F G H I J K L M N
None PMA (30) PMA+Arginine(5) PMA+Sphingosine(5) PMA + Staurosporine (5) Arginine (8) PMA+Streptomycin(10) PMA + Streptomycin+ Arginine (4) PMA+Gentamicin(4) PMA+Gentamicin+Arginine(4) PMA + Ciprofloxacin(4) PMA + Ciprofloxacin+ Arginine (4) PMA+SOD(3) PMA+SOD+Catalase(3)
Chemiluminescence (mean +_SEM) 0.0_0.0 2.6_+0.18 3.6_+0.17 1.1 +__0.09 0.0 +-0.0 1.8_+0.07 5.6+_0.2 7.6 __0.31 6.2_+0.3 8.7+_0.34 2.3 +-0.1 3.2 _+0.09 0.8+_0.05 0.1 +_0.001
P P P P P P P P P P P P
< < < < < < < < > < < <
0.03 0.001 0.001 0.03 0.001 0.001 0.001 0.001 0.05 0.01 0.001 0.001
Time (rain) Fig. 4. Response of FOR and NO in rat cochlea. Rat cochleas were isolated and the oxidation of luminol assayed in the chemiluminometer as described in the Material and methods section. Pane[ A shows the inhibition by PKC inhibitors The upper trace is the response obtained by 50 nM PMA added at the time indicated by the line. The other traces show the chemiluminescencerate after PMA stimulation of the rat cochlea incubated in the presence of 5 #M sphingosine and 0.1 #M staurosporine. Panel B shows the arginine-dependent chemiluminescence.Rat cochleas were incubated in the presence (lower and upper traces) and in the absence (middle trace) of 0.5 mM arginine and at the time indicated by the arrow, stimulated with 50 nM PMA (upper and middle traces) or the solvent DMSO (lower trace). Pane[ C shows the inhibition by NMA of arginine-dependent chemiluminescence.Rat cochleas were analysed followingthe sequential stimulation (see arrows) with: 50 nM PMA (trace a), 50 nM PMA and 0.5 mM arginine (trace b), 50 nM PMA and 0.5 mM arginine and 2mM NMA (trace c). The traces of separate experiments were superimposed to show the decline of the chemiluminescencewhen arginine was omitted and when NMA was added. Panel D shows the effect of peroxidase on arginine-dependent chemiluminescence.Experiments were carried out on rat cochleas incubated in the reaction mixture supplemented as follows: none (lower trace), 0.5 mM L-arginine(arginine), 2 units/ml horseradish peroxidase (POD) and arginine plus POD (POD + arginine). At the time pointed by the arrow 50 nM PMA was injected and the chemiluminescencerate recorded. Chemiluminescencerate values are givenin counts per second (c.p.s.). Results in each panel are representative of two to ten other experiments. Additional details of these results are given in Table 1.
A set of experiments sought the dependence of chemiluminescence o n exogenously added peroxidase that provides the catalytic c o m p o n e n t for l u m i n o l o x i d a t i o n by HaO2. The results in Fig. 4, p a n e l D show the d e p e n d e n c e o n peroxidase for chemiluminescence response i n d u c e d by P M A . It is w o r t h p o i n t i n g o u t that in some cochlear p r e p a r a t i o n s P M A - d e p e n d e n t chemiluminescence was triggered even w i t h o u t exogenously added peroxidase. This v a r i a t i o n is p r o b a b l y due to the extent of 0 2 prod u c t i o n following P M A activation. A r g i n i n e stimulated the P M A - d e p e n d e n t o x i d a t i o n of l u m i n o l by the rat cochlea even in the absence of peroxidase, a l t h o u g h the maxi-
Rat cochleas were preincubated at 37°C for 4 min in the presence of none (A, B, F), 0.5 mM arginine (C), 5/~M sphingosine (D), 0.1 #M staurosporine (E), 100 ng/ml streptomycin (G, H), gentamicin (I, J), ciprofloxacin (K, L), 20 #g/ml superoxide dismutase, SOD, (M, N) and 20 #/ml catalase (N). Afterwards, the tubes were supplemented with 50 nM PMA, except control tubes (A) that received the solvent. The chemiluminescence rate was registered in a chart recorder and the maximal rate was calculated in cm/min from the slope of the traces. File F refers to results obtained in PMA activated cells when 0.5 mM arginine was added at the point of maximal chemiluminescencerate (Fig. 4 panel C). Number in parentheses refer to the number of separate experiments. Statistical significancewas calculated by the ANOVA test by comparison of each group with B. The effect of arginine in the presence of antibiotics was analysed by comparison of files H, J and L versus G, I and K respectively. The effect of antibiotics in the presence of PMA and arginine was analysed by comparison of H, J and L vs C: streptomycin and gentamicin increased very significantly(P < 0.0001) the chemiluminescencerate whereas the effect of ciprofloxacinwas not significant.
m a l rate was achieved in the presence of b o t h peroxidase a n d arginine (Fig. 4, p a n e l D). The assay of a set of antibiotics showed that streptomycin, g e n t a m i c i n a n d ciprofloxacin failed to induce the p r o d u c t i o n of F O R a n d N O in the rat cochlea. I n P M A activated cochleas, b o t h s t r e p t o m y c i n a n d gent a m i c i n increased very significantly the chemiluminescence rate b o t h in the absence a n d the presence of arginine. By contrast, ciprofloxacin did n o t modify it, even in the presence of P M A a n d arginine (Table 1).
4. D i s c u s s i o n Several reports have dealt with the possible i n v o l v e m e n t of F O R , in the m e c h a n i s m causing ototoxicity by indirect evidence of antioxidants, having a protective role (Garetz et al., 1994). This was the case of a l l o p u r i n o l a n d superoxide dismutase (Seidman et al., 1993). Moreover, sig-
M.A. L6pez-Gonzdlez et al,/Neurochem. Int. 33 (1998) 55-59
nificant activities of two antioxidative enzymes, superoxide dismutase and glutathione peroxidase, have been described in the cochlea of guinea-pigs (Zelck et al., 1993). However, a direct evidence of FOR production has still not been properly described. The sensitive chemiluminescence procedure described in the this article, shows the existence in the rat cochlea of the machinery to produce free radicals. Concerning NO production, a role for NO in the regulation of cochlear blood flow has been suggested (Brechtelsbauer et al., 1994). Moreover, NMA, an inhibitor of NO synthase, protects the guinea-pig cochlea from the cytotoxic effect of the toxin pneumolysin (Amee et al., 1995). The presence of L-arginine in the satellite cells that surround the spiral ganglion (Aoki et al., 1991), suggests the in vivo induction of NO synthase expression in a cell subpopulation of the cochlea. This could support the activation of NO synthase in the rat cochlea exposed in vitro to L-arginine. Our experiments suggest a cell subpopulation expressing NO synthase and the triggering of NO synthesis following the challenging to L-arginine, an effect inhibited by NMA. These results agree with the findings of previous reports on NO synthase activity in extracts of the mammalian cochlea (Fessenden et al., 1994) and in the spiral ganglion cells of the rat (Zdanski et al., 1994). NO synthase in the mammalian cochlea is the neuronal type I isoform and has been localized in the lateral wall and the neuronal elements of the organ of Corti (Fessenden et al., 1994). PKC inhibitors block FOR production and therefore the formation of peroxynitrite that is detectable by the chemiluminescent oxidation of luminol (Kikuchi et al., 1993). Nonetheless, a direct inhibition of NO synthase cannot be excluded since NO is directly involved in the chemiluminescence of PMA activated rat peritoneal macrophages and Kupffer cells (Wang et al., 1993). Our results show the induction by PMA of FOR and the cooperation of PMA and arginine in NO production both in the same and different cell subpopulation of the rat cochlea. The reaction of superoxide anion and NO produces peroxynitrite that increases the chemiluminescent oxidation of luminol. The contribution of superoxide anion and H202 in the overall chemiluminescence produced by the oxidation of luminol may be deduced from the strong inhibition caused by superoxide dismutase and catalase. We provided a sensitive experimental approach that showed for the first time the production of FOR and NO by rat cochlea. The procedure described here is easy to do and may be of further interest to gain greater insight into the mechanism of cell injury by ototoxic agents.
59
This was the case of streptomycin and gentamicin that enhanced PMA-dependent production of FOR and NO.
Acknowledgements This study was supported by grants 94/528-112 from the Direccidn General de Coordinacidn, Docencia e Investigacidn de la Consejerla de Salud de la Junta de Andaluci and 96/0278 from the Fondo de Investigaciones Sanitarias.
References Amee, F.R., Cosmis, S.D., Osborne, M.P., 1995. N~-methyl-L-arginine protects the guinea pig cochlea from the cytotoxic effects of pneumolysin. Acta Otolaryngology (Stockh) 115, 386-39i. Aoki, E., Semba, R., Kashiwamata, S., 1991. Evidence for the presence of L-arginine in the glial components of the peripheral nervous system. Brain Research 559, 159--I62. Brechtetsbauer, P.B., Nuttall, A.L., Miller, J.M., 1994. Basal nitric oxide production in regulation of cochlear cells. Hear Research 77, 38-42. Fessenden, J.D., Coling, D.E., Schacht, J., 1994. Detection and characterization of nitric oxide synthase in the mammalian cochlea. Brain Research 668, 9-15. Garetz, S.L., Rhee, D.J., Schacht, J., 1994. Sulfhydryl compounds and antioxidants inhibit cytotoxicity to outer hair cells of gentamicin metabolite in vitro. Hear Research 77, 75-80. Halliwel, B., Gutteridge, J.M.C., 1995. Oxygen radicals and the nervous system. Trends Neuroscience 79, 22-26. Kikuchi, K., Nagano, T., Hayakawa, H. et al., 1993. Detection of nitric oxide production from a perfused organ by a luminol-H202 system. Analytical Chemistry 65, 1794-1799. Knip:~t, J.A., 1995. Diseases related to oxygen-derived free radicals. Annals of Clinical and Laboratory Science 25, 111-121. Knowles, R.G., Moncada, S., 1992. Nitric oxide as a signal in blood vessels. Trends Biochemistry Science 17, 399-402. Lucas, M., S/mchez-Margalet, V., Pedrera, C., et al., 1995. A chemiluminescence method to analyse phosphatidyl-choline- phospholipase activity in plasma membrane preparations and in intact cells. Analytical Biochemistry 231,277-281. Seidman, M.D., Shivapuja, B.G., Quirk, W.S., 1993. The protective effects of allopurinol and superoxide dismutase on noise-induced cochlear damage. Otolaryngology and Head Neck Surgery 109, 1052-1056. Wang, J.F., Komarov, P., Groot, H., 1993. Luminol chemiluminescence in rat macrophages and granulocytes: The role of NO, Of/H20;, and HOCL. Archives Biochemistry and Biophysics 304, 189-I96. Zdanski, C.J., Prazma, J., Petrusz, P. et al., 1994. Nitric oxide synthase is an active enzyme in the spiral ganglion cells of the rat cochlea. Hear Research 79, 39-47. Zelck, U., Nowak, R., Karnstedt, U. et al., 1993. Specific activities of antioxidative enzymes in the cochlea of guinea pigs at different stages of development. European Archives of Otorhinoiaryngology 250, 2t8-219.