Nitroprusside suppresses cochlear potentials and outer hair cell responses

HWRII RI .JMCH ELSEVIER

Hearing Research 87 (1995) 1-8

Nitroprusside suppresses cochlear potentials and outer hair cell ~r responses Chu Chen a, Anastas Nenov a, Ruth Skellett a, Maureen Fallon a, Latasha Bright a, Charles H. Norris a,b, Richard P. Bobbin a,* a Kresge Hearing Research Laboratory of the South, Department of Otorhinolaryngology and Biocommunication, Louisiana State University Medical Center, 2020 Gravier Street, Suite A, New Orleans, LA 70112-2234, USA Department of Otolaryngology, Tulane University School of Medicine, New Orleans, LA, USA

Received 18 December 1994; revised 15 March 1995; accepted 21 March 1995

Abstract

Biochemical and pharmacological evidence supports a role for nitric oxide (NO) in the cochlea. In the present experiments, we tested sodium nitroprusside (SNP), an NO donor, applied by intracochlear perfusions on sound-evoked responses of the cochlea (CM, cochlear microphonic; SP, summating potential; EP, endocochlear potential; CAP, compound action potential) and in vitro on outer hair celt (OHC) voltage-induced length changes and current responses. In vivo application of SNP in increasing concentrations (10, 33, 100, 330 and 1000/zM) reduced all sound-evoked responses starting at about 300/~M. The responses continued to decline after a postdrug wash. At l mM SNP decreased EP slowly ( = 80 min) whereas at 10 mM it reduced EP more rapidly (-- 20 min). Ferricyanide (1 mM) and S-nitroso-N-acetylpenicillamine (SNAP; 1 mM) had no effect on sound-evoked cochlear potentials. Ferricyanide (1 mM and 10 mM) and ferrocyanide (10 mM) had no effect on EP. In vitro, SNP (10 mM) significantly reduced both OHC voltage-induced length changes and whole-cell outward currents. Results suggest that SNP, possibly acting by released NO, influences cochlear function through effects at the stria vascularis and at the OHCs. Keywords: Nitric oxide; Ferricyanide; Ferrocyanide; S-nitroso-N-acetylpenicillamine;Electromotility; Voltage-clamp

1. I n t r o d u c t i o n

The synthesis of nitric oxide (NO) has been described to occur in various tissues (Moncada et al., 1991; Schuman and Madison, 1994). The NO produced has been proposed to act as a transduction mechanism underlying several physiological responses (Moncada et al., 1991). For instance NO has been demonstrated to regulate receptors, ion channels and contraction (Manzoni et al., 1992; Chen and Schofield, 1993,1994; Blatter and Wier, 1994; Schuman and Madison, 1994). A nitric oxide synthase, N A D P H diaphorase, one of the enzymes responsible for NO synthesis has been found recently in spiral ganglion cells (Zdanski et al., 1994), the auditory nerve, lateral wall of the cochlea, and cochlear neuroepithelium (Fessenden et al., 1994). Brechtelsbauer et

*A preliminary report was presented at the XXXlst Workshop on Inner Ear Biology in Montpellier, France, September, 1994 * Corresponding author: Tel.: (504) 568-4785; Fax: (504) 568-4460. 0378-5955/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0378-5955(95)00071-2

al. (1993) and Ohlsen et al. (1993) reported that the NO donor, sodium nitroprusside (SNP, 100 mM; 3%), applied to the round window suppressed the auditory nerve action potential. Therefore, both biochemical and pharmacological evidence has been presented to suggest that NO may be involved in cochlear function. The purpose of the present study was to explore further the role of NO in the cochlea by examining the effect of perfusion o f SNP through the perilymph compartment on the electrical potentials recorded from the cochlea. To test if NO may have a role in OHCs, we examined the effect of SNP on the voltage-induced change in length and in currents of isolated OHCs. As controls for the non-selective effects of SNP in vivo, we used ferricyanide and ferrocyanide which are structurally similar to SNP (East et al., 1991; Southam and Garthwaite, 1991; Kiedrowski et al., 1992). In addition, we tested S-nitroso-N-acetylpenicillamine (SNAP), an additional NO donor, with a different structure and a potency 100 times that of SNP (Kiedrowski et al., 1992).

2

C. Chen et al./Hearing Research 87 (1995) I 8

2. Methods 2.1. In vivo procedures

Pigmented guinea pigs (250-450 g) of either sex were anesthetized (pentobarbital sodium, Nembutal R, 30 mg/kg, i.p., together with chlorprothixene, Taractan R, 30 mg/kg, i.p., or acepromazine maleate, PromAce R, 0.2 mg/kg), tracheotomized and artificially respirated. The rectal temperature was maintained at 38 + I°C. Additional pentobarbital (30 mg/kg, i.p.) was administered as required to assure a deep level of anesthesia. The right cochlea was exposed ventrolaterally and both middle ear muscles were sectioned. Cochlear perfusions

Perfusion experiments were completed using methods described previously (Kujawa et al., 1994). The cochlea was prepared for perfusion by placing holes in the cochlear basal turn perilymph compartment. A hole was placed in scala tympani for the introduction of the perfusates and a hole was placed in scala vestibuli to allow the effluent to escape. The artificial perilymph (AP) employed in these experiments had a composition of 137 mM NaCI, 5 mM KCI, 2 mM CaC12, 1 mM NaH2PO 4, 11 mM glucose, 12 mM NaHCO 3, with a resulting pH of 7.4. The drugs tested were: SNP (nitroprusside, nitroferricyanide, Naz[Fe(CN)sNO]; Sigma), ferricyanide (K3Fe(CN)6; Baker), ferrocyanide (KaFe(CN)6; Baker) and S-nitrosoN-acetylpenicillamine (SNAP; RBI). Drugs were dissolved in the AP solution just before use (found not to change the pH) and were protected from light as much as possible. SNAP was first dissolved in 100% DMSO before addition to the AP (final DMSO concentration--0.1% in 1 mM SNAP). For the 1 mM and lower concentrations of ferricyanide the KCI in AP was reduced to compensate for the K ÷ in the drug. However, for the 10 mM concentrations of both ferricyanide and ferrocyanide used in the EP studies no adjustment for ions was made since the K ÷ concentration in the AP could not be lowered to the extent necessary. The drug solutions were applied to the cochlea at room temperature by perfusing the perilymph compartment from basal turn scala tympani to basal turn scala vestibuli at 2.5 /zl/min usually for 20 min perfusion periods using a micropipette coupled to a syringe pump. Gross cochlear and auditory nerve potentials

Methods used to monitor the effects of multiple perilymphatic perfusions on sound-evoked potentials were similar to those described previously (Bobbin et al., 1990b). The compound action potential (CAP) of the auditory nerve (CAP amplitude; N1-Pl), N 1 latency, cochlear microphonic (CM) and summating potential (SP) were recorded from the right cochlea utilizing a silver wire (teflon-coated except for the tip) placed in basal turn scala

vestibuli. The evoked responses were amplified (Grass, PI5, gain = 1000), averaged (over 20 trials) and stored on the computer disk. The responses were evoked by l0 kHz tone pips (0.25 ms rise/fall, 10 ms duration, 200 ms interstimulus interval). Stimuli were computer generated (Tucker-Davis TDT System II hardware), transduced by a speaker and delivered through a hollow earbar to the right ear of each animal under computer control. Intensity was increased in 6 dB steps over the range 8-92 dB SPL. Custom software was utilized to display and filter the composite waveform (CAP, NI latency, SP: low pass to 2 kHz; CM: 7.5-15 kHz) for identification of CAP threshold and amplitudes of the CAP, CM and SP components and N I latency across stimulus intensities. The effects of SNP (5 animals), ferricyanide (3 animals), and SNAP (2 animals) on sound-evoked potentials were tested. Intensity functions to the tone bursts were recorded prior to the perfusion experiments (following the surgical exposure of the cochlea and middle ear muscle section), at which time responses were required to meet laboratory norms, and again immediately following each perfusion (within 2 min). A standard perfusion protocol was employed for all drugs except SNAP. The first two perfusions were of the AP solution alone. These were followed by successive perfusions with increasing concentrations of experimental drug (10 /~M, 33 /zM, 100 /xM, 330 /xM, and 1 mM). AP perfusions were employed to wash the drugs from perilymphatic spaces. The drug effects on EP were studied in an additional 9 animals using methods described previously (Bobbin et al., 1990b). EP was recorded in basal turn scala media using a glass micropipette with an internal solution containing 134 mM KCI, 0.5 mM MgC12, 11 mM EGTA, 1 mM CaCI 2, 5 mM HEPES, and was adjusted to a pH of 7.35 with HC1 and had an osmolality of 284 mOsm. Animals in which the EP was recorded were given gallamine triethiodide (Flaxedil, Davis and Geck, 25 mg/kg, i.p.) along with their normal regimen of pentobarbital. For these experiments 20 min perfusions were utilized. 2.2. In vitro procedures Cell isolation procedure

OHCs from adult guinea pigs were acutely isolated as described previously (Bobbin et al., 1990a; Erostegui et al., 1994; Ricci et al., 1994). Briefly, guinea pigs were anesthetized with pentobarbital (30 mg/kg, i.p.), decapitated, and the bulla separated and placed in a modified Hank's balanced saline (HBS). The bone surrounding the cochlea was removed and the organ of Corti was placed in a 200 /xl drop of HBS containing collagenase (1 mg/ml, type IV, Sigma) for 5 min. The cells were then transferred into the dishes containing a 100 /.tl drop of HBS using a microsyringe, and stored at room temperature. OHCs were selected for study if they met several morphological criteria (Ricci et al., 1994).

C Chen et al./Hearing Research 87 (1995) 1-8

3

establishment of the whole-cell configuration, series resistance and cell capacitance compensation were carried out prior to recording, and an 80% series resistance compensation was normally applied. Whole-cell capacitance of OHCs ranged from 20 to 47 pF and series resistance ranged from 4 to 10 M ~ . No subtraction of leakage current was made.

2.3. Whole-cell voltage clamp

Single dispersed OHCs were voltage-clamped using the whole-cell variant of the patch-clamp technique (Hamill et al., 1981) with an Axopatch-lD and Axopatch-200A patch-clamp amplifiers (Axon Instruments). Patch electrodes were fabricated from borosilicated capillary tubing (Longreach Scientific Resources) using a micropipette puller (Sutter Instrument), and fire-polished on a microforge (Narashige Scientific Instrument Lab.) prior to use. Membrane currents were filtered at 5 kHz ( - 3 dB) using a 4-pole low-pass Bessel filter digitized with a 12-bit A / D converter (DMA Interface, Axon Instruments), and stored for off-line analysis using 386 and 486 microcomputers. Voltage paradigms were generated from a 12-bit D / A converter (DMA Interface, Axon Instruments) using pCiamp 5.5 and 6.1 software (Axon Instruments). After

Solutions

The HBS utilized for isolating cells and perfusing the bath contained 137 mM NaCI, 5.4 mM KC1, 2.5 mM CaC12, 0.5 mM MgC12, 10 mM HEPES and 10 mM glucose. External solutions were adjusted to a pH of 7.4 with NaOH and to 300 mOsm with sucrose or NaCI. The internal solution contained 134 mM KCI, 0.5 mM MgC12, 11 mM EGTA, 0.1 mM CaCI 2, 10 mM HEPES, 2 mM Na2ATP, 0.1 mM Na2GTP, and was adjusted to a pH of 7.35 with HC! and had a osmolality of 284 mOsm. All

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Fig. 1. Effect of ferricyanide on CAP, N~ latency, SP and CM as a function of stimulus intensity. Shown are functions recorded after pre-drug artificial perilymph perfusion 2 (AP2), after perfusion with increasing concentrations (10-1000 p.M) of ferricyanide, and after a post-drug wash with artificial perilymph (WI). Data are represented as means + SE across 3 animals.

4

C. Chen et al. / Hearing Research 87 (1995) 1 - 8

experiments were conducted at room temperature (22 =

changes were large enough to see on the video monitor ( > 1 /xm of cell length change or about 1 mm on the monitor). Therefore, raters were used to judge the degree of the shortenings by observing the video tapes and noting the degree of shortening. Cells were observed for a maximum of 20 min. Abolishment of shortening rarely occurred probably because of the large voltage step used. Instead the shortening became visibly less and this was labeled a 'decrease in length change'. If a decrease in length change occurred before 10 min of drug application the cell was said to have responded to the treatment and the time that a decrease in length change was first noticed recorded, otherwise the cell was classified as not undergoing a length change in response to the treatment. In some cells the current response to the voltage steps in the absence and presence of the drugs was recorded. The care and use of the animals reported on in this

24°C). External solutions were delivered from an U-tubing system as described previously (Murase et al., 1989; Erostegui et al., 1994). SNP (1 and 10 mM) and sodium salicylate (10 raM) in isosmotic external solutions were applied to the OHCs by way of the U-tube and ,studied on voltage-induced length changes. Comparisons were made with application of control which was external solution without drug. Effects of the drugs on cell shortening induced by 60 ms voltage steps from - 6 0 mV (V h) to + 10 mV (i.e., electromotility) delivered every second were recorded on video tape. We were unable to stop the video frame at each step. Therefore, we could not obtain objective measurements of the cell length change in response to each voltage step with the computer system used previously (Bobbin et al., 1990a). However, the length

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Fig. 2. Effect of nitroprusside on CAP, Nt latency, SP and CM as a function of stimulus intensity. Shown are functions recorded after pre-drug artificial perilymph perfusion 2 (AP2), after perfusion with increasing concentrations (10-1000 /xM) of sodium nitroprusside (SNP), and after a post-drug wash with artificial perilymph (W1). Data are represented as means + SE across 5 animals.

C. Chen et aL //Hearing Research 87 (1995) 1-8

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Fig. 3. Intensity dependence of sodium nitroprusside (SNP) effects on C A P , N1 latency, SP and CM. Shown are the data representing responses evoked by tone bursts at 38 dB SPL and 92 dB SPL after the control perfusion (AP2), after increasing concentrations of SNP ( 1 0 - 1 0 0 0 / x M ) and after an artificial perilymph wash perfusion ( W I ) . D a t a are displayed as means + SE across n = 5 animals. Values significantly different from AP2 are designated • P < 0.05; * * P < 0.01 ( A N O V A and Newman-Keuls multiple range tests). Responses that fell into the noise floor were assigned the numerical value of the noise floor.

study were approved by the Animal Care and Use Committees of the Medical Centers at Louisiana State University and Tulane University. Data are presented as means -tSE. To determine significance of each treatment on the

sound evoked potentials (CM, SP, CAP, and Nj latency), 1-way (perfusion number) repeated-measures analysis of variance (ANOVA) and Newman-Keuls multiple-range tests were performed. Chi Square was used to determine

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6

c. Chen et al./Hearing Research 87 (1995) 1-8

statistical significance of the incidence of shortening of individual OHCs. To determine statistical significance of the 'time to decrease' of the voltage-induced length change A N O V A and Newman-Keuls was used. P values less than 0.05 were considered statistically significant.

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3. Results 3.1. In viuo measures

Baseline (pre-perfusion) measures of the in vivo electrical responses (CAP, N l latency, CM, SP), obtained following preparation of the cochlea for perfusion revealed results consistent with laboratory norms (data not shown; Bledsoe et al., 1981; Bobbin et al., 1991). When measured following the 2 control (AP) perfusions, slight alterations from pre-perfusion values occasionally were observed. Thus, for most animals, responses measured following the second AP perfusion served as the new post-perfusion baselines to which all drug-related changes were compared. In previous studies, multiple perfusions with the same AP produced little change in the cochlear potentials monitored (e.g., Bobbin et al., 1991). The effects of SNP, ferricyanide, and SNAP were tested on the input-output functions of the various sound-evoked cochlear and auditory nerve potentials. Multiple perfusions of ferricyanide ( 1 0 - 1 0 0 0 /xM) produced no change in the input-output functions (Fig. 1; n = 3 animals). In contrast, multiple perfusions of SNP ( 1 0 - 1 0 0 0 /zM) produced response alterations starting at approximately 100-330 /xM with larger effects following 1 mM (Figs. 2 and 3; n = 5 animals). Additional reductions occurred following a postSNP AP wash (Fig. 2 and Fig. 3). SNAP (1 raM) produced no effect after one 30 min or 40 min perfusion (n = 2 animals; data not shown). In view of the effects of SNP on the various soundevoked responses recorded, especially CM, we tested the effect of several of the drugs on the EP in additional animals. All perfusion periods were 20 rain in duration. Fig. 4A shows the effect of 4 successive 20 min perfusions

Table 1 Sodium nitroprusside (SNP) decreases voltage-induced length change of outer hair cells (OHCs) Number of cells Time of decrease n Decrease/ % (rain) total Control 4/14 SNP (1 mM) 6/10 SNP (10 mM) 8/12 Salicylate (10 raM) 15/15

29 60 67 * 100 * *

5.4 + 1.6 5.4+ 1.2 2.0+0.4 * 1.2+0.3 * *

4 6 8 15

Statistical significance was measured by chi square (i.e., incidence) and ANOVA and Newman-Keuls (i.e., time). * P < 0.05; * * P < 0.01 compared with the control.

SNP (10 mM)

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40 ms Fig. 5. Sodium nitroprusside (SNP; 10 mM) decreases outward current recorded from an OHC. Current was elicited by 60 ms depolarizing steps to + l0 mV from a holding potential of -60 mV. Shown are the whole-cell currents recorded before and following 5 min of extracellular applieation of SNP. of SNP (1 mM; n = 3 animals) and ferricyanide (1 mM; n = 3 animals). The ferricyanide perfusions produced little change in the EP. In contrast the SNP perfusions were associated with a slowly developing decrease in the EP. In the 3 animals in which 1 mM ferricyanide was perfused, we followed with a 20 min 10 mM ferricyanide perfusion and then a 20 rain 10 mM SNP perfusion. In 1 animal multiple AP perfusions were followed by a 20 min 10 mM SNP perfusion. The results from these 10 mM ferricyanide (n = 3) and 10 mM SNP (n = 4) perfusions are combined and presented in Fig. 4B. Fig. 4B illustrates that the ferricyanide increased EP slightly during the first few minutes of the perfusion, whereas SNP (10 mM) rapidly decreased EP. Ferrocyanide (10 mM) was tested in 2 animals and found to give the same results as 10 mM ferricyanide (data not shown). 3.2. In vitro measures Measurement o f O H C length change

Table 1 summarizes our results with the effects of drugs on the voltage-induced shortening of OHCs. Complete abolishment of the shortening rarely occurred. Instead, as described under Methods, the degree of shortening became less and this was attributed to the drug if it occurred within 10 rain of drug application. In control perfused OHCs, shortening became visibly less at an average time of 5 min from the start of perfusion in 4 out of the 14 cells observed

C. Chen et al. /Hearing Research 87 (1995) 1-8

(Table 1). The remaining cells continued to shorten to the same degree throughout and beyond the 10 min observation period. SNP (1 mM) could not be distinguished from control in that the contractions became less in about the same amount of time. On the other hand, SNP (10 mM) and salicylate (10 mM) induced a decrease in the degree of shortening in a larger number of cells and in a much faster time than control (Table 1). In general, the salicylate effect was reversible and the SNP (10 mM) effect was difficult to reverse. No collapse of the OHCs was detected with salicylate or with SNP. Whole-cell c u r r e n t s

While observing the effect of SNP on cell shortening in some cells, whole-cell currents in response to the voltage steps were monitored. Externally applied SNP slowly reduced the outward current over a 5 min period at a concentration of 10 mM (mean + S E , 4 3 _ 2%; n = 6 cells; Fig. 5), but not at 1 mM (mean + SE, 4 _ 7%; n = 5 cells). As NO is assumed to elicit its biologic effect by diffusing intracellularly, we added the NO donor, SNP (I mM; n = 6 cells), into the pipette for intracellular application. This route of administration of the drug to the cell induced no obvious effects on the I - V plots or on the voltage-induced shortening (data not shown).

4. Discussion

Our results confirm the preliminary observation reported by Brechtelsbauer et al. (1993) and Ohlsen et al. (1993) that SNP, a NO donor, affects cochlear potentials. We found that SNP decreased all sound-evoked cochlear potentials (e.g., CAP, CM and SP). CM and SP are generated by the hair cells and the CAP is generated by the auditory nerve. All of these potentials require the presence of a normal EP which is generated by the stria vascularis (for review: Bobbin and Kisiel, 1981). We found that SNP decreased the EP. This decrease in the EP then points to the stria vascularis as one of the primary sites of action for SNP. It suggests also that a proportion of the decrease in the other cochlear potentials is probably due to this decrease in EP. We do not know if the drugs applied to the cochlea affected blood pressure since we did not measure it. However, it is unlikely that the cochlear potential changes we observed were due to drug-induced hypotension. In unpublished studies, one of us (Bobbin) lowered the carotid blood pressure of guinea pigs which were anesthetized and artificially respired to 18 mm Hg with pentobarbital and observed no change in the EP. From this we conclude that blood pressure changes by themselves cannot change cochlear potentials. In addition, Ohlsen et al. (1993) report that application of a 5% solution of SNP to the round window induced no change in systemic blood pressure in

7

guinea pig although neural thresholds were dramatically affected. Thus, SNP does change cochlear potentials without a detectable change in blood pressure. In order to examine whether NO may be involved in the function of structures in the organ of Corti such as the OHCs, we tested the effect of SNP on voltage-induced currents and shortening of the OHCs described by others (Santos-Sacchi and Dilger, 1988; Santos-Sacchi, 1989). We used salicylate for a comparison since others have shown it to reduce voltage-induced shortening in OHCs (Shehata et al., 1991). Both SNP and salicylate reduced the voltage-induced shortening compared to control. The salicylate-induced effect has been suggested to be due to a loss of turgor (Shehata et al., 1991). Since we did not observe a loss of turgor with either salicylate or SNP, then this may not be the mechanism underlying our observations. In addition, since we controlled the voltage, then the block by both drugs is independent of any voltage change induced by the drugs. It has been shown that NO can react with an intracellular thiol (i.e., compounds which contain sulfhydryl groups) to form an S-nitrosothiol (Ignarro, 1990). A suifhydryl group has been suggested to be an important component for electromotility of OHCs because reagents which react with sulfhydryl groups have been demonstrated to inhibit OHC electromotility (Kalinec and Kachar, 1993). Thus one possible mechanism for the SNP-induced decrease of voltage-induced shortening may be that NO released from SNP binds to a thiol in the OHC membrane to form a substance which inhibits electromotility. The mechanism of action of salicylate on electromotility most probably is different than SNP and is unknown. SNP (10 mM) reduced the voltage-induced outward current recorded from OHCs. When applied intracellularly 1 mM SNP had no effect. Assuming less than 1 mM diffuses into the intracellular compartment when 10 mM is applied externally, then it appears the action of the drug was extracellular. Others have demonstrated that SNP will block L-type Ca 2+ channels in smooth muscle cells (Blatter and Wier, 1994). Thus, the mechanism for the observed reduction of current in OHCs may be a block of L-type Ca 2÷ channels with subsequent decrease in activity of Ca2+-dependent K ÷ channels. Both the reduction in voltage-induced shortening and the reduction in outward current may have accounted for some of the alterations in cochlear potentials recorded. SNP forms ferricyanide upon release of NO, so both ferricyanide and ferrocyanide were tested to see if the effects observed with SNP were due to the non-NO portion of the molecule. In other systems, ferricyanide (East et al., 1991) and ferrocyanide (Kiedrowski et al., 1992) mimicked the effects of SNP, so the authors concluded that the effects were not likely to have been caused by NO. In our system, both ferricyanide and ferrocyanide had no effect. This is evidence that the SNP-induced effects are due to the released NO. On the other hand, the lack of effect of SNAP, even after 30-40 min of perfusion, is evidence that

8

c. Chen et al./Hearing Research 87 (1995) 1-8

the e f f e c t o f S N P w a s not d u e to N O . W e o n l y tested SNAP on sound-evoked cochlear potentials, however, since t h e s e p o t e n t i a l s are d e p e n d e n t o n E P t h e n w e c o n c l u d e that S N A P h a d n o e f f e c t o n t h e EP. C o n s i s t e n t w i t h t h e s e results, B r e c h t e l s b a u e r et al. ( 1 9 9 3 ) f o u n d n o effect w i t h L - N A M E , an i n h i b i t o r o f nitric o x i d e s y n t h a s e . In addition, the a m o u n t o f S N P n e c e s s a r y to i n d u c e c h a n g e s in s o u n d e v o k e d c o c h l e a r p o t e n t i a l s , EP, a n d O H C c u r r e n t was large c o m p a r e d to a m o u n t s d e s c r i b e d in the literature (e.g., ICs0 = 6.6 / x M in rat c e r e b e l l a r g r a n u l e cells) ( K i e d r o w s k i et al., 1992). Yet, S o u t h a m a n d G a r t h w a i t e ( 1 9 9 1 ) calculated that 10 m M S N P w o u l d yield a local N O c o n c e n t r a tion o f 1 /xM, w h i c h is a r e a s o n a b l e c o n c e n t r a t i o n for a p h a r m a c o l o g i c a l e f f e c t at the stria or at O H C s . T h u s o u r data is o n l y s u g g e s t i v e o f an e f f e c t o f N O in the c o c h l e a . A n a l t e r n a t i v e e x p l a n a t i o n o f o u r d a t a is that the p h a r m a c o l o g i c a l e f f e c t s w e o b s e r v e d w e r e d u e to the S N P m o l e c u l e b e f o r e r e l e a s e o f N O a n d f o r m a t i o n o f ferric y a n i d e . O t h e r t y p e s o f e x p e r i m e n t s s u c h as the u t i l i z a t i o n of new and improved NO releasing substances may shed l i g h t o n the m e c h a n i s m o f a c t i o n o f S N P . In s u m m a r y S N P , a n N O d o n o r , w a s f o u n d to r e d u c e EP, s o u n d - e v o k e d p o t e n t i a l s r e c o r d e d f r o m the c o c h l e a , electromotility and currents recorded from isolated OHCs. A l t h o u g h the r e s u l t s s u g g e s t t h a t N O h a s a role in the f u n c t i o n o f the O H C s a n d stria vascularis, the results c o u l d also b e i n t e r p r e t e d to m e a n that S N P h a s p h a r m a c o l o g i c a l effects a p a r t f r o m t h a t o f r e l e a s e d N O .

Acknowledgements O u r t h a n k s go to S h a r o n G. K u j a w a , Ph.D., C h r i s t o p h e r L e B l a n c , a n d E l a i n e M c D o n a l d for t h e i r help. S u p p o r t e d by N I H G r a n t R 0 1 - D C 0 0 7 2 2 , D A M D 17-93-V-3013, K a m ' s F u n d for H e a r i n g R e s e a r c h , a n d the L o u i s i a n a Lions Eye Foundation.

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