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Modulation of Voltage-Gated Ca²⁺ Current in Vestibular Hair Cells by Nitric Oxide

Instituto de Fisiología, Universidad Autónoma de Puebla, Puebla, Mexico

Submitted 12 August 2006; accepted in final form 14 December 2006

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
The structural elements of the nitric oxide–cyclic guanosine monophosphate (NO–cGMP) signaling pathway have been described in the vestibular peripheral system. However, the functions of NO in the vestibular endorgans are still not clear. We evaluated the action of NO on the Ca²⁺ currents in hair cells isolated from the semicircular canal crista ampullaris of the rat (P14–P18) by using the whole cell and perforated-cell patch-clamp technique. The NO donors 3-morpholinosydnonimine (SIN-1), sodium nitroprusside (SNP), and (±)-(E)-4-ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexen-1-yl-nicotinamide (NOR-4) inhibited the Ca²⁺ current in hair cells in a voltage-independent manner. The NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO) prevented the inhibitory effect of SNP on the Ca²⁺ current. The selective inhibitor of the soluble form of the enzyme guanylate cyclase (sGC), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), also decreased the SNP-induced inhibition of the Ca²⁺ current. The membrane-permeant cGMP analogue 8-Br-cGMP mimicked the SNP effect. KT-5823, a specific inhibitor of cGMP-dependent protein kinase (PGK), prevented the inhibition of the Ca²⁺ current by SNP and 8-Br-cGMP. In the presence of N-ethylmaleimide (NEM), a sulfhydryl alkylating agent that prevents the S-nitrosylation reaction, the SNP effect on the Ca²⁺ current was significantly diminished. These results demonstrated that NO inhibits in a voltage-independent manner the voltage-activated Ca²⁺ current in rat vestibular hair cells by the activation of a cGMP-signaling pathway and through a direct action on the channel protein by a S-nitrosylation reaction. The inhibition of the Ca²⁺ current by NO may contribute to the regulation of the intracellular Ca²⁺ concentration and hair-cell synaptic transmission.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Nitric oxide (NO) has emerged as an important signaling molecule with a functional role in both central and peripheral systems. Experimental evidence indicates that once synthesized, NO rapidly diffuses into neighboring cells, thus regulating their activity. The modulation of ionic channels has been suggested as one of the mechanisms mediating the physiological functions of NO, including relaxation of vascular smooth muscle (Blatter and Wier 1994; Lewis et al. 2005), neurotransmission, synaptic plasticity, and neurodegenerative processes (Garthwaite and Boulton 1995; Rand and Li 1995; Schuman and Madison 1994). Ionic channel modulation by NO can be produced by S-nitrosylation of target proteins (Ahern et al. 2002; Jaffrey et al. 2001) or indirectly through second messengers involving the activation of sGC (the soluble form of the enzyme guanylate cyclase) and increased levels of cyclic guanosine monophosphate (cGMP) (Bredt and Snyder 1989; Garthwaite and Boulton 1995).

In the acoustico-lateral system there is growing evidence of the presence of a NO–cGMP signaling pathway. There is evidence that neuronal- and endothelial nitric oxide synthases (NOS) and enzymatic activities of sGC, NOS, and cGMP-dependent protein kinase I (PKG-I) are co-localized in supporting cells of the organ of Corti (Fessenden and Schacht 1997; Fessenden et al. 1994; Gosepath et al. 1997; Tian et al. 1999; Zdanski et al. 1994). Activity of the enzyme NADPH-diaphorase (NADPH-d), which is now known to be NOS, was demonstrated in the electroreceptors of a gymnotiform teleost Apteronotus leptorhyncus (Turner and Moroz 1995) and in the neuromasts of the axolotl Ambystoma tigrinum (Vega et al. 2006). These sensory receptors are evolutionary related to the hair cells of the vertebrate inner ear. In the vestibular system of the chinchilla and rat, NADPH-d histochemistry and NOS-I positive immunoreactivity were found in a subpopulation of vestibular-efferent neurons contacting hair cells (Lysakowski and Singer 2000). NOS-I, calretinin, and sGC were co-localized in calyx-ending afferent neurons of the chinchilla and both sGC and calmodulin, an important cofactor of NOS (Bredt 1999), were co-localized in type I and type II hair cells (Desai et al. 2004; Dhaliwal and Lysakowski 2000). NOS was co-localized with sGC and cGMP in the sensory epithelia and the vestibular nerve fibers of the guinea pig and mice (Hess et al. 1998a,b). These results indicate that the elements of the NO–cGMP pathway are expressed in hair cells and efferent and afferent neurons. Functionally, it has been shown that NOS inhibitors diminished the afferent resting discharge in the amphibian vestibular and lateral line systems (Flores et al. 1996, 2001) and had inhibitory–excitatory effects in the cephalopod statocyst (Tu and Budelman 1999, 2000). The production of NO was previously reported in guinea pig vestibular hair cells, which increased after stimulation by L-arginine (Takumida and Anniko 2000).

However, there are only a few reports in the literature, as far as we were able to determine, concerning the action mechanism of NO at the cellular level in the acoustico-lateral system. It was shown that cGMP and NO donors inhibit the low-voltage–activated potassium conductance (I_K,L) specific for type I hair cells, thus increasing the hair cell gain and functioning as a positive feedback mechanism capable of boosting afferent neurotransmitter release (Behrend et al. 1997; Chen and Eatock 2000). In the guinea pig cochlea nitroprusside reduced endocochlear potential, sound-evoked potentials, electromotility, and outward current from isolated outer hair cells (Chen et al. 1995).

Rat vestibular hair cells express L-type Ca²⁺ channels formed by the _1D subunit (Almanza et al. 2003; Bao et al. 2003). Given the evidence of the effects of NO–cGMP on Ca²⁺ channels in different cell types and considering a preliminary report that suggests that NO reduced the open probability of Ca²⁺ channels in bullfrog hair cells (Rodriguez-Contreras et al. 2000), it seems feasible that NO might modulate Ca²⁺ channels and constitute an effective mechanism for the control of the intracellular Ca²⁺ level in hair cells and afferent transmitter release. To evaluate this possibility we studied the action of NO on Ca²⁺ channels in hair cells. Our results show that NO inhibits the voltage-activated Ca²⁺ current in rat vestibular hair cells by the activation of a cGMP-signaling pathway and through a direct action on the channel protein by the S-nitrosylation reaction.

	METHODS

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Experiments were made on young Long–Evans rats (P14–P18) supplied by Claude Bernard animal house of the Autonomous University of Puebla. Animal care and procedures were in accordance with the Reglamento de la Ley General de Salud en Materia de Investigación para la Salud de la Secretaría de Salud de México. All efforts were made to minimize animal suffering and to reduce the number of animals used, as outlined in the Guide for the Care and Use of Laboratory Animals issued by the National Academy of Science.

Cell preparation

Hair cells were enzymatically dissociated from the semicircular canal crista ampullaris of the rat as reported previously (Almanza et al. 2003). In brief, tissue pieces containing the vestibular sensory epithelia were incubated in normal Tyrode solution (see Solutions) containing 0.2 mg/mL IA type collagenase for 7 min at 35°C, followed by cell incubation for 10 min at 35°C in a Ca²⁺- and Mg²⁺-free Tyrode solution with 1 mg/mL porcine-trypsin. Finally, tissue was washed for 10 min at 4°C in a Ca²⁺- and Mg²⁺-free Tyrode solution containing 1 mg/mL serum bovine albumin. All enzymes were from Sigma-Aldrich (St Louis, MO). The tissue was then placed in the recording chamber on an inverted microscope stage (Nikon Diaphot, Tokyo) and cells were isolated by gently triturating the tissue with fire-polished Pasteur pipettes. Cells settled and adhered to the bottom of the recording chamber within 10 min. The recording chamber was continuously perfused with the extracellular solution designed for the Ca²⁺ current recording (see Solutions) at a constant rate (0.5 mL/min) using a peristaltic pump (Microperpex, LKB, Sweden).

Cell identification

Isolated cells were visualized with phase-contrast optics. Type I and type II hair cells were identified using morphological criteria. Previous studies have shown that in hair cells there is a good relation between their morphology and electrophysiological properties, hence cellular types can be distinguished morphologically (Almanza et al. 2003; Chen and Eatock 2000; Rüsch et al. 1998). Cells that exhibited a flask-shaped body with a highly refringent cuticular plate wider than the hair cell neck were considered to be type I hair cells and cells exhibiting cylindrical shapes and cuticular plate hardly distinguishable under phase contrast optics were considered to be type II hair cells.

Recordings

All experiments were done at room temperature (20–22°C). Whole cell recordings were done with the patch-clamp technique according to the method described by Sakmann and Neher (1984). Patch pipettes were pulled from borosilicate glass (WPI, Sarasota, FL) using a Flaming-Brown electrode puller (P80/PC; Sutter Instruments, San Rafael, CA) and had an open tip resistance of 3–5 M. Whole cell current recordings were made with an axopatch 200B amplifier (Molecular Devices, Union City, CA). Signals were low-pass filtered at 2 kHz and digitized at 5 kHz. Command pulse generation and data sampling were controlled by pClamp 8.0 software (Molecular Devices) using a 12-bit AD/DA converter (Digidata 1200, Molecular Devices). Both the membrane capacitance (C_m) and 80% of the series resistance (R_s) were electronically compensated. The average C_m was 7 ± 1 pF and the average R_s was 17 ± 1.8 M (n = 72). The maximum voltage errors caused by uncompensated R_s were 1 mV, corresponding to currents 100 pA, therefore no voltage correction for uncompensated R_s was needed. The junction potential was calculated with the generalized Henderson liquid-junction potential equation (Barry 1994) using pClamp 8.0 software (Molecular Devices). Voltages were corrected for a 7.9 mV liquid-junction potential for the Ba²⁺ extracellular solution and Cs⁺ and TEA intracellular Ca²⁺ solutions (see Solutions). Some experiments were made with permeabilized patch techniques. For these the pipette solution containing 200 µM nystatin was sonicated, then back filled into the recording pipette.

Solutions

The normal Tyrode solution was (in mM) 140 NaCl, 5.4 KCl, 1.2 MgCl₂, 3.6 CaCl₂, 10 HEPES, and 10 Glucose. The Ca²⁺- and Mg²⁺-free Tyrode solution was (in mM) 140 NaCl, 5.4 KCl, 10 HEPES, 10 Glucose, and 1 EGTA. For Ca²⁺ current recording, extracellular Ba²⁺ was used as a current carrier and intracellular Cs⁺-TEA solutions were used to block K⁺ currents. The extracellular solution with Ba²⁺ was (in mM) 23 TEACl, 30 CsCl, 55 NaCl, 0.1 CaCl₂, 20 BaCl₂, 10 HEPES, and 10 Glucose. The patch electrodes were filled with a solution containing either (in mM) 23 TEACl, 69 CsCl, 0.1 CaCl₂, 10 HEPES, 80 NMDG⁺, 10 EGTA, 0.1 GTPNa, and 2 ATPMg for whole cell patch experiments or 138 CsCl, 0.1 CaCl₂, 10 HEPES, and 10 EGTA for the perforated-patch mode. The pH of all external solutions was adjusted to 7.4 with NaOH. The pH of intracellular solutions were adjusted to 7.2 with HCl or CsOH. The osmolarity for all solutions was adjusted to 300 mOsm.

Drugs

The compounds used as NO donors were 3-morpholinosydnonimine (SIN-1; Molecular Probes, Eugene, OR), sodium nitroprusside (SNP; RBI, Natick, MA), (±)-(E)-4-ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexen-1-yl-nicotinamide (NOR-4; Sigma–Aldrich). The standard light beam of the microscope was directed onto the investigated cell to favor the NO production. The compound 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (CPTIO; Molecular Probes) was used as a nitric oxide scavenger. To study the participation of the cGMP-PKG pathway, we used 8-Br-cGMP (Sigma–Aldrich) as a membrane-permeant cGMP analog. An inhibitor of sGC, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; Sigma–Aldrich) and a specific inhibitor of PKG, KT-5823 (Sigma–Aldrich) were also used. N-ethylmaleimide (NEM; Sigma–Aldrich), which prevents S-nitrosylation of proteins, was used to study the participation of a cGMP-independent mechanism. Because SIN-1, SNP, 8-Br-cGMP, and NEM are light sensitive, experiments with them were done in a darkened room.

The compounds SNP, SIN-1, NOR-4, CPTIO, and 8-Br-cGMP were applied by microperfusion with a constant flow of 10 µL/min. For this, after the last control recording, a Teflon microtube was drawn close to the cell being recorded and the desired solution was applied by pressure ejection from an electronic microsyringe (Baby Bee, BAS, Lafayette, IN). The recording chamber was designed to have a uniform solution flow from the inflow to the outflow side. The cells selected for the experiment were recorded sequentially starting from the outflow side of the chamber, thus ensuring that previous perfusion of drugs onto a cell did not affect successive recordings. No cell was exposed to more than one drug except for those experiments designed to study drug interactions. In the experiments done to study sGC inhibition, the ODQ was perfused into the bath for 25 min before the recording to allow it to diffuse into the cells. The KT-5823 and NEM were applied intracellularly directly by the recording pipette.

Data analysis

Recordings were analyzed off-line with pClamp 8.0, SigmaPlot 8.0 (Systat Software, Richmond, CA), and Origin 6.0 software (Microcal Software, Northampton, MA). For the curve-fitting routines a nonlinear least-squares method was used.

To construct the current–voltage relationships (I–V), the Ca²⁺ currents were elicited by 300 ms voltage steps between –100 and +50 mV from a holding potential of –60 mV with 10 mV increments every 5 s. The Ca²⁺ current amplitude was measured between 155 and 175 ms after the start of voltage steps. The current produced between –100 and –70 mV was fitted with a linear function that was subtracted assuming a linear leak throughout the whole I–V relationship (Martinez-Dunst et al. 1997).

A single Boltzmann function was fitted to the I–V relationship between –60 and –10 mV, which corresponds to the growth region of the Ca²⁺ conductance (Bao et al. 2003). The function used was

(1)

where I_max is the maximum current, V is the voltage, V_1/2 is the voltage at half channel activation, and S is the slope of the curve at this midpoint.

The effect of different drugs on the Ca²⁺ current was evaluated by using 300 ms depolarizing test pulses to –10 mV from a holding potential of –60 mV every 4 s. The amplitude of the Ca²⁺ current was measured after 2 min of drug perfusion.

Statistics

Numerical data are presented as the mean ± SE for at least four measurements, unless otherwise stated. Statistical differences were determined using a Student’s t-test. P < 0.05 was used to denote statistical difference between the groups.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Under control conditions, using the Ba²⁺ extracellular solution, depolarizing voltage steps of 300 ms from a holding potential of –60 mV caused an inward current that activated above –60 mV, reached its maximum at –18 mV (n = 36), and showed fast activation kinetics and no significant inactivation. The V_1/2 for the control Ca²⁺ current was –37 ± 0.5 mV and the S was 5 ± 0.1 mV. This current was identified as a Ba²⁺ current entering the cell through voltage-dependent Ca²⁺ channels and thus will be referred to as I_Ca. These characteristics are in agreement with previous results in the rat and chick, indicating that the I_Ca in type I and type II hair cells is a L-type Ca²⁺ current (Almanza et al. 2003; Bao et al. 2003; Masetto et al. 2005). A similar I_Ca was found when recording with the perforated patch-clamp technique (n = 6; data not shown).

Modulation of the Ca²⁺ current by nitric oxide donors

To evaluate the effects of NO on the I_Ca in hair cells, SIN-1, NOR-4, and SNP that release NO were used. Initially we studied the action of SNP in hair cells identified as type I or type II. The application of 100 µM SNP, an inorganic nitroso compound, decreased the I_Ca in type I hair cells (53 ± 5%; n = 5, P = 0.0007) (Fig. 1) with no significant effect on the I_Ca activation or inactivation rates (Fig. 1A, inset). The effect of SNP reached its maximum after about 3 min of its perfusion (Fig. 1B) and was partially reversible, recovering to 75% of the control current after 2 min of drug washout. The V_1/2 was shifted to the right by 2.5 ± 1 mV (100 µM SNP; P = 0.07) (Fig. 1C). In cells identified as type II hair cells the application of 100 µM SNP also decreased the I_Ca (49.7 ± 8%; n = 7, P = 0.002) with no significant effect on the I_Ca activation or inactivation rates (data not shown). No significant difference was found between the I_Ca in type I or type II hair cells nor did the action of SNP show any significant difference (P = 0.7); thus no further distinction of the cell type was done. To avoid dialysis of cytoplasmic elements, such as sGC, during the whole cell recording, experiments using the perforated patch-clamp technique were also made. In this condition, the application of 100 µM SNP decreased the I_Ca in a fashion similar to that found with the whole cell recording (59 ± 8%; n = 6 as compared with 53 ± 5%; n = 5, P = 0.67) (data not shown). Because no significant difference was found between whole cell and perforated recordings, the rest of the experiments were done using the whole cell patch clamp.

View larger version (23K): [in this window] [in a new window]   FIG. 1. Inhibition of the calcium current (ICa) by sodium nitroprusside (SNP). A: 100 µM SNP decreased the control ICa 53%. In this and the following current recordings the dotted line indicates zero current. Inset: current in the presence of SNP was scaled up to match it with the control current trace to show that SNP application had no effect on the ICa shape. B: time course of the effect of 100 µM SNP application (line). Maximum effect of SNP was reached about 3 min after its perfusion and it was partially reversible. C: current–voltage (I–V) relationships under control conditions (, n = 5) and during 100 µM SNP perfusion (, n = 5). Currents were normalized as a function of the maximum control current. Boltzmann fitting of I–V relationships indicates that no significant changes, apart from a reduction in current amplitude, take place. Points represent the means ± SE. D: percentage inhibition of the control Ca2+ current (measured at test pulses of –10 mV) by SNP.

Application of 1 µM SIN-1 significantly decreased the I_Ca (45 ± 5%; n = 4, P = 0.004) (data not shown), with the maximum effect reached 3 min after the SIN-1 application. Washout of the drug produced a partial 60% recovery of the I_Ca after 2 min. The SIN-1 (1 µM) did not significantly modify the V_1/2 or the S. Application of 100 µM SIN-1 diminished the I_Ca (60 ± 9%; n = 9, P = 0.0001) (data not shown) and produced a significant positive shift of V_1/2 from –39 ± 0.4 to –35 ± 1.5 mV (n = 9; P = 0.02). No significant change of the slope was observed (S values of 5.4 ± 0.3 and 5.8 ± 0.3 mV; P = 0.3). The action of 100 µM SIN-1 was irreversible after a 2-min washout of the drug (data not shown).

A nonthiol-based NO donor NOR-4 (Kita et al. 1994) in concentrations of 100 (Fig. 2, A and B) and 1 µM (not shown) significantly decreased the I_Ca (67 ± 8%; n = 3, P = 0.0005 and 38 ± 2%; n = 4, P = 0.01). For both concentrations the NOR-4 effect was irreversible (Fig. 2A). NOR-4 produced a nonsignificant displacement to the right of V_1/2 for I_Ca (Fig. 2C). The above results indicate that the inhibitory action of the three NO donors tested is mediated by the common product of these drugs, nitric oxide. Because SNP also releases cyanide ions that may inhibit Ca²⁺ currents (Biscoe and Duchen 1989), we decided to study the action of SNP concurrently with NO scavenger. The compound CPTIO (300 µM) produced a slight nonsignificant decrease of the I_Ca 1 min after its perfusion (11 ± 3%; n = 6, P = 0.07) (Fig. 3, A and B). In the presence of CPTIO the inhibitory effect of 100 µM SNP on the I_Ca was significantly reduced (12 ± 5%; n = 6, compared with 53 ± 5% caused by SNP alone, P = 0.002) (Fig. 3D). These results indicate that the inhibition of I_Ca by SNP is caused by the production of NO and not by other products of SNP breakdown.

View larger version (9K): [in this window] [in a new window]   FIG. 2. Inhibition of the ICa by the nitric oxide (NO) donor (±)-(E)-4-ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexen-1-yl-nicotinamide (NOR-4). A: 100 µM NOR-4 decreased the control ICa 67%. B: time course of the magnitude of the ICa (same cell as in A) during NOR-4 application (line). NOR-4 effects were irreversible. C: I–V relationships under control conditions (, n = 7) and during 1 µM (, n = 4) and 100 µM (, n = 3) NOR-4 application. Data between –60 and –10 mV were fitted by a Boltzmann function (solid lines). Use of NOR-4 (1 and 100 µM) did not produce significant changes in the V1/2 or S parameters. Points represent the means ± SE.

View larger version (19K): [in this window] [in a new window]   FIG. 3. NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO) prevents the inhibition of the ICa by SNP. A: application of 300 µM CPTIO alone had no significant effect on the ICa, whereas CPTIO significantly reduced the 100 µM SNP-induced inhibition of the ICa. B: time course of the actions of CPTIO and SNP on the ICa amplitude from the same cell as in A. Solid lines here and in C indicate the duration of application for each drug. Dotted line in B indicates the current amplitude averaged during last 30 s of CPTIO perfusion. C: time course of the amplitude of the ICa measured at –10 mV after application of 100 µM SNP in the presence of 10 µM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). In this condition, the inhibition of the ICa induced by SNP was reduced to 11%. D: bar graph of the percentage inhibition ±SE of the ICa measured with test pulses to –10 mV by 100 µM SNP alone, 100 µM SNP applied after 3-min perfusion with 300 µM CPTIO, and 100 µM SNP applied after 25-min perfusion with 10 µM ODQ are shown.

To investigate whether the inhibition of the I_Ca produced by NO is mediated by the activation of sGC and the subsequent increase in the levels of cGMP, the cells were treated for 25 min with 10 µM ODQ, a selective sGC inhibitor (Garthwaite et al. 1995). Subsequent coapplication of 100 µM SNP decreased the I_Ca (11 ± 11%; n = 9, P = 0.02 compared with the inhibition by SNP alone), indicating that the action of SNP implies NO production and subsequent sGC activation (Fig. 3, C and D).

To further characterize the NO–cGMP pathway we investigated the action of the membrane-permeant cGMP analogue 8-Br-cGMP and of a specific inhibitor of the PKG, KT-5823. The 8-Br-cGMP in concentrations of 200 and 400 µM decreased the I_Ca after 3 to 4 min of its application (29 ± 7%; n = 4, P = 0.03 and 50 ± 7%; n = 4, P = 0.006), thus mimicking the action of SNP (Fig. 4, A and B). The washout of the drug produced no significant recovery of the I_Ca during the 2 to 3 min after drug removal (in only two of eight cells did the washout produce a partial recovery of the current magnitude). Boltzmann fitting showed that the V_1/2 of current activation shifts by 2 ± 2 mV (200 µM 8-Br-cGMP; P = 0.03) and 6 ± 3 mV (400 µM 8-Br-cGMP; P = 0.01) (Fig. 4C). Changes in the S parameter were not statistically significant. In the presence of 10 µM KT-5823 the inhibitory effect of 400 µM 8-Br-cGMP on the I_Ca was significantly reduced (31 ± 1%; n = 7, P = 0.02) (data not shown), indicating that the inhibition of the I_Ca produced by 8-Br-cGMP is mediated by the activation of the PKG. The KT-5823 (10 µM) also significantly inhibited the effect of 100 µM SNP (31.5 ± 3.6%; n = 4, P = 0.007 compared with the 53% inhibition by 100 µM SNP), indicating that the Ca²⁺ channel modulation by NO also implies the participation of the NO–cGMP–PKG system (Fig. 4D).

View larger version (24K): [in this window] [in a new window]   FIG. 4. Inhibition of the ICa by a cyclic guanosine monophosphate (cGMP)–dependent mechanism. A: application of 400 µM 8-Br-cGMP reversibly decreased the control ICa. B: time course of the ICa magnitude during 400 µM 8-Br-cGMP perfusion (line). Maximum effect was reached between the third and the fourth minutes of application. C: normalized I–V relationships under control conditions (, n = 8) and during 200 µM (, n = 4) and 400 µM (, n = 4) 8-Br-cGMP application. Currents obtained between –60 and –10 mV were fitted by a Boltzmann function (solid lines). Both concentrations caused a significant shift to the right in the V1/2, whereas S was not significantly changed. Points represent the means ± SE. D: perfusion of 10 µM KT-5823 decreased the inhibitory action of 100 µM SNP on the ICa to 31.5%.

We also investigated whether chemical modification of sulfhydryl groups on Ca²⁺ channels participates in the current inhibition induced by NO donors. For this, the effect of 100 µM SNP was tested in the presence of N-ethylmaleimide (NEM), which is known to covalently modify protein sulfhydryl groups to prevent S-nitrosylation of proteins (Bolotina et al. 1994). NEM (1 mM) was applied intracellularly by the recording pipette. In the presence of intracellular NEM, the effect of 100 µM SNP on the I_Ca was significantly diminished (28 ± 4.5%; n = 6, P = 0.03 compared with the 53% inhibition by SNP alone) (Fig. 5). In this condition, the effect of the SNP was slightly reversible with respect to the control (38 ± 17%). We also tested the action of 1 mM NEM applied extracellularly by microperfusion. Importantly, in this case NEM by itself produced a significant 90% reduction of the I_Ca and thus no further pharmacological interaction was studied in this condition (n = 4; data not shown).

View larger version (16K): [in this window] [in a new window]   FIG. 5. Inhibition of the ICa by S-nitrosylation. N-Ethylmaleimide (NEM) attenuates the effect of the nitric oxide donor SNP. A: recordings were done with 1 mM NEM applied intracellularly by the recording pipette (control). In this condition, the inhibition of the ICa induced by 100 µM SNP was 28%. B: time course of the ICa magnitude during SNP perfusion in the presence of NEM. Data from the same cell shown in A.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

A potential problem with the use of SNP is that in solution it releases cyanide ions that may influence the I_Ca (Biscoe and Duchen 1989). However, the use of NO donors (SIN-1 and NOR-4) that do not produce cyanide indicates that the inhibition of I_Ca is caused by a product common to the three donors: nitric oxide. Additionally, the inhibitory effect of the SNP was blocked by the NO scavenger CPTIO, lending further support to the idea that the effects of the SNP are produced through the generation of NO. Both facilitation and inhibition of the voltage-activated Ca²⁺ channels by NO by cGMP were previously reported. NO inhibits the P/Q-type Ca²⁺ channels in rat insulinoma cells (Grassi et al. 1999) and in the cortical neurons of the rat (Petzold et al. 2005). Inhibition of N-type Ca²⁺ channels was reported in the dorsal root ganglion neurons innervating the urinary bladder of the rat (Yoshimura et al. 2001) and in human neuroblastoma cells (D’Ascenzo et al. 2002), although in neurons of the cervical ganglion of the rat and in the ganglion neurons of salamander retina, facilitation of N-type Ca²⁺ channel activation was reported (Chen and Schofield 1995; Hirooka et al. 2000). Inhibition of L-type Ca²⁺ channels by NO was found in mammalian ventricular myocytes (Campbell et al. 1996) and in bovine chromaffin cells (Carabelli et al. 2002). However, in the salamander retina NO suppressed the Ca²⁺ current in cones but facilitated it in rods (Kourennyi et al. 2004). Because the _1D subunit is expressed in cones and the _1F subunit in rods (Morgans 2001; Morgans et al. 2005; Xu et al. 2002), the opposing actions of NO on the retinal cells may be caused by the different expression of the Ca²⁺ channel subunits. Recent evidence indicates that the rat vestibular hair cells express L-type Ca²⁺ channels formed by the _1D subunit (Almanza et al. 2003; Bao et al. 2003). Our results are consistent with the inhibitory effects of NO targeting _1D channels.

NO may also modulate the Ca²⁺ channels independently of the NO–cGMP–PKG pathway through a direct modification of the channel proteins by means of the S-nitrosylation reaction (Ahern et al. 2002). In the rabbit carotid-body glomus cells and in mammalian ventricular myocytes, NO inhibits the L-type Ca²⁺ current by this cGMP-independent mechanism (Campbell et al. 1996; Summers et al. 1999). Under our experimental conditions, we cannot dismiss that S-nitrosylation participates in the inhibitory effects of NO on the I_Ca. Indeed, our results suggest that this process might contribute to the inhibition of the I_Ca in hair cells because none of the concentrations used of 8-Br-cGMP (400 µM) equaled the inhibition reached with the NO donors. Although 8-Br-cGMP is a membrane-permeable molecule, the diffusion barriers are practically nonexistent for NO, which would favor a greater effect of NO donors. Although the sGC inhibitor ODQ and the PKG inhibitor KT-5823 significantly reduced the effect of SNP, they did not completely eliminate it, leaving open the possibility of a cGMP-independent mechanism activated by NO. This is the reason we made an experimental series to study whether the effects of NO on the I_Ca may in part be caused by a mechanism independent of cGMP. In the presence of NEM, an agent that prevents the S-nitrosylation reaction, the inhibitory effect of SNP on I_Ca was decreased and the inhibition percentage reached under these conditions was similar to the SNP effect on the I_Ca in the presence of a specific inhibitor of PKG, KT-5823. These results suggest that the inhibition of I_Ca by NO also involves a direct S-nitrosylation of the Ca²⁺ channel proteins. This is in agreement with previous reports in bullfrog hair cells that showed that NO reduced the open probability of L-type Ca²⁺ channels by both direct and indirect pathways (Rodriguez-Contreras et al. 2000).

The lack of complete reversibility of the I_Ca inhibition by NO donors may be caused by a combination of factors, including the slow deactivation of NO–sGC (Margulis and Sitaramayya 2000) and the S-nitrosylation of the Ca²⁺ channel process, which stabilizes NO in a biologically active form and that may give rise to disulfide-bond formation (Stamler et al. 1992).

In the vestibular endorgans one of the targets proposed for NO is the low-threshold K⁺ current (I_K,L) expressed in type I hair cells (Rennie and Correia 1994; Rüsch and Eatock 1996a,b). Behrend et al. (1997) found that the cGMP and its membrane-permeant 8-Br-cGMP analog inhibit I_K,L. Later it was reported that NO donors inhibit I_K,L through the sGC–cGMP pathway (Chen and Eatock 2000). From these results, inhibition of I_K,L by NO could substantially affect the excitability of the type I hair cells, shifting the membrane potential toward positive voltages and increasing the cell membrane resistance, thus increasing the cell gain and enhancing the transmitter release. However, our results indicate that the action of NO on the postransductional processing of vestibular information may be more complex. The inhibition of the voltage-activated Ca²⁺ channels would decrease the transmitter release in such a way that NO would function as a part of a synaptic-strength modulatory mechanism. This mechanism would act both pre- and postsynaptically. Inhibition of the voltage-activated Ca²⁺ channels would also decrease the Ca²⁺-activated K⁺ current, which is expressed both in hair cells and afferent neurons (Hudspeth and Lewis 1988; Limón et al. 2005). Consequently, the overall excitatory or inhibitory effect of NO production on the vestibular activity would depend on the relative functional weight of each of these processes.

Postsynaptically, NO production would depend on NO released from the nerve endings after Ca²⁺ enters through N-methyl-D-aspartate receptors and voltage-gated Ca²⁺ channels, and activates NOS I/III (Moncada et al. 1991). Accordingly, NOS I and III were reported in the afferent neurons (Hess et al. 1998a,b). In addition to this, the source of NO might also be presynaptic. Although Hess et al. (1998a,b) found NOS I and III in cochlear but not vestibular hair cells of guinea pig and mice, these enzymes were reported in frog saccular hair cells (Heinrich et al. 2003) and NO production was described in guinea pig vestibular hair cells (Takumida and Anniko 2000). Importantly, the intracellular Ca²⁺ concentration needed to activate NOS might inhibit sGC (Parkinson et al. 1999), meaning that NO acts on neighboring hair cells but not on the hair cell that generated it (Lysakowski and Singer 2000).

The inhibitory effects of NO on I_K,L and on Ca²⁺ channels, although apparently contradictory, may reflect the existence of multiple NO-sensitive mechanisms that would control the hair cell response. The inhibition of the Ca²⁺ current using NO donors and 8-Br-cGMP occurs at the micromolar range, whereas that of the I_K,L current occurs at the millimolar range (Behrend et al. 1997; Chen and Eatock 2000), indicating that the Ca²⁺ current is much more sensitive to NO than I_K,L. We hypothesize that in the vestibular endorgans the action of NO can be dual. At low levels it may have a neuroprotective role constituting an effective mechanism controlling neurotransmitter release from hair cells and at high levels NO may contribute to excitotoxic processes by boosting type I hair cell response (because of I_K,L inhibition). In the vestibular system, nonphysiological stimuli such as the exposure to lipopolysaccharides (Takumida and Anniko 1998), cisplatin (Watanabe et al. 2000), and gentamicin (Takumida and Anniko 2001) induce the expression of NOS II. High levels of NO can be produced by NOS II leading to excitotoxic processes. In contrast, constitutive NOS expressions are involved in the production of low levels of NO that are associated with physiological functions (Bredt 1999; Colasanti and Suzuki 2000).

Finally, postsynaptic effects of NO should also be considered. NO can inhibit or enhance the Na⁺ current (Kawai and Miyachi 2001; Li et al. 1998) and also enhance the activity of the large-conductance Ca²⁺-activated (BK) channels (Klyachko et al. 2001). Studies in mammals showed that the calyx terminals and vestibular-afferent neurons express a TTX-sensitive Na⁺ current (Chabbert et al. 1997; Rennie and Streeter 2006) and that the BK current is also expressed in vestibular-afferent neurons (Limón et al. 2005). Therefore the potential effects of NO on the activity of ionic channels in afferent neurons should also be investigated to attain a complete picture of the participation of NO in the sensory coding of vestibular information. The final result of NO production in the vestibule would be complex depending on the NO levels that are reached and the functional role of pre- and postsynaptic ionic channels that are modulated by NO.

	GRANTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
This material is based on work supported by Consejo Nacional de Ciencia y Tecnología de México (CONACyT) Grant 46511 and Vicerrectoría de Investigacíon–Benemérita Universidad Autónoma de Puebla Grants 19/SAL/06-I and 20/SAL/06-G. A. Almanza received CONACyT fellowship 137444.

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
The authors thank Dr. Ellis Glazier for editing of the English-language manuscript and Dr. Francisco Perez Vizcaino and anonymous reviewers for very constructive suggestions.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Address for reprint requests and other correspondence: E. Soto, Instituto de Fisiología–BUAP, Apartado Postal 406, Puebla, Puebla 72000, Mexico (E-mail: esoto{at}siu.buap.mx)

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