Release and elementary mechanisms of nitric oxide in hair cells

Abstract

The enzyme nitric oxide (NO) synthase, that produces the signaling molecule NO, has been identified in several cell types in the inner ear. However, it is unclear whether a measurable quantity of NO is released in the inner ear to confer specific functions. Indeed, the functional significance of NO and the elementary cellular mechanism thereof are most uncertain. Here, we demonstrate that the sensory epithelia of the frog saccule release NO and explore its release mechanisms by using self-referencing NO-selective electrodes. Additionally, we investigated the functional effects of NO on electrical properties of hair cells and determined their underlying cellular mechanism. We show detectable amounts of NO are released by hair cells (>50 nM). Furthermore, a hair-cell efferent modulator acetylcholine produces at least a threefold increase in NO release. NO not only attenuated the baseline membrane oscillations but it also increased the magnitude of current required to generate the characteristic membrane potential oscillations. This resulted in a rightward shift in the frequency-current relationship and altered the excitability of hair cells. Our data suggest that these effects ensue because NO reduces whole cell Ca(2+) current and drastically decreases the open probability of single-channel events of the L-type and non L-type Ca(2+) channels in hair cells, an effect that is mediated through direct nitrosylation of the channel and activation of protein kinase G. Finally, NO increases the magnitude of Ca(2+)-activated K(+) currents via direct NO nitrosylation. We conclude that NO-mediated inhibition serves as a component of efferent nerve modulation of hair cells.

Fig. 1.

Measurement of nitric oxide (NO) efflux. A: schematic diagram of the NO-sensitive microsensor. The microsensor was constructed by inserting 5-μm carbon fiber and then heating and pulling the glass electrode. Epoxy was backfilled into the electrode to seal the fiber with the glass electrode. A graphite paste was used to cement a copper wire to the carbon fiber. The tip of the electrode was beveled, coated with Nafion, and plated with o-phenylenediamine. B: the electrodes were calibrated with 21 standards with known concentrations of NO and the selectivity was tested against ascorbic acid. The gradient of the linear regression of the calibration line was used to calculate the flux of NO. C: NO flux in relation to the distance from a source pipette (diameter ∼3 μm). Symbols represent measured values and the solid squares (▪) represent the product of a theoretical model developed from the Fick equation. D: data collected from a frog saccule. The excursion distance of the microsensor was 5 μm at a frequency of 0.3 Hz. When in the “epithelium” position the electrode was about 2 μm from the tissue. Background represents data collected at a position 2,000 μm away from the saccule. The mean NO flux was 115 ± 20 nmol·m⁻²·s⁻¹ (n = 15), compared with background reading 1 ± 8 nmol·m⁻²·s⁻¹ (n = 15). E: representative recording from a saccule (dotted line) and on application of 5 μM acetylcholine (ACh). Superimposed on the same plot (solid line) is a depiction of the inhibitory effects of N^G-nitro-l-arginine methyl ester (l-NAME). F: summary data on the effects of N^G-nitro-d-arginine methyl ester (d-NAME; n = 7), L-NAME (n = 8), ACh (n = 9), Ca²⁺-free solution (n = 5), and Ca²⁺-free + ACh (n = 7) on NO efflux.

Fig. 2.

NO donor S-nitroso-N-acetylpenicillamine (SNAP) decreases the membrane gain of hair cells. A: a family of oscillations of hair-cell membrane potentials in the absence and in response to injection of current steps (indicated) for controls (left) and after application of 10 μM SNAP. The NO donor reduces baseline membrane oscillations. B: the resulting effects of the NO donor is a shift in the frequency–current (F–I) relationship to the right. C: the frequency of membrane potential oscillations as a function of injected current for a hair cell. The oscillations at current “on” in solid symbols and oscillations at current “off” are in open symbols. The solid curves were calculated using the equation (Crawford and Fettiplace 1981): F_R(I) = F̂ log_e [1 + μ(I − I_o)], where F_R is the frequency of oscillation at a given current step of amplitude I, I_o is the minimum outward current required to suppress the oscillations, and F̂ and μ are constants for the cell with units of Hz and reciprocal current, respectively. For the example shown, the NO donor appears to shift the characteristic frequency of the cell from about 55 to 40 Hz.

Fig. 3.

Effects of NO and activation of protein kinase G (PKG) on inward Ca²⁺ current in hair cells measured using whole cell patch clamp. A: current traces obtained by 250-ms depolarization pulses from a holding potential of −70 mV to a step potential of −30 mV in the absence (black trace) and the presence of 500 nM NO (as measured with an NO-selective electrode). B: similar recordings were made in the absence, control, and presence of 10 μM dibutyryl-3′,5′-cyclic guanosine monophosphate (cGMP), 10 μM cGMP plus 600 nM NO, and 10 μM cGMP plus 600 nM NO and 1 mM dithiothreitol (DTT). Whereas the effects of NO were reversed by DTT, the effect of cGMP was unaltered by DTT (control = −556 ± 31 pA, cGMP = 389 ± 19 pA; n = 7, P < 0.05; control = −578 ± 42 pA, cGMP + NO = −274 ± 16 pA; n = 8, P < 0.01). C: representative current–voltage (I–V) relations generated from data from 14 cells showing the effects of NO (1 μM) and DTT (1 mM). The peak control Ca²⁺ current, −555 ± 27 pA, was reduced to −347 ± 17 pA (n = 14, P < 0.01) by NO after 3 min exposure. The effects of NO was reversed by application of 1 mM DTT (−519 ± 18 pA; n = 14 P = 0.08). D: steady-state activation curves were generated using the ratio of the tail currents (I/I_peak) against the step potentials. The continuous curves were generated from the Boltzmann function {I/I_peak = [1 + exp(V_1/2 − V)/K_m]}⁻¹. V_1/2 is the half-activation voltage and K_m is the slope factor. The estimated values of V_1/2 and K_m for control currents (●) and currents after application of NO (∘) were (in mV): −64.6 ± 0.5 and 4.3 ± 0.5; and −61.2 + 0.6 and 4.1 ± 1.0 (n = 8 cells), respectively. E: NO inhibition of Ca²⁺ current was dose dependent. The half-blocking concentration of NO was estimated to be 81 ± 7 nM (n = 4). F: group data showing the effects of NO after 3 min (n = 8) and 9 min (n = 6) application, DTT (n = 6) on NO-mediated effects, actions of NO in the presence of KT-5823 (n = 7), a PKG inhibitor, and the effects of DTT on the actions of NO in the presence of the PKG inhibitor (n = 5; *P < 0.05; **P < 0.01).

Fig. 4.

Whole cell recordings of Ca²⁺-activated K⁺ current in hair cells. The amplitude of outward Ca²⁺-activated K⁺ currents was increased by NO. A: outward K⁺ currents traces were elicited at a holding potential (−90 mV), step depolarization of −80 to 40 mV, with change in voltage of 5 mV. The current was recorded in the presence of 5 mM 4-aminopyridine (4-AP) and tetraethylammonium (TEA) to suppress other outward K⁺ currents (see methods). The current was enhanced in response to increased external Ca²⁺ (data not shown). The Ca²⁺-activated K⁺ currents were visibly enhanced on application of NO (600 nM) and the effect was reversed by 1 mM DTT. B: the summary data (n = 9) of the I–V relationship for data obtained from control experiments (∘), after application of NO (●), and reversal effects of 0.2 mM DTT (☐). C: to confirm the enhancement effect of NO on the Ca²⁺-activated K⁺ current, hair cells were held at −70 mV and stepped briefly (25 ms) to −30 mV to activate the inward Ca²⁺ current and then stepped to 5 mV to activated the outward K⁺ current (n = 5). The increase in the outward current was independent of the reduction of inward current. DTT completely reversed the effect of NO on the K⁺ current but not the Ca²⁺ current.

Fig. 5.

Single-channel Ca²⁺ current fluctuations in hair cells plummeted in NO but the effect was reversed by DTT. Representative single-channel traces recorded using 65 mM Ca²⁺ as the charge carrier of (A) control, (B) 2 min after application of 500 nM NO, and (C) 2 min after application of 0.2 mM DTT. The recordings were made in bath and pipette solutions containing 10 μM nimodipine. Ten consecutive traces are shown at the step potential indicated (−50 mV) from a holding potential of −70 mV. D: examples of amplitude histograms (step voltage, −50 mV) used to generate the I–V relationships for control (left) and after DTT (right). E: the I–V relationship of control (∘) and after DTT (●). The single-channel conductances were: control, 16.2 ± 0.8 pS (n = 6) and after DTT, 15.8 ± 1.3 pS (n = 5) (P = 0.6). The insensitivity of the channel to nimodipine and the conductance suggests that it belongs to the non L-type Ca²⁺ channel subtype (Rodriguez-Contreras and Yamoah 2001).

Fig. 6.

NO reduces the open probability of L-type Ca²⁺ channel in hair cells. A–C: recordings showing effects of NO modification on single-channel Ca²⁺ current from a cell-attached patch during baseline (A) and after 3 min of superfusion with external solution containing 500 nM NO (B), and finally after application of external solution with 1 mM DTT (C). Recordings were obtained using 400-ms steps to −10 mV from a holding potential of −70 mV. Bay K 8644 (5 μM) was included in the patch pipette. D: amplitude histograms of the unitary current at −10 mV in control (left), the presence of NO (middle), and after application of DTT (right). E: graph showing single-channel I–V relations obtained from control patches (●, n = 5), NO (∘, n = 5), and DTT-modified (☐, n = 5) patches. The calculated conductances were 13.5 ± 0.7, 13.2 ± 0.8, and 13.6 ± 0.3 pS for control, NO, and DTT-modified patches, respectively. F: diary plot of open probability vs. time during control, after superfusion with NO-containing solution, and after application of DTT.

Fig. 7.

Differential effects of NO and DTT on L- and non L-type Ca²⁺ channels in hair cells. A: family of consecutive single channel fluctuations recorded in a cell-attached patch (charge carrier was 65 mM Ca²⁺). The patch contained 2 distinct single channels, one with openings suggestive of L-type channel (arrow) and the other, the non L-type channel (underlined). The bath and patch electrode contained Bay K 8644 (5 μM). Ten consecutive traces shown were generated from a holding potential of −70 mV to a step potential of −30 mV. B: after superfusion of bath solution containing 500 nM NO, openings of the channel with the non L-type channel were dramatically reduced. However, brief openings of the L-type channel persisted. C: on application of DTT (0.2 mM), a substantial number of openings of the non L-type channel emerged. D: amplitude histograms of the corresponding unitary current for control (left), in the presence of NO (middle), and DTT (right).

Fig. 8.

Effects of NO modification on single-channel Ca²⁺ current kinetics. Currents were obtained from control, NO-modified (100 nM), and DTT-modified (0.5 mM) patches during 400-ms steps to −30 mV (A) and −10 mV (B) from a holding potential of −70 mV. A: open-time histogram plotted as the complement of the distribution. The solid line represents a biexponential fit to the data, with time constants indicated on the plots. The bath solution and pipette contained 10 μM nimodipine and the kinetics and conductance of the channel suggested that it belonged to the non L-subclass. At least one open-time constant was abbreviated in the presence of NO. B: here, Bay K 8644 (5 μM) was included in the bath and the pipette. Plots of open-time distribution in the control (left), NO-modified (middle), and DTT-modified (right) L-type channels are shown. The solid lines represent bi- and triexponential fits to the data, with time constants indicated on the plots. Compared with control, time constants obtained from NO-modified patches were significantly abbreviated by exposure to 100 nM NO [e.g., 2.2 ± 0.01 ms (control) vs. 0.50 ± 0.02 ms (n = 4; P < 0.05) in NO-modified patches, respectively]. Note that DTT (0.2 mM) restored the fast open-time constant (1.1 ± 1.5; n = 3; P = 0.7). Also important, the fast open-time constant was eliminated in NO-modified L-type single channel current kinetics.

Fig. 9.

NO modulates the large conductance single-channel K⁺ current in hair cells. A and B: recordings showing effects of NO modification on single-channel BK channel current from a cell-attached patch during baseline (A) and after 2 min of superfusion with 100 nM NO (B). Recordings were obtained using approximately 500-ms steps to 20 mV from a holding potential of −60 mV. C: graph showing amplitude histogram obtained from unitary currents at 20-mV step potential. D: single-channel I–V relations obtained from control patches (∘, n = 6) and NO-modified patches (●, n = 6) (100 nM NO solution was superfused in the bath). The calculated conductances were 275 ± 11 pS (n = 6) and 280 ± 17 pS (n = 6, P = 0.6) for control and NO-modified patches, respectively. E: diary plot of the open probability of a cell-attached Ca²⁺-activated K⁺ current (BK) channel patch as a complement of time, showing the drastic increase in P_o in the presence of NO vs. control and the reversal effects of DTT (0.2 mM).