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.

Responses of pigeon vestibular hair cells to cholinergic agonists and antagonists

“…NOS subtypes have been immune-labeled in vestibular and cochlear epithelia (Lysakowski and Singer, 2000) and specifically eNOS has been labeled in the type I hair cell calyx (Hess et al, 1998a; Hess et al, 1998b). Lv et al, (2010) using NO-selective electrodes measured a 3 fold release of NO in frog saccule neuroepithelium following application of ACh. In earlier studies, Chen and Eatock (2000) found that application of 4 NO donors inhibited the current I K,L in type I hair cells and patches of their membranes from 33 to 76%.…”

Nitric oxide influences potassium currents in inner hair cells isolated from guinea-pig cochlea

“…In type I hair cells from rat semicircular canals, the addition of NO donors to the external solution reduced the instantaneous currents evoked by voltage steps from À67 mV, which were principally carried by I K,L channels [21]. In hair cells from frog saccules, Ca 2+ -activated K + currents were reversibly enhanced after application of NO by increasing the open probability of BK channels [22]. In posterior pituitary nerve terminals, NO enhanced Ca 2+ -activated K + channel activity by activating guanylate cyclase (GC) and cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) [23].…”

“…In the hair cells of frog saccules, NO reduces whole-cell Ca 2+ currents and drastically decreases the open probability of single-channel events of the L-type and non-L-type Ca 2+ channels [22]. The Ca 2+ channel modulation by NO also implies the participation of the NO-cGMP-PKG system [29].…”

“…The actions of NO may involve direct nitrosylation of the channel since dithiothreitol, a thiol reducing reagent, reversed the effects of NO on whole-cell and single-channel currents [22]. Chen et al [21], however, demonstrated that NO's effect is mediated by intracellular second messengers because perfusion of the cell with 1 mM SNP greatly reduced all channel activity when the patch was cell-attached, but excision of the patch restored channel activity, despite continued perfusion with SNP.…”

“…ACh hyperpolarizes the hair cell membrane by activating a Ca 2+ -activated K + (SK) conductance by releasing Ca 2+ intracellularly via the pathway of muscarine receptor, G-protein and inositol trisphosphate (IP 3 ) [37]. However, it has been reported that the application of ACh caused rapid and sustained release of NO by the sensory epithelia in bullfrog saccules [22]. This Ach-induced NO increase might suppress Ca 2+ -activated K + currents in IHCs and show inhibitory effects against Ca 2+ -induced neurotransmitter release, suggesting another mechanism for Ach-induced inhibition.…”