ATP-induced movement of the stalks of isolated cochlear Deiters' cells.

Deiters' cells, a type of supporting cell in the sensory epithelium of the cochlea, the organ of Corti, have been found to have P2X and P2Y adenosine triphosphate (ATP) receptors on their surfaces. Activation of these receptors may alter the mechanical properties of Deiters' cells and thus of the organ itself. ATP was applied to Deiters' cells isolated from guinea pig cochlea and the tip of the cells' stalk monitored for an ATP induced movement. Application of 100 microM ATP to the cell body of isolated Deiters' cells induced a small, reversible movement of the cells' stalk whereas application of bathing media did not. Results suggest that in vivo endogenous extracellular ATP released from unidentified locations could alter cochlear mechanics.

PPADS, an ATP antagonist, attenuates the effects of a moderately intense sound on cochlear mechanics.

Increasing attention is being given to the role of neurotransmitters and other signaling substances in the damage induced by intense sound to the cochlea. Adenosine triphosphate (ATP) is one example of a putative neurotransmitter that may alter cochlear mechanics during sound exposure. The purpose of the present study was to test the hypothesis that endogenous extracellular ATP has a role in the generation of the changes in cochlear mechanics induced by moderate intense sound exposure. Guinea pigs were exposed to either: (1) a perilymphatic administration of pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 1 mM), an ATP antagonist; (2) a moderately intense sound (6 kHz tone, 95 dB SPL, 15 min); or (3) a combination of the PPADS and the sound. The effects on the cubic distortion product otoacoustic emissions (DPOAEs; 2f1-f2) were monitored using three sets of equal level primaries (f1=9.25 kHz, f2=10.8 kHz, 2f1-f2=7.7 kHz; f1=7.2 kHz, f2=8.4 kHz, 2f1-f2=6 kHz; f1=5.55 kHz, f2=6.5 kHz, 2f1-f2=4.6 kHz). PPADS alone had no effect on the cubic DPOAEs monitored. The intense sound alone suppressed all three cubic DPOAEs. The combination of PPADS with the intense sound induced a suppression of the cubic DPOAEs that was equal to or greater than induced by the intense sound alone at f2=10.8 kHz but was equal to or less than induced by the intense sound at f2=8.4 and 6.5 kHz. After washing the PPADS out of the cochlea with artificial perilymph, all three cubic DPOAEs were suppressed less in the PPADS with intense sound treatment group than in the intense sound alone group. The PPADS appeared to provide protection from the intense sound. Results are consistent with the hypothesis that extracellular ATP is involved in the changes in cochlear mechanics induced by moderately intense sound exposure.

The selective AMPA receptor antagonist GYKI 53784 blocks action potential generation and excitotoxicity in the guinea pig cochlea.

The role of AMPA receptors in cochlear synaptic transmission and excitotoxicity was investigated by comparing the actions of a selective AMPA antagonist GYKI 53784 (LY303070) with additional AMPA/kainate antagonists, GYKI 52466 and DNQX, and the NMDA antagonist, D-AP5, in several electrophysiological, neurotoxicological and histochemical tests. GYKI 53784 had the same potency as DNQX and was 10 times more potent than GYKI 52466 in reducing auditory nerve activity. The NMDA antagonist D-AP5 had no effect on auditory nerve activity. When single-fiber activity was blocked with GYKI 53784, the effects of AMPA or kainate were also antagonized. GYKI 53784 completely blocked excitotoxicity (i.e. destruction of the afferent nerve endings) induced by AMPA and kainate. The histochemical detection of Co(2+) uptake was used to study Ca(2+) influx within the primary auditory nerve cells. Application of AMPA induced no significant Co(2+) uptake into the cells, suggesting that these receptors normally have a very low permeability to Ca(2+). Application of kainate induced significant Co(2+) uptake that was blocked by the AMPA receptor antagonist GYKI 53784 suggesting that kainate stimulated Ca(2+) entry through AMPA receptor channels. Results suggest that AMPA-preferring receptors are functionally located at the sensory cell-afferent synapse whereas NMDA and kainate receptors are not.

Functional expression of three P2X(2) receptor splice variants from guinea pig cochlea.

ATP has been suggested to act as a neurotransmitter or a neuromodulator in the cochlea. The responses to ATP in different cell types of the cochlea vary in terms of the rate of desensitization and magnitude, suggesting that there may be different subtypes of P2X receptors distributed in the cochlea. Recently three ionotropic P2X(2) receptor splice variants, P2X(2-1), P2X(2-2), and P2X(2-3,) were isolated and sequenced from a guinea pig cochlear cDNA library. To test the hypothesis that these different splice variants could be expressed as functional homomeric receptors, the three P2X(2) receptor variants were individually and transiently expressed in human embryonic kidney cells (HEK293). The biophysical and pharmacological properties of these receptors were characterized using the whole cell patch-clamp technique. Extracellular application of ATP induced an inward current in HEK293 cells containing each of the three splice variants in a dose-dependent manner indicating the expression of homomeric receptors. Current-voltage (I-V) relationships for the ATP-gated current show that the three subtypes of the P2X(2) receptor had a similar reversal potential and an inward rectification index (I(50 mV)/I(-50 mV)). However, the ATP-induced currents in cells expressing P2X(2-1) and P2X(2-2) variants were large and desensitized rapidly whereas the current in those cells expressing the P2X(2-3) variant was much smaller and desensitized slower. The order of potency to ATP agonists was 2-MeSATP > ATP > alpha,beta -MeATP for all three expressed splice variants. The ATP receptor antagonists suramin and PPADS reduced the effects of ATP on all three variants. Results demonstrate that three P2X(2) splice variants from guinea pig cochlea, P2X(2-1), P2X(2-2), and P2X(2-3), can individually form nonselective cation receptor channels when these subunits are expressed in HEK293 cells. The distinct properties of these P2X(2) receptor splice variants may contribute to the differences in the response to ATP observed in native cochlear cells.

An interaction between PPADS, an ATP antagonist, and a moderately intense sound in the cochlea.

In the organ of Corti ionotropic receptors for ATP (ATPRs) on cells that are bathed by perilymph have been suggested to modulate cochlear mechanics. The purpose of the present study was to test the hypothesis that endogenous extracellular ATP acting through ATPRs is involved in modulating cochlear mechanics during moderately intense sound exposure. Guinea pigs were exposed to either: (1) a perilymphatic administration of pyridoxal-phosphate-6-azophenyl-2', 4'-disulfonic acid (PPADS, 1 mM), an ATP antagonist; (2) a moderately intense sound (6.7 kHz tone, 95 dB SPL, 15 min); or (3) a combination of both the PPADS and the sound. The effects on cochlear potentials (cochlear microphonic, CM; negative summating potential, SP; compound action potential of the auditory nerve, CAP; and N(1) latency) evoked by a 10 kHz tone pip were monitored. PPADS alone reduced the CAP and the SP and increased N(1) latency. The intense sound alone reduced the CAP and SP. The combination of PPADS with the intense tone induced reversible effects on cochlear potentials that were greater than induced by either treatment alone. The effect on N(1) latency and low intensity CM was a potentiation since the effect was greater than a simple addition of the effect of either treatment alone. The effects of the combination treatment on CAP, SP and high intensity CM were not different from additive. Results are consistent with the hypothesis that ATPRs in the organ of Corti are involved in modulating cochlear mechanics during moderately intense sound exposure.

Phosphorothioate oligodeoxynucleotides can selectively alter neuronal activity in the cochlea.

A growing body of evidence indicates that extracellular adenosine triphosphate (ATP) may have a major role in cochlear function. Antagonists of ionotropic ATP receptors (P2X2) have significant effects on cochlear potentials and distortion product otoacoustic emissions (DPOAEs). We tested whether antisense oligodeoxynucleotides (ODNs) would mimic the functional deficiencies induced by the ATP antagonists through binding to P2X2 ATP receptor mRNA and thereby reduce the number of ATP receptors expressed in the membrane of the cells. Both a phosphorothioate ODN (S-ODN) antisense and a phosphodiester ODN (P-ODN) antisense to the P2X2 sequence and random sense ODNs containing 21 nucleotides were administered chronically (7 days) to the guinea pig cochlea via the perilymph compartment. Sound evoked cochlear potentials (cochlear microphonic; summating potential; compound action potential of the auditory nerve, CAP; latency of the first negative peak in the CAP, N1 latency) and DPOAEs were monitored to assess the effects of the ODNs. Results indicate that the phosphorothioate derivatives of both the antisense and random sense ODNs suppressed the CAP and prolonged the N1 latency with no significant effect on the other parameters. The P-ODNs had no effect. Since both the antisense and random sense S-ODNs had the same effect, we conclude that the S-ODNs affected neuronal function in a manner that did not involve binding to the ATP receptor mRNA.

AMPA-preferring glutamate receptors in cochlear physiology of adult guinea-pig.

1. The present study was designed to determine which glutamate (Glu) receptors are involved in excitatory neurotransmission at the first auditory synapse between the inner hair cells and the spiral ganglion neurons. 2. The Glu receptors present at the membrane level were investigated on isolated spiral ganglion neuron somata from guinea-pigs by whole-cell voltage-clamp measurements. Glu and AMPA induced a fast onset inward current that was rapidly desensitized, while kainate induced only a non-desensitizing, steady-state current. NMDA induced no detectable current. 3. To further discriminate between the AMPA and kainate receptors present, we used the receptor-specific desensitization blockers, cyclothiazide and concanavalin A. While no effect was observed with concanavalin A, cyclothiazide greatly enhanced the Glu-, AMPA- and kainate-induced steady-state currents and potentiated Glu-induced membrane depolarization. 4. To extrapolate the results obtained from the somata to the events occurring in situ at the dendrites, the effects of these drugs were evaluated in vivo. Cyclothiazide reversibly increased spontaneous activity of single auditory nerve fibres, while concanavalin A had no effect, suggesting that the functional Glu receptors on the somata may be the same as those at the dendrites. 5. The combination of a moderate-level sound together with cyclothiazide increased and subsequently abolished the spontaneous and the sound-evoked activity of the auditory nerve fibres. Histological examination revealed destruction of the dendrites, suggesting that cyclothiazide potentiates sound-induced Glu excitotoxicity via AMPA receptors. 6. Our results reveal that fast synaptic transmission in the cochlea is mainly mediated by desensitizing AMPA receptors.

Outward rectifying potassium currents are the dominant voltage activated currents present in Deiters' cells.

Supporting cells in the cochlea are thought to maintain the homeostasis of the organ of Corti and contribute to the electrical and micromechanical environment of the hair cells. Of the different types of supporting cells, Deiters' cells form a structure that holds the outer hair cells (OHCs) at their base and apex. This structure may play an important role in modifying cochlear mechanics by influencing the force produced by sound induced motion of the OHCs which in turn may be modulated by ATP acting on ligand gated cation channels on the Deiters' cells. Also, a glia-like role of buffering external K+ concentration for the Deiters' cells has been suggested. We studied Deiters' cells' electrical properties and ion conductances using the whole cell variant of the patch clamp technique since they must play an important role in the function of these cells. It was found that isolated Deiters' cells possess a large voltage activated, outwardly rectifying K+ selective conductance. Voltage activated Ca2+ currents and non-selective currents were not detected and voltage activated inward currents were very small. The outward K+ currents were found to be dependent on voltage but not on Ca2+ for their activation. Nimodipine and 4-aminopyridine (4-AP) were shown to interact directly with the K+ channels in a voltage dependent manner. It is suggested that the K+ selective channels in Deiters' cells may be similar to the Kv1.5 type channel. However, based on the voltage dependence of the channels that was described by double Boltzmann equation and on the alteration of that dependence by 4-AP, it is possible that more than one type of K+ selective channel exists.

Novel variant of the P2X2 ATP receptor from the guinea pig organ of Corti.

ATP functions as a neurotransmitter and a neuromodulator in various tissues by acting on metabotropic (P2Y) and ionotropic (P2X) receptors. Evidence suggests that ATP activates P2X receptors on several cell types in the organ of Corti of guinea pig including outer hair cells (OHCs), Deiters' cells, Hensen's cells, pillar cells and inner hair cells (IHCs). Determining the sequence and structure of P2X receptors in guinea pig organ of Corti is important for understanding the function of ATP in the cochlea. We screened a guinea pig organ of Corti cDNA library for P2X2 ATP receptors using rat P2X2 cDNA as a probe. We sequenced three P2X2 variants which were found to be abundant in this library. One is a novel P2X2 isoform (P2X2-3) created by a retained intron coding for an additional 27 amino acids (81 bp) in the putative extracellular domain. We have also sequenced a variant (P2X2-2) that lacks both the 81-bp sequence and a 192-bp sequence in the 3' intracellular domain. A third variant (P2X2-1) contains the intracellular 192-bp sequence but not the extracellular 81-bp sequence found in P2X2-3. The multiple transcripts arise from alternative intron and exon splicing events. In situ hybridization with a probe common to the three variants localized P2X2 to many of the cells of the organ of Corti.

P2X receptors in cochlear Deiters' cells.

1. The ionotropic purinoceptors in isolated Deiters' cells of guinea-pig cochlea were characterized by use of the whole-cell variant of the patch-clamp technique. 2. Extracellular application of adenosine 5'-triphosphate (ATP) induced a dose-dependent inward current when the cells were voltage-clamped at -80 mV. The ATP-induced current showed desensitization and had a reversal potential around -4 mV. 3. Increasing intracellular free Ca2+ by decreasing the concentration of EGTA in the pipette solution reduced the amplitude of the ATP-gated current. 4. The order of agonist potency was: 2-methylthioATP (2-meSATP)>ATP>benzoylbenzoyl-ATP (BzATP)>alpha,beta-methyleneATP (alpha,beta,meATP>adenosine 5'-diphosphate (ADP)>uridine 5'-triphosphate (UTP)>adenosine 5'-monophosphate (AMP)=adenosine (Ad). 5. Pretreatment with forskolin (10 microM), 8-bromoadenosine-3',5'-cyclophosphate (8-Br-cyclic AMP, 1 mM), 3-isobutyl-1-methylxanthine (IBMX, 1 mM) or phorbol-12-myristate-13-acetate (PMA, 1 microM) reversibly reduced the ATP-induced peak current. 6. The results are consistent with molecular biological data which indicate that P2X2 purinoceptors are present in Deiters' cells. In addition, the reduction of the ATP-gated current by activators of protein kinase A and protein kinase C indicates that these P2X2 purinoceptors can be functionally modulated by receptor phosphorylation.

Additional pharmacological evidence that endogenous ATP modulates cochlear mechanics.

In the cochlea, outer hair cells (OHCs) generate the active cochlear mechanics whereas the supporting cells, such as Deiters' cells and Hensen's cells, may play a role in both the active and passive cochlear mechanics. The presence of receptors for adenosine triphosphate (ATP) on OHCs, Deiters' cells and Hensen's cells indicates that endogenous ATP may have a role in cochlear mechanics. To explore this possibility, the effects of the ATP antagonist, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), were studied in guinea pig both in vitro on isolated OHCs, Deiters' cells, Hensen's cells and pillar cells using the whole-cell configuration of the patch-clamp technique, and in vivo on sound evoked cochlear potentials (cochlear microphonic, CM; summating potential, SP; compound action potential, CAP) and distortion product otoacoustic emissions (DPOAEs) using cochlear perilymphatic perfusion. Results show that PPADS (100 microM) reduced the inward current evoked by 5-10 microM ATP in OHCs, Deiters' cells, Hensen's cells and pillar cells. This effect of PPADS was slow in onset and was slowly reversed to a varying degree in the different cell types. In vivo application of PPADS in increasing concentrations reduced the sound evoked CAP, SP and increased N1 latency starting at about 0.33 mM (SP) and 1 mM (CAP and N1 latency). PPADS (0.33-1 mM) reversibly suppressed the initial value of the quadratic DPOAE and reversed the 'slow decline' in the quadratic DPOAE that occurs during continuous stimulation with moderate level primaries. These results, together with the similar effects of the ATP antagonist suramin reported previously (Skellett et al., 1997), may be evidence that endogenous ATP acting on cells in the organ of Corti alters cochlear mechanics.

Conditioning the auditory system with continuous vs. interrupted noise of equal acoustic energy: is either exposure more protective?

The purpose of this study was to test the hypothesis that differences exist in the amount of protection provided by prior sound conditioning with continuous vs. interrupted, moderate-level noise. Differences were determined by monitoring the changes that occurred in cubic (2f1-f2) distortion product otoacoustic emission (DPOAE) amplitude growth functions subsequent to a traumatizing noise exposure (105 dB SPL, 1.0-2.0 kHz octave band noise presented 24 h per day for 3 days) in guinea pigs which had been conditioned with either continuous (89 dB SPL, 1.0-2.0 kHz octave band noise presented 24 h per day for 11 days) or interrupted noise (95 dB SPL, 1.0-2.0 kHz octave band noise presented on a 6-h 'on'/18-h 'off' schedule for 11 days) of equal acoustic energy. Results suggest that there are significant differences in the degree of protection provided by prior sound conditioning with the continuous and interrupted schedules of moderate-level noise used in this study. Specifically, the interrupted conditioning protocol afforded some degree of protection against the damaging effects of the traumatizing noise exposure, limited to frequencies above the noise exposure band. Conversely, there was a lack of any consistent and sizable protective effect found across the entire test frequency range for the continuous sound conditioning protocol.

Cytotoxicity and mitogenicity of adenosine triphosphate in the cochlea.

Evidence is accumulating to indicate that extracellular adenosine 5'-triphosphate (ATP) may function as a neurotransmitter, neuromodulator, cytotoxin and mitogen. Many of the cells in the cochlea have ATP receptors, however, their function is unknown. The purpose of the present study was to test whether ATP may act as a cytotoxin in the cochlea. ATP was applied to acutely isolated outer hair cells (OHCs) and their shape changes monitored. In addition, ATP was applied into the cochlea by perfusion of the perilymph compartment for 2 h and the animals allowed to survive 3-4 weeks post drug application. At this time, sound-evoked cochlear potentials and distortion product otoacoustic emissions (DPOAEs) were monitored and the cochleas evaluated histologically. Results indicate that when applied to isolated OHCs, ATP (3-30 mM) induced a bleb formation in the infracuticular region of the cell that burst within a few minutes. Short OHCs were more sensitive to this effect of ATP than long OHCs. 3-4 weeks after the perilymph perfusion of ATP (60 mM; 2 h) cochlear potentials and DPOAEs were abolished, and histologically, cells in the organ of Corti and the stria vascularis were found to have been destroyed. In addition, there was loss of spiral ganglion cells and proliferating connective tissue filled varying proportions of the scala tympani and vestibuli. Application of sodium gluconate, a control, at the same concentrations had no effect either on the isolated OHCs or when applied in vivo. Results suggest that extracellular ATP or a metabolic product may act as a cytotoxin to some epithelial and neural elements in the cochlea and possibly as a mitogen to mesenchymal cells or fibrocytes.

Pharmacological evidence that endogenous ATP modulates cochlear mechanics.

In the cochlea, outer hair cells (OHCs) and Deiters' cells most likely contribute to the generation of active cochlear mechanics. The presence of ATP receptors on these cells indicates that endogenous ATP may have a role in cochlear mechanics. To explore this possibility, the effects of ATP antagonists were studied both in vivo on distortion product otoacoustic emissions (DPOAEs) using cochlear perfusion and in vitro on isolated OHCs and Deiters' cells using the whole-cell configuration of the patch-clamp technique. Results show that extracellular application of 5-10 microM ATP to OHCs and Deiters' cells induced an inward current that was reduced by both suramin (100 microM) and cibacron (100 microM). Cibacron reduced the voltage gated currents in Deiters' cells and increased them in OHCs, while suramin had no effect. In addition, cibacron induced a hyperpolarizing shift of the half activation voltage of the whole cell currents in Deiters' cells. Suramin (0.1-1 mM) reversibly suppressed the 'slow decline' in the quadratic DPOAE that occurs during continuous stimulation with moderate level primaries. This effect of suramin may be evidence that endogenous ATP alters active cochlear mechanics.

Nitrosoglutathione suppresses cochlear potentials and DPOAEs but not outer hair cell currents or voltage-dependent capacitance.

Biochemical and pharmacological evidence support a role for nitric oxide (NO) and glutathione (GSH) in the cochlea. GSH combines with NO in tissue to form nitrosoglutathione (GSNO) that can act as a storage form for GSH and NO. Therefore, we tested GSNO on sound-evoked responses of the cochlea (cochlear microphonic, CM; summating potential, SP; compound action potential, CAP; cubic distortion product otoacoustic emission, DPOAE), on the endocochlear potential (EP), on isolated outer hair cell (OHC) currents and voltage-dependent capacitance, and on Deiters' cell currents. In vivo application of GSNO in increasing concentrations reversibly reduced low-intensity sound-evoked CAP, SP and DPOAEs starting at about 1 mM (CAP) and 3.3 mM (SP, DPOAE). However, even at 10 mM, GSNO had little effect on the EP. In vitro, salicylate (10 mM) but not GSNO (3 and 10 mM) suppressed the early capacitative transients of OHCs. GSNO (3 and 10 mM) had no effect on the whole cell currents of OHCs or Deiters' cells. Results show that GSNO suppresses cochlear function. This suppression may be due to an effect of GSNO on the cochlear amplifier. The actions of GSNO were different from those of other NO donors; therefore, the effects of GSNO may not be mediated by NO. The mechanisms underlying GSNO effects seem to be different from those of salicylate.

Differences in the distribution of responses to ATP and acetylcholine between outer hair cells of rat and guinea pig.

Adenosine 5' triphosphate (ATP) and acetylcholine (ACh) are neurotransmitters (ACh) and/or modulators (ATP) in the mammalian cochlea. In guinea pig, it appears that both neurotransmitters have a similar response distribution, with larger responses being evoked by the ligands in short hair cells compared to long hair cells (e.g., Chen et al., 1995b. Noise exposure alters the response of outer hair cells to ATP. Hear. Res. 88, 215-221.; Erostegui et al., 1994. In vitro pharmacologic characterization of a cholinergic receptor on outer hair cells. Hear. Res. 74, 135 147). The purpose of the present study was to test whether the distribution of responses to ACh and ATP in the OHCs of rat is the same as guinea pig. The ligand-induced current was monitored using the whole-cell configuration of the patch-clamp technique. Results show that in guinea pig OHCs, extracellular application of 100 microM ATP induced a current response in a majority of the same cells that responded to the application of 100 microM ACh. In contrast in rat OHCs, 100 microM ATP did not induce a current in the majority of cells that responded to the application of 100 microM ACh. N-methyl-glucamine (NMG+) substituted for K+ in the pipette solution failed to unmask an ATP-evoked inward current in rat OHCs. In addition, no response was produced in rat or guinea pig OHCs by adenosine, adenosine 5'-monophosphate (AMP) or adenosine 5'-diphosphate (ADP) at 100 microM. Results suggest that in guinea pig ACh-gated channels are present on most of the same OHCs that have ATP-gated ion channels, whereas in rat ACh-gated ion channels are present without ATP-gated channels on some OHCs.

Outwardly rectifying currents in guinea pig outer hair cells.

Studies of K+ conductances in hair cells report that big-conductance Ca(2+)-dependent K+ (BK) channels carry parts of the outwardly rectifying currents. Lin et al. (1995) suggested that in guinea pig outer hair cells (OHCs) a portion of these currents is carried via a voltage-dependent and Ca(2+)-independent K+ channel. The present study tests the hypothesis that there are two separable current components of the outwardly rectifying currents by using patch clamp methods in OHCs to characterize the voltage dependence and sensitivity of the outwardly rectifying currents to channel blockers. Lowering of external Ca2+ caused no change in the currents while charybdotoxin (ChTx; 100 nM), a BK K+ channel blocker, and Cd2+ (200 microM), and L-type calcium channel blocker, abolished about 50% of the currents. Both ChTx and Cd2+ caused a depolarizing shift in the half-activation voltage paralleled by a decrease in the voltage sensitivity. 4-Aminopyridine (4-AP, 0.01 mM), an A-type and delayed rectifier type channel blocker, abolished about 50% of the currents and caused a hyperpolarizing shift in the half-activation voltage together with an increase in the voltage sensitivity. The outwardly rectifying currents were more sensitive to block by 4-AP at membrane voltages around 40 mV compared to voltages around -20 mV. The differences in the current characteristics may be due to two separate channel types, one of which is similar to the delayed rectifier type channels while the other may be similar to the BK Ca(2+)-dependent K+ channels. In addition, the largest outwardly rectifying currents were present in long OHCs with the smallest present in short OHCs.

Acetylcholine response in guinea pig outer hair cells. I. Properties of the response.

The properties of the ACh (acetylcholine) response in guinea pig outer hair cells (OHCs) are not well understood. It has been shown that the response to ACh involves the activation of a Ca2+ dependent K+ selective conductance (referred to as Ksub where sub stands for suberyldicholine). In the present study, we examined the voltage dependence, the time dependence, and the desensitization of the ACh response. In addition, we examined the K+ selectivity of K(sub). These properties are important for aiding in the determination of the type of K+ channels activated by ACh. Patch-clamp technique in the whole-cell mode was used to record from single OHCs isolated from adult pigmented guinea pigs. ACh (100 microM) was applied to the voltage-clamped OHCs and the ACh induced currents (IACh) were measured. A voltage dependence of the ACh response was found with the ACh induced currents decaying monoexponentially at potentials positive to -30 mV. The decay of the ACh induced currents was faster soon after establishing the whole-cell mode of recording when compared to the decay of the currents some time later. This effect, referred to as the time dependence, was different from the desensitization of the response upon prolonged application of ACh. The desensitization of the ACh induced currents was about 50% after 2 min of continuous application of 100 microM ACh. The examined characteristics of the ACh response in guinea pig OHCs indicate a voltage and time dependence of the response and strong K+ selectivity of the Ksub.

Acetylcholine response in guinea pig outer hair cells. II. Activation of a small conductance Ca(2+)-activated K+ channel.

The type of K+ channel involved in the acetylcholine (ACh) evoked response (Ksub; sub stands for suberyldicholine) in guinea pig outer hair cells (OHCs) is still uncertain. The present study tests the hypotheses that Ksub is one of the following: a big conductance Ca(2+)-dependent K+ channel (BK), a small conductance Ca(2+)-dependent K+ channel (SK), a KA type of K+ channel, or a Kn type of K+ channel. Patch-clamp technique in the whole-cell mode was used to record from single guinea pig OHCs. ACh (100 microM) was applied to voltage-clamped OHCs and the ACh-induced currents (IACh) were measured. Charybdotoxin (100 nM) had no effect on IACh, while apamin (1 microM) blocked more than 90% of IACh. Lowering the external Ca2+ concentration caused a hyperpolarizing shift of the IACh monitored as a function of the prepulse voltage. Increasing internal Mg2+ (Mgi2+) concentration caused a reduction in the outward IACh without affecting the inward IACh. The Ksub channel was found to be permeable to Cs+. In Cs+ solutions, IACh was 45% of the IACh in K+ solutions. The block of IACh by apamin, the dependence on extracellular Ca2+, the incomplete block of IACh by Cs+, and the ACh-induced Cs+ currents favor the hypothesis that Ksub belongs to the SK type of channels. An ionotropic/nicotinic nature of the ACh mechanism of action is favored. It is suggested that, in vivo, the amplitude of the ACh-induced hyperpolarization may depend on the Ca2+/Mg2+ ratio inside and outside the cell.

Differences in cholinergic responses from outer hair cells of rat and guinea pig.

A cholinergic receptor on outer hair cells (OHC) in guinea pig cochlea induces a K+ current when it is activated by acetylcholine and suberyldicholine but not by nicotine or muscarine (Bobbin, 1995). This unusual receptor may contain an alpha 9-subunit. However, the pharmacology of the alpha 9-subunit cloned from rat and expressed in Xenopus oocytes does not completely match that obtained for the ACh receptor in guinea pig OHCs. The response to 1,1-dimethyl-4-phenylpiperazinium (DMPP) is large in guinea pig OHCs and small in oocytes containing receptors of the alpha 9-subunit. Therefore, we compared the effects of cholinergic receptor agonists in rat and guinea pig OHCs using the whole-cell variant of the patch-clamp technique. ACh caused the largest outward K+ current in OHCs from both rat and guinea pig. Carbachol- and suberyldicholine-induced responses were similar in magnitude in OHCs of rat and guinea pig. However, DMPP produced a small response in OHCs from rat and a large response in OHCs from guinea pig. At a concentration of 100 microM, muscarine, oxotremorine M, nicotine and cytisine induced little response in guinea pig OHCs and none in rat OHCs. Results suggest that the ACh receptor on rat OHCs is similar to the alpha 9-subunit-containing receptor expressed in oocytes but different from the ACh receptor on guinea pig OHCs.