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Dopamine increases Na+ absorption in the Reissner's membrane of the gerbil cochlea
Auris Nasus Larynx 40 (2013) 266–272
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Auris Nasus Larynx journal homepage: www.elsevier.com/locate/anl
Dopamine increases Na+ absorption in the Reissner’s membrane of the gerbil cochlea Chang-Hee Kim a, Hye-Young Kim b, Ho Sun Lee b, Byung Yoon Choi c, Sun O Chang b,d, Seung-Ha Oh b,d, Jun Ho Lee b,d,* a
Department of Otorhinolaryngology-Head and Neck Surgery, Konkuk University School of Medicine, Republic of Korea Department of Otorhinolaryngology, Seoul National University College of Medicine, Republic of Korea Department of Otorhinolaryngology, Seoul National University College of Medicine, Bundang Hospital, Republic of Korea d Sensory Organ Research Institute, Seoul National University Medical Research Center, Seoul, Republic of Korea b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 April 2012 Accepted 14 September 2012 Available online 9 October 2012
Objective: The purpose of the present study was to investigate the effect of dopamine as a possible regulator of epithelial Na+ channel (ENaC) in the Reissner’s membrane (RM). Methods: RM was freshly dissected from the gerbil cochlea, and short-circuit current (Isc) was measured using the voltage-sensitive vibrating probe technique. The dopamine receptor expression was examined using immunohistochemistry. Results: The results showed that dopamine induced activation of the amiloride-sensitive Isc, but not after pre-treatment with amiloride. The D1-like receptor antagonist SCH-23390, but not the D2-like receptor antagonist sulpiride, decreased the stimulatory effect of dopamine on RM. The effect of dopamine on Na+ transport via ENaC was still observed after blockade of the Na+–K+-ATPase by ouabain. D1 receptor immunoreactivity was observed in RM, stria vascularis and spiral ganglion. Conclusion: Na+ transport in RM is activated by dopamine possibly via D1-like receptors, and intracellular mechanisms other than cAMP-mediated pathway may be involved. ß 2012 Elsevier Ireland Ltd. All rights reserved.
Keywords: Dopamine Reissner’s membrane Epithelial sodium channel Voltage-sensitive vibrating probe Cochlea Gerbil
1. Introduction Na+ transport mechanisms in the cochlea have been much known in recent years. Epithelial cells of the Reissner’s membrane (RM) [1–3], outer sulcus cells [4] and Claudius’ cells [5] have been reported to contribute to endolymphatic homeostasis by active Na+ transport. RM forms the boundary between the scala media filled with K+rich endolymph and the scala vestibuli filled with Na+-rich perilymph in the cochlea. RM consists of two cell layers (tight epithelia which face the endolymph, and mesothelia which face the perilymph) that are separated by a basement membrane and a thin layer of intercellular substance. Recently, evidences that RM contributes to endolymphatic Na+ homeostasis by Na+ absorption via apical epithelial Na+ channel (ENaC) have been reported. Localization of ENaC in the epithelial
* Corresponding author at: Department of Otorhinolaryngology, Seoul National University College of Medicine, Seoul National University Hospital, 28 Yeongondong, Chongro-gu, Seoul 110-744, Republic of Korea. Tel.: +82 2 2072 2445; fax: +82 2 745 2387. E-mail address: [email protected] (J.H. Lee). 0385-8146/$ – see front matter ß 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.anl.2012.06.008
cells of RM has been demonstrated by immunohistochemistry or by in situ hybridization [6]. The a-subunit of the Na+–K+-ATPase, which provides the driving force for Na+ transport via ENaC, was reported to be expressed at the basolateral membrane of RM epithelial cells [7]. Electrogenic transepithelial Na+ transport has been demonstrated in freshly dissected gerbil RM by using the vibrating probe method [1]. The activity of ENaC is regulated by hormones, such as glucocorticoids, aldosterone, vasopressin, and local paracrine or autocrine factors including ATP. The transcripts for three subunits of ENaC are present in rat RM, and glucocorticoid upregulates the transcription of Na+ transport gene. Purinergic receptors also regulate the function of ENaC [3]. Other types of currents, such as an inwardly rectifying chloride current, were identified in the epithelial cells of RM using patch clamp studies [8]. In the inner ear, diverse dopamine receptor subtypes have been identified in areas including spiral ganglion neurons, vestibular hair cells, strial marginal cells, and the contact between lateral efferent fibers and type I afferent neurons beneath the inner hair cells [9]. Dopamine released from lateral olivocochlear efferents is known to interact with dopamine receptors located postsynaptically on the afferent nerve fibers of inner hair cells, where it has been shown to modulate cochlear afferent neurotransmission [10].
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However, modulation of Na+ transport through by dopaminergic signaling in the cochlea has not been demonstrated yet. The present study was undertaken to investigate the effect of dopamine on ENaC function using the vibrating probe technique, and to use immunohistochemistry to measure the corresponding dopamine receptor expression in epithelial cells of gerbil RM.
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reported here refers to the flux at the probe position and represents only a fraction of the current crossing the epithelium. No changes in the relative position of the probe were observed owing to the swelling or shrinking of the tissues during the experimental treatments. 2.3. Immunohistochemistry
2. Materials and methods 2.1. Tissue preparation The gerbils (3–4 weeks old) were anesthetized with sodium pentobarbital (50–100 mg/kg; intraperitoneally) and killed to remove temporal bones. The methods used for dissecting RM have been previously described [1]. The stria vascularis was removed from the lateral wall of the apical cochlear turn and the attached portion of RM was folded over the suprastrial portion of the spiral ligament. The tissue was mounted in a perfusion chamber on the stage of an inverted microscope (Olympus IX70) and continuously perfused at 37 8C at an exchange rate of 3 times/ min. All procedures conformed to the protocols approved by the Institutional Animal Care and Use Committee of Seoul National University. 2.2. Voltage-sensitive vibrating probe The vibrating probe technique was used to measure the transepithelial currents under short-circuit conditions due to the small size of RM epithelium. The diameter of the vibrating probe tip was approximately 20 mm, which permits the detection of voltages in the low nanovolt range. The vibration between the 2 positions within the line of current flow yields voltages that correspond to current flow through the resistive physiological saline [11]. The vibrating probe technique used was identical to a previously described method [11]. Briefly, the short-circuit current (Isc) was monitored by vibrating a platinum–iridium wire microelectrode insulated with parylene-C (Micro Electrodes, Gaithersburg, MD, USA), and coated with platinum-black on its exposed tip. The vibration was approximately 20 mm along the horizontal (X) and vertical (Z) axes. The X-axis was perpendicular to the face of the epithelium, and the probe was positioned 30 mm from the apical surface of the epithelium using computer-controlled, stepper-motor manipulators (Applicable Electronics, Forestdale, MA, USA) and a specialized probe software (ASET version 2.0, Science Wares, East Falmouth, MA, USA). The bath references were 26-gauge platinum-black electrodes. Calibration was performed in physiologic saline (see below) using a glass microelectrode (tip <1-mm outer diameter) filled with 3 mol/l KCl as a point source of current. The frequencies of the vibration used were in the 200–400-Hz range and were well separated for the 2 orthogonal directions. Signals from the oscillators driving the probe were also fed to a dual channel phase-sensitive detector. Asymmetry of probe design yielded different resonant frequencies for the two directions of vibration. X and Z detector signals were connected to a 16 bit analog-to-digital converter (CIO-DAS1602/16, ComputerBoards, Mansfield, MA, USA) in a Pentium IV computer. The sampling interval was 0.6 s, which was the minimum interval allowed by the software. The electrode was positioned where Isc showed a maximum X value and minimum Z value. The data are expressed as the recorded X value and plotted using the Origin version 6.1 software (OriginLab Software, Northampton, MA, USA). The output from the vibrating probe depended not only on the specific short circuit current of the epithelium, but also on the position of the probe relative to the surface of the tissue and on the precise geometry of each tissue sample. The current density
The gerbils at the age of 21 days were transcardially perfused with phosphate buffer solution (PBS) under deep anesthesia, and then with 4% paraformaldehyde in PBS. The cochleas were dissected out and postfixed by immersion in a fresh solution of 4% formaldehyde in PBS for 2 h. After postfixation, the tissues were washed with PBS and transferred to a decalcifying solution (0.12 M EDTA, pH 7.2) for 5 days at 4 8C. The EDTA solution was changed every 24 h. The cochlear tissues were dehydrated in a graded ethanol series and embedded in paraffin. Ten-mm sections of cochlea were obtained and sequentially incubated in 0.1% sodium borohydride (PBS plus 5 mM glycine), 3% hydrogen peroxide (for the avidin–biotin peroxidase method), and 2% normal serum (corresponding to the species in which the secondary antibody was made). The Vector ABC elite protocol was used with 3,30 -diaminobenzidine as the chromogen (BioGenex, San Ramon, CA, USA). The sections were incubated overnight at 4 8C with the primary antibody. The primary antibody used for D1 receptor was the rabbit anti-dopamine D1 receptor which was raised against a 13 amino-acid sequence (amino acids 403–415) from rat D1A (AB1765P, Chemicon, Temecula, CA, USA) diluted at 1:100. The primary antibody used for D2 receptor was the rabbit anti-dopamine D2 receptor which was raised against a 28 amino-acid sequence from the human D2 (AB5084P, Chemicon, Temecula, CA, USA) diluted at 1:100. The specificity of the immunohistochemical stain was controlled by the omission of the primary antibody and preincubation of the antiserum a peptide antigen. 2.4. Solutions and chemicals The perfusate used as control solution was a perilymph-like physiologic saline of pH 7.4, containing (in mM) 150 NaCl, 3.6 KCl, 1 MgCl2, 0.7 CaCl2, 5 glucose, and 10 HEPES. Dopamine (Sigma H8502) was directly dissolved in the control solution just before use. SCH-23390 (R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5tetrahydro-1H-3-benzazepine hydrochloride, Sigma C-0206) and sulpiride (Sigma S-8010) were dissolved in ethanol at 0.01 M stock concentrations. Ethanol at this concentration had no effect on Isc. Bromocriptine (Sigma B-2134) and 8-bromo-cyclic adenosine monophosphate (cAMP, Sigma B-5386) was dissolved in distilled water. Amiloride (Sigma A-7410) was predissolved in dimethyl sulfoxide (DMSO) and then diluted to 0.1% DMSO in the control solution before application. DMSO at this concentration had no effect on Isc. 2.5. Data presentation and statistics The tip of the probe was positioned approximately 20 mm from the apical surface of the RM, and a short-circuit current (Isc) was recorded in the apical-to-basolateral direction. The baseline Isc values in the control solution were obtained by averaging the data for 9 s just before solution change. For the analysis of the effect of each drug, the data were averaged for 9 s after reaching a steadystate. The increases or decreases in Isc were considered significant at the P < 0.05 level. Statistical comparisons between two means were obtained with t-test (Mann–Whitney test, if n < 5). The data shown were expressed as mean SEM values (n = number of tissues) of the Isc.
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Fig. 1. Effect of dopamine (100 mM) on short circuit current (Isc) in the absence or presence of amiloride (10 mM) on the epithelial cells of the Reissner’s membrane. (A) The perfusion of dopamine partially increased amiloride-sensitive Na+ absorption. (B) The perfusion of amiloride abolished most of the baseline Isc. The application of dopamine in the presence of amiloride did not change Isc.
3. Results 3.1. Effect of dopamine on Na+ absorption in the epithelial cells of RM We measured the baseline Isc of RM in the perilymph-like control solution and observed a change in the Isc after application of dopamine (100 mM) or amiloride (10 mM). Negative baseline Isc values were observed in the control solution before the application of dopamine or amiloride (Fig. 1, Table 1). The perfusion of dopamine led to a significant increase in the Isc (28.1 2.6%, n = 4), and the subsequent addition of amiloride inhibited the Isc completely (Fig. 1A, Table 1). However, the perfusion of dopamine after the pretreatment with amiloride which abolished most of the baseline Isc showed no change in Isc (Fig. 1B, Table 1). Dopamine increased amiloride-sensitive Na+ absorption in a dose-dependent manner (from 1 mM to 1 mM). Isc was increased by 4.3 2.1%, 12.4 2.2%, 27.2 1.7%, and 29.6 0.7% after application of dopamine at different concentrations of 1 mM, 10 mM, 100 mM, and 1 mM, respectively (n = 6, Fig. 2). The data were fitted to the Hill equation (Fig. 2B). 3.2. Blockade of dopamine effect by dopamine receptor antagonists We applied dopamine receptor antagonists to determine the subtype of dopamine receptor which is responsible for the dopamine-mediated increase in ENaC function. The effects of dopamine on the Na+ absorption in the absence and presence of
each antagonist were compared (Fig. 3, Table 2). We performed control experiments that consisted of 2 consecutive applications of dopamine (100 mM), with a 3-min interval (Fig. 3A). The increase of Isc by the second application of dopamine was insignificantly reduced (12.0 10.7%, n = 4) compared with that evoked by the first application (Fig. 3A, Table 2). The data obtained from the experiments using dopamine receptor antagonists were statistically compared with those from the control experiments (Fig. 3B and C). The application of the specific D1-like receptor (D1 and D5) blocker, SCH-23390 (100 mM), inhibited most of dopamine’s effect on ENaC function (n = 4; Fig. 3B, Table 2). The magnitude of the dopamine-evoked increase in Isc was reduced by 81.9 10.3% in the presence of SCH-23390 (n = 4; Fig. 3B, Table 2) compared with its absence. This (81.9% reduction) was significantly different from that observed in the control experiments (12.0% reduction). In contrast, the D2-like receptor (D2, D3, and D4) blocker, sulpiride (100 mM), did not significantly block the effect of dopamine on ENaC activity (n = 4; Fig. 3C, Table 2). The perfusion of bromocriptine (100 mM), which is an agonist of D2-like receptors, especially D2 and D3 receptors, did not significantly change Isc (n = 5; Fig. 3D, Table 2). 3.3. Dopamine effect on the ENaC activity after inhibition of the Na+– K+-ATPase by ouabain Previous studies have reported that activation of dopamine receptors has distinct effects on the function of the Na+–K+-ATPase in lung and kidney epithelia [to see review, 12]. The Na+–K+-ATPase
Table 1 Effects of dopamine (100 mM) on Isc (mA/cm2) in the absence or presence of amiloride (10 mM).
Dopamine effect in the absence of amiloride (n = 4) Dopamine effect in the presence of amiloride (n = 3) a * z
Baseline
Dopamine
Amiloride
Amiloride + dopamine
14.1 2.1* 15.3 1.9
19.9 3.1* NT
NTa 1.7 0.7z
0.1 0.3 2.1 0.8z
Not tested. P < 0.05, between baseline Isc and Isc after dopamine application. P > 0.05, between Isc during amiloride perfusion and Isc after dopamine application in the presence of amiloride.
Fig. 2. Dose–response relationship of dopamine. (A) Representative traces of dopamine response at different concentrations from 1 mM to 1 mM. (B) EC50 was 16.8 mM (n = 6), which was obtained by fitting the data to the Hill equation using IGOR Pro 4.01 (WaveMetronics, Lake Oswego, OR).
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Fig. 3. Functional characterization of the dopamine-responsive receptor subtype. (A) The control experiment using a protocol consisting of 2 consecutive applications of dopamine (100 mM) with a 3-min interval. (B) The dopamine response was blocked by 100-mM SCH-23390 (D1-like receptor antagonist). (C) Sulpiride (D2-like receptor antagonist) at 100 mM did not significantly inhibit the dopamine effect. (D) The application of 100-mM bromocriptine (D2-like receptor agonist) did not significantly change Isc. Table 2 Effects of dopamine (100 mM) on Isc (mA/cm2) in the absence or presence of dopamine receptor antagonists (see Fig. 3).
Baseline First dopamine Dopamine washout Antagonist Second dopamine Reduction in the effect of second dopamine compared with first dopamine (%) a b c d jj
Controla n = 4
SCH-23390 (100 mM) n = 3
Sulpiride (100 mM) n = 4
15.3 1.7 19.7 1.1 16.2 1.4 NTb 19.9 1.3 12.0 10.7c
14.1 1.3 18.0 1.6 14.4 1.7 14.3 1.3 15.0 1.0 81.9 10.3d,jj
13.5 1.6 16.3 1.4 13.4 1.5 13.4 1.7 15.8 1.3 16.8 14.8d
This experiment used a protocol that consisted of 2 consecutive applications of dopamine (100 mM) with a 3-min interval. Not tested. 100 {(second dopamine dopamine washout)/(first dopamine baseline)} 100. 100 {(second dopamine antagonist)/(first dopamine baseline)} 100. P < 0.05. This value was significantly different from the value (12.0%) observed in the control experiments.
has been found to be expressed on the basolateral membrane of the epithelial cells of RM [7], and known to provide the driving force for Na+ entry via ENaC. It is conceivable that dopaminergic modulation of Na+–K+-ATPase activity may alter Na+ absorption via ENaC independently of any direct effects of dopamine on ENaC. Therefore, we conducted experiments to determine whether the activation of ENaC by dopamine is still observed after inhibition of the Na+–K+-ATPase. The application of ouabain (1 mM), a Na+–K+ATPase inhibitor, reduced Isc from 15.0 1.4 to 9.6 1.7 mA/ cm2 (n = 4), and the subsequent addition of dopamine resulted in an increase in Isc to 11.7 1.6 mA/cm2 (n = 4, Fig. 4). Thus, dopamine clearly increased ENaC activity even when the Na+–K+-ATPase was inhibited, which indicates that dopamine is capable of regulating ENaC function independently of a change in Na+–K+-ATPase activity. 3.4. Effect of 8-bromo-cAMP on Isc We investigated whether the increase of intracellular cAMP could regulate ENaC activity by using a membrane permeable cAMP analog, 8-bromo-cAMP (200 mM). The results showed that the application of 8-bromo-cAMP did not change Isc (n = 5; Fig. 5).
Fig. 4. Dopamine increases ENaC activity after the Na+–K+-ATPase was blocked by ouabain. After basal Isc had been obtained, 1-mM ouabain was added to the bath solution. When ouabain was perfused long enough for Isc to reach a plateau, dopamine was subsequently added. Isc was significantly increased after the perfusion of dopamine. These results indicate that dopamine’s effect on ENaC was not secondary to its activation of the Na+–K+-ATPase.
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Fig. 5. The cAMP analogue, 8-bromo-cAMP, did not mimic the effects of dopamine, showing no change in Isc after application.
3.5. Immunohistochemical analysis of dopamine receptor expression Immunoreactivity for dopamine D1 receptors was observed in RM (Fig. 6A), and stria vascularis (Fig. 6B). However, RM was not immunopositive for D2 receptors (Fig. 6D). The spiral ganglion neurons showed immunoreactivity for both D1 and D2 receptors (Fig. 6C and E). The anti-D1 antibody pre-absorbed with an excess of antigenic peptide resulted in no staining, and omitting the primary antibody from the procedure also produced negative results (not shown). 4. Discussion In the present study, the application of dopamine significantly increased Isc whereas the subsequent addition of amiloride abolished Isc. On the other hand, in the presence of amiloride, dopamine had no effect on Isc. These results indicate that dopamine
increased the amiloride-sensitive Na+ absorption in the epithelial cells of RM. Dopamine receptors are G protein-coupled receptors, and consist of 5 subtypes (D1 to D5). These subtypes are divided into two major groups: D1-like (D1 and D5) and D2-like receptors (D2, D3, and D4) [13]. In our results, the effect of dopamine on ENaC activity was blocked by SCH-23390, a D1-like receptor blocker, but not by sulpiride, a D2-like receptor blocker [13]. The application of bromocriptine, and agonist of D2-like receptors, had no effect on Isc. Furthermore, immunoreactivity for D1 receptors was observed in RM. Taken together, these results indicate that the effect of dopamine on ENaC is mediated by D1-like receptors. These results are consistent with the observation that stimulation of D1 dopamine receptors in lung alveolar epithelium increased ENaC activity [14]. However, the possibility that the D5 receptor is also involved in the effects of dopamine on RM cannot be ruled out. It is known that dopamine receptors have opposing intracellular signaling pathway, in that D1-like receptors activate adenylyl cyclase (AC) to increase cAMP production, whereas D2-like receptors inhibit AC to decrease cAMP production or to activate phospholipase. However, our results showed that the direct application of 8-bromo-cAMP did not change Isc. This implies the possibility of other signaling mechanisms via D1 receptors, which requires further investigation. Interestingly, the presence of a high activity of AC and a high steady-state level of cAMP in RM has been reported [15]. It has been proposed that a primary function of RM may be Cl transport under the control of cAMP [16]. The perilymphatic perfusion of forskolin, which activates AC leading to an increase in cAMP levels, evoked changes in the electrical resistance of the perilymph-endolymph barrier, endocochlear potential, and endolymphatic Cl concentration, which was interpreted as the presence of electrogenic Cl transport in RM under the control of cAMP [16]. However, in the present study, there was no change in Isc in response to the dopamine application in the presence of amiloride (Fig. 1B), which indicates that application of dopamine does not elicit cAMP-mediated Cl transport. These results are in line with previous findings in
Fig. 6. Immunohistochemical localization of the D1 receptors in the cochlea. Immunoreactivity was observed in the Reissner’s membrane (A) and stria vascularis (B). (C) Spiral ganglion neurons were immunopositive for the D1 receptors. (D) D2 receptors were not immunolocalized in the Reissner’s membrane. (E) Spiral ganglion showed immunoreactivity for D2 receptors. Scale bars = 50 mm.
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which Isc was shown to be insensitive to forskolin, an activator of cAMP production via AC, and genistein known to stimulate cAMPdependent Cl currents via cystic fibrosis transmembrane conductance regulator (CFTR) Cl channels when amiloride was co-perfused [1]. However, these findings could be attributable to the experimental conditions used in the previously published reports and the present study, which would not support the involvement of cAMP-related cascades; therefore, this pathway requires further investigation. In lung alveolar epithelial cells, the activity of ENaC at the apical membrane and Na+–K+-ATPase at the basolateral membrane is increased by the stimulation of dopamine receptors, which synergistically facilitates clearance of alveolar fluid [to see review, 12]. The Na+–K+-ATPase provides the driving force for Na+ influx via ENaC by actively transporting Na+ out of the cell through the basolateral membrane. Stimulation of dopamine receptors in the lung or kidney epithelia has opposing effects on the function of the Na+–K+-ATPase, in that dopamine increases Na+–K+-ATPase activity in the lung, whereas it decreases Na+–K+-ATPase activity in the kidney epithelia [12]. The presence of the Na+–K+-ATPase in RM was reported [7], and application of ouabain, a Na+–K+-ATPase inhibitor, significantly reduced Isc [1]. Therefore, we determined whether the change in Isc in response to dopamine application was caused entirely by an alteration of Na+–K+-ATPase activity. The present study shows that the dopamine-mediated increase in ENaC activity was not a secondary effect of the Na+–K+-ATPase stimulation by dopamine, as dopamine-mediated ENaC activation was still observed after the blockade of Na+–K+-ATPase activity by ouabain (Fig. 4). In the cochlea, dopamine plays an important role in the sensory process by modulating afferent auditory nerve activity [9]. Dopamine is released from the terminals of lateral olivocochlear efferent fibers, and is known to serve a protective function against acoustic trauma, hypoxia, and ototoxicity [10]. Recently, it has been proposed that the activation of dopamine receptors at efferent/afferent synapses has a level-dependent response to stimulation, such that D2 receptors may dominate to enhance inhibition during high sound levels [10], whereas D1 receptors may dominate to enhance excitation during low sound levels [9]. The distribution of dopamine receptors in the inner ear, however, has been reported to be more diversely localized not only in the postsynaptic region of efferent fibers, but also in spiral ganglion neurons and probably also in strial marginal cells [9], which is in agreement with our immunohistochemical results. Neither the natural sources of dopamine, nor its concentration in the cochlear fluid has been reported. The baseline level of dopamine in lung tissue is quite high, even higher than that reported in the adrenal gland. It has been suggested that dopamine is important in lung liquid removal at birth because the plasma level of dopamine increases 10-fold immediately before delivery [17]. The lung alveolar epithelia and epithelia of kidney proximal tubules are important sources of extraneuronal dopamine, and the concentration of dopamine in blood circulation is increased postprandially. In kidney epithelia, L-dopa, the dopamine precursor, is transported into the epithelium, and then converted into dopamine by L-amino acid dopa decarboxylase before dopamine is secreted [18]. At present, it is not known whether dopamine is locally formed and exerts its action in an autocrine or paracrine manner in parasensory epithelia in the inner ear, as has been demonstrated in the kidney. Although RM and basilar membrane in mammals have been known to be avascular structures, there have been some reports demonstrating the presence of unusual blood vessels in RM [19]. On the other hand, efferent nerve terminals located in the perilymphatic space beneath the inner and vestibular hair cells are so far the only
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anatomical sites of dopamine release as a neurotransmitter in the inner ear [9]. Small molecules, such as dopamine, might enter the endolymphatic space from the perilymphatic space by pinocytosis through epithelial cells lining the endolymphatic surface, such as RM epithelia, Claudius’ cells, and Hensen’s cells [20]. Unfortunately, we were unable to identify the sub-cellular localization of the D1 receptor in the epithelial cells of RM, as RM is an extremely thin, 2-cell layer tissue. In fact, owing to this specific feature of RM, we were also unable to obtain a sufficient amount of tissue to extract RNA for RT-PCR in this small epithelial domain. The findings of the present study might provide an important role for dopamine in maintaining endolymphatic homeostasis. When Na+ moves across RM from the endolymphatic space, water follows Na+ and, consequently, is cleared from the endolymphatic space, a process that can be enhanced by dopamine. This effect may be counteracted and balanced by the activity of P2Y4 purinergic receptor which was shown to inhibit Na+ absorption in gerbil RM [3]. However, the concentration of dopamine in the endolymphatic fluid has not been reported yet, and plasma concentration of dopamine is known not to exceed tens of nanomolar level [21]. The concentrations of dopamine used in the present study (1 mM to 1 mM) are quite high and conditions that could elevate dopamine level in the endolymph have not been reported, therefore, the physiologic role of this response in vivo is still unclear. In conclusion, dopamine increased Na+ absorption in freshly dissected RM possibly via D1-like receptors and the intracellular mechanisms remain to be elucidated. Conflict of interest None. Acknowledgement This work was supported by a grant of the Korean Health Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A100630). References [1] Lee JH, Marcus DC. Endolymphatic sodium homeostasis by Reissner’s membrane. Neuroscience 2003;119:3–8. [2] Kim CH, Kim HY, Chang OS, Oh SH, Lee JE, Lee JH. Developmental change of Na(+)-absorptive function in Reissner’s membrane epithelia. Neuroreport 2009;20:1275–8. [3] Kim CH, Kim HY, Lee HS, Chang SO, Oh SH, Lee JH. P2Y4-mediated regulation of Na+ absorption in the Reissner’s membrane of the cochlea. J Neurosci 2010;30:3762–9. [4] Lee JH, Chiba T, Marcus DC. P2X2 receptor mediates stimulation of parasensory cation absorption by cochlear outer sulcus cells and vestibular transitional cells. J Neurosci 2001;21:9168–74. [5] Yoo JC, Kim HY, Han KH, Oh SH, Chang SO, Marcus DC, et al. Na+ absorption by Claudius’ cells is regulated by purinergic signaling in the cochlea. Acta Otolaryngol Suppl 2012;132:103–8. [6] Couloigner V, Fay M, Djelidi S, Farman N, Escoubet B, Runembert I, et al. Location and function of the epithelial Na channel in the cochlea. Am J Physiol Renal Physiol 2001;280:F214–22. [7] Iwano T, Yamamoto A, Omori K, Akayama M, Kumazawa T, Tashiro Y. Quantitative immunocytochemical localization of Na+,K+-ATPase alpha-subunit in the lateral wall of rat cochlear duct. J Histochem Cytochem 1989;37:353–63. [8] Kim KX, Marcus DC. Inward-rectifier chloride currents in Reissner’s membrane epithelial cells. Biochem Biophys Res Commun 2010;394:434–8. [9] Niu X, Canlon B. The signal transduction pathway for the dopamine D1 receptor in the guinea-pig cochlea. Neuroscience 2006;137:981–90. [10] d’Aldin C, Puel JL, Leducq R, Crambes O, Eybalin M, Pujol R, et al. Effects of a dopaminergic agonist in the guinea pig cochlea. Hear Res 1995;90: 202–11. [11] Marcus DC. Vibrating probes: new technology for investigation of endolymph homeostasis. Keio J Med 1996;45:301–5. [12] Bertorello AM, Sznajder JI. The dopamine paradox in lung and kidney epithelia: sharing the same target but operating different signaling networks. Am J Respir Cell Mol Biol 2005;33:432–7.
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Dopamine increases Na+ absorption in the Reissner’s membrane of the gerbil cochlea Chang-Hee Kim a, Hye-Young Kim b, Ho Sun Lee b, Byung Yoon Choi c, Sun O Chang b,d, Seung-Ha Oh b,d, Jun Ho Lee b,d,* a
Department of Otorhinolaryngology-Head and Neck Surgery, Konkuk University School of Medicine, Republic of Korea Department of Otorhinolaryngology, Seoul National University College of Medicine, Republic of Korea Department of Otorhinolaryngology, Seoul National University College of Medicine, Bundang Hospital, Republic of Korea d Sensory Organ Research Institute, Seoul National University Medical Research Center, Seoul, Republic of Korea b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 April 2012 Accepted 14 September 2012 Available online 9 October 2012
Objective: The purpose of the present study was to investigate the effect of dopamine as a possible regulator of epithelial Na+ channel (ENaC) in the Reissner’s membrane (RM). Methods: RM was freshly dissected from the gerbil cochlea, and short-circuit current (Isc) was measured using the voltage-sensitive vibrating probe technique. The dopamine receptor expression was examined using immunohistochemistry. Results: The results showed that dopamine induced activation of the amiloride-sensitive Isc, but not after pre-treatment with amiloride. The D1-like receptor antagonist SCH-23390, but not the D2-like receptor antagonist sulpiride, decreased the stimulatory effect of dopamine on RM. The effect of dopamine on Na+ transport via ENaC was still observed after blockade of the Na+–K+-ATPase by ouabain. D1 receptor immunoreactivity was observed in RM, stria vascularis and spiral ganglion. Conclusion: Na+ transport in RM is activated by dopamine possibly via D1-like receptors, and intracellular mechanisms other than cAMP-mediated pathway may be involved. ß 2012 Elsevier Ireland Ltd. All rights reserved.
Keywords: Dopamine Reissner’s membrane Epithelial sodium channel Voltage-sensitive vibrating probe Cochlea Gerbil
1. Introduction Na+ transport mechanisms in the cochlea have been much known in recent years. Epithelial cells of the Reissner’s membrane (RM) [1–3], outer sulcus cells [4] and Claudius’ cells [5] have been reported to contribute to endolymphatic homeostasis by active Na+ transport. RM forms the boundary between the scala media filled with K+rich endolymph and the scala vestibuli filled with Na+-rich perilymph in the cochlea. RM consists of two cell layers (tight epithelia which face the endolymph, and mesothelia which face the perilymph) that are separated by a basement membrane and a thin layer of intercellular substance. Recently, evidences that RM contributes to endolymphatic Na+ homeostasis by Na+ absorption via apical epithelial Na+ channel (ENaC) have been reported. Localization of ENaC in the epithelial
* Corresponding author at: Department of Otorhinolaryngology, Seoul National University College of Medicine, Seoul National University Hospital, 28 Yeongondong, Chongro-gu, Seoul 110-744, Republic of Korea. Tel.: +82 2 2072 2445; fax: +82 2 745 2387. E-mail address: [email protected] (J.H. Lee). 0385-8146/$ – see front matter ß 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.anl.2012.06.008
cells of RM has been demonstrated by immunohistochemistry or by in situ hybridization [6]. The a-subunit of the Na+–K+-ATPase, which provides the driving force for Na+ transport via ENaC, was reported to be expressed at the basolateral membrane of RM epithelial cells [7]. Electrogenic transepithelial Na+ transport has been demonstrated in freshly dissected gerbil RM by using the vibrating probe method [1]. The activity of ENaC is regulated by hormones, such as glucocorticoids, aldosterone, vasopressin, and local paracrine or autocrine factors including ATP. The transcripts for three subunits of ENaC are present in rat RM, and glucocorticoid upregulates the transcription of Na+ transport gene. Purinergic receptors also regulate the function of ENaC [3]. Other types of currents, such as an inwardly rectifying chloride current, were identified in the epithelial cells of RM using patch clamp studies [8]. In the inner ear, diverse dopamine receptor subtypes have been identified in areas including spiral ganglion neurons, vestibular hair cells, strial marginal cells, and the contact between lateral efferent fibers and type I afferent neurons beneath the inner hair cells [9]. Dopamine released from lateral olivocochlear efferents is known to interact with dopamine receptors located postsynaptically on the afferent nerve fibers of inner hair cells, where it has been shown to modulate cochlear afferent neurotransmission [10].
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However, modulation of Na+ transport through by dopaminergic signaling in the cochlea has not been demonstrated yet. The present study was undertaken to investigate the effect of dopamine on ENaC function using the vibrating probe technique, and to use immunohistochemistry to measure the corresponding dopamine receptor expression in epithelial cells of gerbil RM.
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reported here refers to the flux at the probe position and represents only a fraction of the current crossing the epithelium. No changes in the relative position of the probe were observed owing to the swelling or shrinking of the tissues during the experimental treatments. 2.3. Immunohistochemistry
2. Materials and methods 2.1. Tissue preparation The gerbils (3–4 weeks old) were anesthetized with sodium pentobarbital (50–100 mg/kg; intraperitoneally) and killed to remove temporal bones. The methods used for dissecting RM have been previously described [1]. The stria vascularis was removed from the lateral wall of the apical cochlear turn and the attached portion of RM was folded over the suprastrial portion of the spiral ligament. The tissue was mounted in a perfusion chamber on the stage of an inverted microscope (Olympus IX70) and continuously perfused at 37 8C at an exchange rate of 3 times/ min. All procedures conformed to the protocols approved by the Institutional Animal Care and Use Committee of Seoul National University. 2.2. Voltage-sensitive vibrating probe The vibrating probe technique was used to measure the transepithelial currents under short-circuit conditions due to the small size of RM epithelium. The diameter of the vibrating probe tip was approximately 20 mm, which permits the detection of voltages in the low nanovolt range. The vibration between the 2 positions within the line of current flow yields voltages that correspond to current flow through the resistive physiological saline [11]. The vibrating probe technique used was identical to a previously described method [11]. Briefly, the short-circuit current (Isc) was monitored by vibrating a platinum–iridium wire microelectrode insulated with parylene-C (Micro Electrodes, Gaithersburg, MD, USA), and coated with platinum-black on its exposed tip. The vibration was approximately 20 mm along the horizontal (X) and vertical (Z) axes. The X-axis was perpendicular to the face of the epithelium, and the probe was positioned 30 mm from the apical surface of the epithelium using computer-controlled, stepper-motor manipulators (Applicable Electronics, Forestdale, MA, USA) and a specialized probe software (ASET version 2.0, Science Wares, East Falmouth, MA, USA). The bath references were 26-gauge platinum-black electrodes. Calibration was performed in physiologic saline (see below) using a glass microelectrode (tip <1-mm outer diameter) filled with 3 mol/l KCl as a point source of current. The frequencies of the vibration used were in the 200–400-Hz range and were well separated for the 2 orthogonal directions. Signals from the oscillators driving the probe were also fed to a dual channel phase-sensitive detector. Asymmetry of probe design yielded different resonant frequencies for the two directions of vibration. X and Z detector signals were connected to a 16 bit analog-to-digital converter (CIO-DAS1602/16, ComputerBoards, Mansfield, MA, USA) in a Pentium IV computer. The sampling interval was 0.6 s, which was the minimum interval allowed by the software. The electrode was positioned where Isc showed a maximum X value and minimum Z value. The data are expressed as the recorded X value and plotted using the Origin version 6.1 software (OriginLab Software, Northampton, MA, USA). The output from the vibrating probe depended not only on the specific short circuit current of the epithelium, but also on the position of the probe relative to the surface of the tissue and on the precise geometry of each tissue sample. The current density
The gerbils at the age of 21 days were transcardially perfused with phosphate buffer solution (PBS) under deep anesthesia, and then with 4% paraformaldehyde in PBS. The cochleas were dissected out and postfixed by immersion in a fresh solution of 4% formaldehyde in PBS for 2 h. After postfixation, the tissues were washed with PBS and transferred to a decalcifying solution (0.12 M EDTA, pH 7.2) for 5 days at 4 8C. The EDTA solution was changed every 24 h. The cochlear tissues were dehydrated in a graded ethanol series and embedded in paraffin. Ten-mm sections of cochlea were obtained and sequentially incubated in 0.1% sodium borohydride (PBS plus 5 mM glycine), 3% hydrogen peroxide (for the avidin–biotin peroxidase method), and 2% normal serum (corresponding to the species in which the secondary antibody was made). The Vector ABC elite protocol was used with 3,30 -diaminobenzidine as the chromogen (BioGenex, San Ramon, CA, USA). The sections were incubated overnight at 4 8C with the primary antibody. The primary antibody used for D1 receptor was the rabbit anti-dopamine D1 receptor which was raised against a 13 amino-acid sequence (amino acids 403–415) from rat D1A (AB1765P, Chemicon, Temecula, CA, USA) diluted at 1:100. The primary antibody used for D2 receptor was the rabbit anti-dopamine D2 receptor which was raised against a 28 amino-acid sequence from the human D2 (AB5084P, Chemicon, Temecula, CA, USA) diluted at 1:100. The specificity of the immunohistochemical stain was controlled by the omission of the primary antibody and preincubation of the antiserum a peptide antigen. 2.4. Solutions and chemicals The perfusate used as control solution was a perilymph-like physiologic saline of pH 7.4, containing (in mM) 150 NaCl, 3.6 KCl, 1 MgCl2, 0.7 CaCl2, 5 glucose, and 10 HEPES. Dopamine (Sigma H8502) was directly dissolved in the control solution just before use. SCH-23390 (R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5tetrahydro-1H-3-benzazepine hydrochloride, Sigma C-0206) and sulpiride (Sigma S-8010) were dissolved in ethanol at 0.01 M stock concentrations. Ethanol at this concentration had no effect on Isc. Bromocriptine (Sigma B-2134) and 8-bromo-cyclic adenosine monophosphate (cAMP, Sigma B-5386) was dissolved in distilled water. Amiloride (Sigma A-7410) was predissolved in dimethyl sulfoxide (DMSO) and then diluted to 0.1% DMSO in the control solution before application. DMSO at this concentration had no effect on Isc. 2.5. Data presentation and statistics The tip of the probe was positioned approximately 20 mm from the apical surface of the RM, and a short-circuit current (Isc) was recorded in the apical-to-basolateral direction. The baseline Isc values in the control solution were obtained by averaging the data for 9 s just before solution change. For the analysis of the effect of each drug, the data were averaged for 9 s after reaching a steadystate. The increases or decreases in Isc were considered significant at the P < 0.05 level. Statistical comparisons between two means were obtained with t-test (Mann–Whitney test, if n < 5). The data shown were expressed as mean SEM values (n = number of tissues) of the Isc.
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Fig. 1. Effect of dopamine (100 mM) on short circuit current (Isc) in the absence or presence of amiloride (10 mM) on the epithelial cells of the Reissner’s membrane. (A) The perfusion of dopamine partially increased amiloride-sensitive Na+ absorption. (B) The perfusion of amiloride abolished most of the baseline Isc. The application of dopamine in the presence of amiloride did not change Isc.
3. Results 3.1. Effect of dopamine on Na+ absorption in the epithelial cells of RM We measured the baseline Isc of RM in the perilymph-like control solution and observed a change in the Isc after application of dopamine (100 mM) or amiloride (10 mM). Negative baseline Isc values were observed in the control solution before the application of dopamine or amiloride (Fig. 1, Table 1). The perfusion of dopamine led to a significant increase in the Isc (28.1 2.6%, n = 4), and the subsequent addition of amiloride inhibited the Isc completely (Fig. 1A, Table 1). However, the perfusion of dopamine after the pretreatment with amiloride which abolished most of the baseline Isc showed no change in Isc (Fig. 1B, Table 1). Dopamine increased amiloride-sensitive Na+ absorption in a dose-dependent manner (from 1 mM to 1 mM). Isc was increased by 4.3 2.1%, 12.4 2.2%, 27.2 1.7%, and 29.6 0.7% after application of dopamine at different concentrations of 1 mM, 10 mM, 100 mM, and 1 mM, respectively (n = 6, Fig. 2). The data were fitted to the Hill equation (Fig. 2B). 3.2. Blockade of dopamine effect by dopamine receptor antagonists We applied dopamine receptor antagonists to determine the subtype of dopamine receptor which is responsible for the dopamine-mediated increase in ENaC function. The effects of dopamine on the Na+ absorption in the absence and presence of
each antagonist were compared (Fig. 3, Table 2). We performed control experiments that consisted of 2 consecutive applications of dopamine (100 mM), with a 3-min interval (Fig. 3A). The increase of Isc by the second application of dopamine was insignificantly reduced (12.0 10.7%, n = 4) compared with that evoked by the first application (Fig. 3A, Table 2). The data obtained from the experiments using dopamine receptor antagonists were statistically compared with those from the control experiments (Fig. 3B and C). The application of the specific D1-like receptor (D1 and D5) blocker, SCH-23390 (100 mM), inhibited most of dopamine’s effect on ENaC function (n = 4; Fig. 3B, Table 2). The magnitude of the dopamine-evoked increase in Isc was reduced by 81.9 10.3% in the presence of SCH-23390 (n = 4; Fig. 3B, Table 2) compared with its absence. This (81.9% reduction) was significantly different from that observed in the control experiments (12.0% reduction). In contrast, the D2-like receptor (D2, D3, and D4) blocker, sulpiride (100 mM), did not significantly block the effect of dopamine on ENaC activity (n = 4; Fig. 3C, Table 2). The perfusion of bromocriptine (100 mM), which is an agonist of D2-like receptors, especially D2 and D3 receptors, did not significantly change Isc (n = 5; Fig. 3D, Table 2). 3.3. Dopamine effect on the ENaC activity after inhibition of the Na+– K+-ATPase by ouabain Previous studies have reported that activation of dopamine receptors has distinct effects on the function of the Na+–K+-ATPase in lung and kidney epithelia [to see review, 12]. The Na+–K+-ATPase
Table 1 Effects of dopamine (100 mM) on Isc (mA/cm2) in the absence or presence of amiloride (10 mM).
Dopamine effect in the absence of amiloride (n = 4) Dopamine effect in the presence of amiloride (n = 3) a * z
Baseline
Dopamine
Amiloride
Amiloride + dopamine
14.1 2.1* 15.3 1.9
19.9 3.1* NT
NTa 1.7 0.7z
0.1 0.3 2.1 0.8z
Not tested. P < 0.05, between baseline Isc and Isc after dopamine application. P > 0.05, between Isc during amiloride perfusion and Isc after dopamine application in the presence of amiloride.
Fig. 2. Dose–response relationship of dopamine. (A) Representative traces of dopamine response at different concentrations from 1 mM to 1 mM. (B) EC50 was 16.8 mM (n = 6), which was obtained by fitting the data to the Hill equation using IGOR Pro 4.01 (WaveMetronics, Lake Oswego, OR).
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Fig. 3. Functional characterization of the dopamine-responsive receptor subtype. (A) The control experiment using a protocol consisting of 2 consecutive applications of dopamine (100 mM) with a 3-min interval. (B) The dopamine response was blocked by 100-mM SCH-23390 (D1-like receptor antagonist). (C) Sulpiride (D2-like receptor antagonist) at 100 mM did not significantly inhibit the dopamine effect. (D) The application of 100-mM bromocriptine (D2-like receptor agonist) did not significantly change Isc. Table 2 Effects of dopamine (100 mM) on Isc (mA/cm2) in the absence or presence of dopamine receptor antagonists (see Fig. 3).
Baseline First dopamine Dopamine washout Antagonist Second dopamine Reduction in the effect of second dopamine compared with first dopamine (%) a b c d jj
Controla n = 4
SCH-23390 (100 mM) n = 3
Sulpiride (100 mM) n = 4
15.3 1.7 19.7 1.1 16.2 1.4 NTb 19.9 1.3 12.0 10.7c
14.1 1.3 18.0 1.6 14.4 1.7 14.3 1.3 15.0 1.0 81.9 10.3d,jj
13.5 1.6 16.3 1.4 13.4 1.5 13.4 1.7 15.8 1.3 16.8 14.8d
This experiment used a protocol that consisted of 2 consecutive applications of dopamine (100 mM) with a 3-min interval. Not tested. 100 {(second dopamine dopamine washout)/(first dopamine baseline)} 100. 100 {(second dopamine antagonist)/(first dopamine baseline)} 100. P < 0.05. This value was significantly different from the value (12.0%) observed in the control experiments.
has been found to be expressed on the basolateral membrane of the epithelial cells of RM [7], and known to provide the driving force for Na+ entry via ENaC. It is conceivable that dopaminergic modulation of Na+–K+-ATPase activity may alter Na+ absorption via ENaC independently of any direct effects of dopamine on ENaC. Therefore, we conducted experiments to determine whether the activation of ENaC by dopamine is still observed after inhibition of the Na+–K+-ATPase. The application of ouabain (1 mM), a Na+–K+ATPase inhibitor, reduced Isc from 15.0 1.4 to 9.6 1.7 mA/ cm2 (n = 4), and the subsequent addition of dopamine resulted in an increase in Isc to 11.7 1.6 mA/cm2 (n = 4, Fig. 4). Thus, dopamine clearly increased ENaC activity even when the Na+–K+-ATPase was inhibited, which indicates that dopamine is capable of regulating ENaC function independently of a change in Na+–K+-ATPase activity. 3.4. Effect of 8-bromo-cAMP on Isc We investigated whether the increase of intracellular cAMP could regulate ENaC activity by using a membrane permeable cAMP analog, 8-bromo-cAMP (200 mM). The results showed that the application of 8-bromo-cAMP did not change Isc (n = 5; Fig. 5).
Fig. 4. Dopamine increases ENaC activity after the Na+–K+-ATPase was blocked by ouabain. After basal Isc had been obtained, 1-mM ouabain was added to the bath solution. When ouabain was perfused long enough for Isc to reach a plateau, dopamine was subsequently added. Isc was significantly increased after the perfusion of dopamine. These results indicate that dopamine’s effect on ENaC was not secondary to its activation of the Na+–K+-ATPase.
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Fig. 5. The cAMP analogue, 8-bromo-cAMP, did not mimic the effects of dopamine, showing no change in Isc after application.
3.5. Immunohistochemical analysis of dopamine receptor expression Immunoreactivity for dopamine D1 receptors was observed in RM (Fig. 6A), and stria vascularis (Fig. 6B). However, RM was not immunopositive for D2 receptors (Fig. 6D). The spiral ganglion neurons showed immunoreactivity for both D1 and D2 receptors (Fig. 6C and E). The anti-D1 antibody pre-absorbed with an excess of antigenic peptide resulted in no staining, and omitting the primary antibody from the procedure also produced negative results (not shown). 4. Discussion In the present study, the application of dopamine significantly increased Isc whereas the subsequent addition of amiloride abolished Isc. On the other hand, in the presence of amiloride, dopamine had no effect on Isc. These results indicate that dopamine
increased the amiloride-sensitive Na+ absorption in the epithelial cells of RM. Dopamine receptors are G protein-coupled receptors, and consist of 5 subtypes (D1 to D5). These subtypes are divided into two major groups: D1-like (D1 and D5) and D2-like receptors (D2, D3, and D4) [13]. In our results, the effect of dopamine on ENaC activity was blocked by SCH-23390, a D1-like receptor blocker, but not by sulpiride, a D2-like receptor blocker [13]. The application of bromocriptine, and agonist of D2-like receptors, had no effect on Isc. Furthermore, immunoreactivity for D1 receptors was observed in RM. Taken together, these results indicate that the effect of dopamine on ENaC is mediated by D1-like receptors. These results are consistent with the observation that stimulation of D1 dopamine receptors in lung alveolar epithelium increased ENaC activity [14]. However, the possibility that the D5 receptor is also involved in the effects of dopamine on RM cannot be ruled out. It is known that dopamine receptors have opposing intracellular signaling pathway, in that D1-like receptors activate adenylyl cyclase (AC) to increase cAMP production, whereas D2-like receptors inhibit AC to decrease cAMP production or to activate phospholipase. However, our results showed that the direct application of 8-bromo-cAMP did not change Isc. This implies the possibility of other signaling mechanisms via D1 receptors, which requires further investigation. Interestingly, the presence of a high activity of AC and a high steady-state level of cAMP in RM has been reported [15]. It has been proposed that a primary function of RM may be Cl transport under the control of cAMP [16]. The perilymphatic perfusion of forskolin, which activates AC leading to an increase in cAMP levels, evoked changes in the electrical resistance of the perilymph-endolymph barrier, endocochlear potential, and endolymphatic Cl concentration, which was interpreted as the presence of electrogenic Cl transport in RM under the control of cAMP [16]. However, in the present study, there was no change in Isc in response to the dopamine application in the presence of amiloride (Fig. 1B), which indicates that application of dopamine does not elicit cAMP-mediated Cl transport. These results are in line with previous findings in
Fig. 6. Immunohistochemical localization of the D1 receptors in the cochlea. Immunoreactivity was observed in the Reissner’s membrane (A) and stria vascularis (B). (C) Spiral ganglion neurons were immunopositive for the D1 receptors. (D) D2 receptors were not immunolocalized in the Reissner’s membrane. (E) Spiral ganglion showed immunoreactivity for D2 receptors. Scale bars = 50 mm.
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which Isc was shown to be insensitive to forskolin, an activator of cAMP production via AC, and genistein known to stimulate cAMPdependent Cl currents via cystic fibrosis transmembrane conductance regulator (CFTR) Cl channels when amiloride was co-perfused [1]. However, these findings could be attributable to the experimental conditions used in the previously published reports and the present study, which would not support the involvement of cAMP-related cascades; therefore, this pathway requires further investigation. In lung alveolar epithelial cells, the activity of ENaC at the apical membrane and Na+–K+-ATPase at the basolateral membrane is increased by the stimulation of dopamine receptors, which synergistically facilitates clearance of alveolar fluid [to see review, 12]. The Na+–K+-ATPase provides the driving force for Na+ influx via ENaC by actively transporting Na+ out of the cell through the basolateral membrane. Stimulation of dopamine receptors in the lung or kidney epithelia has opposing effects on the function of the Na+–K+-ATPase, in that dopamine increases Na+–K+-ATPase activity in the lung, whereas it decreases Na+–K+-ATPase activity in the kidney epithelia [12]. The presence of the Na+–K+-ATPase in RM was reported [7], and application of ouabain, a Na+–K+-ATPase inhibitor, significantly reduced Isc [1]. Therefore, we determined whether the change in Isc in response to dopamine application was caused entirely by an alteration of Na+–K+-ATPase activity. The present study shows that the dopamine-mediated increase in ENaC activity was not a secondary effect of the Na+–K+-ATPase stimulation by dopamine, as dopamine-mediated ENaC activation was still observed after the blockade of Na+–K+-ATPase activity by ouabain (Fig. 4). In the cochlea, dopamine plays an important role in the sensory process by modulating afferent auditory nerve activity [9]. Dopamine is released from the terminals of lateral olivocochlear efferent fibers, and is known to serve a protective function against acoustic trauma, hypoxia, and ototoxicity [10]. Recently, it has been proposed that the activation of dopamine receptors at efferent/afferent synapses has a level-dependent response to stimulation, such that D2 receptors may dominate to enhance inhibition during high sound levels [10], whereas D1 receptors may dominate to enhance excitation during low sound levels [9]. The distribution of dopamine receptors in the inner ear, however, has been reported to be more diversely localized not only in the postsynaptic region of efferent fibers, but also in spiral ganglion neurons and probably also in strial marginal cells [9], which is in agreement with our immunohistochemical results. Neither the natural sources of dopamine, nor its concentration in the cochlear fluid has been reported. The baseline level of dopamine in lung tissue is quite high, even higher than that reported in the adrenal gland. It has been suggested that dopamine is important in lung liquid removal at birth because the plasma level of dopamine increases 10-fold immediately before delivery [17]. The lung alveolar epithelia and epithelia of kidney proximal tubules are important sources of extraneuronal dopamine, and the concentration of dopamine in blood circulation is increased postprandially. In kidney epithelia, L-dopa, the dopamine precursor, is transported into the epithelium, and then converted into dopamine by L-amino acid dopa decarboxylase before dopamine is secreted [18]. At present, it is not known whether dopamine is locally formed and exerts its action in an autocrine or paracrine manner in parasensory epithelia in the inner ear, as has been demonstrated in the kidney. Although RM and basilar membrane in mammals have been known to be avascular structures, there have been some reports demonstrating the presence of unusual blood vessels in RM [19]. On the other hand, efferent nerve terminals located in the perilymphatic space beneath the inner and vestibular hair cells are so far the only
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anatomical sites of dopamine release as a neurotransmitter in the inner ear [9]. Small molecules, such as dopamine, might enter the endolymphatic space from the perilymphatic space by pinocytosis through epithelial cells lining the endolymphatic surface, such as RM epithelia, Claudius’ cells, and Hensen’s cells [20]. Unfortunately, we were unable to identify the sub-cellular localization of the D1 receptor in the epithelial cells of RM, as RM is an extremely thin, 2-cell layer tissue. In fact, owing to this specific feature of RM, we were also unable to obtain a sufficient amount of tissue to extract RNA for RT-PCR in this small epithelial domain. The findings of the present study might provide an important role for dopamine in maintaining endolymphatic homeostasis. When Na+ moves across RM from the endolymphatic space, water follows Na+ and, consequently, is cleared from the endolymphatic space, a process that can be enhanced by dopamine. This effect may be counteracted and balanced by the activity of P2Y4 purinergic receptor which was shown to inhibit Na+ absorption in gerbil RM [3]. However, the concentration of dopamine in the endolymphatic fluid has not been reported yet, and plasma concentration of dopamine is known not to exceed tens of nanomolar level [21]. The concentrations of dopamine used in the present study (1 mM to 1 mM) are quite high and conditions that could elevate dopamine level in the endolymph have not been reported, therefore, the physiologic role of this response in vivo is still unclear. In conclusion, dopamine increased Na+ absorption in freshly dissected RM possibly via D1-like receptors and the intracellular mechanisms remain to be elucidated. Conflict of interest None. Acknowledgement This work was supported by a grant of the Korean Health Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A100630). References [1] Lee JH, Marcus DC. Endolymphatic sodium homeostasis by Reissner’s membrane. Neuroscience 2003;119:3–8. [2] Kim CH, Kim HY, Chang OS, Oh SH, Lee JE, Lee JH. Developmental change of Na(+)-absorptive function in Reissner’s membrane epithelia. Neuroreport 2009;20:1275–8. [3] Kim CH, Kim HY, Lee HS, Chang SO, Oh SH, Lee JH. P2Y4-mediated regulation of Na+ absorption in the Reissner’s membrane of the cochlea. J Neurosci 2010;30:3762–9. [4] Lee JH, Chiba T, Marcus DC. P2X2 receptor mediates stimulation of parasensory cation absorption by cochlear outer sulcus cells and vestibular transitional cells. J Neurosci 2001;21:9168–74. [5] Yoo JC, Kim HY, Han KH, Oh SH, Chang SO, Marcus DC, et al. Na+ absorption by Claudius’ cells is regulated by purinergic signaling in the cochlea. Acta Otolaryngol Suppl 2012;132:103–8. [6] Couloigner V, Fay M, Djelidi S, Farman N, Escoubet B, Runembert I, et al. Location and function of the epithelial Na channel in the cochlea. Am J Physiol Renal Physiol 2001;280:F214–22. [7] Iwano T, Yamamoto A, Omori K, Akayama M, Kumazawa T, Tashiro Y. Quantitative immunocytochemical localization of Na+,K+-ATPase alpha-subunit in the lateral wall of rat cochlear duct. J Histochem Cytochem 1989;37:353–63. [8] Kim KX, Marcus DC. Inward-rectifier chloride currents in Reissner’s membrane epithelial cells. Biochem Biophys Res Commun 2010;394:434–8. [9] Niu X, Canlon B. The signal transduction pathway for the dopamine D1 receptor in the guinea-pig cochlea. Neuroscience 2006;137:981–90. [10] d’Aldin C, Puel JL, Leducq R, Crambes O, Eybalin M, Pujol R, et al. Effects of a dopaminergic agonist in the guinea pig cochlea. Hear Res 1995;90: 202–11. [11] Marcus DC. Vibrating probes: new technology for investigation of endolymph homeostasis. Keio J Med 1996;45:301–5. [12] Bertorello AM, Sznajder JI. The dopamine paradox in lung and kidney epithelia: sharing the same target but operating different signaling networks. Am J Respir Cell Mol Biol 2005;33:432–7.
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