Apical membrane P2Y4 purinergic receptor controls K+ secretion by strial marginal cell epithelium

Background It was previously shown that K+ secretion by strial marginal cell epithelium is under the control of G-protein coupled receptors of the P2Y family in the apical membrane. Receptor activation by uracil nucleotides (P2Y2, P2Y4 or P2Y6) leads to a decrease in the electrogenic K+ secretion. The present study was conducted to determine the subtype of the functional purinergic receptor in gerbil stria vascularis, to test if receptor activation leads to elevation of intracellular [Ca2+] and to test if the response to these receptors undergoes desensitization. Results The transepithelial short circuit current (Isc) represents electrogenic K+ secretion and was found to be decreased by uridine 5'-triphosphate (UTP), adenosine 5'-triphosphate (ATP) and diadenosine tetraphosphate (Ap4A) but not uridine 5'-diphosphate (UDP) at the apical membrane of marginal cells of the gerbil stria vascularis. The potencies of these agonists were consistent with rodent P2Y4 and P2Y2 but not P2Y6 receptors. Activation caused a biphasic increase in intracellular [Ca2+] that could be partially blocked by 2-aminoethoxy-diphenyl borate (2-APB), an inhibitor of the IP3 receptor and store-operated channels. Suramin (100 μM) did not inhibit the effect of UTP (1 μM). The ineffectiveness of suramin at the concentration used was consistent with P2Y4 but not P2Y2. Transcripts for both P2Y2 and P2Y4 were found in the stria vascularis. Sustained exposure to ATP or UTP for 15 min caused a depression of Isc that appeared to have two components but with apparently no chronic desensitization. Conclusion The results support the conclusion that regulation of K+ secretion across strial marginal cell epithelium occurs by P2Y4 receptors at the apical membrane. The apparent lack of desensitization of the response is consistent with two processes: a rapid-onset phosphorylation of KCNE1 channel subunit and a slower-onset of regulation by depletion of plasma membrane PIP2.


Background
A high concentration of K + is maintained in the lumen of the cochlea via electrogenic secretion by the strial mar-ginal cell epithelium [1]. One pathway of regulation is the coupling of purinergic receptors on the apical membrane of these cells to the apical potassium channels (IKs) which mediate secretion [2]. These receptors are responsive to both ATP and UTP as agonists and they were found to exert their action via the phospholipase C -protein kinase C intracellular signal pathway [3]. At the time of the original investigations, the purinergic receptor field recognized only 1 receptor responding to uracil nucleotides (P2U, now P2Y 2 receptor). The known members of the mammalian metabotropic, G protein-coupled, purinergic receptors has since grown to P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12 , P2Y 13 and P2Y 14 [4]. In addition to P2Y 2 , both the P2Y 4 and P2Y 6 receptors also respond to uracil nucleotides. On the basis of pharmacologic agonist and antagonist profiles, P2Y 6 could be distinguished from P2Y 2 and P2Y 4 by the greater potency of UDP over UTP [5,6]. The pharmacologic distinction between P2Y 2 and P2Y 4 was more ambiguous and the accepted criteria changed rapidly [7].
The question of the subtype of P2Y receptor mediating regulation of K + secretion by strial marginal cells has been addressed by immunohistochemistry, which suggests the presence of P2Y 4 in the apical marginal cell membrane [8]. However, a functional demonstration was lacking, the time-course of activation was not investigated and an independent demonstration of gene expression was not available.
The present study was conducted with the goals of 1) obtaining dose-response profiles for potentially definitive agonists of P2Y 2 , P2Y 4 and P2Y 6 receptors; 2) refining the profile by the use of antagonists, 3) testing for desensitization of the response to agonist and 4) determining the presence of transcripts for pyridine-sensitive P2Y receptors. The results support the conclusion that regulation of K + secretion by strial marginal cells occurs by apical P2Y 4 receptors.

Results
ATP perfused for 30 s at the apical side of strial marginal cell epithelium caused a monophasic decrease in I sc (Fig.  1A), consistent with previous findings [2]. Removal of ATP led to a recovery of I sc after an initial overshoot. Similar responses were seen from perfusion of other agonists, such as UTP and Ap4A (Fig. 1B &1C).
The P2Y 6 agonist, UDP, was tested after removal of contamination by UTP in the commercial product [6]. Contaminating UTP was digested by hexokinase in the presence of glucose. The enzymatically-purified UDP produced little effect on I sc (Fig. 2).
Concentration-response curves for the purinergic agonists are summarized in Figure 2. The potency order was UTP ≥ ATP > Ap4A >> UDP with EC 50 values for UTP and ATP of about 2.3 and 1.2 × 10 -5 M. The relative potencies of the agonists are consistent with an action on P2Y 2 and/or P2Y 4 receptors.
The purinergic receptor antagonist, suramin, was tested for effectiveness in blocking the response to apical UTP. Suramin at 100 µM had no inhibitory effect on the action of the agonist, UTP (Fig. 3). UTP (1 µM) caused a decrease in I sc by 20.6 ± 5.4% in the absence of suramin and by 21.9 ± 5.3% (P > 0.05, n = 5) in the presence of suramin.
Both P2Y 2 and P2Y 4 receptor subtypes have been reported to undergo desensitization within 5-15 min [9,10]. Surprisingly, we found that sustained exposure (15 min) of the apical membrane to ATP led to a biphasic but sustained inhibition of I sc (Fig. 4). The recovery of I sc after removal of agonist was much slower, incomplete and without overshoot compared to acute exposure to agonist.  Perfusion of UTP (10 µM) for 30 s led to an increase in intracellular [Ca 2+ ] that had both a peak and plateau phase and was repeatable (Fig. 5, top). Both the peak and plateau were significantly reduced by 2-APB (75 µM) (to 26.5 ± 3.9% and 25.1 ± 4.6%; Fig. 5, middle). 2-APB is an inhibitor of the IP3 receptor at this concentration [11,12] and can also inhibit other transporters including storeoperated Ca 2+ channels [13]. Store operated channels, however, would not be activated under the present experimental conditions, since the Ca 2+ stores were not emptied prior to measurement. In the absence of external Ca 2+ , both the peak and plateau responses to UTP remained but were significantly reduced (to 68.9 ± 3.0% and 60.5 ± 4.2%; Fig. 5, bottom). The reductions, however, were likely the result of a general decrease in intracellular [Ca 2+ ] seen after removal of bath Ca 2+ prior to perfusion of UTP. These findings taken together suggest that intracellular stores are the primary source of the increase in intracellular [Ca 2+ ] induced by UTP.
We tested for the presence of transcripts for P2Y 2 and P2Y 4 receptors in stria vascularis. Primers proven to recognize gerbil P2Y 2 and P2Y 4 [14] were used to amplify single, gene-specific bands in stria vascularis (Fig. 6); 301 base pairs (bp) for P2Y 2 and 447 bp for P2Y 4 . Controls in which the reactions were run in the absence of reverse transcriptase (-RT) demonstrated the absence of contributions from genomic DNA. PCR products were analyzed for their sequence to confirm the identity of the bands. Sequences were the same as found previously in the gerbil vestibular labyrinth [14] (GenBank accession numbers: P2Y 2 , AF313448; P2Y 4 , AF313447).

Discussion
Purinergic agonists control strial marginal cell K + secretion, a process observed as a transepithelial short circuit current, I sc [1,2]. Strial marginal cells secrete K + by a constellation of transporters previously described [1]. K + is taken up across the basolateral membrane by the Na + , K + -ATPase and the Na + , K + , 2Cl --cotransporter. Na + carried into the cell on the cotransporter is removed by the Na +pump and Clcarried into the cell on the cotransporter leaves by passive diffusion across a large Clconductance in the basolateral membrane [15][16][17] [6]. In fact, ATP acts as a competitive antagonist at the hP2Y 4 receptor [25]. However, these results alone do not identify the strial marginal cell apical purinergic receptor as P2Y 2 since it was subsequently found that rP2Y 4 has an agonist potency sequence similar to rat and human P2Y 2 [7,25]. Since the gerbil is expected to be more closely related to other rodents such as the rat, than to human, our finding is consistent with the contribution of P2Y 4 and/or P2Y 2 . Transcripts for both P2Y 2 and P2Y 4 were found in stria vascularis. The identity of the cell type(s) within the stria that contain these transcripts is not certain from these findings alone. Recent immunohistochemical findings show staining for the P2Y 4 receptor at the apical membrane of strial marginal cells, while the antibody for P2Y 2 stained at the basolateral region of the marginal cells and/or the intermediate cells [8], consistent with the functional evidence for apical P2Y 4 shown here. The transcript expression confirms the immunohistochemical findings by an independent means and thereby provides an important verification of the specificity of the P2Y antibodies used in the previous study [8].
Ap4A was found to be a potent agonist at the hP2Y 2  This conclusion based on function and pharmacology is consistent with the recent report of immunohistochemical localization of P2Y 4 at the apical membrane of strial marginal cells [8]. We reported earlier that the response to ATP is increased in the absence of divalent cations [2]. This increased effect suggests that the apical receptor is preferentially activated by an uncomplexed form of ATP, as in aortic endothelial cells [30].
The overshoot of I sc after removal of purinergic agonists from the apical perfusate ( Fig. 1) is most likely due to a release of K + accumulated in the epithelial cells during the inhibition of secretion across the apical membrane. The basolateral K + uptake mechanisms continue to operate for a time after inhibition of apical IKs channel complexes by activation of purinergic receptors, bringing the cytosolic K + concentration to a level higher in electrochemical potential above the apical bath than in the absence of agonist. Upon removal of agonist this "extra pool" of K + is suddenly released, resulting in the observed overshoot of I sc . The same phenomenon was reported previously [31] when K + secretion was first diminished by raising the apical K + concentration, thereby reducing the outward gradient across the apical cell membrane. Suddenly returning the apical perfusate K + concentration to the original low level led to an overshoot of I sc , as observed with the purinergic agonists.
The decrease in I sc observed in response to purinergic agonists could be due a priori to a reduction of electrogenic K + secretion but could also be accounted for by a stimulation of secretion of Clor absorption of Na + . However, it was found in a previous study that the decrease in I sc could be completely accounted for by a decrease in K + secretion. Apical perfusion of 100 µM ATP lead to a decrease of I sc by 37.1 ± 3.7% and of K + secretory flux by 22.2 ± 5.5% [3]. Those cells were derived from guinea pig, the responses were monophasic and UTP was not tested as agonist.
The apparent absence of desensitization (Fig. 4) was a surprising finding in view of reports of rapid desensitization of both cloned P2Y 2 and cloned P2Y 4 purinergic receptors [9,10]. Desensitization has typically been observed as a decrease of inositol phosphate production and/or cytosolic Ca 2+ increase, either directly [34,35] or via the effect of Ca 2+ on transepithelial anion secretion [36].
Since the sustained response to UTP (Figure 4) is biphasic, it therefore may represent the summation of at least 2 processes.  The question arises as to the basis for the observed sustained decrease in IKs. Possible explanations include: 1) The P2Y 4 receptor desensitizes several minutes after the onset of agonist, but the initial activation of P2Y 4 leads to phosphorylation of KCNE1 via PKC [3] and the channel subunit remains phosphorylated due to inhibition of phosphatase activity. 2) Activation of P2Y 4 leads to localized depletion of phosphatidylinositol-4,5-bisphosphate (PIP 2 ) in the plasma membrane and to the consequent deactivation of the PIP 2 -dependent IKs channel.

Concentration-response curves for inhibition of Isc
I sc is controlled by apical P2Y 4 receptors via the protein kinase C pathway by phosphorylation of the beta subunit of the IKs channel complex [3] [vida infra]. The sustained reduction in I sc and the incomplete recovery of I sc after prolonged exposure to agonist can both be explained if there is a decrease of phosphatase activity at the phosphorylation site. The channel subunit would then be expected to remain phosphorylated and the effect on I sc would be sustained even with a desensitization of the receptor. However, dephosphorylation rates have been reported to increase with elevated intracellular [Ca 2+ ] in olfactory sensory neurons [37], arguing against this hypothesis.
The second explanation is more consistent with our findings. It has been found that activation of receptors (such as P2Y 4 ) coupled to G q/11 deplete PIP 2 [38] and that IKs (KCNQ1/KCNE1) activity is controlled by PIP 2 [39]. In fact, the time course of desensitization of another K + channel by phenylephrine induced depletion of PIP 2 via α 1adrenergic receptors is similar to the secondary effects seen here [38].

Physiological significance
Purinergic receptors have been identified on the apical membrane of many of the cells forming the border of the cochlear duct [40][41][42]. Indeed, IKs of strial marginal cells in the cochlea is inhibited by UTP in much the same way as the homologous vestibular dark cells [2,17,27], although the lower potency of agonists on marginal cells suggests a lower density of receptors. The P2Y 4 receptors can thereby provide the strial marginal cells with an autocrine as well as paracrine signaling pathway. Autocrine signaling is important for these cells since they have no gap junction communication [43], an unusual occurrence for epithelial cells. Paracrine signaling is also important in this organ since the rate of K + secretion must be adjusted for variations in K + efflux during stimulation of the cochlea by sound. In fact, the P2 receptors have been proposed to act as a mechanism to protect the inner ear from noise damage [44].

Conclusion
The response to purinergic agonists at the apical side of strial marginal cells was shown here to be functionally mediated by P2Y4 receptors, previously shown to be present by immunostaining. The decrease in electrogenic K + secretion evoked by agonists was a) initially accompa-

ATP
nied by a biphasic increase in intracellular Ca 2+ and b) surprisingly sustained in the continued presence of agonist. The sustained decrease in K + secretion was best explained by rapid-onset phosphorylation of KCNE1 channel subunit and a slower-onset of regulation by depletion of plasma membrane PIP 2 . Impaired PIP 2 and IKs interaction was recently shown to be the basis of several mutations causing long QT syndrome, a genetic disease characterized by cardiac arrhythmias and deafness [45].

Animals and tissues
Gerbils (4-5 week old females) were anesthetized by injection of sodium pentobarbital (50 mg/kg, i.p.) and the temporal bones were removed. The method for dissecting strial marginal cell epithelium from the cochlear lateral wall was described previously [1]. Dissected epithelia were either transferred to a micro-Ussing chamber for measurement of the equivalent short circuit current (I sc ), to a perfusion chamber for [Ca2+] measurement or were frozen in liquid nitrogen within 10 min of death for reverse transcription-polymerase chain reaction (RT-PCR). All procedures conformed to protocols approved by the Institutional Animal Care and Use Committee.

Short-circuit current measurements
The micro-Ussing chamber for inner ear tissues has been described previously [3]. Briefly, the diameter of the aperture separating the apical and basolateral side half-chambers was 80 µm and each side was continuously perfused independently at 37°C with an exchange of solution accomplished within 1 s. I sc was measured with a 4-wire epithelial current clamp and recorded with a computer data acquisition system with 16 bits resolution. Samples were acquired at 32 Hz and decimated by a factor of 10. Perfusion changes were planned and carefully timed so that experiments from each experimental series could be averaged (Figures 1 &4). Results were analyzed and plotted using Origin software (OriginLab, Northampton, MA).
In one series of experiments (Figure 3), the relative I sc was measured with a vibrating probe as described previously [46]. Briefly, the lateral cochlear wall with stria vascularis was mounted in a perfusion chamber on the stage of an inverted microscope (Nikon TE-300) and continuously perfused with the same solution used for the micro-Ussing chamber at 37°C. The I sc relative to the initial control value was monitored by vibrating a platinum-iridium wire microelectrode coated with Pt-black on the tip. The probe was positioned 20-30 µm from the apical surface of the epithelium. The bath references were 26-gauge Ptblack electrodes. The probe tip was vibrated in the range of 200-400 Hz over an excursion of about 20 µm. Vibration between two positions within the line of current flow yields voltages in the low nanovolt range that correspond to current flow through the resistive physiological saline [47]. The output of the probe amplifier was recorded with ASET software (Science Wares, East Falmouth, MA). Although the single perfusate reached the entire stria vascularis, it was determined that brief changes of solution (≤30 s) effectively reached only the apical surface; the extremely tight junctions of the basal cell layer restricted diffusion of large molecules such as agonists and antagonists to the interior of the stria and to the basolateral membrane of marginal cells [1].

Chemicals and solutions
In all experiments, both sides of the epithelium were perfused with a solution containing (in mM) NaCl 150, KH 2 PO 4 0.4, K 2 HPO 4 1.6, MgCl 2 1, CaCl 2 0.7, glucose 5, pH 7.4. All experimental agents were dissolved in this solution immediately before use. Suramin was purchased from Calbiochem (San Diego, CA) and adenosine 5'-tri-

Intracellular Ca 2+ measurements
Calcium was measured as described previously [48]. Briefly, stria vascularis was incubated for 20 min with the indicator dye 5 µM fluo-4-AM at 37°C (Molecular Probes, Eugene, OR) and mounted in the superfusion chamber on the stage of an inverted microscope (Diaphot, Nikon). The stria vascularis was folded into a loop with the marginal cell layer on the outside surface. An optical section of the marginal cells was observed and the recording slit, which defined the field of the Ca 2+ measurement, was restricted to this cell layer.
The preparation was alternately illuminated at 600 and 488 nm (Deltascan, Photon Technology International, South Brunswick, N.J.). The fluorescence signal was detected by a photon-counter (Photon Technology International) at a rate of 4 Hz. Changes in the emission intensity were taken as measures of changes in intracellular Ca 2+ ([Ca 2+ ] i ). The drift of signal due to leakage of dye from the cells was linearly subtracted.
The PCR mixture was incubated as follows: 1 denaturation cycle for 5 min at 98°C; 40 amplification cycles consisting of denaturation for 45 sec at 95°C, annealing for 45 sec at 58°C, and extension for 45 sec at 72°C; and one extension cycle for 7 min at 72°C. PCR products were analyzed by horizontal electrophoresis in 2.0% agarose gels and visualized by ethidium bromide.

Statistics
Data are expressed as the mean ± S.E.M. (n = number of tissues) of the I sc and concentration-response curves from changes in I sc were normalized to the response to 1 mM ATP. The Student's t-test of paired samples was used to determine statistical significance and increases or decreases in I sc were considered significant for P < 0.05.