1 Department of Anatomy and Physiology, Kansas State University, Manhattan, Kansas 66506; and 2 Department of Pharmacology, Creighton School of Medicine, Omaha, Nebraska 68178
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ABSTRACT |
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It was previously shown that K+ secretion by vestibular dark cell epithelium is under control of G protein-coupled receptors of the P2Y family in the apical membrane that are activated by both purine and uridine nucleotides (P2Y2, P2Y4, or P2Y6). The present study was conducted to determine the subtype of purinergic receptor and to test whether these receptors undergo desensitization. The transepithelial short-circuit current represents electrogenic K+ secretion and was found to be reduced by UTP, ATP, and diadenosine tetraphosphate, but not UDP. Neither pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 30 µM) nor suramin (100 µM) inhibited the effect of UTP. The potencies of the agonists were consistent with rodent P2Y4 and P2Y2, but not P2Y6, receptors. The ineffectiveness of suramin was consistent with P2Y4, but not P2Y2. Transcripts for both P2Y2 and P2Y4 were found in vestibular labyrinth. Sustained exposure to ATP or UTP for 15 min caused a constant depression of short-circuit current with no apparent desensitization. The results support the conclusion that regulation of K+ secretion across vestibular dark cell epithelium occurs by P2Y4 receptors without desensitization of the response.
desensitization; inner ear; vestibular labyrinth; gerbil
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INTRODUCTION |
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A HIGH CONCENTRATION of K+ is maintained in the lumen of the vestibular labyrinth via electrogenic secretion by the vestibular dark cell epithelium (15, 17). One pathway of regulation is the coupling of purinergic receptors on the apical membrane of these cells to the apical K+ (IKs) channels, which mediate secretion (13). 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 (19). At the time of the original investigations, the purinergic receptor field recognized only one receptor responding to UTP, termed the "P2U" receptor.
The nomenclature has evolved, and all the G protein-coupled purinergic receptors are grouped as the P2Y family, with subtypes designated by numbers. The original P2U receptor was renamed P2Y2, and additional receptors that respond to uridine nucleotide agonists were discovered: P2Y4 and P2Y6 (25). On the basis of pharmacological agonist and antagonist profiles, the P2Y6 receptor could be distinguished from P2Y2 and P2Y4 receptors by the greater potency of UDP over UTP (4, 24). The pharmacological distinction between P2Y2 and P2Y4 receptors was more ambiguous, and the accepted criteria changed rapidly (2).
The present study was conducted with the goals of 1) obtaining dose-response profiles for potentially definitive agonists of P2Y2, P2Y4, and P2Y6 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 vestibular dark cells occurs by apical P2Y4 receptors.
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METHODS |
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Gerbils (4-5 wk old females) were anesthetized by injection of pentobarbital sodium (50 mg/kg ip), and the temporal bones were removed. The method for dissecting vestibular dark cell epithelium from the ampulla was described previously (15). Dissected epithelia were transferred to a micro-Ussing chamber for measurement of equivalent short-circuit current (Isc) or frozen in liquid nitrogen within 10 min of death for reverse transcription-polymerase chain reaction (RT-PCR). The heart and brain were cut and frozen on dry ice or liquid nitrogen within 5 min of death. Animals were transcardially perfused with 0.1 M phosphate-buffered saline (pH 7.3) before collection of vestibular tissues for RT-PCR. All procedures conformed to protocols approved by the Institutional Animal Care and Use Committee.
The micro-Ussing chamber for inner ear tissues has been described
previously (15). 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.
Isc was measured with a four-wire epithelial
current clamp and recorded with a computer data acquisition system with
16-bit 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 (Figs.
1, 2, and 4-6).
Results were analyzed and plotted using OriginLab software version 6.1 (Northampton, MA).
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In all experiments, both sides of the epithelium were perfused with a solution containing (in mM) 150 NaCl, 0.4 KH2PO4, 1.6 K2HPO4, 1 MgCl2, 0.7 CaCl2, and 5 glucose, pH 7.4. All experimental agents were dissolved in this solution immediately before use, and the solutions were readjusted to pH 7.4. Suramin was purchased from Calbiochem (San Diego, CA), pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) from Tocris Cookson (St. Louis, MO), and adenosine 5'-triphosphate (ATP), uridine 5'-triphosphate (UTP), diadenosine tetraphosphate (Ap4A), and hexokinase from Sigma Chemical (St. Louis, MO).
Values are means ± SE (n = number of tissues) of the Isc and concentration-response curves from changes in Isc normalized to the response to 1 mM ATP. The Student's t-test of paired samples was used to determine statistical significance. Increases or decreases in Isc were considered significant for P < 0.05.
Transcripts for P2Y2 and P2Y4 receptors were assayed by RT-PCR using methods previously described for extraction of total RNA, DNase I treatment, PCR amplification, subcloning, and sequencing (19). First-strand cDNA synthesis was also performed as described previously (19) with the exception that 25 pmol of random hexamers were used to prime the RNA. The sequence of the primers was based on the known sequences in the coding region of the rat, mouse, and human receptors. P2Y2 primers [sense: 5'-GCTTCAACGAGGACTTCAAGTA(C/T)GTGC-3'; antisense: 5'-AGGTGAGGAAGAGGATGCTGCAGTAG-3'] and P2Y4 primers (sense: 5'-CCAGAGGAGTTTGACCACTA-3'; antisense: 5'-CACCAAGGCCAGGGAGGA-3') were expected to yield RT-PCR products of 301 bp for P2Y2 receptors and 447 bp for P2Y4 receptors, which were cloned and sequenced [GenBank accession numbers AF313448 (P2Y2) and AF313447 (P2Y4)].
The PCR mixture was incubated as follows: 1 denaturation cycle for 5 min at 98°C; 40 amplification cycles consisting of denaturation for 45 s at 95°C, annealing for 45 s at 58°C, and extension for 45 s at 72°C; and 1 extension cycle for 7 min at 72°C in a Perkin-Elmer thermocycler (model 2400). PCR products were analyzed by horizontal electrophoresis in 2.0% agarose gels and visualized by ethidium bromide.
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RESULTS |
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ATP perfused at the apical side of vestibular dark cell epithelium caused a monophasic decrease in Isc (Fig. 1A), consistent with previous findings (13). Removal of ATP led to a recovery of Isc after an initial overshoot. Similar responses were seen from perfusion of other agonists, UTP and Ap4A (Fig. 1, B and C). All three acted as full agonists.
The P2Y6 agonist uridine 5'-diphosphate (UDP) was found to
also yield a substantial response of the same type when the agonist was
perfused in its form as commercially supplied. Reports from other
laboratories suggested that commercial preparations of UDP may be
supplied with a minor component of UTP (24). We therefore compared the response to UDP as supplied with the response to UDP after
preincubation with hexokinase in the presence of glucose. The enzyme
converted the contaminating UTP to UDP + PO4. The
enzymatically purified UDP had little effect on
Isc (Fig. 2). Concentration-response curves for
the purinergic agonists are summarized in Fig.
3. The potency order was UTP > ATP > Ap4A UDP, with best-fit EC50 values of
1.8 ± 0.1 × 10
7, 5.5 ± 2.3 × 10
6, and 7.0 ± 0.7 × 10
5 M. The
potencies of the agonists are consistent with an action on
P2Y2 and/or P2Y4 receptors.
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The purinergic receptor antagonists suramin and PPADS were tested for
effectiveness in blocking the response to apical UTP. Neither suramin
at 100 µM (Fig. 4A) nor
PPADS at 30 µM (Fig. 4B) had any inhibitory effect on the
action of the agonist UTP. UTP (1 µM) caused a decrease of
Isc by 65.0 ± 1.8% in the absence of
suramin and by 62.7 ± 2.4% (P > 0.05, n = 5) in the presence of suramin. UTP (1 µM) caused
a decrease of Isc by 59.3 ± 2.4% in the
absence of PPADS and by 62.6 ± 2.7% (P > 0.05, n = 6) in the presence of PPADS.
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Both receptor subtypes have been reported to undergo desensitization
within 5-15 min (26, 27). Surprisingly, we found that
sustained exposure of the apical membrane to ATP or UTP led to a
sustained inhibition of Isc (Fig.
5). The recovery of
Isc after removal of agonist was much slower,
incomplete, and without overshoot.
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We investigated this phenomenon further by making multiple applications
of a high concentration of agonist in the absence and presence of a
sustained moderate concentration of agonist (Fig.
6). Three brief (20-s) pulses of a
maximal concentration of ATP (1 mM) led to nearly identical levels of
inhibition of Isc (Fig. 6A). The
first, second, and third pulses of ATP led to a decrease in the
Isc by 66.8 ± 2.2, 64.1 ± 3.1, and
60.5 ± 3.1% (n = 6), respectively, compared with
the initial Isc. The decrease from the third
pulse reached numerical statistical significance compared with the
decrease from the first pulse, but we ascribe no physiological
significance to this 4.7% difference. Interestingly, the speed of
recovery after each ATP pulse was reduced with each repetition. The
rates of recovery for the first, second, and third pulses of ATP were
39.2 ± 4.9, 24.9 ± 3.3, and 15.8 ± 2.2 µA · cm2 · s
1
(n = 6). The second and third rates differed
significantly from the first (P < 0.05). There was a
progressive decrease in Isc after each
withdrawal of ATP (Fig. 6). The initial Isc was
927 ± 89 µA/cm2; after the first, second, and third
pulses, Isc was 810 ± 71, 684 ± 64, and 594 ± 49 µA/cm2, respectively.
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A sustained presence of a moderate concentration of ATP (10 µM) had
no influence on the peak response to repeated applications of maximal
ATP. Pulsatile, brief perfusion of 1 mM ATP again led to the same level
of inhibition of Isc, and again the
recovery phase was slowed at subsequent repetitions and acute recovery was only partial (Fig. 6B). The first, second, and third
pulses of ATP led to a decrease in the Isc by
62.9 ± 3.1, 64.2 ± 3.6, and 64.6 ± 3.9%
(n = 6), respectively, compared with the initial Isc (before any ATP). The decreases from the
second and third pulses are not significantly different from the first
pulse. Similar to the previous case without sustained perfusion of 10 µM ATP, the speed of recovery after each ATP pulse was reduced with
each repetition. In addition, the rates were much slower than in the previous series. The rates of recovery for the first, second, and third
pulses of ATP were 12.0 ± 2.4, 7.6 ± 1.4, and 5.7 ± 1.2 µA · cm2 · s
1,
respectively (n = 6). The successive differences were
again statistically significant.
We tested for the presence of transcripts for P2Y2 and
P2Y4 receptors in gerbil vestibular labyrinth. Primers
designed on the basis of known sequences in other mammals were used to
isolate single gene-specific bands in gerbil heart and brain (Fig.
7A): 301 bp for
P2Y2 and 447 bp for P2Y4. Controls in which the
reactions were run in the absence of reverse transcriptase demonstrated the absence of contributions from genomic DNA. Strong single bands of
the same size were found for P2Y2 and P2Y4
receptors in vestibular labyrinth (Fig. 7B). Controls
(without reverse transcriptase) were again negative.
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PCR products were analyzed for their sequence to determine the identity
of the bands. The 301-bp cDNA sequence and its deduced amino acid
sequence correlated closely to those for rat (rP2Y2), mouse, and human P2Y2 (hP2Y2) receptors (Fig.
8). Similarly, the 447-bp cDNA sequence
and its deduced amino acid sequence correlated closely to those for rat
(rP2Y4), mouse, and human P2Y4
(hP2Y4) receptors (Fig. 9).
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DISCUSSION |
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Purinergic agonists control vestibular dark cell K+
secretion, a process observed as a transepithelial
Isc (15, 17). Vestibular dark cells
secrete K+ by a constellation of transporters previously
described (29). 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 Cl
carried into the cell
on the cotransporter leaves by passive diffusion across a large
Cl
conductance in the basolateral membrane (20,
30). K+ taken up by the Na+ pump and
cotransporter is secreted across the apical membrane by diffusion
through IKs channels (16), consisting of KvLQT1
-subunits and IsK or minK
-regulatory subunits (1,
28). The epithelium regulates K+ secretion by a
variety of signal pathways, including those initiated by apical
purinergic receptors, that converge at the IKs channel complex
(14).
Purinergic receptor subtypes have been problematic to define by agonist and antagonist potencies because of contamination of commercial supplies of agonists and interconversion of agonists by ectoenzymes (24). Indeed, we found that our source of UDP had ~1% contamination of UTP and needed to be treated with hexokinase. The agonist potency order defining the cloned purinergic receptors has changed because of these issues and because of strong differences recently discovered among species, including mammals, e.g., human and rat P2Y4 receptors.
The previous demonstration of control of Isc in
vestibular dark cell epithelium by P2U receptors included
determination of a concentration response to ATP and comparative
activities of other nucleotides (UTP, 2-methylthio-ATP, and
,
-methATP) at 1 µM (13). Full
concentration-response curves were obtained in the present study to
better define the active subtype. ATP, UTP, and Ap4A were found to be
full agonists, with the potency for ATP similar to that for UTP, much
less potency for Ap4A, and virtually no activity from enzyme-cleaned UDP.
The lack of response to UDP eliminated P2Y6 as a candidate subtype (7, 23, 31). The similar potency of ATP and UTP is consistent with hP2Y2 (23) and rP2Y2 (7), but not hP2Y4, receptors (24). In fact, ATP acts as a competitive antagonist at the hP2Y4 receptor (11). However, our result does not identify the dark cell apical purinergic receptor as P2Y2, since it was subsequently found that rP2Y4 has an agonist potency sequence similar to rP2Y2 and hP2Y2 (2, 11). Because the gerbil is expected to be more closely related to other rodents, such as the rat, than to humans, our finding is consistent with the contribution of P2Y4 and/or P2Y2.
Ap4A was found to be a potent agonist at the hP2Y2 receptor (23) but much less potent than ATP at the hP2Y4 (9) and rP2Y4 (31) receptors, similar to our finding in gerbil dark cells. Despite this similarity, uncertainty arises in the identification of the apical receptor in dark cells as P2Y4 on this basis alone, since there is also a conflicting report from Bogdanov et al. (2), who found that Ap4A and ATP were equally potent at rP2Y4 receptors.
Suramin inhibits several P2 receptors, but it was found recently that at high concentration (100 µM) it can be used to distinguish both rP2Y2 (7) and hP2Y2 (5) from both rP2Y4 (2) and hP2Y4 (5). This criterion applied to the present results points to the apical P2Y purinergic receptor in gerbil vestibular dark cells as the P2Y4 subtype.
PPADS is a purinergic receptor antagonist that potently inhibits P2Y1 receptors but has no effect on rP2Y4 receptors (2). This finding is consistent with the lack of effect of PPADS observed here on the apical P2Y receptor of vestibular dark cells. PPADS also has no effect on hP2Y2 receptors (6); there are no reports on the effect of PPADS on rP2Y2 receptors.
The overshoot of Isc 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 purinergic agonists, bringing the cytosolic K+ concentration to a level higher in electrochemical potential above the apical bath than in the absence of agonist. On removal of agonist, this "extra pool" of K+ is suddenly released, resulting in the observed overshoot of Isc. The same phenomenon was reported previously (21) 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 Isc, as observed with the purinergic agonists.
We reported earlier that the response to ATP is increased in the absence of divalent cations (13). This increased effect suggests that the apical receptor is preferentially activated by an uncomplexed form of ATP, as in aortic endothelial cells (22).
The decrease in Isc 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 Cl or absorption of
Na+. However, it was found in a previous study that the
decrease in Isc could be completely accounted
for by a decrease in K+ secretion. Apical perfusion of 100 µM ATP led to a decrease of Isc by 40.8 ± 3.0% and of K+ secretory flux by 40.9 ± 9.2%
(19).
The apparent absence of desensitization (Figs. 5 and 6) was a surprising finding in view of reports of rapid desensitization of cloned P2Y2 and cloned P2Y4 receptors (26, 27). Desensitization has typically been observed as a decrease of inositol phosphate production and/or cytosolic Ca2+ increase, either directly (3, 10) or via the effect of Ca2+ on transepithelial anion secretion (8).
The question arises whether the apparent absence of desensitization of
dark cell Isc represents a feature specific to
gerbil P2Y4 receptor or whether a desensitization of gerbil
P2Y4 receptor is simply not reflected in the end effect.
Our findings suggest the latter answer, although the signaling
mechanisms involved are not at all clear. Isc is
controlled by apical P2Y4 receptors via the protein kinase
C pathway by phosphorylation of the -subunit of the IKs channel
complex (19). The progressive slowing of the recovery of
Isc on removal of the agonist and the incomplete recovery of Isc after prolonged exposure to
agonist can be explained if there is a rundown of phosphatase activity
at the phosphorylation site. The channel subunit would then be expected
to remain phosphorylated and the effect on Isc
sustained, even with a desensitization of the receptor. The apparent
coordination of receptor desensitization and reduction in phosphatase
activity would occur through an as yet undetermined mechanism.
Similarly, incomplete recovery of Isc after
repeated acute exposure to agonist (Fig. 6) may be due to poorly
reversible phosphorylation of the
-subunit of the IKs channel
complex. Alternatively, key proteins in the desensitization pathway may
not be expressed in these cells.
Transcripts for P2Y2 and P2Y4 receptors were found in vestibular labyrinth. The identity of the cell type(s) within the vestibular labyrinth that contains these transcripts is not certain from these findings alone. Recent immunohistochemical findings show staining for both receptors in vestibular dark cells: the P2Y4 antibody reaction at the apical border and the P2Y2 antibody reaction toward the basolateral aspect (unpublished observations).
Physiological significance. Purinergic receptors have been identified on the apical membrane of many of the cells forming the border of the cochlear duct and likely also occur on the homologous vestibular cells. Indeed, IKs of strial marginal cells in the cochlea is inhibited by uridine nucleotide agonists in much the same way as the homologous vestibular dark cells (13, 18). This can provide the dark cells with an autocrine as well as a paracrine signaling pathway. Autocrine signaling is important for these cells, since they have no gap junction communication (12), 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 vestibular organ by acceleration. Variations in luminal P2 agonist concentrations have not yet been investigated but are clearly deserving of attention.
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NOTE ADDED IN PROOF |
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Identically to our results in gerbil vestibular dark cells, suramin has been found to inhibit mouse as well as rat and human P2Y4 receptor (Suarez-Huerta N, Pouillon V, Boeynaems J, and Robaye B. Molecular cloning and characterization of the mouse P2Y4 nucleotide receptor. Eur J Pharmacol 416: 197-202, 2001). In contrast to our results, Suarez-Huerta et al. found PPADS to be a strong inhibitor of both mouse and rat P2Y4 expressed in 1321N1 cells. The authors found that the agonist potency order for ATP and UTP was similar in the mouse, rat, and gerbil P2Y4 receptor in contrast to that for the human P2Y4 receptor.
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ACKNOWLEDGEMENTS |
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We thank Jianzhong Liu for technical assistance.
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FOOTNOTES |
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This work was supported by National Institute on Deafness and Other Communication Disorders Grant 5R01-DC-00212 to D. C. Marcus.
Address for reprint requests and other correspondence: D. C. Marcus, Dept. of Anatomy and Physiology, Kansas State University, 1600 Denison Ave., Manhattan, KS 66506 (E-mail: marcus{at}ksu.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 2 January 2001; accepted in final form 2 February 2001.
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