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Apical Membrane P2Y4 Purinergic Receptor Controls K+ Secretion By Cell Communication and Signaling BioMed Central Research Open Access Apical membrane P2Y4 purinergic receptor controls K+ secretion by strial marginal cell epithelium Daniel C Marcus*1, Jianzhong Liu1, Jun Ho Lee1, Elias Q Scherer2, Margaret A Scofield3 and Philine Wangemann2 Address: 1Cellular Biophysics Laboratory, Dept. Anatomy & Physiology, Kansas State University, Manhattan, KS 66506 USA, 2Cell Physiology Laboratory, Dept. Anatomy & Physiology, Kansas State University, Manhattan, KS 66506 USA and 3Molecular Pharmacology Laboratory, Dept. Pharmacology, Creighton School of Medicine, Omaha, NE 68178 USA Email: Daniel C Marcus* - [email protected]; Jianzhong Liu - [email protected]; Jun Ho Lee - [email protected]; Elias Q Scherer - [email protected]; Margaret A Scofield - [email protected]; Philine Wangemann - [email protected] * Corresponding author Published: 02 November 2005 Received: 09 June 2005 Accepted: 02 November 2005 Cell Communication and Signaling 2005, 3:13 doi:10.1186/1478-811X-3-13 This article is available from: http://www.biosignaling.com/content/3/1/13 © 2005 Marcus et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract 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 µ (1 M). 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 ginal cell epithelium [1]. One pathway of regulation is the A high concentration of K+ is maintained in the lumen of coupling of purinergic receptors on the apical membrane the cochlea via electrogenic secretion by the strial mar- of these cells to the apical potassium channels (IKs) which Page 1 of 8 (page number not for citation purposes) Cell Communication and Signaling 2005, 3:13 http://www.biosignaling.com/content/3/1/13 The question of the subtype of P2Y receptor mediating 1500 ATP A regulation of K+ secretion by strial marginal cells has been addressed by immunohistochemistry, which suggests the ) 2 1000 presence of P2Y4 in the apical marginal cell membrane [8]. However, a functional demonstration was lacking, the time-course of activation was not investigated and an (µA/cm 500 independent demonstration of gene expression was not sc I available. 0 The present study was conducted with the goals of 1) obtaining dose-response profiles for potentially definitive B 1500 UTP 30 s agonists of P2Y2, P2Y4 and P2Y6 receptors; 2) refining the ) profile by the use of antagonists, 3) testing for desensitiza- 2 1000 tion of the response to agonist and 4) determining the presence of transcripts for pyridine-sensitive P2Y recep- (µA/cm tors. The results support the conclusion that regulation of 500 sc + I K secretion by strial marginal cells occurs by apical P2Y4 receptors. 0 Results C 1500 Ap4A ATP perfused for 30 s at the apical side of strial marginal cell epithelium caused a monophasic decrease in Isc (Fig. ) 2 1A), consistent with previous findings [2]. Removal of 1000 ATP led to a recovery of Isc after an initial overshoot. Sim- ilar responses were seen from perfusion of other agonists, (µA/cm 500 such as UTP and Ap4A (Fig. 1B &1C). sc I The P2Y6 agonist, UDP, was tested after removal of con- 0 tamination by UTP in the commercial product [6]. Con- taminating UTP was digested by hexokinase in the SummaryginalFigure cells 1 recordings of Isc (Ussing chamber) of strial mar- presence of glucose. The enzymatically-purified UDP pro- Summary recordings of Isc (Ussing chamber) of strial duced little effect on I (Fig. 2). marginal cells during apical perfusion of A) ATP (10-3 M; 30 sc s; n = 6), B) UTP (10-4 M; 30 s; n = 7) and C) Ap4A (3 × 10-4 Concentration-response curves for the purinergic agonists M; 60 s; n = 4). Vertical bars are SEM (not all shown; spaced ≥ for clarity). are summarized in Figure 2. The potency order was UTP ATP > Ap4A >> UDP with EC50 values for UTP and ATP of about 2.3 and 1.2 × 10-5 M. The relative potencies of the mediate secretion [2]. These receptors are responsive to agonists are consistent with an action on P2Y2 and/or both ATP and UTP as agonists and they were found to P2Y4 receptors. exert their action via the phospholipase C – protein kinase C intracellular signal pathway [3]. At the time of the orig- The purinergic receptor antagonist, suramin, was tested inal investigations, the purinergic receptor field recog- for effectiveness in blocking the response to apical UTP. nized only 1 receptor responding to uracil nucleotides Suramin at 100 µM had no inhibitory effect on the action µ (P2U, now P2Y2 receptor). The known members of the of the agonist, UTP (Fig. 3). UTP (1 M) caused a decrease mammalian metabotropic, G protein-coupled, purinergic in Isc by 20.6 ± 5.4% in the absence of suramin and by 21.9 receptors has since grown to P2Y1, P2Y2, P2Y4, P2Y6, ± 5.3% (P > 0.05, n = 5) in the presence of suramin. P2Y11, P2Y12, P2Y13 and P2Y14 [4]. In addition to P2Y2, both the P2Y4 and P2Y6 receptors also respond to uracil Both P2Y2 and P2Y4 receptor subtypes have been reported nucleotides. On the basis of pharmacologic agonist and to undergo desensitization within 5–15 min [9,10]. Sur- antagonist profiles, P2Y6 could be distinguished from prisingly, we found that sustained exposure (15 min) of P2Y2 and P2Y4 by the greater potency of UDP over UTP the apical membrane to ATP led to a biphasic but sus- [5,6]. The pharmacologic distinction between P2Y2 and tained inhibition of Isc (Fig. 4). The recovery of Isc after P2Y4 was more ambiguous and the accepted criteria removal of agonist was much slower, incomplete and changed rapidly [7]. without overshoot compared to acute exposure to agonist. Page 2 of 8 (page number not for citation purposes) Cell Communication and Signaling 2005, 3:13 http://www.biosignaling.com/content/3/1/13 Perfusion of UTP (10 µM) for 30 s led to an increase in comparative activities of other nucleotides (UTP, 2-MeS- intracellular [Ca2+] that had both a peak and plateau ATP and α, β-meth-ATP) at 1 µM [2]. Full concentration- phase and was repeatable (Fig. 5, top). Both the peak and response curves were obtained in the present study to bet- plateau were significantly reduced by 2-APB (75 µM) (to ter define the active subtype. The lack of response to UDP 26.5 ± 3.9% and 25.1 ± 4.6%; Fig. 5, middle). 2-APB is an eliminated P2Y6 as a candidate subtype [22-24]. The sim- inhibitor of the IP3 receptor at this concentration [11,12] ilar potency of ATP and UTP is consistent with hP2Y2 [23], and can also inhibit other transporters including store- rP2Y2 [22] but not hP2Y4 [6]. In fact, ATP acts as a compet- 2+ operated Ca channels [13]. Store operated channels, itive antagonist at the hP2Y4 receptor [25]. However, these however, would not be activated under the present exper- results alone do not identify the strial marginal cell apical 2+ imental conditions, since the Ca stores were not emp- purinergic receptor as P2Y2 since it was subsequently tied prior to measurement. In the absence of external found that rP2Y4 has an agonist potency sequence similar 2+ Ca , both the peak and plateau responses to UTP to rat and human P2Y2 [7,25]. Since the gerbil is expected remained but were significantly reduced (to 68.9 ± 3.0% to be more closely related to other rodents such as the rat, and 60.5 ± 4.2%; Fig. 5, bottom). The reductions, however, than to human, our finding is consistent with the contri- were likely the result of a general decrease in intracellular bution of P2Y4 and/or P2Y2. [Ca2+] seen after removal of bath Ca2+ prior to perfusion of UTP. These findings taken together suggest that intrac- The potency order of the agonists UTP, ATP, Ap4A and ellular stores are the primary source of the increase in UDP in regulation of Isc from strial marginal cells is the 2+ intracellular [Ca ] induced by UTP.
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