Abstract
Nucleotides are released from bovine chromaffin cells and take part in a feedback loop to inhibit further exocytosis. To identify the nucleotide receptors involved, we measured the effects of a range of exogenous nucleotides and related antagonists on voltage-operated calcium currents (ICa), intracellular calcium concentration ([Ca2+]i), and membrane capacitance changes. In comparative parallel studies, we also cloned the bovine P2Y12 receptor from chromaffin cells and determined its properties by coexpression in Xenopus laevis oocytes with inward-rectifier potassium channels made up of Kir3.1 and Kir3.4. In both systems, the agonist order of potency was essentially identical (2-methylthio-ATP ≈ 2-methylthio-ADP ≫ ATP ≈ ADP > UDP). αβ-Methylene-ATP and adenosine were inactive. UTP inhibited ICa in chromaffin cells (pEC50 = 4.89 ± 0.11) but was essentially inactive at the cloned P2Y12 receptor. The relatively nonselective P2 antagonist pyridoxal-phosphate-6-azophenyl-2′,4′ disulfonic acid blocked nucleotide responses in both chromaffin cells and X. laevis oocytes, whereas the P2Y12- and P2Y13-selective antagonist N6-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-β,γ-dichloromethylene ATP (ARC69931MX) blocked responses to ATP in both chromaffin cells and X. laevis oocytes but not to UTP in chromaffin cells. These results identify the P2Y12 purine receptor as a key component of the nucleotide inhibitory pathway and also demonstrate the involvement of a UTP-sensitive Gi/o -coupled pyrimidine receptor.
Given the profusion of P2 nucleotide receptors in the nervous system and the many pathways for nucleotide release, the potential for extracellular nucleotides to play a major modulatory role in neurotransmission is high. Because of their structure and signaling mechanisms, P2 receptors are classified either as ligand-gated P2X1–7 cation channels or as metabotropic P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, P2Y14, and P2Y15 receptors coupled to heterotrimeric G proteins (North, 2002; Abbracchio et al., 2003; Inbe et al., 2004). Activation of P2X receptors by ATP leads directly to membrane depolarization and calcium entry both via the P2X channels themselves and by the subsequent activation of voltage-operated calcium channels (VOCCs) (North, 2002). P2Y receptors have a wider agonist profile than the P2X receptors responding to purines, pyrimidines, and UDP-glucose. These receptors can be divided into two subgroups based on their molecular structure and coupling to Gα-subunits, with P2Y12, P2Y13, and P2Y14 making up one group that signals via PTX-sensitive Gi/o proteins and P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, and P2Y15 making up the second group that couples to phospholipase C and G proteins of the Gq class (Abbracchio et al., 2003; Inbe et al., 2004).
In the sympathetic nervous system, the effects of presynaptic purine receptors on neurotransmission have been well-documented; facilitation of catecholamine release is mediated by P2X receptors, whereas inhibition is mediated by the activation of an unidentified P2Y receptor (Von Kügelgen et al., 1989; Boehm and Kubista, 2002). Evidence for inhibitory presynaptic P2Y receptor(s)-regulating release of catecholamine as well as other neurotransmitters in the central nervous system is also accumulating (Cunha and Ribeiro 2000; Zhang et al., 2003). Identifying the nucleotide receptor subtypes that mediate presynaptic inhibition has been complicated by the inaccessibility of the majority of mammalian nerve terminals, complexity arising from neural circuits in which multiple P2 receptors may be activated, stimulation of P1 adenosine receptors after breakdown of purines by ectonucleotidases, and the limited availability of P2 receptor subtype-selective agonists and antagonists.
Adrenal chromaffin cells are embryonically derived from precursors of sympathetic neurons; they also release catecholamines and ATP by Ca2+-regulated exocytosis and express inhibitory P2 receptors that couple to neuronal VOCCs (Diverse-Pierluissi et al., 1991; Gandia et al., 1993; Currie and Fox, 1996). Moreover, evidence for an autocrine feedback loop similar to that proposed for sympathetic neurons involving an inhibitory P2Y-like receptor has been reported (Carabelli et al., 1998). In a previous study, we used combined Cm measurements and voltage-clamp recordings to examine the mechanisms underlying purinergic inhibition of exocytosis in chromaffin cells (Powell et al., 2000). We showed that the purine analog 2-methylthio-ATP (2-MeSATP) inhibits Ca2+ entry through N- and P/Q-type VOCCs and, consequently, stimulus-evoked changes in Cm through a PTX-sensitive G protein. The aim of this study was to expand on this finding by determining the molecular identity of the P2Y receptor(s) involved. Here, we provide evidence for two inhibitory PTX-sensitive Gi/o-coupled P2Y receptors in bovine chromaffin cells. One of these receptors shows a pharmacology similar but not identical (ATP being a full agonist and equipotent to ADP) with the human P2Y12 receptor, whereas the second receptor is UTP-sensitive and hence shows a pharmacology not matching any of the known Gi/o-coupled P2Y receptors. To confirm the role of P2Y12 in VOCC inhibition, we cloned the bovine P2Y12 receptor from bovine chromaffin cells and expressed this receptor in Xenopus laevis oocytes coexpressing inward-rectifier potassium channels made up of rat Kir3.1 and Kir3.4. The pharmacological properties of this cloned receptor closely mirrored the pharmacology observed in chromaffin cells except that UTP was a very weak partial agonist. We therefore conclude that P2Y12 and another yet-unidentified Gi/o-protein–coupled UTP-sensitive receptor inhibit VOCCs and exocytosis in chromaffin cells. These findings support the view that Gi/o-coupled P2Y receptors may also act as presynaptic inhibitory receptors in other neuronal systems to regulate neurotransmitter release.
Materials and Methods
Chromaffin Cell Culture. Chromaffin cells were prepared by collagenase digestion of bovine adrenal glands as described previously (Powell et al., 2000). Adrenal glands from 18- to 24-month-old cows were obtained from a local abattoir and were retrogradely perfused at 25 ml/min for 30 min at 37°C with the digestive enzymes collagenase type 2 (0.03%) (Worthington Biochemicals, Freehold, NJ) and DNase I (0.0013%) (Roche Diagnostics, Indianapolis, IN) added to Locke's solution [154.2 mM NaCl, 2.6 mM KCl, 2.2 mM K2HPO4, 0.85 mM KH2PO4, 10 mM glucose, 5 mM HEPES, and 0.0005% phenol red (Invitrogen, Paisley, UK); pH adjusted to 7.2 with NaOH]. After surgical removal of the cortex, the medulla was dissected, cut into small pieces, placed in a trypsinization flask with fresh enzyme solution, and stirred at slow speed for 30 min at 37°C. Cells were washed twice with Earle's balanced salt solution (Invitrogen) and resuspended in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with 44 mM NaHCO3, 15 mM HEPES, 10% fetal calf serum (Invitrogen), 1% glutamine, 1% penicillin/streptomycin solution, 2.5 mg/ml gentamycin, 0.5 mg/ml 5′-fluorodeoxyuridine, and 0.01 mg/ml cytosine-β-δ-arabino-furanoside. Cells were plated on glass coverslips coated with Matrigel (BD Biosciences Discovery Labware, Bedford, MA) at an approximate density of 800 cells/mm2. Approximately 80% of the media was replaced 24 h after plating, and cells were maintained for up to 7 days in a humidified atmosphere of 95% O2/5% CO2 at 37°C.
[Ca2+]i Measurements in Bovine Chromaffin Cells. Cells were loaded with the Ca2+ indicator Fura 2/acetoxymethyl ester by the addition of 5 μM Fura 2/acetoxymethyl ester (Molecular Probes, Eugene, OR) to DMEM and incubated for 25 min at 37°C. Cells were then washed with fresh DMEM and incubated a further 15 min at 37°C. Isolated fluorescent chromaffin cells were alternately illuminated at 340 and 380 nm using a monochromator (TILL Photonics, Gräfelfing, Germany) controlled by the data acquisition software. Emission >430 nm was collected with a photomultiplier tube (TILL Photonics) and sampled approximately every 12 ms. Data were stored on personal computers, and ratios of 340/380 nm were calculated offline (AxoBASIC-written software; Axon Instruments Inc., Union City, CA).
Electrophysiological Recordings in Bovine Chromaffin Cells. A coverslip carrying chromaffin cells was placed in a microperfusion chamber (∼200-μl volume) on the stage of an inverted phasecontrast microscope (Diaphot 200; Nikon, Tokyo, Japan). Cells were continuously superfused with an external solution consisting of 130 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM CaCl2, 10 mM glucose, and 10 mM HEPES adjusted to pH 7.2 with NaOH; osmolarity, ∼280 mOsM. Special care was taken to superfuse cells at a high rate (∼3 ml/min) throughout the experiment and to select well-isolated single cells for recording to avoid compounding effects of endogenously released modulators (Carabelli et al., 1998). Ionic currents were recorded in whole-cell or perforated patch-clamp configuration using borosilicate glass electrodes coated with Sylgard 184 (Dow Corning, Midland, MI) and fire-polished on a microforge to a resistance of 1 to 2 MΩ. Electrodes were filled with an internal solution consisting of 145 mM cesium-glutamate (Calbiochem, Nottingham, UK), 10 mM HEPES, 9.5 mM NaCl, 0.3 mM BAPTA (Molecular Probes), adjusted to pH 7.2 with CsOH (MP Biomedicals, Irvine, CA); osmolarity, ∼280 mOsM. For whole-cell recording experiments, 2 mM Mg-ATP was added to the internal solution to prevent rundown of VOCCs and exocytosis. Gramicidin D (Sigma Chemical, Poole, Dorset, UK) at a final concentration of 9.7 μg/ml was used for perforation. For both whole-cell and perforated-patch recordings, series resistance was less than 12 MΩ and compensated (typically >70%) electronically with the patch-clamp amplifier (Axopatch 200B; Axon Instruments). Voltage protocol generation and data acquisition were performed using custom data acquisition software (kindly provided by Dr. A. P. Fox, University of Chicago) running on a Pentium computer equipped with a Digidata 1200 acquisition board (Axon Instruments). Current traces were low pass-filtered at 5 kHz using the 4-pole Bessel filter of the amplifier and digitized at 10 kHz. Chromaffin cells were voltage-clamped at –90 mV, and Cm was sampled with a resolution of 12 ms using a software-based phase-tracking method as described previously (Fidler and Fernandez, 1989; Powell et al., 2000). Data were stored on the computer hard drive and analyzed offline using custom (AxoBASIC; Axon Instruments) and commercial (Origin; OriginLab Corporation, Northampton, MA) software. All experiments were performed at ambient temperature (21–25°C).
Cloning of the Bovine P2Y12 Receptor. The strategy used to clone the bovine P2Y12 receptor consisted of three sequential rounds of cloning. First, a conserved central region of the receptor was amplified by polymerase chain reaction (PCR) with degenerate primers (PCR1). Second, 5′- and 3′-rapid amplification of cDNA ends (RACE) primers were designed from the sequence obtained from PCR product 1 and used to amplify the 5′ and 3′ ends of the receptor by 5′- and 3′-RACE, respectively (PCRs 2 and 3). Finally, the sequence obtained from PCR products 2 and 3 was used to design primers to amplify the full-length receptor from bovine chromaffin cell cDNA by RT-PCR. A proofreading polymerase (Bio-X-Act; Bioline Ltd., London, England) was used for all PCR reactions. Total RNA was prepared from bovine chromaffin cells, and 5 μg was used in a first-strand cDNA reaction using RoRidT(17) primer (Harvey and Darlison, 1991) and Superscript II reverse transcriptase according to the manufacturer's instructions (Amersham Biosciences UK, Ltd., Little Chalfont, Buckinghamshire, UK). The degenerate primers for PCR1, y12degF (5′-TTTCTGTTGYCATCTGGCCMTTCATG-3′) and y12degR (5′-GGTCACCACCWTCYTGTYCTTTYTTC-3′) were designed from homologous regions of the human mouse and rat P2Y12 sequences (accession numbers NM_022788, AK013804, and NM_022800, respectively). 5′ RACE (PCR2) was performed using a SMART RACE kit (BD Biosciences Clontech, Palo Alto, CA) according to the manufacturer's instructions with the sequence-specific primer TEW81 (5′-GCCAAACCAGACCAAACTCTGACTTCAG-3′) designed from the sequence of PCR1. 3′ RACE (PCR3) was performed using the primers Ro (Harvey and Darlison, 1991) and bY12RACEfor (5′-GGTGCTGGCAAAGTCCCCAAGAA-3′). The primers 2ndby12fullfor (5′-GACGGAAATACAGTGTCTGC-3′) and 2ndby12fullrev (5′-CTTGCCTTTGGGGAGTT-3′) were designed from the sequence obtained from PCRs 1 and 2 and were used in RT-PCR to amplify the full-length P2Y12 receptor from first-strand cDNA prepared from bovine chromaffin cells. PCR products were cloned into the plasmid pCDNA3 (Invitrogen, Carlsbad, CA) and two independent colonies sequenced on both strands (automated ABI sequencing service, Protein and Nucleic Acid Laboratory, University of Leicester, Leicester, UK).
RT-PCR Analysis. RT-PCR was performed on first-strand cDNA prepared from bovine chromaffin cells as described above. The only published bovine P2Y sequence available for primer design was that of P2Y1 (Henderson et al., 1995). BLAST homology searches of the bovine expressed sequence tag database found sequences corresponding to the bovine P2Y2, P2Y6, and P2Y14 receptors (accession numbers BM031311, BI680595, and CB429080, respectively), allowing for the presence of transcripts for P2Y1, P2Y2, P2Y6, P2Y12, and P2Y14 to be analyzed in addition to the published bovine P2Y1 (primer sequences in Table 1).
Electrophysiological Recordings inX. laevisOocytes.X. laevis oocytes expressing the rat inwardly rectifying potassium channels Kir 3.1 and Kir 3.4 were used to assess the function of the cloned bovine P2Y12 receptor in a system similar to that used by Hollopeter and coworkers (2001) to characterize the human P2Y12 receptor. Plasmids for rat Kir 3.1 and Kir 3.4 were a kind gift from Dr. M. Boyett (University of Leeds, Leeds, UK). cRNA was transcribed from linearized P2Y12, Kir 3.1, and Kir 3.4 plasmids using the mMessage mMachine system (Ambion, Austin, TX) according to the manufacturer's instructions. Defolliculated stage-V to -VI X. laevis oocytes were injected with 1.25 ng of Kir 3.1, 1.25 ng of Kir 3.4, and 50 pg of P2Y12 cRNA in a total volume of 50 nl using an INJECT + MATIC microinjector (J. Alejandro Gaby, Geneva). Oocytes were stored at 18°C in ND96 buffer (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM sodium pyruvate, and 5 mM HEPES, pH 7.6) before use 3 to 7 days later.
Two-electrode voltage-clamp recordings were made from oocytes using a Turbo TEC 10C amplifier (NPI Electronic GmbH, Tamm, Germany) with a Digidata 1200 analog-to-digital converter (Axon Instruments) and WinWCP acquisition software (Dr. J. Dempster, University of Strathclyde, Glasgow, Scotland). Agonists and antagonists were bath-perfused using a custom-built rapid-exchange perfusion system. Oocytes were initially perfused with ND96 buffer to obtain a value for the resting membrane potential (typically ∼–70 mV for Kir-injected oocytes and ∼–20 mV for noninjected or P2Y12 only-injected oocytes) before exchange to a solution of 20 mM NaCl, 70 mM KCl, 3 mM MgCl2, and 5 mM HEPES for recording of agonist-evoked membrane currents. Agonist-evoked currents could be measured in oocytes clamped constantly at –60 mV; however, the current required (∼5 μA) to clamp at this potential tended to kill cells after ∼15 to 30 min. Therefore, to obtain full-concentration response-curve data for individual cells, oocytes were clamped at 0 mV during the initial period of agonist application and in recovery periods between applications. One minute after agonist application commenced, the holding potential was stepped down to –60 mV for 5 s and the peak current was recorded. A ramp from –60 mV to +90 mV over 2 s was applied after the 5-s recording period to visualize the inward rectification from the Kir channels, verifying that the cell was still in a healthy condition. Currents in the presence of agonist were normalized to the mean of recordings taken 5 min before and 5 min after in the absence of agonist.
All drugs were made up as concentrated stock solutions in distilled water and stored in aliquots at –20°C until use. Stocks were thawed once and diluted into the superfusing solution. Nucleotide analogs were obtained from Sigma. 2-MeSATP and pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) were obtained from Tocris Cookson Inc.(Bristol, UK). PTX (Sigma) was dissolved in 50% glycerol containing 50 mM Tris, 10 mM glycine, and 0.5 M NaCl, pH 7.5.
Data Analysis. Ca2+ entry into chromaffin cells was determined by integration of ICa. The left limit was set ∼3 ms into the voltage pulse to exclude the major portion of the contaminating Na+ current. ΔCm measurements were performed as described previously (Powell et al., 2000).
Concentration-response data obtained from individual cells were fitted with the Hill equation Y = ((X)nH · M)/((X)nH + (EC50)nH), where Y is the response, X is the agonist concentration, nH is the Hill coefficient, M is maximum response, and EC50 is the concentration of agonist evoking 50% of the maximum response. pEC50 is the –log10 of the EC50 value. Data are presented as mean ± S.E.M., and differences between means were tested using either paired or independent Student's t test, as appropriate.
Results
Measurements of ICa in Chromaffin Cells. The effects of adenosine and uridine nucleotides on ICa in bovine chromaffin cells were examined (Fig. 1). Currents were activated every 30 s with step depolarizations to +20 mV from a holding potential of –90 mV. Superfusion with ATP or the P2Y-selective analog 2-MeSATP had no effect on the holding current but reversibly inhibited ICa by 45 ± 3% (100 μM ATP, n = 13) and 44 ± 4% (100 nM 2-MeSATP, n = 11). UTP (100 μM) also inhibited ICa (36 ± 6%, n = 11) without changing the holding current. Preceding the test pulse by a 20-ms depolarizing prepulse to 120 mV reduced the inhibitory effect of 2-MeSATP to 15.5 ± 3.5% (n = 3) and of UTP to 8 ± 2% (n = 3) (Fig. 1C). The voltage-dependence of the inhibitory effect was also observed by examining the effect of 2-MeSATP on the current-voltage relationship (data not shown). Inhibition of ICa by 2-MeSATP was significantly reduced at potentials positive to +30 mV. Furthermore, 2-MeSATP produced a significant depolarizing shift in the activation curve (V50 control, 14.8 ± 3.7 mV; 2-MeSATP, 22.4 ± 4.7 mV; n = 6, p < 0.05). The voltage-sensitivity of the inhibitory effects of the purines and pyrimidines on ICa is consistent with a signaling pathway that involves direct modulation of the channels by Gβγ subunits (Dolphin, 2003). Treatment of chromaffin cells with PTX (250 ng/ml for 24 h) completely blocked the effect of both 2-MeSATP (3.5 ± 0.8%, n = 4) and UTP (2.8 ± 1.0%, n = 4) (Fig. 1, D and E), confirming the sole involvement of Gi/o-coupled P2Y receptor(s) in the modulation of ICa. In contrast to heterologously expressed P2Y4 receptors (Filippov et al., 2003), the inhibition produced by UTP in chromaffin cells was not sensitive to cell dialysis; application of 30 μM UTP (∼EC50 concentration) inhibited ICa recorded in the whole-cell configuration by 15.2 ± 2.1% (n = 6) and by 16.3 ± 3.1% (n = 8) in the perforated-patch configuration.
Involvement of Ca2+Mobilizing P2Y or P2X Receptors in VOCC Inhibition. To investigate the possible contribution from Ca2+-mobilizing P2 receptors to inhibition of VOCCs, we loaded chromaffin cells with the Ca2+-sensitive dye Fura 2 to monitor [Ca2+]i. In 10 of 12 cells examined, neither ATP (100 μM) nor UTP (100 μM) produced any increase in [Ca2+]i; in these same cells, histamine (100 μM) and angiotensin II (300 nM), agonists known to activate PLC-coupled receptors in chromaffin cells (Cheek et al., 1993; Teschemacher and Seward, 2000), produced robust increases in [Ca2+]i (Fig. 2). Superfusion with 2-MeSATP (100 nM) also failed to produce any significant change in basal [Ca2+]i (n = 4) (also see Fig. 1 in Powell et al., 2000). In 2 of 12 cells, ATP produced a small increase in [Ca2+]i (mean, 186% of control). However, both of these cells were found to be relatively unresponsive to histamine (mean value of 124% of control compared with 279% for ATP/UTP-nonresponsive cells) and had approximately half the diameter of chromaffin cells usually selected for electrophysiological investigation (mean membrane capacitance, 7.7 ± 0.5 pF, n = 20, corresponding to a diameter of ∼15 μm). Whether this minor population of cells corresponds to noradrenergic or cortical cells, which make up 10 to 20% of adrenal medullary cultures, was not investigated further. From these results we can conclude that neither Ca2+-mobilizing P2X receptors nor PLC-coupled P2Y1, P2Y2, P2Y4, and P2Y6 receptors are functionally detectable in the majority of chromaffin cells.
Agonist Profile of P2Y Receptors in Chromaffin Cells. The relative paucity of high-affinity subtype-selective ligands complicates the unambiguous identification of P2Y receptors within intact tissues. The method most commonly used to identify native P2Y receptors is to examine the relative order of potency of numerous purine and pyrimidine analogs. We examined the efficacy of a number of commonly used purine and pyrimidine analogs to inhibit ICa in chromaffin cells. Agonist-profiling yielded an agonist order of potency of 2-MeSATP ≈ 2MeSADP ≫ ATP ≈ ADP ≥ ATPγS > UTP ≥ UDP (Fig. 3A). αβ-Methylene-ATP and adenosine were inactive (100 μM, data not shown). 2-MeSATP, 2-MeSADP, ATP, ADP, and ATPγS were full agonists with mean EC50 values of 0.49 nM (n = 4), 0.73 nM (n = 4), 347 nM (n = 4), 387 nM (n = 3), and 1170 nM (n = 3) and Hill slopes of approximately 1 (Table 2).
The uridine nucleotides were less potent than the adenosine analogs (Table 2), and UTP was incapable of exerting a full inhibitory response (Fig. 3A), suggesting that it may act as a partial agonist on a receptor with mixed purine/pyrimidine sensitivity. To test this possibility, the effects of coapplication of a submaximal concentration of UTP (10 μM) with 2-MeSATP (10 pM to 10 nM) were tested. If UTP was acting as a partial agonist at the same receptor population as those activated by 2-MeSATP, coapplication would be expected to cause a rightward shift in the concentration-response curve and a depression of the maximal response. The IC50 values for 2-MeSATP alone and in the presence of UTP (10 μM), however, were similar (0.22 and 0.16 nM) (Fig. 3B), and although a slight decrease in the maximal response was observed (2-MeSATP alone, 48 ± 3%, and 2-MeSATP and UTP, 45 ± 6%; n = 4), this was not significant (p = 0.66).
In response to prolonged activation, many G-protein–coupled receptors undergo desensitization; UTP-preferring P2Y4 receptors can be distinguished from UDP-preferring P2Y6 receptors in that they show rapid desensitization (Brinson and Harden, 2001). Thus, to further characterize the UTP receptor in chromaffin cells, we examined the rate and cross-desensitization of nucleotide inhibition of ICa (Fig. 4). The general protocol to study desensitization was an initial 3-min application of either 2-MeSATP or UTP, to evaluate the control inhibition. Subsequent to this, either 2-MeSATP or UTP was applied for 15 min and then washed out briefly before 2-MeSATP and UTP were reapplied to check for cross-desensitization. With prolonged superfusion of UTP, the inhibition of ICa was reduced from 35 ± 9% to 11 ± 6% (Fig. 4A). In the same cells, application of 2-MeSATP inhibited ICa by 48 ± 6% and 49 ± 6% (n = 4) before and after perfusion with UTP, showing that the decline in ICa inhibition seen during perfusion with UTP was not caused by rundown of the channels but rather desensitization of the receptor. Moreover, because the response to 2-MeSATP was unaffected by desensitization of the UTP response, we can conclude that there is no cross-desensitization of the purine- and pyrimidine-preferring receptors in these cells. The desensitized response of UTP did not recover after an 11-min wash period. In the converse experiment, it was noted that the inhibitory effect of 2-MeSATP on ICa did not undergo such pronounced desensitization (Fig. 4B). 2-MeSATP maximally inhibited ICa by 43 ± 3%. During the 15-min superfusion, the mean inhibition was reduced to 29 ± 4% (n = 4). The response to a second application of 2-MeSATP after an 11-min wash period was back to 37 ± 6%. The size of the response to UTP (100 μM) was slightly decreased on the second application (28 ± 13% versus 19 ± 9%; n = 4); however, this was not significant.
Antagonist Sensitivity of P2Y Receptors in Chromaffin Cells. The agonist selectivity and PTX sensitivity of the P2Y receptor(s) expressed in bovine adrenal chromaffin cells do not match that reported for any single cloned mammalian P2Y receptor. We therefore proceeded to examine the antagonist selectivity of the receptor(s). PPADS has been shown to be an antagonist at P2Y1, P2Y2, P2Y6, and P2Y13 receptors (Marteau et al., 2003) but not at P2Y4 receptors (Boyer et al., 1994; Charlton et al., 1996) or human P2Y12 receptors (Takasaki et al., 2001). PPADS antagonized the inhibitory effects of 2-MeSATP (1 nM) and UTP (30 μM) in a reversible manner. Schild analysis of PPADS antagonism of 2-MeSATP inhibition of ICa showed that the antagonist was acting in a competitive manner, with an apparent pA2 value of 6.42 ± 0.33 (Fig. 5A). Examination of whether PPADS produced competitive antagonism of the UTP-induced inhibition of ICa was not carried out because of the low potency of UTP. Finally, we examined the ability of the antithrombotic drug ARC69931MX, reported to be selective for P2Y12 and P2Y13 receptors (Ingall et al., 1999; Marteau et al., 2003), to antagonize the regulation of ICa in chromaffin cells. Superfusion with ARC69931MX (1 μM) for 1 to 3 min had no effect on ICa (106 ± 12% of control, n = 4), but in the same cells it largely abolished the inhibition produced by ATP (100 μM) from 49 ± 4% to 6 ± 5% (n = 4). Schild analysis of ARC69931MX antagonism of 2-MeSADP inhibition of ICa gave an apparent pA2 value of 9.90 ± 0.06. Inhibition of ICa by 30 μM UTP, however, persisted in the presence of ARC69931MX (mean, 20 ± 9%; n = 3), supporting the notion that distinct ATP and UTP receptors are expressed by chromaffin cells.
Measurements of Exocytosis in Chromaffin Cells. An increase in Cm follows vesicle fusion after Ca2+ entry and provides a measurement of exocytosis corresponding to 2 fF per vesicle fusion. We therefore used Cm measurements to determine the effects of VOCC inhibition by nucleotides on exocytosis from bovine chromaffin cells (Fig. 5, B and C). Application of ATP (100 μM) resulted in a marked decrease in vesicle fusion (Fig. 5B). This inhibitory effect of ATP on exocytosis was completely blocked by the P2Y12-specific antagonist ARC69931MX (Fig. 5C).
Cloning of the Bovine P2Y12 Receptor. Taken together, the pharmacological data obtained from bovine chromaffin cells suggest that the purine receptor responsible for inhibition of ICa and exocytosis is most similar to that of P2Y12 or P2Y13 except that ATP was a full agonist rather than a weak partial agonist (Marteau et al., 2003) and PPADS is an antagonist. Unlike the pharmacology of the nucleotide responses observed in the P2Y receptors expressed in bovine chromaffin cells, human P2Y13 is unresponsive to both 2-MeSATP and ATP (Communi et al., 2001). Human P2Y12, however, is responsive to 2-MeSATP in the nanomolar range and ATP in the micromolar range (Takasaki et al., 2001). We therefore cloned the bovine P2Y12 receptor to compare its pharmacology with the purine-sensitive Gi/o-coupled P2Y receptor expressed in bovine chromaffin cells. A PCR product of 1145 base pairs was amplified from bovine chromaffin cell cDNA using the primers 2ndby12fullfor and 2ndby12fullrev. This sequence is available in the European Molecular Biology Laboratory database under the accession number AJ623293. The sequence contained an open-reading frame of 339 amino acids with a consensus Kozak sequence at the starting methionine. CLUSTAL alignment of the deduced bovine P2Y12 amino acid sequence with human, rat, and mouse P2Y receptors confirmed that this sequence corresponds to P2Y12 and not to a related receptor such as P2Y13 or P2Y14 (Fig. 6A). The cloned bovine receptor showed strong sequence identity to the known mammalian P2Y12 sequences with percentage identities of 89.4, 84.4, and 85.9% for human, rat, and mouse P2Y12 sequences, respectively. Alignment of the human and bovine P2Y12 amino acid sequences (Fig. 6B) demonstrates the positions of the 37 residues that differ between species. There are no differences in amino acid sequence in the region from TM6 through to TM7, a region that has been implicated previously in agonist binding in the P2Y2 receptor (Erb et al., 1995).
Measurements of Potassium Currents inX. laevisOocytes Expressing the Bovine P2Y12 Receptor. Coexpression in X. laevis oocytes of the cloned cardiac inward-rectifier subunits Kir 3.1 and Kir 3.4 resulted in robust expression of an inwardly rectifying potassium channel (Fig. 7A). Activation of this channel by Gβγ release was used to characterize the pharmacology of the cloned bovine P2Y12 receptor. To confirm the absence of endogenous oocyte channels or receptors that could interfere with results by coupling to the exogenous bovine P2Y12 receptor or rat Kir channels, noninjected oocytes, and oocytes injected with cRNA for the bovine P2Y12 receptor only or only the Kir 3.1 and Kir 3.4 cRNAs were tested. These oocytes showed no nucleotide-evoked currents (data not shown). When oocytes were coinjected with cRNAs for the bovine P2Y12 receptor (50 pg) and rat Kir 3.1 + 3.4 channels (1.25 ng each), nucleotide-evoked currents were observed. These currents reached a peak within 30 s, did not desensitize with the continued agonist application, and decayed back to baseline within 3 min of agonist removal.
Concentration-response data were obtained by using the voltage protocol depicted in Fig. 7A (and described in detail under Materials and Methods). Similar to the agonist profile obtained in bovine chromaffin cells, ATP and ADP were essentially equipotent at the cloned bovine P2Y12 receptor, showing EC50 values of 3.74 μM (pEC50 = 5.47 ± 0.10) and 1.56 μM (pEC50 = 5.97 ± 0.17), respectively (Fig. 7B). 2-MeSADP and 2-MeSATP were considerably more potent, with EC50 values of 0.28 nM (pEC50 = 9.55 ± 0.10) and 0.84 nM (pEC50 = 9.31 ± 0.21), respectively. Uridine nucleotides showed varying degrees of potency with UDP, a full agonist (EC50 = 105.5 μM, pEC50 = 4.12 ± 0.64), UTP, a very weak partial agonist (∼10% maximal UDP response with 10 mM UTP), and UMP, inactive.
The effects of the antagonists ARC69931MX and PPADS were also determined at the cloned P2Y12 receptor in X. laevis oocytes (Fig. 7C). The P2Y12-specific antagonist ARC69931MX completely blocked responses of the bovine P2Y12 receptor to 10 μM ADP (IC50 = 0.78 nM, pIC50 = 8.67 ± 0.06) and 10 μM ATP (IC50 = 2.1 nM, pIC50 = 9.14 ± 0.44). Furthermore, the responses to 1 mM UDP, a concentration normally eliciting a 100% response, was blocked completely by 1 μM ARC69931MX (n = 4 oocytes) (data not shown). PPADS, a nonspecific P2 receptor antagonist, blocked responses to 1 nM 2MeSATP with an IC50 value of 1.71 μM (pIC50 = 5.80 ± 0.05).
Detection of P2Y Receptor mRNA in Bovine Chromaffin Cells by RT-PCR. Of the nine known mammalian P2Y receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, P2Y14, and P2Y15) it was possible to design sequence-specific primers for bovine P2Y1, P2Y2, P2Y6, P2Y12, and P2Y14 (Table 1). Amplicons of the expected size were obtained for all primer pairs when PCR was performed on bovine genomic DNA (data not shown). Three primer pairs (P2Y1, P2Y12, and P2Y14) gave amplicons of the expected size when RT-PCR was performed on first-strand cDNA prepared from isolated bovine chromaffin cells (Fig. 8). Faint bands were observed in amplifications using P2Y2 and P2Y6 primers. However, these bands were not of the correct size and are therefore likely to correspond to nonspecific amplifications. No bands were observed in control reactions minus reverse transcriptase, confirming the absence of contaminating genomic DNA. Thus, from the RT-PCR analysis, transcripts for P2Y1, P2Y12, and P2Y14 but not P2Y2 or P2Y6 could be detected in bovine chromaffin cells.
Discussion
Like other classical neurotransmitters, it is now clear that postsynaptic receptors for nucleotides exist as either ligand-gated ion channels (ATP-sensitive P2X receptors), ideally suited to rapid neurotransmission, or G-protein–coupled P2Y receptors, suited to slower modulatory roles. Evidence for presynaptic nucleotide receptors in the peripheral and central nervous systems is also accumulating (Cunha and Ribeiro, 2000; Boehm and Kubista, 2002; Zhang et al., 2003). However, positive identification of the receptor subtypes mediating presynaptic effects of nucleotides has been complicated by a lack of selective pharmacological tools and a paucity of data from receptor knockout studies. We have shown previously that activation of a PTX-sensitive Gi/o-protein–coupled P2Y receptor in adrenal chromaffin cells inhibits exocytosis and Ca2+ entry through N-type and P/Q-type VOCCs (Powell et al., 2000); a similar mechanism is believed to underlie purinergic presynaptic inhibition of sympathetic neurotransmission. In this study, we have identified one of the inhibitory receptors in chromaffin cells as P2Y12. In addition, we have found evidence for a second UTP-preferring receptor that acts in a similar manner. The modulation of VOCCs in chromaffin cells by both nucleotide receptors showed all the characteristic properties of Gi/o signaling, namely sensitivity to voltage and PTX (Dolphin, 2003). This signaling pathway is known to be membrane-delimited, to be independent of diffusible second messengers, and to involve direct coupling between the receptor, Gi/o βγ subunits, and intracellular domains found on the α1A and α1B pore-forming subunits that make up neuronal N- and P/Q-VOCCs.
Similar inhibition of N-type VOCCs by heterologously expressed P2Y12 and the closely related P2Y13 receptor also has been reported (Simon et al., 2002; Kubista et al., 2003; Wirkner et al., 2004). In the chromaffin-like pheochromocytoma (PC-12) cell line, inhibition of N-type channels by a P2Y12-like receptor is found at the cell soma (Vartian and Boehm, 2001; Kubista et al., 2003) as well as in processes (Kulick and von Kügelgen, 2002) in which it contributes to an autocrine-paracrine inhibitory loop regulating exocytotic nucleotide release (Moskvina et al., 2003). One notable difference between the PC-12 receptor and bovine P2Y12 receptor is sensitivity to PPADS. We found it to be a competitive antagonist of the bovine receptor, whereas in PC-12 cells, which are rat-derived, it is reported to be ineffective (Vartian and Boehm, 2001; Kulick and von Kügelgen, 2002; Unterberger et al., 2002). Species differences in the pharmacology of other P2Y receptor subtypes have been reported previously (Sak and Webb, 2002).
In this study, we describe the cloning and characterization of a new member of the mammalian P2Y receptor family: bovine P2Y12. At the amino acid level, the bovine receptor is similar to human P2Y12, with 89% of residues identical between species (compared with 85% between human and rat P2Y12). However, the agonist selectivity of the receptor showed some slight differences from that reported for the human P2Y12 receptor, most notably for ATP and UDP. Whether ATP acts as an agonist at human P2Y12 is unclear and most likely depends on cell type and receptor density. At purified and reconstituted human P2Y12, in which nucleotide breakdown has been eliminated, ATP is not an agonist but is rather a low-affinity antagonist (Bodor et al., 2003). However, recombinant human P2Y12 expressed in Chinese hamster ovary cells shows an EC50 value for ATP (∼1 μM) similar to that reported for native rat P2Y12 in brain endothelial, capillary cells (Simon et al., 2002) and to values reported here for bovine P2Y12 in chromaffin cells and oocytes. In those studies in which ATP was reported as a P2Y12 agonist, ATP potency is an order of magnitude lower than ADP (Simon et al., 2002). At bovine P2Y12, however, in both native bovine chromaffin cells and X. laevis oocytes coexpressing recombinant bovine P2Y12 with rat inwardly rectifying potassium channels, ATP acts as a full agonist equipotent to ADP. Nucleotide breakdown of ATP to ADP can be excluded as an explanation of the bovine P2Y12 ATP response, because for this to be the case, it would require that 100% of ATP be instantaneously broken down by X. laevis oocytes and bovine chromaffin cells in a constant perfusion system, and in any case, in the bovine chromaffin cell system, ATP was actually slightly more potent than ADP.
A second difference in agonist selectivity between bovine and human P2Y12 was the sensitivity to UDP. UDP is inactive at human P2Y12 (Hollopeter et al., 2001; Takasaki et al., 2001). However, at bovine P2Y12 expressed in X. laevis oocytes, UDP is a full agonist, albeit with a low potency (EC50 ∼100 μM). Although unlikely, a contamination of the commercial UDP stocks used in this study with 1% ADP or ATP would be enough to explain the UDP sensitivity observed. To rule out this possibility, we performed high-performance liquid chromatography on UDP alone and on UDP spiked with ATP and ADP. No contaminating peak in the sample was greater than 0.01%, and no contaminating peak corresponded to either ATP or ADP. We also observed a small response to UTP in bovine P2Y12 expressed in X. laevis oocytes (∼10% maximal response to 10 mM UTP). At such high concentrations of UTP, the possibility that responses were caused by a breakdown of UTP to UDP could not be ruled out.
The UTP receptor inhibiting ICa and exocytosis in chromaffin cells has not been identified at the molecular level. UTP does not seem to be acting as a partial agonist at the bovine P2Y12 receptor because it was insensitive to ARC69931MX, caused no shift in the 2-MeSATP concentration-response curve, showed no cross-desensitization with the purine receptor in chromaffin cells, and had a very low potency at the cloned receptor. Heterologously expressed P2Y2, P2Y4, and P2Y6 receptors have also been shown to inhibit ICa in a neuronal expression system (Filippov et al., 1999, 2003). However, there are notable differences between the results from the expression studies and those found with the endogenous receptor in chromaffin cells; thus, even when overexpressed in neurons, P2Y2, P2Y4, and P2Y6 maintain their ability to couple to PTX-insensitive Gq proteins and inhibit M potassium currents, which would lead to an increase in [Ca2+]i. UTP does not cause calcium mobilization or entry in chromaffin cells, indicating that it is not acting through a Gq- or PLC-coupled receptor in these cells. Furthermore, inhibition of ICa by heterologously expressed P2Y4 is lost in whole-cell recording of neurons, whereas the receptor in chromaffin cells showed no such sensitivity to intracellular dialysis. Because a positive RT-PCR result was obtained for P2Y14 expression in bovine chromaffin cells (Fig. 8), we considered the possibility that UTP could be acting as a low-potency agonist at the bovine P2Y14 receptor in chromaffin cells. However, UDP-glucose, the cognate ligand for human P2Y14, gave no response when tested in bovine chromaffin cells (E. Seward, unpublished data). Thus, either bovine P2Y14 does not couple to VOCCs in bovine chromaffin cells or the P2Y14 RT-PCR product originated from nontranslated mRNA in chromaffin cells or from P2Y14 in a contaminating cell type. It is interesting to note that UTP inhibition of VOCCs has been observed in parasympathetic neurons (Abe et al., 2003), and evidence for a presynaptic inhibitory UTP receptor on sympathetic nerves of the rat and mouse vas deferens has also been reported (Von Kügelgen et al., 1989; Forsyth et al., 1991). Further studies will be required to determine whether one of the still-orphaned G-protein–coupled receptors that share significant sequence identity with the P2Y12 receptor represent a pyrimidine-selective Gi/o-coupled P2Y receptor in the ever-growing P2 receptor family.
Finally, the results from this study confirm that P2Y12 receptors, the targets of antithrombotic agents, are not restricted to platelets but are also expressed in neuroendocrine cells, in which they act as inhibitory receptors to regulate the activity of neuronal VOCCs and vesicular neurotransmitter release. Expression of these receptors at nerve terminals could serve as an important autocrine inhibitory feedback loop to regulate neurotransmission in the periphery and mediate heterosynaptic suppression in the central nervous system.
Acknowledgments
We thank Dr. Tania Webb for help and advice with the cloning the bovine P2Y12 receptor and preparation of the manuscript, Prof. Mark Boyett for providing the Kir 3.1 and Kir 3.4 plasmids, Prof. Mike Boarder for help with high-performance liquid chromatography analysis of nucleotides, Prof. Alan North for critical review of the manuscript, and AstraZeneca for the kind donation of ARC69931MX.
Footnotes
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This work was supported by a project grant from the Biotechnology and Biological Sciences Research Council (to S.J.E.), a studentship from the Medical Research Council (to A.D.P.), and a project grant from the Wellcome Trust (to E.P.S.).
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S.J.E. and A.D.P. contributed equally to the work.
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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doi:10.1124/mol.104.000224.
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ABBREVIATIONS: VOCC, voltage-operated calcium channel; ICa, calcium current; Cm, membrane capacitance; PPADS, pyridoxal-phosphate-6-azophenyl-2′,4′ disulfonic acid; 2-MeSATP, 2-methylthio-ATP; 2-MeSADP, 2-methylthio-ADP; PTX, pertussis toxin; ARC69931MX, N6-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-β,γ-dichloromethylene ATP; DMEM, Dulbecco's modified Eagle's medium; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; RT-PCR, reverse transcriptase-polymerase chain reaction; TM, transmembrane; PLC, phospholipase C; ATPγS, adenosine-5′-O-(3-thio)triphosphate; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid.
- Received March 8, 2004.
- Accepted May 28, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
References
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