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CELLULAR AND MOLECULAR

Involvement of P2Y₁ and P2Y₁₁ Purinoceptors in Parasympathetic Inhibition of Colonic Smooth Muscle

Departments of Physiology (B.F.K.) and Biochemistry and Molecular Biology (A.T.-N.), University College London, London, United Kingdom

Received for publication September 3, 2007
Accepted November 28, 2007.

	Abstract

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Purinergic signaling was first recognized in the guinea pig (Cavia porcellus) taenia coli, where relaxation of smooth muscle by nerve-released ATP may involve the activation of P2Y₁ and P2Y₁₁ receptors, and where transcripts for both genes have been found. A partial sequence for P2Y₁₁ protein was identified; the full-length P2Y₁ sequence has already been described. P2Y₁ and P2Y₁₁ proteins were localized by immunohistochemistry in smooth muscle cells. P2X₂ and P2X₃ proteins were also localized in motoneurons of the myenteric plexus. β-Methylene-ATP (βmeATP) and dibenzoyl-ATP (BzATP) evoked fast relaxations in the taenia, and they were inhibited by the P2Y₁ receptor antagonist 2'-deoxy-N⁶-methyladenosine 3',5'-bisphosphate (MRS2179). However, βmeATP and BzATP may stimulate neuronal P2X receptors to release ATP, which then acts on P2Y₁ receptors. In accordance, fast relaxations evoked by βmeATP and BzATP were inhibited by the P2X₃ and P2X_2/3 receptor antagonist 5-({[3-phenoxybenzyl][(1S)-1,2,3,4-tetrahydro-1-naphthalenyl] amino} carbonyl)-1,2,4-benzene-tricarboxylic acid (A317491). When P2Y₁, P2X₃, and P2X_2/3 receptors were blocked and adenosine was removed enzymatically, βmeATP and BzATP evoked slow relaxations that were inhibited by Reactive Red. Fast and slow relaxations involve small and large conductance calcium-activated potassium channels; the latter are dependent on intracellular cyclic AMP levels, which altered the duration and amplitude of relaxations. βmeATP and BzATP were confirmed as agonists, and Reactive Red as an antagonist, of human P2Y₁₁ receptors. In summary, G_q-coupled P2Y₁ receptors are involved mainly in fast relaxations, whereas G_qand G_s-coupled P2Y₁₁ receptors are involved in both fast and slow relaxations. These P2Y receptor subtypes, plus neuronal P2X receptors, may explain the phenomenon of parasympathetic inhibition first described by Langley (1898).

For the gastrointestinal tract, either ATP, nitric oxide (NO), vasoactive intestinal peptide (VIP), and/or the structurally related pituitary adenylyl cyclase-activating peptide (PACAP), or an admix of all four, is thought to mediate parasympathetic/nonadrenergic, noncholinergic inhibition of gut SMCs (Lecci et al., 2002). Of these autonomic neurotransmitters, ATP alone directly activates membrane surface receptors (P2Y receptors), which couple strongly to the G_q/11/phospholipase Cβ inositol 1,4,5-triphosphate pathway (King et al., 1998) and rapidly mobilize intracellular calcium ions (Ca²⁺_i) to open SK_Ca, BK_Ca, and intermediate conductance Ca²⁺-activated potassium channels (Lecci et al., 2002). In contrast, nitrergic (NO) and peptidergic (VIP/PACAP) transmission involves the generation of cGMP and cAMP followed by protein kinase Gand protein kinase A-based downstream phosphorylation of potassium ion channels, including TREK-1 and BK_Ca channels (Koh et al., 2001; Lecci et al., 2002).

It has been proposed that two relaxant P2Y receptors are activated by ATP in gut SMCs (Bültmann et al., 1996; Lecci et al., 2002). Eight mammalian P2Y subtypes (namely, P2Y_{1,2,4,6,11,12,13,14}) have been cloned, and of these subtypes, only P2Y_1,2,4,6,11 can couple efficiently to the G_q/11/phospholipase Cβ inositol 1,4,5-triphosphate pathway (Abbracchio et al., 2006). P2Y₂, P2Y₄, and P2Y₆ are activated by either UTP or UDP, which are poor relaxants of SMCs; in accordance, these P2Y receptors are usually excluded from consideration (Ralevic and Burnstock, 1998). The remaining P2Y₁ and P2Y₁₁ receptors are activated fully by ATP; as a result, they can rapidly mobilize Ca²⁺_i. Although sharing these common attributes, P2Y₁ and P2Y₁₁ receptor subtypes also differ in a number of their signaling and pharmacological properties. For example, human P2Y₁ receptors couple exclusively to G_q/11 protein (Abbracchio et al., 2006), they are antagonized by dibenzoyl-ATP (BzATP) (Vigne et al., 1999), and they are not activated by ,β-methylene-ATP (βmeATP) (King et al., 1998). By contrast, human P2Y₁₁ receptors can couple both to G_s and G_q/11 proteins (Abbracchio et al., 2006), and they are activated fully by either BzATP or βmeATP (van der Weyden et al., 2000a,b; Abbracchio et al., 2006).

We have investigated how parasympathetic inhibition is mediated by ATP, and we focused on the involvement of P2Y₁ and P2Y₁₁ receptors in SMC relaxation. We have used the guinea pig (Cavia porcellus) taenia coli as a test model, because purinergic transmission was first established here (Burnstock et al., 1970). We have also used BzATP, βmeATP, and some recently developed selective P2 receptor antagonists to investigate the relative contribution made by P2Y₁ and P2Y₁₁ receptors in the relaxation of the taenia.

	Materials and Methods

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Receptor Localization: Immunohistochemistry. The distribution of ATP receptor (P2X and P2Y) subtypes in guinea pig gastrointestinal tissues was determined by immunohistochemistry using the method of Poole et al. (2002), with modifications; namely, tissue sections were incubated postfixation in sodium citrate (10 mM, pH 6.0, at 80°C for 10 min) to allow antigen retrieval. P2X₂ and P2X₃ antibodies (Millipore Bioscience Research Reagents, Temecula, CA) were used at a dilution of 1:400. Smooth muscle cells in the guinea pig taenia coli were dispersed using the method of Albert and Large (2001). P2Y₁ and P2Y₁₁ antibodies (Abcam plc, Cambridge, UK) were also used at a dilution of 1:400.

Reverse Transcription-Polymerase Chain Reaction. Total RNA was isolated from guinea pig taenia using TRIzol Reagent (Invitrogen, Paisley, UK). Forward and reverse primers for P2Y₁ (AY048684 [GenBank] ) were nucleotides 639 to 662 (5'-AAAACCATCACCTGCTATGACACC-3') and 817 to 797 (5'-TTCCTCCTCAAAGGCGAGTTG-3'), respectively. Forward and reverse primers for P2Y₁₁ were 5'-CGGCTGTGGTCTTCTCGGC-3' and 5'-AGGCTGATGCAGGTGAGGAA-3', respectively. Polymerase chain reaction (PCR) was performed with the AccuPrime GC-rich DNA Polymerase kit (Invitrogen), using either 1/10 of the reverse transcription reaction (first amplification reaction) or 1/25 of the first PCR amplification (second amplification reaction) in a total volume of 25 µl containing 5 pmol of each of the forward and reverse primers, 1x reaction buffer A (for P2Y₁₁) or B (for P2Y₁), and 1 µl of DNA polymerase. Cycling conditions for the first round of amplification were 1 min at 94°C, followed by 30 cycles of (15 s at 94°C, 20 s at 63°C, and 15 s at 68°C), with a final 3 min at 68°C. Cycling conditions for the second round of amplification were as follows: 3 min at 94°C followed by 30 cycles of 15 s at 94°C, 15 s at either 63°C (P2Y₁) or 70°C (P2Y₁₁), and 15 s at 72°C followed by 3 min at 72°C. The amplified fragments were purified and cloned for DNA sequence analysis. In total, nine independent P2Y₁ fragment clones and 10 independent P2Y₁₁ fragment clones were sequenced.

Organ Bath Pharmacology. All animals were housed and cared for in accordance with Home Office (UK) regulations. Guinea pigs (450 –700 g) were killed by an approved schedule 1 method (cervical dislocation), after which the taenia coli were removed surgically. Strips of guinea pig taenia coli approximately 15 mm in length were suspended vertically in a 5-ml organ bath, with the upper end of the strip attached to an isometric force transducer (FT03; Gould Instrument Systems Inc., Cleveland, OH). The bathing solution contained 133 mM NaCl, 4.7 mM KCl, 1.4 mM NaH₂PO₄, 16.4 mM NaHCO₃, 0.6 mM MgSO₄, 7.7 mM D-glucose, and 2.5 mM CaCl₂, bubbled continuously with 95% O₂, 5% CO₂. Muscle strips were allowed to equilibrate for approximately 1 h at 37°C before being placed under 1-g resting tension. Tension recordings were stored using a PC and analyzed offline using AcqKnowledge III (BIOPAC Systems, Inc., Goleta, CA). Pharmacological data are expressed as the mean EC₅₀ value. Nonlinear regression and Schild analyses were performed using Prism 3.0 (GraphPad Software Inc., San Diego, CA). Significant differences (p < 0.05) between data points were determined by Student's t test using Instat 2.05A (GraphPad Software Inc.).

Signal Transduction Assays: Heterologous Expression. Human P2Y₁₁ cDNA was transfected into human astrocytoma 1321N1 cells using Lipofectamine LF2000 (Invitrogen) and stably selected using 400 µg · ml^–1 Geneticin (G-418; Invitrogen). Cells were grown in Phenol Red-free Dulbecco's modified Eagle's medium supplemented with 2 mM L-glutamine and 10% fetal bovine serum. Changes in the levels of intracellular calcium were assayed using a fluorometric imaging plate reader (FLIPR; Molecular Devices, Sunnyvale, CA). Cells were plated in poly-D-lysine-coated, black-walled, clear-bottomed 96-well tissue culture plates at a density of 10⁵ cells per well, and cells were allowed to grow to confluence (approximately 48 h). Cells were loaded using the Calcium Plus Assay dye (Molecular Devices) for 30 min at 37°C without washing, before they were assayed. Cell fluorescence was monitored every second for 10 s before agonist addition. Agonists were added as 10x stocks, and fluorescence was monitored for a further 170 s. Antagonists, where included, were preincubated for a 30-min period before agonist addition. Concentration-response curves were generated using peak height (maximal response–minimal response) values. Nonlinear regression and Schild analysis were performed using Prism 3.0 (GraphPad Software Inc.).

	Results

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Molecular Identification of P2Y₁ and P2Y₁₁ Receptor Transcripts. Whole genome shotgun C. porcellus sequences, submitted to the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov) trace archive database (version 4.0), were queried using human and canine P2Y₁₁ receptor sequences. Four overlapping guinea pig P2Y₁₁ genomic clones were identified (trace identifier nos. 816740877, 775645507, 835465461, and 816939529), and they were used to design gene-specific primers. Primers for P2Y₁ were derived from the published sequence of guinea pig P2Y₁ (AY048684 [GenBank] ).

Single fragments of the expected size for both P2Y₁ (179 base pairs) and P2Y₁₁ (187 base pairs) were observed after PCR amplification. No amplified products were detected in the control reactions (Fig. 1A), indicating that single P2Y₁ and P2Y₁₁ products were derived from reverse transcribed taenia coli mRNA and not from contaminating genomic DNA. The identity of the 179-base pair P2Y₁ and 187-base pair P2Y₁₁ amplification products was confirmed by DNA sequencing. The predicted amino acid sequence of the P2Y₁₁ amplification product is 81 and 78% identical to the same region (amino acid residues 64 –124) of the human and canine P2Y₁₁ receptor proteins, respectively (Fig. 1B). This is the first reported P2Y₁₁ sequence from a rodent-like (lagomorph) species. The nucleotide sequences of the guinea pig, human, and canine P2Y₁₁ receptors are highly conserved at 71.7, 66.8, and 64.7%, respectively. The full nucleotide sequence for guinea pig P2Y₁ has already been reported (AY048684 [GenBank] ).

View larger version (37K): [in this window] [in a new window]   Fig. 1. P2Y1 (lane 2) and P2Y11 (lane 5) receptor transcripts were detected using polymerase chain reaction amplification of reverse transcribed RNA isolated from guinea pig taenia coli. No amplification was seen in the control reactions (lanes 1 and 4). The fragment sizes of molecular weight markers (lane 3) are indicated to the right of the panel in base pairs. Below A, the predicted translation product of the amplified guinea pig P2Y11 fragment (gpP2Y11) is aligned with the corresponding region of the human (hP2Y11) and canine P2Y11 (dP2Y11) receptor proteins. B, asterisks, under the alignments, indicate residues conserved in all three P2Y11 receptor proteins.

Cellular Localization of P2Y₁ and P2Y₁₁ Receptor Proteins. Enzymatically dispersed SMCs from the guinea pig taenia coli were stained with the nuclear dye 4',6-diamidino-2-phenylindole (DAPI), and then they were stained again for P2Y₁ and P2Y₁₁ immunoreactivity (Fig. 2). SMCs were spindle shaped and measured 300 to 400 µm in length and 10 to 15 µm in diameter, and the DAPI-stained nucleus was centrally located (Fig. 2, top). Immunopositive staining for either P2Y₁ or P2Y₁₁ protein was seen in single SMCs seeded on separate coverslips (Fig. 2, bottom). No significant immunolabeling occurred where SMCs were processed without the primary P2Y antisera.

View larger version (77K): [in this window] [in a new window]   Fig. 2. Enzymatically dispersed SMCs from guinea pig taenia coli were processed for DAPI nuclear staining and for P2Y1 and P2Y11 immunostaining thereafter. Isolated SMCs were viewed under phase-contrast microscopy to help observe cell shape (phase + DAPI; top). By fluorescence microscopy, the presence of P2Y1 and P2Y11 immunoreactivity (P2Y1-ir and P2Y11-ir; lower panels) was confirmed in the same pair of cells (top). Scale bar, 50 µm.

View larger version (87K): [in this window] [in a new window]   Fig. 3. Intrinsic motoneurons of the myenteric plexus showed P2X3-ir in a spread preparation of the taenia (left). In addition, consecutive sections (10 µm in thickness) of the external smooth muscle layers of the guinea pig caecum showed colocalization of peroxidase-labeled P2X3and P2X2-immunoreactivity in a series of myenteric ganglia (right, arrows). The longitudinal layer (taenia) was sectioned transversely and lay in the top half of micrographs (right). Scale bars, 10 and 100 µm.

Nucleotide-Evoked Relaxations of Taenia Coli. Muscle strips from the guinea pig taenia coli were stimulated with 30 nM carbachol, which produced approximately 80% of maximal tone and established a state of persistent contraction (Fig. 4A). ATP, BzATP, and βmeATP caused brief relaxations of increasing size, in a concentration-dependent manner, in carbachol-precontracted taenia muscle strips (Fig. 4, A and B). Analysis of relaxant responses showed that BzATP and βmeATP were equipotent, with mean EC₅₀ values of 2.96 and 2.00 µM, respectively (see control data in Table 1). Their C/R curves were steep (n_H 2), and they rose from minimum to maximum over a single log₁₀ unit of concentration. Some form of co-operativity may have caused this acute sensitivity to these relaxants, and potential molecular targets for such co-operativity include the P2X receptors found in motoneurons of the myenteric plexus. By contrast, the C/R curve for ATP was shallow and biphasic, indicating that ATP exerted two effects and by two separate P2Y receptors. The two mean EC₅₀ values for ATP were 0.04 and 10.3 µM.

View larger version (23K): [in this window] [in a new window]   Fig. 4. Nucleotides (ATP, BzATP, and βmeATP; at their maximal concentration) briefly relaxed the taenia precontracted with carbachol (Carb; 30 nM) (A). Muscle tone was reduced in a concentration-dependent manner by ATP, BzATP, and βmeATP (B); data are expressed as mean ± S.E.M. (n = 4). Concentration/response curves for BzATP and βmeATP were steep and monophasic; the curve for ATP was shallow and biphasic. Potency indices and Hill coefficients are given in Table 1.

Inhibition and Potentiation of Nucleotide-Evoked Relaxations. Two antagonists were used to change the tissue sensitivity to the above-mentioned three relaxant nucleotides (see antagonist data in Table 1). The first was MRS2179 (100 µM), which blocks P2Y₁ receptors, and the second was A317491 (100 µM), which blocks P2X₃ and P2X_2/3 receptors. The potency of ATP, BzATP, and βmeATP was significantly reduced by the P2Y₁ receptor antagonist MRS2179, although ATP alone can activate P2Y₁ receptors. Instead, BzATP and βmeATP may act through P2X receptors on inhibitory motor nerves to release ATP. This possibility was supported in experiments showing that the potency of ATP, BzATP, and βmeATP was further reduced by blocking P2X₃ receptors and P2X_2/3 receptors with A317491 in the presence of MRS 2179 (see antagonist data in Table 1).

The relaxant potency of ATP, BzATP, and βmeATP was augmented by adding adenosine deaminase (ADA) at a final concentration of 2 U ml^–1 to the organ baths, in the added presence of MRS2179 and A317491 (see deaminase data in Table 1). ADA rapidly removes adenosine in the bathing solution, and it may prevent the activation of inhibitory adenosine receptors present on nerve and muscle. With ADA present, both ATP and BzATP were found to be equipotent (mean EC₅₀ values of 5.7 and 6.6 µM, respectively), whereas βmeATP was 10-fold less potent than either (mean EC₅₀ of 66.6 µM).

In control experiments, isoprenaline (0.01–100 µM) was used to see whether MRS2179, A317491, and ADA exerted any nonspecific effects on smooth muscle relaxations. EC₅₀ values (mean ± S.E.M.; n = 4) were isoprenaline (ISO) alone, 1.02 ± 0.25 µM; ISO + MRS2179, 0.75 ± 0.17 µM; ISO + MRS2179/A317491, 0.58 ± 0.07 µM; and ISO + MRS2179/A317491/ADA, 0.97 ± 0.22 µM. None of these EC₅₀ values was significantly different.

Fast-to-Slow Conversion of Nucleotide-Evoked Relaxations. The combination of MRS2179 and A317491 (with, or without, ADA present) led to a second phenomenon—a conversion of fast relaxations to slow relaxations (Fig. 5A, i–iii). This was true for all three nucleotides tested (Fig. 5B) and particularly for the nonhydrolyzable βmeATP (Fig. 5A). Measuring the time to recover 50% of the original muscle tone from the onset of relaxations (T₅₀ value), relaxations evoked by βmeATP lasted for 38 ± 2 s (mean ± S.E.M.; n = 8) in the absence of antagonists, and, in their presence, they persisted for more than 10 min (600 s), after which the nucleotide was removed by washout to prevent P2 receptor desensitization. Reactive Red (100 µM) antagonized the slow relaxations evoked by BzATP in a competitive manner, with apK_B value of 4.86 ± 0.14 (Fig. 5C).

View larger version (22K): [in this window] [in a new window]   Fig. 5. Recovery from relaxations was considerably faster in taenia muscle strips studied under control conditions (A, i) than in the presence of the P2 antagonists MRS2179 (100 µM) and A317491 (100 µM) and enzyme ADA (2 U ml–1) (A, ii and iii). A slow recovery rate was observed for all nucleotides under test conditions (antagonists + ADA; B), and slow relaxations evoked by BzATP (C) were antagonized competitively by Reactive Red (pKB = 4.86 ± 0.14; n = 4).

View larger version (16K): [in this window] [in a new window]   Fig. 6. Forskolin relaxed taenia muscle strips in a concentration-dependent manner (pEC50 of 5.90 ± 0.03; n = 4) (A). In addition, a low concentration of forskolin (FSK; 0.1 µM) significantly increased the amplitude and duration of relaxations evoked by βmeATP (100 µM) (B). A phosphodiesterase inhibitor (PDEi; Ro-20-1724; 20 µM) had similar potentiating effects on βmeATP-evoked relaxations (C). In contrast, an adenylate cyclase inhibitor (9-cPA; 9-cyclopentyladenine; 20 µM) significantly reduced the amplitude of near-maximal relaxations evoked by BzATP (100 µM) (D). Significant differences assessed by Student's paired t test (*, p < 0.05; **, p < 0.01; N.S., not significant).

View larger version (19K): [in this window] [in a new window]   Fig. 7. The amplitude of fast relaxations of the taenia was unaffected by dequalinium (DEQ; 20 µM), and the amplitude of slow relaxations was also unaffected by iberiotoxin (IBTX; 0.1 µM) (A). However, dequalinium increased and iberiotoxin decreased the duration of relaxations (B). The size and duration of slow relaxations were reduced significantly by a combination of dequalinium and iberiotoxin (DEQ + IBTX), and equally reduced by the general K+ channel blocking agent tetraethylammonium ions (TEA; 0.6 mM) (A, inset). Data are expressed as mean ± S.E.M. (n = 4).

Human P2Y₁₁ Receptor Expressed in 1321N1 Cells. Human P2Y₁₁ cDNA (AF030335 [GenBank] ) was transfected into human astrocytoma 1321N1 cells maintained in Phenol Red-free Dulbecco's modified Eagle's medium. Changes in levels of intracellular calcium were assayed using a FLIPR (Molecular Devices) in cells loaded with Calcium Plus Assay dye. ATP, BzATP, and βmeATP transiently elevated calcium levels in a concentration-dependent manner in transfected human astrocytoma 1321N1 cells (Fig. 8, A and B), whereas nontransfected 1321N1 cells failed to respond. For Ca²⁺ assays, the EC₅₀ values for ATP, BzATP, and βmeATP agonist potency were 3.31, 1.70, and 7.24 µM, respectively. These nucleotides also increased cAMP production in a concentration-dependent manner in transfected 1321N1 cells (data not shown).

View larger version (32K): [in this window] [in a new window]   Fig. 8. Calcium responses were measured using FLIPR fluorimetry for hP2Y11-1321N1 astrocytoma cells stimulated by extracellular nucleotides, either at the single concentration (100 µM; A) or over a range of concentrations (0.01–1000 µM; B). Antagonists, added to 96-well plates 30 min before the addition of a nucleotide (BzATP; 10 µM), inhibited calcium responses in a concentration-dependent manner (C and D). Data (A and C) were from 50,000 cells per well, whereas other data (B and D) are mean values from six wells per drug concentration. Mean pEC50 and pIC50 values are given in parentheses.

	Discussion

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Our experiments show that SMCs in the guinea pig taenia possess P2Y₁ and P2Y₁₁ receptors that mediate fast and slow purinergic relaxations. In fact, earlier work had already pointed to two unidentified P2Y receptors in this tissue (Dudeck et al., 1995; Bültmann et al., 1996; Lecci et al., 2002). Historically, the first P2Y subtype was known to be activated by ATP, 2-methylthio-ATP, and adenosine-5'-O-(3-thio)triphosphate (Dudeck et al., 1995) and blocked by suramin, Reactive Blue 2, and isoPPADS (Bültmann et al., 1996), which mirrors the pharmacological profile of P2Y₁ receptors (King et al., 1998). ATP and the P2Y₁ agonist 2-methylthio-ATP activate SK-channels in dispersed SMCs of the taenia, and this action is antagonized by the P2Y₁ antagonist PPADS (Kong et al., 2000). P2Y₁ receptors acting via SK-channels were shown to mediate fast hyperpolarization (IJPs) in SMCs of the guinea pig ileum (Wang et al., 2007) and human colon (Gallego et al., 2006). However, we now report that P2Y₁₁ receptors represent the second, unidentified P2Y subtype in the taenia. This second P2Y subtype was activated by βmeATP and other methylene phosphonates, and it was inhibited by suramin and Reactive Red (Dudeck et al., 1995; Bültmann et al., 1996). Of the now known recombinant P2Y subtypes, we find that human P2Y₁₁ receptors are uniquely activated by βmeATP and inhibited by suramin and Reactive Red.

In the present study, we found both P2Y₁ and P2Y₁₁ transcripts in the taenia and immunopositive P2Y₁ and P2Y₁₁ proteins in dispersed smooth muscle cells. We found that the selective P2Y₁ antagonist MRS2179 potently inhibited ATP-mediated fast relaxations in the taenia. This compound does not block the tetrodotoxin-resistant relaxations produced by the neuropeptides VIP and PACAP or by the NO donor sodium nitroprusside in human colon (Gallego et al., 2006). In addition, it does not inhibit recombinant human P2Y₁₁ receptors. However, MRS2179 puzzlingly inhibited the fast relaxations evoked by βmeATP and BzATP. Neither of these nucleotides is an agonist of P2Y₁ receptors (King and Townsend-Nicholson, 2003); furthermore, BzATP is an antagonist of human and rat P2Y₁ receptors (Vigne et al., 1999). However, both βmeATP and BzATP are potent agonists of P2X receptors, especially at P2X₃ and P2X_2/3 receptors (Jarvis et al., 2001). We looked for P2X₂ and P2X₃ protein in the taenia and found them in myenteric neurons, as have other investigators who placed these proteins alongside the enzyme nitric-oxide synthase in the majority (>80%) of inhibitory motoneurons (Castelucci et al., 2002; Poole et al., 2002). The activation of neuronal P2X₃ and P2X_2/3 receptors by βmeATP and BzATP may release significant amounts of ATP (and NO) to inhibit the taenia. We have not measured ATP release and its inhibition by MRS2179. Instead, we found that the highly selective P2X₃ and P2X_2/3 receptor antagonist A317491 inhibited fast relaxations to βmeATP and BzATP either when used alone or in tandem with MRS2179. Blocking P2X₃, P2X_2/3, and P2Y₁ receptors with A317491 and MRS2179 resulted in a new twist to this study: fast relaxations were replaced by slow relaxations. This new phenomenon seems to involve postjunctional P2Y₁₁ receptors.

We confirmed that both βmeATP and BzATP activate human P2Y₁₁ receptors, resulting in calcium release from intracellular stores and increased cAMP production. βmeATP has already been already shown to activate human P2Y₁₁ receptors expressed in human leukemic cell lines (van der Weyden et al., 2000a,b), although this observation seems to have been overlooked. We chose the frequently used human astrocytoma 1321N1 cell line to express human P2Y₁₁ receptors, at which βmeATP is only 2and 4-fold less potent than ATP and BzATP, respectively. In hindsight, it seems unremarkable that βmeATP is a P2Y₁₁ agonist, because the structurally related methylene phosphonate derivative ARC67085 is the most potent of the known P2Y₁₁ agonists (Abbracchio et al., 2006). In hindsight, it is also unremarkable to link βmeATP-evoked relaxations with P2Y₁₁ activation, because an extended series of methylene phosphonate derivatives (βmeATP, βCl₂meATP, and 2-methylthio-βCl₂meATP), which are structurally related to the P2Y₁₁ agonist ARC67085 are also highly potent relaxants of the taenia (Cusack et al., 1987). Furthermore, βmeATP can directly open apamin sensitive K⁺ channels in the taenia, even in the absence of extracellular calcium, by stimulating a suramin-sensitive P2Y receptor that mobiles intracellular Ca²⁺ stores (Den Hertog et al., 1985). We confirmed that suramin can antagonize Ca²⁺ responses mediated by BzATP-stimulated human P2Y₁₁ receptors, and we also showed that Reactive Red can do the same. In turn, Reactive Red competitively antagonized the slow relaxations evoked by BzATP in the taenia and also acted as a competitive antagonist of human P2Y₁₁ receptors.

We have found P2Y₁₁ transcripts in the taenia and also found P2Y₁₁ mRNA in esophagus, stomach, small intestine, and colon of human and guinea pig (our unpublished data). In humans, a short form (2-kilobase mRNA) of P2Y₁₁ transcripts occurs in leukocytes and spleen-derived tissue, but elsewhere mRNA exists in a series of larger chimeric forms (up to 5.6 kilobases) involving the readthrough of contiguous P2Y₁₁ and SSF1 genes on chromosome 19 (Abbracchio et al., 2006). Chimeric P2Y₁₁ transcripts are ubiquitously distributed throughout the body, and they are abundant in brain and pituitary, but they are also present in the gastrointestinal tract (Abbracchio et al., 2006). At present, we are unable to comment on the genomic structure and resulting transcription products of the guinea pig P2Y₁₁ receptor gene. A 187-base pair cDNA amplification product isolated from reverse transcribed RNA is homologous to the nucleotide sequences for human and canine P2Y₁₁ genes. However, the full gene sequence for guinea pig has eluded us, in part due to the high GC-rich content of the gene and the fact that only a low-coverage (1.92x) preliminary assembly of the guinea pig genome is currently available.

EC₅₀ values for nucleotide-evoked slow relaxations were initially unrelated to published values for the same nucleotides activating heterologously expressed human P2Y₁₁ receptors (Abbracchio et al., 2006), native P2Y₁₁ receptors in HL-60 cells (Conigrave et al., 1998), or our own data on ATP, βmeATP, and BzATP activation of human P2Y₁₁ receptors. Circumstances changed when we began to use ADA to prevent the buildup of adenosine in the taenia. Thereafter, the potency of ATP and BzATP increased significantly, and each nucleotide gave EC₅₀ values similar to those identified for P2Y₁₁ receptor activation. ADA did not exert nonspecific effects on the taenia, because it did not alter the relaxant potency of the β-adrenoceptor agonist isoprenaline. However, ADA did increase the potency of βmeATP in the taenia, and this observation was a cause for concern, because βmeATP does not readily generate adenosine. We suspect that βmeATP may stimulate a subtype of neuronal P2X receptors that cannot be blocked by either MRS2179 or A317491. One possibility is the P2X₂ receptor subtype (Spelta et al., 2002), which is present in the taenia and could be activated by the very high βmeATP concentrations we used to evoke slow relaxations. In turn, the stimulation of a neuronal P2X₂ receptor population may lead to ATP release, and, ultimately, to the production of adenosine on which ADA would act. We have already shown that low concentrations of adenosine inhibit the purinergic IJP by limiting the amount of transmitter released by motoneurons in guinea pig ileum (King, 1994).

P2Y₁₁ receptors can couple efficiently to both G_s and G_q/11 signaling proteins (Abbracchio et al., 2006). We found that the production of cAMP (via G_s stimulation), in tandem with the mobilization of intracellular Ca²⁺ (via G_q/11 stimulation), was critically important in the taenia, because the duration and amplitude of relaxations were altered by procedures that either raised or lowered intracellular cAMP levels. Cyclic AMP acts as a molecular switch for the -subunit of BK_Ca channels, and via subunit phosphorylation, it causes a leftward displacement of the voltage activation curve for BK_Ca channels and increases the probability of channel opening (Zhou et al., 2001). Because of their voltage sensitivity (typically active at –40 mV or lower), BK_Ca channels seem to make little or no contribution to the purinergic IJP when it is studied at the resting membrane potential of smooth muscles under investigation. However, the electrical and mechanical activities of agonist-stimulated smooth muscle are significantly potentiated by blocking BK_Ca channels, which seem to subserve the regulatory role of dampening intense excitatory stimulation (Carl et al., 1995; Vogalis et al., 1996; Liu et al., 1999). P2Y₁₁-based parasympathetic inhibition may only be fully effective when smooth muscle is excited and continuously depolarized, to maximize the P2Y₁₁-evoked responses of increased cAMP production and Ca²⁺ store mobilization on BK_Ca channel activation.

In summary, our study revealed that two different P2Y receptors occur on colonic SMCs and that they elicit fast or slow relaxations depending on local conditions. P2X receptors on neurons play a facilitatory role, mainly by stimulating ATP release, whereas adenosine receptors seem to do the opposite. The pharmacology of P2Y₁₁ receptors has been extended, further elucidating the actions of βmeATP in smooth muscle. The implication of this pharmacological observation may be wide ranging, because βmeATP is also capable of evoking prolonged vasorelaxation, and this action is more easily attributable to P2Y receptor activation than to P2X.

	Acknowledgements

 
We thank Prof. Trevor Smart (Department of Pharmacology, University College London) for the use of some equipment and Dr. Michael J. Jarvis (Abbott Laboratories, Abbott Park, IL) who provided A317491. We also thank Prof. William A. Large (St. George's Medical School, London, UK) for advice on dispersal of smooth muscle cells and Linda Churchill (Department of Physiology, University College London) who assisted in some immunohistochemical experiments.

	Footnotes

 
A.T.-N. is funded by British Heart Foundation Grants FD/99049 and PG2001052.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.131169.

ABBREVIATIONS: SMC, smooth muscle cell; IJP, inhibitory junction potential; SK_Ca, small conductance calcium-activated potassium channel; BK_Ca, large conductance calcium-activated potassium channel; NO, nitric oxide; VIP, vasoactive intestinal peptide; PACAP, pituitary adenylyl cyclase-activating peptide; BzATP, dibenzoyl-ATP; βmeATP, ,β-methylene-ATP; PCR, polymerase chain reaction; FLIPR, fluorometric imaging plate reader; C/R, concentration-response; DAPI, 4',6-diamidino-2-phenylindole; MRS2179, 2'-deoxy-N⁶-methyladenosine 3',5'-bisphosphate; A317491, 5-({[3-phenoxybenzyl][(1S)-1,2,3,4-tetrahydro-1-naphthalenyl] amino} carbonyl)-1,2,4-benzene-tricarboxylic acid; ADA, adenosine deaminase; ISO, isoprenaline; Ro-20-1724, 4-(3-butoxy-4-methoxyphenyl)methyl-2-imidazolidone; PPADS, pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid; ARC67085, 2-propylthio-βCl₂meATP; ir, immunoreactivity.

Address correspondence to: Dr. Brian F. King, Department of Physiology (Hampstead Campus), Medical School, University College London, Rowland Hill St., London, NW3 2PF, UK. E-mail: b.king{at}medsch.ucl.ac.uk

	References

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