Present address: X. Fang, Division of Gastroenterology, Peking Union Medical College Hospital, Beijing, China.
The P2Y1 purinergic receptor expressed by enteric neurones in guinea-pig intestine
Article first published online: 23 JAN 2006
Neurogastroenterology & Motility
Volume 18, Issue 4, pages 316–323, April 2006
How to Cite
Gao, N., Hu, H.-z., Zhu, M. X., Fang, X., Liu, S., Gao, C. and Wood, J. D. (2006), The P2Y1 purinergic receptor expressed by enteric neurones in guinea-pig intestine. Neurogastroenterology & Motility, 18: 316–323. doi: 10.1111/j.1365-2982.2005.00754.x
- Issue published online: 31 JAN 2006
- Article first published online: 23 JAN 2006
- Received: 20 October 2005 Accepted for publication: 1 December 2005
- enteric nervous system;
- intestinal secretion;
- purinergic neurotransmission;
- submucosal plexus
Abstract Electrophysiological recording methods provided evidence for presynaptic release of ATP from enteric neurones and postganglionic sympathetic fibres in the enteric nervous system (ENS) of guinea-pig intestine (J Physiol Lond 2003; 550: 493–504). The released ATP acted at postsynaptic P2Y1 receptors to evoke slow synaptic excitation in neurones in the submucosal division of the ENS. Here, we report the cloning and characterization of the P2Y1 receptor, which was found in the guinea-pig submucosal layer. A 1178 bp cDNA clone was isolated from guinea-pig submucosal RNA by reverse transcription polymerase chain reaction (RT–PCR). The cDNA contained an open-reading frame of 1119 bp, encoding a 373 amino acid polypeptide of the same length and with 95% identity to the human P2Y1 receptor. Stable expression of the guinea-pig cDNA in human embryonic kidney (HEK)293 cells was accompanied by a marked increase in sensitivity for elevation of free intracellular calcium evoked by ATP or related nucleotides. The potency order for ATP and its analogues was: 2-methio-adenosine diphosphate > 2-methio-adenosine triphosphate > ADP > ATP-γ-S > ATP. The selective P2Y1 receptor antagonist, MRS2179, was a competitive antagonist for the receptor with a pA2 value of 6.5. The results add to existing evidence for expression of a functional P2Y1 purinergic receptor in neurones of the submucosal division of the ENS.
Neurones in the submucosal division of the enteric nervous system (ENS) in guinea-pig intestine receive fast excitatory synaptic input, slow excitatory synaptic input (slow EPSP) and excitatory paracrine input from endocrine and inflammatory/immune cells.1,2 A specialized kind of slow EPSP in a discrete subpopulation of neurones in the guinea-pig submucosal plexus is mediated by the synaptic release of ATP.3 Application of ATP mimics the slow EPSP with an EC50 of 1.5 ± 0.2 μmol L−1 and the selective P2Y1 receptor antagonist, MRS2179, suppresses both the EPSP and the slow EPSP-like action of ATP. MRS2179 suppresses the slow EPSP-like action of ATP in a competitive manner with a pA2 value of 6.18 in this neuronal population.3
The purinergic excitatory input to the identified subpopulation of submucosal neurones is derived from neighbouring neurones in the same plexus, from neurones in the myenteric division of the ENS and from sympathetic postganglionic neurones.3 The neighbouring purinergic neurones in the submucosal plexus themselves express serotonergic 5-HT3 receptors that might be exposed to serotonin released from mucosal enterochromaffin cells. The ATP-mediated slow EPSPs occur coincident with fast nicotinic synaptic potentials that are evoked by projections from the myenteric plexus and with inhibitory postsynaptic potentials (IPSPs) evoked by release of noradrenaline from sympathetic fibres.
The P2Y1 receptor expressed by the identified neuronal subpopulation has been identified as a metabotropic receptor linked to activation of phospholipase C (PLC), synthesis of inositol-1,4,5-trisphosphate (IP3) and mobilization of Ca2+ from intracellular stores.3 The ionic channels underlying the P2Y1-mediated excitatory responses in the neurones have been identified tentatively as belonging to the transient receptor potential (TRP)6 family of ionic channels.4,5
The present study was designed to acquire additional information on the functional characteristics of the P2Y1 receptor in the guinea-pig submucosal plexus by cloning the receptor and analysing its behaviour after heterologous expression in human embryonic kidney (HEK)293 cells. A preliminary report has appeared in abstract form.6
Adult male guinea-pigs (Albino-Hartley, 300–600 g) were killed by stunning followed immediately by exsanguination from the cervical vessels according to procedures reviewed and approved by the Ohio State University Laboratory Animal Care and Use Committee and USA Department of Agriculture veterinary inspectors. Ten centimetres of ileum was pinned flat with the mucosal side up to Sylgard 184® encapsulating resin (Dow Corning, Midland, MI, USA) in a dissection dish containing ice-cold Krebs solution. Fine forceps were used to remove the mucosa and expose the submucosal plexus. The submucosal plexus, within its matrix of connective tissue, was dissected free and homogenized in TRIzol (Life Tech., Gaithersburg, MD, USA) with 100 mg of tissue per 1 mL TRIzol. Total RNA was extracted according to the manufacturer's protocol and dissolved in diethylpyrocarbonate (DEPC)-treated water. The extracted RNA was treated with DNase and stored at −70 °C until use.
For cloning of the P2Y1 receptor, degenerate oligonucleotide primers [sense: 5′-CTTTTCCGATGC(G/T)(C/T)GCTG-3′; antisense: 5′-TCT(G/T)GTGCCTTCACA(A/G)CT-3′] were synthesized based on the most conserved segments between the mouse and human P2Y1 receptor sequences (GenBank accession numbers: U42030 and AJ245636). Reverse transcription polymerase chain reaction (RT–PCR) was carried out with the Titan one tube RT–PCR kit (Roche, Indianapolis, IN, USA) in a 0.2 mL tube containing 1 μg of total RNA, 0.2 mmol L−1 each dNTP, 5 mmol L−1dithiothreitol, 5 units RNase inhibitor, 0.4 μg each of the above primers (0.4 μg), 1X RT–PCR buffer and a 1-μL enzyme mix in a final volume of 50 μL. Reverse transcription was performed according to the manufacturer's protocol. Amplification conditions were: 94 °C for 2 min; 10 cycles of 94 °C for 30 s, 55 °C for 30 s and 68 °C for 45 s; 25 cycles of 94 °C for 30 s, 55 °C for 30 s, 68 °C for 45 s with cycle elongation of 5 s for each cycle. An extension step was then performed at 68 °C for 7 min. The PCR product was subcloned into a pT-Adv vector using the Advantage® PCR cloning kit (Clontech, Palo Alto, CA, USA). The plasmid was then isolated and used for DNA sequencing. An EcoRI fragment containing the whole P2Y1 receptor cDNA insert was cut out and subcloned into pIRESneo (Clontech) for mammalian cell expression.
The distribution of the P2Y1 receptor in guinea-pig organs other than the gut was studied by preparing cDNA from total RNA isolated in TRIzol for the different tissues with the First-strand cDNA Synthesis Kit for avian myeloblastosis virus reverse transcriptase (Roche Diagnostics Corporation). PCR Master (Roche Diagnostics Corporation) was used for PCR. Oligonucleotides: 5′-AAGACGGGCTTCCAGTT(C/T)TACTAC-3′ and 5′-CATCGTTTTCATCACATGGA-3′ were used as degenerate sense and antisense PCR primers, respectively, for the amplification of the P2Y1 receptor. The primers were synthesized based on the most conserved segments between the mouse and human P2Y1 receptor sequences (GenBank accession numbers: U42030 and AJ245636). The β-actin primers, 5′-GATCTGGCACCACACCTTTT-3′ and 5′-TCCTTGATGTCACGCACAAT-3′, were used to amplify a 390 bp actin fragment for normalization of the amounts of cDNA across the different tissues. Conditions for PCR were: 94 °C for 2 min; 30 cycles of 94 °C for 1 min, 56.5 °C for 2 min and 72 °C for 3 min. An extension step was then performed at 72 °C for 7 min. The final products were run on 1.5% agarose gels containing ethidium bromide and visualized with an ultraviolet light source. Negative controls were performed without RT.
Heterologous expression in HEK293 cells
The HEK293 cells were grown in Dulbecco's modified Eagle's medium containing 4.5 mg mL−1 glucose, 10% heat-inactivated fetal bovine serum, 50 units mL−1 penicillin and 50 μg mL−1 streptomycin. For transfection, 2.8 × 106 cells were cultured in a 10-cm tissue culture dish for 20 h before 40 μg of the full-length guinea-pig P2Y1 receptor cDNA in the pIRESneo vector and the Lipofectamine Plus® reagent were added to the cells following the manufacturer's protocol (Life Tech.). After 24 h, the HEK293 cells were harvested, suspended in the same medium supplemented with 400 μg mL−1 G418, and transferred into the 96-well plates in three serial dilutions of 1 : 4. The medium was replaced once in every 4 days until colonies appeared in the wells. G418-resistant transformants that appeared to arise from single colonies were transferred to 12-well plates for growth and further analysis. The stable cell lines were diluted twice weekly and maintained in the medium supplemented with 400 μg mL−1 G418.
Measurement of intracellular calcium
HEK293 cells, which were expressing the guinea-pig P2Y1 receptor or a control vector, were seeded onto 10 mm diameter glass coverslips that were coated with poly-d-lysine (10 μg mL−1) on the day prior to study. The HEK293 cells were loaded with 2 μmol L−1 fura2-AM in an extracellular medium consisting of: 140 mmol L−1 NaCl, 5 mmol L−1 KCl, 1 mmol L−1 MgCl2, 1.8 mmol L−1 CaCl2, 10 mmol L−1 glucose, 0.1% bovine serum albumin and 15 mmol L−1 HEPES, pH 7.4 at 37 °C for 30 min. After two washes with the same medium, the cells were placed at room temperature in the dark for no more than 6 h prior to use. The coverslips were mounted at the bottom of a perfusion chamber (RC-25F, Warner Instruments Inc., New Heaven, CT, USA), which was placed on the stage of a Nikon Eclipse TE200 (Nikon, Inc., Melville, NY, USA) inverted epifluorescence microscope. The cells were superfused with a Rainin circulating pump at a flow rate of 3 mL min−1. Changes in intracellular Ca2+ in a small population of 10–15 cells were monitored at room temperature by fluorescence photometry. Alternating excitation wavelengths of 340 and 380 nm were provided by a DeltaRAM monochromator and emitted fluorescence at 520 nm was recorded by a photomultiplier (Photon Technology International, Inc., Lawrenceville, NJ, USA). Known concentrations of ATP and its analogues were applied in the superfusion solution. The Felix Program (Photon Technology International, Inc.) was used to calculate the ratios of the emitted fluorescence for intensities at 340 and 380 nm.
Data are presented as the mean ± SEM. Agonist concentration–response curves were fit by computer to a simple logistic equation and EC50 values for the agonist actions of nucleotides on increases in intracellular free Ca2+ in the HEK293 cells were determined with sigmaplot non-linear curve-fitting software (SPSS, Inc., Chicago, IL, USA). The pA2 values for antagonists were obtained from Schild plots of the data. Concentration–response data sets were obtained for each of at least five coverslips. Curves were then fitted to pooled mean data points with the Hill equation , where y is the normalized response; ymax is maximum response; x is ligand concentration (nmol L−1); K is EC50 of the agonist (nmol L−1) and nH is the Hill coefficient. Data for changes 2-methio-adenosine triphosphate (2-Me-ATP)-evoked increases in intracellular Ca2+ were normalized to the maximal response evoked by the maximal concentration of 2-Me-ATP.
Fura2-AM was purchased from Molecular Probes (Eugene, OR, USA). ATP, 2-methio-adenosine diphosphate (2-Me-ADP), ADP, ATP-γ-S, UTP, UDP, α,β-methylene-ATP, ryanodine, U73122, U73343 and suramin were purchased from Sigma (St Louis, MO, USA). 2-Methio-adenosine triphosphate was purchased from Research Biochemical (Natick, MA, USA). 2-Aminoethoxydiphenyl borate (2-APB) was purchased from Torcris (Ballwin, MO, USA).
Reverse transcription polymerase chain reaction was carried out with total RNA from the submucosal plexus layer and with a pair of primers, which were designed to correspond with the homologous regions between the mouse and human P2Y1 receptor. This method generated a 1222 bp PCR product, which contained an open-reading frame that was 90%, 91%, 87%, 86% and 79% identical to the cDNA obtained from human, bovine, rat, mouse and chicken P2Y1 receptors, respectively. The deduced polypeptide contained 373 amino acids, which was the same for all mammalian P2Y1 receptors known to date. Analysis of the multiple sequence alignment with the clustal w Program (http://align.genome.jp) revealed that the P2Y1 receptor from the guinea-pig was most closely related to the bovine and human counterparts with the guinea-pig receptor sharing 96% and 95% sequence identity, respectively, at the amino acid level (Fig. 1A,B). The amino-terminal regions of P2Y1 receptors exhibit the most overall diversity across all species. When compared with the human, amino acid substitutions in the guinea-pig receptor were found mostly at the N-terminus (i.e. 11 of the 20 total substitutions). The C-terminus and extracellular loops 1 and 2 each had one substitution. Extracellular loop 3 and transmembrane regions I and IV each had two substitutions (Fig. 1B). Therefore, most of the amino acid substitutions for the guinea-pig receptor were found in the extracellular extension of the receptor.
Semiquantitative multiple tissue RT–PCR was used to investigate the relative distribution of the P2Y1 receptor among the various guinea-pig organs. We selected this method because mRNA transcripts for P2Y receptors are frequently found at concentrations detectable by RT–PCR, but not by Northern blot analysis.7 TRIzol reagent was used to prepare total RNA from the different tissues. Primers were designed to amplify a 711-bp fragment (273–983) of the guinea-pig P2Y1 receptor. The RT–PCR yielded a single 711-bp band from all tissues tested (Fig. 2A,B). All RT–PCR products were purified and confirmed by sequencing. No product was found after PCR for RNA controls. For non-nervous tissues, the P2Y1 transcript was more abundant in the spleen and heart than in other tissues. In the nervous system, the P2Y1 transcript was found in brain, spinal cord, dorsal root ganglia and the submucosal and myenteric divisions of the ENS. Compared with other nervous tissues, the guinea-pig brain expressed a relatively smaller amount of the P2Y1 transcript (Fig. 2A). The widespread distribution is supporting evidence that the P2Y1 receptor sequence we isolated from the submucosal layer is the true guinea-pig P2Y1 receptor, which is expressed ubiquitously and to varying degrees throughout guinea-pig tissues.
Expression in HEK293 cells
The cDNA was subcloned into a mammalian expression vector and transfected into HEK293 cells for functional and pharmacological study of the behaviour of the newly cloned guinea-pig P2Y1 gene. Stable clonal cell lines were established after limiting dilution and selection in a medium containing G418. Changes in intracellular Ca2+ in response to purinergic agonists in fura2-loaded cells were the end points for assessment of the functional and pharmacological properties of the receptor.
Because the HEK293 cells endogenously express the human P2Y1 receptor, we compared the concentration dependence for responses to 2-Me-ATP in HEK293 cells that were transfected with the pIRESneo vector alone (i.e. controls) to the responses of cells in which the guinea-pig P2Y1 receptor was overexpressed. Concentration–response data were obtained for 10–30 individual cells and averaged values were used to calculate EC50 values. The overexpressed guinea-pig P2Y1 receptor was activated by concentrations of 2-Me-ATP that were 1 × 104-fold lower than the concentrations required for activation of the endogenously expressed human P2Y1 receptors in the HEK293 cells. For non-transfected HEK293 cells, the EC50 for 2-Me-ATP was 579.8 ± 70.35 nmol L−1, the threshold was ≅100 nmol L−1 and the maximum response was obtained at 30 μmol L−1 (Fig. 3A). For HEK293 cells, which overexpressed the guinea-pig P2Y1 receptor, the EC50 for 2-Me-ATP-evoked responses was 0.28 ± 0.02 nmol L−1, the threshold was ≅0.01 nmol L−1 and the maximum response occurred at 3 nmol L−1 (Fig. 3B). This leftward shift of the concentration–response curve was typical for overexpressed receptors. The finding that the overexpressed guinea-pig receptor was maximally activated by 3 nmol L−1 2-Me-ATP, which was more than 30 times lower than the minimal concentration required to activate the endogenous human P2Y1 receptors expressed by the HEK293 cells, made possible study of the pharmacology of the heterologously expressed guinea-pig P2Y1 receptor at concentrations of 2-Me-ATP lower than 10 nmol L−1. This could be carried out because the endogenously expressed human P2Y1 receptors were not activated at concentrations <10 nmol L−1.
ATP or some of its analogues were applied in differing concentrations to evaluate specificity of the cloned P2Y1 receptor for different nucleotides when expressed in HEK293 cells. The 2-Me-ADP was the most potent agonist with an EC50 of 0.045 ± 0.003 nmol L−1. The EC50 values for 2-Me-ATP, ADP, ATP-γ-S and ATP were, 0.28 ± 0.02, 0.75 ± 0.07, 1.93 ± 0.16 and 6.32 ± 0.18 nmol L−1 respectively (Fig. 4). The potency order was therefore 2-Me-ADP > 2-Me-ATP > ADP > ATP-γ-S > ATP. UTP, UDP, adenosine or AMP were ineffective at concentrations up to 100 μmol L−1 (data not shown). These values are consistent with the potency order reported for other mammalian P2Y1 receptors.8
The selective P2Y1 receptor antagonist, MRS2179 (0.3–10 μmol L−1), suppressed elevation of intracellular Ca2+ evoked by 2-Me-ATP (0.1–100 μmol L−1). The nature of the antagonism was determined by generating a series of concentration–response curves for 2-Me-ATP in the presence of different concentrations of MRS2179. The concentration–response curves for 2-Me-ATP were shifted rightward by increasing concentrations of MRS2179 (Fig. 5A). Schild analysis confirmed that the MRS2179 antagonism was competitive with a pA2 value of 6.50 (Fig. 5B). Elevations of intracellular Ca2+ evoked by 2-Me-ATP were also suppressed by 73.5 ± 12.1% by the non-selective purinergic receptor antagonist, suramin (300 μmol L−1), in five trials with 10–30 HEK293 cells (data not shown).
Inhibition of ryanodine receptors by high concentrations of ryanodine (10 μmol L−1) did not change 2-Me-ATP-evoked responses. 2-APB, which is a documented inhibitor of IP3 receptors and capacitative Ca2+ entry,9 suppressed 2-Me-ATP-evoked increases to 9.4 ± 1.6% of control. The PLC inhibitor U73122 (10 μmol L−1), but not its inactive analogue U73343 (10 μmol L−1), suppressed 2-Me-ATP-evoked responses to 12.5 ± 7.8% of control (Fig. 6B). These findings are consistent with our electrophysiological findings for purinergic slow EPSPs and slow EPSP-like responses to ATP, which suggested that postreceptor signalling for the guinea-pig P2Y1 receptor involves stimulation of PLC, activation of intracellular IP3 receptors and subsequent Ca2+ release from intracellular stores.3
The amino acid sequence obtained for the cloned guinea-pig submucosal P2Y1 receptor in the present study displayed close homology with other mammalian P2Y1 receptors, especially in the transmembrane domains. The pharmacological behaviour of the cloned receptor, when expressed in HEK293 cells, was essentially the same as for the P2Y1 receptor expressed by neurones in the guinea-pig submucosal plexus. Nucleotides activate P2Y1 receptors expressed by submucosal neurones and our cloned P2Y1 receptor with the same potency order of 2-Me-ADP > 2-Me-ATP > ADP > ATP-γ-S > ATP.3 This potency order for the cloned guinea-pig receptor is consistent with the order of potency reported for cloned and functionally expressed P2Y1 receptors elsewhere.8
MRS2179 is a competitive antagonist at the P2Y1 receptor expressed by neurones in the guinea-pig submucosal plexus.3 MRS2179 behaved in like manner as a competitive antagonist at the cloned submucosal P2Y1 receptor when expressed in HEK293 cells. The pA2 value of 6.5 for MRS2179 in the HEK293 cells is close to the pA2 value of 6.18 obtained for MRS2179 with electrophysiological recording in S-type neurones in the whole mount submucosal plexus.3 These results overall, define a correlate at the molecular level for the excitatory action of ATP on the subpopulation of submucosal neurones, which are characterized by S-type electrophysiological behaviour, uniaxonal morphology, immunoreactivity for vasoactive intestinal peptide and inhibitory synaptic input mediated by α2 noradrenergic receptors.1,3,10 Moreover, the results add support for purinergic slow excitatory synaptic transmission mediated by the P2Y1 receptor subtype in the submucosal plexus of guinea-pig intestine.
The pharmacological evidence, obtained in the present study, suggests that the postreceptor signal transduction cascade for the cloned P2Y1 receptor in HEK293 cells is the same as the signal transduction cascade for purinergic slow EPSPs and the slow EPSP-like action of exogenously applied ATP in S-type submucosal neurones.3 Signal transduction for the P2Y1 receptor follows the generalized theme for metabotropic signal transduction in enteric S-type neurones with uniaxonal morphology, which involves activation of PLC and a Ca2+-calmodulin second messenger system.2,3,11–15 Receptors for the slow EPSP in S-type neurones are G protein-coupled to activation of phosphatidal inositol (PI)-PLC. The PI-PLC catalyses the formation of IP3 and diacylglycerol. Once released, IP3 acts as a second messenger to release Ca2+ from intraneuronal membrane stores. Binding of the released Ca2+ to calmodulin activates calmodulin kinases II and IV (CaMKII–IV). Diacylglycerol together with Ca2+ and anionic phospholipids activates conventional isoforms of PKC (i.e. α-, βI-, βII- and γ-isoforms). The PKC phosphorylates cation conductance channels that open, when phosphorylated, to increase cationic conductance and thereby depolarize the membrane potential. Limited evidence suggests that the cationic conductance channels might be TRP-6 channels.4,5 Opening of the cationic conductance channels accounts for the decreased input resistance and reversal potentials near zero that are observed while recording with microelectrodes during the depolarization phase of the EPSP and the slow EPSP-like actions of ATP6 and other mimetics such as mast cell proteases11 and bradykinin.15,16 Termination of the EPSP probably results from activation of the intraneuronal phosphatase, calcineurin, which catalyses dephosphorylation of the cationic channels.12,17
The study was supported by the National Institutes of Health RO1 DK 68258, RO1 DK 37238 to J.D.W. and a Pharmaceutical Manufacturers of America Foundation Postdoctoral Fellowship to S.L.
- 4Transient receptor potential channel 6 in guinea-pig small intestinal submucosal plexus. Neurogastroenterol Motil 2002; 14: 590–1., , et al.
- 5Transient receptor potential canonical 6 (TRPC6) channel in guinea-pig enteric neurons. Gastroenterology 2004; 126: A93., , et al.
- 6Purinergic slow excitatory synaptic transmission in guinea-pig small intestinal submucosal plexus. Gastroenterology 2002; 122: A8., , et al.
- 10Cellular neurophysiology of enteric neurons. In: BarrettKE, GhishanFK, JohnsonLR, MerchantJL, SaidHM, WoodJD, eds. Physiology of the Gastrointestinal Tract, 4th edn. San Diego, USA: Elsevier, 2006 (in press)..
- 17Calmodulin (CaM) and CaM kinase signaling in the enteric nervous system. Neuroscience (Abstr) 2001; 27: 839.11., , , , , .