ACCELERATED COMMUNICATION Loss of Nucleotide Regulation of Epithelial Chloride Transport in the Jejunum of P2Y4-Null Mice

The P2Y4 receptor is responsive to UTP in human and to ATP and UTP in rodents. With the aim of identifying its pharmacotherapeutic interest, we generated P2Y4-null mice by a classic gene targeting method. The proportion of genotypes was consistent with X-linked Mendelian transmission. Gene inactivation was checked by the complete disappearance of P2Y4 receptor mRNA from liver, stomach, and intestine. The P2Y4-null mice had a grossly normal behavior, growth, and reproduction. Chloride secretion by the jejunal epithelium was assessed in Ussing chambers by the measurement of the short circuit current in the presence of phlorizin. We show here that the UTPand ATPinduced chloride secretory responses observed in wild-type mice are abolished in P2Y4-null mice. This is the first clearcut demonstration of a biological role of the P2Y4 receptor. The P2Y4 receptor is a member of the P2Y family of G-protein-coupled receptors for extracellular nucleotides. Cloning of the human P2Y4 receptor was reported in 1995 (Communi et al., 1995; Nguyen et al., 1995; Stam et al., 1996) and was followed by the cloning of the rat (Bogdanov et al., 1998; Webb et al., 1998) and mouse (Lazarowski et al., 2001; Suarez-Huerta et al., 2001) orthologs. The human P2Y4 receptor is a selective UTP receptor, whereas the rodent ones are activated equipotently by UTP and ATP. No physiological role of the P2Y4 receptor has yet been established, and its pharmacotherapeutic potential thus remains uncertain. P2Y4 mRNA has been detected in human placenta (Communi et al., 1995) and in murine stomach, intestine, and liver (Suarez-Huerta et al., 2001). In the adult rat, they were detected in multiple organs but at a low level, whereas the expression was higher in neonatal animals (Webb et al., 1998). The presence of P2Y4 mRNA and/or protein, combined with functional responses to UTP, supports the expression of the P2Y4 receptor in 6CFSMEosubmucosal cells derived from human lung (Communi et al., 1999), in the vestibular dark cell epithelium of gerbil inner ear (Marcus and Scofield, 2001; Sage and Marcus, 2002), and in rat aortic smooth muscle cells (Harper et al., 1998). Expression in endothelial cells (Jin et al., 1998) has also been reported and might be related to the chemotactic and mitogenic actions of UTP on guinea pig coronary endothelial cells (Satterwhite et al., 1999). In addition, the persistence of a Cl secretory response to UTP and ATP in P2Y2 / mice led to the suggestion that this response could be mediated by the P2Y4 receptor (Cressman et al., 1999). To evaluate the physiological role and pharmacotherapeutic potential of the P2Y4 receptor we have generated P2Y4-deficient mice. In the work reported here, we focused our attention on the regulation by ATP and UTP of the Cl secretion in the jejunal epithelium with the aim of identifying the P2Y receptor mediating this control. This work was supported by the Fonds Forton, an Action de Recherche Concertée of the Communauté Française de Belgique, the Belgian Program on Interuniversity Poles of Attraction initiated by the Belgian State, Prime Minister’s Office, Federal Service for Science, Technology and Culture, grants of the Fonds de la Recherche Scientifique Médicale, and the Fonds Emile DEFAY. ABBREVIATIONS: bp, base pair(s); PCR, polymerase chain reaction; RT, reverse transcription; HPRT, hypoxanthine phosphoribosyltransferase; SSC, short-circuit current; 8-p-SPT, 8-(para-sulfophenyl)theophylline; ES, embryonic stem; CFTR, cystic fibrosis transmembrane conductance regulator. 0026-895X/03/6304-777–783$7.00 MOLECULAR PHARMACOLOGY Vol. 63, No. 4 Copyright © 2003 The American Society for Pharmacology and Experimental Therapeutics 2212/1044991 Mol Pharmacol 63:777–783, 2003 Printed in U.S.A. 777 at A PE T Jornals on A ril 3, 2017 m oharm .aspeurnals.org D ow nladed from Materials and Methods Generation of P2Y4-Deficient Mice. To generate the targeting vector, a 5 -SalI-XbaI-3 4900-bp fragment, with the XbaI site located 700 bp upstream from the initiation codon of the P2Y4 gene, was inserted in the pFlox vector upstream from the neomycin gene (Fig. 1A). A 2300-bp fragment, beginning at base 399 of the coding sequence of the gene and obtained by PCR, was inserted in the BamHI and XhoI sites of the targeting plasmid (i.e., downstream from the neomycin resistance cassette). Upon integration of the targeting vector, 1100 bp of the recombinant locus containing the first 398 bp of the published P2Y4 coding sequence are deleted. After electroporation of this targeting vector in R1 ES cells, G-418–resistant colonies were isolated. Classically extracted genomic DNA was analyzed by EcoRI digestion followed by Southern blotting using a DNA probe located outside the targeting vector, downstream from the P2Y4 gene. This probe detected a 7-kilobase pair fragment of the wild-type locus and a 8.6-kilobase pair fragment of the mutated locus. R1 P2Y4 / ES cells were cocultured overnight with CD1 morulae. Blastulae were then transferred into CD1 pseudopregnant female mice to generate chimeric mice. Offspring genotype was analyzed by Southern blotting as described above or by PCR. The actual repartition of genotypes was compared with the theoretical repartition expected from a binomial distribution with equal frequency of each allele. Total RNA Isolation, Reverse Transcription, and PCR Procedure. The expression of the P2Y4 receptor mRNA was studied on tissues extracted from wild-type and P2Y4-null male mice. Total mouse RNA was isolated from tested organs using TriPure reagent following the manufacturer’s instructions. One microgram of total RNA was submitted to OneStep reverse transcription PCR according to the manufacturer’s recommendations. Oligonucleotide amplification primers were designed according to the mouse P2Y4 sequence: sense primer, 5 -gactccttgctattcacat-3 ; antisense primer, 5 -agtagaggttccagtagaaa-3 . OneStep RT-PCR was performed with 0.6 M concentrations of each primer, with Q solution, and with 0.6 units of RNase inhibitor under the following conditions: 30 min at 50°C for the reverse transcription, 15 min at 95°C for the inactivation of the reverse transcriptase and the initial PCR activation step, followed by 3-step cycles (30 or 40 times) of 60 s at 94°C, 60 s at 52°C, 60 s at 72°C, and a final extension of 10 min at 72°C. Genomic DNA contamination was checked by omitting reverse transcription. The expression of HPRT mRNA was used for internal control of expression. Oligonucleotide amplification primers were designed according to the mouse HPRT sequence: sense primer, 5 -gctggtgaaaaggacctct-3 ; antisense primer, 5 -cacaggactagaacacctgc-3 . The protocol was the same as reported above, except that the PCR conditions were: (30 s at 94°C, 45 s at 57°C, 60 s at 72°C) 30 cycles, and a final extension of 10 min at 72°C. Amplification products were resolved on 1.5% (w/v) agarose gel by electrophoresis and visualized under UV light after ethidium bromide coloration. Bioelectric Measurements in Isolated Mouse Jejunum. The technique will be fully described in another article that will cover the theoretical aspect of the impedance analysis performed. Briefly, mice aged 2 to 5 months were sacrificed by intraperitoneal pentobarbital (10 mg/kg). The midportion of the jejunum, extending 10 cm after the ligament of Treitz, was dissected, opened, and washed with Krebs bicarbonate solution. The jejunal mucosa was stripped from the adjacent muscularis layer and sealed on the basolateral side to a fixation ring with an opening diameter of 3 mm. This ring was placed between the halves of an Ussing chamber. KCl electrodes connected to the solution via a short agar bridge were used for measuring potential difference and passing current. Impedance analysis was used to determine the resistance of the epithelium and bathing solution between the voltage electrodes. The impedance spectrum represented in a Nyquist diagram consisted of a single semicircle. This behavior is caused by the paracellular conductance related to the structure of the epithelium and/or possible damage of the epithelium caused by stripping the mucosa. Given these parameters, the epithelium can be represented by a lumped model consisting of a parallel circuit of a capacitance and resistance in series with the solution resistance between the voltage electrodes (Rsol). The resistance shunting the capacitance represents the transepithelial resistance (Repi). In the Nyquist diagram, the intercepts of the semicircle at high and low frequencies correspond to Rsol and Rsol Repi, respectively. In this series of experiments, the mean values were: Rsol 25 3 /cm 2 and Repi 14 2 /cm . Rsol attenuates the current recorded by the voltage clamp (SCCrec), and the short circuit current (SCC) expected for an ideal voltage clamp across the epithelium can be calculated as: SCC SCCrec (Repi Rsol)/Repi. The volume of each compartment bathing the jejunal mucosa was 2 ml, and Krebs bicarbonate solution, pre-equilibrated with a gas mixture of 5% CO2/95% O2 at 37°C, was flowing in each compartment at a rate of 20 ml/min. The composition of the Krebs bicarbonate solution was 140 mM Na, 5.2 mM K, 1.2 mM Mg, 1.2 mM Ca, 120 mM Cl, 2.8 mM PO4, 25 mM HCO3, and 11.5 mM glucose, pH 7.4. Study of Cl Secretory Response in Jejunum. Preliminary experiments in jejuna from control mice indicated that the shortcircuit current can be divided into two components: 1) a sodium absorptive component caused by the operation of the sodium-glucose cotransporter at the apical border of villus enterocytes and 2) a chloride secretory component linked to the existence of apical chloride channels in crypt cells. The first component could be eliminated by addition of 1 mM phlorizin to the apical bath, whereas the latter component was quantitatively accounted for by chloride secretion, because it was abolished in chloride-free solutions. Furthermore, the maneuvers of stripping the mucosa from its adjacent muscularis and mounting it in a small Ussing chamber induce the release of prostaglandins, which constitute a stimulus to chloride secretion, as first demonstrated by Pierce et al. (1971), and could therefore obscure other stimuli. Therefore, jejunal chloride secretion was assessed as the SSC after addition of phlorizin (1 mM) to the mucosal side as well as of indomethacin to both bathing media (100 M). UTP or ATP were added at 100 M to the apical solution and in some experiments Fig. 1. A, schematic representation of the targeting vector, endogenous allele and mutated allele after homologous integration. X, XbaI; E, EcoRI; S, SalI. B, Southern blot analysis of genomic DNA extracted from two recombinant clones of ES cells. After extraction, genomic DNA of ES clones was analyzed by EcoRI digestion and Southern blotting as described under Materials and Methods. The 7-kilobase pair signal corresponds to the wild-type allele and the 8.6-kilobase pair signal to the targeted allele. 778 Robaye et al. at A PE T Jornals on A ril 3, 2017 m oharm .aspeurnals.org D ow nladed from after preincubation in the presence of 8-(para-sulfophenyl)theophylline (8-p-SPT). Forskolin was added to the basolateral solution at the concentration of 10 M in ethanol 0.1%; this concentration of ethanol does not affect SCC (data not shown). The SCC was expressed in microamperes per square centimeter. Data are expressed as mean S.E.M. Statistical analysis was performed using unpaired t test. Materials. OneStep reverse transcription PCR was from QIAGEN (Westburg, Leusden, The Netherlands). RNase inhibitor was from Invitrogen (Merelbeke, Belgium). TriPure kit was from Roche Diagnostics (Basel, Switzerland). UTP, ATP, adenosine, indomethacin, forskolin, phlorizin, and 8-p-SPT were purchased from Sigma (Merelbeke, Belgium). R1 ES cells were a generous gift of Dr. A. Nagy (Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada). CD1 mice were purchased from Iffa Credo Belgium s.a. and Harlan France.