Expression and Trans-synaptic Regulation of P2x4 and P2z Receptors for Extracellular ATP in Parotid Acinar Cells
1998; Elsevier BV; Volume: 273; Issue: 41 Linguagem: Inglês
10.1074/jbc.273.41.26799
ISSN1083-351X
AutoresLalitha Tenneti, Simon J. Gibbons, Barbara R. Talamo,
Tópico(s)Pancreatic function and diabetes
ResumoTrans-synaptic regulation of muscarinic, peptidergic, and purinergic responses after denervation has been reported previously in rat parotid acinar cells (McMillian, M. K., Soltoff, S. P., Cantley, L. C., Rudel, R., and Talamo, B. R. (1993) Br. J. Pharmacol. 108, 453–461). Characteristics of the ATP-mediated responses and the effects of parasympathetic denervation were further analyzed through assay of Ca2+ influx, using fluorescence ratio imaging methods, and by analysis of P2x receptor expression. ATP activates both a high affinity and a low affinity response with properties corresponding to the recently described P2x4 and the P2z (P2x7)-type purinoceptors, respectively. Reverse transcription-polymerase chain reaction analysis reveals mRNA for P2x4 as well as P2x7 subtypes but not P2x1, P2x2, P2x3, P2x5, or P2x6. P2x4 protein also is detected by Western blotting. Distribution of the two types of ATP receptor responses on individual cells was stochastic, with both high and low affinity responses on some cells, and only a single type of response on others. Sensitivity to P2x4-type activation also varied even among cells responsive to low concentrations of ATP. Parasympathetic denervation greatly enhanced responses, tripling the proportion of acinar cells with a P2x4-type response and increasing the fraction of highly sensitive cells by 7-fold. Moreover, P2x4 mRNA is significantly increased following parasympathetic denervation. These data indicate that sensitivity to ATP is modulated by neurotransmission at parasympathetic synapses, at least in part through increased expression of P2x4mRNA, and suggest that similar regulation may occur at other sites in the nervous system where P2x4 receptors are widely expressed. Trans-synaptic regulation of muscarinic, peptidergic, and purinergic responses after denervation has been reported previously in rat parotid acinar cells (McMillian, M. K., Soltoff, S. P., Cantley, L. C., Rudel, R., and Talamo, B. R. (1993) Br. J. Pharmacol. 108, 453–461). Characteristics of the ATP-mediated responses and the effects of parasympathetic denervation were further analyzed through assay of Ca2+ influx, using fluorescence ratio imaging methods, and by analysis of P2x receptor expression. ATP activates both a high affinity and a low affinity response with properties corresponding to the recently described P2x4 and the P2z (P2x7)-type purinoceptors, respectively. Reverse transcription-polymerase chain reaction analysis reveals mRNA for P2x4 as well as P2x7 subtypes but not P2x1, P2x2, P2x3, P2x5, or P2x6. P2x4 protein also is detected by Western blotting. Distribution of the two types of ATP receptor responses on individual cells was stochastic, with both high and low affinity responses on some cells, and only a single type of response on others. Sensitivity to P2x4-type activation also varied even among cells responsive to low concentrations of ATP. Parasympathetic denervation greatly enhanced responses, tripling the proportion of acinar cells with a P2x4-type response and increasing the fraction of highly sensitive cells by 7-fold. Moreover, P2x4 mRNA is significantly increased following parasympathetic denervation. These data indicate that sensitivity to ATP is modulated by neurotransmission at parasympathetic synapses, at least in part through increased expression of P2x4mRNA, and suggest that similar regulation may occur at other sites in the nervous system where P2x4 receptors are widely expressed. reverse transcription-polymerase chain reaction glyceraldehyde-3-phosphate dehydrogenase base pair(s) guanosine 5′-(β-thio)diphosphate pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid dihydro-4,4′-diisothiocyano 2,2′ stilbenedisulfonate. Extracellular ATP acts as a signaling molecule through the interaction of ATP with ligand-gated ion channels (P2x) as well as metabotropic receptors (P2y) (for reviews see Refs.2Harden T.K. Boyer J.L. Nicholas R.A. Annu. Rev. Pharmacol. Toxicol. 1995; 35: 541-579Crossref PubMed Google Scholar, 3North R.A. Barnard E.A. Curr. Opin. Neurobiol. 1997; 7: 346-357Crossref PubMed Scopus (426) Google Scholar, 4Turner, J. T., Landon, L. A., Gibbons, S. 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Sympathetic and parasympathetic nerves control the secretion and composition of parotid saliva (25Schneyer L.H. Emmelin N. Jacobson E.D. Shambour L.L. Gastrointestinal Physiology. Butterworths, London1974: 183-226Google Scholar). Physiologically mediated changes in tonic patterns of nerve activity elicited by surgical denervation or by dietary manipulation alter the rat parotid gland, modifying acinar cell proliferation, cell size, and sensitivity of parotid secretion to neurotransmitters (26Ekström J. Acta Physiol. Scand. 1980; 108: 253-261Crossref PubMed Scopus (56) Google Scholar, 27Ekström J. Wahlestedt C. Acta Physiol. Scand. 1982; 115: 437-446Crossref PubMed Scopus (58) Google Scholar, 28Ekström J. Mansson B. Tobin G. Acta Physiol. Scand. 1983; 119: 169-175Crossref PubMed Scopus (92) Google Scholar, 29Schneyer C.A. Humphreys-Beher M.G. Hall H.D. Proc. Soc. Exp. Biol. Med. 1992; 200: 127-132Crossref PubMed Scopus (12) Google Scholar, 30Ekström J. Templeton D. Acta Physiol. Scand. 1977; 101: 329-335Crossref PubMed Scopus (16) Google Scholar). Parasympathetic denervation enhances secretory responses elicited in vivo by activation of calcium-mobilizing neurotransmitter receptors linked to phospholipase C (26Ekström J. Acta Physiol. Scand. 1980; 108: 253-261Crossref PubMed Scopus (56) Google Scholar, 27Ekström J. Wahlestedt C. Acta Physiol. Scand. 1982; 115: 437-446Crossref PubMed Scopus (58) Google Scholar, 28Ekström J. Mansson B. Tobin G. Acta Physiol. Scand. 1983; 119: 169-175Crossref PubMed Scopus (92) Google Scholar). Our previous denervation studies also established that trans-synaptic regulation of sensitivity to these neurotransmitters can be demonstrated in vitro in dissociated cell suspensions and showed for the first time that sensitivity to ATP is increased very dramatically (1McMillian M.K. Soltoff S.P. Cantley L.C. Rudel R. Talamo B.R. Br. J. Pharmacol. 1993; 108: 453-461Crossref PubMed Scopus (58) Google Scholar, 31McMillian M.K. Talamo B.R. Peptides. 1989; 10: 721-727Crossref PubMed Scopus (10) Google Scholar). This suggests that P2x purinoceptors are modulated by changes in synaptic activity and that ATP plays a role in the physiologically important regulation of food intake and metabolism. Here, we further characterize and identify the high and low affinity ATP receptors in parotid acinar cells and determine whether the receptors are co-expressed or independently distributed. P2x receptor types were classified in individual cells by pharmacology of the Ca2+iresponse to ATP, as analyzed by fluorescence ratio imaging of cells loaded with Fura-2. The properties of the responses are consistent with those observed for homomeric P2x4 receptors and P2x7 receptors in expression systems. RT-PCR1amplification provided evidence for expression of the cognate mRNAs. Imaging experiments established that 1) the high and low affinity responses are independently expressed across acinar cells and likely mediated by P2x4 and P2x7 receptors, respectively; 2) denervation produces an increase in sensitivity to ATP (decrease in threshold) in individual cells; and 3) denervation leads to an increase in the number (proportion) of cells with high affinity ATP responses. Both responses to denervation could be explained, at least in part, by quantitative RT-PCR data showing that P2x4 receptor mRNA increases following denervation. These data suggest that elevated P2x4 receptor protein contributes to the observed increases in the sensitivity and total magnitude of the glandular response. Dissociated parotid acinar cells were prepared by trypsin and collagenase treatment as described previously (32McMillian M.K. Soltoff S.P. Cantley L.C. Talamo B.R. Biochem. Biophys. Res. Commun. 1987; 149: 523-530Crossref PubMed Scopus (36) Google Scholar). The cell pellet was suspended in oxygenated HEPES/Ringer buffer of the following composition (in mm) 120 NaCl, 5 KCl, 2.2 MgCl2, 1 CaCl2, 20 HEPES, 5 β-hydroxybutyrate, 10 glucose, and 0.1% bovine serum albumin, pH 7.4. This preparation is composed of single cells and small clumps containing up to five cells; acinar cells comprise 85% or more of the dissociated cell preparation. Unilateral deafferentation of one parotid gland of Sprague-Dawley rats (50–100 g) was carried out surgically by avulsion of the right auriculotemporal nerve, which carries post-ganglionic parasympathetic nerve fibers. Tetracycline HCl (Polyotic, for veterinary use; 500 mg/liter drinking water; American Cyanamid, Wayne, NJ) was provided in drinking water ad libitum for 4 days following surgery. The contralateral gland served as control. Dissociated acinar cells were prepared 2–3 weeks after denervation. Ca2+i was assayed in single cells using the fluorescent calcium indicator dye Fura-2 (Molecular Probes). Cells were loaded with Fura-2 acetoxymethyl ester (1.5 μm) for 60 min at room temperature as described previously (32McMillian M.K. Soltoff S.P. Cantley L.C. Talamo B.R. Biochem. Biophys. Res. Commun. 1987; 149: 523-530Crossref PubMed Scopus (36) Google Scholar). Washed cells were suspended in Mg2+-free HEPES/Ringer buffer, and 150 μl of Fura-2 loaded cells were plated on an acid-washed glass coverslip coated with concanavalin A (1 mg/ml) 5–10 min before mounting in the perfusion chamber for Ca2+i measurements. Ca2+i was estimated by the ratio method, using a K of 224 nm for Ca2+ binding to Fura-2 (33Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). Maximum and minimum fluorescence values were obtained by the addition of ionomycin and EGTA, respectively, to the perfusion chamber at the end of experiments. In most cases, calcium responses are indicated as the ratio of the fluorescence values at 340 and 380 nm excitation. Rapid exchange of solutions was achieved by superfusion (4 ml/min) of cells in an open chamber. Cells were imaged using a Nikon Diaphot inverted fluorescence microscope equipped with a Xenon light source, Fura-2 barrier and emission filter sets, and a Cohu CCD camera. A computerized filter wheel (Sutter Instruments) equipped with an electronic shutter regulated excitation at 340 or 380 ± 10 nm. Data were collected on a 486 computer equipped with an Itex 100 frame-grabbing board. Software was developed to collect data simultaneously from as many as 50 cells, by placing circles of about 80 pixels in area over intracellular regions of fluorescent cells imaged on a 512 × 512 video monitor. Fluorescent cells were visualized using a Nikon fluor 40× oil immersion objective and excitation light at 380 nm. Cells with the morphology of polarized acinar cells were selected for analysis, including both fully dissociated individual cells as well as polarized cells organized in clusters of three to five cells around a lumen. Strings of duct cells were not analyzed. Ratio-pair data from all selected areas were usually collected every 5–7 s during the experiment. Data from a single frame were saved with no averaging. Background fluorescence was determined by placing a data collection circle in an area without cells; this value was subtracted for each data point before calculating the ratio of 340:380 measurements. Fluorescence images of cells with superimposed data collection circles as well as bright-field (Nomarski) images were saved and printed for each experimental run. Data were analyzed off-line. Reagents were of analytical grade and were obtained from Sigma unless otherwise noted. Total RNA was prepared by a single step method using Ultraspec, a commercially available isolation system (Biotecx, Houston, TX). The parotid glands were denervated as described above, removed 2–3 weeks later, and immediately immersed in liquid nitrogen. Control and denervated samples were crushed in liquid nitrogen and then homogenized in 4 ml of Ultraspec. Purified total RNA was quantified by absorbance at 260 nm, and 10 μg was run on a formaldehyde gel to confirm the integrity of the RNA as indicated by the preservation of the 28 and 18 S rRNA. First strand cDNA was synthesized from 10 μg total RNA using Moloney murine leukaemia virus reverse transcriptase (Life Technologies, Inc.) with random hexamers as primers (Promega). Rat brain and rat dorsal root ganglia cDNAs were prepared from total RNA obtained in the same manner. Genomic DNA was prepared from rat liver by a standard procedure (34Moore D. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1995: 2.1.1-2.1.9Google Scholar). Briefly, the liver was excised, minced, frozen in liquid nitrogen, and crushed to a fine powder. The powdered tissue was suspended in digestion buffer containing 100 mm NaCl, 10 mm Tris-Cl, 25 mm EDTA, 0.8% sodium dodecyl sulfate, 0.1 mg/ml proteinase K, pH 8.0, and then incubated with shaking at 50 °C for 18 h. An equal volume of phenol/chloroform/isoamyl alcohol was added, and the nucleic acids were extracted. The DNA was precipitated from the upper layer with 1.07m ammonium acetate and 57% ethanol and resuspended in Tris/EDTA buffer (pH 8). The P2x-receptor and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNAs were amplified with specific primers using Taq DNA polymerase (Life Technologies, Inc.) according to the manufacturer's instructions. The 50-μl reaction mixture contained 10 μl of DNA template, 1 × Taq buffer, 800 μm dNTPs, 1.5 mm magnesium, 50 pmol of each primer, and 2.5 units of Taq polymerase. The products were visualized by electrophoresis on a 2% agarose gel by ethidium bromide staining. The primer sequences and expected sizes of the products are shown in Table I. The identity of the products was confirmed by sequencing the amplified cDNA (Tufts DNA Sequencing Facility, Boston).Table IOligonucleotide primers used to detect P2x-receptor subtypes by RT-PCRForward primerReverse primerExpected size (bp)P2x1GGGTGGGTGTTTGTCTATGAAGCTGATGCTGTTTTTGATGAA447P2x2TGGGACTACGAGACGCCTAAGATGGTGGGAATGAGACTGAAT953P2x3TTTGTGGGGTGGGTTTTCTTGTAATGGTGGGGATGATGTTGA856P2x4(up/lo)AACATCCTCCCCAACATCACCCCATCTCTCCCCATCTTTCTG618P2x4(A/B)AACACCTCTCAGCTTGGATTCAGGTAGGACGTGGTGATGTTG418P2x4(A/Far)AACACCTCTCAGCTTGGATTCAAAGACCCTGCTCGTAGTC927P2x5AAGCCAATGTACTAGAAACAGTCATCCTGACGAACCCTCTCC613P2x6GCTGGGGGTTTCTGGATTACAGCTCTTGCCTCTTCATACTTG1081P2x7(Trunc)CTTTACAGAGGTGGCAGTTCATGGTGGAGAGAGAGGGAGAGC578P2x7(C-term)GTTTTGACATCCTGGTTTTTGCTGCGGTTCTCTGGTAGTTGA529P2x7(3′-UTR)GTTTTGACATCCTGGTTTTTGGGCGGCTTTTAGTGGTTTCTG1877GAPDHCCATCACCATCTTCCAGGAGCCTGCTTCACCACCTTCTTG595 Open table in a new tab The basis for competitive PCR is the inclusion of a known quantity of a synthetic internal standard that is amplified by the same primers as the cDNA of interest. Specific competitive templates were constructed to determine the amounts of GAPDH and P2x4-receptor cDNAs in the RT-DNA samples. For each experiment, a standard curve is obtained by adding known quantities of the internal standard to the PCR mixture containing the cDNA of interest. The level of amplification of each template depends upon its relative abundance in the mixture. The product from the internal standard is shorter than the product from the cDNA, and they can be separated by gel electrophoresis (see Fig. 7 a). The ratio of the intensities of the two bands is derived by densitometric measurements of the ethidium bromide-stained gel. Ratios from data of Fig. 7 a are plotted in Fig. 7 b. At a ratio of 1.0, the intensity of the two bands is the same, and the quantity of mRNA in the original sample may be read on the xaxis. The competitive template for P2x4 was constructed using a plasmid (pcDNA3, Invitrogen) containing the P2x4-receptor cDNA (courtesy of G. Buell, Glaxo (Ref.12Buell G. Lewis C. Collo G. North R.A. Surprenant A. EMBO J. 1996; 15: 55-62Crossref PubMed Scopus (376) Google Scholar)). Two HaeII restriction endonuclease sites within the sequence amplified by PCR with primer set P2x4 (up/lo) were employed to remove 200 bp from the cDNA. The fragments were then ligated back into the empty vector to create the template, P2x4-ΔHaeII. A similar template was generated from a plasmid containing the human GAPDH cDNA (pHcGAP; ATCC, Rockville, MD) by restriction digest with XbaI and BstXI to remove 95 bp. The resulting product was then blunted with T4 DNA polymerase (New England Biolabs) and ligated to form GAPDH ΔX/B. The nucleotide sequence recognized by the primers is the same for both the human and rat GAPDH cDNA. Identical primers were used for competitive templates and the cDNA samples, and products differed only slightly in size, so both were amplified with the same efficiency. In some experiments, annealing between the heterologous strands of the competitor and cDNA products was observed. The third band generated in this manner was not included in the analysis because it contains equal quantities of both products and did not affect the quantification. Immunoblotting of P2x4-receptor protein was carried out using samples from rat parotid gland and, as a control, human embryonic kidney 293 cells (293 cells), which heterologously expressed the receptor. A plasmid containing the full-length cDNA sequence for the rat P2x4 receptor (p464) was transfected into the 293 cells using the calcium phosphate precipitation method of Okayama and Chen, as modified by Pritchett et al. (35Pritchett D.B. Sontheimer H. Gorman C.M. Kettenmann H. Seeburg P.H. Schofield P.R. Science. 1988; 242: 1306-1308Crossref PubMed Scopus (192) Google Scholar). Protein samples from parotid glands were prepared by freezing and pulverizing the tissue in liquid nitrogen followed by direct transfer into SDS sample buffer containing 62.5 mm Tris-HCl, 10% (v/v) glycerol, 2% (w/v) sodium dodecyl sulfate, 5% (v/v) 2-mercaptoethanol, 0.01% (w/v) bromphenol blue, 0.1 mg/ml aprotinin, 10 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride, and 2 mmEDTA, pH 6.8. The sample was then homogenized for 30 s using a Brinkman polytron and boiled for 5 min. Extracts from 293 cells were prepared by scraping the cells from 100-mm tissue culture dishes in SDS sample buffer and homogenizing the sample as described above. Total protein was separated by 10 or 12% SDS-polyacrylamide gel electrophoresis, and the P2x4 receptor was detected by immunoblotting with an antibody (final concentration, 2 μg/ml; kindly donated by Gary Buell, Glaxo, Geneva) against the COOH-terminal of the rat protein. Crude antiserum and affinity-purified antibody were used in these experiments. Some blots were also reacted with antibody preabsorbed for 2 h at 20 °C with the antigenic peptide (20 μg/ml). Protein concentrations were determined by the Coomassie protein reagent assay (Pierce). P2x4-receptor-like immunoreactivity was visualized by the enhanced chemiluminescence detection method (Pierce). The basis for increased sensitivity of denervated parotid acinar cells to ATP was investigated by characterizing Ca2+i responses in individual cells and by molecular analysis. The properties of the Ca2+iresponses have been partially determined in previous studies and can be evaluated through Fura-2 analysis. Although muscarinic, α-adrenergic, and substance P receptors in parotid acinar cells elevate Ca2+i through G protein-coupled activation of phospholipase C and subsequent production of inositol 1,4,5, trisphosphate (32McMillian M.K. Soltoff S.P. Cantley L.C. Talamo B.R. Biochem. Biophys. Res. Commun. 1987; 149: 523-530Crossref PubMed Scopus (36) Google Scholar, 36McMillian M.K. Soltoff S.P. Lechleiter J.D. Cantley L.C. Talamo B.R. Biochem. J. 1988; 255: 291-300PubMed Google Scholar, 37Merritt J.E. Rink T.J. J. Biol. Chem. 1987; 262: 14912-14916Abstract Full Text PDF PubMed Google Scholar), our results show that the ATP responses do not seem to be of the metabotropic type. Both ATP responses required extracellular Ca2+. ATP is not effective in stimulating inositol phosphate formation in this preparation (however, see also Ref. 38Jorgensen T.D. Gromada J. Tritsaris K. Nauntofte B. Dissing S. Biochem. J. 1995; 312: 457-464Crossref PubMed Scopus (37) Google Scholar), and whole cell patch clamp recordings demonstrate that activation of whole cell currents by ATP does not involve a GDPβS-sensitive step (36McMillian M.K. Soltoff S.P. Lechleiter J.D. Cantley L.C. Talamo B.R. Biochem. J. 1988; 255: 291-300PubMed Google Scholar). P2z responses in parotid cells resemble those in other cell types, requiring relatively high concentrations of ATP (100 μm or greater) to stimulate influx of both Na+ and Ca2+ (39Tenneti L. Talamo B.R. Biochem. J. 1993; 295: 255-261Crossref PubMed Scopus (14) Google Scholar). 3′-O-(4-benzoyl)benzoyl-ATP is the most potent and effective agonist, and the response is inhibited by high concentrations of divalent cations such as Mg2+, Brilliant Blue G, reactive blue 2, and H2DIDS (36,40). The second type of response to ATP is of higher affinity (EC50 less than 10 μm), insensitive to nucleotides UTP, GTP, adenosine, and α,β-methylene ATP and weakly responsive to 2-methylthio-ATP. Further, it is neither activated by 3′-O-(4-benzoyl)benzoyl-ATP nor inhibited by high concentrations of divalent cations, Brilliant Blue G, reactive blue 2, and H2DIDS (1McMillian M.K. Soltoff S.P. Cantley L.C. Rudel R. Talamo B.R. Br. J. Pharmacol. 1993; 108: 453-461Crossref PubMed Scopus (58) Google Scholar, 36McMillian M.K. Soltoff S.P. Lechleiter J.D. Cantley L.C. Talamo B.R. Biochem. J. 1988; 255: 291-300PubMed Google Scholar, 40Soltoff S.P. McMillian M.K. Talamo B.R. Biochem. Biophys. Res. Commun. 1989; 165: 1279-1285Crossref PubMed Scopus (48) Google Scholar). An additional distinguishing feature of the two responses to ATP is that they are differentially modulated by protein kinase inhibitors (39Tenneti L. Talamo B.R. Biochem. J. 1993; 295: 255-261Crossref PubMed Scopus (14) Google Scholar). Ca2+i is elevated in parotid acinar cells by at least four different neurotransmitters, including agonists of the metabotropic G protein-coupled receptors and P2 purinergic receptors for extracellular ATP. To determine whether the muscarinic and P2 receptors are differentially distributed across acinar cells, Fura-2 loaded cells were perfused sequentially with maximally effective concentrations of the agonists carbachol (30 or 100 μm) and ATP (30 or 300 μm). Between agonist doses, cells were washed with buffer until Ca2+i values returned to basal levels. Almost all cells responded to maximal concentrations of carbachol, and cells that did not respond to carbachol rarely showed responses to ATP. Only the cells responding to carbachol were analyzed. Among the carbachol-responsive cells, 93% (75/81) also responded to 300 μm ATP (Fig. 1), a concentration that would activate both high and low affinity receptors. This indicated that the majority of cells had P2xreceptors. Addition of a maximal dose of carbachol rapidly elevated Ca2+i to a peak value that then declined to a slightly lower steady-state level followed by recovery to a basal value when agonist was washed out. Previous studies using cell suspensions demonstrated that the response to ATP is biphasic and that there are two pharmacologically distinct responses. However, under those assay conditions, the signal is a composite of responses from a large population of cells, and it is difficult to determine whether the two responses arise from subsets of cells with different receptors or whether the two receptors are homogeneously distributed across all responding cells. In the present studies, the distribution of the two distinct receptor types was characterized on individual cells. Cells were assayed in buffer without added Mg2+ to optimize the P2z response. Perfusion with both low and high concentrations of ATP identified some cells that displayed only the low affinity or only the high affinity response as defined below, but many cells showed both responses (Fig. 1). In 73% of the cells with responses to 300 μm ATP (164/225 cells), a small, rapid elevation in Ca2+i was detected at low concentrations of ATP (1–30 μm, high affinity response), and Ca2+i usually reversed to basal level when agonist was washed out (Fig. 1). At higher concentrations of ATP (300 μm), Ca2+i increased more slowly and reached higher levels (low affinity response). After exposure to 300 μm ATP, the elevated Ca2+i did not always reverse to basal level after washing out the ATP (Fig. 1). In parotid acinar cell suspensions, the large increase in Ca2+iin response to a high concentration of ATP (300 μm) is primarily mediated by P2z receptors. Thus, Mg2+ or high concentrations of Ca2+ would be expected to reverse or block this response in cells with low affinity receptors (see for example, Ref. 41Virginio C. Church D. North R.A. Surprenant A. Neuropharmacology. 1997; 36: 1285-1294Crossref PubMed Scopus (269) Google Scholar). To test this, single cells were first exposed to
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