John T. Turner

G protein-coupled P2 purinoceptors: from molecular biology to functional responses

Nucleotides such as ATP and ADP act as intercellular messengers and exert a widespread influence on cellular function by acting on a variety of cell surface receptors. Until recently, progress has been restrained, in part, by a lack of cloned receptors. Now, however, the successful cloning of a variety of P2 purinoceptors is holding out the prospect of rapid advances in the understanding of this diverse group of receptors and the potent therapeutic resource they represent. In this article, Michael Boarder and colleagues summarize the findings of recent cloning studies, and assess the impact of these on the understanding of the function of the G protein-coupled P2 purinoceptors in several types of cells and tissues.

PPADS and suramin as antagonists at cloned P2Y- and P2U-purinoceptors.

1 The effect of suramin and pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) on the stimulation of phospholipase C in 1321N1 cells transfected with the human P2U‐purinoceptor (h‐P2U‐1321N1 cells) or with the turkey P2Y‐purinoceptor (t‐P2Y‐1321N1 cells) was investigated. 2‐Methylthioadenosine triphosphate (2MeSATP) was used as the agonist at t‐P2Y‐1321N1 cells and uridine triphosphate (UTP) at h‐P2U‐1321N1 cells. 2 Suramin caused a parallel shift to the right of the concentration‐response curves for 2MeSATP in the t‐P2Y‐1321N1 cells, yielding a Schild plot with a slope of 1.16 ± 0.08 and a pA2 value of 5.77 ± 0.11. 3 Suramin also caused a shift to the right of concentration‐response curves for UTP in the h‐P2U‐1321N1 cells, and on Schild plots gave a slope different from unity (1.57 ± 0.19) and an apparent pA2 value of 4.32 ± 0.13. Suramin was therefore a less potent antagonist at the P2U‐purinoceptor than the P2Y‐purinoceptor. 4 In the presence of the ectonucleotidase inhibitor, ARL 67156 (6‐N,N‐diethyl‐β,γ‐dibromomethylene‐D‐ATP) there was no significant difference in the EC50 or shapes of curves with either cell type, and no difference in pA2 values for suramin. 5 PPADS caused an increase in the EC50 for 2MeSATP in the t‐P2Y‐1321N1 cells. The Schild plot had a slope different from unity (0.55 ± 0.15) and an X‐intercept corresponding to an apparent pA2 of 5.98 ± 0.65. 6 PPADS up to 30 μm had no effect on the concentration‐response curve for UTP with the h‐P2U‐1321N1 cells. 7 In conclusion, suramin and PPADS show clear differences in their action at the 2 receptor types, in each case being substantially more effective as an antagonist at the P2Y‐purinoceptor than at the P2U‐purinoceptor. Ectonucleotidase breakdown had little influence on the nature of the responses at the two receptor types, or in their differential sensitivity to suramin.

Functional P2Y2 nucleotide receptors mediate uridine 5′-triphosphate-induced intimal hyperplasia in collared rabbit carotid arteries

Background—Extracellular uridine 5′-triphosphate (UTP) induces mitogenic activation of smooth muscle cells (SMCs) through binding to P2Y2 nucleotide receptors. P2Y2 receptor mRNA is upregulated in intimal lesions of rat aorta, but it is unclear how this G-protein–coupled receptor contributes to development of intimal hyperplasia. Methods and Results—This study used a silicone collar placed around rabbit carotid arteries to induce vascular injury and intimal thickening. Collar placement caused rapid upregulation of P2Y2 receptor mRNA in medial SMCs before appearance of neointima. Fura-2 digital imaging of single SMCs was used to measure changes in myoplasmic calcium concentration (Cam) in response to P2Y receptor agonists. In contrast to UDP, activation by UTP or adenosine 5′-triphosphate (ATP) greatly increased Cam, which indicates upregulation of functional P2Y2 receptors at which UTP and ATP are equipotent agonists. The number of responsive cells was significantly greater for freshly dispersed SMCs from collared arteries than for controls. Perivascular infusion of UTP (100 &mgr;mol/L) within the collar significantly enhanced neointimal development. Intimas that resulted from UTP exposure were infiltrated by macrophages. Moreover, increased expression of osteopontin occurred in response to in situ application of UTP. ATP or UTP also stimulated osteopontin expression in cultured SMCs in a dose-dependent manner. Furthermore, P2Y2 antisense oligonucleotide inhibited osteopontin expression induced by UTP. Conclusions—These findings indicate for the first time a role for the UTP/ATP receptor, P2Y2, in development of intimal hyperplasia associated with atherosclerosis and restenosis.

Cloned and transfected P2Y4 receptors: characterization of a suramin and PPADS-insensitive response to UTP

The P2Y family of receptors are G protein‐coupled receptors for ATP, ADP, UTP and UDP. Recently several members of this family have been cloned, including the P2Y4, which is activated by UTP but not by ATP. In the present report, using receptors stably transfected into 1321N1 cells, we show that suramin acts as an antagonist at cloned P2Y1 and (less potently) P2Y2 receptors, but not at the cloned P2Y4 receptor. Furthermore, PPADS (pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid), a potent antagonist at the P2Y1 receptor, is a relatively inneffective antagonist at the cloned P2Y4 receptor. This work moves us closer to the goal of classifying the native P2Y receptors on the basis of agonist and antagonist profiles.

A fragment of vasoactive intestinal peptide, VIP(10–28), is an antagonist of VIP in the colon carcinoma cell line, HT29

The 19 amino acid carboxyl terminus fragment of vasoactive intestinal peptide (VIP), VIP(10-28), inhibits [125I]VIP binding in intact HT29 colonic adenocarcinoma cells and in membranes from these cells. However, VIP(10-28) alone has no effect on adenylate cyclase activity (membranes) or cyclic AMP synthesis (intact cells) in HT29 cells although VIP receptor agonists are markedly stimulatory. The indicated antagonist character of VIP(10-28) was confirmed by rightward parallel shifts of VIP dose response curves in the presence of VIP(10-28) in adenylate cyclase and cyclic AMP synthesis experiments. The equilibrium dissociation constant values for VIP(10-28) from these experiments agree with values from inhibition binding studies. The lack of effect of VIP(10-28) on forskolin dose response curves in HT29 adenylate cyclase assays indicates the specificity of the VIP(10-28) antagonism, thus suggesting that VIP(10-28) may be a useful tool in studying VIP receptor regulation and other aspects of the mechanisms of VIP action. The potential regulatory role of a proteolytically generated fragment of VIP acting antagonistically at VIP receptors is discussed.

The P2 Nucleotide Receptors

Part I. Historical Perspectives of P2 Nucleotide Receptors. History of Extracellular Nucleotides and Their Receptors, G. Burnstock. Part II. Pharmacological and Molecular Characterization of P2 Nucleotide Receptors. P2x Receptors: Cloned and Expressed, R. J. Evans, A. Surprenant, and R. A. North. The Cloning and Expression of G Protein-Coupled P2Y Nucleotide Receptors, G. A. Weisman, L. Erb, R. C. Garrad, J. T. Turner, and F. A. Gonzalez. Structure Activity Relationships of P2 Receptor Agonists and Antagonists, K. A. Jacobson, Y.-C. Kim, E. Camaioni, and A. M. van Rhee. Pharmacological Selectivities of Molecularly Defined Subtypes of P2Y Receptors, T. K. Harden, R. A. Nicholas, J. R. Schachter, E. R. Lazarowski, and J. L. Boyer. P2 Receptor Modeling and Identification of Ligand Binding Sites, A. M. van Rhee. Part III. Mechanisms of P2 Nucleotide Receptor Action. Nucleotide Receptors Coupling to the Phospholipase C Signaling Pathway, J.-M. Boeynaems, D. Communi, R. Janssens, S. Motte, B. Robaye, and S. Pirotton. Cyclic AMP and Tyrosine Kinase Cascades in the Regulation of Cellular Function by P2Y Nucleotide Receptors, M. R. Boarder. Characteristics of Ligand-Gated Ion Channel P2 Nucleotide Receptors, C. Kennedy, S. E. Hickman, and S. C. Silverstein. Cell Responses Initiated by Ecto-kinases, J. S. Fedan. Part IV. Physiological Roles for P2 Nucleotide Receptors. Functions of Extracellular Nucleotides in Peripheral and Central Neuronal Tissues, E. M. Silinsky, I. von Kugelgen, A. D. Smith and D. P. Westfall. The Role of Purinoceptors in the Regulation of Lung Surfactant Secretion, S. A. Rooney. The Role of Nucleotide Receptors in the Cardiovascular System, S. Pirotton, C. Desgranges, G. Vassort, and J.-M. Boeynaems. Physiological Roles for P2 Receptors in Platelets, Visceral Smooth Muscle, and the Immune and Endocrine Systems, S. Hourani, F. DiVirgilio, and M.-M. Loubatieres-Mariani. Therapeutic Applications for Nucleotides in Lung Disease, S. H. Donaldson and R. C.Boucher. Part V. Future Vistas. P2 Nucleotide Receptors: Future Vistas, J. S. Fedan, J. T. Turner, and G. A. Weisman. Index.

Development and characterization of SV40 immortalized rat parotid acinar cell lines.

SummaryRat parotid salivary gland acinar cells were transfected by CaPO4 precipitation using a plasmid containing a replication-defective simian virus (SV40) genome. Out of 30 clonal cell lines, 2 were shown to have moderate to high levels of cytodifferentiation and salivary gland acinar cell function. Functional studies with the two cell lines indicated that the β-adrenergic agonist (isoproterenol), vasoactive intestinal peptide prostaglandin E1, and forskolin were effective activators of intracellular cyclic adenosine 3′:5′-cyclic monophosphate production. Phenylephrine, carbamylcholine, and UTP were effective in increasing inositol phosphate production and intracellular free calcium levels, whereas substance P was without affect. Utilizing indirect immunofluorescence analysis, both cell lines were shown to express the SV40 large T antigen. Electron microscopic evaluation documented moderate to high levels of cytodifferentiation with the maintenance of tripartite junctional complexes, cellular polarization, and presence of moderate amounts of secretory granules and rough endoplasmic reticulum. The two cell lines had doubling times of 22 and 36 h, respectively.

Development and characterization of SV40 immortalized rat submandibular acinar cell lines

SummaryRat submandibular salivary gland acinar cells were transfected by CaPO4 precipitation using a plasmid containing a replication-defective simian virus (SV40) genome. Out of 27 clonal cell lines, two were shown to have moderate to high levels of cytodifferentiation and salivary gland acinar cell function. Functional studies with the two cell lines indicated that the β-adrenergic agonist, isoproterenol, vasoactive intestinal peptide, and prostaglandin E1 were effective activators of intracellular cyclic AMP production. Epinephrine, norepinephrine, phenylephrine, acetylcholine, and P2U-purinoceptor agonists were effective in increasing inositol phosphate production and intracellular free calcium levels, whereas substance P was without effect. Utilizing indirect immunofluorescence analysis, both cell lines were shown to express glutamine/glutamic acid-rich proteins, a submandibular acinar cell specific secretory protein family. Electron microscopic evaluation documented the maintenance of tripartite junctional complexes, cellular polarization, and the presence of moderate amounts of secretory granules and rough endoplasmic reticulum. The two cell lines had doubling times of 25 h.

Mechanisms of agonist-dependent and -independent desensitization of a recombinant P2Y2 nucleotide receptor

UTP activates P2Y2 receptors in both 1321N1 cell transfectants expressing the P2Y2 receptor and human HT-29 epithelial cells expressing endogenous P2Y2 receptors with an EC50 of 0.2- 1.0 μM. Pretreatment of these cells with UTP diminished the effectiveness of a second dose of UTP (the IC50 for UTP-induced receptor desensitization was 0.3 - 1.0 μM for both systems). Desensitization and down-regulation of the P2Y2 nucleotide receptor may limit the effectiveness of UTP as a therapeutic agent. The present studies investigated the phenomenon of P2Y2 receptor desensitization in human 1321N1 astrocytoma cells expressing recombinant wild type and C-terminal truncation mutants of the P2Y,2 receptor. In these cells, potent P2Y2 receptor desensitization was observed after a 5 min exposure to UTP. Full receptor responsiveness returned 5-10 min after removal of UTP. Thapsigargin, an inhibitor of Ca2+-ATPase in the endoplasmic reticulum, induced an increase in the intracellular free calcium concentration, [Ca2+]i, after addition of desensitizing concentrations of UTP, indicating that P2Y2 receptor desensitization is not due to depletion of calcium from intracellular stores. Single cell measurements of increases in [Ca2+]i induced by UTP in 1321N1 cell transfectants expressing the P2Y2 receptor indicate that time- and UTP concentration-dependent desensitization occurred uniformly across a cell population. Other results suggest that P2Y2 receptor phosphorylation/dephosphorylation regulate receptor desensitization/resensitization. A 5 min preincubation of 1321N1 cell transfectants with the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), reduced the subsequent response to UTP by about 50% whereas co-incubation of PMA with UTP caused a greater inhibition in the response. The protein phosphatases - 1 and -2A inhibitor, okadaic acid, partially blocked resensitization of the receptor. Furthermore, C-terminal truncation mutants of the P2Y2 receptor that eliminated several potential phosphorylation sites including two for PKC were resistant to UTP-, but not phorbol ester-induced desensitization. Down regulation of protein kinase C isoforms prevented phorbol ester-induced desensitization but had no effect on agonist-induced desensitization of wild type or truncation mutant receptors. These results suggest that phosphorylation of the C-terminus of the P2Y2 receptor by protein kinases other than protein kinase C mediates agonist-induced receptor desensitization. A better understanding of the molecular mechanisms of P2Y2 nucleotide receptor desensitization may help optimize a promising cystic fibrosis pharmacotherapy based on the activation of anion secretion in airway epithelial cells by P2Y2 receptor agonists.

Identification and characterization of P2Y2 nucleotide receptors in human retinal pigment epithelial cells

P2 nucleotide receptor expression in cultured human retinal pigment epithelial (RPE) cells was investigated using the photoaffinity ATP analog BzATP, polymerase chain reaction of reverse‐transcribed RNA (RT‐PCR) and fura‐2 fluorescence measurement of changes in intracellular free calcium concentration ([Ca2+]i). In experiments carried out in RPE cells at passage 10–15, addition of micromolar concentrations of ATP, UTP, and ATPγS to RPE cells resulted in a rapid, transient 3.5‐fold increase in [Ca2+]i followed by a prolonged elevation that was twofold above the original baseline. Similar results were obtained from cells at passage 2. Characteristics of nucleotide‐stimulated calcium mobilization in RPE cells, including partial inhibition by pertussis toxin, suggest that a G protein‐coupled receptor mediates this response. Consistent with the expression of a P2Y2 nucleotide receptor subtype in RPE cells, [α‐32P]BzATP labeled a 53‐kDa protein in plasma membranes, and RT‐PCR revealed the presence of P2Y2 receptor RNA. Adenosine had no effect on [Ca2+]i in RPE cells, indicating that the A2 subtype of P1 receptor described previously in human RPE is not involved in the response to nucleotides. Together the results indicate that human RPE cells express functional P2Y2 nucleotide receptors. J. Neurosci. Res. 49:43–52, 1997. Published 1997 Wiley‐Liss Inc. This article was prepared by a group of United States government employees and non‐United States government employees, and as such is subject to 17 U.S.C. Sec. 105.