Activation of a P2Y4-like purinoceptor triggers an increase in cytosolic [Ca2+] in the red blood cells of the lizard Ameiva ameiva (Squamata, Teiidae)

Sartorello, R.; Garcia, C.R.S.

doi:10.1590/S0100-879X2005000100002

Abstract

An increasing number of pathophysiological roles for purinoceptors are emerging, some of which have therapeutic potential. Erythrocytes are an important source of purines, which can be released under physiological and physiopathological conditions, acting on purinergic receptors associated with the same cell or with neighboring cells. Few studies have been conducted on lizards, and have been limited to ATP agonist itself. We have previously shown that the red blood cells (RBCs) of the lizard Ameiva ameiva store Ca2+ in the endoplasmic reticulum (ER) and that the purinergic agonist ATP triggers a rapid and transient increase of [Ca2+]c by mobilization of the cation from internal stores. We also reported the ability of the second messenger IP3 to discharge the ER calcium pool of the ER. Here we characterize the purinoceptor present in the cytoplasmic membrane of the RBCs of the lizard Ameiva ameiva by the selective use of ATP analogues and pyrimidine nucleotides. The nucleotides UTP, UDP, GTP, and ATPgammaS triggered a dose-dependent response, while interestingly 2MeSATP, 2ClATP, alpha, ß-ATP, and ADP failed to do so in a 1- to 200-µm con- centration. The EC50 obtained for the compounds tested was 41.77 µM for UTP, 48.11 µM for GTP, 53.11 µM for UDP, and 30.78 µM for ATPgammaS. The present data indicate that the receptor within the RBCs of Ameiva ameiva is a P2Y4-like receptor due to its pharmacological similarity to the mammalian P2Y4 receptor.

Red blood cells; Calcium; Teiidae; Ameiva ameiva; Purinoceptor; UTP

Braz J Med Biol Res, January 2004, Volume 38(1) 5-10

Activation of a P2Y ₄ -like purinoceptor triggers an increase in cytosolic [Ca ²⁺ ] in the red blood cells of the lizard Ameiva ameiva (Squamata, Teiidae)

R. Sartorello and C.R.S. Garcia

Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP, Brasil

References

Acknowledgments ^{Correspondence and Footnotes} Acknowledgments

Abstract

An increasing number of pathophysiological roles for purinoceptors are emerging, some of which have therapeutic potential. Erythrocytes are an important source of purines, which can be released under physiological and physiopathological conditions, acting on purinergic receptors associated with the same cell or with neighboring cells. Few studies have been conducted on lizards, and have been limited to ATP agonist itself. We have previously shown that the red blood cells (RBCs) of the lizard Ameiva ameiva store Ca²⁺ in the endoplasmic reticulum (ER) and that the purinergic agonist ATP triggers a rapid and transient increase of [Ca²⁺]_c by mobilization of the cation from internal stores. We also reported the ability of the second messenger IP₃ to discharge the ER calcium pool of the ER. Here we characterize the purinoceptor present in the cytoplasmic membrane of the RBCs of the lizard Ameiva ameiva by the selective use of ATP analogues and pyrimidine nucleotides. The nucleotides UTP, UDP, GTP, and ATPgS triggered a dose-dependent response, while interestingly 2MeSATP, 2ClATP, a, ß-ATP, and ADP failed to do so in a 1- to 200-µm con- centration. The EC₅₀ obtained for the compounds tested was 41.77 µM for UTP, 48.11 µM for GTP, 53.11 µM for UDP, and 30.78 µM for ATPgS. The present data indicate that the receptor within the RBCs of Ameiva ameiva is a P2Y₄-like receptor due to its pharmacological similarity to the mammalian P2Y₄ receptor.

Key words: Red blood cells, Calcium, Teiidae, Ameiva ameiva, Purinoceptor, UTP

Introduction

Calcium regulates a myriad of physiological processes in organisms ranging from protozoa (1-5) to vertebrates (6,7) by an orchestrated cytosolic elevation and/or calcium influx (8). Since the early recognition of the potent extracellular actions of ATP and adenosine by Drury and Szent-Györgyi, in 1929 (9), considerable knowledge has been accumulated about the receptors involved in transducing nucleotide signals (10,11).

Extracellular purines (adenosine, ADP, ATP) as well as pyrimidines (UDP and UTP) play an important role in signaling events, mediating many biological effects through receptors on the cell surface, including smooth muscle contraction, neurotransmission, exocrine and endocrine secretion, the immune response, inflammation, plaque aggregation, pain, and modulation of cardiac function. These receptors are named purinergic receptors (10).

Purinergic receptors are divided into two major families, P1 or adenosine receptors, and P2, which recognize ATP, UTP and UDP. P2 receptors are subdivided into P2Y, the G protein coupled receptors, and P2X, coupled to ionic channels. In mammals, 8 subtypes of P2Y receptors (P2Y₁, P2Y₂, P2Y₄, P2Y₆, P2Y₁₁) (12) and, recently, P2Y₁₂ (13), P2Y₁₃ (14) and P2Y₁₄ (15) have been identified thus far.

Metabotropic P2Y_1-14 receptors are characterized by a subunit topology involving an extracellular N terminus and intracellular C terminus, the latter possessing consensus binding motifs for protein kinases, and seven transmembrane-spanning regions, which help to form the ligand-docking pocket. Each P2Y receptor binds to a single heterotrimeric G protein (typically G_q/11) although P2Y₁₁ can couple both with G_q/11 and G_s, whereas P2Y₁₂ and P2Y₁₃ couple with G_i and P2Y₁₄ couples with G_i/0 (16).

The recently cloned P2Y receptors P2Y₁₃ are present mainly in spleen, brain, lymph nodes, and bone marrow, whereas P2Y₁₄ receptors are present in placenta, adipose tissue, stomach, intestine, and discrete brain regions (17).

An increasing number of pathophysiological roles for purinoceptors are emerging, some of which have therapeutic potential (18-21). Bladder incontinence, contraception and fertility, skin diseases, diabetes, thrombosis, gut motility disorders, cardiopulmonary diseases, cancer, diseases of the ear, diseases of the eye, behavioral disorders, bone disorders, and pain (19,20) are related in one way or other to responses to purinergic receptors.

Most of the studies concerning the functional distribution of purinoceptors were carried out on mammalian preparations even though purinoceptors are widespread throughout the evolutionary scale. Evidence of their presence is found even in protozoa, where ATP was found to have an inhibitory effect on ameboid movement (10).

Few studies have been conducted on lizards, and have been limited to ATP agonist itself: an excitatory innervation has been found in the ileum of the lizard Tiliqua rugosa (11) and in the rectum and portal vein of the lizard Agama agama (22,23). In all cases, the subtype of the receptors involved was unknown.

Erythrocytes are an important source of purines, which can be released in physiological and physiopathological conditions, acting on purinergic receptors associated with the same cell or with neighboring cells (24).

Red blood cells (RBCs) of the lizard Ameiva ameiva loaded with the fluorescent Ca²⁺ indicator Fluo-3 AM store Ca²⁺ in the endoplasmic reticulum and in one or more acidic pools endowed with an H⁺ pump sensitive to the inhibitors 7-chloro-4-nitrobenz-2-oxa-1,3-diazole and bafilomycin. Moreover, the internal Ca²⁺ pools of the RBCs of the lizard are sensitive to surface receptor stimulation since the purinergic agonist ATP stimulates the release of Ca²⁺ in a process that is hardly affected by the removal of external Ca²⁺ but is inhibited by suramin and PPADS (pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate), non-specific purinergic inhibitors (25,26). These data strongly suggest the presence of a G-coupled purinergic receptor in RBCs of the lizard Ameiva ameiva.

The objective of the present study was to investigate the subtype of this receptor by challenging the RBCs of Ameiva ameiva with ATP analogues (ATPgS, 2-methylthioATP (2MeSATP), a, ß-ATP, and 2-chloroATP (2ClATP)), ADP, GTP, and

pyrimidine nucleotides (UDP, UTP) and monitoring the concentration of calcium by spectrofluorometric measurement of Fluo-3 AM.

Material and Methods

Material

2MeSATP, 2-ClATP, ADP, ATP, UDP, UTP, and GTP were purchased from Sigma, St. Louis, MO, USA. Fluo-3 AM was purchased from Molecular Probes, Eugene, OR, USA.

Lizards

The lizards Ameiva ameiva were captured with Tomahawk traps in the town of Barretos (20º 33' S, 48º 30' W), State of São Paulo, Brazil, and by hand in the town of Lajeado (10º 43' S, 48º 24' W), State of Tocantins, Brazil. The blood, collected from the lizard's tail with a syringe, was centrifuged at 1500 g for 5 min and washed in phosphate-buffered isotonic saline (7.5 mM sodium phosphate and 137 mM NaCl, pH 7.2). Leukocytes were removed from RBCs by blood filtration through a cellulose powder column (long fibers; Whatman CF11, Madstone, Kent, UK) according to the method of Homewood and Neame (27).

RBC loading with the calcium indicator Fluo-3 AM

RBCs were washed twice in buffer (116 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO₄, 5.5 mM D-glucose, and 50 mM Mops, pH 7.2) and resuspended at 10⁶ cells/ml in the same buffer containing 1.8 mM probenecid, an inhibitor of organic anion transport (28), to prevent fluorochrome release and sequestration (29). Fluo-3 AM was added at a final concentration of 5 µM. The suspension was incubated for 1 h at 37ºC followed by three washes with buffer to remove the extracellular dye. In each experiment an aliquot of 100 µl (10⁶ cells) was placed in a thermostated cuvette equipped with magnetic stirring. Control experiments with the solvent alone showed no measurable change in fluorescence.

Spectrofluorometric measurements with Fluo-3 AM were performed using a model F-4500 Hitachi spectrofluorometer (Tokyo, Japan) with excitation at 505 nm and emission at 530 nm. The excitation and emission slits were 1 mm.

The curves for free Ca²⁺ concentration were calculated from fluorescence measured using the Ca²⁺ software F-4500 Intracellular Cation Measurement System-Version 1.02 (Copyright^© Hitachi, 1994-1995), which takes into account that [Ca²⁺] = K_d (F - F_min/(F_max - F), where the K_d utilized for Fluo-3 is 390, F is the fluorescence intensity measured under the conditions of the experiment, F_max the fluorescence in the presence of digitonin, and F_min the fluorescence in the presence of 8 mM EGTA. Unless otherwise specified, all experiments were performed at 37ºC.

The results are reported as means ± SEM (N = 3) and data were analyzed statistically by two-way ANOVA.

Results and Discussion

Figure 1 shows that the different concentrations (100 and 800 µM UTP) were able to trigger the increase of [Ca²⁺]_c in RBCs of the lizards Ameiva ameiva loaded with 5 µM Fluo-3 AM.

As shown in Figure 1D, with the aid of confocal microscopy we were able to observe the same effect of calcium mobilization by a purinergic agonist, in this case, 100 µM UTP. Moreover, the addition of increasing concentrations of ATPgS, GTP, UDP, and UTP (from 1 to 800 µM) led to a dose-dependent increase of [Ca²⁺]_c (data not shown).

The EC₅₀ was 41.77 µM for UTP (Figure 2A), 48.11 µM for GTP (Figure 2B), 53.11 µM for UDP (Figure 2C), and 30.78 µM for ATPgS (Figure 2D).

The ATP analogues tested, 2MeSATP, a, ß-ATP, 2ClATP, as well ADP in a range of 1 to 200 µM failed to promote an increase of [Ca²⁺]_c in the RBCs (data not shown).

We have pharmacologically characterized the P2Y receptor in Ameiva ameiva lizard RBCs. For this purpose, a set of purinergic analogues were tested (Table 1). Dose-response curves were obtained for the agonists which responded: ATPgS, GTP, UDP, UTP, 2MeSATP, 2ClATP, a, ß-ATP and ADP. The subtype identification has been inferred on the basis of sensitivity to different types of agonists, the potency relationships among them and the ability to inhibit the response. The receptor under study was pharmacologically characterized according to the criteria described below.

A typical trait of P2Y receptors is that they respond differently to natural agonists such as ATP and UDP and their diphosphate and triphosphate analogues (11).

The P2Y₁, P2Y₁₁ and P2Y₁₂ receptors are selective for adenine nucleotides, while the others can be stimulated by uracil nucleotides. The P2Y₁ and P2Y₁₂ receptors are equally responsive to 2MeSATP and ATP.

The P2Y₂ and P2Y₄ receptors are equally responsive to ATP and UTP, and are not responsive to 2MeSATP (21). The P2Y₆ receptor is selectively activated by UDP (15). Suramin is a non-selective P2 receptor antagonist which competitively antagonizes P2Y₁ and P2Y₂. The PPADS antagonist competitively antagonizes P2Y₁ and P2Y₄ receptors, but not P2Y₂ (30).

The P2Y₁₂ receptor, recently cloned from human platelets, is stimulated by ADP and ATP and selectively blocked by 2MeSATP (31).

Since addition of the uracil nucleotides UTP and UDP to lizard RBCs elicits a Ca²⁺ response, the presence of the receptors P2Y₁, P2Y₁₁ and P2Y₁₂ can be eliminated. Furthermore, since similar to adenine nucleotides (ATP), UDP also promotes a Ca²⁺ response in these cells, the P2Y₆ receptor subtype can also be ruled out. Since PPADS were able to antagonize the response promoted by ATP addition, we excluded the participation of P2Y₂ receptor signaling in these cells. Finally, P2Y₄ seems to be the receptor involved in transducing the signal within the RBCs of Ameiva ameiva lizards.

However, all of these considerations are based on mammalian models, and the denomination P2Y₄-like receptor seems to be more appropriate to designate the receptor found on the surface of Ameiva ameiva RBCs until structural information is available.

Figure 1.
Effect of UTP on lizard red blood cells. Cytosolic calcium concentration (A, B, C) was measured fluorometrically with Fluo-3 AM. A, 100 µM UTP; B, 800 µM UTP in nominally calcium-free medium; C, 800 µM UTP in 2 mM EGTA medium; D, 100 µM UTP in Fluo-3 AM-loaded lizard red blood cells observed by confocal microscopy and reported in arbitrary fluorescence units (AU). The fluorescence data are qualitative. Note that the ordinate scales of panels A, B and C are not equal: B > A > C. Cytosolic calcium concentration is reported in nM.

Figure 2.
Dose-response curves for A, UTP; B, GTP; C, UDP, and D, ATPgS in Fluo-3 AM-loaded red blood cells of the lizard Ameiva Ameiva. [Ca²⁺]_c was measured by loading red blood cells with Fluo-3 AM as described in Methods. Data are reported as means ± SD for three measurements for panels A, B and D.

Thumbnail

Table 1.
Calcium response of red blood cells of the lizard Ameiva Ameiva to nucleotide agonists.

We thank Regina P. Markus, Departamento de Fisiologia, USP, São Paulo, SP, Brazil, for helpful criticism, and Miguel T. Rodrigues, Departamento de Zoologia, USP, São Paulo, SP, Brazil, for providing the conditions for an expedition to Tocantins to collect lizards.

Correspondence and Footnotes

Address for correspondence: C.R.S. Garcia, Departamento de Fisiologia, Instituto de Biociências, USP, Rua do Matão, Travessa 14, 321, 05508-900, São Paulo, SP, Brasil. Fax: +55-11-3091-7422. E-mail: cgarcia@usp.br

C.R.S. Garcia was supported by FAPESP. Received May 21, 2004. Accepted October 7, 2004.

1. Passos AP & Garcia CR (1998). Inositol 1,4,5-trisphosphate induced Ca²⁺ release from chloroquine-sensitive and insensitive intracellular stores in the intraerythrocytic stage of the malaria parasite P. chabaudi Biochemical and Biophysical Research Communications, 245: 155-160.
2. Garcia CR, Ann SE, Tavares ES, Dluzewski AR, Mason WT & Paiva FB (1998). Acidic calcium pools in intraerythrocytic malaria parasites. European Journal of Cell Biology, 76: 133-138.
3. Hotta CT, Gazarini ML, Beraldo FH, Varotti FP, Lopes C, Markus RP, Pozzan T & Garcia CR (2000). Calcium-dependent modulation by melatonin of the circadian rhythm in malarial parasites. Nature Cell Biology, 2: 466-468.
4. Varotti FP, Beraldo FH, Gazarini ML & Garcia CR (2003). Plasmodium falciparum malaria parasites display a THG-sensitive Ca²⁺ pool. Cell Calcium, 33: 137-144.
5. Gazarini ML, Thomas AP, Pozzan T & Garcia CR (2003). Calcium signaling in a low calcium environment: how the intracellular malaria parasite solves the problem. Journal of Cell Biology, 161: 103-110.
6. Pozzan T, Rizzuto R, Volpe P & Meldolesi J (1994). Molecular and cellular physiology of intracellular calcium stores. Physiological Reviews, 74: 595-636.
7. Berridge MJ, Bootman MD & Roderick HL (2003). Calcium signalling: dynamics, homeostasis and remodeling. Nature Reviews. Molecular Cell Biology, 4: 517-529.
8. Carafoli E (1987). Intracellular calcium homeostasis. Annual Review of Biochemistry, 56: 395-433.
9. Drury AN & Szent-Györgyi A (1929). The physiological activity of adenine compounds with especial reference to their action upon the mammalian heart. Journal of Physiology, 68: 213-237.
10. Ralevic V & Burnstock G (1998). Receptors for purines and pyrimidines. Pharmacological Reviews, 50: 413-492.
11. Burnstock G (1996). P2 purinoceptors: historical perspective and classification. Ciba Foundation Symposium 1, 98: 1-28 (Discussion 29-34).
12. Communi D & Boeynaems JM (1997). Receptors responsive to extracellular pyrimidine nucleotides. Trends in Pharmacological Sciences, 18: 83-86.
13. Communi D, Motte S, Boeynaems JM & Pirotton S (1996). Pharmacological characterization of the human P2Y4 receptor. European Journal of Pharmacology, 317: 383-389.
14. Marteau F, Le Poul E, Communi D, Communi D, Labouret C, Pierre Savi, Boeynaems J & Gonzalez NS (2003). Pharmacological characterization of the human P2Y₁₃ receptor. Molecular Pharmacology, 64: 104-112.
15. Abbracchio MP, Boeynaems JM, Barnard EA et al. (2003). Characterization of the UDP-glucose receptor (re-named here the P2Y14 receptor) adds diversity to the P2Y receptor family. Trends in Pharmacological Sciences, 24: 52-55.
16. Abbracchio MP & Burnstock G (1998). Purinergic signalling: pathophysiological roles. Japanese Journal of Pharmacology, 78: 113-145.
17. Cheung KK, Ryten M & Burnstock G (2003). Abundant and dynamic expression of G protein-coupled P2Y receptors in mammalian development. Developmental Dynamics, 228: 254-266.
18. Fischer Y, Becker C & Loken C (1999). Purinergic inhibition of glucose transport in cardiomyocytes. Journal of Biological Chemistry, 274: 755-761.
19. Agteresch HJ, Dagnelie PC, van den Berg WJ & Wilson JH (1999). Adenosine triphosphate: established and potential clinical applications. Drugs, 58: 211-232.
20. Williams M & Jarvis MF (2000). Purinergic and pyrimidinergic receptors as potential drug targets. Biochemical Pharmacology, 59: 1173-1185.
21. Communi D, Janssens R, Suarez-Huerta N, Reobaye B & Boeynaems JM (2000). Advances in signalling by extracellular nucleotides. The role and transduction mechanisms of P2Y receptors. Cellular Signalling, 12: 351-360.
22. Ojewole JA (1983). Effects of drugs and electrical stimulation on rainbow lizard (Agama agama Linn.) isolated gastrointestinal tract smooth muscles. Methods and Findings in Experimental and Clinical Pharmacology, 5: 299-310.
23. Savage AO & Atanga GK (1985). Effect of adenosine 5'triphosphate (ATP) on the isolated rectum of the rainbow lizard Agama agama General Pharmacology, 16: 235-240.
24. Ellsworth ML, Forrester T, Ellis CG & Dietrich HH (1995). The erythrocyte as a regulator of vascular tone. American Journal of Physiology, 269: 2155-2161.
25. Beraldo FH, Sartorello R, Lanari RD & Garcia CR (2001). Signal transduction in red blood cells of the lizards Ameiva ameiva and Tupinambis merianae (Squamata, Teiidae). Cell Calcium, 29: 439-445.
26. Beraldo FH, Sartorello R, Gazarini ML, Caldeira W & Garcia CR (2002). Red blood cells of the lizards Ameiva ameiva (Squamata, Teiidae) display multiple mechanisms to control cytosolic calcium. Cell Calcium, 31: 79-87.
27. Homewood CA & Neame KD (1976). A comparison of methods used for the removal of white cells from malaria-infected blood. Annals of Tropical Medicine and Parasitology, 70: 249-251.
28. Di Virgilio F, Steinberg TH & Silverstein SC (1990). Inhibition of Fura-2 sequestration and secretion with organic anion transport blockers. Cell Calcium, 11: 57-62.
29. Di Virgilio F, Steinberg TH & Silverstein SC (1989). Organic-anion transport inhibitors to facilitate measurement of cytosolic free Ca²⁺ with fura-2. Methods in Cell Biology, 31: 453-462.
30. Charlton SJ, Brown CA, Weisman GA, Turner JT, Erb L & Boarder MR (1996). Cloned and transfected P2Y4 receptors: characterization of a suramin and PPADS-insensitive response to UTP. British Journal of Pharmacology, 119: 1301-1303.
31. Li Q, Olesky M, Palmer RK, Harden TK & Nicholas RA (1998). Evidence that the p2y3 receptor is the avian homologue of the mammalian P2Y6 receptor. Molecular Pharmacology, 54: 541-546.

Acknowledgments

Publication Dates

Publication in this collection
10 Feb 2006
Date of issue
Jan 2005

History

Accepted
07 Oct 2004
Received
21 May 2004

This work is licensed under a Creative Commons Attribution 4.0 International License.

[1] 1. Passos AP & Garcia CR (1998). Inositol 1,4,5-trisphosphate induced Ca²⁺ release from chloroquine-sensitive and insensitive intracellular stores in the intraerythrocytic stage of the malaria parasite P. chabaudi Biochemical and Biophysical Research Communications, 245: 155-160.

[2] 2. Garcia CR, Ann SE, Tavares ES, Dluzewski AR, Mason WT & Paiva FB (1998). Acidic calcium pools in intraerythrocytic malaria parasites. European Journal of Cell Biology, 76: 133-138.

[3] 3. Hotta CT, Gazarini ML, Beraldo FH, Varotti FP, Lopes C, Markus RP, Pozzan T & Garcia CR (2000). Calcium-dependent modulation by melatonin of the circadian rhythm in malarial parasites. Nature Cell Biology, 2: 466-468.

[4] 4. Varotti FP, Beraldo FH, Gazarini ML & Garcia CR (2003). Plasmodium falciparum malaria parasites display a THG-sensitive Ca²⁺ pool. Cell Calcium, 33: 137-144.

[5] 5. Gazarini ML, Thomas AP, Pozzan T & Garcia CR (2003). Calcium signaling in a low calcium environment: how the intracellular malaria parasite solves the problem. Journal of Cell Biology, 161: 103-110.

[6] 6. Pozzan T, Rizzuto R, Volpe P & Meldolesi J (1994). Molecular and cellular physiology of intracellular calcium stores. Physiological Reviews, 74: 595-636.

[7] 7. Berridge MJ, Bootman MD & Roderick HL (2003). Calcium signalling: dynamics, homeostasis and remodeling. Nature Reviews. Molecular Cell Biology, 4: 517-529.

[8] 8. Carafoli E (1987). Intracellular calcium homeostasis. Annual Review of Biochemistry, 56: 395-433.

[9] 9. Drury AN & Szent-Györgyi A (1929). The physiological activity of adenine compounds with especial reference to their action upon the mammalian heart. Journal of Physiology, 68: 213-237.

[10] 10. Ralevic V & Burnstock G (1998). Receptors for purines and pyrimidines. Pharmacological Reviews, 50: 413-492.

[11] 11. Burnstock G (1996). P2 purinoceptors: historical perspective and classification. Ciba Foundation Symposium 1, 98: 1-28 (Discussion 29-34).

[12] 12. Communi D & Boeynaems JM (1997). Receptors responsive to extracellular pyrimidine nucleotides. Trends in Pharmacological Sciences, 18: 83-86.

[13] 13. Communi D, Motte S, Boeynaems JM & Pirotton S (1996). Pharmacological characterization of the human P2Y4 receptor. European Journal of Pharmacology, 317: 383-389.

[14] 14. Marteau F, Le Poul E, Communi D, Communi D, Labouret C, Pierre Savi, Boeynaems J & Gonzalez NS (2003). Pharmacological characterization of the human P2Y₁₃ receptor. Molecular Pharmacology, 64: 104-112.

[15] 15. Abbracchio MP, Boeynaems JM, Barnard EA et al. (2003). Characterization of the UDP-glucose receptor (re-named here the P2Y14 receptor) adds diversity to the P2Y receptor family. Trends in Pharmacological Sciences, 24: 52-55.

[16] 16. Abbracchio MP & Burnstock G (1998). Purinergic signalling: pathophysiological roles. Japanese Journal of Pharmacology, 78: 113-145.

[17] 17. Cheung KK, Ryten M & Burnstock G (2003). Abundant and dynamic expression of G protein-coupled P2Y receptors in mammalian development. Developmental Dynamics, 228: 254-266.

[18] 18. Fischer Y, Becker C & Loken C (1999). Purinergic inhibition of glucose transport in cardiomyocytes. Journal of Biological Chemistry, 274: 755-761.

[19] 19. Agteresch HJ, Dagnelie PC, van den Berg WJ & Wilson JH (1999). Adenosine triphosphate: established and potential clinical applications. Drugs, 58: 211-232.

[20] 20. Williams M & Jarvis MF (2000). Purinergic and pyrimidinergic receptors as potential drug targets. Biochemical Pharmacology, 59: 1173-1185.

[21] 21. Communi D, Janssens R, Suarez-Huerta N, Reobaye B & Boeynaems JM (2000). Advances in signalling by extracellular nucleotides. The role and transduction mechanisms of P2Y receptors. Cellular Signalling, 12: 351-360.

[22] 22. Ojewole JA (1983). Effects of drugs and electrical stimulation on rainbow lizard (Agama agama Linn.) isolated gastrointestinal tract smooth muscles. Methods and Findings in Experimental and Clinical Pharmacology, 5: 299-310.

[23] 23. Savage AO & Atanga GK (1985). Effect of adenosine 5'triphosphate (ATP) on the isolated rectum of the rainbow lizard Agama agama General Pharmacology, 16: 235-240.

[24] 24. Ellsworth ML, Forrester T, Ellis CG & Dietrich HH (1995). The erythrocyte as a regulator of vascular tone. American Journal of Physiology, 269: 2155-2161.

[25] 25. Beraldo FH, Sartorello R, Lanari RD & Garcia CR (2001). Signal transduction in red blood cells of the lizards Ameiva ameiva and Tupinambis merianae (Squamata, Teiidae). Cell Calcium, 29: 439-445.

[26] 26. Beraldo FH, Sartorello R, Gazarini ML, Caldeira W & Garcia CR (2002). Red blood cells of the lizards Ameiva ameiva (Squamata, Teiidae) display multiple mechanisms to control cytosolic calcium. Cell Calcium, 31: 79-87.

[27] 27. Homewood CA & Neame KD (1976). A comparison of methods used for the removal of white cells from malaria-infected blood. Annals of Tropical Medicine and Parasitology, 70: 249-251.

[28] 28. Di Virgilio F, Steinberg TH & Silverstein SC (1990). Inhibition of Fura-2 sequestration and secretion with organic anion transport blockers. Cell Calcium, 11: 57-62.

[29] 29. Di Virgilio F, Steinberg TH & Silverstein SC (1989). Organic-anion transport inhibitors to facilitate measurement of cytosolic free Ca²⁺ with fura-2. Methods in Cell Biology, 31: 453-462.

[30] 30. Charlton SJ, Brown CA, Weisman GA, Turner JT, Erb L & Boarder MR (1996). Cloned and transfected P2Y4 receptors: characterization of a suramin and PPADS-insensitive response to UTP. British Journal of Pharmacology, 119: 1301-1303.

[31] 31. Li Q, Olesky M, Palmer RK, Harden TK & Nicholas RA (1998). Evidence that the p2y3 receptor is the avian homologue of the mammalian P2Y6 receptor. Molecular Pharmacology, 54: 541-546.