Ren LM et al / Acta Pharmacol Sin 2002 Aug; 23 (8): 721-726

Distribution of functional P2X₁-like receptor in isolated rabbit arteries¹

REN Lei-Ming², ZHANG Miao

Department of Pharmacology, School of Pharmacy, Hebei Medical University, Shijiazhuang 050017, China

¹ Project supported by the Hebei Natural Science Foundation, ¡í 398294.

² Correspondence to Prof REN Lei-Ming. Phn 86-311-604-4121, ext 5170. Fax 86-311-604-8177. E-mail ren-leiming@263.net

Received 2001-11-19 Accepted 2002-05-13

KEY WORDS arteries; purinergic P2 receptors; rabbits; muscle contraction

ABSTRACT

AIM: To clarify the distribution of functional P2X₁-like receptors in rabbit arteries. METHODS: Isometric contractile responses to noradrenaline (NA) and ¦Á,¦Â-methylene ATP (¦Á,¦Â-meATP) were observed in the arteries isolated from rabbit renal (Re), femoral (Fe), saphenous (Sa), mesenteric (Me), splenic (Sp), and ear (Ea). RESULTS: The maximal responses to NA (E_max·NA) varied among the arteries in an order of Re>Fe>Sa>Me>Sp>Ea. After standardization by the maximal response to KCl (E_max·KCl), however, the values of E_max·NA/E_max·KCl in the six kinds of arteries were almost the same. EC₅₀ values of NA in arteries were different, and the EC₅₀ value of NA in Me artery was 54 times that of Fe artery (P<0.01). The maximal response to ¦Á,¦Â-meATP (E_{max·¦Á,¦Â-meATP} ) varied among the arteries in an order of Re>Sa=Fe>Ea>Sp=Me, and the values of E_{max·¦Á,¦Â-meATP} /E_max·KCl were still different (Fe<Re<Sa=Ea=Sp=Me). The EC₅₀ values of ¦Á,¦Â-meATP in regional arteries were almost the same (0.23-0.77 mmol/L). The vasoconstrictive responses induced by ¦Á,¦Â-meATP in the Re, Fe, Sa, Me, Sp, and Ea arteries were subject to tachyphylaxis. CONCLUSION: There are functional P2X₁-like receptors in the six kinds of arteries used, and the vasoconstrictive responses regulated via the receptors are Fe<Re<Sa=Ea=Sp=Me, which is consistent with the sympathetic innervation of the arteries.

INTRODUCTION

The neurogenic vasoconstrictive responses induced by electrical field stimulation consist of purinergic (prazosin-resistant) component and adrenergic (prazosin-sensitive) component in many different regional blood vessels including the rabbit ear^[1], saphenous^[2], hepatic^[3] and splenic artery^[4]. Adenosine 5'-triphosphate (ATP) is considered as the purinergic cotransmitter released together with the classical transmitter of noradrenaline (NA) from the sympathetic nerve endings supplying those arteries^[1-4]. The proportion of purinergic component to adrenergic component varied considerably among various arteries. In the rabbit ear artery the prazosin-resistant (purinergic) component is less apparent in comparison with the adrenergic component^[1], however, a large component of the vasoconstriction in the rabbit saphenous artery or splenic artery in response to sympathetic nerve stimulation is puri-nergic component^[2-4]. Regional differences of endogenous ATP release in the rabbit arteries have been reported^[5]. But the contribution of functional P2X₁-like receptors to the rabbit regional vascular smooth muscle has not been known.

The present study was designed to investigate the contribution of functional a₁-adrenoceptor and P2X₁-like receptor to the vascular smooth muscle by observing contractile responses to NA (a-adrenoceptor agonist) and ¦Á,¦Â-methylene ATP (¦Á,¦Â-meATP, P2X₁ receptor agonist^[6]) in the rabbit isolated renal, femoral, saphenous, mesenteric, splenic, and ear arteries. In this study, we first reported the ¦Á,¦Â-meATP-induced contractile responses in the rabbit isolated renal, femoral, and mesenteric arteries.

MATERIALS AND METHODS

Rabbits Male New-Zealand white rabbits (2.5-3.5 kg) were obtained from Experimental Animal Center of Hebei Medical University (Certificate ¡í 0059).

Chemicals ¦Á,¦Â-Methylene adenosine 5'-triphosphate (¦Á,¦Â-meATP), (-)-noradrenaline (NA), and acetylcholine (ACh) were all obtained from Sigma Chemical Co. All drugs were dissolved in distilled water.

Arterial preparation Rabbits were stunned by a blow, then exsanguinated. The renal (Re), femoral (Fe), saphenous (Sa), mesenteric (Me), splenic (Sp), and ear (Ea) arteries were excised and cleaned of excess connective tissue and fat. The vascular endothelium was removed by gently rubbing the lumen with a scored polythene cannula that was slightly smaller in external diameter than the internal diameter of the vessels^[7]. Ring segments (4-mm in length) with or without endothelium were mounted horizontally in a 10 mL organ bath by carefully inserting a tungsten wire through the lumen of the vessel ring and anchoring it to a stationary support. Another wire similarly inserted, was connected to an isometric tension transducer, and responses were recorded on a polygraph (ERT-884, Youlin Electron Co, Kaifeng). Optimal preloads of 3.0 g, 3.0 g, 2.5 g, 1.5 g, 2.0 g and 2.0 g were applied to the Re, Fe, Sa, Me, Sp, and Ea arterial segments, respec-tively. The preparations were allowed to equilibrate for 1 h in physiological solution of the following composition (mmol/L)^[4]: NaCl 133, KCl 4.7, NaH₂PO₄ 1.35, NaHCO₃ 16.3, MgSO₄ 0.61, glucose 7.8, and CaCl₂ 2.52, pH 7.2. The solution was maintained at 37 ¡æ and aerated with 95 % O₂ and 5 % CO₂. A successful removal of the arterial endothelium was confirmed by the loss of relaxation response to ACh (1 mmol/L) in precontracted arterial rings^[8].

Drug administration NA (0.001-100 mmol/L) was added cumulatively to the organ bath to construct concentration-response curves. The concentration-response curve for NA was repeated twice in each preparation at 35 min interval. The first set of data was not used in analysis.

A selective P2X₁ receptor agonist ¦Á,¦Â-meATP (0.03-10 mmol/L) was added non-cumulatively at 1 h intervals^[4], because it rapidly desensitized its own receptor^[6]. Only one concentration-response curve for ¦Á,¦Â-meATP was generated per preparation. ¦Á,¦Â-MeATP 3 mmol/L was added to the preparation 1 h before constructing the concentration-response curve for ¦Á,¦Â-meATP. A single concentration (120 mmol/L) of KCl was added to the preparation in the end of each experiment in order to standardize the vasoconstrictive responses induced by NA or ¦Á,¦Â-meATP in the different arteries^[9,10]. In the experiment of desensitizing P2X₁-like receptor related response, the preparations of the Re, Fe, Sa, Me, Sp, and Ea arteries were exposed to 1 mmol/L ¦Á,¦Â-meATP for 20 min, and then the second administration of the same concentration of ¦Á,¦Â-meATP was repeated in the preparations without washing.

Statistical analysis Data were expressed as mean±SD. The EC₅₀ values were calculated with the equation: lgE/(E_max-E)=lgC -lgK (E, response; E_max, maximal response; C, agonist concentration; K, equilibrium dissociation constant). Duncan's multiple range test (a computer program of PHARM/PCS-Version 4) was used to evaluate the differences of E_max or EC₅₀ values of NA and ¦Á,¦Â-meATP in the six arteries. If the statistic test was significant, we compared the individual datum by unpaired t -test. P values less than 0.05 were considered statistically significant.

RESULTS

Contractile responses to KCl in the arteries with or without endothelium The maximal contractile responses to KCl 120 mmol/L (E_max·KCl) in the Sa, Sp, and Ea arteries without endothelium were significantly increased, in comparison with the arteries with endothelium (Fig 1). Endothelium removal did not modify the contraction in Re, Fe, and Me arteries (Fig 1). In the arteries without endothelium, the order of KCl-induced contraction magnitude was Re>Fe>Sa>Me>Sp>Ea (t tests).

Fig 1. Maximal contractile responses to KCl (120 mmol/L) in the rabbit renal (Re), femoral (Fe), saphenous (Sa), splenic (Sp), ear (Ea), and mesenteric (Me) arteries with or without endothelium. n=5-10. Mean±SD. ^aP>0.05, ^cP<0.01 vs the arteries with endothelium.

Contractile responses to NA in the arteries without endothelium NA (0.001-100 ¦Ìmol/L) produced vasoconstrictive responses in Re, Fe, Sa, Me, Sp, and Ea arteries in a concentration-dependent manner (Fig 2). The maximal contractile responses to NA (E_max·NA) in the six arteries were significantly different in the contraction magnitude, Re>Fe>Sa>Me>Sp>Ea (t-tests). After standardization of E_max·NA by E_max·KCl, however, the values of E_max·NA/E_max·KCl were almost the same in the six arteries (P>0.05, Duncan's new multiple range test, Tab 1). On the other hand, the EC₅₀ value of NA in Me artery was 54 times that of Fe artery (P<0.01, Tab 1).

Fig 2. Concentration dependent response curves for NA (A, n=6) and ¦Á,¦Â-meATP (B, n=4) in the rabbit renal, femoral, saphenous, splenic, ear, and mesenteric arteries without endothelium. Mean±SD.

Tab 1. Values of EC₅₀ and E_max for NA or ¦Á,¦Â-meATP in the rabbit arteries without endothelium. n=6 for NA. n=4 for ¦Á,¦Â-meATP. Mean±SD.

Contractile responses to ¦Á,¦Â-meATP in the arteries without endothelium ¦Á,¦Â-MeATP (0.03-10 ¦Ìmol/L) produced vasoconstrictive responses in Re, Fe, Sa, Me, Sp, and Ea arteries in a concentration-dependent manner (Fig 2). The maximal contractile responses to ¦Á,¦Â-meATP (E_{max·¦Á,¦Â-meATP}) in the six arteries were different in the contraction magnitude, Re>Sa=Fe>Ea>Sp=Me (t-tests). After standardization of E_{max·¦Á,¦Â-meATP} with E_max·KCl, however, the values of E_{max·¦Á,¦Â-meATP}/E _max·KCl were still different, Fe<Re<Sa=Ea=Sp=Me (t-tests). The EC₅₀ values of ¦Á,¦Â-meATP in the six arteries were 0.23-0.77 mmol/L (Tab 1).

Vasoconstrictive responses (g) induced by the first exposure to ¦Á,¦Â-meATP 3 ¦Ìmol/L, which was added to the organ bath before constructing the concentration-response curve, were 4.9±0.7, 2.8±0.6, 3.0±0.3, 1.6±0.4, 2.4±0.5, and 2.35±0.06 in the Re, Fe, Sa, Me, Sp, and Ea arteries, and which were not significantly different from the vascular responses (g) induced by the second exposure to ¦Á,¦Â-meATP 3 mmol/L in the values of 4.9±0.5, 3.0±0.6, 3.2±0.4, 1.7±0.4, 2.4±
0.5, and 2.33±0.05 (n=4, P>0.05).

Contractile responses to KCl in the arteries without endothelium for both groups of NA and ¦Á,¦Â-meATP After constructing the concentration-dependent response curves for NA or ¦Á,¦Â-meATP in each experiment with six kinds of arteries, the contractile responses to KCl 120 mmol/L were observed in order to standardize the vascular responses to agonists NA or ¦Á,¦Â-meATP. The vasoconstrictive responses (g) induced by KCl (120 mmol/L) between the two experimental groups (NA and ¦Á,¦Â-meATP) in the six kinds of arteries were not significantly different from each other (P>0.05), and which were 5.4±0.4, 4.7±0.7, 3.9±0.4, 2.6 ±0.5, 2.35±0.23, and 2.3±0.4 (n=6) in NA group; and 5.9±0.4, 4.9±0.6, 3.8±0.6, 2.0±0.4, 2.5±0.7, and 2.4±0.1 (n=4) in ¦Á,¦Â-meATP group.

Contractile responses to ¦Á,¦Â-meATP in the arteries before and after desensitizing P2X₁-like receptor In the preparations of the Re, Fe, Sa, Me, Sp and Ea arteries without endothelium, ¦Á,¦Â-meATP 1 mmol/L caused obvious contractile responses (g) in the values of 4.4±0.3, 2.3±0.4, 2.5±0.3, 1.5±0.4, 2.0±0.6, and 1.70±0.22 (n=4), respectively. The contractile responses to ¦Á,¦Â-meATP diminished rapidly in the unwashed-preparations continually exposed to the first administration of ¦Á,¦Â-meATP for 20 min, and the second administration of the same concentration of ¦Á,¦Â-meATP did not produce any responses (n=4).

DISCUSSION

It is widely accepted that the P2X₁-like receptor seems to be the most important P2X subtype in the vascular smooth muscle^[11], though this concept is not a result from functional study. Nori et al^[12] observed the coexpression of mRNAs of three P2X receptor subtypes (P2X₁, P2X₂, and P2X₄) in the rat vascular smooth muscle. However, it was reported that P2X₄ subtype did not couple to a vasomotor response^[13], and that P2X₂ receptor was mainly located on nerves and arterial endothelial cells, only low density on the smooth muscle cells^[14]. Moreover, ¦Á,¦Â-meATP was inactive as an agonist at the recombinant P2X₂ receptor^[15]. EC₅₀ values of ¦Á,¦Â-meATP to activate the P2X₁ receptor were reported to be approximately 0.5-5 mmol/L^[13]. In the present study, the EC₅₀ values of ¦Á,¦Â-meATP in the six kinds of arteries were 0.23-0.77 mmol/L which were consistent with the reported EC₅₀ values of ¦Á,¦Â-meATP, and the vasoconstrictive responses induced by ¦Á,¦Â-meATP in the Re, Fe, Sa, Me, Sp, and Ea arteries were subject to tachyphylaxis, indicting that the functional P2X receptors in the smooth muscle of the rabbit regional arteries were dominantly P2X₁-like subtype.

The vasoconstrictive responses to KCl (120 mmol/L) are usually used to assess the contractile ability of the vascular smooth muscle^[9,10,16], and agonists-induced vasoconstrictions are often standardized by E_max·KCl in order to eliminate an influence of the different thickness of muscle layer in larger and smaller arteries. In the present experiments, because the vasoconstriction induced by KCl 120 mmol/L in the Sa, Sp, and Ea arteries without endothelium were much bigger than that with endothelium (Fig 1), we used the arterial ring preparations without endothelium. In the end of experiments constructing the concentration-dependent response curves for both NA and ¦Á,¦Â-meATP, KCl-induced vasoconstrictive responses (E_max·KCl) of the same kind of artery in the two experimental groups were not significantly different from each other. Therefore, it was reasonable to standardize the responses to NA or ¦Á,¦Â-meATP with E_max·KCl in the present study.

The values of E_max·NA or E_{max·¦Á,¦Â-meATP} varied significantly among the regional arteries, ie, Re>Fe>Sa>Me>Sp>Ea arteries for NA or Re>Sa=Fe>Ea>Sp=Me arteries for ¦Á,¦Â-meATP. When the vascular responses were standardized by E_max·KCl, however, the values of E_max·NA /E_max·KCl were almost the same in the six kinds of arteries (Tab 1). On the other hand, the values of E_{max·¦Á,¦Â-meATP}/E _max·KCl were still different (Fe<Re<Sa=Ea=Sp=Me).

It has been known that the releases of NA induced by electrical field stimulation from Ea, Fe, Re, and pulmonary (Pu) arteries of the rabbit are different in characteristics from the releases of ATP^[5]. The amounts of NA released by electrical stimulation from the four arteries were almost equal, but the amounts of ATP released by electrical stimulation varied significantly, Ea>Re>Fe>>Pu^[5]. Moreover, it is well known that the density of sympathetic innervation increases with the decrease in lumen diameter of artery^[17]. In the present experiments, the standardized maximal responses to ¦Á,¦Â-meATP in the large arteries (Fe and Re) were significantly smaller than those in smaller arteries (Sa, Ea, Sp, and Me), which was consistent with the experimental results obtained from both transmitter release study^[5] and histological study^[17].

In contrast with the EC₅₀ values of ¦Á,¦Â-meATP, those of NA in the rabbit regional arteries were not the same, eg, the EC₅₀ values in Me artery was 54 times that of Fe artery. Satoh et al^[18] reported that there were different EC₅₀ values of NA to produce vasoconstrictive responses in the rabbit aortic, mesenteric, renal, and iliac arteries, indicating an existence of different ¦Á₁ adrenoceptor subtypes, and they further confirmed that in addition to ¦Á_1A and ¦Á_1B subtypes, other subtypes such as ¦Á_1D existed in the rabbit arteries. Similar results had been obtained from canine arteries^[19] and veins^[20]. Hence, functional P2X₁-like receptor holds an important action in the control of blood pressure, especially in the rapid and powerful vasoconstriction of the defense reaction.

REFERENCES

• 1 Kennedy C, Saville VL, Burnstock G. The contributions of noradrenaline and ATP to the response of the rabbit central ear artery to sympathetic nerve stimulation depend on the parameters of stimulation. Eur J Pharmacol 1986; 122: 291-300.
• 2 Burnstock G, Warland JJ. A pharmacological study of the rabbit saphenous artery in vitro: a vessel with a large purinergic contractile response to sympathetic nerve stimulation. Br J Pharmacol 1987; 90: 111-20.
• 3 Brizzolara AL, Burnstock G. Evidence for noradrenergic-purinergic cotransmission in the hepatic artery of the rabbit. Br J Pharmacol 1990; 99: 835-9.
• 4 Ren LM, Burnstock G. Prominent sympathetic purinergic vasoconstriction in the rabbit splenic artery: potentiation by 2,2'-pyridylisatogen tosylate. Br J Pharmacol 1997; 120: 530-6.
• 5 Ishii R, Shinozuka K, Kunitomo M, Hashimoto T, Takeuchi K. Regional differences of endogenous ATP release in rabbit arteries. Comp Biochem Physiol (C) 1996; 113: 387-91.
• 6 Burnstock G, Kennedy C. Is there a basis for distinguishing two types of P₂-purinoceptor. Gen Pharmacol 1985; 16: 433-40.
• 7 O'Connor SE, Wood BE, Leff P. Characterization of P_2x-receptors in rabbit isolated ear artery. Br J Pharmacol 1990; 101: 640-4.
• 8 Leff P, Wood BE, O'Connor SE. Suramin is a slowly -equilibrating but competitive antagonist at P_2X-receptors in the rabbit isolated ear artery. Br J Pharmacol 1990; 101: 645-9.
• 9 Vails AJ, Crowe R, Burnstock G. A neuromodulatory role for neuronal nitric oxide in the rabbit renal artery. Br J Pharmacol 1997; 121: 213-20.
• 10 Bo X, Sexton A, Xiang Z, Nori SL, Burnstock G. Pharmacological and histochemical evidence for P2X receptors in human umbilical vessels. Eur J Pharmacol 1998; 353: 59-65.
• 11 Valera S, Hussy N, Evans RJ, Adami N, North RA, Surprenant A, et al. A new class of ligand gated ion channel defined by P2X receptor for extracellular ATP. Nature 1994; 371: 516-9.
• 12 Nori S, Fumagalli L, Bo X, Bogdanov Y, Burnstock G. Coexpression of mRNAs for P2X1, P2X2 and P2X4 receptors in rat vascular smooth muscle: an in situ hybridization and RT-PCR study. J Vasc Res 1998; 35: 179-85.
• 13 Ralevic V, Burnstock G. Receptors for purines and pyrimidines. Pharmacol Rev 1998; 50: 413-92.
• 14 Hansen MA, Dutton JL, Balcar VJ, Barden JA, Bennett MR. P2X (purinergic) receptor distributions in rat blood vessels. J Auton Nerv Syst 1999; 75: 147-55.
• 15 Brake AJ, Wagenbach MJ, Julius D. New structural motif for ligand-gated ion channels defined by an ionotropic ATP receptor. Nature 1994; 371: 519-23.
• 16 Garcia-Villalon AL, Garcia JL, Fernandez N, Monge L, Gomez B, Dieguez G. Regional differences in the arterial response to vasopressin: role of endothelial nitric oxide. Br J Pharmacol 1996; 118: 1848-54.
• 17 Chen MQ, Cao JM. The individualization of activities of blood vessels. Prog Physiol Sci 1996; 27: 203-9.
• 18 Satoh M, Enomoto K, Niwano H, Fujimura H, Toyama Y, Takayanagi I, et al. Regional differences in a₁-adrenoceptor subtypes and mechanisms in rabbit arteries. Eur J Pharmacol 1998; 350: 67-73.
• 19 Griendling KK, Sastre A, Milnor WR. Regional differences in a₁-adrenoceptor numders and responses in canine aorta. Am J Physiol 1984; 247: H928-35.
• 20 Takayanagi I, Koike K, Noguchi R, Ogishima M, Takiguchi F, Sato T. A regional difference in a₁-adrenoceptor mechanism in canine veins. Arch Int Pharmacodyn Ther 1987; 289: 106-17.