Role of different subtypes of P2 purinoceptor on cytosolic Ca2+ levels in rat aortic smooth muscle

European Journal of Pharmacology - Molecular Pharmacology Section, 266 (1994) 263-267

263

© 1994 Elsevier Science B.V. All rights reserved 0922-4106/94/$07.00

EJPMOL 90578

Role of different subtypes of P2 purinoceptor on cytosolic C a 2+ levels in rat aortic smooth muscle S a t o s h i K i t a j i m a *, H i r o s h i O z a k i a n d H i d e a k i K a r a k i Department of Veterinary Pharmacology, Faculty of Agriculture, The University of Tokyo, Bunkyo-Ku, Yayoi 1-1-1, Tokyo 113, Japan

Received 26 July 1993; revised MS received 5 October 1993; accepted 22 October 1993

The role of different subtypes of P2 purinoceptors on cytosolic Ca 2+ level ([Ca2+]i) was examined in vascular smooth muscle of rat aorta, a/3-Methylene-ATP (P2x agonist), 2-methylthio-ATP (Pzv agonist), UTP and ATPyS (P2u agonists), and ATP (nonselective P2 agonist) induced a transient increase followed by a small sustained increase in [Ca2+] i in a concentration dependent manner. Among these agonists, a/3-methylene-ATP was the most potent. In the absence of extracellular Ca 2÷ (with 0.5 mM EGTA), ATP, UTP and ATPyS induced a transient increase in [Ca2+] i whereas a/3-methylene-ATP and 2-methylthioATP were ineffective. ATPyS showed the highest potency in Ca2+-free solution. After desensitization of P2x purinoceptor, ATP, UTP and ATPyS induced a rapid increase in [Ca2+] i followed by a sustained increase while a/3-methylene-ATP and 2-methylthio-ATP were ineffective. These results suggest that Ca 2+ release from the intracellular Ca 2+ store is mediated by P2u purinoceptor whereas Ca 2÷ influx is mediated by both P2x and P2u purinoceptors in the rat aortic smooth muscle.

P2 Purinoceptor; P2u Purinoceptor; Ca 2+ release; Ca 2+ influx; Smooth muscle, vascular

1. Introduction

Extracellular A T P acts on purinoceptors and plays an important role in the regulation of vascular tone (Burnstock and Kennedy, 1986; Olsson and Pearson, 1990) by directly changing contractility of smooth muscle cells (Kennedy and Burnstock, 1985a; Kennedy et al., 1985; Ralevic et al., 1991) and also by releasing e n d o t h e l i u m - d e p e n d e n t relaxing factor ( E D R F ) (Burnstock and Kennedy, 1985; Ralevic and Burnstock, 1991). Purinoceptors are classified into P1 and P2 subtypes. In the cardiovascular system, Pz receptors are further classified into Pzx and PzY subtypes (Burnstock and Kennedy, 1985). Recently, a nucleotide receptor, now termed P2u receptor, has also been described (Abbracchio et al., 1993). In various types of smooth muscle, Pzx receptor has been reported to activate non-selective, CaZ+-permeable, cation channels (Ben h a m and Tsien, 1987) and to induce contraction by an increase in Ca 2+ influx (Burnstock and Kennedy, 1985; Rembold et al., 1991). On the other hand, P2v and Pzu purinoceptors have been reported to exist on vascular endothelial cells and mediate the release of E D R F

* Corresponding author. Tel.: +81-3-3812-2111 (Ext. 5395); Fax: + 81-3-5802-2959. SSDI 0922-4106(93)E0174-I

(O'Connor et al., 1991; O'Connor, 1992). These purinoceptors are suggested to be linked to phospholipase C and to increase [Ca2+] i by activation of Ca z+ release via inositol 1,4,5-trisphosphate (Abbracchio et al., 1993). Although PzY and Pzu purinoceptors exist also in vascular smooth muscles (Kennedy and Burnstock, 1985b; Chinellato et al., 1992), coupling of these receptor subtypes to the signal transduction remains unknown. Recently, we have reported that A T P induces not only Ca 2 ÷ influx but also Ca 2 + release from intracellular Ca 2÷ store in rat aortic smooth muscle (Kitajima et al, 1993). The purpose of the present study was to identify the purinoceptors mediating Ca 2÷ release and Ca 2÷ influx in rat aortic smooth muscle. For this purpose we compared the effects of specific purinoceptor agonists including a/3-methylene-ATP (P2x agonist), 2-methylthio-ATP (P2Y agonist), U T P (Pzu agonist) and A T P y S (P2u agonist) (see O'Connor, 1992).

2. Materials and methods 2.1. Preparation, solutions and measurement o f cytosolic Ca 2+ level

Male Wistar rats (250-300 g) were stunned and bled and the thoracic aorta was dissected. After removing

264 fat and connective tissues, the aorta was cut into helical strips approximately 2 mm in width and 8 mm in length and placed in normal physiological salt solution (PSS) which contained (mM): NaC1 136.9, KC1 5.4, CaCI 2 1.5, MgCI 2 1.0, NaHCO 3 23.8, E D T A 0.01 and glucose 5.5. Solution with elevated K ÷ was made by replacing NaCI with equimolar KC1. Ca2+-free solution was made by removing CaC1 z and adding 0.5 mM EGTA. These solutions were maintained at 37°C and pH 7.4 aerated with 95% 0 2 and 5% CO 2. Endothelium was removed by gently rubbing the intimal surface with a finger moistened with PSS. This procedure changed neither the magnitude of high K÷-induced contraction nor the threshold concentration of KCI to induce contraction, suggesting that smooth muscle layer was not damaged. In such tissue, carbachol (1 /zM), a releaser of endothelium-derived relaxing factor, did not change the contraction induced by 0.1 /zM norepinephrine, suggesting that endothelium was removed. [Ca2+] i was measured according to the method described by Ozaki et al. (1987) and Sato et al. (1988) using the fluorescent Ca 2÷ indicator fura-2 (Grynkiewicz et al., 1985). Muscle strips were exposed to the acetoxymethyl ester of fura-2 (5/zM) in the presence of 0.02% cremophor EL for 3 - 4 h at room temperature. The muscle strip was then transferred to the muscle bath integrated in the fluorimeter (CAF-100) and illuminated alternately (48 Hz) with two excitation wave lengths (340 nm and 380 nm). Fluorescence at 500 nm was measured, and the ratio of the fluorescence induced by these two wavelengths was calculated and used as an indicator of [Ca2+]i . Absolute [Ca2+] i was not calculated because the dissociation constant of fura-2 for Ca 2÷ may change in smooth muscle ceils (Konishi et al., 1988; Karaki, 1989). Ratios of fluorescence in the resting muscle and that in the depolarized muscle with elevated external K ÷ (72.4 mM) were considered as 0 and 100%, respectively. 2.2. Chemicals Chemicals used were adenosine-5'-triphosphate (ATP) (Yamasa Shoyu, Tokyo, Japan), a/3-methyleneadenosine-5'-triphosphate (o~/3-methylene-ATP), uridine-5'-triphosphate (UTP), carbamylcholine chloride (carbachol) (Sigma Chemicals, St. Louis, USA), adenosine-5'-O-(3-thiotriphosphate) ( A T P y S ) (Boehringer Mannheim Y a m a n o u c h i , T o k y o , J a p a n ) , 2(methylthio)-adenosine-5'-(tetrahydrogen triphosphate) (2-methylthio-ATP) (Research Biochemicals, Natick, USA), norepinephrine bitartrate (Wako Pure Chemicals, Osaka, Japan), acetoxymethyl esters of fura-2 (fura-2 AM) (Dojindo Laboratories, Kumamoto, Japan) and cremophor E L (Nacarai Tesgue, Tokyo, Japan). The pH of stock solutions of ATP, afl-methylene-ATP, UTP, 2-methylthio-ATP, A T P y S was adjusted to approximately 7.4 by adding NaOH.

2.3. Statistics

Since some of the agonists did not induce the maximum response even at the highest concentrations examined (10-4-10 -3 M), it was not possible to calculate the concentration needed to induce a half-maximum response. Instead, we compared the potency of these agonists by calculating the concentrations (in - l o g M) needed to increase [Ca2+] i to a half-maximum level of the high K÷-stimulated response (ECsoK). Since all the experiments were done by single application of agonist, mean ECs0 K values and S.E.M. were calculated by first order regression. Differences were evaluated by Student's t-test and the P value equal to or less than 0.05 was considered to be statistically significant.

3. Results 3.1. In the presence o f extracellular Ca 2 +

In rat aorta, ATP (1 mM) induced a rapid increase in [Ca2+] i followed by a small sustained increase which reached a plateau after about 10 min (Fig. 1A). P2x agonist, a/3-methylene-ATP (0.01-100 /xM), P2Y agonist, 2-methylthio-ATP (1-100/zM), and P2tJ agonists, UTP (1-100/zM) and A T P y S (1-100/xM), also showed biphasic increases in [Ca2+] i. The levels of the peak [Ca2+] i (Caoeak) and steady state [Ca2+] i measured at 10 min (Cal0 min ) a r e plotted against the concentrations of P2 agonists in Fig. lB. Peak [Ca2+] i and steady state [Ca2+] i increased with the increase in the concentrations of P2 agonists. The ECs0 K values (see Materials and methods) measured at the Capeak were 6.74 + 0.08 (n = 4) for a/3-methylene-ATP, 5.73 + 0.08 (n = 4) for ATP3~S, 5.25 + 0.11 (n = 4) for 2-methythio-ATP, 4.62 + 0.17 (n = 4) for ATP and 4.58 + 0.15 (n = 4) for UTP. Thus, the rank order of agonist potency at the Capeak was a/3-methylene-ATP > A T P y S ( P < 0.01 vs. a/3-methylene-ATP) > 2-methylthio-ATP ( P < 0.01 vs. A T y S ) > ATP ( P < 0.05 vs. 2-methylthio-ATP) = UTP ( P > 0.05 vs. ATP). In contrast, the ECs0 K values measured at Cal0min were 4.63 + 0.08 (n = 4) for A T P y S , 4.32 + 0.18 (n = 4) for UTP and > 3 (n = 4) for ATP, 2-methylthio-ATP and a/3-methylene-ATP. Thus, the rank order of agonist potency at the C a l 0 m i n w a s A T P y S = UTP ( P > 0.05 vs. A T y S ) > ATP = 2methylthio-ATP = a/3-methylene-ATP ( P < 0.01 vs. UTP). 3.2. In the absence o f extracellular Ca 2 +

Muscle strips were treated with high K ÷ (72.4 mM) for 5 min to load Ca 2÷ in storage sites (see Karaki et al., 1979). After the Ca 2÷ loading, external Ca e÷ was

265

3.3. Effects of P2 agonists after desensitization of P2x purinoceptor

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It has b e e n shown that a / 3 - m e t h y l e n e - A T P desensitizes P2x purinoceptors (Burnstock and Kennedy, 1985). As d e m o n s t r a t e d in Fig. 3, a / 3 - m e t h y l e n e - A T P (10 /zM) transiently increased [Ca2+] i (40.0 q-6.5% at the peak, n = 4). A f t e r p r e t r e a t m e n t with aft-methyleneA T P (10 /zM) for 7 rain, the second application of a / 3 - m e t h y l e n e - A T P (10 ~ M ) had little effect on [Ca z+ ]i. A d d i t i o n of the P2v agonist, 2-methylthio-ATP (100 /zM), in the presence of a / 3 - m e t h y l e n e - A T P induced a small and slow additional increase in [Ca2+]i (28.8 + 5.2%, n = 4 of the high K+-induced increase in [CaZ+] i) (Fig. 3A). This value is only 21% of the p e a k [Ca2+]i

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Fig. 1. The effects of the P2 purinoceptor agonists on [Ca2+ ]i in the

presence of external Ca2+. A: Typical recording of ATP-induced response. ATP induced a rapid and transient increase in [Ca2+]i (defined as Capeak) followed by a small sustained increase which reached a plateau after about 10 min (Cal0min). Other P2 agonists, a/3-methylene-ATP, 2-methylthio-ATP, UTP and ATPyS showed a similar response to ATP. B and C: Concentration-response curves for the P2 purinoceptor agonists on Capeak and Cal0min, respectively (©, ATP; o, afl-methylene-ATP (a/3meATP); v, 2-methylthio-ATP (2meSATP); [3, UTP; ,7, ATPyS). Each point represents mean+_ S.E.M. of 4 experiments. r e m o v e d (with 0.5 m M E G T A ) for 2 min which decreased [Ca2+]i below the resting level. T h e n the P2 agonists were applied to the muscle strips. As shown in Fig. 2A, A T P (1 m M ) induced a transient increase in [Ca2+] i without sustained phase in the absence of Ca 2+. As shown in Fig. 2B, p e a k [Ca2+]i increased with the increase in the c o n c e n t r a t i o n of A T P (10-1000 /zM), U T P ( 1 - 1 0 0 0 / z M ) and A T P y S ( 1 - 1 0 0 / x M ) . T h e ECs0 K values were 5.62_+ 0.09 (n = 4) for A T P y S , 4.56 + 0.16 (n = 4) for U T P and 3.75 + 0.09 (n = 4) for A T P . Thus, the rank o r d e r of agonist p o t e n c y was A T P y S > U T P ( P < 0.01 vs. A T P y S ) > A T P ( P < 0.01 vs. UTP). In contrast, a / 3 - m e t h y l e n e - A T P ( 1 0 / z M ) and 2-methylthio-ATP ( 1 0 0 / z M ) were almost ineffective.

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Fig. 2. The effects of the P2 purinoceptor agonists on [Ca2+ ]i in the absence of external Ca2+. A: Typical recording of ATP-induced response. After Ca2÷ loading with high K ÷ (72.7 mM) for 5 min, external medium was replaced with CaZ÷-free normal PSS (with 0.5 mM EGTA) for 2 min. Addition of ATP induced a rapid and transient increase in [Ca2+]i followed by a decrease below the resting level. Other P2 agonists, except a/3-methylene-ATP and 2-methylthio-ATP, showed a similar response to ATP. B: Concentration-response curves for the P2 agonists on Capeak, respectively (o, ATP; n, a/3-methylene-ATP (a/3meATP); v, 2-methylthio-ATP (2meSATP); v, UTP; o, ATPyS). Each point represents mean_+ S.E.M. of 4 experiments.

266 5 rain

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ATPyS. These results indicate that the pretreatment with afl-methylene-ATP did not change the effects of ATP and UTP and slightly but significantly inhibited the effect of ATPyS (P < 0.05).

I I 101~M 20 aM (x~meATP

4. Discussion ATP 1 mM

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In the present study, we found that ATP and other

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Fig. 3. Effectsof 2-methylthio-ATP(2meSATP)(A),ATP(B),UTP (C) and ATPyS(D) on [Ca2+]i in the presenceof a/3-methyleneATP (a/3meATP)(10 /zM).After pretreatmentwith 10 /xM a/3methylene-ATPfor 7 min, the same concentrationof a/3-methyleneATP was cumulativelyapplied. Three minutes after the second applicationof a/3-methylene-ATP,2-methylthio-ATP(100/xM),ATP (1 mM),UTP (100/~M) or ATPyS(100/zM)was added.

due to 2-methylthio-ATP without a/~-methylene-ATP pretreatment (137.9 + 15.8%, n = 4, Fig. 1). Fig. 3B-D shows that, even in the presence of a/3-methylene-ATP (i.e. after desensitization of P2x purinoceptor), the P2u agonists, UTP (100 /zM) and ATPyS (100 /zM), and the non-selective P2 agonist, ATP (1 mM), induced additional rapid increase in [Ca2+]i followed by a sustained increase. The additional increase in peak [Ca2+] i and that in sustained [Ca2+] i (measured 7 min after the addition of agonists) were respectively 139.3 + 9.9% and 41.0 + 5.3%, n = 4, for ATP; 111.7 + 7.4% and 60.1 + 6.9%, n = 4, for UTP and 141.8+9.3% and 71.9+3.4%, n = 4 , for

ence of extracellular Ca 2+. Among the P2 purinoceptor agonists examined, the P2x agonist, a/3-methyleneATP, showed the highest potency when measured at the Capeak. Increase in [Ca/+] i induced by a/3-methylene-ATP was completely suppressed by the removal of external Ca 2+. These results suggest that activation of P2x purinoceptor increases [Ca2+]i by increasing Ca 2+ influx but not Ca 2+ release. 2-Methylthio-ATP has been suggested to selectively activate P2Y purinoceptor (Abbracchio et al., 1993). In the present experiment, we found that 2-methylthioATP increased [Ca2+]i in the presence of Ca 2+ but not in the absence of Ca 2+. Desensitization of P2x purinoceptor inhibited the peak [Ca2+] i due to 2-methylthio-ATP by 79%. These results suggest that 2-methylthio-ATP increases Ca 2+ influx by activating P2x purinoceptor in the rat aorta. We also found that ATP, UTP and ATPyS increased [Ca2+]i even in the absence of extracellular Ca 2+ (with 0.5 mM EGTA) which is due to Ca 2+ release from the intracellular Ca 2+ store. Among these P2 purinoceptor agonists, the P2u agonist, ATP~/S, showed the highest potency. In contrast, the P2Y agonist, 2-methylthio-ATP, failed to increase [Ca2+] i in the absence of external Ca 2+. These results suggest that P2u purinoceptor, but not P2Y purinoceptor, is responsible for Ca 2+ release in the rat aortic smooth muscle. In the muscle in which P2x purinoceptor had been desensitized, ATP, UTP and ATPyS induced a rapid increase in [Ca2+]i followed by a sustained increase. Since the first phase of increase in [Ca2+]i induced by these agonists is due mainly to Ca 2+ release, P2u purinoceptor may be responsible for Ca 2+ release. In the muscle in which P2x purinoceptor was not desensitized, ATPyS and UTP showed the highest potency to induce a sustained increase in [Ca2+] i (Cal0min). The sustained increase in [Ca2+]i induced by these agonists is completely abolished in the absence of external Ca 2+. These result suggest that Pzu purinoceptor is responsible for Ca 2+ influx. In conclusion, Ca 2+ release from the intracellular Ca 2+ store is mediated by P2u purinoceptor whereas Ca 2+ influx is mediated both by P2x and P2u purinoceptors in the rat aortic smooth muscle.

267

Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.

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Kennedy, C. and G. Burnstock, 1985b, Evidence for two types of P2-purinoceptors in longitudinal muscle of the rabbit portal vein, Eur. J. Pharmacol. 111, 49. Kennedy, C., D. Delbro and G. Burnstock, 1985, P2-Purinoceptors mediate both vasodilation (via the endothelium) and vasoconstriction of the isolated rat femoral artery, Eur. J. Pharmacol. 107, 161. Kitajima, S., H. Ozaki and H. Karaki, 1993, The effects of ATP and ~fl-methylene-ATP on cytosolic Ca 2+ level and force in isolated rat aorta, Br. J. Pharmacol. 110, 263. Konishi, M., A. Olson, S. Hollingworth and S.M. Baylor, 1988, Myoplasmic binding of fura-2 investigated by steady state fluorescence and absorbance measurement, Biophys. J. 54, 1089. O'Connor, S.E., 1992, Recent developments in the classification and functional significance of receptors for ATP and UTP, evidence for nucleotide receptors, Life Sci. 50, 1657. O'Connor, S.E., I.A. Dainty and P. Left, 1991, Further subclassification of ATP receptors based on agonist studies, Trends Pharmacol. Sci. 12, 137. Olsson, R.A. and J.D. Pearson, 1990, Cardiovascular purinoceptors, Physiol. Rev. 70, 761. Ozaki, H., K. Sato and H. Karaki, 1987, Simultaneous recordings of calcium signals and mechanical activity using fluorescent dye fura 2 in isolated strips of vascular smooth muscle, Jpn. J. Pharmacol. 45, 429. Ralevic, V. and G. Burnstock, 1991, Effects of purines and pyrimidines on the rat mesenteric arterial bed, Circ. Res. 69, 1583. Ralevic, V., R.T. Mathie, B. Alexander and G. Burnstock, 1991, Characterization of Pzx- and 2wpurinoceptors in the rabbit hepatic arterial vasculature, Br. J. Pharmacol. 103, 1108. Rembold, C.M., B.A. Weaver and J. Linden, 1991, Adenosine triphosphate induces a low [Ca 2+ ]i sensitivity of phosphorylation and an unusual form of receptor desensitization in smooth muscle, J. Biol. Chem. 266, 5407. Sato, K., H. Ozaki and H. Karaki, 1988, Changes in cytosolic calcium level in vascular smooth muscle strip measured simultaneously with contraction using fluorescent calcium indicator fura-2, J. Pharmacol. Exp. Ther. 246, 294.