Pyridylisatogen Tosylate of ATP-Responses at a Recombinant P2y1 Purinoceptor 1B.F
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British Journal of Pharmacology (I996) 117, 1111 1118 1996 Stockton Press All rights reserved 0007-1188/96 $12.00 0 Potentiation by 2,2'-pyridylisatogen tosylate of ATP-responses at a recombinant P2y1 purinoceptor 1B.F. King, *C. Dacquet, A.U. Ziganshin, tD.F. Weetman, G. Burnstock, *P.M. Vanhoutte & *M. Spedding Department ofAnatomy and Developmental Biology, University College London, Gower Street, London WClE 6BT; *Institute de Recherches Servier, 125 Chemin de Ronde, Croissy sur Seine, 72890, France and tDepartment of Pharmacology, University of Sunderland, Tyne and Wear SRI 3SD 1 2,2'-Pyridylisatogen tosylate (PIT) has been reported to be an irreversible antagonist of responses to adenosine 5'-triphosphate (ATP) at metabotropic purinoceptors (of the P2Y family) in some smooth muscles. When a recombinant P2Y purinoceptor (derived from chick brain) is expressed in Xenopus oocytes, ATP and 2-methylthioATP (2-MeSATP) evoke calcium-activated chloride currents (IC1,ca) in a concentration-dependent manner. The effects of PIT on these agonist responses were examined at this cloned P2Y purinoceptor. 2 PIT (0.1-100 rM) failed to stimulate P2y, purinoceptors directly but, over a narrow concentration range (0.1-3 tM), caused a time-dependent potentiation (2-5 fold) of responses to ATP. The potentiation of ATP-responses by PIT was not caused by inhibition of oocyte ecto-ATPase. At high concentrations (3-100 gM), PIT irreversibly inhibited responses to ATP with a IC_0 value of 13 +9 gM (pKB= 4.88 + 0.22; n = 3). PIT failed to potentiate inward currents evoked by 2-MeSATP and only inhibited the responses to this agonist in an irreversible manner. 3 Known P2 purinoceptor antagonists were tested for their ability to potentiate ATP-responses at the chick P2Y purinoceptor. Suramin (IC,0 = 230 + 80 nM; n = 5) and Reactive blue-2 (IC50 = 580 + 130 nM; n = 6) reversibly inhibited but did not potentiate ATP-responses. Coomassie brilliant blue-G (0.1- 3 ,M) potentiated ATP-responses in three experiments, while higher concentrations (3-100 gM) irreversibly inhibited ATP-responses. The results indicated that potentiation and receptor antagonism were dissociable and not a feature common to all known P2 purinoceptor antagonists. 4 In radioligand binding assays, PIT showed a low affinity (pKi < 5) for a range of membrane receptors, including: a,, CX2-adrenoceptors, 5-HTIA, 5-HTIB, 5-HT2, 5-HT3, D,, D2, muscarinic, central benzodiazepine, HI, M-opioid, dihydropyridine and batrachotoxin receptors. PIT showed some affinity (pKi= 5.3) for an adenosine (Al) receptor. 5 In guinea-pig isolated taenia caeci, PIT (12.5-50 gM) irreversibly antagonized relaxations to ATP (3-1000 gM); PIT also directly relaxed the smooth muscle and histamine was used to restore tone. Relaxations to nicotine (10-100 gM), evoked by stimulating intrinsic NANC nerves of taenia caeci preparations in the presence of hyoscine (0.3 pM) and guanethidine (17 gM), were not affected by PIT (50 gM, for 25-60 min). 6 These experiments indicate that PIT causes an irreversible antagonism of ATP receptors but, for recombinant chick P2Y purinoceptors, this effect is preceded by potentiation of ATP agonism. The initial potentiation by PIT (and by Coomassie brilliant blue-G) of ATP-responses raises the possibility of designing a new class of modulatory drugs to enhance purinergic transmission at metabotropic purinoceptors. Keywords: 2,2'-Pyridylisatogen; P2 purinoceptor; ATP; adenosine; purinergic transmission; recombinant P2y purinoceptor; metabotropic purinoceptor; Xenopus oocyte Introduction The classification of ATP receptors (P2 purinoceptors) is based laxations to adenosine 5'-triphosphate (ATP) in guinea-pig predominantly on the rank potency of agonists, since there isolated taenia caeci (Spedding et al., 1975), in a time- and remains a lack of potent and selective antagonists to dis- concentration-dependent manner consistent with an irrever- criminate between P2 purinoceptor subtypes (Burnstock, 1978; sible antagonism of postjunctional ATP receptors on smooth Burnstock & Kennedy, 1985; Burnstock et al., 1994; Dalziel & muscle. Responses to adenosine were unaffected by PIT and, Westfall, 1994). Recently, ATP receptors have been reclassified to this extent, this compound provided a pharmacological into two extended families, P2X,-4 and P2Y, purinoceptors, means to differentiate receptors for ATP and adenosine based on agonist activity of novel purine analogues, trans- (Spedding & Weetman, 1976). However, relaxations to the duction mechanisms and molecular structure (Abbracchio & stimulation of intramural non-cholinergic, non-adrenergic Burnstock, 1994). (NANC) nerves of taenia caeci were unaffected by PIT 2,2'-Pyridylisatogen tosylate (PIT) is one of a series of isa- (Spedding et al., 1975) and this result raised doubts about PIT togen analogues which, originally, were designed to be selective as an effective pharmacological tool to test for purinergic antagonists of ATP receptors. PIT (10-50 gM) reduced re- transmission. The early use of PIT to distinguish receptors for ATP and adenosine was complicated by the fact that the compound relaxed smooth muscle directly, an effect secondary to inhibi- 'Author for correspondence. tion of oxidative phosphorylation (Foster et al., 1978; Sped- 1112 B.F. King et al Modulation of ATP agonism ding & Weetman, 1978a,b). This effect meant that spasmogens Oocyte ecto-A TPase assays were needed to recontract the smooth muscle tissues (Spedding & Weetman, 1978b). Furthermore, PIT was not an effective The enzymatic degradation of ATP by ecto-ATPase was antagonist of ATP responses in all smooth muscles (Spedding measured by the Fiske/Subbarow (1925) determination of in- & Weetman, 1978a), suggesting its use as an antagonist might organic phosphate (Pi) production, as described for oocytes by be limited to certain subtypes of P2 purinoceptors. Also, PIT Ziganshin and colleagues (1995). Briefly, folliculated oocytes was reported to potentiate, but not antagonize, responses to were placed in 24-well dishes, three oocytes per well, in 300 PI ATP in the guinea-pig terminal ileum (Kazic & Milosavljevic, of Ringer solution and washed for 30 min, shaking con- 1977). tinuously. This wash solution was replaced with 250 Ml of The present study was designed to re-investigate the phar- Ringer solution containing ATP (100 gM). After 30 min in- macology ofPIT on a recombinant P2Y purinoceptor where the cubation (at 20 + 10C), the reaction was halted by removing a actions and efficacy ofPIT can be studied in isolation. The P2y sample (100 Pl) of the bathing solution and adding it to so- purinoceptor derived from chick brain was chosen for this dium dodecyl sulphate (0.9 ml, 2.5% w/v solution). Then purpose since it is activated by ATP and ADP but by no other ammonium molybdate (1 ml, 1.25% w/v solution) and Fiske/ naturally-occurring nucleotides or nucleosides (Webb et al., Subbarow Reducer (0.1 ml, 0.16% w/v solution) were added to 1993; King et al., 1994; Simon et al., 1995). This recombinant samples and the final solution left at room temperature for purinoceptor is activated strongly by 2-MeSATP but weakly 30 min to develop colour. The reaction product was measured by a,f-meATP and, therefore, resembles P2Y purinoceptors spectrophotometrically at 700 nm. KH2PO4 solutions were found on many smooth muscles. Also, the chick P2y, pur- used as phosphate standards to calibrate the spectro- inoceptor is structurally- and pharmacologically-identical to photometer (Beckman Du-65). The resting velocity of Pi pro- two P2Y purinoceptors cloned recently from vascular en- duction by oocyte ecto-ATPase was compared with the enzyme dothelial cells (Henderson et al., 1995; Leon et al., 1995). The activity of oocytes incubated with PIT (1- 100 gM) for 45 min. findings demonstrate that, over a narrow range of concentra- tions, PIT potentiates the responses to ATP at the P2Y pur- Radioligand binding assays inoceptor expressed in Xenopus oocytes, then inhibits responses to ATP and 2-MeSATP at higher concentrations. Binding experiments to study the affinity of PIT for different Other P2 purinoceptor antagonists were tested and revealed receptors were carried out as described previously (see Table that the potentiating effect is not a feature of these drugs, 1). Inhibition constants (IC50 values) were calculated using except for Coomassie brilliant blue-G. The biphasic actions of non-linear regression analysis (Lundon Software) and Ki va- PIT at a recombinant P2Y punnoceptor might explain some of lues were computed according to the Cheng & Prussoff (1973) the discrepancies seen earlier with PIT on ATP receptors in equation (Ki=IC50/(1+ Kd)), where L is the concentration of isolated smooth muscle preparations. Part of this study has radioligand and Kd is the apparent dissociation constant. For been communicated to the British Pharmacological Society each receptor, data (pKi values) are presented for a key re- (King et al., 1994; Spedding et al., 1994). ference ligand and for PIT (see Table 1). Smooth muscle assays Methods Taenia caeci preparations were taken from female guinea-pigs Electrophysiology (weight range: 250-600 g). Isolated preparations were sus- pended in 10 ml organ baths filled with McEwen's solution Defolliculated Xenopus oocytes (stages V and VI) were cytosol- (McEwen, 1956) which was maintained at 35 + 1°C and gassed injected (40- 80 nl, at 2 Mg Ml- ') with a cRNA (transcribed in with 95% 02 and 5% CO2. After an equilibration period of vitro from pSG5-803, capped and polyadenylated, as described 30 min, contractile responses were recorded isotonically (load, in Webb et al., 1993), then stored in a modified Barth's solu- 1.5 g) (Spedding et al., 1975). tion and incubated at 18°C for 48 h to express P2Y pur- Cumulative concentration-response curves (Van Rossum, inoceptors. Thereafter, twin-electrode voltage clamp 1963) for purine bases and noradrenaline relaxing taenia caeci recordings were carried out on cRNA-injected oocytes, using strips were determined at 20 min intervals. Each dose of drug an Axoclamp 2A (Axon Instruments Inc.) amplifier to control was allowed to produce its full effect (5-40 s contact) before intracellular voltage and measure membrane currents.