BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.

242, 272276 (1998)

RC977949

2-2 -Pyridylisatogen, a Selective Allosteric Modulator of P2 Receptors, Is a Spin Trapping Agent

F. Nepveu,* J.-P. Souchard,* Y. Rolland, G. Dorey, and M. Spedding,,,1
Institut de Recherches Internationales Servier, 192 avenue Charles de Gaulle, 92200 Neuilly-sur-Seine, France; *Universite Paul Sabatier, Faculte des Sciences Pharmaceutiques, 35 Chemin des Marachers, 31062 Toulouse Cedex-4, France, Les Laboratoires Servier, 22 rue Garnier, 92200 Neuilly-sur-Seine, France; and Institute de Recherche Servier, Croissy sur Seine, 78290, France

Received October 17, 1997

2-2 -Pyridylisatogen (PIT) has been reported to be a relatively selective irreversible antagonist of responses to adenosine 5 -triphosphate (ATP) in some smooth muscle preparations and to be an allosteric modulator of responses to ATP at recombinant P2Y receptors from chick brain. PIT is also a potent inhibitor of mitochondrial oxidative phosphorylation. However, the compound has a unique nitrone structure, so PIT was compared with dimethyl-pyrroline-N-oxide (DMPO) as a spin trapping agent for superoxide and hydroxyl radicals using electron spin resonance (ESR). PIT was found to be a potent spin trapper of both hydroxyl and superoxide radicals. PIT was more potent than DMPO to trap the hydroxyl radical forming an adduct which was more stable than the DMPO adduct in aqueous media. PIT was an effective spin trap of hydroxyl radical in aqueous buffer at pH 7.4. PIT more slowly trapped the superoxide anion but at concentrations where DMPO trapped none. 1998
Academic Press

Key Words: 2-2 -Pyridylisatogen; P2 receptors; spin trap; hydroxyl; superoxide.

The classication of receptors for ATP is hampered because of the lack of potent and selective antagonists, despite the recent availability of recombinant receptors (1). 2-2 -Pyridylisatogen (PIT; 10-50 mM) has been shown to antagonise the relaxant responses to adenosine 5-triphosphate (ATP) in taenia caeci preparations from the guinea-pig (2) in a time- and concentration-dependent manner, suggesting an irreversible antagonism. Responses to adenosine were unaffected allowing the rst clear differentiation of distinct receptors (3). Thus the compound was the rst selective antagonist at P2 recepTo whom correspondence should be addressed. Fax: 33146417655; E-mail: spedding@servier.fr. 0006-291X/98 $25.00
Copyright 1998 by Academic Press All rights of reproduction in any form reserved.
1

tors, and more recent compounds are not more selective (1). However, the use of PIT in high concentrations in receptor classication is complicated by the fact that the compound relaxes smooth muscle directly, secondarily to inhibition of oxidative phosphorylation (4). Futhermore PIT is not an effective antagonist of ATP in all tissues (4) and potentiates responses to ATP in guinea-pig terminal ileum (4). PIT may specically potentiate responses to ATP in xenopus oocytes expressing the P2Y receptor (5) by acting as an allosteric modulator. As PIT may interact with sulphydryl groups, then this may be an important factor in the mechanism of action, particularly as some P2 receptors may comprise up to 50 sulphydryl groups in the extracellular medium. There is now considerable interest in the question of whether P2 receptors may mediate neuroprotection in some experimental situations. However, nitrone spin traps are potent neuroprotective agents (711) and attenuate mitochondrial dysfunction after ischaemia (11). In this respect, PIT shares structural characteristics with nitrone spin trappers such as dimethyl-pyrroline-N-oxide (DMPO) or a-phenyl-tertbutyl-nitrone (tBPN) (Figure 1), but has unique chemical characteristics. Thus we have studied the ability of PIT to act as a spin trapping agent. Electron spin resonance spectroscopy (ESR) is the only technique capable of detecting directly the presence of short-lived reactive free radicals and although the technique is reasonably sensitive, it is insufcient to detect the presence of highly reactive unstable species. Thus the ability of compounds to trap radicals in a stable species, generating high concentrations of the species, is a critical determinant of the capacity to measure the presence of radicals in in vivo systems (12). We show that PIT forms one of the most stable spin trap adducts known, capable of trapping both hydroxyl and superoxide radicals. MATERIALS AND METHODS
Reagents. PIT was synthetized as described previously (2-4). DMPO, activated charcoal, Xanthine, Xanthine oxydase, were ob-

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FIG. 1. Structure of DMPO, tBPN and PIT.

tained from Sigma Chemical Co, Na2PO4 , NaHPO4 , H2O2 and {(NH4)2Fe(SO4)2 , 6H2O} (Mohrs salt) were purchased from Prolabo. DMPO was puried as reported in the literature (13), aliquoted, stored frozen and kept protected from light. ESR hydroxyl scavenging experiments. A 1 ml solution was prepared in a glass tube by adding the reagents in the following order: DMPO (0.5 mM or 3 mM) or PIT (0.5 mM or 3 mM) or BPN (0.5 mM or 3 mM), H2O2 (10 mM), and Fe2/ (Mohrs salt, 10 mM) in aquous medium or in phosphate buffer (pH 7.4) containing ethylenediaminetetraacetic acid (10,5 mM). ESR superoxide scavenging experiments. A 1 ml solution was prepared in a glass tube at 37 C by adding the reagents in the following order: DMPO (0.5 mM or 3 mM) or PIT (0.5 mM or 3 mM) or BPN (0.5 mM or 3 mM), Xanthine (500mM), xanthine oxidase (50 mU/mL) in phosphate buffer (pH 7.4) containing diethylenetriaminepentacacetic acid (1 mM). ESR mesurements. The ESR spectra were recorded at room temperature in a at quartz cell inserted in a TM 110 Bruker cavity with an ER 200 D Bruker ESR spectrophotometer by starting a 3 minutes scan, 3 or 6 minutes or more for time course experiments after mixing the reagents. EPR spectrometer recording conditions were: central eld, 3500 G; scan range eld, 200 G; microwave frequency, 9.66 GHz; frequency modulation, 100 KHz; modulation amplitude, 1 G; time constant 0.5 s and mirowave power 10mW.

RESULTS 2-2 -Pyridylisatogen (0.5 mM; Figure 2) showed a characteristic ESR spectrum in the presence of hydroxyl radical generated by the Fenton reaction with three coupling constants (AN1: 10.0 G, AN2: 3.2 G, AH: 1.16 G). At 0.5 mM PIT gave an intensive ESR signal when no signal could be detected with DMPO (Figure 3). Thus PIT was a more sensitive spin trap than the classic spin trap, DMPO, which yielded its classic spectrum only at 3 mM (Figure 3). tBPN did not yield a spectrum under these conditions (Figure 3), which may be due to the short half life of the corresponding adduct (14). Spin traps are usually less effective under physiological conditions (e.g. Figure 5), but PIT (3 mM) showed its characteristic spectrum in phosphate buffer, at pH 7.4, whereas DMPO (3 mM) did not show detectable spin trapping under these conditions (Figure 4). The stability of the PIT and DMPO adducts was followed in a time course experiment. In all cases the spectra were almost fully developped at the rst time

FIG. 2. Spectrum of the PIT-OH spin adduct formed by incubation of PIT (0.5 mM) with H2O2/Fe2/, 10 mM/10 mM, in aqueous solution. 273

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FIG. 3. Spectrum of the hydroxyl spin adduct formed by incubation of PIT (A1: 0.5 mM; A2: 3 mM), DMPO (B1: 0.5 mM ; B2: 3 mM), or tBPN (C1: 0.5 mM ; C2: 3 mM) with H202/Fe2/, 10 mM/10 mM in aqueous solution.

FIG. 4. Spectrum of the hydroxyl spin adduct formed by incubation of PIT (A: 3 mM), DMPO (B: 3 mM), with H202/Fe2/, 10 mM/10 mM, in phosphate buffer, pH 7.4. 274

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FIG. 5. Kinetics of the formation of the hydroxyl spin adduct formed by incubation of PIT (3 mM) in aqueous solution (/) or phosphate buffer (1, pH 7.4), DMPO (*, 3 mM) in aqueous solution. The ESR signal intensity is obtained from the average of the height of the two highest peaks for DMPO-OH and of the three highest peaks for PIT-OH.

point (2 minutes), but the spectrum of DMPO-OH (3 mM in aqueous media) declined over 20 min indicating that the formed adduct was not fully stable. In contrast, the PIT-OH radical (3 mM in either aqueous media, or in phosphate buffer, pH 7.4) showed no decline in ESR signal indicating a stable adduct (Figure 5). When PIT (3 mM) and DMPO (3 mM) were coincubated in the presence of hydroxyl radicals, the DMPO-OH ESR signal declined rapidly (Figure 6), indicating that PIT

could compete with DMPO for trapping the hydroxyl radical, forming a very stable spin adduct. The ability of PIT to interact with superoxide radicals was studied by generating radicals with the xanthine (0.5 mM) / xanthine oxidase (0.05 U/ml) system in a phosphate buffer (pH 7.4) at 37 C. Under these conditions the ESR spectrum intensity of the PIT-OOH adduct (3 mM) increased slowly, but became clear at 15 min (Figure 7).

FIG. 6. Spectrum of the hydroxyl spin adduct formed by co-incubation of PIT (3 mM) and DMPO (3 mM), with H202/Fe2/, 10 mM/10 mM, in aqueous solution for 3 (A) or 6 (B) min. Note the time-dependent reduction of the peak attributed to DMPO-OH (arrow). 275

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angles to the isatogen moiety, inducing a stabilised structure of the adduct. However, it is not clear whether the spin trapping properties of PIT contribute to the antagonism and potentiation of responses at the ATP receptor (2,3) or to the effects on mitochondrial function, which have a distinct structure/activity relationship (4). Preliminary studies indicate that spin trappers are not necessarily antagonists at P2Y receptors (15). However, the remarkable neuroprotective effects of spin-trapping agents (10) argue for a study of the effects of PIT, compared with other spin trappers and P2 antagonists, in a range of neurodegenerative models. ACKNOWLEDGMENTS
We thank D. Billington, P. Casara, G. Dorey for synthesising samples of PIT.

REFERENCES
FIG. 7. Spectrum of the superoxide spin adduct formed by incubation of PIT (3 mM), with xanthine (0.5 mM)/xanthine oxidase (0.05 U/ml) in phosphate buffer, pH 7.4, after 12 min incubation at 37 C. 1. Fredholm, B. B., Abbracchio, M. P., Burnstock, G., Daly, J. W., Harden, T. K., Jacobson, K. A., Leff, P., and Williams, M. (1994) Pharmacol. Rev. 46, 143156. 2. Spedding, M., Sweetman, A. J., and Weetman, D. F. (1975) Br. J. Pharmac. 53, 575583. 3. Spedding, M., and Weetman, D. F. (1976) Br. J. Pharmac. 57, 305310. 4. Spedding, M., and Weetman, D. F. (1978) J. Pharm. Pharmac. 30, 335336. 5. Kazic, T., and Milosavljevic, D. (1977) J. Pharm. Pharmac. 29, 542545. 6. King, B. F., Dacquet, C., Ziganshin, A. U., Weetman, D. F., Burnstock, G., Vanhoutte, P. M., and Spedding M. (1996). Br J Pharmacol. 117, 11111118. 7. Phillis, J. W., and Clough-Helfman, C. (1990). Med. Sci. Res. 18, 403. 8. Cao, X., and Phillis, J. W. (1994). Brain Res. 644, 267271. 9. Zhao, Q., Pahlmark, K., Smith, M.-L., and Siesjo, B. K. (1994) Acta Phys. Scand. 152, 349352. 10. Thomas, C. E. (1997) in Neuroprotection in CNS Diseases (Bar, P. R., and Beal, M. F., Eds.), pp. 183204, Marcel Dekker, New York. 11. Kuroda, S., Katsura, K. I., Hillered, L., Bates, T. E., and Siesjo, B. K. (1996) Neurobiol. Dis. 3, 149157. 12. Kaur, H. (1996) Free Rad. Res. 24, 409420. 13. Buettner, G. R., and Oberley, L. W. (1978) Biochem. Biophys. Res. Comm. 83, 6974. 14. Tomasi, A., and Iannone, A. (1993) in Biological Magnetic Resonance (Berliner, L. J., and Reuben, J., Eds.), Vol. 13, pp. 53 384, Plenum Press, New York. 15. Menton, K., Morgan, R. M, Spedding, M., and Markham, A. (1997). Br J Pharmac, in press

CONCLUSION 2-2 -Pyridylisatogen (PIT) forms highly stable adducts with hydroxyl and superoxide radicals. PIT is more efcient in trapping HO and O20 than the well known spin traps DMPO and tBPN. Moreover, PITOH adduct is more stable than the corresponding DMPO-OH adduct. Such a chemical prole may be of considerable use by forming stable adducts in biological media, which can be followed over long periods of time. Spin traps which can be used in physiological conditions are particularly valuable, as attempts to trap important physiological radicals such as nitric oxide have not been totally convincing (12). Current experiments are ongoing in order to dene the interactions with nitric oxide. Nitrones such as PIT can form stable adducts because of delocalisation of the electron, and PIT may be particularly favoured in this respect. The structure of PIT is unique among spin trappers on the basis that there is no hydrogen on the a carbon to the nitrone function, and the nitrone may be highly conjugated with the ketone and the 2 pyridyl group. The reactive species will also force the pyridine ring to be at right

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