Br. J. Pharmacol. (1994), 113, 349 - 354

Blockade of P2X-purinoceptors by trypan blue in rat vas

deferens'Ralph Biiltmann, Marie Trendelenburg & Klaus Starke

Pharmakologisches Institut, Universitit Freiburg, Hermann-Herder-Strasse 5, D-79104 Freiburg i.Br., Germany

1 The possibility of an antagonist effect of trypan blue at P2X-purinoceptors was studied in rat vas

deferens.2 Trypan blue (3.2-320 tLM) shifted the concentration-contraction response curve of a,-methyleneATP (aj-MeATP) to the right and simultaneously increased the maximum of the curve by up to 40%.The Schild plot had a slope not significantly different from unity and yielded a pA2 value of 5.3 (KB4.9 fLM).3 Suramin (32 jLM) also shifted the concentration-response curve of ax,-MeATP to the right, KB 2.6 LLM,

and increased the maximum by 31%. In the presence of suramin (32 gM), trypan blue (32 JLM) did notchange the concentration-response curve of x,4-MeATP.4 1-Amino-8-naphthol-3, 6-disulphonate (H-acid) 10 mM, the sulphonic acid-carrying moiety of trypanblue, shifted the concentration-response curve of a,4-MeATP to the right, KB 1.4 mM, and increased themaximum by 33%.5 Trypan blue did not change contractions elicited by high K+ and noradrenaline.6 Trypan blue attenuated the purinergic component of neurogenic contractions, IC50 44.9 fLM, but didnot change the adrenergic component.7 It is concluded that trypan blue blocks P2X-purinoceptors in rat vas deferens. The increase of themaximum of the a,4-MeATP concentration-response curve is similar in mechanism to the increaseproduced by suramin.

Keywords: Rat vas deferens; trypan blue; suramin; a,-methylene ATP; P2X-purinoceptor; P2-purinoceptor antagonists; co-

transmission; purinergic transmission

Introduction

Reactive blue 2 (Kerr & Krantis, 1979) and suramin (Dunn& Blakeley, 1988) are perhaps the best characterized P2-purinoceptor antagonists at present (see Cusack, 1993, for areview on purinoceptor classification). Reactive blue 2 isfairly selective for P2Y-purinoceptors (see Kennedy, 1990),whereas suramin possesses similar affinity for several P2 sub-types (see Cusack, 1993). Other substances have been testedonly on a few preparations. Cibacron blue 3GA, an isomerof reactive blue 2, and brilliant blue G act as antagonists atthe P2Z-purinoceptors of rat parotid acinar cells (Soltoff etal., 1989) and the P2Y-like receptors on sympathetic nerveterminals (Fuder & Muth, 1993; von Kfigelgen et al., 1994).4,4'-Diisothiocyanatostilbene-2,2'-disulphonate (DIDS) alsoblocks the rat parotid P2Z-purinoceptor (McMillian et al.,1988; Soltoff et al., 1993) and in addition the P2X-purino-ceptor in rat vas deferens (Biiltmann & Starke, 1994a).Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PP-ADS; Lambrecht et al., 1992) and the 2',5'-disulphonic acidisomer iso-PPADS block P2X-purinoceptors in a variety ofsmooth muscle tissues (McLaren et al., 1993; Ziganshin et al.,1993; Bultmann & Starke, 1994b). Finally, we have recentlyshown that Evans blue is a P2x antagonist in rat vas deferens(Biltmann & Starke, 1993).Trypan blue, which like suramin was introduced as a

trypanocide early in this century, differs from Evans blue inthe position of the two sulphonic acid residues at the naph-thalene rings. We examined whether trypan blue blocks P2X-purinoceptors in rat vas deferens.

Methods

Male Wistar rats (240-300 g) were decapitated and the vasadeferentia removed and cleaned of adherent tissue. The

' Author for correspondence.

medium used for incubation and superfusion contained(mM): NaCl 118, KCl 4.8, CaCl22.5, MgSO4 1.2, KH2PO40.9, NaHCO3 25, glucose 11, ascorbic acid 0.3 and disodiumEDTA 0.03. It was saturated with 95% 02/5% CO2 and keptat 37°C.Whole vasa deferentia or (in experiments with ap-

methylene ATP, a,3-MeATP) prostatic thirds were suspendedvertically in a 5.7 ml organ bath. The lower end was fixedand the upper end attached to an isometric force transducer(K30, Hugo Sachs Elektronik, Hugstetten, Germany) underan initial tension of 9.8 mN (Graphtec thermal pen recorder,Ettlingen, Germany). The medium was replaced every15 min. Tissues relaxed to about 3 mN during a 60 minequilibration period. This final resting tension remained con-stant for the remainder of the experiments.

Contractions were elicited by a,-MeATP, high K+,noradrenaline or electrical field stimulation. High K+ wasadded as 35 mM KCl (final K+ concentration therefore40.7 mM) without osmotic compensation. Agonists and highK+ were washed out immediately after contractions hadpeaked. Field stimulation (single pulses, 0.3 ms pulse width,100 mA) was applied via platinum electrodes located at thetop and the bottom of the organ bath (Stimulator II, HugoSachs Elektronik). Concentration-response data were analys-ed by logistic curve fitting to the weighted mean contractionvalues using equation No. 25 of Waud (1976) and non-linearregression. The calculation yielded the maximal effect and theEC50 or ICs for each curve, EC50 or IC50 being the concen-tration producing 50% of the maximum of that curve.

Antagonist effects on concentration-response curves of aj,-MeATP and noradrenaline were examined as follows. Twoconcentration-response curves of the agonist were determinedin each vas deferens, the first before, the second 60 min afteraddition of the antagonist or its solvent. The ECm for eachcurve was obtained as mentioned. The shift caused by theantagonist was then quantified as the ratio 'EC50 in second

'." Macmillan Press Ltd, 1994

350 R. BOLTMANN et al.

curve (with antagonist)' over 'ECm in first curve (beforeantagonist)', corrected for the shift in solvent controls.Antagonist KB values were calculated from the shifts eitherby means of the method of Arunlakshana & Schild (1959) orby means of equation No. 4 of Furchgott (1972). The proce-

a Solvent

dure was analogous when the effect of trypan blue on theconcentration-response curve of a,,-MeATP was studied intissues exposed to suramin throughout the experiment.

a,P-Methylene ATP lithium salt (a,-MeATP), trypan blue,(-)-noradrenaline bi-(+)-tartrate (Sigma, Deisenhofen, Ger-

b

40 -

30 F

zE 20 -

10 I

0

C Trypan blue 10 pM

40 F

30 FzE 20 I-

10 -

0

Trypan blue 3.2 pM

d Trypan blue 32 pM

a... .3IAtid.IU Ia ad aaa

Trypan blue 100 MM

.p..niI ..am.anI .d .......aI

Trypan blue 320 pM

aa....a ad a..

1 10 100 1000 1 10 100 1000

a, P-Methylene ATP (pM)

Figure I Effect of trypan blue on the concentration-response curve of a,p-methylene ATP. Increasing concentrations of a,-methylene ATP were added every 30 min and washed out immediately after the contraction had peaked. Two concentration-response curves were determined in each tissue. Solvent (a) or trypan blue (b-f) was added to the medium after completion of thefirst curve (0) and the second curve was determined 60 min later (0). Abscissae, agonist concentration. Ordinates showcontraction (mN). Means ± s.e.mean from 4 to 9 experiments.

S

40 1

30 -

zE 20 F

10 I

0

f

TRYPAN BLUE BLOCKS P2x-PURINOCEPTORS 351

many) and suramin (Bayer, Wuppertal, Germany) were dis-solved in distilled water. KCl for high K+ was dissolved inmedium. I-Amino-8-naphthol-3, 6-disulphonic acid mono-sodium salt (H-acid; Aldrich, Steinheim, Germany) was dis-solved in medium with addition of the same molar amount ofNaOH. Solutions of drugs were added to the organ bath inaliquots not exceeding 32 pl.Data are expressed as either the arithmetic mean ± s.e.

mean; or the EC_0 or ICo and maximal effect with thestandard error as defined by Waud (1976) in the case of fittedcurves; or the pA2 with 95% confidence interval. Differencesbetween means were tested for significance by the Mann-Whitney test. Differences between fitted curves were testedaccording to p. 371 of Motulsky & Ransnas (1987). P<0.05was taken as the limit of statistical significance.

Results

In initial experiments, contractions were elicited by K+(35 mM), noradrenaline (30 pM) and xj-MeATP (3 iM) ineach single tissue, given at 5 min intervals and repeated after70 and 140 min. Contractions were 14.1 ± 1.3, 10.9 ± 0.9 and10.8 + 0.6 mN, respectively, upon the first exposure (n = 13)and increased by 13 to 52% upon the second and thirdexposure 70 and 140min later in solvent controls (n=6).Compared with these controls, responses to high K+ andnoradrenaline were not changed 70 min after addition oftrypan blue 100lM, whereas responses to aP-MeATP weregreatly reduced (by 96 ± 2%; second exposure; n = 7;P<0.005). The response to aP-MeATP recovered to controllevels after 70 min of washout of trypan blue (third exposure;n = 7).The effect of trypan blue on the concentration-response

curve of x,4-MeATP was then determined. Increasing con-centrations of aj-MeATP elicited contractions with a max-imum of 24.3 ± 1.7 mN and an EC50 of 4.2 ± 0.9 JiM (n = 37;all first concentration-response curves pooled; open circles inFigures 1 and 3). A second curve after addition of solventwas very similar to the first one (solid circles in Figure la).Trypan blue (3.2-320 pM) caused two changes: it increasedthe maximal effect of a,-MeATP, and it shifted the con-centration-response curve progressively to the right (Figurelb-f). The greatest increase of the maximum (by 40%) was

obtained with trypan blue 100 jaM. A plot according to Arun-lakshana & Schild (1959) yielded a slope of 1.09, notsignificantly different from unity, and a pA2 value of 5.31(5.19 to 5.44, 95% confidence interval; KB 4.91M). It shouldbe noted, however, that the simultaneous increase of themaximum, here as well as in the cases of antagonism des-cribed below, leaves some doubt in the validity of the KBcalculations; the results obtained should be considered as'apparent' KB values.

Concentration-response curves of noradrenaline (0.1-1,000AM) were also determined using the protocol of Figure 1. TheEC50 was 6.7 ± 2.0 AM and the maximal contraction 22.3 +1.5 mN. Trypan blue (320 tM) changed neither the maximumnor the EC50 (n = 4 and 6 controls).An increase of the maximum response to a,43-MeATP,

40 F

30 -

zE 20 V

10 F

0

!111111 I II Rid I osf ItI*

1 10 100 1000

a, P-Methylene ATP (pM)

Figure 3 Effect of l-amino-8-naphthol-3,6-disulphonic acid (H-acid)on the concentration:response curve of a,,-methylene ATP. Increas-ing concentrations of a,p-methylene ATP were added every 30 minand washed out immediately after the contraction had peaked. Twoconcentration-response curves were determined in each tissue. H-acidwas added to the medium after completion of the first curve (0) andthe second curve was determined 60 min later (0). Abscissae,agonist concentration. Ordinates show contraction (mN). Means± s.e.mean from 4 experiments.

Trypan blue 32 pM

1 10 100 1000a I id i it. n al .i

1 10 100 1000

a, P-Methylene ATP (pM)

Figure 2 Effect of trypan blue on the concentration-response curve of a,P-methylene ATP in the presence of suramin. The mediumcontained suramin 32 JM from the beginning. Increasing concentrations of a,-methylene ATP were added every 30 min andwashed out immediately after the contraction had peaked. Two concentration-response curves were determined in each tissue.Solvent (a) or trypan blue 32 1AM (b) was added to the medium after completion of the first curve (0) and the second curve wasdetermined 60 min later (0). Abscissae, agonist concentration. Ordinates show contraction (mN). Means ± s.e.mean from 4experiments each.

a Solvent40 F

30F

b

E 20

10

0

352 R. BOLTMANN et al.

1 s

710 mN

0 . . a . .

1 3.2 10 32 100 320

Trypan blue (gM)

Figure 4 Effect of trypan blue on neurogenic contractions. Tissues were electrically stimulated by single pulses every 60 min (dots).Trypan blue was added at increasing concentrations immediately after the first and all following stimulations. Representativetracings from 5 experiments.

0

-

C

0

C

0b-

4-._

0

100

80

60

40

20

0

1 10

Antagonist (pM)100

Figure 5 Effect of trypan blue and suramin on the purinergic com-ponent of neurogenic contractions. The medium contained prazosin0.3 gM from the beginning. Tissues were electrically stimulated bysingle pulses every 60 min. Trypan blue (0) or suramin (@) wasadded at increasing concentrations immediately after the first and allfollowing stimulations. Abscissae, antagonist concentration. Ordin-ates show contraction as a percentage of the first, pre-antagonist,contraction, corrected for any change observed in controls (solvent).Means ± s.e.mean from 4 to 5 experiments.

similar to that caused by trypan blue, has also been reportedfor suramin (e.g. von Kugelgen et al., 1990). We studied theinteraction of trypan blue with suramin in order to find outwhether the mechanism might be the same. Suramin (32 jaM)alone, when added like trypan blue in the experiments ofFigure 1, shifted the concentration-response curve of a,,-MeATP to the right by a factor of 13 and increased themaximum by 31% (n = 4). A KB value of 2.6 gM was cal-culated from the shift. In subsequent experiments, suramin32 gM, 12.3 times KB, was present from the beginning, i.e.60 min before the first aP-MeATP concentration-responsecurve, and either solvent or trypan blue 32 gM, 6.5 times KB,was administered after the first curve. The maximal effect ofczji-MeATP was 31.9 ± 2.1 mN in the presence of suraminand the EC" was 41.9 ± 8.1 gM (first concentration-responsecurves; n = 8; both values are higher than in the absence ofsuramin, P< 0.005). A second concentration-response curve

in the presence of suramin alone, after addition of solvent,was very similar to the first one (solid circles in Figure 2a).When tested in the presence of suramin (32 f4M), trypan blue(32 I4M) failed to change the concentration-response curve ofa,-MeATP: the maximum was no longer increased, and themeasured shift to the right, corrected for the solvent controls,was only 1.1 fold (compare Figure 2a and b).

1-Amino-8-naphthol-3, 6-disulphonate (H-acid) is thenaphthalene derivative which in the trypan blue moleculecarries the sulphonic acid residues. At a concentration of1O mm, H-acid shifted the concentration-response curve ofoi,P-MeATP to the right and increased the maximum by 33%(Figure 3). From the shift to the right a KB value of 1.4 mMwas calculated.

Electrical stimulation of vasa deferentia with single pulsesevery 60 min elicited biphasic contractions (Figure 4) whichremained approximately constant in solvent controls (n = 5).Trypan blue reduced the rapid purinergic phase in aconcentration-dependent manner but did not alter the slowadrenergic phase (Figure 4). When the adrenergic phase wassuppressed by prazosin 0.3 tiM (Builtmann et al., 1993), con-tractions became monophasic and amounted to 8.0 ± 0.6 mN(first contraction; n = 15). These purinergic responses againremained approximately constant upon repeated stimulationin solvent controls (n = 6). Trypan blue progressively reducedand eventually abolished the purinergic contractions (IC5o44.9 ± 4.2 jsM), as did suramin (IC5o 9.9 ± 1.3 Jsm; Figure 5).

Discussion

a,P-Methylene ATP (a,P-MeATP) elicits contraction of therat vas deferens by activation of P2x-purinoceptors (Bult-mann & Starke, 1994b). Trypan blue reversibly inhibitedthese contractions. The lack of effect against noradrenalineand high K+ indicates some degree of selectivity, althoughblockade of other P2-purinoceptors (which also exist in ratvas deferens: Bultmann & Starke, 1994b) was not ruled out.The apparent affinity of trypan blue (KB 4.9 j.M) was slightlylower than that of suramin (KB 2.6 g1m; compare Hoyle et al.,1990; Leff et al., 1990; von Kugelgen et al., 1990). Theinteraction of trypan blue with suramin (Figure 2) is inaccord with an identical site of their antagonist action:

i

TRYPAN BLUE BLOCKS P2X-PURINOCEPTORS 353

trypan blue, when given at 6.5 times its KB, caused only a 1.1fold, non-significant, shift of the concentration-responsecurve of m,P-MeATP beyond the shift caused by suraminalone, given at 12.3 times its KB; from the law of mass action,the shift should be 1.5 fold, experimentally indistinguishablefrom the 1.1 found, if a,4-MeATP, suramin and trypan blueacted at the same site.Trypan blue produced an additional change: it increased

the maximum of the concentration-response curve of aP-MeATP. The same was observed, and has previously beenobserved (Hoyle et al., 1990; von Kugelgen et al., 1990;Blakeley et al., 1991; Mallard et al., 1992; Trezise et al., 1993;Venkova & Krier, 1993), for suramin. The potentiation bytrypan blue of the maximal response to ax,,-MeATP dis-appeared when suramin was present throughout, indicatingthat the potentiation mechanism was the same for bothantagonists. Since suramin (Figures 5 and 6 of von Kugelgenet al., 1990) and trypan blue enhanced the maximal effect ofot,P-MeATP over the same range of concentrations that shiftedthe x,,-MeATP curve to the right, it seems possible that theyproduced enhancement of the maximum and antagonismagainst aB-MeATP at the same site. For example, the an-tagonists might interfere with a process of fast desensitizationlimiting the contraction response to high agonist concentra-tions (von Kfigelgen et al., 1990). Another possibility is thatsuramin and trypan blue abolished an inherent relaxant effectof high concentrations of x,4-MeATP (von Kugelgen et al.,1990), possibly mediated by P2Y-purinoceptors (Blakeley etal., 1991; see also Boland et al., 1992).

Evans blue, previously shown to be a P2x antagonist in ratvas deferens (Bultmann & Starke, 1993), differs chemicallyfrom trypan blue in the position of the two sulphonic acidresidues at the terminal naphthalene rings. Pharmacologi-

cally, there were two differences. First, Schild analysis of theantagonism of Evans blue against x,P-MeATP yielded a slopedifferent from unity. Second, Evans blue enhanced responsesto noradrenaline and high K+ in addition to enhancing themaximum of the a,P-MeATP concentration-response curve.The position of the sulphonic acid residues, hence, deter-mines the mode of interaction with the P2x-purinoceptor aswell as the occurrence of non-specific effects.The basic pharmacological properties of trypan blue (MR

873) were retained in H-acid (MR 319), the naphthalenemoiety which carries the sulphonic acid residues and ofwhich two occur per molecule of trypan blue. However,H-acid was about 300 fold less potent than trypan blue,indicating that the proper positioning of two naphthalenedisulphonate groups in one molecule, connected by a centraltolidine residue, greatly enhances the affinity for P2X-purinoceptors.

Like suramin (Mallard et al., 1992) and DIDS (Bultmann& Starke, 1994a), trypan blue also acted selectively againstthe components of neurogenic contractions in rat vasdeferens: the purinergic, P2X-receptor-mediated component ofsingle pulse-evoked twitches was decreased, whereas theadrenergic component was unchanged, thus confirmingnoradrenaline-ATP co-transmission. The ICW values ofsuramin (9.9 fM) and trypan blue (44.9 ELM) were higher thantheir P2X-purinoceptor KB values (2.6 and 4.9 pM, respec-tively), possibly because very high concentrations of purin-ergic transmitter are generated upon single pulse stimulationin the vicinity of postjunctional P2x-purinoceptors.

This study was supported by the Deutsche Forschungsgemeinschaft(SFB 325). We thank Bayer for suramin.

References

ARUNLAKSHANA, 0. & SCHILD, H.O. (1959). Some quantitative usesof drug antagonists. Br. J. Pharmacol. Chemother., 14, 48-58.

BLAKELEY, A.G.H., BROCKBANK, J.E., KELLY, S.S. & PETERSEN,S.A. (1991). Effects of suramin on the concentration-responserelationship of ax,-methylene ATP on the mouse vas deferens. J.Auton. Pharmacol., 11, 45-49.

BOLAND, P., HIMPENS, B., VINCENT, M.F., GILLIS, J.M. & CAST-EELS, R. (1992). ATP activates Ph-contracting and P2y-relaxingpurinoceptors in the smooth muscle of mouse vas deferens. Br. J.Pharmacol., 107, 1152-1158.

BOLTMANN, R. & STARKE, K. (1993). Evans blue blocks P2x-purinoceptors in rat vas deferens. Naunyn-Schmied. Arch. Phar-macol., 348, 684-687.

BtLTMANN, R. & STARKE, K. (1994a). Blockade by 4,4'-diisothio-cyanatostilbene-2,2'-disulphonate (DIDS) of P2X-purinoceptors inrat vas deferens. Br. J. Pharmacol., 112, 650-654.

BOLTMANN, R. & STARKE, K. (1994b). P2-purinoceptor antagonistsdiscriminate three contraction-mediating receptors for ATP in ratvas deferens. Naunyn-Schmied. Arch. Pharmacol., 349, 74-80.

BOLTMANN, R., SZABO, B. & STARKE, K. (1993). Inhibition byethanol of contractions of rat vas deferens: no evidence forselective blockade of P2x-purinoceptors. Naunyn-Schmied. Arch.Pharmacol., 347, 527-533.

CUSACK, N.J. (1993). P2 Receptor: subclassification and structure-activity relationships. Drug Dev. Res., 28, 244-252.

DUNN, P.M. & BLAKELY, A.G.H. (1988). Suramin: a reversible P2-purinoceptor antagonist in the mouse vas deferens. Br. J. Phar-macol., 93, 243-245.

FUDER, H. & MUTH, U. (1993). ATP and endogenous agonistsinhibit evoked [3H]-noradrenaline release in rat iris via A, andP2y-like purinoceptors. Naunyn-Schmied. Arch. Pharmacol., 348,352-357.

FURCHGOTT, R.F. (1972). The classification of adrenoceptors(adrenergic receptors). An evaluation from the standpoint ofreceptor theory. In Catecholamines. Handbook of ExperimentalPharmacology, vol 33. ed. Blaschko, H. & Muscholl, E. pp.283-335. Berlin Heidelberg New York: Springer.

HOYLE, C.H.V., KNIGHT, G.E. & BURNSTOCK, G. (1990). Suraminantagonizes responses to P2-purinoceptor agonists and purinergicnerve stimulation in the guinea-pig urinary bladder and taeniacoli. Br. J. Pharmacol., 99, 617-621.

KENNEDY, C. (1990). P,- and P2-purinoceptor subtypes - an update.Arch. Int. Pharmacodyn., 303, 30-50.

KERR, D.I.B. & KRANTIS, A. (1979). A new class of ATP antagonist.Proc. Austr. Physiol. Pharmacol. Soc., 10, 156P.

LAMBRECHT, G., FRIEBE, T., GRIMM, U., WINDSCHEIF, U., BUN-GARDT, E., HILDEBRANDT, C., BAUMERT, H.G., SPATZ-KCM-BEL, G. & MUTSCHLER, E. (1992). PPADS, a novel functionallyselective antagonist of P2 purinoceptor-mediated responses. Eur.J. Pharmacol., 217, 217-219.

LEFF, P., WOOD, B.E. & O'CONNOR, S.E. (1990). Suramin is a slowly-equilibrating but competitive antagonist at P2X-receptors in therabbit isolated ear artery. Br. J. Pharmacol., 101, 645-649.

MALLARD, N., MARSHALL, R., SITHERS, A. & SPRIGGS, B. (1992).Suramin: a selective inhibitor of purinergic neurotransmission inrat isolated vas deferens. Eur. J. Pharmacol., 220, 1-10.

McLAREN, G.J., SNEDDON, P., KENNEDY, C., LAMBRECHT, G.,BURNSTOCK, G., MUTSCHLER, E., BAUMERT, H.G. & HOYLE,C.H.V. (1993). Investigation of a putative P2A-purinoceptorantagonist in guinea-pig isolated vas deferens. Br. J. Pharmacol.,109, l09P.

MCMILLIAN, M.K., SOLTOFF, S.P., LECHLEITER, J.D., CANTLEY,L.C. & TALAMO, B.R. (1988). Extracellular ATP increases freecytosolic calcium in rat parotid acinar cells. Biochem. J., 255,291-300.

MOTULSKY, H.J. & RANSNAS, L.A. (1987). Fitting curves to datausing nonlinear regression: a practical and nonmathematicalreview. FASEB J., 1, 365-374.

SOLTOFF, S.P., McMILLIAN, M.K. & TALAMO, B.R. (1989). Coomas-sie brilliant blue G is a more potent antagonist of P2 purinergicresponses than reactive blue 2 (cibacron blue 3GA) in rat parotidacinar cells. Biochem. Biophys. Res. Commun., 165, 1279-1285.

354 R. BULTMANN et al.

SOLTOFF, S.P., MCMILLIAN, M.K., TALAMO, B.R. & CANTLEY, L.C.(1993). Blockade of ATP binding site of P2 purinoceptors in ratparotid acinar cells by isothiocyanate compounds. Biochem. Phar-macol., 45, 1936-1940.

TREZISE, D.J., KENNEDY, I. & HUMPHREY, P.P.A. (1993). Charac-terization of purinoceptors mediating depolarization of ratisolated vagus nerve. Br. J. Pharmacol., 110, 1055-1060.

VENKOVA, K. & KRIER, J. (1993). Stimulation of lumbar sym-pathetic nerves evokes contractions of cat colon circular musclemediated by ATP and noradrenaline. Br. J. Pharmacol., 110,1260-1270.

VON KOGELGEN, I., BOLTMANN, R. & STARKE, K. (1990). Interac-tion of adenine nucleotides, UTP and suramin in mouse vasdeferens: suramin-sensitive and suramin-insensitive componentsin the contractile effect of ATP. Naunyn-Schmied. Arch. Phar-macol., 342, 198-205.

VON KOGELGEN, I., KURZ, K. & STARKE, K. (1994). P2-purino-ceptor-mediated autoinhibition of sympathetic transmitter releasein mouse and rat vas deferens. Naunyn-Schmied. Arch. Phar-macol., 349, 125-132.

WAUD, D.R. (1976). Analysis of dose-response relationships. InAdvances in General and Cellular Pharmacology, vol. 1. ed.Narahashi, T. & Bianchi, C.P. pp. 145-178. London: Plenum.

ZIGANSHIN, A.U., HOYLE, C.H.V., BO, X., LAMBRECHT, G., MUT-SCHLER, E., BAUMERT, H.G. & BURNSTOCK, G. (1993). PPADSselectively antagonizes P2x-purinoceptor-mediated responses inthe rabbit urinary bladder. Br. J. Pharmacol., 110, 1491-1495.

(Received March 3, 1994Revised April 27, 1994Accepted May 6, 1994)