135

Fundam Clin fharmacol(1992) 6,135-144 0 Elsevier, Paris

Contractile effect of a,P-methylene ATP on the guinea-pig isolated trachea ML Candenas 1

1,

P Devillier

1.2,

E Naline

1,

C Advenier

1*

Laboratoire de Pharmacologie, Faculti de Midecine Paris-Ouesf, 15, rue de l’f?cole de Midecine, F-75270 Paris Cedex 06, 2 Direction Mt!dicale, Roussel-Uclaj 102, Route de Noisy, F-93230 Romainville, France (Received 6 December 1991; accepted 28 February 1992)

Summary - The effects of a,pmethylene adenosine 5’-triphosphate (a,pmethylene ATP). 2-chloroadenosine and R-PIA (“5-(L-2phenylisopropy1)adenosine) and their interaction with dipyridamole, indomethacin, 8-phenyl-theophylline, diazepam and other agonists of central or peripheral benzodiazepine receptors were studied on the guinea-pig isolated trachea. a&methylene ATP exerted contractile effects on the guinea-pig isolated trachea; - log EC, and Em values were 8.86 f 0.19 and 31.3 f 2.3 (n = 31) (% vs acetylcholine 10-3 M)respectively. In comparison with other purinergic receptor agonists, the rank order of potency was: a,pmethylene ATP > 2-chloroadenosine > R-PIA. a,/3-niethylene ATP and 2-chloroadenosine had significantly (P< 0.05) greater efficacy (Em) than R-PIA. lndomethacin (3 x 10“ M) and 8-phenyltheophylline to 10-5 M)significantly reduced the contractile effect of 2chloroadenosine and R-PIA but did not affect a,pmethylene ATP-induced contraction. Conversely, dipyridamole significantly reduced (1W7M) or suppressed ( 1 W M ) the contractile effects of qpmethylene ATP whereas it only partially reduced (1W M)the contractile effects of high concentrations of 2-chloroadenosine or R-PIA. Diazepam potentiated the efficacy of a,pmethylene ATP. The Em (% vs acetylcholine 1C3M)values were 26.1 f 2.0 (n = 10) in control conditions and 45.9 f 4.6 (n= 5 ; f < 0.05) in the presence of diazepam 10-5 M.Diazepam did not modify the contractile effects of 2-chloroadenosine or R-PIA. Ro5-4864 (10-7 to 10-5 M),an agonist of peripheral benzodiazepine receptors, potentiated the contractile effects of gpmethylene ATP to the same extent as diazepam. Clonazepam, an agonist of central benzodiazepine receptors, did not modify these effects. Antagonists of central (flumazenil) or peripheral (RP 52028) benzodiazepine receptors had no influence on the interaction between diazepam or Ro5-4864 and a,/hethylene ATP. In conclusion, a,pmethylene ATP exem on guinea-pig isolated trachea a contractile effect which is not modified by indomethacin and 8-phenyltheophylline, but is reduced by dipyridamole. It is suggested that this effect might involve P,, receptor stimulation. gpmethylene ATP / purinergic receptors / peripheral benzodiazepine receptors / guinea-pig isolated trachea

Introduction

The purinergic receptors involved in the action of adenosine on the guinea-pig isolated trachea have classically been classified according to Burnstock (1978) and to Van Calker el rtl(1979) as type P,A2

* Correspondence and reprints

for relaxation (Brown and Collis, 1982; Farmer et al, 1988) and type P,A, for contraction (Ghai et al, 1987; Farmer et al, 1988). However, Burnstock and Kennedy (1985a) put the stress on P, receptors and showed on blood vessels that stimulation of P,, receptors may induce smooth muscle contrac-

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ML Candenas et a1

tion, whereas stimulation of P,, receptors may result in endothelium-dependent relaxation (Burnstock and Kennedy, 1985b). Since purines are involved in the regulation of airway smooth muscle tone, we have studied the effects of a$methylene adenosine 5'-triphosphate (@-methylene ATP), a P, receptor agonist (Burnstock and Kennedy, 198Sa), in an attempt to further characterize the purinergic receptors present in the guineapig trachea. We have compared the effects of a,p methylene ATP to those of 2-chloroadenosine and R-PIA (N6-(L-2 phenylisopropy1)-adenosine) which are both considered agonists to the P,A, adenosine receptor subtype for their contractile effect (Bruns et al, 1980; Paton, 1981; Collis, 1983; Ghai et al, 1987; Farmer er al, 1988). We then studied the modulatory effects of 8-phenyltheophylline, a P,-receptor antagonist (Brown and Collis, 1982; Farmer et al, 1988) on the action of the P, receptor agonist and the two P, receptor agonists on the guinea-pig isolated trachea. In addition, we studied the modulatory effects of other drugs: 1). indomethacin, since it has been shown that the contractile action of ATP and adenosine on the guinea-pig trachea is at least partly mediated through prostaglandin synthesis induction (Kamikawa and Shimo. 1976; Advenier et al, 1982); 2), dipyridamole, an inhibitor of adenosine uptake (Maguire and Satchell, 1979; Kolassa et al. 1990) potentiating the action of adenosine (Advenier et 01, 1982; Brown and Collis, 1982; Satchell and Smith, 1984); and 3), several benzodiazepine compounds that have also been shown to potentiate the effects of adenosine on the guinea-pig trachea (Advenier et ttl, 1990). Benzodiazepines have been shown to potentiate the relaxation induced by adenosine (10-5 to 10-3 M) on the guinea-pig isolated trachea. It was hypothesized that this effect involved a peripheral benzodiazepine receptor site because the rank order of potency of benzodiazepines was Ro5-4864 (an agonist of peripheral benzodiazepine receptors) > diazepam > clonazepam (an agonist of central benzodiazepine receptors) and because PK 11195 (or RP S2028), an antagonist of peripheral benzodiazepine receptors, specifically inhibits these effects (Advenier et al, 1990).

Materials and methods Tissue preparation Male guinea-pigs (25C350 g) were killed by a blow to the head and exsanguinated. The trachea was removed and placed in Krebs-Henseleit solution (composition in mM: NaCl 114, KCl 4.7. CaClz 2.5. KH2P0, 1.2, MgSO, 1.2. NaHCO, 25.0, glucose 11.7). Following removal of adhering fat and connective tissue, the trachea was cut into 6-43 rings. The rings were placed in parallel in 10-ml organ baths containing Krebs-Henseleit solution at 37OC, gassed with 95% 0, and 5% CO, and tied to transducers. The tissues were equilibrated under an initial tension of 1.80 g and washed every 15 min before the start of the experiments. After equilibration for 1.25 h, the resting tension was between 0.8 and 1.6 g. Under these conditions, responses to agonists were reproducible. Tensions were measured isometrically with Celaster strain gauges and were displayed on Linseis recorders. Protocols In ail experiments, tracheal rings were first contracted to maximal tension with acetylcholine l W M and then relaxed with theophylline (3 X 10-3 M).After 1 h 30 min rest, with washing every 15 min, 30-min pretreatments with saline or drugs were made and cumulative concentration-response curves to qpmethylene ATP, R-PIA or 2-chloroadenosine were obtained by increasing the concentration of these drugs at 10-15-min intervals in logarithmic increments. When the concentrationresponse curves were completed, acetylcholine 10-3 M was added to the bath to determine maximal contraction. Owing to the development of tachyphylaxis. only one concentration-response curve was made on each tracheal preparation. The contractions induced by qPmethylene ATP, RPIA and 2-chloro-adenosine were expressed as a percentage of the maximal effect of acetylcholine 1 6 3 M. The log EC,, values (defined as the negative log of the drug concentration that caused 50% of maximal effect) were derived from the log concentration-effect curves. These values were evaluated graphically from each experiment.

Statistical analysis of results Statistical analysis of the results was performed using a 2-way analysis of variance and the Student's t-test for

a.pMethylene ATP and guinea-pig isolated trachea

137

paired or unpaired data. All values in the text, tables and figures are expressed as mean f SEM; n is the number of animals.

produced no direct effect on basal tone or on responses to adenosine.

Drugs

Results

The drugs used were: 2-chloroadenosine. R-PIA (N6(L-2-phenylisopropyl)-adenosine),gPmethylene ATP (dilithium salt), indomethacin and 8-phenyl-theophylline (Sigma, St Louis, USA), dipyridamole (Boehringer-Ingelheim. Reims. France), theophylline sodium anisate (Bruneau. Paris, France), diazepam, Ro5-4864 (7-chlor-5-(4-chlorphenyl)-1.3-dih ydro-1-methyl- 2H1.4-benzodiazepine-2-0n). flumazenil (ethyl 8-fluoroS,6-dihydro-5-methy1-6-0~0-4H-imidazo [ 1.5-a] [ 1.41 benzodiazepine-3-carboxylate)(Roche. Basel, Switzerland), RP 52028 (or PK 11195) (N-methyl-N-(methyl-1propyl) (chloro-2 pheny1)- 1 isoquinoleine-3 carboxamide) (RhBne-Poulenc. Vitry-sur-Seine. France). Theophylline was used as proprietary injectable solution (Theophylline Bruneau); dipyridamole and indomethacin were dissolved daily in ethanol and the solutions were further diluted with Krebs solution. Diazepam and the other benzodiazepines were dissolved in sulfolane (tetramethylene sulfone; Aldrich Chemie, Steinheim. Germany). The appropriate concentration of drugs was added to the organ baths so that at no time did the volume of sulfolane or ethanol exceed 0.04 ml (0.4% by volume). The solvents in such maximal concentrations

Contractile effects of a$-methylene ATP, 2-chloroadenosine and R-PIA on guinea-pig isolated trachea

h

0

50

a

z

s

1 0 9 8 7 6 a , b-me ATP(-IogM)

a,P-Methylene ATP, 2-chloroadenosine and R-PIA exerted a contractile effect on the guineapig isolated trachea (fig 1; control values). The -log EC,, values (table I) showed that the rank order of potency of these substances was a,Pmethylene ATP 2 2-chloroadenosine 2 R-PIA, a,P-methylene ATP and 2-chloroadenosine had significantly ( P c 0.05) ( n = 31 and 18) greater efficacy (Em=)than R-PIA (table I). Influence of indomethacin, dipyridamole and 8phenyltheophylline on the contractile effects of a,p - met hy 1e n e A TP, 2 - ch 1o ro a de n o s i n e and R-PIA Indomethacin (3 x 10-6 M) significantly reduced the contractile effect of 2-chloroadenosine and

t

501

T

1

0

9

8

7

6

2-CHLORO ADENOSINE (-1ogM)

1 1 1 0 9 8 7 R - PIA (-log M )

6

Fig 1. Effects of indornethacin (3 x 10" M) on the contractile concentration-response curves to a,pmethylene ATF', 2-chloroadenosine and R-PIA on the guinea-pig isolated trachea. Control ( 0 ) ;indornethacin 3 x 10" M (m). Data are the means f SEM of 4-6 experiments. Significant differences from control are (')P < 0.05; (t)P< 0.01.

138

ML Candenas et a1

R-PIA on the isolated guinea-pig trachea but did not significantly modify the a:/?-rnethylene ATPinduced contraction (fig 1). Conversely, dipyridaM)or suppressed (1WM) the mole reduced (le7 contractile effects of a,P-methylene ATP, but at M it only reduced the the concentration of effects of 2-chloroadenosine or R-PIA (fig 2). Finally, 8-phenyltheophylline significantly suppressed the maximal responses to 2-chloroadenosine and R-PIA without affecting the response to &/3-methylene ATP (fig 3).

Table I. -Log EC, and maximal effect (Em) of @-methylene ATP, 2-chloroadenosine and R-PIA on the guinea-pig isolated trachea.

Em7

Treatment n

-log

EC,,

E(%vsACh

(mg)

10;'

M)

a.pmethylene ATP 31 8.86 f 0.19 798 f 86 31.3 f 2.3* 2-Chloroadenosine 18 8.74 f 0.16 615 f 79 32.1 f 2.8* R-PIA 21 8.21 fO.15 462f46 23.1 f 2 . 1 n = number of experiments; R-PIA ( P < 0.05).

* significantly

different from

Influence of diazepam, Ro5-4864 and clonazepam on the contractile effects of a$-methylene ATP, 2-chloroadenosine and R-PIA

Diazepam ( M) increased the efficacy of a,P-methylene ATP as shown by the increase observed in Em, (table 11; fig 4) but it had no effect on the potency of a,&methylene ATP since the -log EC,, values of these substances remained unmodified (table 11). Diazepam did not significantly modify the concentration-responsecurves to 2-chloroadenosine or R-PIA. Ro5-4864, a structural analogue of diazepam, which specifically stimulates peripheral benzodiazepine receptors, increased a,/3-methylene ATP effects in a manner similar to diazepam whereas clonazepam. which stimulates central benzodiazepine receptors, did not significantly modify the contractile effect of &/3-methyleneATP (fig 5). Influence of benzodiazepine receptor antagonists on the potentiution of the effects of a,P-methylene ATP by diazepam and Ro5-4864

In the presence or the absence of diazepam M) or RoS-4864 M), the contractile effects of a,P-methylene ATP (fig 6) were not

25

* I

0

0

2 V 0

9 8 7 6 5 a , P - m eATP (-1ogM)

1 0 9 8 7 6 2-CHLOROADENOSINE (-log M )

1 1 1 0 9 8 7 R-PIA (-lOgM)

6

Fig 2. Effects of dipyridamole on the contractile concentration-response curves to a,/.%methyleneATP, 2-chloroadenosine and R-PIA on the guinea-pig isolated trachea. Control (0);dipyridamole 10-7 M (m) and 1W M (0).Data are the means f SEM of 4-6 experiments. Significant differences from control are (*)P< 0.05;W < 0.01.

139

u.PMethylene ATF’ and guinea-pig isolated trachea

z

-E

50-

50 -

50 -

25 -

25

v)

0-

I

-

0-

Fig 3. Effects of 8-phenyltheophylline (lo-’ to M) on the contractile concentration-response curves to a,gmethylene AT”, 2-chloroadenosine and R-PIA on the guinea-pig isolated trachea. Control ( 0 ) ;8-phenyltheophylline: 10-7 M (0).10-6 M (M), M (0)Data . are the means f SEM of 4-6 experiments. Significant differences from control are (*)Pc 0.05;(W < 0.01.

Table 11. Effects of diazepam, R05-4864 and clonazepani on the contractile response of the guinea-pig isolated trachea to a&methylene ATP. Trentmcnt

u,&tethylenc ATP

Em -log EC,,

n

(% versus ACh

10”M) Control Diazepam

Ro5-4864

Clonazepam

10 IO-’M 6 1 0 4 ~ a 1 0 - 5 ~ s 1 0 - 7 ~ s 10“M 5 10-sM 6 104M 5 1 0 - 5 ~

s

104M

5

= number of experiments; control (P< 0.05) n

8.45 f 0.38 8.38f0.17 8.33 f 0 . 1 4 8.30 f 0.25 8.51 f 0.27 8.68 f 0.22 a.48 f 0.12 8.43 f 0.18 8.45 f 0.10 8.21 f 0.44

*

26.1 f 2.0 22.1 f 5.0 28.8 f 3.5 45.9 f 4.6+ 24.0 f 4.7 30.4 f 3.2 42.2 f 4.9* 25.1 f 4 . 8 32.5 f 4.7 36.5 f 6.9

= significant difference from

modified by flumazenil (10-5 M), an antagonist of central benzodiazepine receptors, or by RP 52028 (or PK 11195) (10-5 M), an antagonist of peripheral benzodiazepine receptors.

Discussion and Conclusion

a;P-methylene ATP exerted a contractile effect on the guinea-pig isolated trachea under resting tension. The contractile effect of cqPmethylene ATP was, in terms of potency, similar to those exerted by 2-chloroadenosine and R-PIA, but cqpmethylene ATP and 2-chloroadenosine had significantly greater efficacy (Emm)than R-PIA (N6-(L-2-phenylisopropy1)-adenosine. The previously reported contractile effect of adenosine (Adevnier el al, 1982; Fredholm et al, 1979; Satchel and Smith, 1984), or R-PIA and 2-chloroadenosine (Caparrotta et al, 1984; Ghai et al, 1987; Farmer et al, 1988) on the same preparation has been attributed to stimulation of P,A, receptors associated with a decrease in CAMP(Burnstock, 1978; Van Calker et al, 1979: Bumstock and Buckley, 1985; Farmer et al, 1988). Our results show that qpmethylene ATP, a P, receptor agonist (Burnstock and Kennedy, 1985a), induces contraction of the tracheal smooth muscle as do P,A, receptor agonists. However, the characteristics of the contractile activity of a,P-methylene ATP, were different from those of P,A, agonists. In fact, the contractile effects of PIAl agonists were reduced in the pres-

140

ML Candenas et nl

T

I

9 W

I

I

I

I

. -

I

1 0 9 8 7 2-CHLORO ADENOSINE (-logM)

8 7 6 5 a , 0 -me ATP ( - l o g M )

1 1 1 0 9 8 7 R-PIA (-IogM)

6

Fig 4. Effects of dazeparn on the contractile concentration-response curves to a,pmethylene A", 2-chloroadenosine and R-PIA on the guinea-pig isolated trachea. Control ( 0 ) ;diazepam 10-' M (O), M (m), lC5M (0)and lo4 M (X). Data are the means f SEM of 4-8 experiments. significant differences from control are (*)P< 0.05.

C L 0 NA Z E PA M

RO 5-4864

50 T T

4 I U

I

9 8 a , p - m e ATP (-log M )

I

I

1

7

6

5

Fig 5. Effects of Ro5-4684 and clonazepam on the contractile concentration-response curves to a,Pmethylene A" on the guinea-pig isolated trachea. Control ( 0 ) ;Ro5-4864 lo-' M @), 10-6 M (01, le5M (A);clonazepam 10-5 M (m) and lo4 M (A). Data are the means f SEM of 5-7 experiments. Significant differences from control are (')P < 0.05.

ence of indomethacin, as previously reported in the literature (Kamikawa and Shimo. 1976; Advenier et al, 1982) and also in the presence of 8-phenyltheophylline, a non-specific P1-receptor antagonist (Farmer et a l , 1988). B y contrast, the

contractile effect of a,P-methylene ATP was unmodified by these 2 drugs. With regard to indomethacin, our results are in agreement with those obtained in the guinea-pig isolated tracheal chain where ATP stimulates prostaglandin synthesis

141

u,PMethylene ATP and guinea-pig isolated trachea

a

b 50

T

-

0 9

8

7

s/sA

6

9

8

7

6

0 -= 6 9 8 a , p-meATP(-logM)

7

6

d

C

5c ,T

-

9

8

7

Fig 6. Effects of flumazenil ( a h ) and RF' 52028 (PK 1 1 195)(c,d) on the potentiating effects of diazepam (a,c) and Ro5-4684(b,d) on the a&methylene ATP-induced contractions of the guinea-pig isolated trachea. Control concentration-response curves ( 0 ) ;flumazenil 10-5 M ( 0 ) ;diazepam 1 0 - 5 M (m), flumazenil 1 0 - 5 M +diazepam 10-5 M (0). RP 52028 10-5 M (0);RP 52028 1 0 - 5 M +diazepam 10-5 M (A); Ro5-4864 10-5 M (v);RoS-4864 1 0 - 4 M + flumazenil 10-5 M (V);RP 52028 10-5 M + Ro5-4864I P 5 M (+). Data are the means f SEM of 4 4 experiments.

while the methylene isosteres of ATP, Gp-methylene ATP and p, ymethylene ATP, are unable to do so (Brown and Burnstock, 1981). Moreover, dipyridamole was more potent in suppressing the contractile activity of Gpmethylene ATP than in suppressing the contractile activity of P,Al agonists. In addition, diazepam increased the contractile respon-

se to aPmethylene ATP whereas it has no effect on the responses induced by the PIAl agonists. It therefore clearly appears that alp-methylene ATP induces contraction of the guinea-pig isolated trachea through the activation of a receptor that is different from the PIAl receptor. The adenosine receptor subtype involved in the effect of

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ML Candenas et nl

a;Pmethylene ATP on the guinea-pig isolated trachea seems to be a P,, receptor, since, in addition to its strong P,, receptor action (Hopwood and Burnstock, 1987), a,/?-methylene ATP does not activate P, receptor in guinea-pig isolated trachea (Brown and Burnstock, 1981). Moreover, activation of P,, receptors results in direct contraction of numerous tissues like the rat femoral or coronary arteries and mesenteric bed (Kennedy et al, 1985; Burnstock and Warland, 1987; Hopwood and Burnstock, 1987; Ralevic and Burnstock, 1988). the rat and human pulmonary arteries and veins (Liu et al, 1989a, b), the rat vas deferens (Taylor el al, 1983). the rabbit isolated ear artery (O’Connor et al, 1990) and isolated strips of human urinary bladder detrusor muscle (Hoyle el n l , 1989), whereas activation of P2y receptors causes relaxation of the taenia coli or indirect endothelial celldependent relaxation of the vasculature (Burnstock and Kennedy, 198Sa). We have previously suggested that diazepam and other benzodiazepines may act, like dipyridamole, by inhibiting adenosine uptake and increasing therefore extracellular concentrations of adenosine (Advenier et cd, 1990; Candenas et al, 1991), as also reported in studies in the guinea-pig isolated atrium (Clanachan and Marshall, 1980; Kenakin, 1982). Indeed, we have demonstrated that the P,A,-mediated relaxant effect of adenosine is not only potentiated by diazepam and Ro54864 ( l W to 10-4 M) but also by dipyridamole with no additive effects of the compounds (Advenier et al, 1990; Candenas et u l , 1991). With regard to the contraction of the guinea-pig isolated trachea induced by aPmethylene ATP, the opposite effects of dipyridamole and diazepam or Ro54864 suggest a different mode of action for the 2 classes of compounds. This is supported by the report by Woo et nl (1977) that ATP is not transported in isolated frog skeletal muscle, suggesting that dipyridamole and benzodiazepines might not act by inhibiting a,/?-methylene ATP uptake. A different mode of action is also strengthened by the different pharmacological profile of the benzodiazepine-binding sites involved respectively in the increase in the relaxant effect of adenosine and

in the contractile effect of a;Pmethylene ATP. In fact, our results obtained with benzodiazepine agonists suggest that peripheral benzodiazepine receptors might be involved since the effects of aPmethylene ATP were potentiated by Ro5-4864 (parachlorodiazepam), a peripheral receptor agonist chemically related to the benzodiazepines (Marangos et al, 1982). and since clonazepam, a specific agonist of central benzodiazepine receptors, did not potentiate the contractile effects of a,D-methylene ATP. However, these data were not confirmed by experiments conducted with cenual and peripheral benzodiazepine receptor antagonists. As expected, flumazenil, a central benzodiazepine receptor antagonist (Brogen and Goa. 1988). had no influence on the potentiation of the a;Pmethylene ATP-induced contraction by diazepam or Ro54864, but contrary to expectation neither did RP 52028 (PK 11 195), a peripheral benzodiazepine receptor antagonist (Mestre er al. 1985). On the other hand. the same compound RP 52028 has been reported to inhibit the potentiation by diazepam of the relaxant effect of adenosine on the guinea-pig isolated trachea (Advenier et al, 1990). Therefore, the type of the benzodiazepine site involved in the potentiation of the a@-methylene ATP induced contraction of the guinea-pig isolated trachea as well as the mode of action of dipyridamole and the benzodiazepine compounds need further investigation to be fully characterized. In conclusion, our results show that a,P-methylene ATP can induce a contraction of the guineapig isolated trachea which is inhibited by dipyridamole, potentiated by diazepam or Ro5-4864 and unaffected by indomethacin or 8-phenyltheophilline. It is suggested that this effect might involve P,, receptors.

Acknowledgments This work was supported by a grant from the scientific council of the Facult6 de Mkdecine Paris-Ouest. ML Candenas was the recipient of a fellowship from Ministerio de Educacidn y Ciencia, Spain.

a,bMethylene ATP and guinea-pig isolated trachea

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