European Journal of Pharmacology, 214 (1992) 67-74 (~_~ ' 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$(15.0(I
67
EJP 52399
Influence of benzodiazepines on the response of the guinea-pig isolated trachea to the contractile action of adenosine P h i l i p p e Devillier, M a r i a - L u z C a n d e n a s , E m m a n u e l N a l i n e a n d C h a r l e s A d v e n i e r Laboratoire de Pharmacolo~,,ie, Faculu; de M&h'cine Paris-Ouest, 15 Rue de l'Ecoh, de Mddecine, F-75270 P.ris ('edex 00, France Received 10 January 1992. accepted 28 January. 1992
The effects of diazepam and other agonists of central or peripheral benzodiazepinc receptors were studied on thc contractile action of adenosine, 2-chloroadenosine and R-PIA (N6-(L-2-phenylisopropyl)-adenosine) on the guinea-pig isolated trachea. These effects were compared to those of dipyridamole. Diazepam 10 7 to 10 s M potentiated the efficacy of adenosine; the maximal contractile effect of adenosine (% vs. acetylcholine 10 -3 M) was 20.4 + 4.2 (n = 21) in control conditions and 45.5 +_3.7 (n = 6; P < 0.001) in the presence of diazepam 10 5 M. Ro5-4864 (10 7 to 10 5 M) or alpidem (10 v to 10 5 M), both agonists of peripheral benzodiazepine receptors, potentiated the contractile effects of adenosine to the same extent as diazepam. Clonazepam and zopiclone, both agonists of central benzodiazepine receptors, did not modify these effects. Antagonists of central (flumazenil) or peripheral (RP 52028) benzodiazepinc receptors had no influence on the interaction between diazepam or Ro5-4864 and adenosine. Conversely, dipyridamole significantly reduced (10 7 M) or suppressed (10 ~' M) the contractile effects of adenosine. The contractile effects of 2-chloroadenosine and R-PIA were weakly affected in presence of high concentrations of diazepam and dipyridamole. Epithelium removal potentiated the contractile effect of adenosine on the guinea-pig isolated trachea and increased the potentiating cffect of diazepam. It is concluded that benzodiazepines and related compounds can potentiate the contractile effect of adenosinc on the guinea-pig isolated trachea through the activation of a peripheral receptor for the benzodiazepines and the resulting inhibition of adenosine uptakc. Adenosinc; Purinoceptors; Benzodiazepine receptors (peripheral): Trachea (guinea-pig, isolated)
1. Introduction Non-neuronal high-affinity binding sites for benzodiazepines have been found in several peripheral tissues (Verma and Snyder, 1989), including bronchial smooth muscle (Mak and Barnes, 1991), and have been designated as 'peripheral' type benzodiazepine receptors. On the guinea-pig isolated trachea, benzodiazepines have been shown to potentiate the relaxation induced by adenosine (10 5 to 10 - 3 M ) by inhibition of adenosine uptake a n d / o r metabolism (Advenier et al., 1990). Although this effect was observed at concentrations above 1 /xM, it was hypothesized to involve a peripheral benzodiazepine receptor site because the rank order of potency of benzodiazepines was Ro5-4864 (parachlorodiazepam, a specific agonist of peripheral benzodiazepine receptors) > diazepam > clonazepam (an agonist of central benzodiazepine receptors) and because RP 52028 (or PK 11195), an antagonist of
Correspo,~dence to: C. Advenier. Facult& de M6decine Paris-Ouest, 15 Rue de l'Ecole de M6decine, F-75270 Paris Cedex 06. France.
peripheral benzodiazepine receptors, specifically inhibits these effects (Advenier et al., 1990). In contrast to its relaxant effect, adenosine induced contractions of the guinea-pig isolated trachea at low concentrations (10 -~ to 10 -5 M). The purpose of the present study was therefore to investigate the effects of three benzodiazepines (diazepam, Ro5-4864 and clonazepam) and of two substances chemically different from the benzodiazepines, but that behave as agonists of either central (zopiclone) (Blanchard et al., 1979, 1983) or peripheral (alpidem) (Langer and Arbilla, 1988) benzodiazepine receptors, on the contractile effect of adenosine on the guinea-pig isolated tracheal smooth muscle. The effects of these substances were compared with those of dipyridamole, a compound which inhibits adenosine uptake (Maguire and Satchell, 1979; Kolassa et al., 1970) and, similarly but not additively to the benzodiazepines, potentiates the relaxant effect of adenosine (Advenier et al., 1990; Candenas et al., 1991). Furthermore, in order to better characterize the type of adenosine receptor and the mechanism of potentiation by the benzodiazepine receptor agonists and dipyridamole, we studied the effects of these corn-
68
pounds on the contractile effects of two agonists, R-PIA (N6-(L-2-phenylisopropyl)-adenosine) and 2-chloroadenosine, which are more selective for the A~ adenosine receptor subtype and are not substrates for the uptake mechanism. Finally, since the epithelium of guinea-pig isolated trachea may modify the effccts of adenosine (Holroyde, 1986, Farmer et al., 1986; Advenier et al., 1988), we evaluated the influence of epithelium removal on the interaction between adenosine and benzodiazepines.
2. Materials and methods
2.1. Tissue preparation Male guinea-pigs (250-350 g) were killed by a blow' to the head and exsanguinated. The trachea was removed and placed in K r e b s - H e n s e l e i t solution (composition raM: NaCI 114, KCI 4.7, CaCI 2 2.5, K H 2 P O 4 1.2, MgSO 4 1.2, N a H C O s 25.0, glucose 11.7). Following removal of adhering fat and connective tissue the trachea was cut into six to eight rings. The rings were placed in parallel in 10-ml organ baths containing K r e b s - H e n s e l e i t solution at 37°C, gassed with 95% O , and 5% CO~ and tied to transducers. The tissues were equilibrated under an initial tension of 1.80 g and washed every 15 min up to the start of the experiments. After equilibration for 1.25 h, the resting tension was between 0.8 and 1.6 g. The responses to agonists were reproducible under these conditions. Tensions were measured isometrically with Celaster strain gauges and amplifiers and were displayed on Linseis recorders. In some experiments the epithelium was removed by gently rubbing the luminal surface (over both smooth muscle and cartilage areas) with a cotton-tipped applicator (Advenier et al., 1988: Devillier et al., 1988). The efficiency of this method of epithelium removal had been verified both histologically and pharmacologically (Devillier et al., 1988).
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2.2. Proto
67
EJP 52399
Influence of benzodiazepines on the response of the guinea-pig isolated trachea to the contractile action of adenosine P h i l i p p e Devillier, M a r i a - L u z C a n d e n a s , E m m a n u e l N a l i n e a n d C h a r l e s A d v e n i e r Laboratoire de Pharmacolo~,,ie, Faculu; de M&h'cine Paris-Ouest, 15 Rue de l'Ecoh, de Mddecine, F-75270 P.ris ('edex 00, France Received 10 January 1992. accepted 28 January. 1992
The effects of diazepam and other agonists of central or peripheral benzodiazepinc receptors were studied on thc contractile action of adenosine, 2-chloroadenosine and R-PIA (N6-(L-2-phenylisopropyl)-adenosine) on the guinea-pig isolated trachea. These effects were compared to those of dipyridamole. Diazepam 10 7 to 10 s M potentiated the efficacy of adenosine; the maximal contractile effect of adenosine (% vs. acetylcholine 10 -3 M) was 20.4 + 4.2 (n = 21) in control conditions and 45.5 +_3.7 (n = 6; P < 0.001) in the presence of diazepam 10 5 M. Ro5-4864 (10 7 to 10 5 M) or alpidem (10 v to 10 5 M), both agonists of peripheral benzodiazepine receptors, potentiated the contractile effects of adenosine to the same extent as diazepam. Clonazepam and zopiclone, both agonists of central benzodiazepine receptors, did not modify these effects. Antagonists of central (flumazenil) or peripheral (RP 52028) benzodiazepinc receptors had no influence on the interaction between diazepam or Ro5-4864 and adenosine. Conversely, dipyridamole significantly reduced (10 7 M) or suppressed (10 ~' M) the contractile effects of adenosine. The contractile effects of 2-chloroadenosine and R-PIA were weakly affected in presence of high concentrations of diazepam and dipyridamole. Epithelium removal potentiated the contractile effect of adenosine on the guinea-pig isolated trachea and increased the potentiating cffect of diazepam. It is concluded that benzodiazepines and related compounds can potentiate the contractile effect of adenosinc on the guinea-pig isolated trachea through the activation of a peripheral receptor for the benzodiazepines and the resulting inhibition of adenosine uptakc. Adenosinc; Purinoceptors; Benzodiazepine receptors (peripheral): Trachea (guinea-pig, isolated)
1. Introduction Non-neuronal high-affinity binding sites for benzodiazepines have been found in several peripheral tissues (Verma and Snyder, 1989), including bronchial smooth muscle (Mak and Barnes, 1991), and have been designated as 'peripheral' type benzodiazepine receptors. On the guinea-pig isolated trachea, benzodiazepines have been shown to potentiate the relaxation induced by adenosine (10 5 to 10 - 3 M ) by inhibition of adenosine uptake a n d / o r metabolism (Advenier et al., 1990). Although this effect was observed at concentrations above 1 /xM, it was hypothesized to involve a peripheral benzodiazepine receptor site because the rank order of potency of benzodiazepines was Ro5-4864 (parachlorodiazepam, a specific agonist of peripheral benzodiazepine receptors) > diazepam > clonazepam (an agonist of central benzodiazepine receptors) and because RP 52028 (or PK 11195), an antagonist of
Correspo,~dence to: C. Advenier. Facult& de M6decine Paris-Ouest, 15 Rue de l'Ecole de M6decine, F-75270 Paris Cedex 06. France.
peripheral benzodiazepine receptors, specifically inhibits these effects (Advenier et al., 1990). In contrast to its relaxant effect, adenosine induced contractions of the guinea-pig isolated trachea at low concentrations (10 -~ to 10 -5 M). The purpose of the present study was therefore to investigate the effects of three benzodiazepines (diazepam, Ro5-4864 and clonazepam) and of two substances chemically different from the benzodiazepines, but that behave as agonists of either central (zopiclone) (Blanchard et al., 1979, 1983) or peripheral (alpidem) (Langer and Arbilla, 1988) benzodiazepine receptors, on the contractile effect of adenosine on the guinea-pig isolated tracheal smooth muscle. The effects of these substances were compared with those of dipyridamole, a compound which inhibits adenosine uptake (Maguire and Satchell, 1979; Kolassa et al., 1970) and, similarly but not additively to the benzodiazepines, potentiates the relaxant effect of adenosine (Advenier et al., 1990; Candenas et al., 1991). Furthermore, in order to better characterize the type of adenosine receptor and the mechanism of potentiation by the benzodiazepine receptor agonists and dipyridamole, we studied the effects of these corn-
68
pounds on the contractile effects of two agonists, R-PIA (N6-(L-2-phenylisopropyl)-adenosine) and 2-chloroadenosine, which are more selective for the A~ adenosine receptor subtype and are not substrates for the uptake mechanism. Finally, since the epithelium of guinea-pig isolated trachea may modify the effccts of adenosine (Holroyde, 1986, Farmer et al., 1986; Advenier et al., 1988), we evaluated the influence of epithelium removal on the interaction between adenosine and benzodiazepines.
2. Materials and methods
2.1. Tissue preparation Male guinea-pigs (250-350 g) were killed by a blow' to the head and exsanguinated. The trachea was removed and placed in K r e b s - H e n s e l e i t solution (composition raM: NaCI 114, KCI 4.7, CaCI 2 2.5, K H 2 P O 4 1.2, MgSO 4 1.2, N a H C O s 25.0, glucose 11.7). Following removal of adhering fat and connective tissue the trachea was cut into six to eight rings. The rings were placed in parallel in 10-ml organ baths containing K r e b s - H e n s e l e i t solution at 37°C, gassed with 95% O , and 5% CO~ and tied to transducers. The tissues were equilibrated under an initial tension of 1.80 g and washed every 15 min up to the start of the experiments. After equilibration for 1.25 h, the resting tension was between 0.8 and 1.6 g. The responses to agonists were reproducible under these conditions. Tensions were measured isometrically with Celaster strain gauges and amplifiers and were displayed on Linseis recorders. In some experiments the epithelium was removed by gently rubbing the luminal surface (over both smooth muscle and cartilage areas) with a cotton-tipped applicator (Advenier et al., 1988: Devillier et al., 1988). The efficiency of this method of epithelium removal had been verified both histologically and pharmacologically (Devillier et al., 1988).
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2.2. Proto
