Rabbit isolated vas deferens possess A1 and A2 adenosine receptors

Gen. Pharmac. Vol. 23, No. 4, pp. 631-635, 1992 Printed in Great Britain. All rights reserved

0306-3623/92 $5.00 + 0.00 Copyright © 1992 Pergamon Press Ltd

RABBIT ISOLATED VAS DEFERENS POSSESS Al AND ADENOSINE RECEPTORS

A2

AHMAD REZA DEHPOUR* and FATEMEHVAZIFEHSHENAS Department of Pharmacology, Faculty of Medicine, Tehran University of Medical Sciences, P.O. Box 13145-784, Tehran, Iran (Received 6 January 1992)

Abstract--1. The effects of adenosine, 5'-N-ethylcarboxamidoadenosine (NECA), 2-chloroadenosine, N6-phenylisopropyladenosine (L-PIA and t>-PIA) and N6-cyclohexyladenosine (CHA) were examined on the rabbit isolated vas deferens. 2. All the analogues in a concentration-dependent manner inhibited contractile response to electrical stimulation. 3. 8-Phenyltheophylline caused a rightward shift of all the adenosine and its analogues' concentration-response curves. 4. The order of potency for the adenosine and its analogues on the rabbit isolated vas deferens was: CHA = NECA > L-PIA > 2-chloroadenosine > D-PIA > adenosine. 5. It is concluded that CHA, NECA, L-PIA, 2-chloroadenosine, D-PIA and adenosine mediate their inhibitory effects on the rabbit isolated muscle via both A~ and A 2 adenosine receptors.

INTRODUCTION Adenosine receptors have been classified as A1 or A2 depending on whether activation produces inhibition or stimulation of adenylyl cyclase (Van Calker et al., 1979). On the other hand, the term Ri-receptor is used corresponding to AI and Ra-receptor corresponding to A2 (Londos et al., 1980). It has been claimed that the A~- and A2-receptors may be differentiated by the use o f N6- and Y-substituted adenosine analogues (Londos et al., 1990). The A~-site prefer N6-substituted compounds such as L-N6-phenylisopropyladenosine (LPIA) and N6-cyclohexyladenosine (CHA), while at the A2-site 5'-substituted analogues such as 5'-N-ethylcarboxamidoadenosine ( N E C A ) are more potent. In addition, at the A~-site, a high degree of stereoselectivity for L-PIA over its isomer (D-PIA) is displayed. However, not all adenosine receptors are linked to this effector system, and a further classification based on the order of potency adenosine analogues has been proposed (Stone, 1985; Burnstock and Bluckley, 1985). In general, the order of potency at A~ receptors is C H A > L - P I A > N E C A and at A2 receptors N E C A > L-PIA > C H A . In the present study we examined the effects of adenosine, D-PIA, 2-chloroadenosine, L-PIA, C H A and N E C A on the rabbit isolated vas deferens by determination ofthese c o m p o u n d s dose-response curves and shifting curves in the presence of 8-phenyltheophylline, an adenosine receptor antagonist. MATERIALS AND METHODS

Preparation Rabbits were killed by a lethal injection of air (10-15 ml) into an ear vein. The vasa deferentia were removed and

placed in Krebs-bicarbonate buffer of the following composition (mM): NaCI, 118.00; KCI, 4.75; CAC12,2.54; KH2PO4, 1.19; NaHCO 3, 25.00; and glucose, 11.00 (Oka et al., 1981). Each vas deferens was placed in an organ bath containing Krebs-bicarbonate buffer maintained at 36°C and aerated with 95% 02 and 5% CO 2. The preparations were attached to Grass FTI0C force transducers and glass supports, 500 mg resting force (Oka et al., 1981) was maintained on each preparation, and force generation was recorded on a Grass model 79C polygraph. Preparations were incubated for 60 min prior to electrical stimulation. The vasa deferentia were stimulated electrically with stimulator of Bioscience 200. Nerve endings were activated by stimulating platinum ring electrodes around the preparations with supramaximal voltage (100V at the electrodes) for a pulse duration of 1 msec at a stimulation frequency of 0.1 Hz (Oka et al., 1981). Effects o f adenosine and its analogues on neurotransmission Adenosine and its analogues were administered cumulatively to the preparations every 5 rain. Adenosine at the concentrations of 0.5-500/z M, NECA at the concentrations of 0.005-0.5 ~uM, 2-chloroadenosine at the concentrations of 0.05-5/~ M, CHA at the concentrations of 0.005-0.5/~ M, L-PIA at the concentrations of 0.005-0.5/~ M and D-PIA at the concentrations of 0.05-5/~M were administered to determine their effects on the contractile response to electrical stimulation. Concentration-response curves of adenosine and its analogues were determined in the presence of 8-phenyltheophylline at a concentration of 10/~M. Stock solutions o f drugs Stock solutions of drugs were made up in the following: CHA--20% ethanol/80% deionized water; L-PIA and D-PIA--50% dimethylsulphoxide/95% deionized water; adenosine, 2-chloroadenosine and NECA were made up in acidic deionized water and its pH was regulated by NaOH. All subsequent dilutions were made in deionized water. Materials Adenosine, 2-chloroadenosine, L-N6-phenylisopropyladenosine, D-N6-phenylisopropyladenosine, 5'-N-ethyl-

*To whom all correspondence should be addressed. 631

AHMADR£ZA I ~ P O U R and FATEMEH VAZXFI~ISHENAS

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100--

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Log of concentration (M) Fig. I. Effects of adenosine (O), D-PIA (C)), 2chloroadenosine (A), L-PIA (ll), CHA (Z~) and NECA (l-l) on neurogenic contraction of rabbit isolated vas deferens. All values are means + SE (n = 6 per group).

Fig. 2. Effect of adenosine on neurogenic contraction of rabbit isolated vas deferens in the absence (O) and presence of 8-PT (0). All values are means + SE (n = 6 per group). 8-PT (10/~M) significantly depressed adenosine effects (*P < 0.001; * ' P < 0.001).

carboxamidoadenosine, N6-cyclohexyladenosine and 8-phenyltheophylline, all were obtained from Sigma. Curves were compared by the analysis of variance for randomised blocks. Individual values were compared by the Student's paired t-test. A P-value of 0.05 or less was considered statistically significant.

on the ICso values was: C H A = N E C A > L-PIA > 2chloroadenosine > D-PIA > adenosine (Fig. I, Table 1). 8-Phenyltheophylline (10/zM), a potent adenosine receptors antagonist, shifted each concentrationresponse curve to the right. 8-Phenyltheophylline significantly antagonised adenosine and its analogues' effects (Figs 2, 3, 4, 5, 6 and 7, Table 1).

RESULTS

DISCUSSION

C H A , N E C A , L-PIA, 2-chloroadenosine, D-PIA and adenosine all caused concentration-dependent inhibition o f contractile response to electrical stimulation. Potency of C H A (selective for A, receptors, IC50 = 0.0345 + 0.0021 # M) was equivalent to potency of N E C A (selective for A2 receptors, ICso = 0.0367 -I0.0074/JM). Adenosine was a least potent with an ICs0 of 17 + 3.5/~M. The rank order of potency based

The results presented show that inhibitory effects of adenosine and its analogues on the rabbit isolated vas deferens are mediated via both A~ and A 2 adenosine receptors. C o m p o u n d s which are substituted at the C5 position of the ribose ring (e.g. N E C A ) are equipotent to compounds which are substituted at the N6 amino position on the purine ring (e.g. C H A ) at inhibition of contractile response

Statistical analysis

Table 1. ICs0s for adenosine and analogues on the vas deferens of the rabbit in the presence of 8-phenyltheophyllin¢(8-PT) (10#M) NECA L-PIA CHA 2-Chloroadenosine D-PIA Adenosine ICs0(pM) 0.0367 -1-0.0074(6) 0.0619+ 0.0292(6) 0.0345+ 0.0021(6) 0.272:t: 0.047(6) 0.516± 0.173(6) 17 -t- 3.5(6) pD2 7.44 7.21 7.46 6.57 6.29 4.77 Relative activity 463 274 493 62.5 32.9 1 In the presence of 8-phenyltheophylllne (IOI~M) IC~o (pM) pD 2 Relative antagoni~n

0.973 :/: 0.295(6) 6.01 26.5

0.88 -t- 0.349(6) 6.05 14.32

0.874 -1-0.173(6) 6.06 25.3

4.77 :/: I. 10(6) 5.32 17.5

15.4 + 2.9(6) 4.81 29.8

240 -t- 75(6) 3.62 14.12

Values ~ S E mean (n). pD2- negative log of ICso. Relative activity - ratio of analogue IC50to adenosine IC~, relative antagonism = ratio of analogue IC50in presence and absence of g-PT.

Adenosine receptors in rabbit vas deferens 100--

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Fig. 3. Effect of ~ P I A on neurogenic contraction of rabbit isolated vas deferens in the absence ((D) and presence of 8-PT (Q). All values are means + SE (n = 6 per group). 8-PT (10/~ M) significantly depressed D-PIA effects (*P < 0.001). 100

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Fig. 5. Effect of L-PIA on neurogenic contraction of rabbit isolated vas deferens in the absence (©) and presence of 8-PT (O). All values are means + SE (n = 6 per group). 8-PT (10/JM) significantly depressed L-PIA effects (*P < 0.02; **P < 0.01).

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Fig. 4. Effect of 2-cldoroadenosine on neurogenic contraction of rabbit isolated vas deferens in the absence (O) and presence of 8-PT ( 0 ) . All values are means :I: SE (n = 6 per group). 8.PT (10/~M) significantly depressed 2-chloroadenosine effects ( ' P < 0.002; " ' P < 0.001).

I

I

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Fig. 6. Effect of NECA on neurogenic contraction of rabbit isolated vas deferens in the absence (O) and presence of 8-PT (O). All values are means + SE (n , - 6 per group). 8-PT (10/~M) significantly depressed NECA effects (*P < 0.001; **P < 0.001).

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AHMAD REZA DEHPOUR a n d FATEMEH VAZIFEHSHENAS 100

displayed for PIA. The changes which occur as a result of adenosine receptor activation with adenyl cyclase remain to be investigated, in order to confirm our proposed classification.

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CHA, NECA, L-PIA, 2-chloroadenosine, D-PIA and adenosine mediate their inhibitory effects on the rabbit isolated vas deferens via both A~ and A2 adenosine receptors.

v C O es t-

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SUMMARY

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Log [CHA] (M) Fig. 7. Effect of CHA on neurogenic contraction of rabbit isolated vas deferens in the absence ((3) and presence of 8-PT (O). All values are means + SE (n = 6 per group). 8-PT (10/~M) significantly depressed CHA effects (*P <0.05; **P < 0.01; ***P < 0.001).

of rabbit isolated vas deferens to electrical stimulation. In addition, our experiments revealed that adenosine receptors in the rabbit vas deferens show marked stereoselectivity for PIA. This stereoselectivity has been claimed to indicate the presence of A~-receptors, since when L-PIA acts on an A~-receptor, it is significantly more potent than D-PIA (Smellie et al., 1979; Bruns et al., 1980). The adenosine-induced relaxation of the guinea-pig taenia coil is mediated via a receptor which closely resembles the A2-receptor as defined in other systems. NECA (selective for A2-receptor) more potent at relaxing the guinea-pig taenia coli than CHA and L-PIA (selective for Al-receptor) (Burnstock et al., 1984). Methyixanthines have been shown to antagonise competitively both A~ and A2 adenosine receptors (Londos et al., 1978; Bruns et al., 1980). Such findings are in agreement with the ability of 8phenyltheophylline, a potent adenosine receptors antagonist (Griffith et al., 1981), to antagonise the responses to adenosine and to all of its analogues in the rabbit isolated vas deferens. In addition, the effects of adenosine analogues are via extracellular adenosine receptors, since methylxanthines don't antagonise the intracellular "P" site (Bruns, 1980). It is proposed that rabbit vas deferens possess A~ and A2 adenosine receptors. Such a proposal is made on the rank order of agonist potency of adenosine analogues in inducing inhibition and stereoselectivity

The effects of adenosine, 5'-N-ethylcarboxamidoadenosine (NECA), 2-chloroadenosine, N6phenylisopropyladenosine (L-PIA and D°PIA) and N6-cyclohexyladenosine (CHA) were examined on the rabbit isolated vas deferens. All the analogues in a concentration-dependent manner inhibited contractile response to electrical stimulation. Adenosine with an ICs0 of 17+3.51aM, NECA with an IC50 of 0.0367 + 0.0074/~M, 2-chloroadenosine with an IC50 of 0.272+0.047/aM, CHA with an ICs0 of 0.0345 + 0 . 0 0 2 1 p M , L-PIA with an IC50 of 0.0619+0.0292/aM and D-PIA with an IC50 of 0.516 + 0.173/~ M inhibited neurogenic contractile response of rabbit was deferens. 8-Phenyltheophylline, an adenosine receptors antagonist, caused a rightward shift of all the adenosine and its analogues concentration-response curves. The order of potency for the adenosine and its analogues on the rabbit isolated vas deferens was: C H A = N E C A > L PIA > 2-chloroadenosine > D-PIA > adenosine. It is concluded that CHA, NECA, L-PIA, 2chloroadenosine, o-PIA and adenosine mediate their inhibitory effects on the rabbit isolated muscle via both A 1 and A: adenosine receptors. Acknowledgements--The authors thank Dr M. H. Ghahre-

mani and Dr P. Ghafouri for statistical analysis. REFERENCES

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