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Opiate receptors in the rat vas deferens
Life Sciences, Vol. 33, Sup. I, 1983, pp. 327-330 Printed in the U.S.A.
Pergamon Pres
OPIATE RECEPTORSIN THE RAT VAS DEFERENS Colin F.C. Smith and Michael J. Rance Reckitt and Colman Pharmaceutical Division, Dansom Lane, Hull HU8 7DS, U.K. (Received in final form June 26, 1983)
Summary The nature of the opiate receptor population in the rat vas deferens (RVD) has been examined by evaluating the interaction of a range of antagonists with prototypic ~ - , ~ - and d-opioid agonists in the tissue. Ke values for 5 antagonists against normorphine in the isolated mouse vas deferens showed excellent correlation with Ke values obtained against the ~agonist RX783030 in the RVD. RX783030 could be effectively antagonised by naltrexone in the RVD b~t note by the ~antagonist ICI 154129 whereas D-Ala~,D-Leu~-enkephalin required both antagonists to yield parallel shifts of its dose response. The lack of agonist activity of morphine is a result of the low intrinsic activity of this agent in the RVD. The~-agonists ethylketocyclazocine, tifluadom and U50488 also showed antagonist properties in the RVD. These results can be rationalised by postulating that the RVD contains a v-receptor population with a high intrinsic activity requirement together with some d-receptors. I t is not necessary to propose the existence of a novel E-receptor in order to rationalise the data reported. The opiate-receptor population in the rat vas deferens (RVD) has been widely studied (1-5) and the results obtained have been rationalised by some groups by postulation of the existence of a noveIE-receptor population in this tissue. Of particular interest has been the observation that Bendorphin shows h i g h potency in the RVD whereas morphine demonstrates l i t t l e or no agonist activity. We have studied the action of a range of opioid agonists and antagonists in the isolated mouse (MVD) and rat vas deferens preparations in an attempt to determine the nature of the receptor population in the latter tissue. METHODS Vasa deferentia from both species were mounted in 50ml organ baths containing Krebs buffer and were stimulated electrically via platinum electrodes with contractions recorded isometrically as described by Handa et al (6). Drugs used: H-~yr-D-A~a-Gly-MePhe NH(CH2)pN(O)Me2 (RX783030; Reckitt and Colman); D-AlaC,D-LeuU-enkephalin (DADL; Cambridge Research Biochemicals); normorphine (NM; Wellcome); morphine (MacFarlan Smith); ethylketocyclazocine(Sterling); t i f l u a d o m (Sandoz); U50488 (Upjohn); naloxone, naltrexone (Endo); MR2266,MR1452, clonidine (Boehringer Ingelheim).
0024-3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.
328
Opiate Receptors in Vas Deferens
Vol. 33, Sup. I, 1983
RESULTS Ke values obtained with a range of antagonists against pro~otypic ago~ists normorphine (~), ethylketocyclazocine (EKC) (~) and D-Ala~, DLeu~-enkephalin (DADL) (~) in the MVD were compared to those obtained in the RVD against the ~-agonist RX783030. Ke values for the antagonists against the p-agonist in the RVD showed an excellent correlation with the values obtained against normorphine in the MVD (Fig 1) but a poor correlation with values obtained a t ~ (r:0.84) and ~ sites (r=0.78) (data not shown).
Nalorphine v
10
z
MR2266/, N ~ a ltOeNxilnl x°ne
l.O .j
|
1.0
I
lO Ke RVD vs RX783030 (nH)
l~O
l
FIG 1 Correlation of Ke values for a range of antagonists in the MVD vs normorphine (NM)with values obtained in the RVD vs RX783030 (r=0.996) The dose response to the p-agonist RX783030 in RVD was displaced to the right with the ~-selective antagonist naltrexone (3nM). No further displacement could be obtained with the d-antagonist ICI 154129 (3pM) suggesting a ~-mediated effect for RX783030. The dose response to DADLwas p a r t i a l l y displaced to the right in a non-parallel manner by naltrexone (3nM). A further s h i f t to the right was obtained with ICI 154129 (3~M) suggesting an involvement for both p and ~-receptors in the agonist effect of DADL (Figure 2). ICI 154129 was ineffective as an antagonist of DADL in the absence of naltrexone.
80 60
20 - , ~
-7
-6
Log DADL conc n (M) FIG 2 Antagonism of DADL (0) by naltrexone (3nM) ( • ) and nal trexone (3nM) + ICI 154129 (3~M) (A) in the RVD. Results are means ± s.e.m, (n=5), In agreement with other workers (1-5) morphine was essentially devoid of agonist activity in the RVD showing at best 10-20% depression of twitch height even at high doses. I t was however an effective antagonist of RX783030 and showedan a f f i n i t y which was not inconsistent with i t s agonist
Vol. 33, Sup. I, 1983
Opiate Receptors in Vas Deferens
329
potency in the guinea pig ileum (Fig 3 and Table 1). This antagonist activity appeared selective for opiate receptors as high concentrations of morphine in the RVD failed to inhibit the agonist activity of the a2-agent clonidine (Table 1). 80
60
/
40
20
• i
-8
-7
-6
Log RX783030 conc n (M) FIG 3
Antagonism of RX783030 (Q) by morphine (8~M) in the RVD ( I ) . Results are means ± s.e.m. (n=5) TABLE l Antagonist activity of morphine and~-agonists in the rat vas deferens Ke (nM) Compound vs RX783030 (u-agonist) Ethylketocyclazocine Tifluadom U50488 Morphine Results are means ± s.e.m.
vs clonidine (~2-agonist)
79 ± 14 (4) 1925 ± 554 (4) 528 ± 168 (5) 1226 ± 181 (5)
>20000 1260 ± 361 (5) >27000
Numbersof determinations in brackets.
Ethylketocyclazocine and the newer ~-agonists tifluadom (7) and U50488 (8) also demonstrated antagonist properties against RX783030 in the RVD (Table 1), results which are consistent with the observations of Gillan et al. (4). U50488 differs from the other agents in that i t also antagonises the ~2agonist clonidine at relatively low concentrations. DISCUSSION The results above are largely consistent with data reported previously on the opioid pharmacology of the RVD. The consistency of data obtained is not however reflected by a single interpretation of their significance. Lemaire et al (I) and Herz et al. (2,3) favour the existence of a new (6) receptor sub-type in the RVD whereas Liao et al. (5) has proposed that the observed data can be rationalised by invoking different intrinsic activities for the agonists tested at a single receptor population. The data of Gillan
330
Opiate Receptors in Vas Deferens
Vol. 33, Sup. I, 1983
et al. (4) would suggest that a population of ~-receptors exists in the RVD at which the benzomorphans have antagonist properties. Our data tend to support and extend the observations of Gillan et al. (4) and Liao et al. (5). The correlations obtained between ~-effects in the MVO and the action of the ~-agonist RX783030 in the RVD support the existence of a ~-receptor population in the l a t t e r tissue. The fact that the ~-selective antagonist naltrexone does not produce a parallel s h i f t in the dose response to DADL whereas the combination of naltrexone and the ~-antagonist ICI 154129 is more effective would suggest that there are ([-receptors in the tissue which, especially at low doses in the presence of ~-receptor blockade, are capable of mediating a DADL agonist response. At higher doses the agonist a c t i v i t y of DADL appears to be largely ~-receptor mediated which suggests that dreceptors have a limited a b i l i t y to i n h i b i t the e l e c t r i c a l l y stimulated response of the RVD. Our results clearly demonstrate a selective antagonist action of morphine against the ~-peptide RX783030 in the RVD. This observation is in agreement with Liao et al (5) and f u l l y explains the limited agonist activity of morphine in this tissue. The action of clonidine was unaffected at concentrations of morphine which were producing substantial antagonism at the opiate receptor. The ~-agonists ethylketocyclazocine, tifluadom and U50488 also demonstrated antagonist actions in the RVD which are most probably mediated through ~-receptors as postulated by Gillan et al. (4). Tifluadom shows selectivity for the opioid receptor system whereas U50488 also shows ~2-antagonist properties. In conclusion, the above evidence is consistent with the RVD containing a ~-receptor population which possesses a high i n t r i n s i c activity requirement for activation such that partial agonists (including morphine) demonstrate only antagonist properties. In addition some d-receptors are present and mediate a component of DADL's agonist action. From this data i t is not possible to rule out the existence of a novel ~receptor with which B-endorphin might interact but i t is not necessary to invoke such an entity to explain f u l l y the pharmacology of the agents used in this study. Acknowledgement: The authors wish to thank Mr. A. Carter and Miss L. Henderson for their technical assistance. REFERENCES l •
2. 3.
4.
5. 6. 7. 8.
S. LEMAIRE, J. MAGNANand D. REGOLI. Br. J. Pharmac. 64, 327-329 (1978). M. WUSTER, R. SCHULZand A. HERZ, Endogenous and Exogenous Opiate Agonists and Antagonists (E.L. Way, ed.) New York Pergamon Press, pp75-78 (1980). R. SCHULZ, E. FASE, M. WUSTERand A. HERZ, Life Sci. 24, 843-850 (1979). M.C.G. GILLAN, H.W. Kosterlitz and J. MAGNAN, Br. J. Pharmac. 72, 1315 (1981). C.S. LIAO, A.R DAY and R.J. FREER, Life Sci. 29, 2617-2622 (198l). B.K. HANDA, A.C. LANE, J.A.H. MORGAN, B.A. LOl~l~M.J. RANCEand C.F.C. SMITH, Eur. J. Pharmac. 70, 531-540 (1981). D. ROMER, H.H. BUSCHER, R.C. HItT, et al. Nature 298, 759. P.F. VONVOIGTLANDER, R.A. LAHTI and J.H. LUDENS, ~TT-Pharmac. Exp. Ther. 224, 7-12.
Pergamon Pres
OPIATE RECEPTORSIN THE RAT VAS DEFERENS Colin F.C. Smith and Michael J. Rance Reckitt and Colman Pharmaceutical Division, Dansom Lane, Hull HU8 7DS, U.K. (Received in final form June 26, 1983)
Summary The nature of the opiate receptor population in the rat vas deferens (RVD) has been examined by evaluating the interaction of a range of antagonists with prototypic ~ - , ~ - and d-opioid agonists in the tissue. Ke values for 5 antagonists against normorphine in the isolated mouse vas deferens showed excellent correlation with Ke values obtained against the ~agonist RX783030 in the RVD. RX783030 could be effectively antagonised by naltrexone in the RVD b~t note by the ~antagonist ICI 154129 whereas D-Ala~,D-Leu~-enkephalin required both antagonists to yield parallel shifts of its dose response. The lack of agonist activity of morphine is a result of the low intrinsic activity of this agent in the RVD. The~-agonists ethylketocyclazocine, tifluadom and U50488 also showed antagonist properties in the RVD. These results can be rationalised by postulating that the RVD contains a v-receptor population with a high intrinsic activity requirement together with some d-receptors. I t is not necessary to propose the existence of a novel E-receptor in order to rationalise the data reported. The opiate-receptor population in the rat vas deferens (RVD) has been widely studied (1-5) and the results obtained have been rationalised by some groups by postulation of the existence of a noveIE-receptor population in this tissue. Of particular interest has been the observation that Bendorphin shows h i g h potency in the RVD whereas morphine demonstrates l i t t l e or no agonist activity. We have studied the action of a range of opioid agonists and antagonists in the isolated mouse (MVD) and rat vas deferens preparations in an attempt to determine the nature of the receptor population in the latter tissue. METHODS Vasa deferentia from both species were mounted in 50ml organ baths containing Krebs buffer and were stimulated electrically via platinum electrodes with contractions recorded isometrically as described by Handa et al (6). Drugs used: H-~yr-D-A~a-Gly-MePhe NH(CH2)pN(O)Me2 (RX783030; Reckitt and Colman); D-AlaC,D-LeuU-enkephalin (DADL; Cambridge Research Biochemicals); normorphine (NM; Wellcome); morphine (MacFarlan Smith); ethylketocyclazocine(Sterling); t i f l u a d o m (Sandoz); U50488 (Upjohn); naloxone, naltrexone (Endo); MR2266,MR1452, clonidine (Boehringer Ingelheim).
0024-3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.
328
Opiate Receptors in Vas Deferens
Vol. 33, Sup. I, 1983
RESULTS Ke values obtained with a range of antagonists against pro~otypic ago~ists normorphine (~), ethylketocyclazocine (EKC) (~) and D-Ala~, DLeu~-enkephalin (DADL) (~) in the MVD were compared to those obtained in the RVD against the ~-agonist RX783030. Ke values for the antagonists against the p-agonist in the RVD showed an excellent correlation with the values obtained against normorphine in the MVD (Fig 1) but a poor correlation with values obtained a t ~ (r:0.84) and ~ sites (r=0.78) (data not shown).
Nalorphine v
10
z
MR2266/, N ~ a ltOeNxilnl x°ne
l.O .j
|
1.0
I
lO Ke RVD vs RX783030 (nH)
l~O
l
FIG 1 Correlation of Ke values for a range of antagonists in the MVD vs normorphine (NM)with values obtained in the RVD vs RX783030 (r=0.996) The dose response to the p-agonist RX783030 in RVD was displaced to the right with the ~-selective antagonist naltrexone (3nM). No further displacement could be obtained with the d-antagonist ICI 154129 (3pM) suggesting a ~-mediated effect for RX783030. The dose response to DADLwas p a r t i a l l y displaced to the right in a non-parallel manner by naltrexone (3nM). A further s h i f t to the right was obtained with ICI 154129 (3~M) suggesting an involvement for both p and ~-receptors in the agonist effect of DADL (Figure 2). ICI 154129 was ineffective as an antagonist of DADL in the absence of naltrexone.
80 60
20 - , ~
-7
-6
Log DADL conc n (M) FIG 2 Antagonism of DADL (0) by naltrexone (3nM) ( • ) and nal trexone (3nM) + ICI 154129 (3~M) (A) in the RVD. Results are means ± s.e.m, (n=5), In agreement with other workers (1-5) morphine was essentially devoid of agonist activity in the RVD showing at best 10-20% depression of twitch height even at high doses. I t was however an effective antagonist of RX783030 and showedan a f f i n i t y which was not inconsistent with i t s agonist
Vol. 33, Sup. I, 1983
Opiate Receptors in Vas Deferens
329
potency in the guinea pig ileum (Fig 3 and Table 1). This antagonist activity appeared selective for opiate receptors as high concentrations of morphine in the RVD failed to inhibit the agonist activity of the a2-agent clonidine (Table 1). 80
60
/
40
20
• i
-8
-7
-6
Log RX783030 conc n (M) FIG 3
Antagonism of RX783030 (Q) by morphine (8~M) in the RVD ( I ) . Results are means ± s.e.m. (n=5) TABLE l Antagonist activity of morphine and~-agonists in the rat vas deferens Ke (nM) Compound vs RX783030 (u-agonist) Ethylketocyclazocine Tifluadom U50488 Morphine Results are means ± s.e.m.
vs clonidine (~2-agonist)
79 ± 14 (4) 1925 ± 554 (4) 528 ± 168 (5) 1226 ± 181 (5)
>20000 1260 ± 361 (5) >27000
Numbersof determinations in brackets.
Ethylketocyclazocine and the newer ~-agonists tifluadom (7) and U50488 (8) also demonstrated antagonist properties against RX783030 in the RVD (Table 1), results which are consistent with the observations of Gillan et al. (4). U50488 differs from the other agents in that i t also antagonises the ~2agonist clonidine at relatively low concentrations. DISCUSSION The results above are largely consistent with data reported previously on the opioid pharmacology of the RVD. The consistency of data obtained is not however reflected by a single interpretation of their significance. Lemaire et al (I) and Herz et al. (2,3) favour the existence of a new (6) receptor sub-type in the RVD whereas Liao et al. (5) has proposed that the observed data can be rationalised by invoking different intrinsic activities for the agonists tested at a single receptor population. The data of Gillan
330
Opiate Receptors in Vas Deferens
Vol. 33, Sup. I, 1983
et al. (4) would suggest that a population of ~-receptors exists in the RVD at which the benzomorphans have antagonist properties. Our data tend to support and extend the observations of Gillan et al. (4) and Liao et al. (5). The correlations obtained between ~-effects in the MVO and the action of the ~-agonist RX783030 in the RVD support the existence of a ~-receptor population in the l a t t e r tissue. The fact that the ~-selective antagonist naltrexone does not produce a parallel s h i f t in the dose response to DADL whereas the combination of naltrexone and the ~-antagonist ICI 154129 is more effective would suggest that there are ([-receptors in the tissue which, especially at low doses in the presence of ~-receptor blockade, are capable of mediating a DADL agonist response. At higher doses the agonist a c t i v i t y of DADL appears to be largely ~-receptor mediated which suggests that dreceptors have a limited a b i l i t y to i n h i b i t the e l e c t r i c a l l y stimulated response of the RVD. Our results clearly demonstrate a selective antagonist action of morphine against the ~-peptide RX783030 in the RVD. This observation is in agreement with Liao et al (5) and f u l l y explains the limited agonist activity of morphine in this tissue. The action of clonidine was unaffected at concentrations of morphine which were producing substantial antagonism at the opiate receptor. The ~-agonists ethylketocyclazocine, tifluadom and U50488 also demonstrated antagonist actions in the RVD which are most probably mediated through ~-receptors as postulated by Gillan et al. (4). Tifluadom shows selectivity for the opioid receptor system whereas U50488 also shows ~2-antagonist properties. In conclusion, the above evidence is consistent with the RVD containing a ~-receptor population which possesses a high i n t r i n s i c activity requirement for activation such that partial agonists (including morphine) demonstrate only antagonist properties. In addition some d-receptors are present and mediate a component of DADL's agonist action. From this data i t is not possible to rule out the existence of a novel ~receptor with which B-endorphin might interact but i t is not necessary to invoke such an entity to explain f u l l y the pharmacology of the agents used in this study. Acknowledgement: The authors wish to thank Mr. A. Carter and Miss L. Henderson for their technical assistance. REFERENCES l •
2. 3.
4.
5. 6. 7. 8.
S. LEMAIRE, J. MAGNANand D. REGOLI. Br. J. Pharmac. 64, 327-329 (1978). M. WUSTER, R. SCHULZand A. HERZ, Endogenous and Exogenous Opiate Agonists and Antagonists (E.L. Way, ed.) New York Pergamon Press, pp75-78 (1980). R. SCHULZ, E. FASE, M. WUSTERand A. HERZ, Life Sci. 24, 843-850 (1979). M.C.G. GILLAN, H.W. Kosterlitz and J. MAGNAN, Br. J. Pharmac. 72, 1315 (1981). C.S. LIAO, A.R DAY and R.J. FREER, Life Sci. 29, 2617-2622 (198l). B.K. HANDA, A.C. LANE, J.A.H. MORGAN, B.A. LOl~l~M.J. RANCEand C.F.C. SMITH, Eur. J. Pharmac. 70, 531-540 (1981). D. ROMER, H.H. BUSCHER, R.C. HItT, et al. Nature 298, 759. P.F. VONVOIGTLANDER, R.A. LAHTI and J.H. LUDENS, ~TT-Pharmac. Exp. Ther. 224, 7-12.