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Different modes of opiate interaction in rat vas deferens
Life Sciences, Vol. 31, pp. 1683-1686 Printed in the U.S.A.
Pergamon Press
DIFFERENT MODES OF OPIATE INTERACTION IN RAT VAS DEFERENS
Pilar S~nchez-Bl~zquez, Javier Garzon and Nancy M. Lee
Dept. of Pharmacology, University of California San Francisco, California 94143 USA (Received in final form June 14, 1982)
Summary We have analyzed the effect of arylsulfatase (AS) on electrically-stimulated contractions in the rat vas deferens (RVD) and on the inhibition of the latter by various opiates. Although most of these ligands are pharmacologically inactive in this system, they shift the agonists' dose-response curves to the right and alter the slopes. This indicates a complex interaction not explained by a simple receptor model. Biochemical and pharmacological studies performed with the enzyme arylsulfatase (AS) demonstrated its ability to modify opiate action. In vivo treatment with the enzyme results in a loss of analgesic potency of morphine or B-endorphin but not DADL or FK33824 (I). Binding studies performed in tissues from mice treated in vivo with AS showed a decrease in number of low affinity receptors (i). Finally, mouse vas deferens treated in vitro with this enzyme exhibited modifications in the response to opiates (2). In this study, we have analyzed the effect of AS, morphine, ethylketocyclazocine (EKC) and naloxone on electrically stimulated contraction in the RVD response to the activities of different opiates. Methods A single vas deferens of Long Evans rats (175-200 g) was mounted in a 20 ml organ bath containing Krebs solution at 37°C and bubbled with a mixture of 02-C02 (95%-5%). The upper end was attached to a Grass force displacement transducer FT03 (Grass Instruments Co., MA), connected to a Grass Model 7 polygraph (Grass Instrument Co., MA). A basal resting tension of 750 mg was applied and the tissues were allowed to equilibrate for 60 min. Electrical field stimulation was provided for a Grass $4 stimulator (Grass Instrument Co., MA). Dose-response curves were constructed by increasing bath concentration cumulatively, IC n and the slopes of the dose-response curves were obtained from linear regression between 20-80% inhibition.
0024-3205/82/161683-04503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
1684
Opiate Interaction in Vas Deferens
Vol. 31, No.s 16 & 17, 1982
Results As shown in Table i, AS ~reatme~t (i mg/ml) affected the activity of Bh-endorphin , etorphine, D-ala -D-leu -enkephalin and FK33824 in inhibiting electrically stimulated twitching in rat vas deferens. For DADLE and Table i Effect of AS*** Treatment in the Opiates Dose-Response Curves from Long Evans Rat Vas Deferens
Control IC50 (riM) Slope
Bh-EP Etorphine DADLE FK33824
35.2(10) 20(6) 901(8) 128(8)
55(46-64) 50(39.7-60.3) 48(42.6-53.4) 61(52.7-69.3)
AS IC50 (nM)
17.2(10) 15.5(6) 1800(8)* 197(8)*
Slope
67(58.8-75.2) 62(49.4-74.6) 77(67.5-86.5)** 86.9(75.8-98)**
(n) = number of experiments; *p<0.05, test non-parametric, IC50 cannot give complete information but can be used as representative of the change; **95% confidence limits; ***vas deferens was incubated in the presence of AS (i mg/ml bath capacity) with continuous stimulation for 60 min and washed. Table 2 Effect of Ethylketocyclazocine on B-EP or DADLE Activity in Rat Vas Deferens a
Control IC50 (nM)
Bh-EP DADLE
20.3 1300
Slope
52.9(47.9-59.9) 45(36.5-54.5)
EKC, 500 nM IC50 (nM) Slope
1830 37900
41.3(31.4-51.2) 38(27.8-48.2)
Table 3 Effect of Morphine on Bh-EP or DADLE Activity in Rat Vas Deferens a
Control IC50 (nM) Slope
Bh-EP DADLE
36 908
56(51.3-62.7) 50(43.8-58.2)
Morphine, I0 ~M IC50 (nM) Slope
143 55900
53(43.9-62.1) 51(43.5-58.5)
~Each value is mean of at least 4 vas deferens from different animals.
Vol. 31, No.s 16 & 17, 1982
Opiate Interaction in Vas Deferens
1685
Table 4 Effect of Naloxone on B-EP Activity in Rat Vas Deferens
Naloxone (nM)
0(8) 5(4) 120(4) 400(6) 1000(4)
IC50
58 125 579 3.08 8.89
nM nM nM ~M ~M
Slope
57(51.3-62.7) 54(45.5-62.5) 56(50.7-61.3) 76(68.7-83.3)* 73(66-80)*
Table 5 Effect of Naloxone on DADL Activity in Rat Vas Deferens
Naloxone (nM)
IC50 (~M)
Slope
0(8) 5(4) 40(4) 120(4) 400(4)
0.972 2.8 5.3 37.0 96.7
48(42.8-53.2) 40(30.8-49.2) 56(46.1-65.9) 89(78.8-99.2)* 110(98.2-121.8)*
*p<0.05 from control FK33824, both IC50 and slopes increased; however, a trend to decreasing IC50 was observed for Bh-EP and etorphine. Morphine and ethylketocyclazocine, although by themselves inactive in this system, were able to antagonize B-endorphin or DADLE's activity. Thus, in the presence of 500 nM of EKC, the IC50s for Bh-EP and DADLE were decreased 92- and 29-fold, respectively (Table 2) and with i0 juM morphine, the IC50s for B-EP and DADLE were decreased 4- and 62-fold, respectively (Table 3), no change in slopes was observed. When an antagonist, naloxone, was used to study the system, it was found that as the IC50s increased in the presence of increasing naloxone concentration, the slopes of the inhibitory curves were also increased (Table 4,5), indicating that the inhibition may not be a single competition on B-EP site.
Discussion The rat vas deferens system has often been said to contain only ~ receptor types, since only a few known opiates such as B-EP are active in it. Other opiates such as morphine and enkephallns, are not active. However, it is not known whether the inactivity of morphine or enkephalin is due to a lack of binding sites or to a deficiency in the coupling of such sites to the effector system. In this study, we attempted to distinguish these posslbillties~ employing arylsulfatase as an enzymatic probe, mor-
1686
Opiate Interaction in Vas Deferens
Vol. 31, No.s 16 & 17, 1982
phine and EKC as inactive opiates, and nalo×one as opiate antagonist. We reasoned that if a single receptor type is present, then any drug which has an effect in this system would have to act on this receptor; thus the inhibitory curve would always be shifted in parallel with no change in slopes (3). Morphine or EKC, although both were inactive in this system, can induce a parallel shift in B-EP or DADLE's curves, indicating that the binding sites were available for these seemingly inactive opiates. Our data also revealed, however, that naloxone's antagonism on B-EP or DADLE resulted in a significant change in slopes when naloxone concentration was higher than i00 nM. Furthermore, the AS treatment demonstrated that the active opiates may be affected differently by the enzyme, again indicating the complexity of the system. Although the exact mechanism of these interactions has not been delineated, the evidence obtained in this study strongly suggests that the rat vas deferens receptor may have behaved like a specific B-endorphin receptor, actually is more complicated (3) and the reason that many typical opiates are inactive it is not due to lack of binding to the receptor.
Acknowledgements Pilar S~nchez-Bl~zquez is a recipient of a Fulbright-MUl Postdoctoral Fellowship. Javier Garz~n is a recipient of a NIH Fellowship, FO5-TW-03080. This work was supported in part by NIDA Grant DA-02643.
References i. 2. 3.
J. GARZON, M.F. JEN and N.M. LEE, Biochem. Pharmac., submitted for publication. P. SANCHEZ-BLAZQUEZ, J° GARZON, N.M. LEE and H.H. LOH, FASEB Proc. 41, 1220 (1982). J.M. BOEYNAEMS and J.E. DUMONT, Outlines of Receptor Theory Elsevier/ North-Holland (1980).
Pergamon Press
DIFFERENT MODES OF OPIATE INTERACTION IN RAT VAS DEFERENS
Pilar S~nchez-Bl~zquez, Javier Garzon and Nancy M. Lee
Dept. of Pharmacology, University of California San Francisco, California 94143 USA (Received in final form June 14, 1982)
Summary We have analyzed the effect of arylsulfatase (AS) on electrically-stimulated contractions in the rat vas deferens (RVD) and on the inhibition of the latter by various opiates. Although most of these ligands are pharmacologically inactive in this system, they shift the agonists' dose-response curves to the right and alter the slopes. This indicates a complex interaction not explained by a simple receptor model. Biochemical and pharmacological studies performed with the enzyme arylsulfatase (AS) demonstrated its ability to modify opiate action. In vivo treatment with the enzyme results in a loss of analgesic potency of morphine or B-endorphin but not DADL or FK33824 (I). Binding studies performed in tissues from mice treated in vivo with AS showed a decrease in number of low affinity receptors (i). Finally, mouse vas deferens treated in vitro with this enzyme exhibited modifications in the response to opiates (2). In this study, we have analyzed the effect of AS, morphine, ethylketocyclazocine (EKC) and naloxone on electrically stimulated contraction in the RVD response to the activities of different opiates. Methods A single vas deferens of Long Evans rats (175-200 g) was mounted in a 20 ml organ bath containing Krebs solution at 37°C and bubbled with a mixture of 02-C02 (95%-5%). The upper end was attached to a Grass force displacement transducer FT03 (Grass Instruments Co., MA), connected to a Grass Model 7 polygraph (Grass Instrument Co., MA). A basal resting tension of 750 mg was applied and the tissues were allowed to equilibrate for 60 min. Electrical field stimulation was provided for a Grass $4 stimulator (Grass Instrument Co., MA). Dose-response curves were constructed by increasing bath concentration cumulatively, IC n and the slopes of the dose-response curves were obtained from linear regression between 20-80% inhibition.
0024-3205/82/161683-04503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
1684
Opiate Interaction in Vas Deferens
Vol. 31, No.s 16 & 17, 1982
Results As shown in Table i, AS ~reatme~t (i mg/ml) affected the activity of Bh-endorphin , etorphine, D-ala -D-leu -enkephalin and FK33824 in inhibiting electrically stimulated twitching in rat vas deferens. For DADLE and Table i Effect of AS*** Treatment in the Opiates Dose-Response Curves from Long Evans Rat Vas Deferens
Control IC50 (riM) Slope
Bh-EP Etorphine DADLE FK33824
35.2(10) 20(6) 901(8) 128(8)
55(46-64) 50(39.7-60.3) 48(42.6-53.4) 61(52.7-69.3)
AS IC50 (nM)
17.2(10) 15.5(6) 1800(8)* 197(8)*
Slope
67(58.8-75.2) 62(49.4-74.6) 77(67.5-86.5)** 86.9(75.8-98)**
(n) = number of experiments; *p<0.05, test non-parametric, IC50 cannot give complete information but can be used as representative of the change; **95% confidence limits; ***vas deferens was incubated in the presence of AS (i mg/ml bath capacity) with continuous stimulation for 60 min and washed. Table 2 Effect of Ethylketocyclazocine on B-EP or DADLE Activity in Rat Vas Deferens a
Control IC50 (nM)
Bh-EP DADLE
20.3 1300
Slope
52.9(47.9-59.9) 45(36.5-54.5)
EKC, 500 nM IC50 (nM) Slope
1830 37900
41.3(31.4-51.2) 38(27.8-48.2)
Table 3 Effect of Morphine on Bh-EP or DADLE Activity in Rat Vas Deferens a
Control IC50 (nM) Slope
Bh-EP DADLE
36 908
56(51.3-62.7) 50(43.8-58.2)
Morphine, I0 ~M IC50 (nM) Slope
143 55900
53(43.9-62.1) 51(43.5-58.5)
~Each value is mean of at least 4 vas deferens from different animals.
Vol. 31, No.s 16 & 17, 1982
Opiate Interaction in Vas Deferens
1685
Table 4 Effect of Naloxone on B-EP Activity in Rat Vas Deferens
Naloxone (nM)
0(8) 5(4) 120(4) 400(6) 1000(4)
IC50
58 125 579 3.08 8.89
nM nM nM ~M ~M
Slope
57(51.3-62.7) 54(45.5-62.5) 56(50.7-61.3) 76(68.7-83.3)* 73(66-80)*
Table 5 Effect of Naloxone on DADL Activity in Rat Vas Deferens
Naloxone (nM)
IC50 (~M)
Slope
0(8) 5(4) 40(4) 120(4) 400(4)
0.972 2.8 5.3 37.0 96.7
48(42.8-53.2) 40(30.8-49.2) 56(46.1-65.9) 89(78.8-99.2)* 110(98.2-121.8)*
*p<0.05 from control FK33824, both IC50 and slopes increased; however, a trend to decreasing IC50 was observed for Bh-EP and etorphine. Morphine and ethylketocyclazocine, although by themselves inactive in this system, were able to antagonize B-endorphin or DADLE's activity. Thus, in the presence of 500 nM of EKC, the IC50s for Bh-EP and DADLE were decreased 92- and 29-fold, respectively (Table 2) and with i0 juM morphine, the IC50s for B-EP and DADLE were decreased 4- and 62-fold, respectively (Table 3), no change in slopes was observed. When an antagonist, naloxone, was used to study the system, it was found that as the IC50s increased in the presence of increasing naloxone concentration, the slopes of the inhibitory curves were also increased (Table 4,5), indicating that the inhibition may not be a single competition on B-EP site.
Discussion The rat vas deferens system has often been said to contain only ~ receptor types, since only a few known opiates such as B-EP are active in it. Other opiates such as morphine and enkephallns, are not active. However, it is not known whether the inactivity of morphine or enkephalin is due to a lack of binding sites or to a deficiency in the coupling of such sites to the effector system. In this study, we attempted to distinguish these posslbillties~ employing arylsulfatase as an enzymatic probe, mor-
1686
Opiate Interaction in Vas Deferens
Vol. 31, No.s 16 & 17, 1982
phine and EKC as inactive opiates, and nalo×one as opiate antagonist. We reasoned that if a single receptor type is present, then any drug which has an effect in this system would have to act on this receptor; thus the inhibitory curve would always be shifted in parallel with no change in slopes (3). Morphine or EKC, although both were inactive in this system, can induce a parallel shift in B-EP or DADLE's curves, indicating that the binding sites were available for these seemingly inactive opiates. Our data also revealed, however, that naloxone's antagonism on B-EP or DADLE resulted in a significant change in slopes when naloxone concentration was higher than i00 nM. Furthermore, the AS treatment demonstrated that the active opiates may be affected differently by the enzyme, again indicating the complexity of the system. Although the exact mechanism of these interactions has not been delineated, the evidence obtained in this study strongly suggests that the rat vas deferens receptor may have behaved like a specific B-endorphin receptor, actually is more complicated (3) and the reason that many typical opiates are inactive it is not due to lack of binding to the receptor.
Acknowledgements Pilar S~nchez-Bl~zquez is a recipient of a Fulbright-MUl Postdoctoral Fellowship. Javier Garz~n is a recipient of a NIH Fellowship, FO5-TW-03080. This work was supported in part by NIDA Grant DA-02643.
References i. 2. 3.
J. GARZON, M.F. JEN and N.M. LEE, Biochem. Pharmac., submitted for publication. P. SANCHEZ-BLAZQUEZ, J° GARZON, N.M. LEE and H.H. LOH, FASEB Proc. 41, 1220 (1982). J.M. BOEYNAEMS and J.E. DUMONT, Outlines of Receptor Theory Elsevier/ North-Holland (1980).