- Email: [email protected]
δ-opioid receptors in mice
ELSEVIER
e.jp European Journal of Pharmacology258 (1994) 159-162
Short communication
Mastoparan reduces the supraspinal analgesia mediated by lX/6-opioid receptors in mice Pilar Sfinchez-Blfizquez *, Javier Garzdn Neurofarmacologfa, Instituto Cajal, CSIC, Avenida DoctorArce 37, 28002Madrid, Spare
(Received 27 January 1994;accepted 31 March 1994)
Abstract
Intracerebroventricular (i.c.v.) administration of the venom peptide, mastoparan, to mice decreased to a limited extent opioid-induced supraspinal analgesia in a non-competitive fashion. The/x-opioid receptor agonists, [D-Ala2,N-MePhe4,Gly-olS]enkephalin (DAMGO) and morphine, the /~/~-opioid receptor ligands, human /3-endorphin-(1-31) and [D-Ala2,o-LeuS]enkephalin (DADLE), and the selective ligands of ~-opioid receptors, [D-Pen2'5]enkephalin (DPDPE) and [o-Ala2]deltorphin II, showed an impaired analgesic effect in mice given mastoparan. Mastoparan diminished the analgesic activity of DPDPE and [o-Ala2]deltorphin II to the same extent as observed after giving the 6-opioid receptor-selective antagonist, ICI 174864. The /z-opioid receptor-mediated analgesia that remained after mastoparan was abolished in the presence of the opioid antagonist, naloxone. Mastoparan after binding to Gia/Goot subunits could block opioid antinociception. The existence of a class of G protein functionally coupled to/z-opioid receptors, but resistant to the effect of mastoparan is suggested. Key words: Mastoparan; GTP-binding protein; Supraspinal analgesia; Opioid
1. Introduction
After binding to their receptors in the cellular membrane opioids activate GTP-binding transducer proteins that regulate the corresponding effectors (Ueda et al., 1988; Vogel et al., 1990). A series of studies indicated the diversity of the transducer system regulated b y / z / 6 - o p i o i d receptors to promote supraspinal analgesia. In this respect, bacterial toxins known to alter the function of G proteins, i.e., cholera and pertussis toxin, modified this opioid activity in a differential manner (Sfinchez-Blfizquez and Garz6n, 1988, 1991). Similarly, the i.c.v, injection of immunoglobulins raised against specific sequences of G a subunits also revealed multiple G proteins involved in this characteristic opioid quality (S~inchez-Blfizquez and Garz6n, 1993; S~inchez-Blfizquez et al., 1993). Mastoparan, a peptide toxin from wasp venom, has been reported to bind to the receptor domain of certain G proteins promoting an accelerated exchange of G D P by G T P at G a subunits (Higashijima et al., 1988).
* Corresponding author. Tel. 341 5854733, fax 341 5854754. 0014-2999/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0014-2999(94)00197-F
The cationic amphiphilic characteristics of this venom peptide facilitate access through the cellular membrane to interact directly with the G proteins (Mousli et al., 1990). To further explore the transducer system linked to opioid-mediated effects, mastoparan was i.c.v. administered to mice and the supraspinal antinociception elicited by different opioids was subsequently evaluated in the warm water tail-flick test.
2. Materials and methods 2.1. Animals and evaluation o f analgesia
Albino male CD-1 mice (Charles River, Barcelona, Spain) weighing 22-25 g were used throughout. The animals were kept at 22°C, with a 12 h l i g h t / d a r k cycle (8 a . m . / 8 p.m.) and food and water provided ad libitum. The animals were housed and used strictly in accordance with the guidelines of the European Community about Care and Use of Laboratory Animals. All substances were dissolved in saline, except [DAla2]deltorphin II which was dissolved in 0.1% dimethyl sulphoxide (DMSO), and injected i.c.v, in a
160
P. Sdnchez-Bldzquez, J. Garz6n / European Journal of Pharmacology 258 (1994) 159-162
final volume of 4 /~1 to mice under light ether anaesthesia. Injections were unilateral and subsequent administrations were performed on the same side. The warm water (52°C) tail-flick test was used to evaluate analgesia. Latencies in seconds were measured manually, before (basal latency) and after the administration of the opioid under study (test latency). A cut-off time of 10 s was selected and antinociceptive activity was expressed as a percent of the maximum possible effect (%MPE). The following equation was used: % M P E = 100 X (test latency - b a s a l ) / ( 1 0 - basal). A single dose of saline (control) or mastoparan was injected to the mice and afterwards the antinociceptive effects of the analgesic substances were evaluated. In the case of sequential treatments, the vehicle replaced the substance whose effect was being tested. Statistical significance was determined by analysis of variance ( A N O V A ) followed by Scheffe's test. The level of significance was set at P < 0.05. After the analgesic dose-response curves had been made, the slopes, EDs0 and 95% confidence limits were determined according to the method of Tallarida and Murray (1981).
2.2. Drugs
Time-Course
t
o
0
i
i
control lh
ld
3. Results
3.1. Effect of mastoparan on the supraspinal antinociception induced by opioids in the tail-flick test Mastoparan at the i.c.v, doses of 10 -22 to 10 - 9 m o l / m o u s e did not modify the basal latencies displayed by the mice in the antinociceptive test, and no behavioural disturbances were observed. Animals receiving 1 p m o l / m o u s e mastoparan showed a decreased response to the analgesic activity of i.c.v, opioids. This antagonism was long-lasting, the effect was fully expressed 60 min after a single administration of the venom peptide, being almost identical 24 h later, and declining slowly within a week (Fig. 1). The analgesic effects of D A M G O , a/z-opioid receptor-selective ligand, and D P D P E , a g-opioid receptorselective ligand, were significantly reduced in mice injected with the venom peptide (Fig. 1). The apparent ED50 values ( n m o l / m o u s e ) and 95% confidence limits
i
i
3d
i
i
5d
i
i
i
i
9d
7d
Interval post-Mastoparan (1 pmol/rnouse, i.c,v,)
0
LU
_¢ o0 O ft.
E .-1 E
Control;
r~
+ Mastoparan
DAMGO
JQ
DPDPE
75
75
50
50
25 0
25 I 12
I 11
o~
I 10
I 0 , I 9 9 -tog (mol/moulm, i.c.v.) Opioid
I
I Opioid;
~IB
I 8
+ Mastoparan
0 Morphine
The following substances were used: human /3-endorphin, D A M G O , D A D L E , D P D P E , [D-Ala2] deltorphin II, mastoparan and mastoparan 17 were purchased from Peninsula Laboratories (San Carlos, CA, USA); ICI 174864 from CRB (Cambridge, UK). Morphine sulphate came from Merck (Darmstadt, Germany). Naloxone hydrochloride was obtained from Sigma (Saint Louis, MO, USA).
i
20
f~-Endorphin 2
DADLE
Deltorphin II
0.3
20 nmol
Fig. 1. Antagonism by i.c.v, administered mastoparan on opioid-induced supraspinal antinociception. Mice received a single injection of 1 pmol mastoparan and the time course for the antagonist effect on 20 nmol morphine or I0 nmol DPDPE was studied at the intervals indicated (upper panel). Different groups of mice were used for each post-mastoparan interval studied. The dose-response curves for DAMGO and DPDPE (middle panel), or a single dose for the other opioids (lower panel), were evaluated 24 h after receiving the venom peptide. Antinociception was determined at the peak effect 30 min after morphine and /3-endorphin, 15 min after DAMGO, DADLE and DPDPE and 10 min after [D-Ala2]deltorphin II. Analgesia was expressed as a percent of the maximum possible effect (%MPE). The values are the means:l:S.E.M, from groups of 10-15 mice. * Significantly different from the control group that received saline instead of mastoparan, ANOVA-Scheffe's test, P < 0.05. for antinociception shown by these opioids in salineand mastoparan-treated animals were respectively: D A M G O , 0.08 (0.06-0.10) and 0.27 (0.19-0.38)*; D P D P E , 5.93 (3.95-8.89) and 35.48 (21.50-58.54)* (* significantly different from the control (saline) group, P < 0.05). The potency of the selective 8-opioid receptor ligand, deltorphin II, the/z/~5-opioid receptor agonist, D A D L E , the endogenous opioid,/3-endorphin and the /z-opioid receptor preferential ligand, morphine, were also greatly reduced in mastoparan-treated mice (Fig. 1). Mastoparan 17, an analog devoid of effect on G proteins (Higashijima et al., 1990), lacked activity on opioid antinociception. Comparable results were obtained when opioids were injected 1 or 24 h after mastoparan.
P. S{mchez-Bldzquez, J. Garzdn / European Journal of Pharmacology 258 (1994) 159-162
The analgesic potency of the higher doses of DAMGO and DPDPE appeared lessened, particularly those producing effects above 30% (Fig. 1). Thus, the analgesic dose-response curves of both opioids exhibited a reduced slope in mice receiving the venom peptide. The computed slopes (m) in control and mastoparan-treated mice were respectively: m = -55.61 and m = - 3 2 . 2 4 " , for DAMGO, and m = -51.21 and m = - 2 4 . 2 8 " , for DPDPE (* P<0.05; Tallarida and Murray (1981), test for parallelism). This result and the long-lasting activity that mastoparan displayed on opioid analgesia suggest a non-competitive quality for this antagonism.
3.2. Antagonism of opioid supraspinal antinociception in mastoparan pretreated mice The effect of naloxone (1-80 nmol/mouse i.c.v.), an antagonist at opioid receptors, and ICI 174864 (0.8-8 nmol/mouse i.c.v.), a selective antagonist of the ~ type of opioid receptors, were studied in mice treated with
100
Morphine ~.
--0-
+ 1 nmol + 10 nrnol
~
+ 80 nmol
Naloxone
•
50 25 0
I-
i
i
control 20
100
w n ~
DPDPE
18
~
16
I
:
14
12
+ 0.8 nmol IC1174864 + 80 nmol Naloxone
50 25 z~ 0
F-
zx I
control 22
different doses of mastoparan (from 10 -22 to 10 -12 mol/mouse). Fmol doses of mastoparan produced an antagonist effect on opioid-evoked analgesia that remained almost constant when the venom peptide was injected at doses 10000 times higher (Fig. 2). Naloxone was co-administered with morphine or given 15 min before DPDPE, whereas ICI 174864 was co-injected with DPDPE. The analgesia evoked by DPDPE was reduced by ICI 174864 but only to a limited extent, being 0.8 nmol of the tS-opioid receptor antagonist maximal to this effect (S~nchez-Blfizquez and Garz6n, 1993). This reduction was coincident with that produced by mastoparan (Fig. 2). ICI 174864 used at 0.8 or 8 nmol/mouse did not further decrease the diminishing activity of mastoparan on DPDPE analgesia. However, 80 nmol naloxone practically abolished thc antinociception produced by DPDPE. Mastoparan also lessened morphine analgesia to a limited extent (Fig. 2). Naloxone produced a dose-related antagonism of morphine-elicited analgesia both in controls and in mice receiving the venom peptide, mastoparan.
4. Discussion
75 LU 13.
161
~
1
t
I
I
I
20
18
16
14
12
-log (mol/mouse, i.c.v.) Mastoparan
Fig. 2. Effect of opioid antagonists on morphine- and DPDPE-induced supraspinal antinociception in mice injected with mastoparan. The venom peptide was i.c.v, administered 1 h before 20 nmol morphine or 10 nmol DPDPE. After 30 min (morphine) or 15 min (DPDPE) supraspinal analgesia was evaluated in the warm water tail-flick test. Naloxone at the doses indicated in the figure was co-administered with morphine or given 15 min before DPDPE. ICI 174864 was co-administered with DPDPE. * Significant difference with respect to the corresponding control group not receiving mastoparan. Experimental details as in Fig. 1.
G proteins appear to be conspicuous targets for diverse biological toxins, i.e., pertussis and cholera toxins as well as mastoparan. These agents seem to reach their targets in the cellular membrane after i.c.v. administration thus being useful tools to ascribe G proteins to receptor-mediated effects. The in vivo administration of mastoparan resulted in a long-lasting impairment of the supraspinal antinociception evoked by opioids in mice when evaluated in the tail-flick test. This reducing effect was not shared by the inactive analog, mastoparan 17. The changes brought about by this venom peptide are probably not due to non-specific phenomena as the opioid antagonism remained practically steady from doses of fmol up to 10000 times greater. In in vitro studies mastoparan behaved as an agonist-receptor complex, that is, promoted the dissociation of GDP and the subsequent binding of GTP to the G a subunits of certain transducer proteins (Higashijima et al., 1988, 1990). However, after in vivo administration of this peptide no agonist effect could be observed, i.e., it did not change the basal latencies exhibited by the mice in the antinociceptive test. The antagonism exerted by mastoparan lasted much longer than the receptor-mediated effect produced by an acute dose of opioids. Therefore, it might account for the mastoparan activation of G proteins at doses higher than those required to interfere with opioid receptor regulation of the transducer proteins. The existence of different conformations of this peptide could explain these observations. The direct comparison of the in
162
P. Sdnchez-Bldzquez, J. Garz6n /European Journal of Pharmacology 258 (1994) 159-162
vivo effects of mastoparan with those previously reported for in vitro systems is difficult, as receptors and G proteins were reconstituted in phospholipid vesicles and /zM concentrations of the venom peptide were utilized (Higashijima et al., 1988, 1990). Receptors and G proteins are not continuously associated in the cellular membrane, rather they seem to be highly mobile (Hille, 1992). In this scheme, mastoparan binds to the Got domain where G receptors regulate its function (Higashijima et al., 1988, 1990), and interferes with the formation of receptor-G protein complexes. This idea agrees with the notion that G proteins are crucial for maintaining the high affinity state of the receptor. In fact, in reconstituted systems, mastoparan decreases the affinity of the muscarinic receptor to acetylcholine, to the extent observed after G T P y S treatment (Higashijima et al., 1990). Consequently, mastoparan, by reducing the availability of functional G proteins to the opioid receptor (and decreasing the high-affinity state), would impair the analgesic potency of opioid agonists. The antagonist of opioid receptors, naloxone, reduced the antinociception displayed by the opioids in mastoparan-treated mice. This result agrees with the concept of this peptide acting at a point beyond the opioid binding site. In the presence of the antagonist of ~-opioid receptors, ICI 174864, D P D P E still produced an effect, thus revealing a naloxone-sensitive non-delta component of this analgesia (S~inchez-Bl~izquez and Garz6n, 1993). O f interest was the observation that mastoparan only abolished a portion of the /z-opioid receptor-mediated supraspinal antinociception. This indicates the existence of a /z-opioid receptor-regulated G transducer protein insensitive to the blocking effect of mastoparan. This finding agrees with previous reports suggesting the diversity of the transducer system signalling opioid-evoked supraspinal analgesia. In vivo pertussis toxin, an agent that alters the function of G i / G o transducer proteins, produced a differential attenuation of opioid-mediated supraspinal antinociception (S~inchez-Bl~izquez and Garz6n, 1988, 1991). Analgesia mediated by /z-opioid receptors seems to involve Gi2 and the pertussis toxin-insensitive Gx/z proteins (S,'inchez-Bl~izquez et al., 1993), whereas the Gi3 subtype is mostly regulated by ~-opioid receptors (S~inchez-Blfizquez and Garz6n, 1993).
In conclusion, the analgesia mediated by 8-opioid receptors is mediated by a transduction sensitive to mastoparan. The one produced via/~-opioid receptors showed both a sensitive and an insensitive component regarding the blocking activity of mastoparan. Multiplicity of the transducer system regulated by opioid receptors to produce supraspinal antinociception is therefore suggested.
Acknowledgements This work was supported by funds provided by FIS 93/147 and CITRAN 5/1992.
References Higashijima, T., S. Uzu, T. Nakajima and E.M. Ross, 1988, Mastoparan, a peptide toxin from wasp venom, mimics receptors by activating GTP-binding regulatory proteins (G proteins), J. Biol. Chem. 263, 6491. Higashijima, T., J. Burnier and E.M. Ross, 1990, Regulation of Gi and Go by mastoparan, related amphiphilic peptide, and hydrophobic amines, J. Biol. Chem. 265, 14176. Hille, B., 1992, G protein-coupled mechanisms and nervous signalling, Neuron 9, 187. Mousli, M., J.L. Bueb, Ch. Bronner, R. Rouot and Y. Landry, 1990, G protein activation: a receptor-independent mode of action for cationic amphiphilic neuropeptides and venom peptides, Trends Pharmacol. Sci. 11,358. S~nchez-Bl~izquez, P. and J. Garz6n, 1988, Pertussis toxin differentially reduces the efficacy of opioids to produce supraspinal analgesia in the mouse, Eur. J. Pharmacol. 152, 357. S~nchez-Blhzquez, P, and J. Garz6n, 1991, Cholera toxin and pertussis toxin on opioid- and a2-mediated supraspinal analgesia in mice, Life Sci. 48, 1721. S~nchez-Bl~izquez, P. and J. Garz6n, 1993, 8-Opioid supraspinal antinociception in mice is mediated by Gi3 transducer proteins, Life Sci. 53, PL129. S~inchez-BI;izquez, P., J.L. Juarros, Y. Martlnez-Pefia, M.A. Castro and J. Garz6n, 1993, Gx/z and Gi2 transducer proteins on /z/8 opioid mediated supraspinal antinociception, Life Sci. 53, PL381. Tallarida, R.J. and R.B. Murray, 1981, Manual of Pharmacologic Calculations (Springer-Verlag, New York). Ueda, H., H. Harada, M. Nozaki, T. Katada, M. Ui, M. Satoh and H. Takagi, 1988, Reconstitution of rat brain/z opioid receptors with purified guanine nucleotide-binding regulatory proteins, G i and Go, Proc. Natl. Acad. Sci. USA 85, 7013. Vogel, Z., J. Barg, B. Atali and R. Simantov, 1990, Differential effect of V-, 8, and K ligands on G protein a subunits in cultured brain cells, J. Neurosci. Res. 27, 106.
e.jp European Journal of Pharmacology258 (1994) 159-162
Short communication
Mastoparan reduces the supraspinal analgesia mediated by lX/6-opioid receptors in mice Pilar Sfinchez-Blfizquez *, Javier Garzdn Neurofarmacologfa, Instituto Cajal, CSIC, Avenida DoctorArce 37, 28002Madrid, Spare
(Received 27 January 1994;accepted 31 March 1994)
Abstract
Intracerebroventricular (i.c.v.) administration of the venom peptide, mastoparan, to mice decreased to a limited extent opioid-induced supraspinal analgesia in a non-competitive fashion. The/x-opioid receptor agonists, [D-Ala2,N-MePhe4,Gly-olS]enkephalin (DAMGO) and morphine, the /~/~-opioid receptor ligands, human /3-endorphin-(1-31) and [D-Ala2,o-LeuS]enkephalin (DADLE), and the selective ligands of ~-opioid receptors, [D-Pen2'5]enkephalin (DPDPE) and [o-Ala2]deltorphin II, showed an impaired analgesic effect in mice given mastoparan. Mastoparan diminished the analgesic activity of DPDPE and [o-Ala2]deltorphin II to the same extent as observed after giving the 6-opioid receptor-selective antagonist, ICI 174864. The /z-opioid receptor-mediated analgesia that remained after mastoparan was abolished in the presence of the opioid antagonist, naloxone. Mastoparan after binding to Gia/Goot subunits could block opioid antinociception. The existence of a class of G protein functionally coupled to/z-opioid receptors, but resistant to the effect of mastoparan is suggested. Key words: Mastoparan; GTP-binding protein; Supraspinal analgesia; Opioid
1. Introduction
After binding to their receptors in the cellular membrane opioids activate GTP-binding transducer proteins that regulate the corresponding effectors (Ueda et al., 1988; Vogel et al., 1990). A series of studies indicated the diversity of the transducer system regulated b y / z / 6 - o p i o i d receptors to promote supraspinal analgesia. In this respect, bacterial toxins known to alter the function of G proteins, i.e., cholera and pertussis toxin, modified this opioid activity in a differential manner (Sfinchez-Blfizquez and Garz6n, 1988, 1991). Similarly, the i.c.v, injection of immunoglobulins raised against specific sequences of G a subunits also revealed multiple G proteins involved in this characteristic opioid quality (S~inchez-Blfizquez and Garz6n, 1993; S~inchez-Blfizquez et al., 1993). Mastoparan, a peptide toxin from wasp venom, has been reported to bind to the receptor domain of certain G proteins promoting an accelerated exchange of G D P by G T P at G a subunits (Higashijima et al., 1988).
* Corresponding author. Tel. 341 5854733, fax 341 5854754. 0014-2999/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0014-2999(94)00197-F
The cationic amphiphilic characteristics of this venom peptide facilitate access through the cellular membrane to interact directly with the G proteins (Mousli et al., 1990). To further explore the transducer system linked to opioid-mediated effects, mastoparan was i.c.v. administered to mice and the supraspinal antinociception elicited by different opioids was subsequently evaluated in the warm water tail-flick test.
2. Materials and methods 2.1. Animals and evaluation o f analgesia
Albino male CD-1 mice (Charles River, Barcelona, Spain) weighing 22-25 g were used throughout. The animals were kept at 22°C, with a 12 h l i g h t / d a r k cycle (8 a . m . / 8 p.m.) and food and water provided ad libitum. The animals were housed and used strictly in accordance with the guidelines of the European Community about Care and Use of Laboratory Animals. All substances were dissolved in saline, except [DAla2]deltorphin II which was dissolved in 0.1% dimethyl sulphoxide (DMSO), and injected i.c.v, in a
160
P. Sdnchez-Bldzquez, J. Garz6n / European Journal of Pharmacology 258 (1994) 159-162
final volume of 4 /~1 to mice under light ether anaesthesia. Injections were unilateral and subsequent administrations were performed on the same side. The warm water (52°C) tail-flick test was used to evaluate analgesia. Latencies in seconds were measured manually, before (basal latency) and after the administration of the opioid under study (test latency). A cut-off time of 10 s was selected and antinociceptive activity was expressed as a percent of the maximum possible effect (%MPE). The following equation was used: % M P E = 100 X (test latency - b a s a l ) / ( 1 0 - basal). A single dose of saline (control) or mastoparan was injected to the mice and afterwards the antinociceptive effects of the analgesic substances were evaluated. In the case of sequential treatments, the vehicle replaced the substance whose effect was being tested. Statistical significance was determined by analysis of variance ( A N O V A ) followed by Scheffe's test. The level of significance was set at P < 0.05. After the analgesic dose-response curves had been made, the slopes, EDs0 and 95% confidence limits were determined according to the method of Tallarida and Murray (1981).
2.2. Drugs
Time-Course
t
o
0
i
i
control lh
ld
3. Results
3.1. Effect of mastoparan on the supraspinal antinociception induced by opioids in the tail-flick test Mastoparan at the i.c.v, doses of 10 -22 to 10 - 9 m o l / m o u s e did not modify the basal latencies displayed by the mice in the antinociceptive test, and no behavioural disturbances were observed. Animals receiving 1 p m o l / m o u s e mastoparan showed a decreased response to the analgesic activity of i.c.v, opioids. This antagonism was long-lasting, the effect was fully expressed 60 min after a single administration of the venom peptide, being almost identical 24 h later, and declining slowly within a week (Fig. 1). The analgesic effects of D A M G O , a/z-opioid receptor-selective ligand, and D P D P E , a g-opioid receptorselective ligand, were significantly reduced in mice injected with the venom peptide (Fig. 1). The apparent ED50 values ( n m o l / m o u s e ) and 95% confidence limits
i
i
3d
i
i
5d
i
i
i
i
9d
7d
Interval post-Mastoparan (1 pmol/rnouse, i.c,v,)
0
LU
_¢ o0 O ft.
E .-1 E
Control;
r~
+ Mastoparan
DAMGO
JQ
DPDPE
75
75
50
50
25 0
25 I 12
I 11
o~
I 10
I 0 , I 9 9 -tog (mol/moulm, i.c.v.) Opioid
I
I Opioid;
~IB
I 8
+ Mastoparan
0 Morphine
The following substances were used: human /3-endorphin, D A M G O , D A D L E , D P D P E , [D-Ala2] deltorphin II, mastoparan and mastoparan 17 were purchased from Peninsula Laboratories (San Carlos, CA, USA); ICI 174864 from CRB (Cambridge, UK). Morphine sulphate came from Merck (Darmstadt, Germany). Naloxone hydrochloride was obtained from Sigma (Saint Louis, MO, USA).
i
20
f~-Endorphin 2
DADLE
Deltorphin II
0.3
20 nmol
Fig. 1. Antagonism by i.c.v, administered mastoparan on opioid-induced supraspinal antinociception. Mice received a single injection of 1 pmol mastoparan and the time course for the antagonist effect on 20 nmol morphine or I0 nmol DPDPE was studied at the intervals indicated (upper panel). Different groups of mice were used for each post-mastoparan interval studied. The dose-response curves for DAMGO and DPDPE (middle panel), or a single dose for the other opioids (lower panel), were evaluated 24 h after receiving the venom peptide. Antinociception was determined at the peak effect 30 min after morphine and /3-endorphin, 15 min after DAMGO, DADLE and DPDPE and 10 min after [D-Ala2]deltorphin II. Analgesia was expressed as a percent of the maximum possible effect (%MPE). The values are the means:l:S.E.M, from groups of 10-15 mice. * Significantly different from the control group that received saline instead of mastoparan, ANOVA-Scheffe's test, P < 0.05. for antinociception shown by these opioids in salineand mastoparan-treated animals were respectively: D A M G O , 0.08 (0.06-0.10) and 0.27 (0.19-0.38)*; D P D P E , 5.93 (3.95-8.89) and 35.48 (21.50-58.54)* (* significantly different from the control (saline) group, P < 0.05). The potency of the selective 8-opioid receptor ligand, deltorphin II, the/z/~5-opioid receptor agonist, D A D L E , the endogenous opioid,/3-endorphin and the /z-opioid receptor preferential ligand, morphine, were also greatly reduced in mastoparan-treated mice (Fig. 1). Mastoparan 17, an analog devoid of effect on G proteins (Higashijima et al., 1990), lacked activity on opioid antinociception. Comparable results were obtained when opioids were injected 1 or 24 h after mastoparan.
P. S{mchez-Bldzquez, J. Garzdn / European Journal of Pharmacology 258 (1994) 159-162
The analgesic potency of the higher doses of DAMGO and DPDPE appeared lessened, particularly those producing effects above 30% (Fig. 1). Thus, the analgesic dose-response curves of both opioids exhibited a reduced slope in mice receiving the venom peptide. The computed slopes (m) in control and mastoparan-treated mice were respectively: m = -55.61 and m = - 3 2 . 2 4 " , for DAMGO, and m = -51.21 and m = - 2 4 . 2 8 " , for DPDPE (* P<0.05; Tallarida and Murray (1981), test for parallelism). This result and the long-lasting activity that mastoparan displayed on opioid analgesia suggest a non-competitive quality for this antagonism.
3.2. Antagonism of opioid supraspinal antinociception in mastoparan pretreated mice The effect of naloxone (1-80 nmol/mouse i.c.v.), an antagonist at opioid receptors, and ICI 174864 (0.8-8 nmol/mouse i.c.v.), a selective antagonist of the ~ type of opioid receptors, were studied in mice treated with
100
Morphine ~.
--0-
+ 1 nmol + 10 nrnol
~
+ 80 nmol
Naloxone
•
50 25 0
I-
i
i
control 20
100
w n ~
DPDPE
18
~
16
I
:
14
12
+ 0.8 nmol IC1174864 + 80 nmol Naloxone
50 25 z~ 0
F-
zx I
control 22
different doses of mastoparan (from 10 -22 to 10 -12 mol/mouse). Fmol doses of mastoparan produced an antagonist effect on opioid-evoked analgesia that remained almost constant when the venom peptide was injected at doses 10000 times higher (Fig. 2). Naloxone was co-administered with morphine or given 15 min before DPDPE, whereas ICI 174864 was co-injected with DPDPE. The analgesia evoked by DPDPE was reduced by ICI 174864 but only to a limited extent, being 0.8 nmol of the tS-opioid receptor antagonist maximal to this effect (S~nchez-Blfizquez and Garz6n, 1993). This reduction was coincident with that produced by mastoparan (Fig. 2). ICI 174864 used at 0.8 or 8 nmol/mouse did not further decrease the diminishing activity of mastoparan on DPDPE analgesia. However, 80 nmol naloxone practically abolished thc antinociception produced by DPDPE. Mastoparan also lessened morphine analgesia to a limited extent (Fig. 2). Naloxone produced a dose-related antagonism of morphine-elicited analgesia both in controls and in mice receiving the venom peptide, mastoparan.
4. Discussion
75 LU 13.
161
~
1
t
I
I
I
20
18
16
14
12
-log (mol/mouse, i.c.v.) Mastoparan
Fig. 2. Effect of opioid antagonists on morphine- and DPDPE-induced supraspinal antinociception in mice injected with mastoparan. The venom peptide was i.c.v, administered 1 h before 20 nmol morphine or 10 nmol DPDPE. After 30 min (morphine) or 15 min (DPDPE) supraspinal analgesia was evaluated in the warm water tail-flick test. Naloxone at the doses indicated in the figure was co-administered with morphine or given 15 min before DPDPE. ICI 174864 was co-administered with DPDPE. * Significant difference with respect to the corresponding control group not receiving mastoparan. Experimental details as in Fig. 1.
G proteins appear to be conspicuous targets for diverse biological toxins, i.e., pertussis and cholera toxins as well as mastoparan. These agents seem to reach their targets in the cellular membrane after i.c.v. administration thus being useful tools to ascribe G proteins to receptor-mediated effects. The in vivo administration of mastoparan resulted in a long-lasting impairment of the supraspinal antinociception evoked by opioids in mice when evaluated in the tail-flick test. This reducing effect was not shared by the inactive analog, mastoparan 17. The changes brought about by this venom peptide are probably not due to non-specific phenomena as the opioid antagonism remained practically steady from doses of fmol up to 10000 times greater. In in vitro studies mastoparan behaved as an agonist-receptor complex, that is, promoted the dissociation of GDP and the subsequent binding of GTP to the G a subunits of certain transducer proteins (Higashijima et al., 1988, 1990). However, after in vivo administration of this peptide no agonist effect could be observed, i.e., it did not change the basal latencies exhibited by the mice in the antinociceptive test. The antagonism exerted by mastoparan lasted much longer than the receptor-mediated effect produced by an acute dose of opioids. Therefore, it might account for the mastoparan activation of G proteins at doses higher than those required to interfere with opioid receptor regulation of the transducer proteins. The existence of different conformations of this peptide could explain these observations. The direct comparison of the in
162
P. Sdnchez-Bldzquez, J. Garz6n /European Journal of Pharmacology 258 (1994) 159-162
vivo effects of mastoparan with those previously reported for in vitro systems is difficult, as receptors and G proteins were reconstituted in phospholipid vesicles and /zM concentrations of the venom peptide were utilized (Higashijima et al., 1988, 1990). Receptors and G proteins are not continuously associated in the cellular membrane, rather they seem to be highly mobile (Hille, 1992). In this scheme, mastoparan binds to the Got domain where G receptors regulate its function (Higashijima et al., 1988, 1990), and interferes with the formation of receptor-G protein complexes. This idea agrees with the notion that G proteins are crucial for maintaining the high affinity state of the receptor. In fact, in reconstituted systems, mastoparan decreases the affinity of the muscarinic receptor to acetylcholine, to the extent observed after G T P y S treatment (Higashijima et al., 1990). Consequently, mastoparan, by reducing the availability of functional G proteins to the opioid receptor (and decreasing the high-affinity state), would impair the analgesic potency of opioid agonists. The antagonist of opioid receptors, naloxone, reduced the antinociception displayed by the opioids in mastoparan-treated mice. This result agrees with the concept of this peptide acting at a point beyond the opioid binding site. In the presence of the antagonist of ~-opioid receptors, ICI 174864, D P D P E still produced an effect, thus revealing a naloxone-sensitive non-delta component of this analgesia (S~inchez-Bl~izquez and Garz6n, 1993). O f interest was the observation that mastoparan only abolished a portion of the /z-opioid receptor-mediated supraspinal antinociception. This indicates the existence of a /z-opioid receptor-regulated G transducer protein insensitive to the blocking effect of mastoparan. This finding agrees with previous reports suggesting the diversity of the transducer system signalling opioid-evoked supraspinal analgesia. In vivo pertussis toxin, an agent that alters the function of G i / G o transducer proteins, produced a differential attenuation of opioid-mediated supraspinal antinociception (S~inchez-Bl~izquez and Garz6n, 1988, 1991). Analgesia mediated by /z-opioid receptors seems to involve Gi2 and the pertussis toxin-insensitive Gx/z proteins (S,'inchez-Bl~izquez et al., 1993), whereas the Gi3 subtype is mostly regulated by ~-opioid receptors (S~inchez-Blfizquez and Garz6n, 1993).
In conclusion, the analgesia mediated by 8-opioid receptors is mediated by a transduction sensitive to mastoparan. The one produced via/~-opioid receptors showed both a sensitive and an insensitive component regarding the blocking activity of mastoparan. Multiplicity of the transducer system regulated by opioid receptors to produce supraspinal antinociception is therefore suggested.
Acknowledgements This work was supported by funds provided by FIS 93/147 and CITRAN 5/1992.
References Higashijima, T., S. Uzu, T. Nakajima and E.M. Ross, 1988, Mastoparan, a peptide toxin from wasp venom, mimics receptors by activating GTP-binding regulatory proteins (G proteins), J. Biol. Chem. 263, 6491. Higashijima, T., J. Burnier and E.M. Ross, 1990, Regulation of Gi and Go by mastoparan, related amphiphilic peptide, and hydrophobic amines, J. Biol. Chem. 265, 14176. Hille, B., 1992, G protein-coupled mechanisms and nervous signalling, Neuron 9, 187. Mousli, M., J.L. Bueb, Ch. Bronner, R. Rouot and Y. Landry, 1990, G protein activation: a receptor-independent mode of action for cationic amphiphilic neuropeptides and venom peptides, Trends Pharmacol. Sci. 11,358. S~nchez-Bl~izquez, P. and J. Garz6n, 1988, Pertussis toxin differentially reduces the efficacy of opioids to produce supraspinal analgesia in the mouse, Eur. J. Pharmacol. 152, 357. S~nchez-Blhzquez, P, and J. Garz6n, 1991, Cholera toxin and pertussis toxin on opioid- and a2-mediated supraspinal analgesia in mice, Life Sci. 48, 1721. S~nchez-Bl~izquez, P. and J. Garz6n, 1993, 8-Opioid supraspinal antinociception in mice is mediated by Gi3 transducer proteins, Life Sci. 53, PL129. S~inchez-BI;izquez, P., J.L. Juarros, Y. Martlnez-Pefia, M.A. Castro and J. Garz6n, 1993, Gx/z and Gi2 transducer proteins on /z/8 opioid mediated supraspinal antinociception, Life Sci. 53, PL381. Tallarida, R.J. and R.B. Murray, 1981, Manual of Pharmacologic Calculations (Springer-Verlag, New York). Ueda, H., H. Harada, M. Nozaki, T. Katada, M. Ui, M. Satoh and H. Takagi, 1988, Reconstitution of rat brain/z opioid receptors with purified guanine nucleotide-binding regulatory proteins, G i and Go, Proc. Natl. Acad. Sci. USA 85, 7013. Vogel, Z., J. Barg, B. Atali and R. Simantov, 1990, Differential effect of V-, 8, and K ligands on G protein a subunits in cultured brain cells, J. Neurosci. Res. 27, 106.