- Email: [email protected]
Nipecotic acid and guvacine antagonism on morphine analgesia in rats
Pharmacological Research Communications, Vol. 1 I, IVo. 8, 1979
657
NIPECOTICACID AND GUVACINE ANTAGONISM ON MORPHINE ANALGESIA IN RATS P.Mantegazza, R.Tammiso, L.Vicentini, F.Zambotti and N.Zonta Department of Pharmacology, School of Medicine, University of Milan, I t a l y .
SUMMARY In the attempt to further characterize the role of the GABA-ergic system on morphine analgesia, nipecotic acid and guvacine, inhibitors of GABA reuptake, were administered intraventricularly in rats pretreated with morphine. Under these conditions the animals showed a decreased response to the antinociceptive effect of morphine whencompared to control rats.
INTRODUCTION Several lines of i n d i r e c t investigations tend to implicate GABA in the mediation of the analgesic effect of morphine (Yoneda, Takashima & Kurijama, 1976; Ho, Loh & Way, 1976; Kuriyama & Yoneda, 1978). However'the data available are in many aspects contradictory most probably because of the d i f f i c u l t y of manipulations of central GABA synapses (Biswas & Carlson, 19,7). The i n h i b i t i o n of GABA transaminase by aminooxyacetic acid (AOAA) has been reported to potentiate the analgesic effect of morphine (Yoneda, Takashima & Kuriyama, 1976), while in other studies antagonism was found (Ho, Loh & Way, 1976).
This study was supported by C.N.R. g r a n t n. CT77.01401.04
0031-6989/79/080657-06/~2.00/0
0 1979 ~
ittllln Pl'.lrmll:~oglicll Society
65B
Pharmacological Research Communications, VoL l l, IVo. B, 1979
Biggio, Della Bella, Frigeni & Guidotti (1977) have demonstrated that the peripheral administration of muscimol, a specific GABA agonist at the receptor s i t e (Curtis, Duggan, Felix & Johnston, 1971) potentiated the morphine analgesia while other found no effect (Christensen, Arnt & Scheel-KrUger, 1978). On the contrary we have shown that the i n t r a v e n t r i c u l ar muscimol i n j e c t i o n decreased the morphine antinociceptive effect (Mantegazza, Tammiso, V i c e n t i n i , Zambotti & Zonta, 197g). The aim of our experiments was to examine the e f f e c t of the i n t r a v e n t r i c u l a r i n j e c t i o n s of n i p e c o t i c acid and guvacine, i n h i b i t o r s of GABA reuptake (Johnston, Krogsgaard-Larsen & Stephanson, 1975) on morphine analgesia.
MATERIAL AND METHODS Chronic polyethylene cannula w e r e implanted into both cerebral ventricles in Sprague-Dawley male rats of Charles River strain weighing llO-120 g under barbiturate anesthesia, (Nembutal 30 mg/kg) according to A l t a f f e r , De Balbian Werster, Hall, Long & D'Encarnacao (IgTO). Animals were placed in individual cages. Not less than 7 days l a t e r , nociceptive s e n s i t i v i t y was determined using the t a i l f l i c k test (D'Amour & Smith, 1941). The "reaction time" to heat was measured in tenths of a second; in basal conditions the t y p i c a l nociceptive response was obtained after a time of 35-45 tenths of a second. Each measuring would not be longer than 80 tenths of a second. Without anesthesia the animals were injected into each v e n t r i c l e with 300xug of nipecotic acid and with 300)ug (free base) of guvacine hydrochloride dissolved in lOjul saline. Control animals received an equal volume of saline. The dose of 600~g of i n h i b i t o r s was chosen because in preliminary experiments i t proved to be the most active in e l i c i t i n g the antagonistic e f f e c t on morphine analgesia. Morphine-hydrochloride (2 mg/kg) or saline were administered subcutaneously 15 min a f t e r nipecotic acid or guvacine.
Pharmacological Research Communications, Vol. 11, No. 8, 1979 IUILINI * IALINI
,1I
s t
s~
ere
IQ
/
,/S
I
tI* 4o
.o
eO
O0
30
t
tflt~DTt¢ /C.
t
t~Otmt.INI
Antinociceptive effect of morphine hydrochloride (2 mg/kg s.c.) in rats pretreated with nipecotic acid (300~g/vent r i c l e ) administered 15' before morphine, n= 9 for the sal i n e - s a l i n e group; n = II for the experimental groups. All values are mean + SEM O p L 0.01 compared to saline-saline group P~ 0 . 0 1 refer to differences between saline-morphine and nipecotic acid-morphine group.
The dose of morphine was chosen because in preliminary experiments i t proved to have the a b i l i t y to increase the reaction time about twofold. S t a t i s t i c a l evaluation was done according to the Kramer Test (Kramer, 1956).
RESULTS
In morphine-treated animals the pretreatment with nipecotic acid (300 p g / v e n t r i c l e ) s i g n i f i c a n t l y antagonizes the morphine induced analgesic responses for at least 60 mln after morphine i n j e c t i o n . Nipecotic acid ( 3 0 0 ~ g / v e n t r i c l e ) induced no s i g n i f i c a n t change in the t a i l f l i c k reaction time as compared to control group receiving saline only (Fig. I ) . The administration of guvacine ( 3 0 0 ~ g / v e n t r i c l e ) s i g n i f i c a n t l y decreased the morphine antinociceptive effect at 30 and
659
o~oo
Pharmacological Research Communications, VoL 11, No. 8, 1979
W
T
. - . 4 GW4~WI * ~
.........
.---'"
, , s"
X ,s
.
I.I
~
30
"
oJ
? .
.........
(~It/
. . . .
,-
I~IN)
Fig. Antinociceptive effect of morphine hydrochloride (2 mg/kg s . c . ) in rats pretreated with guvacine (300 ~ g / v e n t r i c l e ) administered 15' before morphine, n = 9 f o r the salinesaline group ; n = I I for the experimental groups. All values are mean + SEM 0 ~ ~ O.Ol compared to saline=saline group P ~ O.Ol refer to differences between saline-morphine and guvacine-morphine group. oU min a f t e r morphine i n j e c t i o n . No differences from the control algesic responses were observed in guvacine-treated animals (Fig. 2). With regard to the behaviour nipecotic acid and guvacine e l i c i t only a s l i g h t muscle relaxation. DISCUSSION Previous work from these laboratories has shown that muscimol, when injected i n t r a v e n t r i c u l a r l y , markedly attenuated the a n t i nociceptive effect of morphine (~antegaZza, Tammiso, V i c e n t i n i , Zambotti & Zonta, 1979). The effect of nipecotic acid and guvacine on morphine analgesia is q u a l i t a t i v e l y s i m i l a r to that observed with muscimo] even i f the l a t t e r was active at much lower doses. We suggested t h a t , among other p o s s i b i l i t i e s , muscimol could antagoniz~ the morphi'ne antinociceptive effect by acting on GABA-
Pharmacological Research Cornmunicat/ons, VoL 11, No. 8, 1979 ergic neurons located in the periaqueductal gray matter (PAG), the principal supraspinal.site where morphine acts to produce antinocicepti'on (Yaksh, Yeung &~Rudy,"1976). This suggestion was supported bY biochemical studies which have demonstrated that a glutamate d'ecarboxylase activ.ity andGABA-ergic neurons are located in the PAG (Be!in, Aguera, Tappaz & Pujol, 1978). A similarmechanism.of action could, be claimed to explain the antagonistic effect of nipecotic acid and guvacine on morphine analgesia. Nevertheless, irrispective of the mechanism involved, the present results show that intraventricular injections of nipecotic acid and guvacine markedly attenuate the antinociceptive effect of morphine. Moreover, the present data seem f u r t h e r l y to confirm our previous results obtained by using muscimol, a GABA agonist, indicating the involvement of the GABA-ergic system in morphine analgesia. REFERENCES .,Itaffer, F.B., De Balbian Herster, F., Hall, S., Long,C.J. & D'Encarnacao, P., Physio1.Behav. 5, 119-121, 1970 Belin, M.F., Aguera, M., Tappaz, M.L. & Pujol, J.F., Neurosci. Lett. Supp. 1 $255 Elsevier North Holland,1978 Biggio, G., Della Bella, D., Frigeni, V. & Guidotti, A. Neuropharmacology, 16_6, 149-150, 1977 Biswas, B, & Carlson, A., Naun. Schmied. Arch. Pharmacol. 29~9, 41-46, 1977 Christensen, A.V., Arnt, J. & Scheel-Kr~ger, J., Europ. J. Pharmaco1. 48 , 459-462, 1978 Curtis, D.R., Duggan, A.W., Felix, D. & Johnston, G.A.R., Brain Res. 32, 69-96, 1971 D'Amour, E.F. & Smith, D.L., J. Pharmacol. Exp. Ther. 72, 74-79, 1941 Ho, I . K . , Loh, H.H. & Way, E.L., Life Sci. 18, 1111-1124,1976
661
662
Pharmacological Research Communications, Vol. 11, No. 8, 1979
Kramer, C.J., Biometrics, 12, 307-310, 1956 Kuriyama, J. & Yoneda, Y.., Brain Res. 148, 163-179, 1978 Johnston, G.A.R., Krogsgaard-Larsen, P. & Stephanson, A.L., Nature, 25.8,, 627-628, 1975 Mantegazza, P., Tammiso, R., Vicentini, L., Zambotti, F. & Zonta,N., Br. J. Pharmac. 1979 in press Scheel-KrE}ger, J., Arnt, J. & Magelund, G., lleurosci, Lett.. 4, 351, 1977 Yaksh, T.L., Yeung, J.C. & Rudy, T.A., Brain Res. 114, 83-104, 1976 Yoneda, Y., Takashima, S. & Kuriyama, K., Biochem. Pharmac.
2s, 2669-2670, 1976
657
NIPECOTICACID AND GUVACINE ANTAGONISM ON MORPHINE ANALGESIA IN RATS P.Mantegazza, R.Tammiso, L.Vicentini, F.Zambotti and N.Zonta Department of Pharmacology, School of Medicine, University of Milan, I t a l y .
SUMMARY In the attempt to further characterize the role of the GABA-ergic system on morphine analgesia, nipecotic acid and guvacine, inhibitors of GABA reuptake, were administered intraventricularly in rats pretreated with morphine. Under these conditions the animals showed a decreased response to the antinociceptive effect of morphine whencompared to control rats.
INTRODUCTION Several lines of i n d i r e c t investigations tend to implicate GABA in the mediation of the analgesic effect of morphine (Yoneda, Takashima & Kurijama, 1976; Ho, Loh & Way, 1976; Kuriyama & Yoneda, 1978). However'the data available are in many aspects contradictory most probably because of the d i f f i c u l t y of manipulations of central GABA synapses (Biswas & Carlson, 19,7). The i n h i b i t i o n of GABA transaminase by aminooxyacetic acid (AOAA) has been reported to potentiate the analgesic effect of morphine (Yoneda, Takashima & Kuriyama, 1976), while in other studies antagonism was found (Ho, Loh & Way, 1976).
This study was supported by C.N.R. g r a n t n. CT77.01401.04
0031-6989/79/080657-06/~2.00/0
0 1979 ~
ittllln Pl'.lrmll:~oglicll Society
65B
Pharmacological Research Communications, VoL l l, IVo. B, 1979
Biggio, Della Bella, Frigeni & Guidotti (1977) have demonstrated that the peripheral administration of muscimol, a specific GABA agonist at the receptor s i t e (Curtis, Duggan, Felix & Johnston, 1971) potentiated the morphine analgesia while other found no effect (Christensen, Arnt & Scheel-KrUger, 1978). On the contrary we have shown that the i n t r a v e n t r i c u l ar muscimol i n j e c t i o n decreased the morphine antinociceptive effect (Mantegazza, Tammiso, V i c e n t i n i , Zambotti & Zonta, 197g). The aim of our experiments was to examine the e f f e c t of the i n t r a v e n t r i c u l a r i n j e c t i o n s of n i p e c o t i c acid and guvacine, i n h i b i t o r s of GABA reuptake (Johnston, Krogsgaard-Larsen & Stephanson, 1975) on morphine analgesia.
MATERIAL AND METHODS Chronic polyethylene cannula w e r e implanted into both cerebral ventricles in Sprague-Dawley male rats of Charles River strain weighing llO-120 g under barbiturate anesthesia, (Nembutal 30 mg/kg) according to A l t a f f e r , De Balbian Werster, Hall, Long & D'Encarnacao (IgTO). Animals were placed in individual cages. Not less than 7 days l a t e r , nociceptive s e n s i t i v i t y was determined using the t a i l f l i c k test (D'Amour & Smith, 1941). The "reaction time" to heat was measured in tenths of a second; in basal conditions the t y p i c a l nociceptive response was obtained after a time of 35-45 tenths of a second. Each measuring would not be longer than 80 tenths of a second. Without anesthesia the animals were injected into each v e n t r i c l e with 300xug of nipecotic acid and with 300)ug (free base) of guvacine hydrochloride dissolved in lOjul saline. Control animals received an equal volume of saline. The dose of 600~g of i n h i b i t o r s was chosen because in preliminary experiments i t proved to be the most active in e l i c i t i n g the antagonistic e f f e c t on morphine analgesia. Morphine-hydrochloride (2 mg/kg) or saline were administered subcutaneously 15 min a f t e r nipecotic acid or guvacine.
Pharmacological Research Communications, Vol. 11, No. 8, 1979 IUILINI * IALINI
,1I
s t
s~
ere
IQ
/
,/S
I
tI* 4o
.o
eO
O0
30
t
tflt~DTt¢ /C.
t
t~Otmt.INI
Antinociceptive effect of morphine hydrochloride (2 mg/kg s.c.) in rats pretreated with nipecotic acid (300~g/vent r i c l e ) administered 15' before morphine, n= 9 for the sal i n e - s a l i n e group; n = II for the experimental groups. All values are mean + SEM O p L 0.01 compared to saline-saline group P~ 0 . 0 1 refer to differences between saline-morphine and nipecotic acid-morphine group.
The dose of morphine was chosen because in preliminary experiments i t proved to have the a b i l i t y to increase the reaction time about twofold. S t a t i s t i c a l evaluation was done according to the Kramer Test (Kramer, 1956).
RESULTS
In morphine-treated animals the pretreatment with nipecotic acid (300 p g / v e n t r i c l e ) s i g n i f i c a n t l y antagonizes the morphine induced analgesic responses for at least 60 mln after morphine i n j e c t i o n . Nipecotic acid ( 3 0 0 ~ g / v e n t r i c l e ) induced no s i g n i f i c a n t change in the t a i l f l i c k reaction time as compared to control group receiving saline only (Fig. I ) . The administration of guvacine ( 3 0 0 ~ g / v e n t r i c l e ) s i g n i f i c a n t l y decreased the morphine antinociceptive effect at 30 and
659
o~oo
Pharmacological Research Communications, VoL 11, No. 8, 1979
W
T
. - . 4 GW4~WI * ~
.........
.---'"
, , s"
X ,s
.
I.I
~
30
"
oJ
? .
.........
(~It/
. . . .
,-
I~IN)
Fig. Antinociceptive effect of morphine hydrochloride (2 mg/kg s . c . ) in rats pretreated with guvacine (300 ~ g / v e n t r i c l e ) administered 15' before morphine, n = 9 f o r the salinesaline group ; n = I I for the experimental groups. All values are mean + SEM 0 ~ ~ O.Ol compared to saline=saline group P ~ O.Ol refer to differences between saline-morphine and guvacine-morphine group. oU min a f t e r morphine i n j e c t i o n . No differences from the control algesic responses were observed in guvacine-treated animals (Fig. 2). With regard to the behaviour nipecotic acid and guvacine e l i c i t only a s l i g h t muscle relaxation. DISCUSSION Previous work from these laboratories has shown that muscimol, when injected i n t r a v e n t r i c u l a r l y , markedly attenuated the a n t i nociceptive effect of morphine (~antegaZza, Tammiso, V i c e n t i n i , Zambotti & Zonta, 1979). The effect of nipecotic acid and guvacine on morphine analgesia is q u a l i t a t i v e l y s i m i l a r to that observed with muscimo] even i f the l a t t e r was active at much lower doses. We suggested t h a t , among other p o s s i b i l i t i e s , muscimol could antagoniz~ the morphi'ne antinociceptive effect by acting on GABA-
Pharmacological Research Cornmunicat/ons, VoL 11, No. 8, 1979 ergic neurons located in the periaqueductal gray matter (PAG), the principal supraspinal.site where morphine acts to produce antinocicepti'on (Yaksh, Yeung &~Rudy,"1976). This suggestion was supported bY biochemical studies which have demonstrated that a glutamate d'ecarboxylase activ.ity andGABA-ergic neurons are located in the PAG (Be!in, Aguera, Tappaz & Pujol, 1978). A similarmechanism.of action could, be claimed to explain the antagonistic effect of nipecotic acid and guvacine on morphine analgesia. Nevertheless, irrispective of the mechanism involved, the present results show that intraventricular injections of nipecotic acid and guvacine markedly attenuate the antinociceptive effect of morphine. Moreover, the present data seem f u r t h e r l y to confirm our previous results obtained by using muscimol, a GABA agonist, indicating the involvement of the GABA-ergic system in morphine analgesia. REFERENCES .,Itaffer, F.B., De Balbian Herster, F., Hall, S., Long,C.J. & D'Encarnacao, P., Physio1.Behav. 5, 119-121, 1970 Belin, M.F., Aguera, M., Tappaz, M.L. & Pujol, J.F., Neurosci. Lett. Supp. 1 $255 Elsevier North Holland,1978 Biggio, G., Della Bella, D., Frigeni, V. & Guidotti, A. Neuropharmacology, 16_6, 149-150, 1977 Biswas, B, & Carlson, A., Naun. Schmied. Arch. Pharmacol. 29~9, 41-46, 1977 Christensen, A.V., Arnt, J. & Scheel-Kr~ger, J., Europ. J. Pharmaco1. 48 , 459-462, 1978 Curtis, D.R., Duggan, A.W., Felix, D. & Johnston, G.A.R., Brain Res. 32, 69-96, 1971 D'Amour, E.F. & Smith, D.L., J. Pharmacol. Exp. Ther. 72, 74-79, 1941 Ho, I . K . , Loh, H.H. & Way, E.L., Life Sci. 18, 1111-1124,1976
661
662
Pharmacological Research Communications, Vol. 11, No. 8, 1979
Kramer, C.J., Biometrics, 12, 307-310, 1956 Kuriyama, J. & Yoneda, Y.., Brain Res. 148, 163-179, 1978 Johnston, G.A.R., Krogsgaard-Larsen, P. & Stephanson, A.L., Nature, 25.8,, 627-628, 1975 Mantegazza, P., Tammiso, R., Vicentini, L., Zambotti, F. & Zonta,N., Br. J. Pharmac. 1979 in press Scheel-KrE}ger, J., Arnt, J. & Magelund, G., lleurosci, Lett.. 4, 351, 1977 Yaksh, T.L., Yeung, J.C. & Rudy, T.A., Brain Res. 114, 83-104, 1976 Yoneda, Y., Takashima, S. & Kuriyama, K., Biochem. Pharmac.
2s, 2669-2670, 1976