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Alterations by captopril of pain reactions due to thermal stimulation of the mouse foot: Interactions with morphine, naloxone and aprotinin
European Journal of Pharmacology, 63 (1980).167--177 © Elsevier/North-Holland Biomedical Press
167
ALTERATIONS BY CAPTOPRIL OF PAIN REACTIONS DUE TO THERMAL STIMULATION OF THE MOUSE FOOT: INTERACTIONS WITH MORPHINE, NALOXONE AND APROTININ * Z, SEVIM ERCAN, MUSTAFA ILHAN and R. KAZIM Tt~RKER **
Departments of Medical and Surgical Research and Pharmacology, Faculty of Medicine, University of Hacettepe and Department of Pharmacology, Faculty of Medicine, University of Ankara, Ankara, Turkey Received 20 September 1979, revised MS received 12 December 1979, accepted 30 January 1980
Z.S. ERCAN, M. ILHAN and R.K. Tt~RKER, Alterations by captopril of pain reactions due to thermal stimulation of the mouse foot: interactions with morphine, naloxone and aprotinin, European J. Pharmacol. 63 (1980) 167--177. The effects of Captopril (SQ 14,225) were studied in mice both by measuring the reaction time and observing the number of jumping mice in the 'hot-plate' test. Subcutaneous injections of captopril (0.1-0.4 mg/kg) produced a significant increase in the reaction time at the 4th hour after the injection while no difference in the reaction time was observed at other times. Captopril caused a clear-cut increase in the number of jumping mice which was found to be greater with lower doses of the compound. Morphine alone did not cause an increase in the number of jumping mice, but increased the reaction time which reached a maximum 1 h after administration and gradually returned to the control level after 6 h. Injection of morphine to the captopril-pretreated mice caused a highly significant increase in reaction time remaining above the control values for 3 h. This effect of morphine was antagonized by naloxone (2 mg/kg, s.c.). Captopril, when given together with morphine, prevented the increase in reaction time over 2 h, but caused a progressive and significant increase within 6 h. Morphine also prevented the jumping behaviour induced by captopril. Aprotinin (50 000 KIU/kg i.p.) when given together with captopril, produced a significant increase in reaction time but almost completely blocked the jumping reaction. Aprotinin also prevented the captopril-induced increase in the number of jumping mice. The possible mechanisms of analgesia and jumping behaviour due to thermal stimulation are discussed in the light of these findings. Hot-plate
Captopril
Morphine
Aprotinin
1. Introduction
There is growing evidence that recently discovered endogenous opioid peptides have some physiological role as hormones or neurotransmitters in the production of analgesia (Hughes, 1975; Terenius and WahlstrSm,
* This work is supported by a grant from Turkish Scientific and Technical Research Council (TAG-
350). ** Send correspondence to: Prof. Dr. R.K. Tfirker, A.U. Tip Fakiiltesi, Farmakoloji Enstitfisii, Sfhhiye, Ankara, Turkey.
Reaction time
Jumping
1975; Goldstein, 1976). These peptides, when injected intravenously (i.v.) (Tseng et al., 1976) or intracerebrally (Beluzzi et al., 1976), induce analgesia and have been proposed as endogenous ligands for opiate receptors (Terenius, 1978). Recently, evidence has also been presented indicating that a brain enkephalinase, which degrades enkephalin, has many characteristics of angiotensin-converting enzyme (ACE) (Swerts et al., 1979b; Benuck and Marks, 1979). Greenberg et al. (1979) observed that captopril which is an ACE inhibitor (Ondetti et al., 1977), inhibits the inactivation of Met-
168 enkephalin in the myenteric plexus of the longitudinal muscle of guinea-pig ileum. However, some recent studies did n o t support these observations (Buckett, 1979; Swerts et al., 1979a). We have recently noted that captopril could potentiate the analgesic effect of morphine in mice in a 'hot-plate' test (Tflrker et al., 1979). It is also well known that captopril can inhibit the activity of kininases (Murthy et al., 1978) which are responsible for the degradation of bradykinin. On the other hand, kinin-peptides have been shown to be the peripheral mediators of pain induced by thermal stimulation in the rat's paw (Rocha E Silva and Antonio, 1960) and the dog's limb (Keele and Armstrong, 1968). Thus, captopril may cause the accumulation of opioid peptides in the brain by inhibiting enkephalinase, but on the other hand, it may cause the accumulation of bradykinin which is a well known algesic peptide. In this study we observed that captopril potentiated the analgesic effect of morphine and also caused variable behavioural reactions to the thermal stimulation of the mouse foot. Possible mechanisms for these effects are discussed.
2. Materials and methods
2.1. General White adult mice of both sexes weighing 20-30 g (25.6 + 0.5, n = 190) from a homogenous strain were used. The animals were fed a standard food and were allowed to drink water ad libitum. The experiments were performed at room temperature (21°C) and all measurements were made between 9 am and 6 pm.
2.2. Hot-plate technique For the thermal stimulation of the mouse foot, the hot-plate m e t h o d of Eddy et al. (1950) was selected and a apparatus with an electrically heated thermostatically controlled
z.s. ERCAN ET AL. metal plate (30 X 30 X 10 cm) was used. The surface was maintained at a temperature of 55.0 -+- 0.5°C. The homogeneity of the surface temperature was controlled before each daily test at 50 different points on t h e plate using a thermocouple transducer (Grass TCT1) and was recorded on a Grass polygraph (Model 79C). The reaction time was designated as the interval between dropping the animals onto the surface of the hot plate and the time when they reacted to the heat. Behavioural responses used as endpoints were either licking of the paws or jumping. In the control studies all mice with the hot plate reacted with paw licking, however, after drug injections some reacted by jumping. The time interval and the number of jumping mice were also counted separately in each group. Cut-off time was 25 sec and all mice reacted within this period.
2.3. Experimental procedure The animals were divided into several groups of 15 mice (about 50% male and 50% female) and placed into separate cages. In a preliminary study sex differences were investigated in 20 male and 20 female mice and no difference in the reaction time to thermal stimulation was observed. After control measurements the animals were injected with saline, captopril, morphine hydrochloride or aprotinin. All injections were made subcutaneously (s.c.) except that of aprotinin. The latter drug was injected intraperitoneally (i.p.) (50000 KIU/ kg). The total volume of injected material was kept constant at 0.1 ml/10 g b.w. Measurements were taken 30 min, 1.0, 2.0, 4.0 and 6.0 h after injections. The experiments were performed in the following order. In the first group, the doseresponse relationship of captopril was studied. Captopril (0.1, 0.2, 0.4 and 0.8 mg/kg, s.c.) was injected to 15 mice for each dose. In the second group, morphine (1 mg/kg, s.c.) and morphine (1 mg/kg, s.c.) together with captopril (0.1 mg/kg, s.c.) were injected to two separate groups of mice. In the third group, aprotinin (50000 KIU/kg, i.p.) was injected
ALTERATION OF THERMAL PAIN REACTIONS BY CAPTOPRIL to 15 mice. In the fourth group, apmtinin (50000 KIU/kg, i.p.) together with captopril (0.1 mg/kg, s.c.) was injected to 15 mice. In the fifth group, administration of aprotinin (50000 KIU/kg, i.p.) followed 30 min after the injection of captopril (0.1 mg/kg, s.c.). In the final group, following initial measurements, 15 mice were injected with captopril (0.1 mg/kg, s.c.). The measurements were repeated after 2 h, then the same animals were injected with morphine (1 mg/kg, s.c.) and the measurements were repeated at the same intervals. After the final measurements at the 6th h, these mice were injected with naloxone ( 2 m g / k g , s.c.). The test was repeated 30 min after the naloxone injection. All measurements were repeated at the same intervals. A saline-injected group served as control. The number of mice which reacted by jumping was also recorded for each group.
2.4. Chemical and statistical analysis Morphine hydrochloride (Macfarland Smith), captopril (SQ 14,225, Squibb), aprotinin (Trasylol®, B a y e r ) a n d Naloxone (Endo) were freshly dissolved in saline before use. Significance was determined in all experiments using Student's t-test (2-tailed). The number of jumping mice in each group was evaluated statistically by using the difference between t w o sample proportions (as percents) (Goldstein, 1964). 3. Results
3.1. Dose-response relation o f cap topril The results obtained in 4 different groups are summarized in table 1. As shown, the reaction time to thermal stimulation of the mouse foot increased slightly but significantly above the control values at 4 h after the administration of captopril (0.1-0.4 mg/kg, s.c.). However, the values obtained at b o t h the fourth and sixth h after the injection of captopril (0.8 mg/kg, s.c.) were significantly different from the corresponding control
169
values. No significant change was noted with saline control injections or with any of the measurements made at various other time intervals following the injection of captopril. In the preliminary control tests, none of the animals reacted with jumping to the thermal stimulation. However, after the injection of captopril, jumping was observed in all the mice tested. Folllowing the injection of captopril (0.1 mg/kg, s.c.), the number of jumping mice gradually increased and finally reached a maximum after 2 h (table 2). The number of jumping mice was significantly higher in the group receiving captopril, 0.1 mg/kg, s.c. than in the other three treated groups. However, a sustained increase in the number of jumping mice was observed in the remaining three groups receiving captopril, 0.2-0.8 mg/kg, s.c., but the results obtained in these groups did not differ significantly from each other. The control group injected with saline also did n o t exhibit any jumping behaviour in response to thermal stimulation. The range of the latency times to jump from the hot plate was found to vary between 3.37.3 sec and no correlation was obtained between these times and the number of jumping mice in the experimental groups. For example, the lowest value for the latency time to jump was obtained in the group receiving captopril 0.8 mg/kg, s.c., 2 h previously. In this group the number of jumping mice was 9 of 15 {60%) animals. However, the highest latency time was obtained in the group receiving captopril 0.1 mg/kg, s.c., 4 h previously. In this group the number of jumping mice was 11 of 15 (80%) animals (table 2).
3.2. Injection morphine
of
captopril
together
with
Morphine ( l m g / k g , s.c.) produced an increase in the reaction time to thermal stimulation. The measured values (fig. 1) were found to be significantly different at 30 min, 1 and 2 h after the injection when
170
Z.S. E R C A N ET AL.
TABLE 1 Dose-response relation o f captopril o n the h o t - p l a t e test. R e a c t i o n time was c o n s i d e r e d to be the interval b e t w e e n the c o n t a c t o f t h e m o u s e w i t h the plate and paw-licking or vertical jumping. Means + SEM. Initial
Time after injection (h) 0.5
1
2
4
6
Saline control (0.1 ml/10 g b.w.) 5.5 + 0.4
5.8 + 0.2
5.7 + 0.3
5.3 +- 0.4
5.6 + 0.4
5.3 + 0.6
(15) '
(15)
(15)
(15)
(15)
(15)
Captopril (0.1 mg/kg) 5.6 + 0.7
6.5 + 0.7
6.4 -+ 1.1
5.3 + 0.7
(15)
(15)
(15)
(15)
7.2 + 0.7 2
5.6 + 0.9
6.0 -+ 1.1 (14)
5.8 + 0.6 (14)
7.8 ± 0.9 2 (14)
7.1 + 1.4 (14)
7.3 + 0.9 2
5.4 + 0.7
(15)
(15)
Captopril (0.2 mg/kg) 4.9 -+ 0.3 (14)
6.4 + 0.9 (14)
Captopril (0.4 mg/kg) 4.7 +- 0.3
5.9 +- 1.1
5.4 + 0.8
7.7 -+ 1.0
(15)
(15)
(15)
(15)
4.1 -+ 0.4 (15)
4.3 + 0.8 (15)
(15)
(15)
Captopril (0.8 mg/kg) 5.0 + 0.7 (15)
6.2 + 0.8 (15)
7.0 -+ 0.9 2 (15)
6.9 + 0.9 2 (15)
I N u m b e r o f e x p e r i m e n t s in p a r e n t h e s e s . 2p < 0.005 w h e n c o m p a r e d with t h e i r initial values. TABLE 2 Dose-response relation o f captopril o n h o t - p l a t e test. R T = r e a c t i o n time was c o n s i d e r e d t o be the interval b e t w e e n the c o n t a c t o f the m o u s e f o o t w i t h the plate and j u m p i n g . The n u m b e r o f j u m p i n g mice in each group was also c o u n t e d and e s t i m a t e d as % o f j u m p i n g mice (J%). Means +- S.E.M. Injections
Initial
Time after injection (h) 0.5
Saline (0.1ml/10 gb.w)
0
Captopril (mg/kg) 0.1 RT J%
0 0
1
0
2
0
4.7 + 1.8 20.0 + 10.3
(3) I
4
0
6
0
0
4.6-+ 1.5 53.3 +- 12.9
5.0-+ 0.8 80.0 +- 10.3
7 . 3 + 0.9 73.3 + 11.4
(8)
(12)
(11)
4.6 + 1.2 80.0 + 10.3 (12)
0.2RT
0
5.4-+ 2.9
4.3+- 1.3
4.8+- 0.9
6.3-+ 1.3
J%
0
28.6 -+ 12.1
42.9 + 13.2
42.9 -+ 13.2
35.7 ± 12.8
0
6.4 + 2.4
5.4 -+ 1.7
6.6 + 2.0
6.5 ± 1.1
0
46.7 -+ 12.9 (7)
46.7 -+ 12.9 (7)
40.0 +- 12.6 (6)
60.0 -+ 12.6 (9)
5.3 +- 0.9 40.0 +- 12.6
3.5 + 0.4 60.0 ± 12.6
3.3 ± 0.5 60.0 + 12.6
4.8 + 0.9 40.0 -+ 12.6
5.6 + 1.6 40.0 + 12.6
(6)
(9)
(9)
(6)
(6)
(4) 0.4 RT J% 0.8 RT J%
0 0
(6)
I N u m b e r o f j u m p i n g mice o u t o f 15 animals in parentheses.
(6)
(5)
5.9 -+ 1.9 57.1 -+ 13.2
(s) 4.2 + 1.1 46.7 -+ 12.9
(7)
A L T E R A T I O N OF TH E R M A L PAIN REACTIONS BY CAPTOPRIL
171
15-
jumping
reaction time
10-
5-
•
,
i
i
I
i
i
i
!
1
i
I
C
0.5
1
2
4
6
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Q5
1
2
"'"iv
~ !
4
@
Fig. 1. The effects of morphine, captopril (SQ 14,225) and morphine plus captopril on the reaction time and the number of jumping mice (%) induced by thermal stimulation (hot-plate test). Reaction time was the interval between the m o m e n t the mouse was placed on the hot plate and the m o m e n t the animal began licking its feet or vertical jumping. (C) initial values; 15 mice were used for each series. Each point represents the mean value of 15 measurements. Vertical bars indicate S.E.M. -" -', Morphine 1 mg/kg s.c.; o . . . . . . o, captopril 0.1 mg/kg s.c.; ix. . . . . . A, morphine 1 mg/kg s.c. + captopril 0.1 mg/kg s.c. Ordinate: reaction time (sec). Abscissa: time after injection (h).
compared with the initial value (P < 0.005). The reaction time at the 4th h was again significantly different ( P < 0.05). However, no significant difference was found for the measurements made 6 h after the injection. Captopril , when injected together with morphine, induced significant analgesia 30 min after the injection. The reaction times at 1 and 2 h after the injection of both drugs were found 'to be significantly lower ( P < 0.05) than the values obtained for morphine alone at t h e same intervals. The reaction times measured 4 and 6 h after the injection, however, were found to be significantly greater than the values for morphine alone ( P < 0.0025) (fig. 1). In the morphine injected group, only one animal reacted with jumping behaviour to the thermal stimulation. Morphine almost completely abolished the jumping reaction when injected together with captopril (fig. 1).
3.3. Interaction between captopril and aprotinin
The effect of aprotinin (50000 KIU/kg, i.p.) on the reaction time to thermal stimulation was investigated in the first group. Fig. 2 shows the results compared with those of captopril (0.1mg/kg, s.c.). A significant increase was obtained for the measurements 4 h after the injection of aprotinin (P < O.OO5). Jumping reaction to the thermal stimulation after the injection of aprotinin was minimal and was the same for all the measurements made (fig. 2). When aprotinin and captopril were injected together, the reaction time was increased at 1, 2, 4 and 6 h (P < 0.025 for 2 h, P < 0.05 for 1 and 4 h, P < 0.0025 for 6 h) when compared with the control value (fig. 3). The percentage of mice jumping decreased signifi-
172
Z.S. E R C A N ET AL. reaction time
10-
100-
5-
~5o-
I
I
I
,
I
i
C
0.5
1
2
4
6
jumping
C
o~
1
2
4
6
Fig. 2. Comparison of the effects of captopril (SQ 14,225) and aprotinin (Trasylol) alone on the reaction time and the n u m b e r o f jumping mice in the hot-plate test. (C) initial values. Each point shows the mean value of 15 measurements. Vertical bars represent S.E.M. -" e, Captopril 0.1 m g / k g s.c.; o . . . . . . o, aprotinin 50,000 K I U / k g i.p. Ordinate: reaction t i m e (sec). Abscissa: time after injection (h).
reaction time. This group of animals then received aprotinin and the measurements were repeated at the same intervals. The results are shown in fig. 4. A significant increase was observed in the reaction time at 2 h (P < 0 . 0 0 2 5 ) and 4 h (P < 0.00125). The jumping reaction measured at the
cantly when compared with the values obtained for captopril alone (fig. 3). In one series of experiments, 15 mice received captopril (0.1 mg/kg, s.c.) following control measurements. 47% of these animals showed the jumping reaction to thermal stimulation. No change was observed in the
reaction time
jumping 10@-
10-
./..! I;
........... E~O-
5-
~6 o~
!
C
I
0.5
I
!
I
!
1
2
4
6
C o6
I
2
•
6
Fig. 3. Change i n t h e reaction time and the n u m b e r o f jumping mice in the hot-plate test after injection of captopril (SQ 14,225) together with aprotinin (Trasylol) and comparison with the data obtained for captopril alone. Each p o i n t indicates the mean value of 15 measurements. Vertical bars represent S.E.M. ~, $, Captopril 0.1 m g / k g s.c.; o . . . . . . o, captopril 0.1 m g / k g s.c. + aprotinin 50,000 K I U / k g i.p. Ordinate: reaction time (sec). Abscissa: time after injection (h).
ALTERATION OF THERMAL PAIN REACTIONS BY CAPTOPRIL
173
reaction time jumping ZOO-
10
g
i
i
i
I
I
!
i
C
0.5
1
2
4
6
C
i
~5
i
i
i
!
1
2
4
6
Fig. 4. The effect of captopril (SQ 14,225) followed by aprotinin (Trasylol) in mice on the hot plate. The tests were repeated 30 rain after injection and these animals were then injected with aprotinin and the measurements repeated at the same intervals. The figure represents the comparison of these data with those obtained for captopril alone. (C) control values, (T) indicates the injection of aprotinin (Trasylol). Each point represents the mean value of 15 measurements + S.E.M. ¢, o, Captopril. 0.1 mg/kg s.c., ©. . . . . . ©, captopril 0.1 mg/kg s.c. + aprotinin 50,000 KIU/kg i.p. Ordinate: reaction time (sec). Abscissa: time after injection (h). s a m e i n t e r v a l s h o w e v e r , d e c r e a s e d significantly after the injection of aprotinin. No ch a n g e was observed in the r e a c t i o n time and the jumping reaction measured at the same intervals in the animals receiving s a l i n e a l o n e (0.1 m l / 1 0 g, b . w . ) .
3.4. Interactions o f captopril with morphine and naloxone I n a f i n a l g r o u p o f 15 m i c e c a p t o p r i l (0.1 m g / k g , s.c.) was first i n j e c t e d a n d t h e r e a c t i o n 15-
Fig. 5. The effect of captopril (SQ14,225) and morphine in mice on the hot plate. The animals were injected with morphine 2 h after injection of captopril, and the measurements were repeated for 4 h at the same intervals. At the 6th h measurement, these animals were injected with naloxone (2 mg/kg s.c.) and the test was repeated 30 min after injection of naloxone. The figure represents the comparison of these data with those obtained for morphine and saline. (M) indicates the injection time of morphine and (N) shows the injection time of naloxone. Each point represents the mean value of 15 measurements. Vertical bars indicate S.E.M. Morphine when injected to the animals pretreated with captopril produced a highly significant increase in the reaction time at the 4th and 6th h when compared with the value obtained for morphine alone at the 1st h (P < 0.001). • -', Morphine 1 mg/kg; o . . . . . -o, saline 0.1 ml/10 g b.w.; A. . . . . ~ , captopril 0.1 mg/kg. Ordinate: reactim, time (sec). Abscissa: time after injection (h).
...'° .~""
10-
5-
C
, 0.5
, 1
i 2
i 2.5
~ 3
w 4
! 6
. 6.5
174
time was measured 2 h after the treatment. No significant increase was observed in the reaction time. However, 33% of the animals showed a jumping reaction to thermal stimulation on the hot plate. Injection of morphine (1 mg/kg, s.c.) to the mice pretreated with captopril produced a highly significant increase in the reaction time for 30 min, 1, 2, 4 and 6 h after the morphine injection. The reaction time was significantly higher than that observed for morphine alone for the measurements at the 4th and 6th h. The jumping behaviour to thermal stimulation disappeared completely after the morphine injection. Injection of naloxone to the captopril + morphine-treated animals caused a significant decrease in the reaction time within 30 min (fig. 5). A b o u t 26% of the mice reacted with jumping to the thermal stimulation following the naloxone injection.
4. Discussion A main finding of this study is that the ACE inhibitor, captopril can enhance the analgesic effect of morphine, confirming our recent results (Tiirker et al., 1979). In order to explain this enhancement we suggest that captopril may inhibit brain enkephalinase and therefore cause the accumulation of enkephalins in the brain; accordingly analgesia induced b y morphine to thermal stimulation may increase. It is clear that this enhancement is not the result of a simple additive effect between the two drugs, because concomittant injections of captopril and morphine were shown to produce a highly significant increase in the reaction time for 4 and 6 h after the injection, whereas the increase in the reaction time induced b y morphine alone was found to return to the initial level within 4 or 6 h. Secondly, when morphine was injected to mice 2 h after the pretreatment with captopril, a highly significant increase was obtained in the reaction time at 4 h, compared with that obtained for morphine alone. Naloxone produced a complete antagonism of
Z.S. E R C A N E T AL.
this increased reaction time within 30 min of its administration, again suggesting a possible opioid peptide mediation of the interaction between captopril and morphine. These results support the hypothesis that opioid peptides are endogenous ligands for morphinelike drugs (Terenius, 1978). However, these results do not necessarily eliminate other possibilities such as direct interaction between captopril and morphine. It needs further research to establish the influence of captopril on the pharmacokinetic properties of morphine. It is well known that captopril inhibits ACE activity (Ondetti et al., 1977) as well as the activity of kininases (Murthy et al., 1978). The inhibition of kininases may cause the accumulation of kinin peptides in the tissue. It is also well known that bradykinin is the peripheral mediator of pain due to thermal stimulation (Rocha E Silva and Antonio, 1960). One can postulate that captopril may have an algesic effect possibly resulting from its inhibitory action on the activity of kininase. The potentiation b y captopril of the algesic effect of exogenously applied bradykinin has recently been seen in the dog (Nakano and Taira, 1979). Thus, this drug could have b o t h algesic and analgesic effects and would interact with an algesic or analgesic in both directions according to conditions. The results of the present study show that morphine caused an immediate increase in the reaction time which reached the maximum and gradually decreased and returned to the control level within 6 h. However, morphine when given together with captopril did not increase the reaction time within the first 2 h . After this period, the reaction time gradually increased and reached the maximum within 6 h. We suggest that captopril can inhibit kininase activity in the paw and thereby cause the accumulation of kinin peptides b y a peripheral mechanism within the first 2 h and thus the morphine analgesia: could be masked. Captopril alone in the doses used did not cause hyperalgesia in the animals tested on the hot plate, indicating that the drug did
ALTERATION OF THERMAL PAIN REACTIONS BY CAPTOPRIL not influence the release of bradykinin by heat, but prevents the degradation of the released bradykinin in the earlier phase. However, in the later phase the drug may penetrate to the central nervous system, reaching a concentration at which the inhibition of enkephalinase may occur and so the opioid peptides may accumulate. Another finding in support of this speculation is that captopril in the doses used produced an increase in the reaction time 4 and 6 h after the injection. Recent investigations concerning the inhibitory effect of captopril on enkephalinase are contradictory. Greenberg et al. (1979) have shown that captopril inhibited Met-enkephalin inactivation in the isolated myenteric plexus longitudinal muscle strip of guinea-pig ileum, whereas Buckett (1979) reported that captopril did not affect the action of enkephalins on the isolated electrically stimulated guineapig ileum and that it had no analgesic property in the mouse. The discrepancy between the results of Buckett (1979) and the present findings is probably due to differences in methods and in doses used. In our experiments captopril was used in doses ranging between 0.1 and 0.8 mg/kg, not 100 mg/kg which is a large dose for enzyme inhibition, especially as Olsen and Arrigoni-Martelli (1979) obtained a highly significant increase in urine kinin excretion using 0.1 mg/kg of captopril in the dog. On the other hand, Buckett (1979) did not' measure the reaction time for a long period of time as we did. We did not obtain any change in the reaction time 30 min after injection of captopril which is in agreement with the findings of Buckett (1979). The participation of the kallikrein-kinin system in the effect of captopril has also been verified using a kallikrein inhibitor, aprotinin (Vogel and Werle, 1970). Aprotinin, when given alone to mice induced an increase in reaction time 4 h after its injection. An analgesic property of aprotinin has previously been described for the dog tooth pain induced by the electrical stimulation of dentine (Tfirker and Tfirker, 1974). Aprotinin, when given
175
together with captopril produced an increase in reaction time to thermal stimt'.lation beginning at the first hour after injection and continuing for at least 6 h. These findings have been taken as further evidence for the analgesic effect of captopril which as probably masked by the accumulation of kinin peptides following injection of the drug. All these results indicate that the analgesic effect of captopril or the potentiation of morphine analgesia by this drug in the 'hotplate' test is dependent upon the activation of two naturally occurring bioactive polypeptide systems; namely, the kallikrein-kinin system through the inhibition of kininases in the early phase of the administration of the drug, and the accumulation of enkephalins, probably through the inhition of enkephalinase in the later phase. An interesting finding of this study is the difference in behavioural reactions of the mice to thermal stimulation following captopril pretreatment. Paw-licking was the general behavioural reaction observed. Only one mouse of 190 showed a jumping reaction to the thermal stimulation within the cut-off time during initial measurements. However, after the injection of captopril in the doses used, the number of jumping mice gradually increased. No increase in jumping was observed in the animals treated with saline, aprotinin or morphine alone. This indicates a close relationship between jumping and captopril treatment. It is also interesting to note that there was an inverse relation between the dose of captopril and the number of jumping mice. The highest percentage (80%) of jumping was observed with 0.1 mg/ kg of captopril whereas 0.8 mg/kg produced 40%. Both morphine and aprotinin could prevent the captopril-induced increase in jumping reaction. The jumping reaction was taken as evidence for hyperalgesia. Naloxone, a pure antagonist of opiate receptors was shown to lower the jumping latency without altering that of paw-licking (Jacob et al., 1974; Grevert and Goldstein, 1977; Kaplan and Glick, 1979). Furthermore, paw-licking
176
was taken as a measure of the sensitivity of perception of a nociceptive stimulus while the jumping behaviour was taken as a reaction to continued perception of a nociceptive stimulus and therefore appears to have more of an emotional component (Frederickson et al., 1977). The increase in the jumping reaction to thermal stimulation is probably due to the accumulation of kinin peptides in the paws caused by heat, because the inhibition of kallikrein activity by aprotinin completely prevented this behavioural reaction. Again the high circulating bradykinin level in the plasma and the inhibition of angiotensin formation by captopril probably caused capillary dilatation in the paw resulting in hypersensitivity to the thermal stimulation. A possible participation of tissue prostaglandins may also be taken into account, because we have recently reported an increased release of prostaglandins following the inhibition of ACE in the lung (Ercan et al., 1979). It is also well known that prostaglandins may contribute to the production of pain by sensitizing the pain receptors to other stimuli (Ferreira et al., 1973). This point is still under investigation. Morphine also prevented the jumping behaviour due to captopril by a central mechanism, probably causing an increase in opioid peptides and/or depressing the emotional interactions.
Acknowledgements The authors would like to express their thanks to Squibb, New Jersey, U.S.A., for the donation of captopril (SQ 14,225).
References Beluzzi, J.D., H. Grant, V. Garsky, D. Sarantakis, C.D. Vise and L. Stein, 1976, Analgesia induced in vivo by central administration of enkephalin in the rat, Nature {London) 260,625. Benuck, M. and N. Marks, 1979, Co-identity of brain angiotensin converting enzyme with a membrane
Z.S. ERCAN ET AL. bound dipeptidylcarboxypeptidase inactivating Met-Enkephalin, Biochem. Biophys. Res. Commun. 88, 215. Buckett, W.R., 1979, The actions of enkephalins are not modified by the 'kininase-II' inhibitor Captopril, European J. Pharmacol. 57,267. Eddy, N.B., C.F. Touchberry and J.E. Lieberman, 1950, Synthetic analgesics, J. Pharmacol. Exp. Ther. 98, 121. Ercan, Z.S., T.A. Bfkesoy and R.K. Tfrker, 1979, Role of endogenous prostaglandins on conversion of angiotensin I to angiotensin II in the pulmonary circulation, Prostaglandins Med. 2,343. Ferreira, S.H., S. Moncada and J.R. Vane, 1973, Prostaglandins and the mechanism of analgesia produced by aspirin-like drugs, Br. J. Pharmacol. 49, 86. Frederickson, R.C.A., V. Burgis and J.D. Edwards, 1977, Hyperalgesia induced by naloxone follows diurnal rhythm in responsivity to painful stimuli, Science 198, 756. Goldstein, A., 1964, Biostatistics: An Introductory Text (MacMillan, New York) p. 101. Goldstein, A., 1976, Opioid peptides (endorphins) in pituitary and brain, Science 193, 1081. Greenberg, R., E.H. O'Keefe and M.J. Antonaccio, 1979, Inhibition of Met-Enkephalin inactivation by captopril (SQ 14,225) in the isolated myenteric plexus longitudinal muscle strip of guinea-pig ileum, Fed. Proc. 38,457. Grevert, P. and A. Goldstein, 1977, Some effects of naloxone on behavior in the mouse, Psychopharmacology 53, 111. Hughes, J., 1975, Isolation an endogenous compound from the brain with pharmacological properties similar to morphine, Brain Res., 88,295. Jacob, J.J., E.C. Tremblay and M.C. Colombel, 1974, Facilitation de r6actions nociceptives par la naloxone chez la souris et chez le rat, Psychopharmacologia {Berlin) 37,217. Kaplan, R. and S.D. Glick, 1979, Prior exposure to footshock induced naloxone hyperalgesia, Life Sci. 24, 2309. Keele, C.A. and D. Armstrong, 1968, Mediators of pain: Pharmacology of pain, Vol. 9, Third International Pharmacological Meeting, eds. R.K.S. Lim, D. Armstrong and E.G. Pardo {Pergamon Press, New York) p. 18. Murthy, V.S., T.L. Valdron and M.E. Goldberg, 1978, Inhibition o f angiotensinconverting enzyme by SQ 14,225 in anaesthetized dogs: Hemodynamic and renal vascular effects, Proc. Soc. Exp. Biol. Med. 157,121. Nakano, T. and N. Taira, 1979, Potentiation of the algogenic action of bradykinin by an inhibitor of angiotensin I converting enzyme, Captopril (SQ 14,225}, Jap. J. Pharmacol. 29, 563.
ALTERATION OF THERMAL PAIN REACTIONS BY CAPTOPRIL Olsen, U.B. and E. Arrigoni-Martelli, 1979, The effects of kininase-II inhibition by SQ 14,225 on kidney kallikrein-kinin and prostaglandin systems in conscious dog, European J. Pharmacol. 54, 229. Ondetti, M.A., B. Rubin and D.W. Cushman, 1977, Design of specific inhibitors of angiotensin converting enzyme: New class o f orally active antihypertensive agents, Science 196,441. Rocha E Silva, M. and A. Antonio, 1960, Release of bradykinin and the mechanism of production of a 'Thermic edema (45°C) ' in the rat's paw, Med. Exp. 3 , 3 7 1 . Swerts, J.P., R. Perdrisot, G. Patey, S. De La Baume and J.C. Schwartz, 1979a, 'Enkephalinase' is distinct from brain 'angiotensin-converting enzyme', European J. Pharmacol. 57,279. Swerts, J.P., R. Perdrisot, B. Malfroy and J.C. Schwartz, 1979b, Is 'enkephalinase' identical with 'angiotensin-converting enzymes'?, European J. Pharmacol. 53,209.
177
Terenius, L., 1978, Endogenous peptides and analgesia, Ann. Rev. Pharmacol. Toxicol. 18,189. Terenius, L. and A. WahlstrSm, 1975, Search for an endogenous ligand for the opiate receptors, Acta Physiol. Scand. 94, 74. Tseng, L.F., H.H. Loh and C.H. Li, 1976, Betaendorphin as a potent analgesic by intravenous injection, Nature (London) 263, 239. Tiirker, M.N. and R.K. Tiirker, 1974, A study on the peripheral mediators of dental pain, Experientia 30,932. Tiirker, R.K., M. Ilhan and Z.S. Ercan, 1979, Potentiation by angiotensin converting enzyme inhibitor, SQ 14,225, of the analgesic effect of morphine in mice, European J. Pharmacol. 58, 99. Vogel, R. and E. Werle, 1970, Kallikrein inhibitors, in: Bradykinin Kallidin and Kallikrein, Handbook of Exptl. Pharmacol., ed. E.G. ErdSs (SpringerVerlag, New York) p. 213.
167
ALTERATIONS BY CAPTOPRIL OF PAIN REACTIONS DUE TO THERMAL STIMULATION OF THE MOUSE FOOT: INTERACTIONS WITH MORPHINE, NALOXONE AND APROTININ * Z, SEVIM ERCAN, MUSTAFA ILHAN and R. KAZIM Tt~RKER **
Departments of Medical and Surgical Research and Pharmacology, Faculty of Medicine, University of Hacettepe and Department of Pharmacology, Faculty of Medicine, University of Ankara, Ankara, Turkey Received 20 September 1979, revised MS received 12 December 1979, accepted 30 January 1980
Z.S. ERCAN, M. ILHAN and R.K. Tt~RKER, Alterations by captopril of pain reactions due to thermal stimulation of the mouse foot: interactions with morphine, naloxone and aprotinin, European J. Pharmacol. 63 (1980) 167--177. The effects of Captopril (SQ 14,225) were studied in mice both by measuring the reaction time and observing the number of jumping mice in the 'hot-plate' test. Subcutaneous injections of captopril (0.1-0.4 mg/kg) produced a significant increase in the reaction time at the 4th hour after the injection while no difference in the reaction time was observed at other times. Captopril caused a clear-cut increase in the number of jumping mice which was found to be greater with lower doses of the compound. Morphine alone did not cause an increase in the number of jumping mice, but increased the reaction time which reached a maximum 1 h after administration and gradually returned to the control level after 6 h. Injection of morphine to the captopril-pretreated mice caused a highly significant increase in reaction time remaining above the control values for 3 h. This effect of morphine was antagonized by naloxone (2 mg/kg, s.c.). Captopril, when given together with morphine, prevented the increase in reaction time over 2 h, but caused a progressive and significant increase within 6 h. Morphine also prevented the jumping behaviour induced by captopril. Aprotinin (50 000 KIU/kg i.p.) when given together with captopril, produced a significant increase in reaction time but almost completely blocked the jumping reaction. Aprotinin also prevented the captopril-induced increase in the number of jumping mice. The possible mechanisms of analgesia and jumping behaviour due to thermal stimulation are discussed in the light of these findings. Hot-plate
Captopril
Morphine
Aprotinin
1. Introduction
There is growing evidence that recently discovered endogenous opioid peptides have some physiological role as hormones or neurotransmitters in the production of analgesia (Hughes, 1975; Terenius and WahlstrSm,
* This work is supported by a grant from Turkish Scientific and Technical Research Council (TAG-
350). ** Send correspondence to: Prof. Dr. R.K. Tfirker, A.U. Tip Fakiiltesi, Farmakoloji Enstitfisii, Sfhhiye, Ankara, Turkey.
Reaction time
Jumping
1975; Goldstein, 1976). These peptides, when injected intravenously (i.v.) (Tseng et al., 1976) or intracerebrally (Beluzzi et al., 1976), induce analgesia and have been proposed as endogenous ligands for opiate receptors (Terenius, 1978). Recently, evidence has also been presented indicating that a brain enkephalinase, which degrades enkephalin, has many characteristics of angiotensin-converting enzyme (ACE) (Swerts et al., 1979b; Benuck and Marks, 1979). Greenberg et al. (1979) observed that captopril which is an ACE inhibitor (Ondetti et al., 1977), inhibits the inactivation of Met-
168 enkephalin in the myenteric plexus of the longitudinal muscle of guinea-pig ileum. However, some recent studies did n o t support these observations (Buckett, 1979; Swerts et al., 1979a). We have recently noted that captopril could potentiate the analgesic effect of morphine in mice in a 'hot-plate' test (Tflrker et al., 1979). It is also well known that captopril can inhibit the activity of kininases (Murthy et al., 1978) which are responsible for the degradation of bradykinin. On the other hand, kinin-peptides have been shown to be the peripheral mediators of pain induced by thermal stimulation in the rat's paw (Rocha E Silva and Antonio, 1960) and the dog's limb (Keele and Armstrong, 1968). Thus, captopril may cause the accumulation of opioid peptides in the brain by inhibiting enkephalinase, but on the other hand, it may cause the accumulation of bradykinin which is a well known algesic peptide. In this study we observed that captopril potentiated the analgesic effect of morphine and also caused variable behavioural reactions to the thermal stimulation of the mouse foot. Possible mechanisms for these effects are discussed.
2. Materials and methods
2.1. General White adult mice of both sexes weighing 20-30 g (25.6 + 0.5, n = 190) from a homogenous strain were used. The animals were fed a standard food and were allowed to drink water ad libitum. The experiments were performed at room temperature (21°C) and all measurements were made between 9 am and 6 pm.
2.2. Hot-plate technique For the thermal stimulation of the mouse foot, the hot-plate m e t h o d of Eddy et al. (1950) was selected and a apparatus with an electrically heated thermostatically controlled
z.s. ERCAN ET AL. metal plate (30 X 30 X 10 cm) was used. The surface was maintained at a temperature of 55.0 -+- 0.5°C. The homogeneity of the surface temperature was controlled before each daily test at 50 different points on t h e plate using a thermocouple transducer (Grass TCT1) and was recorded on a Grass polygraph (Model 79C). The reaction time was designated as the interval between dropping the animals onto the surface of the hot plate and the time when they reacted to the heat. Behavioural responses used as endpoints were either licking of the paws or jumping. In the control studies all mice with the hot plate reacted with paw licking, however, after drug injections some reacted by jumping. The time interval and the number of jumping mice were also counted separately in each group. Cut-off time was 25 sec and all mice reacted within this period.
2.3. Experimental procedure The animals were divided into several groups of 15 mice (about 50% male and 50% female) and placed into separate cages. In a preliminary study sex differences were investigated in 20 male and 20 female mice and no difference in the reaction time to thermal stimulation was observed. After control measurements the animals were injected with saline, captopril, morphine hydrochloride or aprotinin. All injections were made subcutaneously (s.c.) except that of aprotinin. The latter drug was injected intraperitoneally (i.p.) (50000 KIU/ kg). The total volume of injected material was kept constant at 0.1 ml/10 g b.w. Measurements were taken 30 min, 1.0, 2.0, 4.0 and 6.0 h after injections. The experiments were performed in the following order. In the first group, the doseresponse relationship of captopril was studied. Captopril (0.1, 0.2, 0.4 and 0.8 mg/kg, s.c.) was injected to 15 mice for each dose. In the second group, morphine (1 mg/kg, s.c.) and morphine (1 mg/kg, s.c.) together with captopril (0.1 mg/kg, s.c.) were injected to two separate groups of mice. In the third group, aprotinin (50000 KIU/kg, i.p.) was injected
ALTERATION OF THERMAL PAIN REACTIONS BY CAPTOPRIL to 15 mice. In the fourth group, apmtinin (50000 KIU/kg, i.p.) together with captopril (0.1 mg/kg, s.c.) was injected to 15 mice. In the fifth group, administration of aprotinin (50000 KIU/kg, i.p.) followed 30 min after the injection of captopril (0.1 mg/kg, s.c.). In the final group, following initial measurements, 15 mice were injected with captopril (0.1 mg/kg, s.c.). The measurements were repeated after 2 h, then the same animals were injected with morphine (1 mg/kg, s.c.) and the measurements were repeated at the same intervals. After the final measurements at the 6th h, these mice were injected with naloxone ( 2 m g / k g , s.c.). The test was repeated 30 min after the naloxone injection. All measurements were repeated at the same intervals. A saline-injected group served as control. The number of mice which reacted by jumping was also recorded for each group.
2.4. Chemical and statistical analysis Morphine hydrochloride (Macfarland Smith), captopril (SQ 14,225, Squibb), aprotinin (Trasylol®, B a y e r ) a n d Naloxone (Endo) were freshly dissolved in saline before use. Significance was determined in all experiments using Student's t-test (2-tailed). The number of jumping mice in each group was evaluated statistically by using the difference between t w o sample proportions (as percents) (Goldstein, 1964). 3. Results
3.1. Dose-response relation o f cap topril The results obtained in 4 different groups are summarized in table 1. As shown, the reaction time to thermal stimulation of the mouse foot increased slightly but significantly above the control values at 4 h after the administration of captopril (0.1-0.4 mg/kg, s.c.). However, the values obtained at b o t h the fourth and sixth h after the injection of captopril (0.8 mg/kg, s.c.) were significantly different from the corresponding control
169
values. No significant change was noted with saline control injections or with any of the measurements made at various other time intervals following the injection of captopril. In the preliminary control tests, none of the animals reacted with jumping to the thermal stimulation. However, after the injection of captopril, jumping was observed in all the mice tested. Folllowing the injection of captopril (0.1 mg/kg, s.c.), the number of jumping mice gradually increased and finally reached a maximum after 2 h (table 2). The number of jumping mice was significantly higher in the group receiving captopril, 0.1 mg/kg, s.c. than in the other three treated groups. However, a sustained increase in the number of jumping mice was observed in the remaining three groups receiving captopril, 0.2-0.8 mg/kg, s.c., but the results obtained in these groups did not differ significantly from each other. The control group injected with saline also did n o t exhibit any jumping behaviour in response to thermal stimulation. The range of the latency times to jump from the hot plate was found to vary between 3.37.3 sec and no correlation was obtained between these times and the number of jumping mice in the experimental groups. For example, the lowest value for the latency time to jump was obtained in the group receiving captopril 0.8 mg/kg, s.c., 2 h previously. In this group the number of jumping mice was 9 of 15 {60%) animals. However, the highest latency time was obtained in the group receiving captopril 0.1 mg/kg, s.c., 4 h previously. In this group the number of jumping mice was 11 of 15 (80%) animals (table 2).
3.2. Injection morphine
of
captopril
together
with
Morphine ( l m g / k g , s.c.) produced an increase in the reaction time to thermal stimulation. The measured values (fig. 1) were found to be significantly different at 30 min, 1 and 2 h after the injection when
170
Z.S. E R C A N ET AL.
TABLE 1 Dose-response relation o f captopril o n the h o t - p l a t e test. R e a c t i o n time was c o n s i d e r e d to be the interval b e t w e e n the c o n t a c t o f t h e m o u s e w i t h the plate and paw-licking or vertical jumping. Means + SEM. Initial
Time after injection (h) 0.5
1
2
4
6
Saline control (0.1 ml/10 g b.w.) 5.5 + 0.4
5.8 + 0.2
5.7 + 0.3
5.3 +- 0.4
5.6 + 0.4
5.3 + 0.6
(15) '
(15)
(15)
(15)
(15)
(15)
Captopril (0.1 mg/kg) 5.6 + 0.7
6.5 + 0.7
6.4 -+ 1.1
5.3 + 0.7
(15)
(15)
(15)
(15)
7.2 + 0.7 2
5.6 + 0.9
6.0 -+ 1.1 (14)
5.8 + 0.6 (14)
7.8 ± 0.9 2 (14)
7.1 + 1.4 (14)
7.3 + 0.9 2
5.4 + 0.7
(15)
(15)
Captopril (0.2 mg/kg) 4.9 -+ 0.3 (14)
6.4 + 0.9 (14)
Captopril (0.4 mg/kg) 4.7 +- 0.3
5.9 +- 1.1
5.4 + 0.8
7.7 -+ 1.0
(15)
(15)
(15)
(15)
4.1 -+ 0.4 (15)
4.3 + 0.8 (15)
(15)
(15)
Captopril (0.8 mg/kg) 5.0 + 0.7 (15)
6.2 + 0.8 (15)
7.0 -+ 0.9 2 (15)
6.9 + 0.9 2 (15)
I N u m b e r o f e x p e r i m e n t s in p a r e n t h e s e s . 2p < 0.005 w h e n c o m p a r e d with t h e i r initial values. TABLE 2 Dose-response relation o f captopril o n h o t - p l a t e test. R T = r e a c t i o n time was c o n s i d e r e d t o be the interval b e t w e e n the c o n t a c t o f the m o u s e f o o t w i t h the plate and j u m p i n g . The n u m b e r o f j u m p i n g mice in each group was also c o u n t e d and e s t i m a t e d as % o f j u m p i n g mice (J%). Means +- S.E.M. Injections
Initial
Time after injection (h) 0.5
Saline (0.1ml/10 gb.w)
0
Captopril (mg/kg) 0.1 RT J%
0 0
1
0
2
0
4.7 + 1.8 20.0 + 10.3
(3) I
4
0
6
0
0
4.6-+ 1.5 53.3 +- 12.9
5.0-+ 0.8 80.0 +- 10.3
7 . 3 + 0.9 73.3 + 11.4
(8)
(12)
(11)
4.6 + 1.2 80.0 + 10.3 (12)
0.2RT
0
5.4-+ 2.9
4.3+- 1.3
4.8+- 0.9
6.3-+ 1.3
J%
0
28.6 -+ 12.1
42.9 + 13.2
42.9 -+ 13.2
35.7 ± 12.8
0
6.4 + 2.4
5.4 -+ 1.7
6.6 + 2.0
6.5 ± 1.1
0
46.7 -+ 12.9 (7)
46.7 -+ 12.9 (7)
40.0 +- 12.6 (6)
60.0 -+ 12.6 (9)
5.3 +- 0.9 40.0 +- 12.6
3.5 + 0.4 60.0 ± 12.6
3.3 ± 0.5 60.0 + 12.6
4.8 + 0.9 40.0 -+ 12.6
5.6 + 1.6 40.0 + 12.6
(6)
(9)
(9)
(6)
(6)
(4) 0.4 RT J% 0.8 RT J%
0 0
(6)
I N u m b e r o f j u m p i n g mice o u t o f 15 animals in parentheses.
(6)
(5)
5.9 -+ 1.9 57.1 -+ 13.2
(s) 4.2 + 1.1 46.7 -+ 12.9
(7)
A L T E R A T I O N OF TH E R M A L PAIN REACTIONS BY CAPTOPRIL
171
15-
jumping
reaction time
10-
5-
•
,
i
i
I
i
i
i
!
1
i
I
C
0.5
1
2
4
6
C
Q5
1
2
"'"iv
~ !
4
@
Fig. 1. The effects of morphine, captopril (SQ 14,225) and morphine plus captopril on the reaction time and the number of jumping mice (%) induced by thermal stimulation (hot-plate test). Reaction time was the interval between the m o m e n t the mouse was placed on the hot plate and the m o m e n t the animal began licking its feet or vertical jumping. (C) initial values; 15 mice were used for each series. Each point represents the mean value of 15 measurements. Vertical bars indicate S.E.M. -" -', Morphine 1 mg/kg s.c.; o . . . . . . o, captopril 0.1 mg/kg s.c.; ix. . . . . . A, morphine 1 mg/kg s.c. + captopril 0.1 mg/kg s.c. Ordinate: reaction time (sec). Abscissa: time after injection (h).
compared with the initial value (P < 0.005). The reaction time at the 4th h was again significantly different ( P < 0.05). However, no significant difference was found for the measurements made 6 h after the injection. Captopril , when injected together with morphine, induced significant analgesia 30 min after the injection. The reaction times at 1 and 2 h after the injection of both drugs were found 'to be significantly lower ( P < 0.05) than the values obtained for morphine alone at t h e same intervals. The reaction times measured 4 and 6 h after the injection, however, were found to be significantly greater than the values for morphine alone ( P < 0.0025) (fig. 1). In the morphine injected group, only one animal reacted with jumping behaviour to the thermal stimulation. Morphine almost completely abolished the jumping reaction when injected together with captopril (fig. 1).
3.3. Interaction between captopril and aprotinin
The effect of aprotinin (50000 KIU/kg, i.p.) on the reaction time to thermal stimulation was investigated in the first group. Fig. 2 shows the results compared with those of captopril (0.1mg/kg, s.c.). A significant increase was obtained for the measurements 4 h after the injection of aprotinin (P < O.OO5). Jumping reaction to the thermal stimulation after the injection of aprotinin was minimal and was the same for all the measurements made (fig. 2). When aprotinin and captopril were injected together, the reaction time was increased at 1, 2, 4 and 6 h (P < 0.025 for 2 h, P < 0.05 for 1 and 4 h, P < 0.0025 for 6 h) when compared with the control value (fig. 3). The percentage of mice jumping decreased signifi-
172
Z.S. E R C A N ET AL. reaction time
10-
100-
5-
~5o-
I
I
I
,
I
i
C
0.5
1
2
4
6
jumping
C
o~
1
2
4
6
Fig. 2. Comparison of the effects of captopril (SQ 14,225) and aprotinin (Trasylol) alone on the reaction time and the n u m b e r o f jumping mice in the hot-plate test. (C) initial values. Each point shows the mean value of 15 measurements. Vertical bars represent S.E.M. -" e, Captopril 0.1 m g / k g s.c.; o . . . . . . o, aprotinin 50,000 K I U / k g i.p. Ordinate: reaction t i m e (sec). Abscissa: time after injection (h).
reaction time. This group of animals then received aprotinin and the measurements were repeated at the same intervals. The results are shown in fig. 4. A significant increase was observed in the reaction time at 2 h (P < 0 . 0 0 2 5 ) and 4 h (P < 0.00125). The jumping reaction measured at the
cantly when compared with the values obtained for captopril alone (fig. 3). In one series of experiments, 15 mice received captopril (0.1 mg/kg, s.c.) following control measurements. 47% of these animals showed the jumping reaction to thermal stimulation. No change was observed in the
reaction time
jumping 10@-
10-
./..! I;
........... E~O-
5-
~6 o~
!
C
I
0.5
I
!
I
!
1
2
4
6
C o6
I
2
•
6
Fig. 3. Change i n t h e reaction time and the n u m b e r o f jumping mice in the hot-plate test after injection of captopril (SQ 14,225) together with aprotinin (Trasylol) and comparison with the data obtained for captopril alone. Each p o i n t indicates the mean value of 15 measurements. Vertical bars represent S.E.M. ~, $, Captopril 0.1 m g / k g s.c.; o . . . . . . o, captopril 0.1 m g / k g s.c. + aprotinin 50,000 K I U / k g i.p. Ordinate: reaction time (sec). Abscissa: time after injection (h).
ALTERATION OF THERMAL PAIN REACTIONS BY CAPTOPRIL
173
reaction time jumping ZOO-
10
g
i
i
i
I
I
!
i
C
0.5
1
2
4
6
C
i
~5
i
i
i
!
1
2
4
6
Fig. 4. The effect of captopril (SQ 14,225) followed by aprotinin (Trasylol) in mice on the hot plate. The tests were repeated 30 rain after injection and these animals were then injected with aprotinin and the measurements repeated at the same intervals. The figure represents the comparison of these data with those obtained for captopril alone. (C) control values, (T) indicates the injection of aprotinin (Trasylol). Each point represents the mean value of 15 measurements + S.E.M. ¢, o, Captopril. 0.1 mg/kg s.c., ©. . . . . . ©, captopril 0.1 mg/kg s.c. + aprotinin 50,000 KIU/kg i.p. Ordinate: reaction time (sec). Abscissa: time after injection (h). s a m e i n t e r v a l s h o w e v e r , d e c r e a s e d significantly after the injection of aprotinin. No ch a n g e was observed in the r e a c t i o n time and the jumping reaction measured at the same intervals in the animals receiving s a l i n e a l o n e (0.1 m l / 1 0 g, b . w . ) .
3.4. Interactions o f captopril with morphine and naloxone I n a f i n a l g r o u p o f 15 m i c e c a p t o p r i l (0.1 m g / k g , s.c.) was first i n j e c t e d a n d t h e r e a c t i o n 15-
Fig. 5. The effect of captopril (SQ14,225) and morphine in mice on the hot plate. The animals were injected with morphine 2 h after injection of captopril, and the measurements were repeated for 4 h at the same intervals. At the 6th h measurement, these animals were injected with naloxone (2 mg/kg s.c.) and the test was repeated 30 min after injection of naloxone. The figure represents the comparison of these data with those obtained for morphine and saline. (M) indicates the injection time of morphine and (N) shows the injection time of naloxone. Each point represents the mean value of 15 measurements. Vertical bars indicate S.E.M. Morphine when injected to the animals pretreated with captopril produced a highly significant increase in the reaction time at the 4th and 6th h when compared with the value obtained for morphine alone at the 1st h (P < 0.001). • -', Morphine 1 mg/kg; o . . . . . -o, saline 0.1 ml/10 g b.w.; A. . . . . ~ , captopril 0.1 mg/kg. Ordinate: reactim, time (sec). Abscissa: time after injection (h).
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time was measured 2 h after the treatment. No significant increase was observed in the reaction time. However, 33% of the animals showed a jumping reaction to thermal stimulation on the hot plate. Injection of morphine (1 mg/kg, s.c.) to the mice pretreated with captopril produced a highly significant increase in the reaction time for 30 min, 1, 2, 4 and 6 h after the morphine injection. The reaction time was significantly higher than that observed for morphine alone for the measurements at the 4th and 6th h. The jumping behaviour to thermal stimulation disappeared completely after the morphine injection. Injection of naloxone to the captopril + morphine-treated animals caused a significant decrease in the reaction time within 30 min (fig. 5). A b o u t 26% of the mice reacted with jumping to the thermal stimulation following the naloxone injection.
4. Discussion A main finding of this study is that the ACE inhibitor, captopril can enhance the analgesic effect of morphine, confirming our recent results (Tiirker et al., 1979). In order to explain this enhancement we suggest that captopril may inhibit brain enkephalinase and therefore cause the accumulation of enkephalins in the brain; accordingly analgesia induced b y morphine to thermal stimulation may increase. It is clear that this enhancement is not the result of a simple additive effect between the two drugs, because concomittant injections of captopril and morphine were shown to produce a highly significant increase in the reaction time for 4 and 6 h after the injection, whereas the increase in the reaction time induced b y morphine alone was found to return to the initial level within 4 or 6 h. Secondly, when morphine was injected to mice 2 h after the pretreatment with captopril, a highly significant increase was obtained in the reaction time at 4 h, compared with that obtained for morphine alone. Naloxone produced a complete antagonism of
Z.S. E R C A N E T AL.
this increased reaction time within 30 min of its administration, again suggesting a possible opioid peptide mediation of the interaction between captopril and morphine. These results support the hypothesis that opioid peptides are endogenous ligands for morphinelike drugs (Terenius, 1978). However, these results do not necessarily eliminate other possibilities such as direct interaction between captopril and morphine. It needs further research to establish the influence of captopril on the pharmacokinetic properties of morphine. It is well known that captopril inhibits ACE activity (Ondetti et al., 1977) as well as the activity of kininases (Murthy et al., 1978). The inhibition of kininases may cause the accumulation of kinin peptides in the tissue. It is also well known that bradykinin is the peripheral mediator of pain due to thermal stimulation (Rocha E Silva and Antonio, 1960). One can postulate that captopril may have an algesic effect possibly resulting from its inhibitory action on the activity of kininase. The potentiation b y captopril of the algesic effect of exogenously applied bradykinin has recently been seen in the dog (Nakano and Taira, 1979). Thus, this drug could have b o t h algesic and analgesic effects and would interact with an algesic or analgesic in both directions according to conditions. The results of the present study show that morphine caused an immediate increase in the reaction time which reached the maximum and gradually decreased and returned to the control level within 6 h. However, morphine when given together with captopril did not increase the reaction time within the first 2 h . After this period, the reaction time gradually increased and reached the maximum within 6 h. We suggest that captopril can inhibit kininase activity in the paw and thereby cause the accumulation of kinin peptides b y a peripheral mechanism within the first 2 h and thus the morphine analgesia: could be masked. Captopril alone in the doses used did not cause hyperalgesia in the animals tested on the hot plate, indicating that the drug did
ALTERATION OF THERMAL PAIN REACTIONS BY CAPTOPRIL not influence the release of bradykinin by heat, but prevents the degradation of the released bradykinin in the earlier phase. However, in the later phase the drug may penetrate to the central nervous system, reaching a concentration at which the inhibition of enkephalinase may occur and so the opioid peptides may accumulate. Another finding in support of this speculation is that captopril in the doses used produced an increase in the reaction time 4 and 6 h after the injection. Recent investigations concerning the inhibitory effect of captopril on enkephalinase are contradictory. Greenberg et al. (1979) have shown that captopril inhibited Met-enkephalin inactivation in the isolated myenteric plexus longitudinal muscle strip of guinea-pig ileum, whereas Buckett (1979) reported that captopril did not affect the action of enkephalins on the isolated electrically stimulated guineapig ileum and that it had no analgesic property in the mouse. The discrepancy between the results of Buckett (1979) and the present findings is probably due to differences in methods and in doses used. In our experiments captopril was used in doses ranging between 0.1 and 0.8 mg/kg, not 100 mg/kg which is a large dose for enzyme inhibition, especially as Olsen and Arrigoni-Martelli (1979) obtained a highly significant increase in urine kinin excretion using 0.1 mg/kg of captopril in the dog. On the other hand, Buckett (1979) did not' measure the reaction time for a long period of time as we did. We did not obtain any change in the reaction time 30 min after injection of captopril which is in agreement with the findings of Buckett (1979). The participation of the kallikrein-kinin system in the effect of captopril has also been verified using a kallikrein inhibitor, aprotinin (Vogel and Werle, 1970). Aprotinin, when given alone to mice induced an increase in reaction time 4 h after its injection. An analgesic property of aprotinin has previously been described for the dog tooth pain induced by the electrical stimulation of dentine (Tfirker and Tfirker, 1974). Aprotinin, when given
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together with captopril produced an increase in reaction time to thermal stimt'.lation beginning at the first hour after injection and continuing for at least 6 h. These findings have been taken as further evidence for the analgesic effect of captopril which as probably masked by the accumulation of kinin peptides following injection of the drug. All these results indicate that the analgesic effect of captopril or the potentiation of morphine analgesia by this drug in the 'hotplate' test is dependent upon the activation of two naturally occurring bioactive polypeptide systems; namely, the kallikrein-kinin system through the inhibition of kininases in the early phase of the administration of the drug, and the accumulation of enkephalins, probably through the inhition of enkephalinase in the later phase. An interesting finding of this study is the difference in behavioural reactions of the mice to thermal stimulation following captopril pretreatment. Paw-licking was the general behavioural reaction observed. Only one mouse of 190 showed a jumping reaction to the thermal stimulation within the cut-off time during initial measurements. However, after the injection of captopril in the doses used, the number of jumping mice gradually increased. No increase in jumping was observed in the animals treated with saline, aprotinin or morphine alone. This indicates a close relationship between jumping and captopril treatment. It is also interesting to note that there was an inverse relation between the dose of captopril and the number of jumping mice. The highest percentage (80%) of jumping was observed with 0.1 mg/ kg of captopril whereas 0.8 mg/kg produced 40%. Both morphine and aprotinin could prevent the captopril-induced increase in jumping reaction. The jumping reaction was taken as evidence for hyperalgesia. Naloxone, a pure antagonist of opiate receptors was shown to lower the jumping latency without altering that of paw-licking (Jacob et al., 1974; Grevert and Goldstein, 1977; Kaplan and Glick, 1979). Furthermore, paw-licking
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was taken as a measure of the sensitivity of perception of a nociceptive stimulus while the jumping behaviour was taken as a reaction to continued perception of a nociceptive stimulus and therefore appears to have more of an emotional component (Frederickson et al., 1977). The increase in the jumping reaction to thermal stimulation is probably due to the accumulation of kinin peptides in the paws caused by heat, because the inhibition of kallikrein activity by aprotinin completely prevented this behavioural reaction. Again the high circulating bradykinin level in the plasma and the inhibition of angiotensin formation by captopril probably caused capillary dilatation in the paw resulting in hypersensitivity to the thermal stimulation. A possible participation of tissue prostaglandins may also be taken into account, because we have recently reported an increased release of prostaglandins following the inhibition of ACE in the lung (Ercan et al., 1979). It is also well known that prostaglandins may contribute to the production of pain by sensitizing the pain receptors to other stimuli (Ferreira et al., 1973). This point is still under investigation. Morphine also prevented the jumping behaviour due to captopril by a central mechanism, probably causing an increase in opioid peptides and/or depressing the emotional interactions.
Acknowledgements The authors would like to express their thanks to Squibb, New Jersey, U.S.A., for the donation of captopril (SQ 14,225).
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