Lack of involvement of endogenous μ-receptor opioids in the hypothermic effects of clonidine in normotensive and spontaneously hypertensive rats

Neurophormacology Vol. 27, No. 5,

pp. 537-540, 1988

OOZS-3908/88 53.00+ 0.00 Copyright 0 1988Pergamon Press plc

Printed in Great Britain. All rights reserved

LACK OF INVOLVEMENT OF ENDOGENOUS /i-RECEPTOR OPIOIDS IN THE HYPOTHERMIC EFFECTS OF CLONIDINE IN NORMOTENSIVE AND SPONTANEOUSLY HYPERTENSIVE RATS S. J. LEWIS,’ JENNIFER SVEC,~ M. R. FENNESSY~and B. JARROTT’* ‘University of Melbourne, Clinical Pharmacology and Therapeutics Unit, Austin Hospital, Heidelberg, Victoria, 3084 Australia and *Department of Pharmacology, University of Melbourne, Parkville, Victoria, 3052, Australia (Accepted 21 December 1987) Summary-The effects of successive injections of the alpha-adrenoceptor agonist clonidine (25, 50 and lOOug/kg given at hourly intervals) on the body temperature of normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats, previously treated for 48 hr with slow release emulsions (subcutaneous) containing either morphine (morphine SR, 100 mg/kg), naloxone (naloxone SR, 80 mg/kg) or no drug (vehicle SR), were examined. The successive injections of clonidine produced dose-dependent falls in body temperature which were quantitatively similar in the vehicle-treated WKY and spontaneously hypertensive rats. The hypothermic effects of clonidine in the morphine-dependent WKY and spontaneously hypertensive rats, and in the naloxone-treated WKY and spontaneously hypertensive rats, were not different to those of the respective vehicle-treated controls. These results suggest that endogenous p-receptor opioid peptides do not have a major involvement in the hypothermic actions of clonidine, in either normotensive or spontaneously hypertensive rats. Key words:

clonidine,

body temperature,

chronic

morphine,

In rats, the acute injection of the alpha-adrenoceptor agonist clonidine produces a variety of pharmacological effects, including cardiovascular depression, analgesia, abolition of rapid-eye movement (REM)-sleep and hypothermia (see Schmitt, 1977). It

has been reported that the p-receptor opiate antagonist naloxone inhibits the clonidine-induced falls in blood pressure and heart rate in normotensive (Hamilton and Longman, 1982) and spontaneously hypertensive (SHR) (Farsang, Ramirez-Gonzalez, Mucci and Kunos, 1980) rats and clonidine-induced analgesia in the spontaneously hypertensive rat (Lin, Chi, Chandra and Tsay, 1980). However, other studies have demonstrated that naloxone does not modify the hypotensive actions of clonidine in normotensive rats, cats and humans and in hypertensive rats and humans (see Conway, Brown and Dollery, 1984). Nevertheless, the findings in the spontaneously hypertensive rat that clonidine enhanced the release of beta-endorphin from slices of brainstem (Kunos, Fa.rsang and Ramirez-Gonzales, 198 l), increased the release of this peptide from the anterior pituitary (Pettibone and Mueller, 1981) and that the intracerebroventricular (i.c.v.) injection of beta-endorphin antibodies attenuated the hypotensive effects of clonidine (Ramirez-Gonzalez, Tchakarov, Masqueda Garcia and Kunos, 1983) have been interpreted by the Kunos group as an involvement of this endo*To whom

correspondence

should

be addressed. 537

chronic

naloxone,

rats.

geneous p-receptor-selective peptide in the pharmacological actions of clonidine. In addition to hypotension (Petty, De Jong and De Wied, 1982), analgesia and catalepsy (Tseng, Wei, Loh and Li, 1980), the intracerebroventricular injection of beta-endorphin produces bidirectional changes in body temperature in rats which consist of hyperthermia with smaller doses and hypothermia with larger doses (Tseng et al., 1980). In order to investigate further the possibility that endogenous opioid peptides are involved in the pharmacological actions of clonidine, the present study examined whether or not the hypothermic actions of clonidine in normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats were modified in animals treated chronically with naloxone and in those made dependent upon (and tolerant to) morphine. METHODS General procedures

Female WKY (n = 20) and spontaneously hypertensive (n = 20) rats of 20-22 weeks of age were housed in individual cages in a room with a 12 hr light (0800-2000 hrk12 hr dark cycle and an ambient temperature of 22 f 1°C. These rats received slow release emulsions containing either morphine (morphine SR, lOOmg/kg, n = 8 per strain), naloxone (naloxone SR, 80 mg/kg, n = 6 per strain) or no drug (vehicle SR, it = 6 per strain), as described previously

538

S.J. LEWB et

(Laska and Fennessy, 1976). After 48 hr, when the rats were dependent upon morphine, each rat received a series of acute intraperitoneal (i.p.) injections, given at hourly intervals. The regime of injections was as follows: at 1000 hr, saline (0.9% w/v NaCl); at 1100 hr, clonidine (25 pg/kg); at 1200 hr, clonidine (50 pgglkg) and at 1300 hr, clonidine (100 pg/kg). The rectal temperatures were recorded immediately before the injections of saline were given and at 30 min intervals throughout the experiment. The body temperatures were measured by a thermistor probe inserted 67cm into the rectum and recorded by a Yellow Springs Telethermometer (Yellow Springs Instrument Company, U.S.A.). In order to familiarize the rats to the rectal probe, the body temperatures of the animals were recorded 6 times at hourly intervals on the day previous to the experiment and twice before the experiment was to begin. Statistics

All values are expressed as the mean + SEM of group data or differences from initial. The overall effects of clonidine on the body temperatures of the treatment groups were examined by analysis of variance (ANOVA) with repeated measures (BMDP Statistical Package, Department of Mathematics, University of Los Angeles, U.S.A.) followed by Student’s modified t-test with the Bonferoni modification for multiple between group comparisons (Wallenstein, Zucker and Fleiss, 1980). Subsequently, the sums of squares of each group were partitioned into orthogonal comparisons and these analyses demonstrated that the clonidine-induced changes in body temperature and the rate of recovery following the last injection of clonidine were explained almost totally by the linear partitionings. consequently, the linear slopes were derived for each rat. The first slope was that describing the relationship between the change (A) in body temperature from time zero (taken as the time immediately prior to the first dose of clonidine) vs log,, (dose). The second slope was that describing the relationship between the rate of recovery of body temperature following the last injection of clonidine. The significance of the mean slope for each group was determined using the formula t (statistic) = (mean slope x number of animals)/standard deviation of the slopes. Betweengroup compa~sons were performed by an initial oneway ANOVA, followed by Student’s modified i-test with the Bonferoni adjustment (Wallenstein et al., 1980). RESULTS

There were no differences in the body temperatures of the 6 treatment groups at the beginning of the experiment (P > 0.05 for all comparisons). The acute intraperitoneal injection of saline did not modify the mean body temperatures of the treatment goups, as measured 30 and 60min after administration

al.

(P < 0.45 for all comparisons; Fig. 1). The acute injections of clonidine (25, 50 and lOOpg/kg) produced dose-dependent falls in the body temperatures of the vehici~treat~ WKY rats (-0.3 + 0.3O”C, - 1.2 + 0.3”C and -2.3 _t 0.3”C, respectively, F (2, 10) = 171, P < 0.0001). There were no overall differences between the three WKY groups injected with clonidine (F (2, 17) = 1.7, P > 0.10) and the slopes of dose-response relationships (changes from initial body temperature vs log dose) were also similar (Table 1). The acute injections of these doses of clonidine produced somewhat less dose-dependent falls in the body temperatures of the vehicle-treated spontaneously hypertensive rats (-0.8 + 0.2”C, - 1.7 f 0.2”C and - 2.0 + 0.2”C respectively, F (2, 10) = 30.1, P < 0.001). Although there were no differences between the vehicle-treated WKY and spontaneously hypertensive rats injected with clonidine (F (1,lO) = 0.1, P > 0.25) the slope of the dose-response relationship for the spontaneously hy pertensive strain was significantly shallower than for the WKY rats (F (1, 10) = 30.3, P c 0.001, see Table 1). The h~othe~ic effects of the 25 &g/kg dose of

0

60

120

180

240

300

240

300

Time (mln)

0

60

12a

I(10

Time (mln)

Fig 1. The effects of an intraperitoneal injection of saline (0.9% w/v) followed by successive intraperitoneal injections of donidine (25, 50 and l~~~kg) on the body temperatures of WK and spontaneoutsy hypertensive (SHR) rats treated for 48 hr with vehicle, morphine (IOOmgjkg) and naloxone (80 mg/kg). The injections of clonidine produced significant falls in body temperature in all groups (P < 0.001 for all groups). In addition, the hypothermic effects of clonidine were similar in all groups (P > 0.05 for all comparisons).

539

Clonidine hypothermia and brain opioids Table 1. The slopes describing the relationships of change (A) in body temperature from initial vs log,, dose of clonidine (AOC/log,, dose slope I) and the rate of recovery following the last injection of clonidine (A”C/time, slope 2) for the 6 treatment groups. The values in parentheses represent the numbers of animals in each group Pretreatment Strain

SlOpe

Morphine (8)

Vehicle (6)

Naloxone (6)

1

WKY SHR

-3.3 + 0.2A - 1.9 * 0.3ac

- 2.8 * 0.4” -3.5 + 0.3a.b

-3.2 + 0.2a - 3.2 k 0.3P.b

2

WKY SHR

0.5 &O.lM 0.5 i 0.18

0.6 k 0.2” 0.5 + 0.18

0.6 + 0.1’ 0.5 i 0.18

‘P < 0.05 slope significantly different from zero. bP < 0.05 morphine or naloxone vs vehicle. ‘F’ < 0.05 spontaneously hypertensive (SHR) vehicle vs WKY vehicle.

thalamic origin (O’Donnell and Volicer, 1981; clonidine were significant in the vehicle-treated spontaneously hypertensive rats (P < O.Ol), whereas this Wright, Knecht, Badger, Samueloff, Toraason and dose did not modify the body temperature of the Dukes-Dobos, 1978). The present study found that spontaneously hypertensive rats differed from WKY vehicle-treated WKY animals (P > 0.33). Although rats in that the spontaneously hypertensive rats exthe largest dose of clonidine (100 pgg/kg) produced a similar reduction in the body temperatures of hibited a hypothermic response to the smallest dose of clonidine (25 pg/kg) whereas this dose did not both vehicle-treated strains (P > 0.38), the overall produce a significant hypothermia in WKY rats. On difference between the effects of the 25 and 100 pg/kg the other hand, the largest dose of clonidine dose was much less in the spontaneously hypertensive (100 pg/kg) produced a smaller fall in body temrats (Table 2). This resulted in the lower slope values perature of the spontaneously hypertensive rats, comfor clonidine in the vehicle-treated spontaneously hypertensive rats (Table 1). Essentially the same pared to the WKY rats. Overall, the slope of the findings were obtained in two further experiments dose-body temperature response relationship in the (Table 2). Although there were no overall differences vehicle-treated spontaneously hypertensive rats was between the 3 groups of spontaneously hypertensive more shallow than that in the WKY animals. r,ats injected with clonidine (F (2, 17) = 0.4, P > 0.5) Whether or not the different sensitivity of the spontathe slopes of the dose-response relationships for neously hypertensive rats to clonidine was due to clonidine for the morphine- and naloxone-pretreated differences in alpha-adrenoceptors within central spontaneously hypertensive rats were significantly thermoregulatory pathways, or resulted indirectly steeper than that of the vehicle-treated animals injec- from other pharmacological actions of clonidine, ted with clonidine (Table 1). After the last dose of cannot be determined from these studies. As the intraventricular injection of beta-endorphin clonidine (100 pg/kg), the body temperatures of all 6 groups recovered towards preinjection levels at simi- has been reported to alter body temperature (Tseng et al., 1980) and as clonidine has been reported to lar rates (slopes 2, Table 1). release endogenous beta-endorphin in spontaneously hypertensive rats (see introduction), whether or not DISCUSSION the endogenous p-receptor opioid peptides were inSpontaneously hypertensive rats display an in- volved in clonidine-induced falls in body temperature creased sensitivity both to heat stress (O’Donnell and of spontaneously hypertensive and WKY rats was Volicer, 1981; Wright, Iams and Knecht, 1977) and examined. However, the observation that the falls in body temperature in the cold stress (O’Donnell and Volicer, 1981) and these clonidine-induced thermoregulatory dysfunctions are probably of hypo- morphine-dependent and the naloxone-treated WKY Table 2. The changes in body temperature (“C) from initial values following cumulative doses of 25, 50 (not shown) and 100 @g/kg clonidine in vehicle-treated WKY and spontaneously hypertensive (SHR) rats, and the relative differences (PC) between the 25 and lOOpg/kg responses. Each value represents the mean + SEM (n = 6 rats per group) Change from initial WKY Clonidine @g/kg) 25

Exoeriment I 2 3

-0.3 -0.3 -0.2

* 0.3 + 0.2 f 0.2

100 -2.3 -2.6 -2.5

‘P < 0.05, change from initial, paired bP < 0.05, spontaneously hypertensive t-test (one tailed).

+ 0.3’ + 0.4’ + 0.2’

body temperature

(“C) SHR Clonidine @g/kg)

A

25

100

-1.9*0.1 -2.3 k 0.2 -2.2*0.1*

-0.8 + 0.20 -1.2*0.1s - I .o+ 0.2”

-2.0 * 0.2’ -1.7f0.2a -1.8+0.3’

f-test (one tailed). (difference between doses) vs WKY (difference

A -1.1 -0.5 -0.7

+0.2b * O.lb + 0.2b

between doses), paired

540

S. J. LEWISet al.

and spontaneously hypertensive rats were quantitatively similar to those recorded in the respective vehicle

slow-release

controls,

suggests

that

endo-

genous opioid peptides which are active at p-receptors (e.g. beta-endorphin) do not cause, nor do they act in any significant way to limit, the hypothermia induced by clonidine. The present findings with naloxone treated rats are in general agreement with reports that the acute administration of naloxone does not modify the hypotensive effects of clonidine in normotensive and hypertensive experimental animals and humans, but differs from others which have demonstrated that this p-receptor antag onist does diminish the hypotensive effects of clonidine in normotensive and hypertensive animals and humans and the analgesic effects of the alphaadrenoceptor agonist in spontaneously hy~rtensive rats (see introduction). The results from the morphine-dependent rats supports those obtained in the animals treated with slow-release naloxone. A diminished effect of clonidine in morphine-dependent rats would not only indicate cross-tolerance, but would suggest that endogenous eta-endorphin is normally involved in the hypothermic effects of the alpha-adrenoceptor agonist. On the other hand, an enhancement of the hypothermic actions of clonidine in morphine-dependent rats would suggest that betaendorphinergic systems are recruited as part of the reflex designed to limit the extent of the hypothe~ia in non-dependent rats. Indeed, Conway et al. (1984) reported that the hypotensive actions of clonidine were enhanced in morphine-dependent WKY and spontaneously hypertensive rats. In addition, despite the findings that the overall clonidine-induced falls in body temperature were similar in all spontaneouslyhypertensive treatment groups, it was of interest that the slopes of the dose-response relationships were increased in both the morphine-dependent and rats treated with slow-release naloxone, as compared to the animals treated with slow-release vehicle. These results suggest that endogenous actions of clonidine occur in this strain, although this involvement may not be directly mediated through primary thermoregulatory control systems. In summary, despite evidence that endogenous g-receptor opioid peptides, such as beta-endorphin, may be involved in the cardiovascular and analgesic effects of clonidine the results of the present study do not support a maior involvement of these peptides in the thermoregulatory actions of this- alphaadrenoceptor

agonist. authors would like to thank Mr for his help with the body temperature

Acknowledgements-The

Gary Anderson

recordings and Dr Anthony Verbeme for his useful criticisms. This work was supported by a Programme Grant from the National Health and Medical Research Council of Australia. REFERENCES Conway E. L., Brown M. J. and Dollery C. T. (1984) No evidence for involvement of endogenous opioid peptides in effects of clonidine on blood pressure, heart rate and plasma norepinephrine in anaesthetized rats. J. Pharmac. exp. Ther. 229: 803-808.

Farsang C., R~irez-Gon~lez

M. D., Mucci L. and Kunos G. (1980) Possible role of an endogenous opiate in the cardiovascular effects of central alpha adrenoceptor stimulation in spontaneously hypertensive rats. J. Pharmac.

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__

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