0013-7227/91/1286-2799$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 128, No. 6 Printed in U.S.A.
Luteinizing Hormone-Releasing Hormone Alters the Hypothalamic Effects of Morphine in the Rat* LEE ANN BERGLUND AND JAMES W. SIMPKINS Department of Pharmacodynamics and the Center for the Neurobiology of Aging, University of Florida, Gainesville, Florida 32610
ABSTRACT. Female rats exhibit a generalized refractoriness to opiate stimulation during periods of steroid-induced LH secretion. In the present study we evaluated that role of LHRH in this steroid-induced effect on opiate-responsiveness. Central administration to ovariectomized rats of native LHRH or the LHRH agonist [Des-Gly10]LHRH ethylamide causes a dosedependent refractoriness to the hypothermic effects of morphine. The potency relationship of these two LHRH agonists in antagonizing morphine's effect was consistent with their potency in inducing LH release. Treatment of ovariectomized rats with
T
estradiol benzoate and progesterone in a regimen which induces a preovulatory-like LH surge, antagonized morphine-induced hypothermia, and the LHRH antagonist [D-Phe2, Pro3, D-Phe6) LHRH, reversed the effects of the gonadal steroids. These results indicate that the LHRH secretory dynamics associated with the preovulatory surge of LH may serve to modulate opiate responsiveness and thereby could serve to couple behavioral, sensory, and autonomic events with this neuroendocrine response to gonadal steroids. (Endocrinology 128: 2799-2804, 1991)
HE endogenous opiate peptides (EOP) appear to influence reproductive function in mammals through the tonic inhibition of hypothalamic LHRH secretion (1-3). Opiates also interact with gonadal steroids to enhance the sensitivity of the hypothalamus to steroid negative feedback on LHRH secretion (4-6). On proestrous afternoon or during the steroid-induced periods of LHRH hypersecretion (7-9), the LH secretory mechanism is refractory to opiate agonists and antagonists (10-13). Before and after the LH surge, the LH secretory system is sensitive to opiates and can be pharmacologically manipulated by either opiate agonists and antagonists (3, 10-13). Thus, neurochemical changes associated with, and perhaps causative in, the steroidinduced phase of LHRH hypersecretion, rather than the steroid environment itself, appears to be responsible for the refractoriness of the LH secretory mechanism to opiates. We recently observed that in association with the LH surge on proestrus and the LH surge induced by ovarian steroids, alterations in numerous responses to opiates occurs (12, 13). In these rats, locomotor, secretory, temperature, and analgesic responses to morphine administration are reduced or extinguished. As with the LHRH
secretory mechanism, the changes in opiates sensitivity of these responses could not be explained by the steroid environment of the animal, were only exhibited during the LH surge and appeared to be a function of the magnitude of the pituitary LH responses (12, 13). Together, these observations suggested a general reduction in opiates sensitivity during both endogenous and exogenous steroid-induced periods for LH hypersecretion. Although LHRH is recognized for its fundamental role in stimulating pituitary LH synthesis and secretion, this neuropeptide has also been implicated in other central nervous system functions. LHRH has been shown to facilitate lordosis behavior (14) and to modulate neuronal firing rates in regions associated with reproductive behavior (15). LHRH-like immunoreactivity (16, 17) and LHRH receptors (18, 19) have been described in extrahypothalamic regions of the central nervous system, lending anatomical support for a physiological non-LHreleasing role for this neuropeptide. Thus, the hypersecretion of LHRH associated with ovulation may contribute to the decline in opiate sensitivity observed in proestrous and steroid-treated female rats. To address this issue we examined the effect of intracerebroventricular (ICV) administration of LHRH on the responsiveness of rats to the hypothermic effects of morphine.
Received December 10,1990. Address correspondence and requests for reprints to: James W. Simpkins, Department of Pharmacodynamics and the Center for the Neurobiology of Aging, University of Florida, Gainesville, Florida 32610. * Supported by NIH Grant AG-02021.
Materials and Methods Animals Adult female Sprague-Dawley rats obtained from Charles Rivers Breeding Laboratories (Wilmington, MA) were housed
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LHRH-OPIATE INTERACTIONS
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in a temperature (25 ± 1 C) and light-controlled (illuminated 0500-1900 h daily) environment and provided water and Purina laboratory chow (Ralston-Purina, St. Louis, MO) ad libitum. Rats weighed 200-225 g upon arrival and after a 1-week acclimation period were bilaterally ovariectomized (OVX) under light ether anesthesia. All experiments were performed 4-6 weeks after ovariectomy and animals weighed 310-340 g. Rats were used in only one experiment and in no case were animals transferred between experiments. Steroid and drug treatments In most studies, long-term OVX rats were administered ICV, by a single injection, either saline (1 n\) or a neuropeptide agonist or antagonist at 1 h before treatment with a single dose of morphine sulfate (20 mg/kg sc; Merck, St. Louis, MO). The morphine sulfate dose used in these experiments was determined by preliminary range finding studies and the dose chosen was that which induced a marked hypothermic response in OVX (morphine-sensitive) rats (12, 13). A single dose was chosen for evaluation because of limitations in the number of animals which could be processed. The following drugs were administered ICV to OVX rats in 1 n\ saline: Saline (1 MD; native LHRH (10~4 to 10"1 fig); the LHRH agonist, [Des-Glylo]LHRH ethylamide (10~7 to 10~2 Mg); the LHRH antagonist [D-Phe2, Pro3, D-Phe6]LHRH (10~5 Mg); vasopressin (10~4 Mg), or somatostatin (10~4 Mg)- LHRH agonists and antagonists were purchased from Peninsula Laboratory Inc. (Belmont, CA); somatostatin and arginine vasopressin were purchased from Sigma Chemical Co. (St. Louis, MO). In one study, a preovulatory-like LH surge was induced by sc treatment with 7.5 jug estradiol benzoate (EB) at 1000 h 2 days before the experiment. Progesterone (5 mg P) was then administered 48 h after the EB treatment. This steroid regimen induces a surge in LH with onset at 1400-1600 h and maximal testers at about 1730 h. Except where noted, for both OVX and EBP rats, the neuropeptide was administered between 1500 and 1630 h and morphine sulfate was administered exactly 1 h later, between 1600 and 1730 h. Cannulation The cannula were made of 2-cm stainless steel tubing (0.029 in. ID, .035 in. OD; Small Parts Inc., Miami, FL). One to 2 weeks before the experimental rats were placed under 35 to 50 mg/kg BW sodium pentobarbital (Butler, Columbus, OH) anesthesia. The skull was exposed through a dorsal scalp incision and the cannula was stereotaxically placed in the lateral ventricle (incisor bar, - 5 mm; lateral 1.5 mm to left of bregma; ventral —3 mm from dura) and secured to the skull with optical screws and dental acrylic. Each cannula was fitted with a stainless steel stylet (0.022 in. OD) which extended 0.5 mm beyond the ventricular tip of the cannula. ICV injections were slowly made using a 5 MI Hamilton syringe (Hamilton, Reno, NV) which had been modified with PE-100 tubing (0.034 in. ID, Becton Dickinson and Co., Parsippany, NJ) in place of the needle. Animals with cannula that did not freely perfuse were not used in the study. Injection into the lateral ventricles was verified in a preliminary study by evidence during autopsy of methylene blue dye solution, from an antemortem injection, throughout the cerebral ventricles. After consistent perfusions
Endo-1991 Vo! 128-No 6
were achieved, placement of cannulae was confirmed on a random basis by position during autopsy. Core body temperature measurement Animals were lightly restrained in wire cages which were designed to prevent head to tail rotation and thermic insulation of the rats, while accessing the animals for injections and continuous rectal temperature measurements (20). Copper-constantan thermocouples were inserted 5 cm into the rectum and taped to the base of the tail. Temperatures were recorded by a Honeywell strip chart potentiometer (Fort Washington, PA) at 2-min intervals. After a 30-min acclimation period, animals were injected ICV and temperatures were monitored for the next hour, at which time a morphine sulfate injection was given. Both ICV and sc injections were done through ports in the restraining cages. For the next 4 h, core body temperatures were monitored at 2-min intervals. Data collection and statistical analysis The 5.5 h of 2-min interval core temperature recordings for each animal was reduced by calculating temperatures at 10min intervals for the sake of further data reduction and statistical analysis. Basal (time 0 = time of morphine administration) temperatures among all treatment groups were subjected to analysis of variance and Student Newman-Keul's tests and no significant differences were observed. As a result, all data were normalized for each rat to the time zero value which was assigned the value of 0. Core body temperature increases are represented by positive numbers and declines by negative numbers. The maximum change in core body temperature (AT) and the area under the temperature response curve for 240 min (AUC) were determined for each animal and these values were grouped for statistical analysis. The AUC analysis used the trapezoid method (21) and AUC group means as well as AT means were subjected to analysis of variance and Student Newman-Keul's tests. The number of animals per group and the group comparison are reported in the tables and figure legend. Expl Ovx rats were administered, by a single ICV injection, saline (1 fd) native LHRH (10"4 to 10"1 Mg in 1 id saline), the LHRH agonist (10~7 to 10~2 ng in 1 id saline), the LHRH antagonist (10~5 or 10~3 Mg in 1 id saline), the LHRH antagonist (10~5 Mg in 1 MI saline) followed immediately by the LHRH agonist (10~5 Mg in 1 MI saline), vasopressin (10~4 Mg in 1 M! saline) or somatostatin (10~4 Mg i n 1 fd saline). One hour later animals received a single sc injection of morphine sulfate (20 mg/kg). As controls for the ICV injections, OVX rats were injected with saline (1 pi), the LHRH agonist (10~5 or 10"6 Mg in 1 M! saline), or the LHRH antagonist (10~r> or 10"6 Mg in 1id saline). One hour later animals receive a saline injection in place of the morphine sulfate.
Exp2 OVX rats were treated with one of the following steroid regimens and were tested for core body temperature response to morphine sulfate (20 mg/kg):EB (-48 h) plus P, - 8 h (EBP
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LHRH-OPIATE INTERACTIONS rats); oil (-48 h) plus P (-8 h; P rats); EB (-48 h) plus testosterone (5 mg, - 8 h; EBT rats); EB (-48 h) plus oil (-8 h; EB rats); EB (—48 h) and test of morphine responsiveness at 1000 h (EB am rats). Additionally, one group of OVX rats was administered 5 mg rat LH/kg BW (LH group) at 15001630 h and were treated with morphine sulfate 1 h later. To test the role of LHRH in the EBP-induced blunting of the thermic response to morphine, EBP rats were injected with the LHRH antagonist (10~5 Mg in 1 iA saline) 1 h before morphine sulfate administration.
1-
2801 Q— —•• • • •—• n—
-1-
Effects of [Des-Gly10] LHRH ethylamide on morphineinduced hypothermia
o— •— •—•
t-H
3
80
40
o
Results
10-7 ug 10-6 ug 10-5 ug 10-4 ug 10-3 ug 10-2 ug
3
120
160
200
OVX + Saline EBP + Saline EBP + LHRH antagonist
240
g
A single ICV injection of the LHRH agonist [Des-
Glylo]LHRH ethylamide caused a dose-dependent antagonism of the hypothermic effects of morphine. The response curve to morphine was reduced significantly and nearly completely at the 10~4 ug dose (Table 1, Fig. la). Higher doses of the LHRH agonist caused a hyperthermic response to morphine (Table 1, Fig. la). The LHRH antagonist [D-Phe2, Pro3, D-Phe6]LHRH, when administered alone, had no effect on morphine-induced hypothermia, but it antagonized the LHRH agonistinduced blockade of the morphine effect (Table 2). Vasopressin or somatostatin, when administered at a dose of 10~4 fig, were ineffective in changing the response to morphine (Table 2). Additionally, ICV treatment with the LHRH agonist or the antagonist did not modify AT or AUC in rats treated with saline, in place of morphine, indicating that at the doses of the neuropeptides used in OVX rats, neither peptide modified core body temperature (Table 2).
Native LHRH caused a dose-dependent attenuation of the hypothermic response to morphine (Table 1, Fig lc). TABLE 1. Effect of LHRH agonists on the morphine-induced body
temperature response in OVX rats
OVX OVX
OVX
Treatment 1 h before morphine Saline Native LHRH
Dose 1 Mi
AT(C)
'P < 0.05 us. OVX-saline group.
AUC (C X min) (n)
-1.7 ±0.3 -222 ± 65 -74 ± 84 ± 0.4 ± 0.5° -42 ± 71° ± 0.3" 40 ± 54" ±0.2 -233 ± 60 -79 ± 24 ± 0.1 ± 0.7 -17 ± 93 ± 0.3" - 4 ± 75° ± 0.2° 119 ± 111° ± 0.3° 199 ± 82°
10"4Mg -0.6 10"3 Mg -0.1 10"1 Mg -0.1 L H R H agonist 10"7Mg -1.6 10~6Mg -0.9 lO"5 Mg -0.4 10"4 Mg -0.3 lO" 3 Mg -0.1 10"2Mg -0.2
0
Minutes
Effects of native LHRH on morphine-induced hypothermia
Gonadal steroid treatment
***&&**'s*
8 4 7 16 9 5 6 10 7 10
40
80
120
160
Post-Morphine
200
240
Injection
FIG. 1. A, Effects of treatment with an LHRH agonist ([Des-Gly10] LHRH-ethylamide) on the hypothermic response to morphine in OVX rats. Rats were administered ICV saline or the LHRH agonist at doses ranging from 10~7-10"2 Mg in a volume of 1 (A saline. After 1 h, morphine was administered sc and core body temperatures were monitored at 10min intervals for 240 min thereafter. For data presented in A, B, and C the maximal temperature decline, the AUC of the temperature response, group sizes and the statistical treatment are shown in Table 1. SEM bars were omitted for the sake of clarity. The studies presented in A, B, and C were conducted as a single experiment. The control group (OVX + saline) is presented in panel B. B, Effects of an LHRH antagonist ([D-Phe 2 , Pro 3 , D-Phe 6 ]LHRH) on the attenuation in the hypothermic response to morphine induced by treatment of OVX rats with EB and P. E B P treatment completely blocked the hypothermic response to morphine normally observed in OVX rats. Pretreatment with the LHRH antagonist (10~5 Mg, 1 h before morphine) prevented the effects of EBP treatment. Represented are mean ± SEM. C, Effects of treatment with native LHRH on the hypothermic response to morphine in OVX rats. Rats were administered ICV saline or LHRH at doses ranging from 10~4-10~' Mg in a volume of 1 MI saline. After 1 h, morphine was administered sc and core body temperatures were monitored at 10-min intervals for 240 min. SEM bars were omitted for the sake of clarity.
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LHRH-OPIATE INTERACTIONS
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TABLE 2. Effects of neuropeptides and LH on core body temperature and thermic response to morphine in OVX rats Saline challenge Treatment
Dose
M
Saline LHRH agonist
lO" 6 ng 10" 5 fig lO" 6 M g lO" 5 ag
LHRH antagonist
AT(C) 0.3 -0.1 -0.1 -0.1 0.2
± ± ± ± ±
AUC (C x min)
14 ± 3 -24 ± 51 15 ±10 - 5 ±42 14 ± 2 1
0.1 0.2 0.1 0.4 0.3
M
lO" 5 ng
LHRH agonist + LHRHI antagonist Vasopressin Somatostatin
LH
10" 3 Mg 1 0 - 5 tig
lO" 5 ng lO"4 ng 10- 4 Mg 5 mg
-1.7 ± 0.3 - 1 . 2 ± 0.3 - 1 . 7 ± 0.6
-222 ± 65 -171 ± 62 -223 ± 134
-1.1 -1.5 -1.8 -1.4
-105 -222 -255 -155
± ± ± ±
0.3 0.7 0.4 0.1
± ± ± ±
59 135 94 22
TABLE 3. Effects of steroid treatment and the LHRH antagonist, [DPhe 2 , Pro 3 , D-Phe 6 ]LHRH, on temperature response to morphine in OVX rats Gonadal steroid treatment OVX
EBP EBP P EBT EB EB (am) 1 6
Treatment 1 h before morphine
(Cx AT(C)
Saline - 1 . 7 ± 0.3 Saline 0.2 ± 0.1° LHRH Antagonist (10~5 ng) - 1 . 1 ± 0.56 Saline -1.4 ± 0.3 Saline - 0 . 9 ± 0.5 Saline - 1 . 6 ± 0.5 Saline - 2 . 0 ± 0.3
min)
AUC -222 184 -131 -171 -87 -134
± ± ± ± ± ±
challenges in the morning or afternoon, did not antagonize the morphine-induced suppression of core body temperatures (Table 3). Further treatment of OVX rats with 5 mg rat LH/kg BW, at the time of the expected steroidinduced LH surge, did not influence the effects of morphine (Table 2). Discussion
Morphine (20 mg/kg BW) challenge Saline LHRH antagonist
Endo-1991 Voll28«No6
65 68°
996 55 71 64
ND
P < 0.05 vs. OVX-saline group. P < 0.05 us. EBP-saline group.
The AT response to morphine was reduced from -1.7 ± 0.3 in OVX rats to -0.1 ± 0.5 at the 10"3 Mg dose of LHRH (Table 1). Similarly, the AUC of the thermic response to morphine was reduced from —220 C-min in OVX rats to -40 ± 71 C-min at the 10"3 Mg dose of native LHRH indicating that the hypothermic response to morphine was effectively antagonized. Effects of an LHRH antagonist on the ability of EBP to antagonist morphine-induced hypothermia Morphine treatment caused a —1.7 ± 0.3 C decline in AT in OVX rats (Table 3) and an AUC of -222 ± 65 Cmin. In EBP rats this effect of morphine was reduced to 0.2 ± 0.1 C for AT and 184 ± 68 C-min for the AUC (Table 3). Administration of the LHRH antagonist 1 h before the opiate, reinstated the majority of the hypothermic effects of morphine (Table 3, Fig lb). The refractoriness to morphine's hypothermic effects appears to be related to the phase of enhanced secretion of LHRH associated with the LH surge since treatment with P alone, EBT or EB alone, with the morphine
These experiments demonstrate that LHRH agonists can markedly reduce morphine-induced hypothermia and that an LHRH antagonist can negate a similar opiate refractoriness which occurs during the phase of steroidinduced LHRH secretion. These data suggest that endogenously released LHRH serves to modulate the responsiveness of temperature sensitive neurons to opiates. Anatomical support for the physiological interaction of LHRH and opiate receptor mechanisms can be found in the preoptic area-anterior hypothalamus. Morphine-responsive, temperature sensitive neurons have been identified in the preoptic area-anterior hypothalamus (22) and both LHRH-containing terminals and receptors for this decapeptide have been observed in this ventral diencephalic region (16-19). In view of our previous observations that LHRH agonists reduce and an LHRH antagonist enhances the antinociceptive effects of opiates (23), we suggest that LHRH serves as an endogenous modulator of opiate receptor mechanisms. The proposal is consistent with our observations that during the proestrous and the steroid-induced LH surge, locomotor, temperature, analgesic and neuroendocrine responses to morphine are reduced or extinguished (12, 13). The enhanced release of LHRH during the proestrous LH surge and the resulting refractoriness of the opioid-receptor mechanism may help to explain the coupling of body temperature (24), locomotor behavior (25), pain responsiveness (12, 13), and the well known sexual receptivity of proestrous rats to this neuroendocrine event. The LHRH effects on thermic (present study) and analgesic (23) responses to opiates are observed after an ICV dose of 1 ng/rat. This is a contrast to previous evaluations of other neuropeptides which exhibited antinociceptive effects only at higher dose ranges. TRH is ineffective in blocking morphine-induced analgesia at ICV doses as high as 20 Mg/rat (26) whereas somatostatin and cyclic somatostatin analogs were observed to exert modest effects to morphine-induced analgesia at ICV doses of 30-40 ixg/rat (27). In contrast, cholecystokinin was observed to antagonize food shock-induced analgesia at an intrathecal dose of 3.6 ng/rat (28). The high potency of LHRH and its agonists in blocking morphine's effects on body temperature and pain responsiveness (23) argues in favor of a physiological role of endogenously
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LHRH-OPIATE INTERACTIONS
released LHRH as a mediator of the marked decline in opiate responsiveness during steroid-induced LH surges. The observation that the steroid effect on opiate responsiveness can be reversed by the administration of LHRH antagonists or LHRH antisera (23) supports this contention. The present and previous studies have shown that OVX rats are particularly sensitive to the hypothermic and analgesic effects of opiates (12, 13, 23). When the LHRH agonist was administered to these opiate-sensitive rats, it produced a dose-dependent resistance to morphine-induced hypothermia. This resistance was characterized by a reduction in the AT values which ranged from 6% at the 10~7 fig (7.8 nM concentration) dose to 88% at the 10~2 ixg (78 /xM concentration) dose of LHRH. As the dose of LHRH increased, the profile of the morphine-induced temperature change progressed from a hypothermic to a hyperthermic response and AUC values became positive at the two highest doses tested. In this regard, it is well established that in rats, high doses of morphine cause hypothermia whereas low doses of the opiate induce a hyperthermic response (29). Thus, LHRH administration appears to shift the morphineinduced thermic response to that seen at much lower doses of the opiate. A comparison of native LHRH with the LHRH agonist [Des-Gly10]LHRH ethylamide for blockade of morphineinduced hypothermic revealed that the agonist was about 10 times more potent than native LHRH. [Des-Gly10] LHRH ethylamide has been shown to be 6- to 7-fold more potent than native LHRH in effecting reproductive parameters after its peripheral administration (30). Thus, based upon the evaluation of only two agonists the relative potency of LHRH appears similar in a brain and a peripheral mediated test. The possibility that the observed effect of LHRH was secondary to its stimulation of LH release appears to have been eliminated by our observation that the injection of a high dose of rat LH had no effect on morphine-induced hypothermia in OVX rats. The observed effects of LHRH and one of its agonists cannot be attributed to nonspecific peptide or osmotic effects of ICV injection since treatment with two other neuropeptides had no effect on morphine-induced hypothermia. Further the LHRH agonist and antagonist were ineffective at the doses administered in modifying core body temperature, despite their ability to modify morphine effects. Collectively, these studies argue for a specific effect of LHRH on opiate responses. Major questions remained to be answered about the observed interaction between ovarian steroids, LHRH, and opioid systems. First, whereas LHRH-containing neurons and LHRH receptors have been described in extrahypothalamic brain regions (16-19), it has not been
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demonstrated that these LHRH neurons response to ovarian steroid in concert with LHRH neurons which project to the median eminence. Second, the cellular mechanism by which LHRH markedly reduces responses to morphine are unknown. Steroid treatments which result in a hypersecretion of LH have been reported to cause reduced /u-opioid binding in the medial basal hypothalamus and preoptic area (31-33), but the effects of LHRH on opioid receptor subtypes has not been evaluated. Finally, the specific brain regions and opiate-receptor subtypes effected by ovarian steroids and LHRH has not been defined. In summary, we have observed that LHRH administration can markedly reduce the hypothermic effects of morphine and that an LHRH antagonist can reverse the effects of gonadal steroids on morphine-induced temperature decline. In view of a similar involvement of LHRH in antagonizing the antinociceptive effects of morphine, it appears that LHRH may serve as an endogenous modulator of opiate receptor mechanisms and thereby could serve as the means by which the preovulatory surge in LH is coupled to coincident behavioral, sensory, autonomic, and neuroendocrine alterations.
Acknowledgment The authors wish to thank Victoria Redd Patterson for her expert typing and editorial review of this manuscript.
References 1. Barraclough CA, Sawyer CH 1955 Inhibition of the release of pituitary ovulatory hormone in the rat by morphine. Endocrinology 57:329-337 2. Cicero TJ 1980 Effects of exogenous and endogenous opiates on hypothalamic-pituitary-gonadal axis in the male. Fed Proc 39:2551-2554 3. Kalra SP, Simpkins JW, Chen CL 1980 The opioid-catecholamine modulation of gonadotropin and prolactin secretion. In: Functional Correlates of Hormone Receptors in Reproduction, Ed. Mahesh VB, Muldoon TG, Sexena BB, Sadler WA, Elsevier/North Holland Press, New York, pp 135-149 4. Gabriel SM, Simpkins JW, Kalra SP, Kalra PS 1985 Chronic morphine treatment induces hypersensitivity to testosterone negative feedback in castrated male rats. Neuroendocrinology 40:3944 5. Gabriel SM, Berglund LA, Kalra SP, Kalra PS, Simpkins JW 1986 The influence of chronic morphine treatment on the negative feedback regulation of gonadotropin secretion by gonadal steroids. Endocrinology 119:2762-2767 6. Gabriel SM, Berglund LA, Simpkins JW 1987 Chronic morphine treatment enhances the negative and positive feedback effects of estradiol on gonadotropin secretion in ovariectomized rats. Endocrinology 120:1799-1805 7. Sarkar DK, Chiappa SA, Fink G, Sherwood NM 1976 Gonadotropin-releasing hormone surgery in pro-estrous rats. Nature 264:461463 8. deGreef WJ, deKoning J, Tizssen AMI, Karels B 1987 Levels of LH-releasing hormone in hypophyseal stalk plasma during an estrone-stimulated surge of LH in ovariectomized rats. J Endocrinol 112:351-359 9. Levine JE, Ramirez VD 1982 Luteinizing hormone-releasing hormone release during the rat estrous cycle and after ovariectomy,
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LHRH-OPIATE INTERACTIONS
as estimated with push-pull cannulae. Endocrinology 111:1439— 1448 Gabriel SM, Simpkins JW, Kalra SP 1983 Modulation of endogenous opioid influence on LH secretion by progesterone and estrogen. Endocrinology 113:1806-1811 Gabriel SM, Berglund LA, Simpkins JW 1986 A decline in endogenous opioid influence during the steroid-induced hypersecretion of luteinizing hormone in the rat. Endocrinology 118:558-561 Berglund LA, Derendorf H, Simpkins JW 1988 Desensitization of brain opiate receptor mechanisms by gonadal asteroids hormone treatment which stimulates LH secretion. Endocrinology 122:200203 Berglund LA, Simpkins JW 1988 Desensitization of brain opiate receptor mechanisms on proestrous afternoon. Neuroendocrinology 48:394-400 Moss RL, McCann SM, Dudley CA 1975 Releasing hormones and sexual behavior. Prog Brain Res 42:37-46 Moss RL, Dudley CA 1978 Changes in responsiveness of medial preoptic neurons to the microelectrophoresis of releasing hormones as a function of ovarian hormones. Brain Res 149:511-515 Witkin JW, Silverman AJ 1985 Synaptology of luteinizing hormone-releasing hormone neurons in rat preoptic area. Peptides 6:263-271 Silverman AJ 1984 Luteinizing hormone-releasing hormone containing synapses in the diagonal band and preoptic area of the guinea pig. J Comp Neurol 227:452-458 Jennes L, Dalati B, Conn PM 1988 Distribution of gonadotropin releasing hormone agonist binding sites in the rat central nervous system. Brain Res 452:156-164 Badr M, Pelletur G 1987 Characterization and autoradiographic localization of LHRH receptors in the rat brain. Synapse 1:567571 Simpkins JW, Katovich MJ, Song I-C 1983 Similarities between morphine withdrawal in the rat and the menopausal hot flush. Life Sci 32:1957-1966 Tallarida RJ, Murry RB 1981 Manual of Pharmacological Calculations with Computer Programs, Springer Publishing Co., New
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York 22. Lotti VJ, Lomax P, George R 1965 Temperature responses in the rat following intracerebral micro-injection of morphine. J Pharmacol Exp Ther 150:135-139 23. Ratka A, Simpkins JW 1990 A modulatory role for luteinizing hormone-releasing hormone in nociceptive responses of female rats. Endocrinology 127:667-673 24. Marrone BL, Gentry RT, Wade GN 1976 Gonadal hormones and body temperature in rats: effects of oestrus cycles, castration and steroid replacement. Physiol Behav 17:419-425 25. Young WC, Fish WR 1945 The ovarian hormones and spontaneous running activity in the female rat. Endocrinology 36:181-189 26. Holaday JW, Tseng L-F, Loh HH, Li CH 1978 Thyrotropin releasing hormone antagonizes /3-endorphin hypothermia and catalepsy. Life Sci 22:1537-1544 27. Walker JM, Bowen WD, Atkins ST, Hemstreet MK, Coy DH 1987 M-Opiate binding and morphine antagonism by octapeptide analogs of somatostatin. Peptides 8:869-875 28. Faris PL, Komisaruk BR, Watkins LR, Mayer DJ 1982 Evidence for the neuropeptide cholecystokinin as an antagonist of opiate analgesia. Science 219:310-312 29. Cox B, Ary M, Chesarek W, Lomax P 1976 Morphine hyperthermia in the rat: an action on the central thermostats. Eur J Pharmacol 36:33-39 30. Fujino M, Kobayashi S, Obayashi M, Shinagawa S, Fukuda T, Kitada C, Nakayama R, Yamazaki I 1972 Structure-activity relationships in the C-terminal part of luteinizing hormone releasing hormone (LHRH). Biochem Biophys Res Commun 49:863-869 31. Jacobson W, Kalra S 1989 Decreases in mediobasal hypothalamic and preoptic area opioid ([3H] Naloxone) binding are associated with the progesterone-induced luteinizing hormone surge. Endocrinology 124:199-206 32. Wilkinson M, Brawer JR, Wilkinson DA 1985 Gonadal steroidsinduced modification of opiate binding sites in anterior hypothalamus of female rats. Biol Reprod 32:501-506 33. Weiland NG, Wise PM 1990 Estrogen and progesterone regulate opiate receptor binding density in multiple brain regions. Endocrinology 126:804-808
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Vol. 128, No. 6 Printed in U.S.A.
Luteinizing Hormone-Releasing Hormone Alters the Hypothalamic Effects of Morphine in the Rat* LEE ANN BERGLUND AND JAMES W. SIMPKINS Department of Pharmacodynamics and the Center for the Neurobiology of Aging, University of Florida, Gainesville, Florida 32610
ABSTRACT. Female rats exhibit a generalized refractoriness to opiate stimulation during periods of steroid-induced LH secretion. In the present study we evaluated that role of LHRH in this steroid-induced effect on opiate-responsiveness. Central administration to ovariectomized rats of native LHRH or the LHRH agonist [Des-Gly10]LHRH ethylamide causes a dosedependent refractoriness to the hypothermic effects of morphine. The potency relationship of these two LHRH agonists in antagonizing morphine's effect was consistent with their potency in inducing LH release. Treatment of ovariectomized rats with
T
estradiol benzoate and progesterone in a regimen which induces a preovulatory-like LH surge, antagonized morphine-induced hypothermia, and the LHRH antagonist [D-Phe2, Pro3, D-Phe6) LHRH, reversed the effects of the gonadal steroids. These results indicate that the LHRH secretory dynamics associated with the preovulatory surge of LH may serve to modulate opiate responsiveness and thereby could serve to couple behavioral, sensory, and autonomic events with this neuroendocrine response to gonadal steroids. (Endocrinology 128: 2799-2804, 1991)
HE endogenous opiate peptides (EOP) appear to influence reproductive function in mammals through the tonic inhibition of hypothalamic LHRH secretion (1-3). Opiates also interact with gonadal steroids to enhance the sensitivity of the hypothalamus to steroid negative feedback on LHRH secretion (4-6). On proestrous afternoon or during the steroid-induced periods of LHRH hypersecretion (7-9), the LH secretory mechanism is refractory to opiate agonists and antagonists (10-13). Before and after the LH surge, the LH secretory system is sensitive to opiates and can be pharmacologically manipulated by either opiate agonists and antagonists (3, 10-13). Thus, neurochemical changes associated with, and perhaps causative in, the steroidinduced phase of LHRH hypersecretion, rather than the steroid environment itself, appears to be responsible for the refractoriness of the LH secretory mechanism to opiates. We recently observed that in association with the LH surge on proestrus and the LH surge induced by ovarian steroids, alterations in numerous responses to opiates occurs (12, 13). In these rats, locomotor, secretory, temperature, and analgesic responses to morphine administration are reduced or extinguished. As with the LHRH
secretory mechanism, the changes in opiates sensitivity of these responses could not be explained by the steroid environment of the animal, were only exhibited during the LH surge and appeared to be a function of the magnitude of the pituitary LH responses (12, 13). Together, these observations suggested a general reduction in opiates sensitivity during both endogenous and exogenous steroid-induced periods for LH hypersecretion. Although LHRH is recognized for its fundamental role in stimulating pituitary LH synthesis and secretion, this neuropeptide has also been implicated in other central nervous system functions. LHRH has been shown to facilitate lordosis behavior (14) and to modulate neuronal firing rates in regions associated with reproductive behavior (15). LHRH-like immunoreactivity (16, 17) and LHRH receptors (18, 19) have been described in extrahypothalamic regions of the central nervous system, lending anatomical support for a physiological non-LHreleasing role for this neuropeptide. Thus, the hypersecretion of LHRH associated with ovulation may contribute to the decline in opiate sensitivity observed in proestrous and steroid-treated female rats. To address this issue we examined the effect of intracerebroventricular (ICV) administration of LHRH on the responsiveness of rats to the hypothermic effects of morphine.
Received December 10,1990. Address correspondence and requests for reprints to: James W. Simpkins, Department of Pharmacodynamics and the Center for the Neurobiology of Aging, University of Florida, Gainesville, Florida 32610. * Supported by NIH Grant AG-02021.
Materials and Methods Animals Adult female Sprague-Dawley rats obtained from Charles Rivers Breeding Laboratories (Wilmington, MA) were housed
2799
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LHRH-OPIATE INTERACTIONS
2800
in a temperature (25 ± 1 C) and light-controlled (illuminated 0500-1900 h daily) environment and provided water and Purina laboratory chow (Ralston-Purina, St. Louis, MO) ad libitum. Rats weighed 200-225 g upon arrival and after a 1-week acclimation period were bilaterally ovariectomized (OVX) under light ether anesthesia. All experiments were performed 4-6 weeks after ovariectomy and animals weighed 310-340 g. Rats were used in only one experiment and in no case were animals transferred between experiments. Steroid and drug treatments In most studies, long-term OVX rats were administered ICV, by a single injection, either saline (1 n\) or a neuropeptide agonist or antagonist at 1 h before treatment with a single dose of morphine sulfate (20 mg/kg sc; Merck, St. Louis, MO). The morphine sulfate dose used in these experiments was determined by preliminary range finding studies and the dose chosen was that which induced a marked hypothermic response in OVX (morphine-sensitive) rats (12, 13). A single dose was chosen for evaluation because of limitations in the number of animals which could be processed. The following drugs were administered ICV to OVX rats in 1 n\ saline: Saline (1 MD; native LHRH (10~4 to 10"1 fig); the LHRH agonist, [Des-Glylo]LHRH ethylamide (10~7 to 10~2 Mg); the LHRH antagonist [D-Phe2, Pro3, D-Phe6]LHRH (10~5 Mg); vasopressin (10~4 Mg), or somatostatin (10~4 Mg)- LHRH agonists and antagonists were purchased from Peninsula Laboratory Inc. (Belmont, CA); somatostatin and arginine vasopressin were purchased from Sigma Chemical Co. (St. Louis, MO). In one study, a preovulatory-like LH surge was induced by sc treatment with 7.5 jug estradiol benzoate (EB) at 1000 h 2 days before the experiment. Progesterone (5 mg P) was then administered 48 h after the EB treatment. This steroid regimen induces a surge in LH with onset at 1400-1600 h and maximal testers at about 1730 h. Except where noted, for both OVX and EBP rats, the neuropeptide was administered between 1500 and 1630 h and morphine sulfate was administered exactly 1 h later, between 1600 and 1730 h. Cannulation The cannula were made of 2-cm stainless steel tubing (0.029 in. ID, .035 in. OD; Small Parts Inc., Miami, FL). One to 2 weeks before the experimental rats were placed under 35 to 50 mg/kg BW sodium pentobarbital (Butler, Columbus, OH) anesthesia. The skull was exposed through a dorsal scalp incision and the cannula was stereotaxically placed in the lateral ventricle (incisor bar, - 5 mm; lateral 1.5 mm to left of bregma; ventral —3 mm from dura) and secured to the skull with optical screws and dental acrylic. Each cannula was fitted with a stainless steel stylet (0.022 in. OD) which extended 0.5 mm beyond the ventricular tip of the cannula. ICV injections were slowly made using a 5 MI Hamilton syringe (Hamilton, Reno, NV) which had been modified with PE-100 tubing (0.034 in. ID, Becton Dickinson and Co., Parsippany, NJ) in place of the needle. Animals with cannula that did not freely perfuse were not used in the study. Injection into the lateral ventricles was verified in a preliminary study by evidence during autopsy of methylene blue dye solution, from an antemortem injection, throughout the cerebral ventricles. After consistent perfusions
Endo-1991 Vo! 128-No 6
were achieved, placement of cannulae was confirmed on a random basis by position during autopsy. Core body temperature measurement Animals were lightly restrained in wire cages which were designed to prevent head to tail rotation and thermic insulation of the rats, while accessing the animals for injections and continuous rectal temperature measurements (20). Copper-constantan thermocouples were inserted 5 cm into the rectum and taped to the base of the tail. Temperatures were recorded by a Honeywell strip chart potentiometer (Fort Washington, PA) at 2-min intervals. After a 30-min acclimation period, animals were injected ICV and temperatures were monitored for the next hour, at which time a morphine sulfate injection was given. Both ICV and sc injections were done through ports in the restraining cages. For the next 4 h, core body temperatures were monitored at 2-min intervals. Data collection and statistical analysis The 5.5 h of 2-min interval core temperature recordings for each animal was reduced by calculating temperatures at 10min intervals for the sake of further data reduction and statistical analysis. Basal (time 0 = time of morphine administration) temperatures among all treatment groups were subjected to analysis of variance and Student Newman-Keul's tests and no significant differences were observed. As a result, all data were normalized for each rat to the time zero value which was assigned the value of 0. Core body temperature increases are represented by positive numbers and declines by negative numbers. The maximum change in core body temperature (AT) and the area under the temperature response curve for 240 min (AUC) were determined for each animal and these values were grouped for statistical analysis. The AUC analysis used the trapezoid method (21) and AUC group means as well as AT means were subjected to analysis of variance and Student Newman-Keul's tests. The number of animals per group and the group comparison are reported in the tables and figure legend. Expl Ovx rats were administered, by a single ICV injection, saline (1 fd) native LHRH (10"4 to 10"1 Mg in 1 id saline), the LHRH agonist (10~7 to 10~2 ng in 1 id saline), the LHRH antagonist (10~5 or 10~3 Mg in 1 id saline), the LHRH antagonist (10~5 Mg in 1 MI saline) followed immediately by the LHRH agonist (10~5 Mg in 1 MI saline), vasopressin (10~4 Mg in 1 M! saline) or somatostatin (10~4 Mg i n 1 fd saline). One hour later animals received a single sc injection of morphine sulfate (20 mg/kg). As controls for the ICV injections, OVX rats were injected with saline (1 pi), the LHRH agonist (10~5 or 10"6 Mg in 1 M! saline), or the LHRH antagonist (10~r> or 10"6 Mg in 1id saline). One hour later animals receive a saline injection in place of the morphine sulfate.
Exp2 OVX rats were treated with one of the following steroid regimens and were tested for core body temperature response to morphine sulfate (20 mg/kg):EB (-48 h) plus P, - 8 h (EBP
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LHRH-OPIATE INTERACTIONS rats); oil (-48 h) plus P (-8 h; P rats); EB (-48 h) plus testosterone (5 mg, - 8 h; EBT rats); EB (-48 h) plus oil (-8 h; EB rats); EB (—48 h) and test of morphine responsiveness at 1000 h (EB am rats). Additionally, one group of OVX rats was administered 5 mg rat LH/kg BW (LH group) at 15001630 h and were treated with morphine sulfate 1 h later. To test the role of LHRH in the EBP-induced blunting of the thermic response to morphine, EBP rats were injected with the LHRH antagonist (10~5 Mg in 1 iA saline) 1 h before morphine sulfate administration.
1-
2801 Q— —•• • • •—• n—
-1-
Effects of [Des-Gly10] LHRH ethylamide on morphineinduced hypothermia
o— •— •—•
t-H
3
80
40
o
Results
10-7 ug 10-6 ug 10-5 ug 10-4 ug 10-3 ug 10-2 ug
3
120
160
200
OVX + Saline EBP + Saline EBP + LHRH antagonist
240
g
A single ICV injection of the LHRH agonist [Des-
Glylo]LHRH ethylamide caused a dose-dependent antagonism of the hypothermic effects of morphine. The response curve to morphine was reduced significantly and nearly completely at the 10~4 ug dose (Table 1, Fig. la). Higher doses of the LHRH agonist caused a hyperthermic response to morphine (Table 1, Fig. la). The LHRH antagonist [D-Phe2, Pro3, D-Phe6]LHRH, when administered alone, had no effect on morphine-induced hypothermia, but it antagonized the LHRH agonistinduced blockade of the morphine effect (Table 2). Vasopressin or somatostatin, when administered at a dose of 10~4 fig, were ineffective in changing the response to morphine (Table 2). Additionally, ICV treatment with the LHRH agonist or the antagonist did not modify AT or AUC in rats treated with saline, in place of morphine, indicating that at the doses of the neuropeptides used in OVX rats, neither peptide modified core body temperature (Table 2).
Native LHRH caused a dose-dependent attenuation of the hypothermic response to morphine (Table 1, Fig lc). TABLE 1. Effect of LHRH agonists on the morphine-induced body
temperature response in OVX rats
OVX OVX
OVX
Treatment 1 h before morphine Saline Native LHRH
Dose 1 Mi
AT(C)
'P < 0.05 us. OVX-saline group.
AUC (C X min) (n)
-1.7 ±0.3 -222 ± 65 -74 ± 84 ± 0.4 ± 0.5° -42 ± 71° ± 0.3" 40 ± 54" ±0.2 -233 ± 60 -79 ± 24 ± 0.1 ± 0.7 -17 ± 93 ± 0.3" - 4 ± 75° ± 0.2° 119 ± 111° ± 0.3° 199 ± 82°
10"4Mg -0.6 10"3 Mg -0.1 10"1 Mg -0.1 L H R H agonist 10"7Mg -1.6 10~6Mg -0.9 lO"5 Mg -0.4 10"4 Mg -0.3 lO" 3 Mg -0.1 10"2Mg -0.2
0
Minutes
Effects of native LHRH on morphine-induced hypothermia
Gonadal steroid treatment
***&&**'s*
8 4 7 16 9 5 6 10 7 10
40
80
120
160
Post-Morphine
200
240
Injection
FIG. 1. A, Effects of treatment with an LHRH agonist ([Des-Gly10] LHRH-ethylamide) on the hypothermic response to morphine in OVX rats. Rats were administered ICV saline or the LHRH agonist at doses ranging from 10~7-10"2 Mg in a volume of 1 (A saline. After 1 h, morphine was administered sc and core body temperatures were monitored at 10min intervals for 240 min thereafter. For data presented in A, B, and C the maximal temperature decline, the AUC of the temperature response, group sizes and the statistical treatment are shown in Table 1. SEM bars were omitted for the sake of clarity. The studies presented in A, B, and C were conducted as a single experiment. The control group (OVX + saline) is presented in panel B. B, Effects of an LHRH antagonist ([D-Phe 2 , Pro 3 , D-Phe 6 ]LHRH) on the attenuation in the hypothermic response to morphine induced by treatment of OVX rats with EB and P. E B P treatment completely blocked the hypothermic response to morphine normally observed in OVX rats. Pretreatment with the LHRH antagonist (10~5 Mg, 1 h before morphine) prevented the effects of EBP treatment. Represented are mean ± SEM. C, Effects of treatment with native LHRH on the hypothermic response to morphine in OVX rats. Rats were administered ICV saline or LHRH at doses ranging from 10~4-10~' Mg in a volume of 1 MI saline. After 1 h, morphine was administered sc and core body temperatures were monitored at 10-min intervals for 240 min. SEM bars were omitted for the sake of clarity.
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LHRH-OPIATE INTERACTIONS
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TABLE 2. Effects of neuropeptides and LH on core body temperature and thermic response to morphine in OVX rats Saline challenge Treatment
Dose
M
Saline LHRH agonist
lO" 6 ng 10" 5 fig lO" 6 M g lO" 5 ag
LHRH antagonist
AT(C) 0.3 -0.1 -0.1 -0.1 0.2
± ± ± ± ±
AUC (C x min)
14 ± 3 -24 ± 51 15 ±10 - 5 ±42 14 ± 2 1
0.1 0.2 0.1 0.4 0.3
M
lO" 5 ng
LHRH agonist + LHRHI antagonist Vasopressin Somatostatin
LH
10" 3 Mg 1 0 - 5 tig
lO" 5 ng lO"4 ng 10- 4 Mg 5 mg
-1.7 ± 0.3 - 1 . 2 ± 0.3 - 1 . 7 ± 0.6
-222 ± 65 -171 ± 62 -223 ± 134
-1.1 -1.5 -1.8 -1.4
-105 -222 -255 -155
± ± ± ±
0.3 0.7 0.4 0.1
± ± ± ±
59 135 94 22
TABLE 3. Effects of steroid treatment and the LHRH antagonist, [DPhe 2 , Pro 3 , D-Phe 6 ]LHRH, on temperature response to morphine in OVX rats Gonadal steroid treatment OVX
EBP EBP P EBT EB EB (am) 1 6
Treatment 1 h before morphine
(Cx AT(C)
Saline - 1 . 7 ± 0.3 Saline 0.2 ± 0.1° LHRH Antagonist (10~5 ng) - 1 . 1 ± 0.56 Saline -1.4 ± 0.3 Saline - 0 . 9 ± 0.5 Saline - 1 . 6 ± 0.5 Saline - 2 . 0 ± 0.3
min)
AUC -222 184 -131 -171 -87 -134
± ± ± ± ± ±
challenges in the morning or afternoon, did not antagonize the morphine-induced suppression of core body temperatures (Table 3). Further treatment of OVX rats with 5 mg rat LH/kg BW, at the time of the expected steroidinduced LH surge, did not influence the effects of morphine (Table 2). Discussion
Morphine (20 mg/kg BW) challenge Saline LHRH antagonist
Endo-1991 Voll28«No6
65 68°
996 55 71 64
ND
P < 0.05 vs. OVX-saline group. P < 0.05 us. EBP-saline group.
The AT response to morphine was reduced from -1.7 ± 0.3 in OVX rats to -0.1 ± 0.5 at the 10"3 Mg dose of LHRH (Table 1). Similarly, the AUC of the thermic response to morphine was reduced from —220 C-min in OVX rats to -40 ± 71 C-min at the 10"3 Mg dose of native LHRH indicating that the hypothermic response to morphine was effectively antagonized. Effects of an LHRH antagonist on the ability of EBP to antagonist morphine-induced hypothermia Morphine treatment caused a —1.7 ± 0.3 C decline in AT in OVX rats (Table 3) and an AUC of -222 ± 65 Cmin. In EBP rats this effect of morphine was reduced to 0.2 ± 0.1 C for AT and 184 ± 68 C-min for the AUC (Table 3). Administration of the LHRH antagonist 1 h before the opiate, reinstated the majority of the hypothermic effects of morphine (Table 3, Fig lb). The refractoriness to morphine's hypothermic effects appears to be related to the phase of enhanced secretion of LHRH associated with the LH surge since treatment with P alone, EBT or EB alone, with the morphine
These experiments demonstrate that LHRH agonists can markedly reduce morphine-induced hypothermia and that an LHRH antagonist can negate a similar opiate refractoriness which occurs during the phase of steroidinduced LHRH secretion. These data suggest that endogenously released LHRH serves to modulate the responsiveness of temperature sensitive neurons to opiates. Anatomical support for the physiological interaction of LHRH and opiate receptor mechanisms can be found in the preoptic area-anterior hypothalamus. Morphine-responsive, temperature sensitive neurons have been identified in the preoptic area-anterior hypothalamus (22) and both LHRH-containing terminals and receptors for this decapeptide have been observed in this ventral diencephalic region (16-19). In view of our previous observations that LHRH agonists reduce and an LHRH antagonist enhances the antinociceptive effects of opiates (23), we suggest that LHRH serves as an endogenous modulator of opiate receptor mechanisms. The proposal is consistent with our observations that during the proestrous and the steroid-induced LH surge, locomotor, temperature, analgesic and neuroendocrine responses to morphine are reduced or extinguished (12, 13). The enhanced release of LHRH during the proestrous LH surge and the resulting refractoriness of the opioid-receptor mechanism may help to explain the coupling of body temperature (24), locomotor behavior (25), pain responsiveness (12, 13), and the well known sexual receptivity of proestrous rats to this neuroendocrine event. The LHRH effects on thermic (present study) and analgesic (23) responses to opiates are observed after an ICV dose of 1 ng/rat. This is a contrast to previous evaluations of other neuropeptides which exhibited antinociceptive effects only at higher dose ranges. TRH is ineffective in blocking morphine-induced analgesia at ICV doses as high as 20 Mg/rat (26) whereas somatostatin and cyclic somatostatin analogs were observed to exert modest effects to morphine-induced analgesia at ICV doses of 30-40 ixg/rat (27). In contrast, cholecystokinin was observed to antagonize food shock-induced analgesia at an intrathecal dose of 3.6 ng/rat (28). The high potency of LHRH and its agonists in blocking morphine's effects on body temperature and pain responsiveness (23) argues in favor of a physiological role of endogenously
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LHRH-OPIATE INTERACTIONS
released LHRH as a mediator of the marked decline in opiate responsiveness during steroid-induced LH surges. The observation that the steroid effect on opiate responsiveness can be reversed by the administration of LHRH antagonists or LHRH antisera (23) supports this contention. The present and previous studies have shown that OVX rats are particularly sensitive to the hypothermic and analgesic effects of opiates (12, 13, 23). When the LHRH agonist was administered to these opiate-sensitive rats, it produced a dose-dependent resistance to morphine-induced hypothermia. This resistance was characterized by a reduction in the AT values which ranged from 6% at the 10~7 fig (7.8 nM concentration) dose to 88% at the 10~2 ixg (78 /xM concentration) dose of LHRH. As the dose of LHRH increased, the profile of the morphine-induced temperature change progressed from a hypothermic to a hyperthermic response and AUC values became positive at the two highest doses tested. In this regard, it is well established that in rats, high doses of morphine cause hypothermia whereas low doses of the opiate induce a hyperthermic response (29). Thus, LHRH administration appears to shift the morphineinduced thermic response to that seen at much lower doses of the opiate. A comparison of native LHRH with the LHRH agonist [Des-Gly10]LHRH ethylamide for blockade of morphineinduced hypothermic revealed that the agonist was about 10 times more potent than native LHRH. [Des-Gly10] LHRH ethylamide has been shown to be 6- to 7-fold more potent than native LHRH in effecting reproductive parameters after its peripheral administration (30). Thus, based upon the evaluation of only two agonists the relative potency of LHRH appears similar in a brain and a peripheral mediated test. The possibility that the observed effect of LHRH was secondary to its stimulation of LH release appears to have been eliminated by our observation that the injection of a high dose of rat LH had no effect on morphine-induced hypothermia in OVX rats. The observed effects of LHRH and one of its agonists cannot be attributed to nonspecific peptide or osmotic effects of ICV injection since treatment with two other neuropeptides had no effect on morphine-induced hypothermia. Further the LHRH agonist and antagonist were ineffective at the doses administered in modifying core body temperature, despite their ability to modify morphine effects. Collectively, these studies argue for a specific effect of LHRH on opiate responses. Major questions remained to be answered about the observed interaction between ovarian steroids, LHRH, and opioid systems. First, whereas LHRH-containing neurons and LHRH receptors have been described in extrahypothalamic brain regions (16-19), it has not been
2803
demonstrated that these LHRH neurons response to ovarian steroid in concert with LHRH neurons which project to the median eminence. Second, the cellular mechanism by which LHRH markedly reduces responses to morphine are unknown. Steroid treatments which result in a hypersecretion of LH have been reported to cause reduced /u-opioid binding in the medial basal hypothalamus and preoptic area (31-33), but the effects of LHRH on opioid receptor subtypes has not been evaluated. Finally, the specific brain regions and opiate-receptor subtypes effected by ovarian steroids and LHRH has not been defined. In summary, we have observed that LHRH administration can markedly reduce the hypothermic effects of morphine and that an LHRH antagonist can reverse the effects of gonadal steroids on morphine-induced temperature decline. In view of a similar involvement of LHRH in antagonizing the antinociceptive effects of morphine, it appears that LHRH may serve as an endogenous modulator of opiate receptor mechanisms and thereby could serve as the means by which the preovulatory surge in LH is coupled to coincident behavioral, sensory, autonomic, and neuroendocrine alterations.
Acknowledgment The authors wish to thank Victoria Redd Patterson for her expert typing and editorial review of this manuscript.
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LHRH-OPIATE INTERACTIONS
as estimated with push-pull cannulae. Endocrinology 111:1439— 1448 Gabriel SM, Simpkins JW, Kalra SP 1983 Modulation of endogenous opioid influence on LH secretion by progesterone and estrogen. Endocrinology 113:1806-1811 Gabriel SM, Berglund LA, Simpkins JW 1986 A decline in endogenous opioid influence during the steroid-induced hypersecretion of luteinizing hormone in the rat. Endocrinology 118:558-561 Berglund LA, Derendorf H, Simpkins JW 1988 Desensitization of brain opiate receptor mechanisms by gonadal asteroids hormone treatment which stimulates LH secretion. Endocrinology 122:200203 Berglund LA, Simpkins JW 1988 Desensitization of brain opiate receptor mechanisms on proestrous afternoon. Neuroendocrinology 48:394-400 Moss RL, McCann SM, Dudley CA 1975 Releasing hormones and sexual behavior. Prog Brain Res 42:37-46 Moss RL, Dudley CA 1978 Changes in responsiveness of medial preoptic neurons to the microelectrophoresis of releasing hormones as a function of ovarian hormones. Brain Res 149:511-515 Witkin JW, Silverman AJ 1985 Synaptology of luteinizing hormone-releasing hormone neurons in rat preoptic area. Peptides 6:263-271 Silverman AJ 1984 Luteinizing hormone-releasing hormone containing synapses in the diagonal band and preoptic area of the guinea pig. J Comp Neurol 227:452-458 Jennes L, Dalati B, Conn PM 1988 Distribution of gonadotropin releasing hormone agonist binding sites in the rat central nervous system. Brain Res 452:156-164 Badr M, Pelletur G 1987 Characterization and autoradiographic localization of LHRH receptors in the rat brain. Synapse 1:567571 Simpkins JW, Katovich MJ, Song I-C 1983 Similarities between morphine withdrawal in the rat and the menopausal hot flush. Life Sci 32:1957-1966 Tallarida RJ, Murry RB 1981 Manual of Pharmacological Calculations with Computer Programs, Springer Publishing Co., New
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York 22. Lotti VJ, Lomax P, George R 1965 Temperature responses in the rat following intracerebral micro-injection of morphine. J Pharmacol Exp Ther 150:135-139 23. Ratka A, Simpkins JW 1990 A modulatory role for luteinizing hormone-releasing hormone in nociceptive responses of female rats. Endocrinology 127:667-673 24. Marrone BL, Gentry RT, Wade GN 1976 Gonadal hormones and body temperature in rats: effects of oestrus cycles, castration and steroid replacement. Physiol Behav 17:419-425 25. Young WC, Fish WR 1945 The ovarian hormones and spontaneous running activity in the female rat. Endocrinology 36:181-189 26. Holaday JW, Tseng L-F, Loh HH, Li CH 1978 Thyrotropin releasing hormone antagonizes /3-endorphin hypothermia and catalepsy. Life Sci 22:1537-1544 27. Walker JM, Bowen WD, Atkins ST, Hemstreet MK, Coy DH 1987 M-Opiate binding and morphine antagonism by octapeptide analogs of somatostatin. Peptides 8:869-875 28. Faris PL, Komisaruk BR, Watkins LR, Mayer DJ 1982 Evidence for the neuropeptide cholecystokinin as an antagonist of opiate analgesia. Science 219:310-312 29. Cox B, Ary M, Chesarek W, Lomax P 1976 Morphine hyperthermia in the rat: an action on the central thermostats. Eur J Pharmacol 36:33-39 30. Fujino M, Kobayashi S, Obayashi M, Shinagawa S, Fukuda T, Kitada C, Nakayama R, Yamazaki I 1972 Structure-activity relationships in the C-terminal part of luteinizing hormone releasing hormone (LHRH). Biochem Biophys Res Commun 49:863-869 31. Jacobson W, Kalra S 1989 Decreases in mediobasal hypothalamic and preoptic area opioid ([3H] Naloxone) binding are associated with the progesterone-induced luteinizing hormone surge. Endocrinology 124:199-206 32. Wilkinson M, Brawer JR, Wilkinson DA 1985 Gonadal steroidsinduced modification of opiate binding sites in anterior hypothalamus of female rats. Biol Reprod 32:501-506 33. Weiland NG, Wise PM 1990 Estrogen and progesterone regulate opiate receptor binding density in multiple brain regions. Endocrinology 126:804-808
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