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The Effects of Estradiol and Progesterone on Pain Sensitivity and Brain Opioid Receptors in Ovariectomized Rats
Hormones and Behavior 30, 244–250 (1996) Article No. 0029
The Effects of Estradiol and Progesterone on Pain Sensitivity and Brain Opioid Receptors in Ovariectomized Rats Fran T. Gordon and Magdi R. I. Soliman College of Pharmacy, Florida A&M University, Tallahassee, Florida 32307
Previous studies in our laboratory have shown that estradiol and progesterone affect b-endorphin and Met-enkephalin levels in specific brain regions and that these effects are diurnally controlled. The present investigation was conducted to evaluate the effects of estradiol and progesterone on pain latency and brain opioid receptors of ovariectomized rats. Female Sprague–Dawley rats (100– 120 g) adapted to a 12 hr light:12 hr dark illumination cycle were used in these studies. Animals were ovariectomized under pentobarbital anesthesia. After a recovery period of 10–14 days, estradiol (50 mg/kg/day in 0.2 ml olive oil) and progesterone (5 mg/kg/day in 0.1 ml olive oil) were administered subcutaneously in the dorsal neck region alone and in combination at 16:00 hr for 7 days. Control animals received 0.2 ml olive oil. Control and treated groups were evaluated daily for pain latency postinjection using the tailflick and hotplate methods. On Day 7 of drug treatment, animals were sacrificed by decapitiation after pain latency evaluations. Whole brains were removed and immediately frozen at 0707C. Binding and affinity of brain opiate receptors were determined for each treatment group. Results obtained indicate that estradiol and progesterone treatment alone or in combination significantly alter pain latency. This alteration in pain was not accompanied by any change in affinity or number of mu opioid receptors. However, an increase in Kd of kappa opiate receptors was observed following treatment with estradiol, progesterone, or their combination. This increase in Kd of kappa opiate receptors may in part explain the increased hotplate sensitivity following estrogen administration. The present findings suggest that the decrease in pain sensitivity induced by estradiol or progesterone could not be explained by their effects on opioid receptors. The previously reported effects of estradiol and progesterone on brain levels of bendorphin and Met-enkephalin may contribute to the analgesic effects of these steroids. q 1996 Academic Press, Inc.
It has been well established that pregnancy and partition are associated with opioid-mediated elevations in
pain thresholds (Gintzler, 1980; Cogan and Spinnato, 1986; Baron and Gintzler, 1984; Sander, Kream, and Gintzler, 1989; Sander and Gintzler, 1987). The endogenous opioid system appears to be involved in raising maternal thresholds to pain. This maternal analgesia appears to be centrally mediated via the activation of a spinal cord dynorphin/kappa-opioid system (Sander et al., 1989; Sander and Gintzler, 1987; Sander, Portoghese, and Gintzler, 1988). Estrogen and progesterone interact in the regulation of various brain functions including those that control gonadotropin secretion and female sexual behavior. They are also known to be involved in the regulation of analgesia and nociception; however, the extent of their mechanisms in pain sensitivity is not yet fully understood. Nevertheless, estrogen treatment has been shown to modify morphine-induced analgesia (Nomikos, Spyraki, Kazandjian, and Sfikakis, 1987), reduce sensitivity to opioid antagonists (Morley, Levine, Grace, Kneip, and Gosnell, 1984), and induce changes in the levels of b-endorphin (Gordon and Soliman, 1994; Wardlaw, Thoron, and Frantz, 1982). Modifications of binding characteristics of mu opioid receptors have been studied in whole brain of female rats during the different phases of the estrous cycle, which are characterized by different secretory rates of estrogen and progesterone (Limonta, Maggi, Dondi, Martini, and Piva, 1987; Casular, Maggi, Dondi, Limonta, Piva, Motto, and Martini, 1987). The results demonstrated that in the total brain and in the hypothalamus of the female rat, the concentration of mu opioid receptors is significantly altered during the different phases of the estrous cycle. Moreover, the higher concentrations of hypothalamic mu receptors present during the period of the estrous cycle are associated with high serum levels of estrogen while the decrease in the density of the mu sites starts concomitantly with the increase in progesterone secretion. 0018-506X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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The present studies were undertaken to determine whether estrogen and progesterone treatments modulate responsiveness to pain stimuli and whether this treatment might also modify the binding characteristics of brain mu and kappa opioid receptors in ovariectomized rats.
MATERIALS AND METHODS
tus. Their tails were smoothed into the groove with the light source focused as a 1-cm spot on the dorsal surface of the rat’s tail. All groups were tested daily for 7 days at 0, 15, 30, 60, and 120 min. Hotplate method. The temperature of the hotplate apparatus was maintained at 557 { 27C. Rats were placed in the hotplate apparatus and then immediately withdrawn at any indication of an escape attempt and/ or licking of the hind paws. All groups were tested daily at 0, 15, 30, 60, and 120 min.
Animals Female Sprague – Dawley rats (Harlan, Indianapolis, IN) weighing 100 – 120 g were used in this study. They were adapted to a 12 hr light:12 hr dark cycle. Food and water were provided ad libitum. All animals were ovariectomized under pentobarbital anesthesia (50 mg/ kg). After a recovery period of 10 – 14 days, the animals were randomly divided into treatment groups (six animals/group) for the tailflick and hotplate procedures. Separate groups were used for each procedure.
Drug Treatment Treated rats received 17-b-estradiol (50 mg/kg/day dissolved in 0.2 ml of olive oil), progesterone (5 mg/ kg/day in 0.1 ml of olive oil), or estradiol plus progesterone subcutaneously in the dorsal neck region at 16:00 hr for 7 days. All control animals received 0.2 ml olive oil. Control and treated groups were evaluated daily for 7 days for pain threshold at 15, 30, 60, and 120 min postinjection.
Pain Threshold Tailflick method. This is the most commonly used method of pain assessment throughout the stress-induced analgesia and opiate pharmacology literature. The tailflick method tests a reflex that is clearly tied to spinal pain mechanisms (Irwin et al., 1951). In addition, it is relatively insensitive to changes in general activity compared to other pain tests that involve the assessment of gross body movement, such as the hotplate method. The intensity of the light source was controlled by a rheostat to obtain a maximum control latency of 20 sec. In order to avoid injury to the animal’s tail, the light source was automatically terminated by tail movement. Animals’ tails were carefully examined after each session to ensure that no damage had occurred so as not to affect subsequent testing. All animals were held gently by the same experimenter throughout the study. Rats were placed on the platform of the tailflick appara-
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Opioid Receptor Binding Tissue preparation. After 7 days of drug treatment, all animals in each treatment group were sacrificed by decapitation. The whole brains were removed and immediately frozen at 0707C. Three brain tissues were pooled to generate three pools/group. Each group was homogenized in 10 ml each of ice-cold 50 nM Tris – HCl buffer (pH 7.5) containing 5 mM MgCl2 and 1 mM Captopril. All homogenates were centrifuged at 20,000 rpm for 10 min at 47C. The supernatants were discarded. The pellets were resuspended by sonication in 10 ml of buffer, incubated for 40 min at 377C, and then recentrifuged. All membranes were then washed four times at 20,000 rpm for 10 min at 47C. The final pellets were pooled and resuspended in 60 ml of buffer and frozen at 0707C until analysis. Kappa receptor binding assay. Aliquots from each pool of tissue membrane preparations were incubated for 60 min at 377C with increasing concentrations (1.25 – 40 nM ) of [3H]ethylketocyclazine (New England Nuclear, Boston, MA) in the presence of a cold ligand mixture (100 nM DAMGO and 100 nM DADLE; Sigma Chemical Co., St. Louis, MO), which was used to block the mu and delta receptors. All samples were assayed in triplicate. Nonspecific binding for each tissue sample was determined by adding 40 mM naloxone (Sigma Chemical Co.). The incubation was terminated by rapid vacuum filtration using Whatman GF/B filters presoaked in 0.1% polyethylenimine on a Brandel Cell Harvester. Each filter was washed four times with 4 ml of ice-cold 50 nM Tris – HCl buffer. Radioactivity on the filters were determined by liquid scintillation counting. Specific binding was defined as the average binding observed in the absence and presence of 40 mM naloxone hydrochloride. The Kd and Bmax values were calculated using the Lundon-1 binding analysis program. Protein was determined using a Bio-Rad protein assay kit from Bio-Rad Chemical Division (Richmond, CA). Mu receptor binding assay. The mu receptor binding was performed in a manner similar to that described in the kappa receptor assay. The tissue mem-
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FIG. 1. Effect of estradiol, progesterone, and estradiol plus progesterone on pain latency in the tailflick test at Time 0. C, control; E, estradiol; P, progesterone; *, significantly different from control, P õ 0.05.
branes were incubated with increasing concentrations (1.25 – 20 nM ) of [3H]DAMGO (New England Nuclear) in the presence or absence of 1 mM cold DAMGO. Statistical analysis. Statistical significance of differences for pain sensitivity was determined by repeated measures analysis of variance followed by the Tukey – Kramer comparisons test. The receptor binding results were analyzed using the unpaired t test.
FIG. 2. Effect of estradiol, progesterone, and estradiol plus progesterone on pain latency in the tailflick test at Time 30. C, control; E, estradiol; P, progesterone; *, significantly different from control, P õ 0.05.
at Day 4 than at Day 1 (62.8% increase). However, it was significantly lower (P õ 0.01) between Day 4 and Day 5 (61% decrease). By Day 7, a significant rise (P õ 0.05) in pain latency was observed compared to Day 5 (57.3% increase). On Day 3, rats treated with progesterone alone and estradiol plus progesterone produced a significant increase (P õ 0.05) in pain latency compared to controls (69.1 and 63.5% increase, respectively). After administration of estradiol alone on Day 5, there was
RESULTS Effect of Estradiol, Progesterone, and Estradiol Plus Progesterone on Pain Sensitivity Using the Tailflick Method The administration of estradiol and progesterone in combination at Time 0 produced a gradual increase in pain latency, with a significant rise (P õ 0.05) between Day 1 and Day 7 (56.2% increase) (Fig. 1). No significant difference in pain latency was observed with estradiol or progesterone treatment alone. On Day 7, there was a significant increase (P õ 0.05) in pain latency in the rats treated with estradiol plus progesterone compared to controls (54.3% increase). At Time 30, progesterone treatment resulted in a statistically significant increase (P õ 0.05) in pain latency compared to controls on Day 1 (66.4% increase) (Fig. 2). Estradiol treatment alone at Time 120 produced a significant increase (P õ 0.05) in pain latency by Day 7 compared to Day 1 (69.8% increase) (Fig. 3). When progesterone was administered alone, pain latency was significantly higher (P õ 0.01)
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FIG. 3. Effect of estradiol, progesterone, and estradiol plus progesterone on pain latency in the tailflick test at Time 120. C, control; E, estradiol; P, progesterone; *, significantly different from control, P õ 0.05.
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FIG. 4. Effect of estradiol, progesterone, and estradiol plus progesterone on pain latency in the hotplate test at Time 0. C, control; E, estradiol; P, progesterone; *, significantly different from control, P õ 0.05.
a significant increase (P õ 0.05) in pain latency compared to controls (59.2% increase).
Effect of Estradiol, Progesterone, and Estradiol Plus Progesterone on Pain Sensitivity Using the Hotplate Method Rats treated with estradiol alone at Time 0 showed a significantly lower (P õ 0.001) pain latency at Day 3 compared to Day 1 (53.8% decrease) (Fig. 4). This decline did not continue through Day 4, but was observed again at Day 5 (42.3% decrease). However, pain latency was significantly increased (P õ 0.05) between Day 3 and Day 4 (46.7% increase). By Day 7, there was a significant rise (P õ 0.001) in pain latency compared to Day 3 (53.8% increase). Progesterone treatment produced a similar decline in pain latency which started at Day 3 and continued through Day 5 compared to Day 2 (32.1 and 45.3% decrease, respectively). When comparing the different days studied, no significant difference was noted when estradiol and progesterone were administered in combination. On Day 3, estradiol treatment resulted in a significant decline (P õ 0.05) in pain latency (36.8% decrease) compared to controls. As shown in Fig. 5, animals treated with estradiol on Day 3 at Time 15 showed a significant increase (P õ 0.05) in pain latency compared to Day 2 (30.4% increase). However, there was a sudden decline (P õ 0.01) from Day 3 to Day 4 (43.5% decrease), which was followed by a significant increase (P õ 0.001) in pain latency by Day 6 (45.8% increase). A similar decline (P
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õ 0.05) between Day 3 and Day 4 was also observed when estradiol and progesterone were administered in combination (39.1% decrease). The combination treatment also resulted in a significant increase (P õ 0.01) in pain latency by Day 6 (44% increase). Progesterone alone produced a significant increase (P õ 0.01) in pain latency by Day 6 compared to Day 2 (41.3% increase). Estradiol and progesterone alone and in combination significantly decreased (P õ 0.001) pain latency on Day 1 compared to controls (32.3, 40.7, and 37.3% decrease, respectively). Estradiol treatment at Time 30 produced a significant increase (P õ 0.01) in pain latency at Day 6 compared to Day 4 (46% increase) (Fig. 6). When progesterone was administered alone, there was an immediate increase (P õ 0.001) from Day 1 to Day 2 (32.3% increase). A decrease in pain latency was observed at Day 3 through Day 4 (44.4% decrease). By Day 5, there was a rise in pain latency, but it was not significantly higher until Day 6 (38.8% increase). Similarly, estradiol and progesterone in combination resulted in a significant decline (P õ 0.01) from Day 3 to Day 4 (41.2% decrease) followed by a significant rise (P õ 0.01) in pain latency by Day 6 (42.3% increase). On Day 2, progesterone treatment resulted in a significant increase (P õ 0.01) in pain latency compared to controls (38.5% increase). At Time 120, rats treated with estradiol showed a significant decrease (P õ 0.01) in pain latency from Day 1 to Day 2 (48.1% decrease), which was followed by a significant rise (P õ 0.05) from Day 2 to Day 3 (40% increase) (Fig. 7). Administration of estradiol and pro-
FIG. 5. Effect of estradiol, progesterone, and estradiol plus progesterone on pain latency in the hotplate test at Time 15. C, control; E, estradiol; P, progesterone; *, significantly different from control, P õ 0.001.
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FIG. 6. Effect of estradiol, progesterone, and estradiol plus progesterone on pain latency in the hotplate test at Time 30. C, control; E, estradiol; P, progesterone; *, significantly different from control, P õ 0.01.
gesterone alone resulted in a gradual increase which was significantly higher (P õ 0.001) by Day 7 compared to Day 2 and Day 4 (59.7 and 55.2%; 56.2 and 54.8% increase, respectively). A similar effect was also noted when estradiol and progesterone were administered in combination (64.4 and 56.8% increase, respectively). When estradiol plus progesterone were administered, there was a significantly higher (P õ 0.05) pain latency compared to controls on Day 7 (35.1% increase).
FIG. 8. Effect of estradiol, progesterone, and estradiol plus progesterone on the specific binding and affinity of kappa opiate receptors in rat brain. E, estradiol; P, progesterone; *, significantly different from control, P õ 0.05.
Effect of Estradiol, Progesterone, and Estradiol Plus Progesterone on Brain Opioid Receptors Estradiol and progesterone alone and in combination resulted in significant changes in the binding affinities (Kd ) but not binding capacity (Bmax ) of kappa receptors (Fig. 8). There was a significant increase in Kd denoting a decrease in the binding affinity of kappa receptors. However, no significant differences were observed in the Bmax or Kd of mu opiate receptors when estradiol and progesterone were administered alone or in combination (Fig. 9).
DISCUSSION Results obtained in the present study indicate that estradiol and progesterone treatment altered the pain
FIG. 7. Effect of estradiol, progesterone, and estradiol plus progesterone on pain latency in the hotplate test at Time 120. C, control; E, estradiol; P, progesterone; *, significantly different from control, P õ 0.05.
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FIG. 9. Effect of estradiol, progesterone, and estradiol plus progesterone on the specific binding and affinity of mu opiate receptors in rat brain. E, estradiol; P, progesterone.
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latency of ovariectomized rats. In the tailflick method, estradiol treatment produced a significant increase in pain latency at Time 120 on Day 5. When progesterone was administered alone, pain latency was significantly higher than the controls on Day 1 at Time 30 and then again on Day 3 at Time 120. Estradiol plus progesterone also increased pain latency. These effects were observed on Day 3 at Time 120 and again on Day 7 at Time 0. However, in the hotplate method estradiol treatment alone produced a significant decrease in pain latency. This decline was detected only at Time 0 on Day 3 and Time 15 on Day 1. A similar decrease was observed on Day 1 at Time 15 following administration of progesterone and estradiol plus progesterone. By Day 2, progesterone significantly increased pain latency at Time 30. Nevertheless, estradiol and progesterone in combination did not increase pain latency until Day 7 at Time 120. Thus, our results demonstrate that analgesia induced by estradiol, progesterone, or their combination is more pronounced in the tailflick test. Several investigators have analyzed pain sensitivity across the estrus cycle; however, there are inconsistencies in their findings. It has been shown that during the evening of proestrus when estradiol and progesterone levels are rising, tailflick latencies are reduced compared to the metestrus (Frye, Bock, and Kanarek, 1992). On the other hand, Ryan and Maier (1988) did not find a significant difference in pain sensitivity in normal estrus cycling rats. These discrepancies may be due to the different experimental protocols utilized by the researchers. Frye et al. (1992) indicated that tailflick latencies in animals pretreated with estradiol were reduced following administration of progesterone (1.0 mg) compared to controls. Also, these studies demonstrated that as progesterone doses increase in the presence of estradiol, pain sensitivity is more pronounced. These results seem to differ from those obtained in the present study, which shows a significant decrease in pain sensitivity when estradiol and progesterone were administered alone and in combination. The discrepancy in the results may be explained by several reasons. First, the doses of estradiol and progesterone were lower in these studies, which may suggest that a certain level of estradiol and progesterone must be present for nociception to be affected. Moreover, other studies have shown that higher progesterone doses produce analgesia (Meyerson, 1967; Selye, 1941). Second, the inconsistencies in pain sensitivity may be due to whether estradiol and progesterone were administered alone or in combination (Meyerson, 1967; Selye, 1941; Meyerson, 1972). Finally, the possibility that different strains of rats have different endogenous levels of hormones or steroid re-
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ceptor characteristics may contribute to variations in pain latency. Ratka and Simpkins (1991) demonstrated that ovariectomized rats treated with estradiol alone for two days showed significantly increased sensitivity to thermal stimulation. Our findings are consistent with this study, which indicates that estradiol treatment increases pain sensitivity in the hotplate test. However, we observed these effects at Day 1 and Day 3. Some researchers suggest that this hyperalgesia may result from an estradiolinduced increase in the b-endorphin levels in the hypothalamus (Forman, Marqis, and Stevens, 1985) and the reduction of opioid binding sites (Wilkinson, Brawer, and Wilkinson, 1985). The increase in Kd of kappa opiate receptors, observed in the present study, may in part explain the increased hotplate sensitivity following estrogen administration. It has been reported that the number of opioid receptors in the whole brain fluctuates during the estrous cycle (Limonta et al., 1987). Generally, the number of hypothalamic mu opioid receptors is elevated during the morning of diestrus Day 2 and during the day of proestrus and then declines at the end of the day. This decline in the number of mu receptors observed in the proestrus may be due to the rise in serum levels of estradiol and progesterone. Previous studies have shown that ovariectomy increased opioid binding sites (Wilkinson et al., 1985; Weiland and Wise, 1990) but blocked or eliminated opioid analgesia (Ryan and Maier, 1988). However, estradiol treatment has been found to restore opioid analgesia (Ryan and Maier, 1988) and decrease the number of opioid binding sites (Wilkinson et al., 1985). A lower number of mu binding sites were observed following administration of progesterone. Chronic estradiol treatment has been reported to increase the number of opioid binding sites (Wilkinson et al., 1985). Moreover, estradiol plus progesterone treatment resulted in a significant decrease in the number of mu receptors in the brain (Dondi, Maggi, Limonta, and Piva, 1988). In the present study, no effects on brain mu receptors’ maximum binding nor affinity could be detected. Peripheral sex steroids have been shown to influence central opioid activity. Moreover, the changes in steroid hormones during pregnancy modulate a spinal opioid system. This spinal cord dynorphin/kappa-opioid system has been reported to elevate maternal nociceptive thresholds during gestation (Sander et al., 1988, 1989; Sander and Gintzler, 1987). Thus, the levels of estradiol and progesterone during gestation may trigger the activation of this spinal opioid system, which may be responsible for decreased pain sensitivity. Our results have shown that estradiol and progesterone alone and
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in combination produced a significant decrease in the binding affinity of kappa opiate receptors as indicated by an increase in Kd . These findings suggest that the decrease in pain sensitivity induced by estradiol or progesterone could not be explained by their effects on opioid receptors. Other mechanisms may be involved in progesterone-induced analgesia. Frye and Duncan (1994) have reported that progesterone metabolites can attenuate pain sensitivity by interacting with the GABAA . However, the release of endogenous opiates reported in our previous studies (Gordon and Soliman, 1994) may play a role in the analgesic effects of estradiol and progesterone.
REFERENCES Baron, S. A., and Gintzler, A. R. (1984). Effects of hypophysectomy and dexamethasone treatment on plasma b-endorphin and pain threshold during pregnancy. Brain Res. 321, 344 – 346. Casulari, L. A., Maggi, R., Dondi, D., Limonta, P., Piva, F., Motta, M., and Martini, L. (1987). Effect of oestrus cyclicity on the number of brain opioid mu receptors in the rat. Horm. Metab. Res. 19, 549 – 554. Cogan, R., and Spinnato, J. A. (1986). Pain and discomfort thresholds in late pregnancy. Pain 27, 63 – 68. Dondi, D., Maggi, R., Limonta, P., and Piva, F. (1988). Sex steroid positive feedback decreases mu receptors density in the brain of long-term castrated female rats. In Proceedings, 70th Annual Meeting of the Endocrine Society, New Orleans, June 8 – 11 [Abstract No. 1176]. Forman, L. J., Marquis, D. E., and Stevens, R. (1985). The effect of chronic estrogen treatment on immunoreactivity b-endorphin levels in intact female rats. Proc. Soc. Exp. Biol. Med. 179, 217 – 228. Frye, C. A., Bock, B. C., and Kanarek, R. B. (1992). Hormonal milieu affects tailflick latency in females rats and may be attenuated by access to sucrose. Physiol. Behav. 52, 699 – 706. Frye, C. A., and Duncan, J. E. (1994). Progesterone metabolites, effective at the GABAA receptor complex, attenuate pain sensitivity in rats. Brain Res. 643, 194 – 203. Gintzler, A. R. (1980). Endorphin-mediated increases in pain threshold during pregnancy. Science 210, 193 – 195. Gordon, F. T., and Soliman, M. R. I. (1994). Diurnal variation in the
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effects of estradiol and progesterone on b-endorphin and Met-enkephalin levels in specific brain regions of ovariectomized rats. Pharmacology 49, 192 – 198. Irwin, S., Houde, R., Bennett, D., Hendershot, L., and Seevers, M. (1951). The effects of morphine, methadone and meperidine on some reflex responses of spinal animals to nociceptive stimulation. J. Pharmacol. Exp. Ther. 101, 132 – 143. Limonta, P., Maggi, R., Dondi, D., Martini, L., and Piva, F. (1987). Gonadal steroid modulation of brain opioid system. J. Steroid Biochem. 27, 691 – 698. Meyerson, B. J. (1967). Relationship between the anesthetic and gestagenic action and estrous behavior-inducing activity of different progestins. J. Endocrinol. 81, 369 – 371. Meyerson, B. J. (1972). Latency between intravenous injection of progestins and the appearance of estrous behavior in estrogen treated ovariectomized rats. Horm. Behav. 3, 1 – 9. Morley, J., Levine, A., Grace, M., Kneip, J., and Gosnell, B. (1984). The effect of ovariectomy, estradiol, and progesterone on opioid modulation of feeding. Physiol. Behav. 33, 237 – 241. Nomikos, G., Spyraki, C., Kazandjian, A., and Sfikakis, A. (1987). Estrogen treatment to ovariectomized rats modifies morphine-induced behavior. Pharmacol. Biochem. Behav. 27, 611 – 617. Ratka, A., and Simpkins, J. W. (1991). Effects of estradiol and progesterone on the sensitivity to pain and on morphine-induced antinociception in female rats. Horm. Behav. 25, 217 – 228. Ryan, S. M., and Maier, S. F. (1988). The estrous cycle and estrogen modulate stress-induced analgesia. Behav. Neurosci. 102, 371 – 380. Sander, H. W., and Gintzler, A. R. (1987). Spinal cord mediation of the opioid analgesia of pregnancy. Brain Res. 408, 389 – 393. Sander, H. W., Portoghese, P. S., and Gintzler, A. R. (1988). Spinal Kopiate receptor involvement in the analgesia of pregnancy: Effects of intrathecal nor-binalorphimine, a K-selective antagonist. Brain Res. 474, 343 – 347. Sander, H. W., Kream, R. M., and Gintzler, A. R. (1989). Spinal dynorphin involvement in the analgesia of pregnancy: Effect of intrathecal dynorphin antisera. Eur. J. Pharmacol. 159, 205 – 209. Selye, H. (1941). Anesthetic effects of steroid hormones. Proc. Soc. Exp. Biol. Med. 46, 116 – 121. Wardlaw, S., Thoron, L., and Frantz, A. (1982). Effects of sex steroids on brain beta-endorphin. Brain Res. 245, 327 – 331. Weiland, N. G., and Wise, P. M. (1990). Estrogen and progesterone regulate opiate receptor densities in multiple brain regions. Endocrinology 126, 804 – 808. Wilkinson, M., Brawer, J. R., and Wilkinson, D. A. (1985). Gonadal steroid induced modifications of opiate binding sites in anterior hypothalamus of female rats. Biol. Reprod. 32, 501 – 506.
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The Effects of Estradiol and Progesterone on Pain Sensitivity and Brain Opioid Receptors in Ovariectomized Rats Fran T. Gordon and Magdi R. I. Soliman College of Pharmacy, Florida A&M University, Tallahassee, Florida 32307
Previous studies in our laboratory have shown that estradiol and progesterone affect b-endorphin and Met-enkephalin levels in specific brain regions and that these effects are diurnally controlled. The present investigation was conducted to evaluate the effects of estradiol and progesterone on pain latency and brain opioid receptors of ovariectomized rats. Female Sprague–Dawley rats (100– 120 g) adapted to a 12 hr light:12 hr dark illumination cycle were used in these studies. Animals were ovariectomized under pentobarbital anesthesia. After a recovery period of 10–14 days, estradiol (50 mg/kg/day in 0.2 ml olive oil) and progesterone (5 mg/kg/day in 0.1 ml olive oil) were administered subcutaneously in the dorsal neck region alone and in combination at 16:00 hr for 7 days. Control animals received 0.2 ml olive oil. Control and treated groups were evaluated daily for pain latency postinjection using the tailflick and hotplate methods. On Day 7 of drug treatment, animals were sacrificed by decapitiation after pain latency evaluations. Whole brains were removed and immediately frozen at 0707C. Binding and affinity of brain opiate receptors were determined for each treatment group. Results obtained indicate that estradiol and progesterone treatment alone or in combination significantly alter pain latency. This alteration in pain was not accompanied by any change in affinity or number of mu opioid receptors. However, an increase in Kd of kappa opiate receptors was observed following treatment with estradiol, progesterone, or their combination. This increase in Kd of kappa opiate receptors may in part explain the increased hotplate sensitivity following estrogen administration. The present findings suggest that the decrease in pain sensitivity induced by estradiol or progesterone could not be explained by their effects on opioid receptors. The previously reported effects of estradiol and progesterone on brain levels of bendorphin and Met-enkephalin may contribute to the analgesic effects of these steroids. q 1996 Academic Press, Inc.
It has been well established that pregnancy and partition are associated with opioid-mediated elevations in
pain thresholds (Gintzler, 1980; Cogan and Spinnato, 1986; Baron and Gintzler, 1984; Sander, Kream, and Gintzler, 1989; Sander and Gintzler, 1987). The endogenous opioid system appears to be involved in raising maternal thresholds to pain. This maternal analgesia appears to be centrally mediated via the activation of a spinal cord dynorphin/kappa-opioid system (Sander et al., 1989; Sander and Gintzler, 1987; Sander, Portoghese, and Gintzler, 1988). Estrogen and progesterone interact in the regulation of various brain functions including those that control gonadotropin secretion and female sexual behavior. They are also known to be involved in the regulation of analgesia and nociception; however, the extent of their mechanisms in pain sensitivity is not yet fully understood. Nevertheless, estrogen treatment has been shown to modify morphine-induced analgesia (Nomikos, Spyraki, Kazandjian, and Sfikakis, 1987), reduce sensitivity to opioid antagonists (Morley, Levine, Grace, Kneip, and Gosnell, 1984), and induce changes in the levels of b-endorphin (Gordon and Soliman, 1994; Wardlaw, Thoron, and Frantz, 1982). Modifications of binding characteristics of mu opioid receptors have been studied in whole brain of female rats during the different phases of the estrous cycle, which are characterized by different secretory rates of estrogen and progesterone (Limonta, Maggi, Dondi, Martini, and Piva, 1987; Casular, Maggi, Dondi, Limonta, Piva, Motto, and Martini, 1987). The results demonstrated that in the total brain and in the hypothalamus of the female rat, the concentration of mu opioid receptors is significantly altered during the different phases of the estrous cycle. Moreover, the higher concentrations of hypothalamic mu receptors present during the period of the estrous cycle are associated with high serum levels of estrogen while the decrease in the density of the mu sites starts concomitantly with the increase in progesterone secretion. 0018-506X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Estradiol, Progesterone, Pain Sensitivity, and Opioid Receptors
The present studies were undertaken to determine whether estrogen and progesterone treatments modulate responsiveness to pain stimuli and whether this treatment might also modify the binding characteristics of brain mu and kappa opioid receptors in ovariectomized rats.
MATERIALS AND METHODS
tus. Their tails were smoothed into the groove with the light source focused as a 1-cm spot on the dorsal surface of the rat’s tail. All groups were tested daily for 7 days at 0, 15, 30, 60, and 120 min. Hotplate method. The temperature of the hotplate apparatus was maintained at 557 { 27C. Rats were placed in the hotplate apparatus and then immediately withdrawn at any indication of an escape attempt and/ or licking of the hind paws. All groups were tested daily at 0, 15, 30, 60, and 120 min.
Animals Female Sprague – Dawley rats (Harlan, Indianapolis, IN) weighing 100 – 120 g were used in this study. They were adapted to a 12 hr light:12 hr dark cycle. Food and water were provided ad libitum. All animals were ovariectomized under pentobarbital anesthesia (50 mg/ kg). After a recovery period of 10 – 14 days, the animals were randomly divided into treatment groups (six animals/group) for the tailflick and hotplate procedures. Separate groups were used for each procedure.
Drug Treatment Treated rats received 17-b-estradiol (50 mg/kg/day dissolved in 0.2 ml of olive oil), progesterone (5 mg/ kg/day in 0.1 ml of olive oil), or estradiol plus progesterone subcutaneously in the dorsal neck region at 16:00 hr for 7 days. All control animals received 0.2 ml olive oil. Control and treated groups were evaluated daily for 7 days for pain threshold at 15, 30, 60, and 120 min postinjection.
Pain Threshold Tailflick method. This is the most commonly used method of pain assessment throughout the stress-induced analgesia and opiate pharmacology literature. The tailflick method tests a reflex that is clearly tied to spinal pain mechanisms (Irwin et al., 1951). In addition, it is relatively insensitive to changes in general activity compared to other pain tests that involve the assessment of gross body movement, such as the hotplate method. The intensity of the light source was controlled by a rheostat to obtain a maximum control latency of 20 sec. In order to avoid injury to the animal’s tail, the light source was automatically terminated by tail movement. Animals’ tails were carefully examined after each session to ensure that no damage had occurred so as not to affect subsequent testing. All animals were held gently by the same experimenter throughout the study. Rats were placed on the platform of the tailflick appara-
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Opioid Receptor Binding Tissue preparation. After 7 days of drug treatment, all animals in each treatment group were sacrificed by decapitation. The whole brains were removed and immediately frozen at 0707C. Three brain tissues were pooled to generate three pools/group. Each group was homogenized in 10 ml each of ice-cold 50 nM Tris – HCl buffer (pH 7.5) containing 5 mM MgCl2 and 1 mM Captopril. All homogenates were centrifuged at 20,000 rpm for 10 min at 47C. The supernatants were discarded. The pellets were resuspended by sonication in 10 ml of buffer, incubated for 40 min at 377C, and then recentrifuged. All membranes were then washed four times at 20,000 rpm for 10 min at 47C. The final pellets were pooled and resuspended in 60 ml of buffer and frozen at 0707C until analysis. Kappa receptor binding assay. Aliquots from each pool of tissue membrane preparations were incubated for 60 min at 377C with increasing concentrations (1.25 – 40 nM ) of [3H]ethylketocyclazine (New England Nuclear, Boston, MA) in the presence of a cold ligand mixture (100 nM DAMGO and 100 nM DADLE; Sigma Chemical Co., St. Louis, MO), which was used to block the mu and delta receptors. All samples were assayed in triplicate. Nonspecific binding for each tissue sample was determined by adding 40 mM naloxone (Sigma Chemical Co.). The incubation was terminated by rapid vacuum filtration using Whatman GF/B filters presoaked in 0.1% polyethylenimine on a Brandel Cell Harvester. Each filter was washed four times with 4 ml of ice-cold 50 nM Tris – HCl buffer. Radioactivity on the filters were determined by liquid scintillation counting. Specific binding was defined as the average binding observed in the absence and presence of 40 mM naloxone hydrochloride. The Kd and Bmax values were calculated using the Lundon-1 binding analysis program. Protein was determined using a Bio-Rad protein assay kit from Bio-Rad Chemical Division (Richmond, CA). Mu receptor binding assay. The mu receptor binding was performed in a manner similar to that described in the kappa receptor assay. The tissue mem-
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FIG. 1. Effect of estradiol, progesterone, and estradiol plus progesterone on pain latency in the tailflick test at Time 0. C, control; E, estradiol; P, progesterone; *, significantly different from control, P õ 0.05.
branes were incubated with increasing concentrations (1.25 – 20 nM ) of [3H]DAMGO (New England Nuclear) in the presence or absence of 1 mM cold DAMGO. Statistical analysis. Statistical significance of differences for pain sensitivity was determined by repeated measures analysis of variance followed by the Tukey – Kramer comparisons test. The receptor binding results were analyzed using the unpaired t test.
FIG. 2. Effect of estradiol, progesterone, and estradiol plus progesterone on pain latency in the tailflick test at Time 30. C, control; E, estradiol; P, progesterone; *, significantly different from control, P õ 0.05.
at Day 4 than at Day 1 (62.8% increase). However, it was significantly lower (P õ 0.01) between Day 4 and Day 5 (61% decrease). By Day 7, a significant rise (P õ 0.05) in pain latency was observed compared to Day 5 (57.3% increase). On Day 3, rats treated with progesterone alone and estradiol plus progesterone produced a significant increase (P õ 0.05) in pain latency compared to controls (69.1 and 63.5% increase, respectively). After administration of estradiol alone on Day 5, there was
RESULTS Effect of Estradiol, Progesterone, and Estradiol Plus Progesterone on Pain Sensitivity Using the Tailflick Method The administration of estradiol and progesterone in combination at Time 0 produced a gradual increase in pain latency, with a significant rise (P õ 0.05) between Day 1 and Day 7 (56.2% increase) (Fig. 1). No significant difference in pain latency was observed with estradiol or progesterone treatment alone. On Day 7, there was a significant increase (P õ 0.05) in pain latency in the rats treated with estradiol plus progesterone compared to controls (54.3% increase). At Time 30, progesterone treatment resulted in a statistically significant increase (P õ 0.05) in pain latency compared to controls on Day 1 (66.4% increase) (Fig. 2). Estradiol treatment alone at Time 120 produced a significant increase (P õ 0.05) in pain latency by Day 7 compared to Day 1 (69.8% increase) (Fig. 3). When progesterone was administered alone, pain latency was significantly higher (P õ 0.01)
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FIG. 3. Effect of estradiol, progesterone, and estradiol plus progesterone on pain latency in the tailflick test at Time 120. C, control; E, estradiol; P, progesterone; *, significantly different from control, P õ 0.05.
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FIG. 4. Effect of estradiol, progesterone, and estradiol plus progesterone on pain latency in the hotplate test at Time 0. C, control; E, estradiol; P, progesterone; *, significantly different from control, P õ 0.05.
a significant increase (P õ 0.05) in pain latency compared to controls (59.2% increase).
Effect of Estradiol, Progesterone, and Estradiol Plus Progesterone on Pain Sensitivity Using the Hotplate Method Rats treated with estradiol alone at Time 0 showed a significantly lower (P õ 0.001) pain latency at Day 3 compared to Day 1 (53.8% decrease) (Fig. 4). This decline did not continue through Day 4, but was observed again at Day 5 (42.3% decrease). However, pain latency was significantly increased (P õ 0.05) between Day 3 and Day 4 (46.7% increase). By Day 7, there was a significant rise (P õ 0.001) in pain latency compared to Day 3 (53.8% increase). Progesterone treatment produced a similar decline in pain latency which started at Day 3 and continued through Day 5 compared to Day 2 (32.1 and 45.3% decrease, respectively). When comparing the different days studied, no significant difference was noted when estradiol and progesterone were administered in combination. On Day 3, estradiol treatment resulted in a significant decline (P õ 0.05) in pain latency (36.8% decrease) compared to controls. As shown in Fig. 5, animals treated with estradiol on Day 3 at Time 15 showed a significant increase (P õ 0.05) in pain latency compared to Day 2 (30.4% increase). However, there was a sudden decline (P õ 0.01) from Day 3 to Day 4 (43.5% decrease), which was followed by a significant increase (P õ 0.001) in pain latency by Day 6 (45.8% increase). A similar decline (P
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õ 0.05) between Day 3 and Day 4 was also observed when estradiol and progesterone were administered in combination (39.1% decrease). The combination treatment also resulted in a significant increase (P õ 0.01) in pain latency by Day 6 (44% increase). Progesterone alone produced a significant increase (P õ 0.01) in pain latency by Day 6 compared to Day 2 (41.3% increase). Estradiol and progesterone alone and in combination significantly decreased (P õ 0.001) pain latency on Day 1 compared to controls (32.3, 40.7, and 37.3% decrease, respectively). Estradiol treatment at Time 30 produced a significant increase (P õ 0.01) in pain latency at Day 6 compared to Day 4 (46% increase) (Fig. 6). When progesterone was administered alone, there was an immediate increase (P õ 0.001) from Day 1 to Day 2 (32.3% increase). A decrease in pain latency was observed at Day 3 through Day 4 (44.4% decrease). By Day 5, there was a rise in pain latency, but it was not significantly higher until Day 6 (38.8% increase). Similarly, estradiol and progesterone in combination resulted in a significant decline (P õ 0.01) from Day 3 to Day 4 (41.2% decrease) followed by a significant rise (P õ 0.01) in pain latency by Day 6 (42.3% increase). On Day 2, progesterone treatment resulted in a significant increase (P õ 0.01) in pain latency compared to controls (38.5% increase). At Time 120, rats treated with estradiol showed a significant decrease (P õ 0.01) in pain latency from Day 1 to Day 2 (48.1% decrease), which was followed by a significant rise (P õ 0.05) from Day 2 to Day 3 (40% increase) (Fig. 7). Administration of estradiol and pro-
FIG. 5. Effect of estradiol, progesterone, and estradiol plus progesterone on pain latency in the hotplate test at Time 15. C, control; E, estradiol; P, progesterone; *, significantly different from control, P õ 0.001.
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FIG. 6. Effect of estradiol, progesterone, and estradiol plus progesterone on pain latency in the hotplate test at Time 30. C, control; E, estradiol; P, progesterone; *, significantly different from control, P õ 0.01.
gesterone alone resulted in a gradual increase which was significantly higher (P õ 0.001) by Day 7 compared to Day 2 and Day 4 (59.7 and 55.2%; 56.2 and 54.8% increase, respectively). A similar effect was also noted when estradiol and progesterone were administered in combination (64.4 and 56.8% increase, respectively). When estradiol plus progesterone were administered, there was a significantly higher (P õ 0.05) pain latency compared to controls on Day 7 (35.1% increase).
FIG. 8. Effect of estradiol, progesterone, and estradiol plus progesterone on the specific binding and affinity of kappa opiate receptors in rat brain. E, estradiol; P, progesterone; *, significantly different from control, P õ 0.05.
Effect of Estradiol, Progesterone, and Estradiol Plus Progesterone on Brain Opioid Receptors Estradiol and progesterone alone and in combination resulted in significant changes in the binding affinities (Kd ) but not binding capacity (Bmax ) of kappa receptors (Fig. 8). There was a significant increase in Kd denoting a decrease in the binding affinity of kappa receptors. However, no significant differences were observed in the Bmax or Kd of mu opiate receptors when estradiol and progesterone were administered alone or in combination (Fig. 9).
DISCUSSION Results obtained in the present study indicate that estradiol and progesterone treatment altered the pain
FIG. 7. Effect of estradiol, progesterone, and estradiol plus progesterone on pain latency in the hotplate test at Time 120. C, control; E, estradiol; P, progesterone; *, significantly different from control, P õ 0.05.
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FIG. 9. Effect of estradiol, progesterone, and estradiol plus progesterone on the specific binding and affinity of mu opiate receptors in rat brain. E, estradiol; P, progesterone.
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latency of ovariectomized rats. In the tailflick method, estradiol treatment produced a significant increase in pain latency at Time 120 on Day 5. When progesterone was administered alone, pain latency was significantly higher than the controls on Day 1 at Time 30 and then again on Day 3 at Time 120. Estradiol plus progesterone also increased pain latency. These effects were observed on Day 3 at Time 120 and again on Day 7 at Time 0. However, in the hotplate method estradiol treatment alone produced a significant decrease in pain latency. This decline was detected only at Time 0 on Day 3 and Time 15 on Day 1. A similar decrease was observed on Day 1 at Time 15 following administration of progesterone and estradiol plus progesterone. By Day 2, progesterone significantly increased pain latency at Time 30. Nevertheless, estradiol and progesterone in combination did not increase pain latency until Day 7 at Time 120. Thus, our results demonstrate that analgesia induced by estradiol, progesterone, or their combination is more pronounced in the tailflick test. Several investigators have analyzed pain sensitivity across the estrus cycle; however, there are inconsistencies in their findings. It has been shown that during the evening of proestrus when estradiol and progesterone levels are rising, tailflick latencies are reduced compared to the metestrus (Frye, Bock, and Kanarek, 1992). On the other hand, Ryan and Maier (1988) did not find a significant difference in pain sensitivity in normal estrus cycling rats. These discrepancies may be due to the different experimental protocols utilized by the researchers. Frye et al. (1992) indicated that tailflick latencies in animals pretreated with estradiol were reduced following administration of progesterone (1.0 mg) compared to controls. Also, these studies demonstrated that as progesterone doses increase in the presence of estradiol, pain sensitivity is more pronounced. These results seem to differ from those obtained in the present study, which shows a significant decrease in pain sensitivity when estradiol and progesterone were administered alone and in combination. The discrepancy in the results may be explained by several reasons. First, the doses of estradiol and progesterone were lower in these studies, which may suggest that a certain level of estradiol and progesterone must be present for nociception to be affected. Moreover, other studies have shown that higher progesterone doses produce analgesia (Meyerson, 1967; Selye, 1941). Second, the inconsistencies in pain sensitivity may be due to whether estradiol and progesterone were administered alone or in combination (Meyerson, 1967; Selye, 1941; Meyerson, 1972). Finally, the possibility that different strains of rats have different endogenous levels of hormones or steroid re-
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ceptor characteristics may contribute to variations in pain latency. Ratka and Simpkins (1991) demonstrated that ovariectomized rats treated with estradiol alone for two days showed significantly increased sensitivity to thermal stimulation. Our findings are consistent with this study, which indicates that estradiol treatment increases pain sensitivity in the hotplate test. However, we observed these effects at Day 1 and Day 3. Some researchers suggest that this hyperalgesia may result from an estradiolinduced increase in the b-endorphin levels in the hypothalamus (Forman, Marqis, and Stevens, 1985) and the reduction of opioid binding sites (Wilkinson, Brawer, and Wilkinson, 1985). The increase in Kd of kappa opiate receptors, observed in the present study, may in part explain the increased hotplate sensitivity following estrogen administration. It has been reported that the number of opioid receptors in the whole brain fluctuates during the estrous cycle (Limonta et al., 1987). Generally, the number of hypothalamic mu opioid receptors is elevated during the morning of diestrus Day 2 and during the day of proestrus and then declines at the end of the day. This decline in the number of mu receptors observed in the proestrus may be due to the rise in serum levels of estradiol and progesterone. Previous studies have shown that ovariectomy increased opioid binding sites (Wilkinson et al., 1985; Weiland and Wise, 1990) but blocked or eliminated opioid analgesia (Ryan and Maier, 1988). However, estradiol treatment has been found to restore opioid analgesia (Ryan and Maier, 1988) and decrease the number of opioid binding sites (Wilkinson et al., 1985). A lower number of mu binding sites were observed following administration of progesterone. Chronic estradiol treatment has been reported to increase the number of opioid binding sites (Wilkinson et al., 1985). Moreover, estradiol plus progesterone treatment resulted in a significant decrease in the number of mu receptors in the brain (Dondi, Maggi, Limonta, and Piva, 1988). In the present study, no effects on brain mu receptors’ maximum binding nor affinity could be detected. Peripheral sex steroids have been shown to influence central opioid activity. Moreover, the changes in steroid hormones during pregnancy modulate a spinal opioid system. This spinal cord dynorphin/kappa-opioid system has been reported to elevate maternal nociceptive thresholds during gestation (Sander et al., 1988, 1989; Sander and Gintzler, 1987). Thus, the levels of estradiol and progesterone during gestation may trigger the activation of this spinal opioid system, which may be responsible for decreased pain sensitivity. Our results have shown that estradiol and progesterone alone and
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in combination produced a significant decrease in the binding affinity of kappa opiate receptors as indicated by an increase in Kd . These findings suggest that the decrease in pain sensitivity induced by estradiol or progesterone could not be explained by their effects on opioid receptors. Other mechanisms may be involved in progesterone-induced analgesia. Frye and Duncan (1994) have reported that progesterone metabolites can attenuate pain sensitivity by interacting with the GABAA . However, the release of endogenous opiates reported in our previous studies (Gordon and Soliman, 1994) may play a role in the analgesic effects of estradiol and progesterone.
REFERENCES Baron, S. A., and Gintzler, A. R. (1984). Effects of hypophysectomy and dexamethasone treatment on plasma b-endorphin and pain threshold during pregnancy. Brain Res. 321, 344 – 346. Casulari, L. A., Maggi, R., Dondi, D., Limonta, P., Piva, F., Motta, M., and Martini, L. (1987). Effect of oestrus cyclicity on the number of brain opioid mu receptors in the rat. Horm. Metab. Res. 19, 549 – 554. Cogan, R., and Spinnato, J. A. (1986). Pain and discomfort thresholds in late pregnancy. Pain 27, 63 – 68. Dondi, D., Maggi, R., Limonta, P., and Piva, F. (1988). Sex steroid positive feedback decreases mu receptors density in the brain of long-term castrated female rats. In Proceedings, 70th Annual Meeting of the Endocrine Society, New Orleans, June 8 – 11 [Abstract No. 1176]. Forman, L. J., Marquis, D. E., and Stevens, R. (1985). The effect of chronic estrogen treatment on immunoreactivity b-endorphin levels in intact female rats. Proc. Soc. Exp. Biol. Med. 179, 217 – 228. Frye, C. A., Bock, B. C., and Kanarek, R. B. (1992). Hormonal milieu affects tailflick latency in females rats and may be attenuated by access to sucrose. Physiol. Behav. 52, 699 – 706. Frye, C. A., and Duncan, J. E. (1994). Progesterone metabolites, effective at the GABAA receptor complex, attenuate pain sensitivity in rats. Brain Res. 643, 194 – 203. Gintzler, A. R. (1980). Endorphin-mediated increases in pain threshold during pregnancy. Science 210, 193 – 195. Gordon, F. T., and Soliman, M. R. I. (1994). Diurnal variation in the
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effects of estradiol and progesterone on b-endorphin and Met-enkephalin levels in specific brain regions of ovariectomized rats. Pharmacology 49, 192 – 198. Irwin, S., Houde, R., Bennett, D., Hendershot, L., and Seevers, M. (1951). The effects of morphine, methadone and meperidine on some reflex responses of spinal animals to nociceptive stimulation. J. Pharmacol. Exp. Ther. 101, 132 – 143. Limonta, P., Maggi, R., Dondi, D., Martini, L., and Piva, F. (1987). Gonadal steroid modulation of brain opioid system. J. Steroid Biochem. 27, 691 – 698. Meyerson, B. J. (1967). Relationship between the anesthetic and gestagenic action and estrous behavior-inducing activity of different progestins. J. Endocrinol. 81, 369 – 371. Meyerson, B. J. (1972). Latency between intravenous injection of progestins and the appearance of estrous behavior in estrogen treated ovariectomized rats. Horm. Behav. 3, 1 – 9. Morley, J., Levine, A., Grace, M., Kneip, J., and Gosnell, B. (1984). The effect of ovariectomy, estradiol, and progesterone on opioid modulation of feeding. Physiol. Behav. 33, 237 – 241. Nomikos, G., Spyraki, C., Kazandjian, A., and Sfikakis, A. (1987). Estrogen treatment to ovariectomized rats modifies morphine-induced behavior. Pharmacol. Biochem. Behav. 27, 611 – 617. Ratka, A., and Simpkins, J. W. (1991). Effects of estradiol and progesterone on the sensitivity to pain and on morphine-induced antinociception in female rats. Horm. Behav. 25, 217 – 228. Ryan, S. M., and Maier, S. F. (1988). The estrous cycle and estrogen modulate stress-induced analgesia. Behav. Neurosci. 102, 371 – 380. Sander, H. W., and Gintzler, A. R. (1987). Spinal cord mediation of the opioid analgesia of pregnancy. Brain Res. 408, 389 – 393. Sander, H. W., Portoghese, P. S., and Gintzler, A. R. (1988). Spinal Kopiate receptor involvement in the analgesia of pregnancy: Effects of intrathecal nor-binalorphimine, a K-selective antagonist. Brain Res. 474, 343 – 347. Sander, H. W., Kream, R. M., and Gintzler, A. R. (1989). Spinal dynorphin involvement in the analgesia of pregnancy: Effect of intrathecal dynorphin antisera. Eur. J. Pharmacol. 159, 205 – 209. Selye, H. (1941). Anesthetic effects of steroid hormones. Proc. Soc. Exp. Biol. Med. 46, 116 – 121. Wardlaw, S., Thoron, L., and Frantz, A. (1982). Effects of sex steroids on brain beta-endorphin. Brain Res. 245, 327 – 331. Weiland, N. G., and Wise, P. M. (1990). Estrogen and progesterone regulate opiate receptor densities in multiple brain regions. Endocrinology 126, 804 – 808. Wilkinson, M., Brawer, J. R., and Wilkinson, D. A. (1985). Gonadal steroid induced modifications of opiate binding sites in anterior hypothalamus of female rats. Biol. Reprod. 32, 501 – 506.
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