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Coadministration of dextromethorphan during pregnancy and throughout lactation prevents morphine-induced hyperprolactinemia in female rats
Coadministration of dextromethorphan during pregnancy and throughout lactation prevents morphine-induced hyperprolactinemia in female rats Ling-Yi Wu, M.D.,a,b Eagle Yi-Kung Huang, Ph.D.,a,c and Pao-Luh Tao, Ph.D.a,c a
Graduate Institute of Medical Sciences, National Defense Medical Center, b Division of Endocrinology and Metabolism, Department of Internal Medicine, Tri-Service General Hospital, and c Department of Pharmacology, National Defense Medical Center, Taipei, Taiwan, Republic of China
Objective: To investigate whether coadministration of dextromethorphan (DM) could suppress morphine-induced hyperprolactinemia in female rats during pregnancy and throughout lactation. Design: Controlled prospective study. Setting: University research laboratory. Animal(s): One hundred adult female Sprague-Dawley rats. Intervention(s): Rats were randomly divided into four groups and were subcutaneously injected with either saline, morphine, morphine þ DM, or DM alone twice a day, progressively increasing by 1 mg/kg at 7-day intervals from an initial dose of 2 mg/kg for both morphine and DM. Drug administration was continued during pregnancy. After the offspring were born, the doses injected into the dams were increased by 1 mg/kg every 2 weeks. Main Outcome Measure(s): Serum prolactin (PRL) concentration and dopamine turnover rate at the hypothalamus and pituitary. Result(s): Chronic morphine administration induced higher PRL concentrations than the control animals at mating, and at early and late pregnancy. In rats receiving DM coadministration, we did not observe any increase by morphine. Our neurochemical results showed that this effect of DM may be partly through blocking the effect of morphine on inhibition of tuberoinfundibular dopaminergic (TIDA) neuronal activity. Conclusion(s): The use of DM as an adjuvant in females receiving chronic morphine treatment may prevent morphine-induced hyperprolactinemia. (Fertil Steril 2010;93:1686–94. 2010 by American Society for Reproductive Medicine.) Key Words: Dextromethorphan, dopamine, morphine, prolactin, tuberoinfundibular
Heroin addiction during pregnancy may lead to both acute and chronic abnormalities in neonates, such as fetal death, intrauterine growth retardation, prematurity, and withdrawal symptoms (1–4). Malnutrition, venereal disease, hepatitis, pulmonary disorders, preeclampsia, and third-trimester bleeding are the most common maternal complications of heroin addiction (2). Opioids have been proven to influence the hypothalamus-pituitary-gonadal axis and modulate hormonal release, including an increase in the prolactin (PRL) level (5). Prolactin play a key role in female reproduction, and its levels change dramatically during different reproductive stages. In cycling rats, the proestrus PRL surge coincides Received October 3, 2008; revised January 25, 2009; accepted January 26, 2009; published online March 26, 2009. L-Y.W. has nothing to disclose. E.Y-K.H. has nothing to disclose. P-L.T. has nothing to disclose. This study was supported by grants from the National Health Research Institutes (NHRI-EX96-9401NP) and National Science Council (NSC 96-2320-B-016-020-MY3), Taipei, Taiwan, R.O.C. Reprint requests: Pao-Luh Tao, Ph.D., Department of Pharmacology, National Defense Medical Center, Nei-hu, No.161, Section 6, Min-Chuan East Road, Taipei 114, Taiwan, R.O.C. (FAX: 886-2-87923155; E-mail: [email protected]).
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temporally with the preovulatory luteinizing hormone (LH) surge (6, 7). Prolactin is essential for the support of the corpus luteum in rodents. The situation in humans is different: PRL is not luteotropic and, with the exception of lactation, does not have clear effects on most reproductive processes under normal conditions. Although there is no surge of PRL throughout the menstrual cycle in women (8), PRL is secreted by the ovaries and also is found in human follicular fluid (9). Oocyte maturation and successful pregnancy have been found to be associated with high levels of PRL in follicular fluid (10). Therefore, in the human ovary, PRL may play autocrine/paracrine roles at the time of ovulation or beyond. During pregnancy and lactation, the prolonged period of hyperprolactinemia is required for mammary gland development and function and for maternal behavior immediately after parturition in both rodents and humans (11). Prolactin has an important role in the metabolic adaptations of the mother, contributing to the induction of leptin resistance in increased food intake during pregnancy (12–14). Prolactin may also participate in suppression of the stress response (15), in regulation of the activity of oxytocin neurons (16), and in the inhibition of fertility (17). All of these functions are important adaptations mediated by the maternal brain.
Fertility and Sterility Vol. 93, No. 5, March 15, 2010 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/10/$36.00 doi:10.1016/j.fertnstert.2009.01.143
Many studies have reported that these PRL nocturnal surges during early pregnancy (18, 19), the antepartum PRL surge during late pregnancy (20), and suckling-induced PRL secretion during lactation (21, 22) are regulated by opioids and mediated by contemporary stimulation of both kand m-opioid receptors on tuberoinfundibular dopaminergic (TIDA) neurons in the hypothalamus (23). It has been found that k-opioid receptor inhibition of TIDA neuronal activity may predominate during late pregnancy (23–25). In spite of differences in the mechanisms regulating PRL release during each of the reproductive stages, the role of opioids in facilitating PRL secretion seems similar (26). Dextromethorphan (DM), the dextrorotatory isomer of levomethorphan, is best known for its antitussive effect and is without an opiate-like activity. It has widespread binding sites in the central nervous system, including N-methyl-D-aspartate (NMDA) receptors, sigma receptors, and a3b4 nicotinic receptors (27, 28). Wong et al. (29) found that the preoperative administration of DM could improve postoperative pain control in patients undergoing various surgical procedures; therefore, DM may be used as an adjunct with opioids (29–31). It has been reported that coadministration of DM with morphine during pregnancy and throughout lactation could prevent some of the adverse effects associated with chronic morphine treatment in rat offspring; these effects include higher mortality, retardation in body weight gain, development of morphine tolerance and withdrawal, and alterations in NMDA receptor densities and postsynaptic density proteins in the hippocampus (32–34). Dextromethorphan also effectively blocked the increase in the serum PRL level induced by chronic morphine administration in male rats (unpublished data). Dopamine is the principal central inhibitory factor in PRL release (35). Prolactin production and negative feedback regulation are largely controlled by TIDA neurons, which terminate around the capillary loops in the median eminence of the hypothalamus. Tuberohypophyseal dopaminergic (THDA) neurons and periventricular-hypophyseal dopaminergic (PHDA) neurons that reach the anterior lobe of the pituitary contribute to basal regulation of PRL secretion as well (11). Bero and Kuhn (36) reported that morphine-induced stimulation of PRL was related to the reduction of TIDA neuron activity. In view of the therapeutic potential of DM in combination with morphine, we explored the possible effect of DM on morphine-induced hyperprolactinemia in female rats as well as determined whether the underlying mechanism of DM was through the TIDA system and/or the THDA/PHDA system. MATERIALS AND METHODS Animals Adult female Sprague-Dawley rats were purchased from the National Experimental Animal Centre, Taipei, Taiwan. The animals were housed one or two per cage in a room maintained at a temperature of 23 2 C with a 12-hour light/ dark cycle. Food and water were available ad libitum Fertility and Sterility
throughout the experiment. The care of the animals was carried out in accordance with institutional and international standards (U.S. National Institutes of Health principles of laboratory animal care), and the protocol was approved by the Institutional Animal Care and Use Committee of the National Defense Medical Center, Taiwan. Protocols and Drug Dosages The animals were acclimated for at least 3 days before the experiments. All blood samples were obtained between the hours of 0930 and 1300. The rats were randomly divided into four groups, with at least eight rats in each group. The rats underwent subcutaneous injection of saline (1 mL/kg, control group), morphine (morphine group), morphine þ DM (morphine þ DM group), or DM (DM group) twice a day (0900 and 1700), progressively increasing by 1 mg/ kg at 7-day intervals from an initial dose of 2 mg/kg for both morphine and DM. The rats were mated between days 7 and 8. Administration of drugs continued during pregnancy. After rat offspring were born, the doses of morphine or DM injected into the maternal rats were increased by 1 mg/kg every 2 weeks until 21 days after delivery, when the offspring were already weaned. The final doses of morphine and DM were both 7 to 8 mg/kg before weaning. Blood samples were collected from all of the animals 1 week before saline or drug administration to obtain a baseline PRL level for comparison. The successful mating was established by the vaginal plug or the sperm formation in the vaginal smears (37). The day of mating was termed as day 1 of pregnancy. On day 7 (early pregnancy), 14 (late pregnancy), 21 (parturition), 28 (early lactation), 35 (late lactation), and 42 (weaning), blood samples were collected 20 minutes after the morning injection of saline or drug(s). The sera were quickly separated and collected via centrifugation (500 g, 15 minutes) at 4 C and then frozen at –80 C until the PRL level was measured. During the entire course, these animals were not implanted with any cannula. On day 1 (mating) or 42 (weaning), rats were killed by decapitation 20 minutes after saline or drug injection, and blood samples were collected. The brains were rapidly removed and placed on dry ice immediately. The hypothalamus (including the dorsal part of the median eminence) and pituitary were dissected out and frozen in liquid nitrogen until use for neurochemical analysis. Enzyme-linked Immunoassay (EIA) for Prolactin Serum levels of PRL were measured by a specific enzymelinked immunoassay kit (SPI-BIO, Massy, France) for the determination of rat PRL levels (38). The limit of sensitivity for the assay was below 1 pg/mL. High-Performance Liquid Chromatography Analysis of Dopamine and Dopamine Metabolites The high-performance liquid chromatography system was composed of a reverse-phase C-18 column (MD-150, 1687
RP-C-18, 3 mm, length: 15 cm; ESA Biosciences, Chelmsford, MA) and a high-pressure pump (LC-10AD; Shimadzu, Kyoto, Japan) and connected with an electrochemical detector (ECD) coupled with three electrodes (Coulochem II; ESA Biosciences). The electrode of the guard cell was set at 40 mV, and electrodes 1 and 2 (for detection) were set at 250 and 350 mV, respectively. Under an isocratic condition, the mobile phase solvent (MD-TM; ESA Biosciences) was circulated at a flow rate of 0.5 mL/minute in the system. To quantify the sample peaks, each chemical (dopamine [DA], 3,4-dihydroxyphenylacetic acid [DOPAC], and homovanillic acid [HVA]) was compared with the external standards, which were freshly prepared and injected every five-sample run. The dopamine turnover rate was calculated and obtained as ([DOPAC] þ [HVA])/[DA]. Statistical Analysis The data were expressed as mean standard error of the mean. One-way or two-way analysis of variance (ANOVA) followed by the Bonferroni test was used to analyze the significance of the differences between groups as well as the differences between the time points. P<.05 was considered statistically significant. Chemicals Morphine hydrochloride was purchased from the National Bureau of Controlled Drugs, National Health Administration, Taipei, Taiwan. Dextromethorphan and all other chemicals were purchased from Sigma Chemical (St. Louis, MO). RESULTS Serum PRL Profile during Different Reproductive Stages with Saline Injection The basal level of serum PRL in the control group was 43.72 2.08 ng/mL compared with 202.06 46 ng/mL after mating (versus basal level, P<.01), 71.78 16.47 ng/mL in early pregnancy (P¼ not statistically significant [NS]), 93.03 13.56 ng/mL in late pregnancy (P¼NS), 196.01 61.45 ng/mL during parturition (P<.01), 242.67 55.31 ng/mL in early lactation (P<0.001), 61.50 8.28 ng/mL in late lactation (P¼NS), and 52.23 5.30 ng/mL during weaning (P¼NS) (Fig. 1). Effect of Chronic Morphine Administration on Serum PRL Concentration during Different Reproductive Stages Chronic morphine administration resulted in a statistically significant increase in serum PRL concentration in mating, and in early and late pregnancy. The basal PRL concentration was 55.96 3.57 ng/mL for the morphine group, which is not statistically significantly different from the control (saline) group (43.72 2.08 ng/mL). After mating, the PRL concentration showed a statistically significant increase to 348.76 28.21 ng/mL in response to morphine administration, and was statistically significantly higher than that of the control 1688
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group (202.06 46.00 ng/mL, P<.05). Chronic morphine administration resulted in a statistically significant increase in PRL release in early pregnancy (325.52 13.10 ng/mL, in the morphine group versus 71.78 16.47 ng/mL, in the control group, P<.001); late pregnancy (240.39 46.74 ng/mL in the morphine group versus 93.03 13.56 ng/mL in the control group, P<.05); late lactation (196.34 27.36 ng/mL in the morphine group versus 61.50 8.28 ng/mL in the control group, P<.05); and during weaning (185.67 18.78 ng/mL in the morphine group versus 52.23 5.30 ng/mL in the control group, P<.05) (see Fig. 1). No statistically significant change was found in serum PRL concentration with chronic morphine exposure in early lactation (325.50 13.10 ng/mL in the morphine group versus 242.67 55.31 ng/mL in the control group, P¼NS). It was noteworthy that there was a statistically significant decrease in the serum PRL concentration after parturition (65.71 7.67 ng/mL in the morphine group versus 196.01 61.45 ng/mL in the control group, P<.05) (see Fig. 1). No Effect of DM Administration on Serum PRL Concentration Compared with Saline Injection from Mating to Weaning The results of serum PRL concentration under chronic DM administration revealed no statistically significant difference from the control group during pregnancy or throughout lactation. The basal level of serum PRL in the DM group was 45.76 1.10 ng/mL versus 43.72 2.08 ng/mL in the control group (P¼NS). After mating, the serum PRL level was 187.06 47.86 ng/mL in the DM group versus 202.06 46.00 ng/mL in the control group (P¼NS). In early pregnancy, the serum PRL level was 53.48 14.98 ng/mL in the DM group versus 71.78 16.47 ng/mL in the control group (P¼NS). In late pregnancy, the serum PRL level was 39.41 12.24 ng/mL in the DM group versus 93.03 13.56 ng/mL in the control group (P¼NS). After parturition, the serum PRL level was 164.16 3.15 ng/mL in the DM group versus 196.01 61.45 ng/mL in the control group (P¼NS). In early lactation, the serum PRL level was 171.11 46.91 ng/mL in the DM group versus 242.67 55.31 ng/mL in the control group (P¼NS). In late lactation, the serum PRL level was 66.78 8.49 ng/mL in the DM group versus 61.50 8.28 ng/mL in the control group (P¼NS). During weaning, the serum PRL level was 64.92 13.28 ng/mL in the DM group versus 52.23 5.30 ng/ mL in the control group (P¼NS) (Fig. 2). Effect of DM on Morphine-induced Further Increase of PRL Release after Mating, Early and Late Pregnancy, Early and Late Lactation, and Weaning Chronic administration of morphine during pregnancy and throughout lactation caused a onefold to fivefold increase in serum PRL concentration when compared with the control (saline) group. No statistically significant difference was found in PRL concentrations after coadministration of DM
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FIGURE 1 Effect of morphine on serum prolactin (PRL) concentration during different reproductive stages. Animals received subcutaneous injections of saline or morphine twice per day, and the dose of morphine was progressively increased. Each group contained at least eight rats. Data are presented as mean standard error of mean. Twoway analysis of variance followed by the Bonferroni test was used to analyze the statistical significance of the differences between groups as well as time points of the saline group. DM ¼ dextromethorphan; E ¼ early pregnancy; EL ¼ early lactation; L ¼ late pregnancy; LL ¼ late lactation; M ¼ morphine. **P< .01, ***P< .001 versus basal level; #P< .05, ###P< .001 versus control (saline) group.
Wu. Dextromethorphan cures morphine HyperPRL. Fertil Steril 2010.
with morphine when compared with the control group during pregnancy or throughout lactation (Fig. 3). In the morphine þ DM group, however, a statistically significantly lower level of PRL was observed when compared with the morphine group. The PRL level was as follows: after mating (196.08 28.79 ng/mL in the morphine þ DM group versus 348.76 28.21 ng/mL in the morphine group, P<.001); early pregnancy (42.53 3.32 ng/mL in the morphine þ DM group versus
325.52 13.10 ng/mL in the morphine group, P<.001); late pregnancy (37.04 3.55 ng/mL in the morphine þ DM group versus 240.39 46.74 ng/mL in the morphine group, P<.001); early lactation (190.96 47.25 ng/mL in the morphine þ DM group versus 325.50 13.10 ng/mL in the morphine group, P<.01); late lactation (61.93 5.3 ng/mL in the morphine þ DM group versus 196.34 27.36 ng/mL in the morphine group, P<.01); and weaning (40.8 2.72 ng/mL
FIGURE 2 Effect of dextromethorphan (DM) on serum prolactin (PRL) concentration during different reproductive stages. Animals received subcutaneous injections of saline or dextromethorphan (DM) twice per day, and the dose of DM was progressively increased. Each group contained at least eight rats. Data are presented as mean standard error of the mean. There was no statistically significant difference between these two groups after two-way analysis of variance. E ¼ early pregnancy; EL ¼ early lactation; L ¼ late pregnancy; LL ¼ late lactation.
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FIGURE 3 Effect of coadministration of dextromethorphan (DM) with morphine (M þ DM) on hyperprolactinemia induced by chronic morphine administration. Each group contained at least eight rats. Data are presented as mean standard error of mean. Two-way analysis of variance followed by the Bonferroni test was used to analyze the statistical significance of the differences between the M and M þ DM groups. **P< .01, ***P< .001, M versus M þ DM group. E ¼ early pregnancy; EL ¼ early lactation; L ¼ late pregnancy; LL ¼ late lactation.
Wu. Dextromethorphan cures morphine HyperPRL. Fertil Steril 2010.
in the morphine þ DM group versus 185.67 18.78 ng/mL in the morphine group, P<.01) (see Fig. 3). Dopamine Turnover Rate in the Hypothalamus during Mating and Weaning After mating, the dopamine turnover rate in the hypothalamus was 0.21 0.01 in the morphine group, which was statistically significantly lower than that in the control group (0.41 0.03, P<.001). Dextromethorphan by itself did not change the dopamine turnover rate in the hypothalamus (0.38 0.02). When DM was coadministered with morphine (morphine þ DM group), the dopamine turnover rate in the hypothalamus (0.29 0.01) was still statistically significantly lower than that in the control group (0.41 0.03, P<.01) but was higher than that in the morphine group (0.21 0.01, P<.05) (Fig. 4A). We may reasonably conclude that DM partially reverses the morphine-induced decrease in the dopamine turnover rate in the hypothalamus after mating. During weaning, chronic morphine administration caused a statistically significant decrease in the dopamine turnover rate (1.53 0.19) when compared with the control group (4.83 0.84, P<.05). When DM was coadministered with morphine (morphine þ DM group), the dopamine turnover rate in the hypothalamus (4.17 0.81) was not statistically significantly different from that in the control group (4.83 0.84) (see Fig. 4B). It seems reasonable to conclude that DM can reverse the morphine-induced decrease in the dopamine turnover rate in the hypothalamus during weaning. Dopamine Turnover Rate in the Pituitary during Mating and Weaning After mating, the dopamine turnover rate in the pituitary was 0.31 0.03 in the morphine group, which was statistically 1690
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significantly lower than that of the control group (0.64 0.04, P<.001). In the morphine þ DM group, the dopamine turnover rate in the pituitary was also decreased (0.28 0.03, P<.001) (Fig. 5A). Thus, DM did not reverse the decrease in the dopamine turnover rate in the pituitary caused by chronic morphine administration. During weaning, chronic morphine administration caused a statistically significant decrease in the dopamine turnover rate (4.54 0.8) when compared with that of the control group (9.54 0.5) (P<.01). In the morphine þ DM group, the dopamine turnover rate in the pituitary (10.14 1.05) was not statistically significantly different from that of the control group (9.54 0.5) (see Fig. 5B). Thus, DM could reverse the morphine-induced decrease in the dopamine turnover rate in the pituitary during weaning.
DISCUSSION Our study reports on the inhibitory effects of DM on morphine-induced hyperprolactinemia in female rats. First, we demonstrated a regular change of serum PRL level at different reproductive stages in female rats. Acute significant increases in PRL after mating appear to be related to vaginocervical stimulation. It has been reported that this PRL surge could occur in cycling exposure to males between the hours of 1400 on proestrus and 0300 on the day of estrus (39– 41). The response was 5-fold to 10-fold above the baseline PRL level and was noted at 8 to 12 hours after mating (42), which was consistent with our results. The first 10 to 12 days of rat pregnancy are dominated by daily nocturnal (0300 to 0500 h) and diurnal (1700 to 2000 h) PRL surges (6, 43); these surges cease during the second half of pregnancy (44). In our present results, no statistically significant difference of the serum PRL level was found between the
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FIGURE 4
FIGURE 5
Effect of morphine (M), dextromethorphan (DM), or coadministration of dextromethorphan with morphine (M þ DM) on the dopamine turnover rate in the hypothalamus after (A) mating or (B) weaning. Each group contained at least eight rats. Data are presented as mean standard error of mean. Oneway analysis of variance followed by the Bonferroni test was used to analyze the statistical significance of the differences between groups. ***P< .001; **P< .01; *P< .05 when compared with the control (C) group; #P< .05 when compared with the morphine group.
Effect of morphine (M), dextromethorphan (DM), or coadministration of dextromethorphan with morphine (M þ DM) on the dopamine turnover rate in the pituitary after (A) mating or (B) weaning. Each group contained at least eight rats. Data are presented as mean standard error of mean. Oneway analysis of variance followed by the Bonferroni test was used to analyze the statistical significance of the difference between groups. ***P< .001; **P< .01 when compared with the control (C) group.
Wu. Dextromethorphan cures morphine HyperPRL. Fertil Steril 2010. Wu. Dextromethorphan cures morphine HyperPRL. Fertil Steril 2010.
seems that all of the suckling turned out to be weaker at this stage in our animal model. basal state and early/late pregnancy, which should be due to our experimental design to avoid the PRL surges by sampling the blood at the time between the hours of 0930 and 1300. Our data also showed a statistically significant increase of serum PRL after parturition, which is consistent with the results of other reports (44–46). During the 3 weeks of lactation, stimulation of PRL was maintained to a considerable extent by the suckling reflex (46, 47). We found that the serum PRL level of early lactation was truly higher, but the PRL level of late lactation was close to the basal value. In fact, we adjusted the litter size (8 to 10) for each dam to be similar to keep the consistent extent of suckling because the suckling reflex dominated the secretion of PRL in lactation. Although it is difficult to explain the phenomenon at late lactation, it Fertility and Sterility
After parturition, PRL secretion becomes closely linked to the suckling stimulus, where the extent of suckling by the offspring results in a parallel increase of PRL secretion, acting through a decrease of TIDA neuronal activity (11). After parturition, our data showed a statistically significant increase in serum PRL concentration, but chronic morphine administration caused a statistically significant decrease when compared with the control (saline) group. These results imply that chronic morphine administration could impair the sucking reflex of offspring and increase TIDA neuronal activity temporarily after parturition. This could be a consequence of morphine withdrawal in pups after birth. Although the statistical analysis did not show significance, the coadministration of DM tended to reverse this adverse effect by morphine. 1691
Chronic morphine administration resulted in a statistically significant further increase in serum PRL concentration after mating, and also at early and late pregnancy. Apart from parturition, the serum PRL level was statistically significantly higher in the morphine group than the control group at the early and late lactation stages and at weaning. This indicated a clear hyperprolactinemia caused by chronic morphine administration. Coadministration of DM with morphine statistically significantly blocked the increase in the serum PRL level caused by chronic morphine administration during different reproductive stages except parturition. Chronic administration of morphine caused a 1.5-fold to 5-fold increase in the serum PRL level compared with the control level. These increases in PRL level were almost completely blocked by the coadministration of DM with the same dose and injection protocol as that of morphine. Nevertheless, DM by itself did not cause any statistically significant changes in the serum PRL level when compared with the control. To investigate the possible mechanism(s) underlying the actions of morphine and DM in PRL release, we dissected the hypothalamus (including the median eminence) and the pituitary and determined the dopamine turnover rate at these two brain regions. Because the dopamine turnover rate is well recognized as an indicator of dopamine neuronal activity at the nerve terminal (48, 49), the dopamine turnover rates in the hypothalamus and the pituitary may represent the neuronal output intensity of the TIDA neurons and THDA/PHDA neurons, respectively. During weaning, we found that there was 10-fold to 15-fold increase of the dopamine turnover rate in both the hypothalamus and the pituitary when compared with the rate after mating in the control group. Because PRL has been proven to be under the inhibitory regulation of dopamine, this is consistent with the serum PRL concentration showing a parallel drop after weaning. In addition, chronic morphine administration caused a statistically significant decrease in the dopamine turnover rate in both the hypothalamus and the pituitary at two different reproductive stages, mating and weaning. From these results, we speculated that the effects of chronic morphine-induced further hyperprolactinemia after mating and at the different reproductive stages may be related to its inhibitory effect on the TIDA system as well as the THDA and PHDA systems. After mating, coadministration of DM with morphine partially reversed the inhibitory effect of morphine on the dopamine turnover rate in the hypothalamus but did not reverse the effect of morphine in the pituitary. However, during weaning, coadministration of DM with morphine completely reversed the inhibitory effect of morphine on the dopamine turnover rate in both of the hypothalamus and the pituitary. These results suggest that DM may act through certain unknown mechanisms to suppress the effect of morphine on the TIDA, THDA, and PHDA systems after mating and during weaning. In female rats, the other NMDA receptor antagonists, such as MK-801 and CGS-19755 decreased the dopamine turnover rate in the median eminence (50). This suggests that 1692
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these compounds alone are blocking the actions of endogenous excitatory amino acid neurotransmitters at the NMDA receptor, which tonically activate the TIDA neurons. However, the same report demonstrated that MK-801 markedly decreased basal PRL secretion in female rats. This seems to be controversial but actually could be explained by the direct inhibitory effect of MK-801 on the lactotroph (51). Blocking the feedback stimulatory effect of PRL on TIDA neurons did not affect the inhibitory effect of MK-801 on TIDA neurons. On the other hand, removal of the tonic stimulatory effects of endogenous PRL in female rats decreases TIDA neuronal activity (52). Taken together, these results could indicate that the inhibitory effect of MK-801 on TIDA neurons occurs independent of its inhibitory effect on PRL secretion. Unlike MK-801, our data showed that DM by itself did not have any effect on the PRL secretion or the activity of TIDA neurons in our animal model. Because DM behaved differently from the other NMDA receptor antagonists, we propose that DM acts at targets (e.g., sigma and a3b4 nicotinic receptors) other than NMDA receptors to suppress morphineinduced hyperprolactinemia. We found no studies in the literature showing the existence of a3b4 nicotinic receptors within the pituitary and hypothalamus. Thus, it is more likely that the action of DM is mediated by sigma receptors because they have been identified in the anterior and intermediate lobes of pituitary and in the hypothalamus (53). Eaton et al. (54) also found that a relatively selective sigma receptor ligand, rimcazole, decreased PRL secretion, which seems to be mediated by dopaminergic and nondopaminergic mechanisms. However, it should be noted that we used the schedule of escalating dosage of DM to examine its chronic effects, which may also cause certain differences from the reports investigating the acute effects of NMDA receptor antagonists. In our model, the administration of DM covered the whole reproductive cycle, and the analysis of PRL was performed at different stages. This should be more accurate to evaluate the effect of DM in detail. Still, the exact target and mechanism of DM in affecting morphine-induced hyperprolactinemia, whether acting through the NMDA receptor and/or independent from the TIDA system, merit further investigation. Hyperprolactinemia is reported to cause recurrent spontaneous pregnancy loss in humans (55). Hirahara et al. (56) found that maintaining an appropriate level of serum PRL level (PRL level <10 ng/mL) during early pregnancy is an important therapeutic strategy, especially in patients with recurrent pregnancy loss and overt hyperprolactinemia. Elevated PRL is also responsible for 20% of secondary amenorrhea. Hyperprolactinemia in women is often associated with anovulation, reduced libido, and orgasmic dysfunction. The production of viable offspring requires successful fertilization and implantation, a pregnancy that supports the most desirable fetal development, well-timed parturition, and a supply of milk for neonatal nutrition. These are all crucially regulated by PRL whereas hyperprolactinemia may cause
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infertility and abortion in females. Although it seems to be less unfavorable for hyperprolactinemia during lactation, the drastic PRL increase caused by morphine was pathological when compared with the physiological increase in the control group. During lactation, we believe this much higher increase of PRL should not be left untreated. There could be a possibility that morphine-induced overproduction of PRL may lead to more severe postpartum mastalgia and a higher risk of breast carcinoma. In clinics, morphine-induced hyperprolactinemia is commonly overlooked by clinicians prescribing opioids. Coadministration of DM with morphine in pregnant mothers who require chronic morphine administration may be beneficial to both the mother and the offspring. We have demonstrated that DM prevents chronic morphine-induced hyperprolactinemia without causing hypoprolactinemia in female rats at different reproductive stages. This finding expands our knowledge on the beneficial effects of the coadministration of DM with morphine for the endocrine system. The beneficial effect of DM could be of great therapeutic potential in female patients who experience pain or opiate addiction but require continuous use of opioids during pregnancy and lactation. REFERENCES 1. Fricker HS, Segal S. Narcotic addiction, pregnancy, and the newborn. Am J Dis Child 1978;132:360–6. 2. Bashore RA, Ketchum JS, Staisch KJ, Barrett CT, Zimmermann EG. Heroin addiction and pregnancy. West J Med 1981;134:506–14. 3. Slamberova R. Drugs during pregnancy—effects on the mother and next generation. Cesk Fysiol 2003;52:15–21. 4. Naeye RL, Blanc W, Leblanc W, Khatamee MA. Fetal complications of maternal heroin addiction: abnormal growth, infections, and episodes of stress. J Pediatr 1973;83:1055–61. 5. Ballantyne JC, Mao J. Opioid therapy for chronic pain. N Engl J Med 2003;349:1943–53. 6. Freeman ME, Kanyicska B, Lerant A, Nagy G. Prolactin: structure, function, and regulation of secretion. Physiol Rev 2000;80:1523–631. 7. Ben-Jonathan N, Arbogast LA, Hyde JF. Neuroendocrine regulation of prolactin release. Prog Neurobiol 1989;33:399–447. 8. Jaffe RB, Yuen BH, Keye WR Jr, Midgley AR Jr. Physiologic and pathologic profiles of circulating human prolactin. Am J Obstet Gynecol 1973;117:757–73. 9. Mendoza C, Ruiz-Requena E, Ortega E, Cremades N, Martinez F, Bernabeu R, et al. Follicular fluid markers of oocyte developmental potential. Hum Reprod 2002;17:1017–22. 10. Laufer N, Botero-Ruiz W, DeCherney AH, Haseltine F, Polan ML, Behrman HR. Gonadotropin and prolactin levels in follicular fluid of human ova successfully fertilized in vitro. J Clin Endocrinol Metab 1984;58:430–4. 11. Grattan DR, Steyn FJ, Kokay IC, Anderson GM, Bunn SJ. Pregnancy-induced adaptation in the neuroendocrine control of prolactin secretion. J Neuroendocrinol 2008;20:497–507. 12. Augustine RA, Grattan DR. Induction of central leptin resistance in hyperphagic pseudopregnant rats by chronic prolactin infusion. Endocrinology 2008;149:1049–55. 13. Grattan DR, Ladyman SR, Augustine RA. Hormonal induction of leptin resistance during pregnancy. Physiol Behav 2007;91:366–74. 14. Naef L, Woodside B. Prolactin/Leptin interactions in the control of food intake in rats. Endocrinology 2007;148:5977–83. 15. Torner L, Neumann ID. The brain prolactin system: involvement in stress response adaptations in lactation. Stress 2002;5:249–57.
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16. Kokay IC, Bull PM, Davis RL, Ludwig M, Grattan DR. Expression of the long form of the prolactin receptor in magnocellular oxytocin neurons is associated with specific prolactin regulation of oxytocin neurons. Am J Physiol Regul Integr Comp Physiol 2006;290: R1216–25. 17. Grattan DR, Jasoni CL, Liu X, Anderson GM, Herbison AE. Prolactin regulation of gonadotropin-releasing hormone neurons to suppress luteinizing hormone secretion in mice. Endocrinology 2007;148: 4344–51. 18. Sagrillo CA, Voogt JL. Endogenous opioids mediate the nocturnal prolactin surge in the pregnant rat. Endocrinology 1991;129:925–30. 19. Hou Y, Voogt JL. Effects of naloxone infusion on nocturnal prolactin secretion and Fos/FRA expression in pregnant rats. Endocrine 1999;10: 145–52. 20. Grattan DR, Averill RL. Role of the placenta in the control of the antepartum surge of prolactin in the rat. J Endocrinol 1991;130:401–7. 21. Arbogast LA, Voogt JL. Endogenous opioid peptides contribute to suckling-induced prolactin release by suppressing tyrosine hydroxylase activity and messenger ribonucleic acid levels in tuberoinfundibular dopaminergic neurons. Endocrinology 1998;139:2857–62. 22. Selmanoff M, Gregerson KA. Suckling-induced prolactin release is suppressed by naloxone and simulated by beta-endorphin. Neuroendocrinology 1986;42:255–9. 23. Andrews ZB, Grattan DR. Opioid receptor subtypes involved in the regulation of prolactin secretion during pregnancy and lactation. J Neuroendocrinol 2003;15:227–36. 24. Ieiri T, Chen HT, Campbell GA, Meites J. Effects of naloxone and morphine on the proestrous surge of prolactin and gonadotropins in the rat. Endocrinology 1980;106:1568–70. 25. Minami M, Satoh M. Molecular biology of the opioid receptors: structures, functions and distributions. Neurosci Res 1995;23:121–45. 26. Andrews ZB. Neuroendocrine regulation of prolactin secretion during late pregnancy: easing the transition into lactation. J Neuroendocrinol 2005;17:466–73. 27. Hernandez SC, Bertolino M, Xiao Y, Pringle KE, Caruso FS, Kellar KJ. Dextromethorphan and its metabolite dextrorphan block alpha3beta4 neuronal nicotinic receptors. J Pharmacol Exp Ther 2000;293:962–7. 28. Klein M, Musacchio JM. High affinity dextromethorphan binding sites in guinea pig brain. Effect of sigma ligands and other agents. J Pharmacol Exp Ther 1989;251:207–15. 29. Wong CS, Wu CT, Yu JC, Yeh CC, Lee MM, Tao PL. Preincisional dextromethorphan decreases postoperative pain and opioid requirement after modified radical mastectomy. Can J Anaesth 1999;46: 1122–6. 30. Wu CT, Yu JC, Liu ST, Yeh CC, Li CY, Wong CS. Preincisional dextromethorphan treatment for postoperative pain management after upper abdominal surgery. World J Surg 2000;24:512–7. 31. Lin SL, Chao PC, Yeh CC, Li CY, Ho ST, Jao SW, et al. Oral slow release dextromethorphan premedication provides a pethidine spare effect on posthemorrhoidectomy pain management. Acta Anaesthesiol Taiwan 2004;42:87–91. 32. Tao PL, Yeh GC, Su CH, Wu YH. Co-administration of dextromethorphan during pregnancy and throughout lactation significantly decreases the adverse effects associated with chronic morphine administration in rat offspring. Life Sci 2001;69:2439–50. 33. Yang SN, Liu CA, Chung MY, Huang HC, Yeh GC, Wong CS, et al. Alterations of postsynaptic density proteins in the hippocampus of rat offspring from the morphine-addicted mother: beneficial effect of dextromethorphan. Hippocampus 2006;16:521–30. 34. Yeh GC, Tao PL, Chen JY, Lai MC, Gao FS, Hu CL. Dextromethorphan attenuates morphine withdrawal syndrome in neonatal rats passively exposed to morphine. Eur J Pharmacol 2002;453:197–202. 35. Ben-Jonathan N, Hnasko R. Dopamine as a prolactin (PRL) inhibitor. Endocr Rev 2001;22:724–63. 36. Bero LA, Kuhn CM. Role of serotonin in opiate-induced prolactin secretion and antinociception in the developing rat. J Pharmacol Exp Ther 1987;240:831–6.
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37. Bennett LL. The Long and Evans monograph on the estrous cycle in the rat. Endocrinology 1991;129:2812–4. 38. Duhau L, Grassi J, Grouselle D, Enjalbert A, Grognet JM. An enzyme immunoassay for rat prolactin: application to the determination of plasma levels. J Immunoassay 1991;12:233–50. 39. Butcher RL, Fugo NW, Collins WE. Semicircadian rhythm in plasma levels of prolactin during early gestation in the rat. Endocrinology 1972;90:1125–7. 40. Linkie DM, Niswender GD. Serum levels of prolactin, luteinizing hormone, and follicle stimulating hormone during pregnancy in the rat. Endocrinology 1972;90:632–7. 41. Spies HG, Niswender GD. Levels of prolactin, LH and FSH in the serum of intact and pelvic-neurectomized rats. Endocrinology 1971;88:937–43. 42. Rowe DW, Erskine MS. c-Fos proto-oncogene activity induced by mating in the preoptic area, hypothalamus and amygdala in the female rat: role of afferent input via the pelvic nerve. Brain Res 1993;621:25–34. 43. Gunnet JW, Freeman ME. The mating-induced release of prolactin: a unique neuroendocrine response. Endocr Rev 1983;4:44–61. 44. Bast JD, Melampy RM. Luteinizing hormone, prolactin and ovarian 20alpha hydroxysteroid dehydrogenase levels during pregnancy and pseudopregnancy in the rat. Endocrinology 1972;91:1499–505. 45. Grattan DR, Averill RL. Effect of ovarian steroids on a nocturnal surge of prolactin secretion that precedes parturition in the rat. Endocrinology 1990;126:1199–205. 46. Amenomori Y, Chen CL, Meites J. Serum prolactin levels in rats during different reproductive states. Endocrinology 1970;86:506–10. 47. Lu KH, Chen HT, Huang HH, Grandison L, Marshall S, Meites J. Relation between prolactin and gonadotropin secretion in postpartum lactating rats. J Endocrinol 1976;68:241–50.
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48. Lookingland KJ, Jarry HD, Moore KE. The metabolism of dopamine in the median eminence reflects the activity of tuberoinfundibular neurons. Brain Res 1987;419:303–10. 49. Lindley SE, Gunnet JW, Lookingland KJ, Moore KE. 3,4-Dihydroxyphenylacetic acid concentrations in the intermediate lobe and neural lobe of the posterior pituitary gland as an index of tuberohypophysial dopaminergic neuronal activity. Brain Res 1990;506:133–8. 50. Wagner EJ, Moore KE, Lookingland KJ. Sexual differences in N-methylD-aspartate receptor-mediated regulation of tuberoinfundibular dopaminergic neurons in the rat. Brain Res 1993;611:139–46. 51. Login IS. Direct stimulation of pituitary prolactin release by glutamate. Life Sci 1990;47:2269–75. 52. Toney TW, Pawsat DE, Fleckenstein AE, Lookingland KJ, Moore KE. Evidence that prolactin mediates the stimulatory effects of estrogen on tuberoinfundibular dopamine neurons in female rats. Neuroendocrinology 1992;55:282–9. 53. Wolfe SA Jr, Culp SG, De Souza EB. Sigma-receptors in endocrine organs: identification, characterization, and autoradiographic localization in rat pituitary, adrenal, testis, and ovary. Endocrinology 1989;124: 1160–72. 54. Eaton MJ, Lookingland KJ, Moore KE. The sigma receptor ligand rimcazole alters secretion of prolactin and alpha-melanocyte stimulating hormone by dopaminergic and non-dopaminergic mechanisms. Eur J Pharmacol 1996;299:171–7. 55. Yazigi RA, Quintero CH, Salameh WA. Prolactin disorders. Fertil Steril 1997;67:215–25. 56. Hirahara F, Andoh N, Sawai K, Hirabuki T, Uemura T, Minaguchi H. Hyperprolactinemic recurrent miscarriage and results of randomized bromocriptine treatment trials. Fertil Steril 1998;70:246–52.
Dextromethorphan cures morphine HyperPRL
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Graduate Institute of Medical Sciences, National Defense Medical Center, b Division of Endocrinology and Metabolism, Department of Internal Medicine, Tri-Service General Hospital, and c Department of Pharmacology, National Defense Medical Center, Taipei, Taiwan, Republic of China
Objective: To investigate whether coadministration of dextromethorphan (DM) could suppress morphine-induced hyperprolactinemia in female rats during pregnancy and throughout lactation. Design: Controlled prospective study. Setting: University research laboratory. Animal(s): One hundred adult female Sprague-Dawley rats. Intervention(s): Rats were randomly divided into four groups and were subcutaneously injected with either saline, morphine, morphine þ DM, or DM alone twice a day, progressively increasing by 1 mg/kg at 7-day intervals from an initial dose of 2 mg/kg for both morphine and DM. Drug administration was continued during pregnancy. After the offspring were born, the doses injected into the dams were increased by 1 mg/kg every 2 weeks. Main Outcome Measure(s): Serum prolactin (PRL) concentration and dopamine turnover rate at the hypothalamus and pituitary. Result(s): Chronic morphine administration induced higher PRL concentrations than the control animals at mating, and at early and late pregnancy. In rats receiving DM coadministration, we did not observe any increase by morphine. Our neurochemical results showed that this effect of DM may be partly through blocking the effect of morphine on inhibition of tuberoinfundibular dopaminergic (TIDA) neuronal activity. Conclusion(s): The use of DM as an adjuvant in females receiving chronic morphine treatment may prevent morphine-induced hyperprolactinemia. (Fertil Steril 2010;93:1686–94. 2010 by American Society for Reproductive Medicine.) Key Words: Dextromethorphan, dopamine, morphine, prolactin, tuberoinfundibular
Heroin addiction during pregnancy may lead to both acute and chronic abnormalities in neonates, such as fetal death, intrauterine growth retardation, prematurity, and withdrawal symptoms (1–4). Malnutrition, venereal disease, hepatitis, pulmonary disorders, preeclampsia, and third-trimester bleeding are the most common maternal complications of heroin addiction (2). Opioids have been proven to influence the hypothalamus-pituitary-gonadal axis and modulate hormonal release, including an increase in the prolactin (PRL) level (5). Prolactin play a key role in female reproduction, and its levels change dramatically during different reproductive stages. In cycling rats, the proestrus PRL surge coincides Received October 3, 2008; revised January 25, 2009; accepted January 26, 2009; published online March 26, 2009. L-Y.W. has nothing to disclose. E.Y-K.H. has nothing to disclose. P-L.T. has nothing to disclose. This study was supported by grants from the National Health Research Institutes (NHRI-EX96-9401NP) and National Science Council (NSC 96-2320-B-016-020-MY3), Taipei, Taiwan, R.O.C. Reprint requests: Pao-Luh Tao, Ph.D., Department of Pharmacology, National Defense Medical Center, Nei-hu, No.161, Section 6, Min-Chuan East Road, Taipei 114, Taiwan, R.O.C. (FAX: 886-2-87923155; E-mail: [email protected]).
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temporally with the preovulatory luteinizing hormone (LH) surge (6, 7). Prolactin is essential for the support of the corpus luteum in rodents. The situation in humans is different: PRL is not luteotropic and, with the exception of lactation, does not have clear effects on most reproductive processes under normal conditions. Although there is no surge of PRL throughout the menstrual cycle in women (8), PRL is secreted by the ovaries and also is found in human follicular fluid (9). Oocyte maturation and successful pregnancy have been found to be associated with high levels of PRL in follicular fluid (10). Therefore, in the human ovary, PRL may play autocrine/paracrine roles at the time of ovulation or beyond. During pregnancy and lactation, the prolonged period of hyperprolactinemia is required for mammary gland development and function and for maternal behavior immediately after parturition in both rodents and humans (11). Prolactin has an important role in the metabolic adaptations of the mother, contributing to the induction of leptin resistance in increased food intake during pregnancy (12–14). Prolactin may also participate in suppression of the stress response (15), in regulation of the activity of oxytocin neurons (16), and in the inhibition of fertility (17). All of these functions are important adaptations mediated by the maternal brain.
Fertility and Sterility Vol. 93, No. 5, March 15, 2010 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/10/$36.00 doi:10.1016/j.fertnstert.2009.01.143
Many studies have reported that these PRL nocturnal surges during early pregnancy (18, 19), the antepartum PRL surge during late pregnancy (20), and suckling-induced PRL secretion during lactation (21, 22) are regulated by opioids and mediated by contemporary stimulation of both kand m-opioid receptors on tuberoinfundibular dopaminergic (TIDA) neurons in the hypothalamus (23). It has been found that k-opioid receptor inhibition of TIDA neuronal activity may predominate during late pregnancy (23–25). In spite of differences in the mechanisms regulating PRL release during each of the reproductive stages, the role of opioids in facilitating PRL secretion seems similar (26). Dextromethorphan (DM), the dextrorotatory isomer of levomethorphan, is best known for its antitussive effect and is without an opiate-like activity. It has widespread binding sites in the central nervous system, including N-methyl-D-aspartate (NMDA) receptors, sigma receptors, and a3b4 nicotinic receptors (27, 28). Wong et al. (29) found that the preoperative administration of DM could improve postoperative pain control in patients undergoing various surgical procedures; therefore, DM may be used as an adjunct with opioids (29–31). It has been reported that coadministration of DM with morphine during pregnancy and throughout lactation could prevent some of the adverse effects associated with chronic morphine treatment in rat offspring; these effects include higher mortality, retardation in body weight gain, development of morphine tolerance and withdrawal, and alterations in NMDA receptor densities and postsynaptic density proteins in the hippocampus (32–34). Dextromethorphan also effectively blocked the increase in the serum PRL level induced by chronic morphine administration in male rats (unpublished data). Dopamine is the principal central inhibitory factor in PRL release (35). Prolactin production and negative feedback regulation are largely controlled by TIDA neurons, which terminate around the capillary loops in the median eminence of the hypothalamus. Tuberohypophyseal dopaminergic (THDA) neurons and periventricular-hypophyseal dopaminergic (PHDA) neurons that reach the anterior lobe of the pituitary contribute to basal regulation of PRL secretion as well (11). Bero and Kuhn (36) reported that morphine-induced stimulation of PRL was related to the reduction of TIDA neuron activity. In view of the therapeutic potential of DM in combination with morphine, we explored the possible effect of DM on morphine-induced hyperprolactinemia in female rats as well as determined whether the underlying mechanism of DM was through the TIDA system and/or the THDA/PHDA system. MATERIALS AND METHODS Animals Adult female Sprague-Dawley rats were purchased from the National Experimental Animal Centre, Taipei, Taiwan. The animals were housed one or two per cage in a room maintained at a temperature of 23 2 C with a 12-hour light/ dark cycle. Food and water were available ad libitum Fertility and Sterility
throughout the experiment. The care of the animals was carried out in accordance with institutional and international standards (U.S. National Institutes of Health principles of laboratory animal care), and the protocol was approved by the Institutional Animal Care and Use Committee of the National Defense Medical Center, Taiwan. Protocols and Drug Dosages The animals were acclimated for at least 3 days before the experiments. All blood samples were obtained between the hours of 0930 and 1300. The rats were randomly divided into four groups, with at least eight rats in each group. The rats underwent subcutaneous injection of saline (1 mL/kg, control group), morphine (morphine group), morphine þ DM (morphine þ DM group), or DM (DM group) twice a day (0900 and 1700), progressively increasing by 1 mg/ kg at 7-day intervals from an initial dose of 2 mg/kg for both morphine and DM. The rats were mated between days 7 and 8. Administration of drugs continued during pregnancy. After rat offspring were born, the doses of morphine or DM injected into the maternal rats were increased by 1 mg/kg every 2 weeks until 21 days after delivery, when the offspring were already weaned. The final doses of morphine and DM were both 7 to 8 mg/kg before weaning. Blood samples were collected from all of the animals 1 week before saline or drug administration to obtain a baseline PRL level for comparison. The successful mating was established by the vaginal plug or the sperm formation in the vaginal smears (37). The day of mating was termed as day 1 of pregnancy. On day 7 (early pregnancy), 14 (late pregnancy), 21 (parturition), 28 (early lactation), 35 (late lactation), and 42 (weaning), blood samples were collected 20 minutes after the morning injection of saline or drug(s). The sera were quickly separated and collected via centrifugation (500 g, 15 minutes) at 4 C and then frozen at –80 C until the PRL level was measured. During the entire course, these animals were not implanted with any cannula. On day 1 (mating) or 42 (weaning), rats were killed by decapitation 20 minutes after saline or drug injection, and blood samples were collected. The brains were rapidly removed and placed on dry ice immediately. The hypothalamus (including the dorsal part of the median eminence) and pituitary were dissected out and frozen in liquid nitrogen until use for neurochemical analysis. Enzyme-linked Immunoassay (EIA) for Prolactin Serum levels of PRL were measured by a specific enzymelinked immunoassay kit (SPI-BIO, Massy, France) for the determination of rat PRL levels (38). The limit of sensitivity for the assay was below 1 pg/mL. High-Performance Liquid Chromatography Analysis of Dopamine and Dopamine Metabolites The high-performance liquid chromatography system was composed of a reverse-phase C-18 column (MD-150, 1687
RP-C-18, 3 mm, length: 15 cm; ESA Biosciences, Chelmsford, MA) and a high-pressure pump (LC-10AD; Shimadzu, Kyoto, Japan) and connected with an electrochemical detector (ECD) coupled with three electrodes (Coulochem II; ESA Biosciences). The electrode of the guard cell was set at 40 mV, and electrodes 1 and 2 (for detection) were set at 250 and 350 mV, respectively. Under an isocratic condition, the mobile phase solvent (MD-TM; ESA Biosciences) was circulated at a flow rate of 0.5 mL/minute in the system. To quantify the sample peaks, each chemical (dopamine [DA], 3,4-dihydroxyphenylacetic acid [DOPAC], and homovanillic acid [HVA]) was compared with the external standards, which were freshly prepared and injected every five-sample run. The dopamine turnover rate was calculated and obtained as ([DOPAC] þ [HVA])/[DA]. Statistical Analysis The data were expressed as mean standard error of the mean. One-way or two-way analysis of variance (ANOVA) followed by the Bonferroni test was used to analyze the significance of the differences between groups as well as the differences between the time points. P<.05 was considered statistically significant. Chemicals Morphine hydrochloride was purchased from the National Bureau of Controlled Drugs, National Health Administration, Taipei, Taiwan. Dextromethorphan and all other chemicals were purchased from Sigma Chemical (St. Louis, MO). RESULTS Serum PRL Profile during Different Reproductive Stages with Saline Injection The basal level of serum PRL in the control group was 43.72 2.08 ng/mL compared with 202.06 46 ng/mL after mating (versus basal level, P<.01), 71.78 16.47 ng/mL in early pregnancy (P¼ not statistically significant [NS]), 93.03 13.56 ng/mL in late pregnancy (P¼NS), 196.01 61.45 ng/mL during parturition (P<.01), 242.67 55.31 ng/mL in early lactation (P<0.001), 61.50 8.28 ng/mL in late lactation (P¼NS), and 52.23 5.30 ng/mL during weaning (P¼NS) (Fig. 1). Effect of Chronic Morphine Administration on Serum PRL Concentration during Different Reproductive Stages Chronic morphine administration resulted in a statistically significant increase in serum PRL concentration in mating, and in early and late pregnancy. The basal PRL concentration was 55.96 3.57 ng/mL for the morphine group, which is not statistically significantly different from the control (saline) group (43.72 2.08 ng/mL). After mating, the PRL concentration showed a statistically significant increase to 348.76 28.21 ng/mL in response to morphine administration, and was statistically significantly higher than that of the control 1688
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group (202.06 46.00 ng/mL, P<.05). Chronic morphine administration resulted in a statistically significant increase in PRL release in early pregnancy (325.52 13.10 ng/mL, in the morphine group versus 71.78 16.47 ng/mL, in the control group, P<.001); late pregnancy (240.39 46.74 ng/mL in the morphine group versus 93.03 13.56 ng/mL in the control group, P<.05); late lactation (196.34 27.36 ng/mL in the morphine group versus 61.50 8.28 ng/mL in the control group, P<.05); and during weaning (185.67 18.78 ng/mL in the morphine group versus 52.23 5.30 ng/mL in the control group, P<.05) (see Fig. 1). No statistically significant change was found in serum PRL concentration with chronic morphine exposure in early lactation (325.50 13.10 ng/mL in the morphine group versus 242.67 55.31 ng/mL in the control group, P¼NS). It was noteworthy that there was a statistically significant decrease in the serum PRL concentration after parturition (65.71 7.67 ng/mL in the morphine group versus 196.01 61.45 ng/mL in the control group, P<.05) (see Fig. 1). No Effect of DM Administration on Serum PRL Concentration Compared with Saline Injection from Mating to Weaning The results of serum PRL concentration under chronic DM administration revealed no statistically significant difference from the control group during pregnancy or throughout lactation. The basal level of serum PRL in the DM group was 45.76 1.10 ng/mL versus 43.72 2.08 ng/mL in the control group (P¼NS). After mating, the serum PRL level was 187.06 47.86 ng/mL in the DM group versus 202.06 46.00 ng/mL in the control group (P¼NS). In early pregnancy, the serum PRL level was 53.48 14.98 ng/mL in the DM group versus 71.78 16.47 ng/mL in the control group (P¼NS). In late pregnancy, the serum PRL level was 39.41 12.24 ng/mL in the DM group versus 93.03 13.56 ng/mL in the control group (P¼NS). After parturition, the serum PRL level was 164.16 3.15 ng/mL in the DM group versus 196.01 61.45 ng/mL in the control group (P¼NS). In early lactation, the serum PRL level was 171.11 46.91 ng/mL in the DM group versus 242.67 55.31 ng/mL in the control group (P¼NS). In late lactation, the serum PRL level was 66.78 8.49 ng/mL in the DM group versus 61.50 8.28 ng/mL in the control group (P¼NS). During weaning, the serum PRL level was 64.92 13.28 ng/mL in the DM group versus 52.23 5.30 ng/ mL in the control group (P¼NS) (Fig. 2). Effect of DM on Morphine-induced Further Increase of PRL Release after Mating, Early and Late Pregnancy, Early and Late Lactation, and Weaning Chronic administration of morphine during pregnancy and throughout lactation caused a onefold to fivefold increase in serum PRL concentration when compared with the control (saline) group. No statistically significant difference was found in PRL concentrations after coadministration of DM
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FIGURE 1 Effect of morphine on serum prolactin (PRL) concentration during different reproductive stages. Animals received subcutaneous injections of saline or morphine twice per day, and the dose of morphine was progressively increased. Each group contained at least eight rats. Data are presented as mean standard error of mean. Twoway analysis of variance followed by the Bonferroni test was used to analyze the statistical significance of the differences between groups as well as time points of the saline group. DM ¼ dextromethorphan; E ¼ early pregnancy; EL ¼ early lactation; L ¼ late pregnancy; LL ¼ late lactation; M ¼ morphine. **P< .01, ***P< .001 versus basal level; #P< .05, ###P< .001 versus control (saline) group.
Wu. Dextromethorphan cures morphine HyperPRL. Fertil Steril 2010.
with morphine when compared with the control group during pregnancy or throughout lactation (Fig. 3). In the morphine þ DM group, however, a statistically significantly lower level of PRL was observed when compared with the morphine group. The PRL level was as follows: after mating (196.08 28.79 ng/mL in the morphine þ DM group versus 348.76 28.21 ng/mL in the morphine group, P<.001); early pregnancy (42.53 3.32 ng/mL in the morphine þ DM group versus
325.52 13.10 ng/mL in the morphine group, P<.001); late pregnancy (37.04 3.55 ng/mL in the morphine þ DM group versus 240.39 46.74 ng/mL in the morphine group, P<.001); early lactation (190.96 47.25 ng/mL in the morphine þ DM group versus 325.50 13.10 ng/mL in the morphine group, P<.01); late lactation (61.93 5.3 ng/mL in the morphine þ DM group versus 196.34 27.36 ng/mL in the morphine group, P<.01); and weaning (40.8 2.72 ng/mL
FIGURE 2 Effect of dextromethorphan (DM) on serum prolactin (PRL) concentration during different reproductive stages. Animals received subcutaneous injections of saline or dextromethorphan (DM) twice per day, and the dose of DM was progressively increased. Each group contained at least eight rats. Data are presented as mean standard error of the mean. There was no statistically significant difference between these two groups after two-way analysis of variance. E ¼ early pregnancy; EL ¼ early lactation; L ¼ late pregnancy; LL ¼ late lactation.
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FIGURE 3 Effect of coadministration of dextromethorphan (DM) with morphine (M þ DM) on hyperprolactinemia induced by chronic morphine administration. Each group contained at least eight rats. Data are presented as mean standard error of mean. Two-way analysis of variance followed by the Bonferroni test was used to analyze the statistical significance of the differences between the M and M þ DM groups. **P< .01, ***P< .001, M versus M þ DM group. E ¼ early pregnancy; EL ¼ early lactation; L ¼ late pregnancy; LL ¼ late lactation.
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in the morphine þ DM group versus 185.67 18.78 ng/mL in the morphine group, P<.01) (see Fig. 3). Dopamine Turnover Rate in the Hypothalamus during Mating and Weaning After mating, the dopamine turnover rate in the hypothalamus was 0.21 0.01 in the morphine group, which was statistically significantly lower than that in the control group (0.41 0.03, P<.001). Dextromethorphan by itself did not change the dopamine turnover rate in the hypothalamus (0.38 0.02). When DM was coadministered with morphine (morphine þ DM group), the dopamine turnover rate in the hypothalamus (0.29 0.01) was still statistically significantly lower than that in the control group (0.41 0.03, P<.01) but was higher than that in the morphine group (0.21 0.01, P<.05) (Fig. 4A). We may reasonably conclude that DM partially reverses the morphine-induced decrease in the dopamine turnover rate in the hypothalamus after mating. During weaning, chronic morphine administration caused a statistically significant decrease in the dopamine turnover rate (1.53 0.19) when compared with the control group (4.83 0.84, P<.05). When DM was coadministered with morphine (morphine þ DM group), the dopamine turnover rate in the hypothalamus (4.17 0.81) was not statistically significantly different from that in the control group (4.83 0.84) (see Fig. 4B). It seems reasonable to conclude that DM can reverse the morphine-induced decrease in the dopamine turnover rate in the hypothalamus during weaning. Dopamine Turnover Rate in the Pituitary during Mating and Weaning After mating, the dopamine turnover rate in the pituitary was 0.31 0.03 in the morphine group, which was statistically 1690
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significantly lower than that of the control group (0.64 0.04, P<.001). In the morphine þ DM group, the dopamine turnover rate in the pituitary was also decreased (0.28 0.03, P<.001) (Fig. 5A). Thus, DM did not reverse the decrease in the dopamine turnover rate in the pituitary caused by chronic morphine administration. During weaning, chronic morphine administration caused a statistically significant decrease in the dopamine turnover rate (4.54 0.8) when compared with that of the control group (9.54 0.5) (P<.01). In the morphine þ DM group, the dopamine turnover rate in the pituitary (10.14 1.05) was not statistically significantly different from that of the control group (9.54 0.5) (see Fig. 5B). Thus, DM could reverse the morphine-induced decrease in the dopamine turnover rate in the pituitary during weaning.
DISCUSSION Our study reports on the inhibitory effects of DM on morphine-induced hyperprolactinemia in female rats. First, we demonstrated a regular change of serum PRL level at different reproductive stages in female rats. Acute significant increases in PRL after mating appear to be related to vaginocervical stimulation. It has been reported that this PRL surge could occur in cycling exposure to males between the hours of 1400 on proestrus and 0300 on the day of estrus (39– 41). The response was 5-fold to 10-fold above the baseline PRL level and was noted at 8 to 12 hours after mating (42), which was consistent with our results. The first 10 to 12 days of rat pregnancy are dominated by daily nocturnal (0300 to 0500 h) and diurnal (1700 to 2000 h) PRL surges (6, 43); these surges cease during the second half of pregnancy (44). In our present results, no statistically significant difference of the serum PRL level was found between the
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FIGURE 4
FIGURE 5
Effect of morphine (M), dextromethorphan (DM), or coadministration of dextromethorphan with morphine (M þ DM) on the dopamine turnover rate in the hypothalamus after (A) mating or (B) weaning. Each group contained at least eight rats. Data are presented as mean standard error of mean. Oneway analysis of variance followed by the Bonferroni test was used to analyze the statistical significance of the differences between groups. ***P< .001; **P< .01; *P< .05 when compared with the control (C) group; #P< .05 when compared with the morphine group.
Effect of morphine (M), dextromethorphan (DM), or coadministration of dextromethorphan with morphine (M þ DM) on the dopamine turnover rate in the pituitary after (A) mating or (B) weaning. Each group contained at least eight rats. Data are presented as mean standard error of mean. Oneway analysis of variance followed by the Bonferroni test was used to analyze the statistical significance of the difference between groups. ***P< .001; **P< .01 when compared with the control (C) group.
Wu. Dextromethorphan cures morphine HyperPRL. Fertil Steril 2010. Wu. Dextromethorphan cures morphine HyperPRL. Fertil Steril 2010.
seems that all of the suckling turned out to be weaker at this stage in our animal model. basal state and early/late pregnancy, which should be due to our experimental design to avoid the PRL surges by sampling the blood at the time between the hours of 0930 and 1300. Our data also showed a statistically significant increase of serum PRL after parturition, which is consistent with the results of other reports (44–46). During the 3 weeks of lactation, stimulation of PRL was maintained to a considerable extent by the suckling reflex (46, 47). We found that the serum PRL level of early lactation was truly higher, but the PRL level of late lactation was close to the basal value. In fact, we adjusted the litter size (8 to 10) for each dam to be similar to keep the consistent extent of suckling because the suckling reflex dominated the secretion of PRL in lactation. Although it is difficult to explain the phenomenon at late lactation, it Fertility and Sterility
After parturition, PRL secretion becomes closely linked to the suckling stimulus, where the extent of suckling by the offspring results in a parallel increase of PRL secretion, acting through a decrease of TIDA neuronal activity (11). After parturition, our data showed a statistically significant increase in serum PRL concentration, but chronic morphine administration caused a statistically significant decrease when compared with the control (saline) group. These results imply that chronic morphine administration could impair the sucking reflex of offspring and increase TIDA neuronal activity temporarily after parturition. This could be a consequence of morphine withdrawal in pups after birth. Although the statistical analysis did not show significance, the coadministration of DM tended to reverse this adverse effect by morphine. 1691
Chronic morphine administration resulted in a statistically significant further increase in serum PRL concentration after mating, and also at early and late pregnancy. Apart from parturition, the serum PRL level was statistically significantly higher in the morphine group than the control group at the early and late lactation stages and at weaning. This indicated a clear hyperprolactinemia caused by chronic morphine administration. Coadministration of DM with morphine statistically significantly blocked the increase in the serum PRL level caused by chronic morphine administration during different reproductive stages except parturition. Chronic administration of morphine caused a 1.5-fold to 5-fold increase in the serum PRL level compared with the control level. These increases in PRL level were almost completely blocked by the coadministration of DM with the same dose and injection protocol as that of morphine. Nevertheless, DM by itself did not cause any statistically significant changes in the serum PRL level when compared with the control. To investigate the possible mechanism(s) underlying the actions of morphine and DM in PRL release, we dissected the hypothalamus (including the median eminence) and the pituitary and determined the dopamine turnover rate at these two brain regions. Because the dopamine turnover rate is well recognized as an indicator of dopamine neuronal activity at the nerve terminal (48, 49), the dopamine turnover rates in the hypothalamus and the pituitary may represent the neuronal output intensity of the TIDA neurons and THDA/PHDA neurons, respectively. During weaning, we found that there was 10-fold to 15-fold increase of the dopamine turnover rate in both the hypothalamus and the pituitary when compared with the rate after mating in the control group. Because PRL has been proven to be under the inhibitory regulation of dopamine, this is consistent with the serum PRL concentration showing a parallel drop after weaning. In addition, chronic morphine administration caused a statistically significant decrease in the dopamine turnover rate in both the hypothalamus and the pituitary at two different reproductive stages, mating and weaning. From these results, we speculated that the effects of chronic morphine-induced further hyperprolactinemia after mating and at the different reproductive stages may be related to its inhibitory effect on the TIDA system as well as the THDA and PHDA systems. After mating, coadministration of DM with morphine partially reversed the inhibitory effect of morphine on the dopamine turnover rate in the hypothalamus but did not reverse the effect of morphine in the pituitary. However, during weaning, coadministration of DM with morphine completely reversed the inhibitory effect of morphine on the dopamine turnover rate in both of the hypothalamus and the pituitary. These results suggest that DM may act through certain unknown mechanisms to suppress the effect of morphine on the TIDA, THDA, and PHDA systems after mating and during weaning. In female rats, the other NMDA receptor antagonists, such as MK-801 and CGS-19755 decreased the dopamine turnover rate in the median eminence (50). This suggests that 1692
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these compounds alone are blocking the actions of endogenous excitatory amino acid neurotransmitters at the NMDA receptor, which tonically activate the TIDA neurons. However, the same report demonstrated that MK-801 markedly decreased basal PRL secretion in female rats. This seems to be controversial but actually could be explained by the direct inhibitory effect of MK-801 on the lactotroph (51). Blocking the feedback stimulatory effect of PRL on TIDA neurons did not affect the inhibitory effect of MK-801 on TIDA neurons. On the other hand, removal of the tonic stimulatory effects of endogenous PRL in female rats decreases TIDA neuronal activity (52). Taken together, these results could indicate that the inhibitory effect of MK-801 on TIDA neurons occurs independent of its inhibitory effect on PRL secretion. Unlike MK-801, our data showed that DM by itself did not have any effect on the PRL secretion or the activity of TIDA neurons in our animal model. Because DM behaved differently from the other NMDA receptor antagonists, we propose that DM acts at targets (e.g., sigma and a3b4 nicotinic receptors) other than NMDA receptors to suppress morphineinduced hyperprolactinemia. We found no studies in the literature showing the existence of a3b4 nicotinic receptors within the pituitary and hypothalamus. Thus, it is more likely that the action of DM is mediated by sigma receptors because they have been identified in the anterior and intermediate lobes of pituitary and in the hypothalamus (53). Eaton et al. (54) also found that a relatively selective sigma receptor ligand, rimcazole, decreased PRL secretion, which seems to be mediated by dopaminergic and nondopaminergic mechanisms. However, it should be noted that we used the schedule of escalating dosage of DM to examine its chronic effects, which may also cause certain differences from the reports investigating the acute effects of NMDA receptor antagonists. In our model, the administration of DM covered the whole reproductive cycle, and the analysis of PRL was performed at different stages. This should be more accurate to evaluate the effect of DM in detail. Still, the exact target and mechanism of DM in affecting morphine-induced hyperprolactinemia, whether acting through the NMDA receptor and/or independent from the TIDA system, merit further investigation. Hyperprolactinemia is reported to cause recurrent spontaneous pregnancy loss in humans (55). Hirahara et al. (56) found that maintaining an appropriate level of serum PRL level (PRL level <10 ng/mL) during early pregnancy is an important therapeutic strategy, especially in patients with recurrent pregnancy loss and overt hyperprolactinemia. Elevated PRL is also responsible for 20% of secondary amenorrhea. Hyperprolactinemia in women is often associated with anovulation, reduced libido, and orgasmic dysfunction. The production of viable offspring requires successful fertilization and implantation, a pregnancy that supports the most desirable fetal development, well-timed parturition, and a supply of milk for neonatal nutrition. These are all crucially regulated by PRL whereas hyperprolactinemia may cause
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infertility and abortion in females. Although it seems to be less unfavorable for hyperprolactinemia during lactation, the drastic PRL increase caused by morphine was pathological when compared with the physiological increase in the control group. During lactation, we believe this much higher increase of PRL should not be left untreated. There could be a possibility that morphine-induced overproduction of PRL may lead to more severe postpartum mastalgia and a higher risk of breast carcinoma. In clinics, morphine-induced hyperprolactinemia is commonly overlooked by clinicians prescribing opioids. Coadministration of DM with morphine in pregnant mothers who require chronic morphine administration may be beneficial to both the mother and the offspring. We have demonstrated that DM prevents chronic morphine-induced hyperprolactinemia without causing hypoprolactinemia in female rats at different reproductive stages. This finding expands our knowledge on the beneficial effects of the coadministration of DM with morphine for the endocrine system. The beneficial effect of DM could be of great therapeutic potential in female patients who experience pain or opiate addiction but require continuous use of opioids during pregnancy and lactation. REFERENCES 1. Fricker HS, Segal S. Narcotic addiction, pregnancy, and the newborn. Am J Dis Child 1978;132:360–6. 2. Bashore RA, Ketchum JS, Staisch KJ, Barrett CT, Zimmermann EG. Heroin addiction and pregnancy. West J Med 1981;134:506–14. 3. Slamberova R. Drugs during pregnancy—effects on the mother and next generation. Cesk Fysiol 2003;52:15–21. 4. Naeye RL, Blanc W, Leblanc W, Khatamee MA. Fetal complications of maternal heroin addiction: abnormal growth, infections, and episodes of stress. J Pediatr 1973;83:1055–61. 5. Ballantyne JC, Mao J. Opioid therapy for chronic pain. N Engl J Med 2003;349:1943–53. 6. Freeman ME, Kanyicska B, Lerant A, Nagy G. Prolactin: structure, function, and regulation of secretion. Physiol Rev 2000;80:1523–631. 7. Ben-Jonathan N, Arbogast LA, Hyde JF. Neuroendocrine regulation of prolactin release. Prog Neurobiol 1989;33:399–447. 8. Jaffe RB, Yuen BH, Keye WR Jr, Midgley AR Jr. Physiologic and pathologic profiles of circulating human prolactin. Am J Obstet Gynecol 1973;117:757–73. 9. Mendoza C, Ruiz-Requena E, Ortega E, Cremades N, Martinez F, Bernabeu R, et al. Follicular fluid markers of oocyte developmental potential. Hum Reprod 2002;17:1017–22. 10. Laufer N, Botero-Ruiz W, DeCherney AH, Haseltine F, Polan ML, Behrman HR. Gonadotropin and prolactin levels in follicular fluid of human ova successfully fertilized in vitro. J Clin Endocrinol Metab 1984;58:430–4. 11. Grattan DR, Steyn FJ, Kokay IC, Anderson GM, Bunn SJ. Pregnancy-induced adaptation in the neuroendocrine control of prolactin secretion. J Neuroendocrinol 2008;20:497–507. 12. Augustine RA, Grattan DR. Induction of central leptin resistance in hyperphagic pseudopregnant rats by chronic prolactin infusion. Endocrinology 2008;149:1049–55. 13. Grattan DR, Ladyman SR, Augustine RA. Hormonal induction of leptin resistance during pregnancy. Physiol Behav 2007;91:366–74. 14. Naef L, Woodside B. Prolactin/Leptin interactions in the control of food intake in rats. Endocrinology 2007;148:5977–83. 15. Torner L, Neumann ID. The brain prolactin system: involvement in stress response adaptations in lactation. Stress 2002;5:249–57.
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