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N-methyl-d,l-aspartate stimulates growth hormone and prolactin but inhibits luteinizing hormone secretion in the pig
Vol. 9(3):225-232,1992
DOMESTICANIMAL ENDOCRINOLOGY
N-METHYL-D,L-ASPARTATE STIMULATES GROWTH HORMONE AND PROLACTIN BUT INHIBITS LUTEINIZING HORMONE SECRETION IN THE PIG C.R. Barb,* G.M. Derochers,** 8. Johnson,* R.V. Utley,** W.J. Chang,** G.B. Rampacek,** R.R. Kraeling* *Animal Physiology Unit Richard B. Russell Agricultural Research Center USDA, ARS, Athens, GA 30613 and **Animal and Dairy Science Department University of Georgia Athens, GA 30602 Received March 19, 1992
ABSTRACT The effects of n-methyl-d,l-aspartate (NMA), a neuroexcitatory amino acid agonist, on luteinizing hormone (LH), prolactin (PRL) and growth hormone (GH) secretion in gilts treated with ovarian steroids was studied. Mature gilts which had displayed one or more estrous cycles of 18 to 22 d were ovariectomized and assigned to one of three treatments administered in: corn oil vehicle (V; n=6); 10 pg estradiol-17b/kg BW given 33 hr before NMA (E; n=6); .85 mg progesterone/kg BW given twice daily for 6 d prior to NMA (P,; n=6). Blood was collected via jugular cannulae every 1.5 min for 6 hr. Pigs received 10 mg NMA/kg BW i.v. 2 hr after blood collection began and a combined synthetic [Ala15]-h GH releasing factor (l-29)-NH, (GRF; 1 pg/kg BW) and gonadotropin releasing hormone (GnRH; .2 &kg BW) challenge given i.v. 3 hr after NMA. NMA did not alter LH secretion in E gilts. However, NMA decreased (Pc.02) serum LH concentrations in V and P, gilts. Serum LH concentrations increased (Pc.01) after GnRH in all gilts. NMA did not alter PRL secretion in P, pigs, but increased (Pc.01) serum PRL concentrations in V and E animals. Treatment with NMA increased (Pc.01) GH secretion in all animals while the GRF challenge increased (Pc.01) serum GH concentrations in all animals except in V treated pigs. NMA increased (Pc.05) cortisol secretion in all treatment groups. These results indicate that NMA inhibits LH secretion and is a secretagogue of PRL, GH and cortisol secretion with ovarian steroids modulating the LH and PRL response to NMA.
INTRODUCTION N-methyl-d,l-aspartate (NMA) is a potent agonist of the neuroexcitatory amino acids aspartate and glutamate. NMA treatment increased serum luteinizing hormone (LH) concentrations in rats (1) and in both juvenile (2,3) and adult monkeys (4). Moreover, NMA is a potent secretagogue of prolactin (PRL) and growth hormone (GH) in the monkey (2,4). When administered to rodents, excitatory amino acids stimulated PRL secretion and altered the pulsatile pattern of GH secretion (5). In wethers, NMA treatment enhanced GH secretion, but failed to increase LH secretion (6). Similarly, in the ovariectomized (OVX) ewe, NMA stimulated GH secretion (7) but did not alter LH secretion (8). However, when ewes were pretreated with estradiol, NMA stimulated LH secretion (8). In a recent study, Reyes et al. (9) observed an inhibition of LH secretion following NMA in the OVX monkey. However, treatment with ovarian steroids resulted in a rapid reversal of this phenomena (10). Little is known about the endocrinological consequences of NMA administration in the pig. Therefore, the objective of this stucljr was to determine the effect of NMA on LH, GH and PRL secretion in the OVX pig in the absence and presence of ovarian steroids. Copyright 0 1992 Butterworth-Heinemann
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MATERIALS AND METHODS Eighteen gilts, weighing 121 f 2 kg and which had displayed one or more estrous cycles of 18-22 d, were OVX. All gilts were housed in individual pens in an environmentally controlled building and exposed to a constant ambient temperature of 22 C and an artificial photoperiod of 12: 12 hr 1ight:dark. Pigs were fed between 0700 and 0800 hr a corn-soybean meal ration (14% crude protein) supplemented with vitamins and minerals, according to Nutrition Research Council (11) guidelines. Starting approximately 2 weeks after OVX, the experiment was conducted in two replicates of 3 pigs/treatment group in each replicate, with the following treatment groups: corn oil vehicle im (V; n=6); 10 pg estradiol-17P,/kg BW im 33 hr before NMA injection (E; n = 6); .85 mg progesterone/kg BW im twice daily for six d prior to NMA (P,; n=6). Gilts in the V and E groups received corn oil injection twice daily for six d prior to NMA. Progesterone treatment was employed in order to simulate luteal phase concentrations of P, (12), while the dose of E was used to simulate late follicular phase concentrations of E (12). On the day prior to NMA treatment, an indwelling catheter was placed into the jugular vein of each pig (13). Blood samples were collected every 15 min for 2 hr prior to and 3 hr after iv injection of NMA’ (10 mg/kg SW) in .9% saline and for 1 hr after a combined GnRH (.2 pg/kg BW) and synthetic [AlaIs]-h GH-releasing factor (l-29)-NH, (GRF; 1 @kg BW) iv challenge. Before initiation of the experiment, a dose-response study (0,5, 10 and 15 mg/ kg BW) was performed with NMA in OVX gilts (n = 4/dose). There was no difference between doses with regard to LH secretion. Therefore, the 10 m&kg dose was chosen, since it produced consistent GH and PRL responses. Based on previous observations, the doses of GnRH and GRF employed in the present study were optimal for assessing differences in pituitary sensitivity (14). Blood samples were allowed to clot at 4 C for 24 hr and serum was harvested after centrifugation and stored at -20 C. Hormone Assays. Serum concentrations of LH ( 15), PRL ( 16) and GH ( 17) were quantified by radioimmunoassay (RIA) on all samples. Since a PRL secretagogue challenge was not given during the last hr of the sampling period, these samples were not included for PRL assay. The sensitivities of the assays were .15 ng/ml of LH, 1.O ng/ml of PRL and .4 ng/ml of GH. The intraassay and interassay coefficients of variation were 4.8% and 9.0% for LH, 16.3% and 15.2% for PRL and 3.2% and 13.6% for GH, respectively. Serum P, (18) and E (19) were quantified by RIA on the first sample collected from each gilt. The sensitivity of the P, and E assays were .5 rig/ml and .6 pg/ml, respectively. Intraassay and interassay coefficients of variation were 9.3% and 15.0% for P, and 13.6% and 19.1% for E, respectively. Serum cortisol concentrations were determined on samples collected every 30 min starting at time of NMA challenge. Cortisol concentrations were quantified by RIA as reported for the bovine (20), except for the following validation changes. Addition of 10,50, 100,250, 500 or 1000 pg of cortisol to 5 and 10 pl aliquots of porcine serum resulted in 113% recovery. Dose response curves from 5 to 50 pl of pooled porcine serum were parallel (fi.50) to the standard curve. Sensitivity of the assay was 1 ng/ml and intraassay and interassay coefficients of variation were 1.4% and 4.4%, respectively. Statistical Analysis. To determine the effect of steroid treatment on LH, PRL and GH response to NMA, data were divided into five periods. Period one represented the mean of samples collected prior to NMA treatment. The remainder of the sampling time was divided into four 1-hr periods, except for PRL in which sampling time was divided into three 1-hr periods. Cortisol data was not divided into hourly periods, since samples were collected every 30 min starting at time of NMA challenge. Data were then subjected to the general linear model split plot-in-time analysis of variance procedure of the Statistical Analysis System (21). The statistical model included treatment, period, replicate, treatment x replicate, and treatment x period interactions. Effects of treatment and replicate were tested using animal
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within treatment x replicate as the error term. Period, treatment x replicate and treatment x period were tested using treatment x period x replicate as the error term. Total area under the curve (AUC) was calculated using trapezoidal summation method for serum PRL and GH response to NMA after post-treatment concentrations were adjusted for pre-treatment levels. Data were then subjected to a one-way analysis of variance. Difference between treatment means were determined by least-squares contrasts (21). If a significant treatment x period interaction was detected, then differences between treatments within a period or between periods within a treatment were determined by least-squares contrasts (21). RESULTS
There was no treatment x replicate interaction detected for any of the hormones studied. Therefore, data was pooled across replicates. Serum E concentrations were nondetectable in V and P4 gilts, but averaged 30 + 5 pg/ml for the E gilts. Serum P, was nondetectable in V and E gilts, but averaged 24.7 + 3.8 r&ml in P, gilts. NMA increased (Pc.05) cortisol secretion in all treatment groups (Figure 1). A treatment x period (P<.OOOl)interaction was detected for LH. Profiles of serum LH concentrations of a representative gilt for each treatment group are depicted in Figure 2. Pretreatment serum LH concentrations were lower (Pc.05) in P, and E gilts compared to V animals (table 1). Serum LH concentrations decreased (Pc.02) after NMA in V and P4 treated gilts from 1.1 f . 1 ng/ml to .7 f . 1 @ml and from .9 + . 1 rig/ml to .5 + .l rig/ml, respectively (table 1). Luteinizing hormone secretion was not altered by NMA treatment in E treated animals (table 1). The LH response to GnRH was lower (Pc.02) in E pigs than in V animals, while this response was similar between V and P, gilts (table 1). Treatment x period (Pc.01) interactions were detected for PRL and GH. Mean serum PRL concentrations increased (Pc.01) after NMA in V and E treated gilts (table 2). However, NMA failed to alter PRL concentrations in P, treated gilts. The PRL response to NMA as determined by AUC was similar for V (2.2 + .5 ng*hr/ml) and E (2.5 + .5 ng+r/ml) animals but lower (Pc.05) in P, (.5 + .5 ng*hr/ml) pigs compared to V treated gilts. NMA in-
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Figure I. Mean serum cortisol concentrations (mean f SE) after NMA (treatment = time 0) for gilts treated with vehicle (V), progesterone (PJ, or estradiol (E). Bars with different superscripts differ (P<.O5).
BARB ET AL.
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MINUTES Figure 2. Serum luteinizing hormone (LH) concentrations before and after N-methyl-d,l-aspartate (treatment = Time 0) and gonadotropin-releasing hormone (indicated by arrows) treatment for a gilt treated with oil vehicle (V), progesterone (PJ. or estradiol (E).
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TABLE I. MEAN SERUMLUTEINIZING HORMONE(LH) CONCENTRATIONS FORGILTS TREATEDWITH VEHICLE(V) PR~CESTERONE (P,), OR ESTRADIOL(E) FORPERIODS1, 2, 3.4 AND 5” Serum LH (@ml) Periodb
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TABLE 2. MEAN SERUMPROLACTIN(PRL) CONCENTRATION; FORGILTS TREATEDWITHVEHICLE(V), PROGES~RONE(P,), OR ESTRADIOL(E) FORPERIODS1,2,3 Serum Treatment
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TABLE 3. MEAN SERUMGROWTHHORMONE(GH) CONCENTRATIONS FORGILTS TREATEDWITHVEHICLE(V), PROGESIERONE (PA), OR ESTRADIOL(E) FORPERIODS1, 2, 3.4 Serum Treatment
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creased (Pc.01) serum GH concentrations in all treatment groups (table 3). The GH response to NMA as measured by AUC was similar for all treatments and averaged 41 + 5,40 k 5, and 30 k 5 ngWm1 for V, P, and E treated gilts, respectively. The GH response to GRF was greater (Pc.05) in E and P, pigs than in V treated animals (table 3).
DISCUSSION In the present study, LH secretion decreased following a bolus injection of NMA in both V and P, treated animals but LH secretion was not altered in E treated pigs. These results are surprising since, in a recent study, Reyes et al. (10) reported that treatment with either
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E alone or incombination with P, reversed inhibition of LH secretion by NMA in monkeys. Similarly, in the OVX ewe (8), NMA failed to stimulate LH release. However, pretreatment with E instituted the stimulatory influence of NMA on LH secretion. In wethers, although an immediate effect of NMA on LH secretion was not observed, serum LH concentrations were lower the day following NMA treatment (6). Therefore, presence of E appears to be a necessary antecedent for the excitatory action of NMA on LH secretion in some species. However, in the present study E did not sensitize the GnRH/LH secretory system to NMA. A recent report by Sesti and Britt (22) demonstrated that administration of NMA to lactating sows increased LH secretion. This apparent dichotomy between the present study and that of Sesti and Britt (22) is difficult to explain. We believe that physiological state may influence effectiveness of NMA to activate the GnRH/LH secretory system. In support of this idea we previously reported that the opioid antagonist, naloxone, stimulated PRL secretion in luteal phase gilts (12), while naloxone attenuated the suckling-induced increase in PRL secretion in lactating sows (23). Moreover, in mature female rats NMA treatment resulted in an acute increase in serum LH concentrations. However, this phenomenon was not demonstrable in lactating rats (24). Therefore, we suggest a similar paradigm may exist for excitatory amino acid modulation of LH secretion in the pig. It is generally accepted that NMA acts at the central nervous system (CNS) to release GnRH (2,3,25). Moreover, since NMA is a general excitatory amino acid which stimulates release of anterior pituitary hormones (2,4,6,26) it is conceivable that other neural factors which are inhibitory to LH secretion are released in response to NMA. However, action of NMA on LH secretion may also occur at the pituitary gland (27). The observation that exogenously administered GnRH increased LH secretion during inhibition by NMA in the present study eliminates this idea. However, increased cortisol secretion after NMA suggests activation of the hypothalamic-pituitary-adrenal axis. Similar increases in cortisol secretion after NMA treatment were reported in the monkey (2,9,10). Moreover, the increase in cortisol secretion after NMA may have resulted from an elevation in hypothalamic CRF and P-endorphin. Farah et al. (26) observed increased secretion of ACTH and P-endorphin after NMA in the male rat. Recently, Reyes et al. (9) reported that inhibition of LH secretion by NMA in the OVX monkey is the result of CRF and endogenous opioid peptide (EOP) secretion. In the present study, inhibition of LH secretion by NMA may be the result of both elevated cortisol secretion and increased release of EOP. We previously reported that the presence of P, is necessary for initiation of endogenous opioid modulation of LH secretion (28). In addition, activation of the adrenal axis inhibited both pulsatile and surge secretion of LH in the pig (13,29). Therefore, both cortisol and EOP may be acting in concert to inhibit LH secretion in the P, treated gilts. This may explain why P, treatment failed to reverse the inhibitory action of NMA on LH secretion as reported by Reyes et al. (10). The failure of NMA to influence LH secretion in E treated animals, may in part be due to the already suppressed levels of LH due to negative feedback action of E on LH secretion (30). Secondly, if the action of NMA on LH secretion is via GnRH release, then E associated changes in pituitary responsiveness to GnRH may have attenuated the LH response to NMA. This idea is supported by the fact that pituitary responsiveness was compromised by E treatment, since serum LH concentrations were lower following GnRH challenge in E treated compared to V and P4 treated gilts. As in the monkey (2) and wether (6), NMA markedly increased GH secretion without regard to steroid treatment in the present study. Although the current investigation did not address mechanisms responsible for stimulation by NMA, it is reasonable to suggest that this stimulation of GH secretion is due to a release of GRF and/or suppression of somatostatin secretion from the hypothalamus. Moreover, this response may be mediated by EOP, since stimulation of EOP receptors increased GH secretion in pigs (31) and NMA evoked EOP
LH, PROLACTIN AND GH SECRETION AFTER NMA
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secretion (6,9). In contrast to this idea, naloxone, an EOP antagonist, failed to alter GH response to NMA in OVX ewes (7). In the present study, NMA increased PRL secretion in V and E treated animals, but not in P, treated pigs. Wilson and Knobil(4) reported that NMA treatment increased serum or plasma PRL concentrations during both the late luteal and early follicular phase of the menstrual cycle in the Rhesus monkey. In addition, other reports demonstrated that administration of NMA to male and female rats (24,32) elicited marked increase in PRL secretion, whereas NMA failed to stimulate PRL secretion during lactation (24). Furthermore, if administered when PRL concentrations were elevated during suckling NMA acutely suppressed PRL release (24). Therefore, results of the present study and above reports indicate that NMA involvement in central nervous system regulation of PRL secretion may occur through activation of both PRL simulating and inhibiting systems depending on the physiological state or steroid milieu. We previously reported the existence of a P, dependent EOP system which inhibits PRL secretion in the pig (12,31). Therefore, lack of a PRL response to NMA in P, treated gilts may in part be due to P, induced EOP inhibition of the PRL secretory system. In light of the diverse effects of NMA on pituitary hormone secretion between the present study and others, future studies should focus on whether neuroexcitatory amino acids play a physiological role in the control of pituitary function via the use of specific agonist and antagonist. ACKNOWLEDGMENTS/FOOTNOTES Theauthors wish to thank Mr. John B. Barrett, Ms. Elizabeth A. Taras, Ms. Amanda Latimer and Ms. Donna Slavin for their technical assistance; Ruel L. Wilson, biometrician, Southern Region, ARS for his statistical advice; Dr. D.J. Bolt, USDA, Beltsville, MD for providing pituitary hormones used in radioimmunoassay; Dr. Myron Brown, Ceva Laboratory, Overland Park, KS, for the generous gift of GnRH, and Drs. Robert M. Campbell and Thomas F. Mowles, Hoffmann-LaRoche, Nutley, NJ, for the generous gift of synthetic [Ala”]hGRF( l -29)-NH,. ‘Sigma Chemical Co., St. Louis, MO Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the USDA and does not imply its approval to the exclusion of other products that may be suitable. Corresponding Author: Dr. C. Richard Barb, USDA, ARS, Animal Physiology, R.B. Russell Research Center, P.O. Box 5677, Athens, GA 30613. REFERENCES 1. Urbanski
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HF. Ojeda SR. Activation of luteinizing hormone-releasing hormone release advances the onset of female puberty. Neuroendocrinology 46:273-276, 1987. Gay VL, Plant TM. N-methyl-D,L-aspartate elicits hypothalamic gonadotropin-releasing hormone release in prepubertal male Rhesus monkeys (Macaca mulatta). Endocrinology 120:2289-2296, 1987. Gay VL, Plant TM. Sustained intermittent release of gonadotropinreleasing hormone in the prepubertal male Rhesus monkey induced by N-methyl-D,L-aspartic acid. Neuroendocrinology 48: 147-152, 1988. Wilson RC, Knobil E. Acute effects of N-methyl-D,L-aspartate on the release of pituitary gonadotropins and prolactin in the adult female rhesus monkey. Brain Res 248:177-179, 1982. Terry LC, Martin JB. The effects of lateral hypothalamic media1 forebrain stimulation and somatostatin antiserum on pulsatile growth hormone secretion in freely behaving rats. Endocrinology 190~622-627, 1981. Estienne MJ, Schillo KK, Green MA, Hileman SM, Boling JA. N-methyl-d,l-aspartate stimulates growth hormone but not luteinizing hormone secretion in the sheep. Life Sci 44:1527-1533, 1989. Estienne MJ, Schillo KK, Green MA, Hileman SM. Growth hormone release after N-methyl-D, L-aspartate in sheep: dose response and effect of an opioid antagonist. J Anim Sci 68:3198-3203, 1990. Estienne MJ, Schillo KK, Hileman SM. Green MA, Hayes SH. Effects of N-methyl-D,L-aspartate on luteinizing hormone secretion in ovariectomized ewes in the absence and presence of estradiol. Biol Reprod 42:126-130, 1990. Reyes A, Luckhaus J, Ferin M. Unexpected inhibitory action of N-methyl-D,L-aspartate on luteinizing hormone release in adult ovariectomized Rhesus monkeys: A role of the hypothalamic-adrenal axis. Endocrinology 127:724-729, 1990. Reyes A, Xia L, Ferin M. Modulation of the effects of n-methyl-d,l-aspartate on luteinizing hormone by the ovarian steroids in the adult Rhesus monkey. Neuroendocrinology 54:405-4 11, 199 1.
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11. Nutrition Research Council, Nurrienr Requirements of&vine, Washington,D.C.:National Academy Press, 1988. Ed. 9th 12. Barb CR, Kraeling RR, Rampacek GB, Whisnant CS. Influence of stage of the estrous cycle on endogenous opioid modulation of luteinizing hormone, prolactin and cortisol secretion in the gilt. Biol Reprod 35: 11621167, 1986. 13. Barb CR, Kraeling RR, Rampacek GB, Fonda ES, Kiser TE. Inhibition of ovulation and LH secretion in the gilt after treatment with ACTH or hydrocortisone. J Reprod Fertil64:85-92, 1982. 14. Barb CR, Kraeling RR, Barrett JB, Rampacek GB, Campbell RM, Mowles TF. Serum glucose and free fatty acids modulate growth hormone and luteinizing hormone secretion in the pig. Proc Sot Exp Biol Med 198:636-642, 1991. 15. Kesner JS, Kraeling RR, Rampacek GB, Johnson B. Absence of an estradiol-induced surge of luteinizing hormone in pigs receiving unvarying pulsatile gonadotropin-releasing hormone stimulation. Endocrinology 121:1862-1869, 1987. 16. Kraeling RR, Rampacek GB, Cox NM, Kiser TE. Prolactin and luteinizing hormone secretion after bromocryptine (CB-154) treatment in lactating sows and ovariectomized gilts. J Anim Sci 54:1212-1220, 1982. 17. Barb CR, Estienne MJ, Kraeling RR, Marple DN, Rampacek GB, Rahe CH, Sartin JL. Endocrine changes in sows exposed to elevated ambient temperature during lactation. Domest Anim Endocrinol 8: 117-127, 1991. 18. Kraeling RR, Rampacek GB, Kiser TE. Corpus luteum function after indomethacin treatment during the estrous cycle and following hysterectomy in the gilt. Biol Reprod 2S:5 1 l-5 18, 1981. 19. Barb CR, Rampacek GB, Kraeling RR, Estienne MJ, Taras E, Estienne CE, Whisnant CS. Absence of brain opioid peptide modulation of luteinizing hormone secretion in the prepubertal gilt. Biol Reprod 39:603609,1988. 20. Byerley DJ, Bertrand JK, Berardinelli JG, Kiser TE. Testosterone and luteinizing hormone response to GnRH in yearling bulls of different libido. Theriogenology 34:1041-1049, 1990. 21. SAS, SAS User’s Guide, Gary, N.C.:Statistical Analysis Systems Institute, Inc., 1982. 22. Sesti LAC, Britt JH. Agonist-induced release of GnRH, LH and FSH and their associations with basal secretion of LH and FSH in lactating sows. Society for the Study of Reproduction 44 [Suppl. 1]:105, 1991. 23. Barb CR, Kraeling RR, Rampacek GB, Leshin LS. Gpioid modulation of follicle stimulating hormone (FSH) and prolactin (PRL) secretion in the postpartum sow. In: Mahesh VB, Dhindsa DS, Anderson E and Kalra SP (eds). Advances in Experimental Medicine and Biology, Plenum Press, New York, p. 647-652, 1987. 24. Pohl CR, Lee LR, Smith MS. Qualitative changes in luteinizing hormone and prolactin responses to nmethyl-aspartic acid during lactation in the rat. Endocrinology 124 (4):1905-1911, 1989. 25. Ondo JG, Wheeler DD, Dom RM. Hypothalamic site of action on N-methyl-D-aspartate (NMDA) on LH secretion. Life Sci 43:2283-2286, 1988. 26. Farah JM, Mick SJ, Michel J, Rao TS, Iyengar S. N-methyl-D,L-aspartic acid treatment increases circulating ACTH and immunoreactive B-endorphin in rats. Program of the 71st Annual Meeting of The Endocrine Society.Seatt1e.WA.p 180 1989. 27. Messi E, Zanisi M, Martini L. Excitatory amino acids stimulate LH release with a dual mechanism. Neuroendocrinology 52 [Suppl.l]:95:P2.93, 1990. 28. Barb CR, Kraeling RR, Rampacek GB. Opioid modulation of gonadotropin and prolactin secretion in domestic farm animals. Domest Anim Endocrinol8: 15-27, 199 1. 29. Fonda ES, Rampacek GB, Kraeling RR. The effect of adrenocorticotropin or hydrocortisone on serum luteinizing hormone concentrations after adrenalectomy and/or ovariectomy in the prepuberal gilt. Endocrinology 114:268-273, 1984. 30. Kraeling RR, Barb CR. Hypothalamic control of gonadotropin and prolactin secretion in pigs. J Reprod Fertil40:3-17, 1990. 3 1. Barb CR, Kraeling RR, Rampacek GB. Gpioid modulation of FSH, growth hormone and prolactin secretion in the prepuberal gilt. J Endocrinol 133:13-19, 1992. 32. Arslan M, Pohl CR, Smith MS, Plant TM. Studies of the role of the n-methyl-d-aspartate (NMDA) receptor in the hypothalamic control of prolactin secretion. Life Sci 50:295-300, 1992.
DOMESTICANIMAL ENDOCRINOLOGY
N-METHYL-D,L-ASPARTATE STIMULATES GROWTH HORMONE AND PROLACTIN BUT INHIBITS LUTEINIZING HORMONE SECRETION IN THE PIG C.R. Barb,* G.M. Derochers,** 8. Johnson,* R.V. Utley,** W.J. Chang,** G.B. Rampacek,** R.R. Kraeling* *Animal Physiology Unit Richard B. Russell Agricultural Research Center USDA, ARS, Athens, GA 30613 and **Animal and Dairy Science Department University of Georgia Athens, GA 30602 Received March 19, 1992
ABSTRACT The effects of n-methyl-d,l-aspartate (NMA), a neuroexcitatory amino acid agonist, on luteinizing hormone (LH), prolactin (PRL) and growth hormone (GH) secretion in gilts treated with ovarian steroids was studied. Mature gilts which had displayed one or more estrous cycles of 18 to 22 d were ovariectomized and assigned to one of three treatments administered in: corn oil vehicle (V; n=6); 10 pg estradiol-17b/kg BW given 33 hr before NMA (E; n=6); .85 mg progesterone/kg BW given twice daily for 6 d prior to NMA (P,; n=6). Blood was collected via jugular cannulae every 1.5 min for 6 hr. Pigs received 10 mg NMA/kg BW i.v. 2 hr after blood collection began and a combined synthetic [Ala15]-h GH releasing factor (l-29)-NH, (GRF; 1 pg/kg BW) and gonadotropin releasing hormone (GnRH; .2 &kg BW) challenge given i.v. 3 hr after NMA. NMA did not alter LH secretion in E gilts. However, NMA decreased (Pc.02) serum LH concentrations in V and P, gilts. Serum LH concentrations increased (Pc.01) after GnRH in all gilts. NMA did not alter PRL secretion in P, pigs, but increased (Pc.01) serum PRL concentrations in V and E animals. Treatment with NMA increased (Pc.01) GH secretion in all animals while the GRF challenge increased (Pc.01) serum GH concentrations in all animals except in V treated pigs. NMA increased (Pc.05) cortisol secretion in all treatment groups. These results indicate that NMA inhibits LH secretion and is a secretagogue of PRL, GH and cortisol secretion with ovarian steroids modulating the LH and PRL response to NMA.
INTRODUCTION N-methyl-d,l-aspartate (NMA) is a potent agonist of the neuroexcitatory amino acids aspartate and glutamate. NMA treatment increased serum luteinizing hormone (LH) concentrations in rats (1) and in both juvenile (2,3) and adult monkeys (4). Moreover, NMA is a potent secretagogue of prolactin (PRL) and growth hormone (GH) in the monkey (2,4). When administered to rodents, excitatory amino acids stimulated PRL secretion and altered the pulsatile pattern of GH secretion (5). In wethers, NMA treatment enhanced GH secretion, but failed to increase LH secretion (6). Similarly, in the ovariectomized (OVX) ewe, NMA stimulated GH secretion (7) but did not alter LH secretion (8). However, when ewes were pretreated with estradiol, NMA stimulated LH secretion (8). In a recent study, Reyes et al. (9) observed an inhibition of LH secretion following NMA in the OVX monkey. However, treatment with ovarian steroids resulted in a rapid reversal of this phenomena (10). Little is known about the endocrinological consequences of NMA administration in the pig. Therefore, the objective of this stucljr was to determine the effect of NMA on LH, GH and PRL secretion in the OVX pig in the absence and presence of ovarian steroids. Copyright 0 1992 Butterworth-Heinemann
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MATERIALS AND METHODS Eighteen gilts, weighing 121 f 2 kg and which had displayed one or more estrous cycles of 18-22 d, were OVX. All gilts were housed in individual pens in an environmentally controlled building and exposed to a constant ambient temperature of 22 C and an artificial photoperiod of 12: 12 hr 1ight:dark. Pigs were fed between 0700 and 0800 hr a corn-soybean meal ration (14% crude protein) supplemented with vitamins and minerals, according to Nutrition Research Council (11) guidelines. Starting approximately 2 weeks after OVX, the experiment was conducted in two replicates of 3 pigs/treatment group in each replicate, with the following treatment groups: corn oil vehicle im (V; n=6); 10 pg estradiol-17P,/kg BW im 33 hr before NMA injection (E; n = 6); .85 mg progesterone/kg BW im twice daily for six d prior to NMA (P,; n=6). Gilts in the V and E groups received corn oil injection twice daily for six d prior to NMA. Progesterone treatment was employed in order to simulate luteal phase concentrations of P, (12), while the dose of E was used to simulate late follicular phase concentrations of E (12). On the day prior to NMA treatment, an indwelling catheter was placed into the jugular vein of each pig (13). Blood samples were collected every 15 min for 2 hr prior to and 3 hr after iv injection of NMA’ (10 mg/kg SW) in .9% saline and for 1 hr after a combined GnRH (.2 pg/kg BW) and synthetic [AlaIs]-h GH-releasing factor (l-29)-NH, (GRF; 1 @kg BW) iv challenge. Before initiation of the experiment, a dose-response study (0,5, 10 and 15 mg/ kg BW) was performed with NMA in OVX gilts (n = 4/dose). There was no difference between doses with regard to LH secretion. Therefore, the 10 m&kg dose was chosen, since it produced consistent GH and PRL responses. Based on previous observations, the doses of GnRH and GRF employed in the present study were optimal for assessing differences in pituitary sensitivity (14). Blood samples were allowed to clot at 4 C for 24 hr and serum was harvested after centrifugation and stored at -20 C. Hormone Assays. Serum concentrations of LH ( 15), PRL ( 16) and GH ( 17) were quantified by radioimmunoassay (RIA) on all samples. Since a PRL secretagogue challenge was not given during the last hr of the sampling period, these samples were not included for PRL assay. The sensitivities of the assays were .15 ng/ml of LH, 1.O ng/ml of PRL and .4 ng/ml of GH. The intraassay and interassay coefficients of variation were 4.8% and 9.0% for LH, 16.3% and 15.2% for PRL and 3.2% and 13.6% for GH, respectively. Serum P, (18) and E (19) were quantified by RIA on the first sample collected from each gilt. The sensitivity of the P, and E assays were .5 rig/ml and .6 pg/ml, respectively. Intraassay and interassay coefficients of variation were 9.3% and 15.0% for P, and 13.6% and 19.1% for E, respectively. Serum cortisol concentrations were determined on samples collected every 30 min starting at time of NMA challenge. Cortisol concentrations were quantified by RIA as reported for the bovine (20), except for the following validation changes. Addition of 10,50, 100,250, 500 or 1000 pg of cortisol to 5 and 10 pl aliquots of porcine serum resulted in 113% recovery. Dose response curves from 5 to 50 pl of pooled porcine serum were parallel (fi.50) to the standard curve. Sensitivity of the assay was 1 ng/ml and intraassay and interassay coefficients of variation were 1.4% and 4.4%, respectively. Statistical Analysis. To determine the effect of steroid treatment on LH, PRL and GH response to NMA, data were divided into five periods. Period one represented the mean of samples collected prior to NMA treatment. The remainder of the sampling time was divided into four 1-hr periods, except for PRL in which sampling time was divided into three 1-hr periods. Cortisol data was not divided into hourly periods, since samples were collected every 30 min starting at time of NMA challenge. Data were then subjected to the general linear model split plot-in-time analysis of variance procedure of the Statistical Analysis System (21). The statistical model included treatment, period, replicate, treatment x replicate, and treatment x period interactions. Effects of treatment and replicate were tested using animal
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within treatment x replicate as the error term. Period, treatment x replicate and treatment x period were tested using treatment x period x replicate as the error term. Total area under the curve (AUC) was calculated using trapezoidal summation method for serum PRL and GH response to NMA after post-treatment concentrations were adjusted for pre-treatment levels. Data were then subjected to a one-way analysis of variance. Difference between treatment means were determined by least-squares contrasts (21). If a significant treatment x period interaction was detected, then differences between treatments within a period or between periods within a treatment were determined by least-squares contrasts (21). RESULTS
There was no treatment x replicate interaction detected for any of the hormones studied. Therefore, data was pooled across replicates. Serum E concentrations were nondetectable in V and P4 gilts, but averaged 30 + 5 pg/ml for the E gilts. Serum P, was nondetectable in V and E gilts, but averaged 24.7 + 3.8 r&ml in P, gilts. NMA increased (Pc.05) cortisol secretion in all treatment groups (Figure 1). A treatment x period (P<.OOOl)interaction was detected for LH. Profiles of serum LH concentrations of a representative gilt for each treatment group are depicted in Figure 2. Pretreatment serum LH concentrations were lower (Pc.05) in P, and E gilts compared to V animals (table 1). Serum LH concentrations decreased (Pc.02) after NMA in V and P4 treated gilts from 1.1 f . 1 ng/ml to .7 f . 1 @ml and from .9 + . 1 rig/ml to .5 + .l rig/ml, respectively (table 1). Luteinizing hormone secretion was not altered by NMA treatment in E treated animals (table 1). The LH response to GnRH was lower (Pc.02) in E pigs than in V animals, while this response was similar between V and P, gilts (table 1). Treatment x period (Pc.01) interactions were detected for PRL and GH. Mean serum PRL concentrations increased (Pc.01) after NMA in V and E treated gilts (table 2). However, NMA failed to alter PRL concentrations in P, treated gilts. The PRL response to NMA as determined by AUC was similar for V (2.2 + .5 ng*hr/ml) and E (2.5 + .5 ng+r/ml) animals but lower (Pc.05) in P, (.5 + .5 ng*hr/ml) pigs compared to V treated gilts. NMA in-
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TABLE I. MEAN SERUMLUTEINIZING HORMONE(LH) CONCENTRATIONS FORGILTS TREATEDWITH VEHICLE(V) PR~CESTERONE (P,), OR ESTRADIOL(E) FORPERIODS1, 2, 3.4 AND 5” Serum LH (@ml) Periodb
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TABLE 2. MEAN SERUMPROLACTIN(PRL) CONCENTRATION; FORGILTS TREATEDWITHVEHICLE(V), PROGES~RONE(P,), OR ESTRADIOL(E) FORPERIODS1,2,3 Serum Treatment
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TABLE 3. MEAN SERUMGROWTHHORMONE(GH) CONCENTRATIONS FORGILTS TREATEDWITHVEHICLE(V), PROGESIERONE (PA), OR ESTRADIOL(E) FORPERIODS1, 2, 3.4 Serum Treatment
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creased (Pc.01) serum GH concentrations in all treatment groups (table 3). The GH response to NMA as measured by AUC was similar for all treatments and averaged 41 + 5,40 k 5, and 30 k 5 ngWm1 for V, P, and E treated gilts, respectively. The GH response to GRF was greater (Pc.05) in E and P, pigs than in V treated animals (table 3).
DISCUSSION In the present study, LH secretion decreased following a bolus injection of NMA in both V and P, treated animals but LH secretion was not altered in E treated pigs. These results are surprising since, in a recent study, Reyes et al. (10) reported that treatment with either
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E alone or incombination with P, reversed inhibition of LH secretion by NMA in monkeys. Similarly, in the OVX ewe (8), NMA failed to stimulate LH release. However, pretreatment with E instituted the stimulatory influence of NMA on LH secretion. In wethers, although an immediate effect of NMA on LH secretion was not observed, serum LH concentrations were lower the day following NMA treatment (6). Therefore, presence of E appears to be a necessary antecedent for the excitatory action of NMA on LH secretion in some species. However, in the present study E did not sensitize the GnRH/LH secretory system to NMA. A recent report by Sesti and Britt (22) demonstrated that administration of NMA to lactating sows increased LH secretion. This apparent dichotomy between the present study and that of Sesti and Britt (22) is difficult to explain. We believe that physiological state may influence effectiveness of NMA to activate the GnRH/LH secretory system. In support of this idea we previously reported that the opioid antagonist, naloxone, stimulated PRL secretion in luteal phase gilts (12), while naloxone attenuated the suckling-induced increase in PRL secretion in lactating sows (23). Moreover, in mature female rats NMA treatment resulted in an acute increase in serum LH concentrations. However, this phenomenon was not demonstrable in lactating rats (24). Therefore, we suggest a similar paradigm may exist for excitatory amino acid modulation of LH secretion in the pig. It is generally accepted that NMA acts at the central nervous system (CNS) to release GnRH (2,3,25). Moreover, since NMA is a general excitatory amino acid which stimulates release of anterior pituitary hormones (2,4,6,26) it is conceivable that other neural factors which are inhibitory to LH secretion are released in response to NMA. However, action of NMA on LH secretion may also occur at the pituitary gland (27). The observation that exogenously administered GnRH increased LH secretion during inhibition by NMA in the present study eliminates this idea. However, increased cortisol secretion after NMA suggests activation of the hypothalamic-pituitary-adrenal axis. Similar increases in cortisol secretion after NMA treatment were reported in the monkey (2,9,10). Moreover, the increase in cortisol secretion after NMA may have resulted from an elevation in hypothalamic CRF and P-endorphin. Farah et al. (26) observed increased secretion of ACTH and P-endorphin after NMA in the male rat. Recently, Reyes et al. (9) reported that inhibition of LH secretion by NMA in the OVX monkey is the result of CRF and endogenous opioid peptide (EOP) secretion. In the present study, inhibition of LH secretion by NMA may be the result of both elevated cortisol secretion and increased release of EOP. We previously reported that the presence of P, is necessary for initiation of endogenous opioid modulation of LH secretion (28). In addition, activation of the adrenal axis inhibited both pulsatile and surge secretion of LH in the pig (13,29). Therefore, both cortisol and EOP may be acting in concert to inhibit LH secretion in the P, treated gilts. This may explain why P, treatment failed to reverse the inhibitory action of NMA on LH secretion as reported by Reyes et al. (10). The failure of NMA to influence LH secretion in E treated animals, may in part be due to the already suppressed levels of LH due to negative feedback action of E on LH secretion (30). Secondly, if the action of NMA on LH secretion is via GnRH release, then E associated changes in pituitary responsiveness to GnRH may have attenuated the LH response to NMA. This idea is supported by the fact that pituitary responsiveness was compromised by E treatment, since serum LH concentrations were lower following GnRH challenge in E treated compared to V and P4 treated gilts. As in the monkey (2) and wether (6), NMA markedly increased GH secretion without regard to steroid treatment in the present study. Although the current investigation did not address mechanisms responsible for stimulation by NMA, it is reasonable to suggest that this stimulation of GH secretion is due to a release of GRF and/or suppression of somatostatin secretion from the hypothalamus. Moreover, this response may be mediated by EOP, since stimulation of EOP receptors increased GH secretion in pigs (31) and NMA evoked EOP
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secretion (6,9). In contrast to this idea, naloxone, an EOP antagonist, failed to alter GH response to NMA in OVX ewes (7). In the present study, NMA increased PRL secretion in V and E treated animals, but not in P, treated pigs. Wilson and Knobil(4) reported that NMA treatment increased serum or plasma PRL concentrations during both the late luteal and early follicular phase of the menstrual cycle in the Rhesus monkey. In addition, other reports demonstrated that administration of NMA to male and female rats (24,32) elicited marked increase in PRL secretion, whereas NMA failed to stimulate PRL secretion during lactation (24). Furthermore, if administered when PRL concentrations were elevated during suckling NMA acutely suppressed PRL release (24). Therefore, results of the present study and above reports indicate that NMA involvement in central nervous system regulation of PRL secretion may occur through activation of both PRL simulating and inhibiting systems depending on the physiological state or steroid milieu. We previously reported the existence of a P, dependent EOP system which inhibits PRL secretion in the pig (12,31). Therefore, lack of a PRL response to NMA in P, treated gilts may in part be due to P, induced EOP inhibition of the PRL secretory system. In light of the diverse effects of NMA on pituitary hormone secretion between the present study and others, future studies should focus on whether neuroexcitatory amino acids play a physiological role in the control of pituitary function via the use of specific agonist and antagonist. ACKNOWLEDGMENTS/FOOTNOTES Theauthors wish to thank Mr. John B. Barrett, Ms. Elizabeth A. Taras, Ms. Amanda Latimer and Ms. Donna Slavin for their technical assistance; Ruel L. Wilson, biometrician, Southern Region, ARS for his statistical advice; Dr. D.J. Bolt, USDA, Beltsville, MD for providing pituitary hormones used in radioimmunoassay; Dr. Myron Brown, Ceva Laboratory, Overland Park, KS, for the generous gift of GnRH, and Drs. Robert M. Campbell and Thomas F. Mowles, Hoffmann-LaRoche, Nutley, NJ, for the generous gift of synthetic [Ala”]hGRF( l -29)-NH,. ‘Sigma Chemical Co., St. Louis, MO Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the USDA and does not imply its approval to the exclusion of other products that may be suitable. Corresponding Author: Dr. C. Richard Barb, USDA, ARS, Animal Physiology, R.B. Russell Research Center, P.O. Box 5677, Athens, GA 30613. REFERENCES 1. Urbanski
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11. Nutrition Research Council, Nurrienr Requirements of&vine, Washington,D.C.:National Academy Press, 1988. Ed. 9th 12. Barb CR, Kraeling RR, Rampacek GB, Whisnant CS. Influence of stage of the estrous cycle on endogenous opioid modulation of luteinizing hormone, prolactin and cortisol secretion in the gilt. Biol Reprod 35: 11621167, 1986. 13. Barb CR, Kraeling RR, Rampacek GB, Fonda ES, Kiser TE. Inhibition of ovulation and LH secretion in the gilt after treatment with ACTH or hydrocortisone. J Reprod Fertil64:85-92, 1982. 14. Barb CR, Kraeling RR, Barrett JB, Rampacek GB, Campbell RM, Mowles TF. Serum glucose and free fatty acids modulate growth hormone and luteinizing hormone secretion in the pig. Proc Sot Exp Biol Med 198:636-642, 1991. 15. Kesner JS, Kraeling RR, Rampacek GB, Johnson B. Absence of an estradiol-induced surge of luteinizing hormone in pigs receiving unvarying pulsatile gonadotropin-releasing hormone stimulation. Endocrinology 121:1862-1869, 1987. 16. Kraeling RR, Rampacek GB, Cox NM, Kiser TE. Prolactin and luteinizing hormone secretion after bromocryptine (CB-154) treatment in lactating sows and ovariectomized gilts. J Anim Sci 54:1212-1220, 1982. 17. Barb CR, Estienne MJ, Kraeling RR, Marple DN, Rampacek GB, Rahe CH, Sartin JL. Endocrine changes in sows exposed to elevated ambient temperature during lactation. Domest Anim Endocrinol 8: 117-127, 1991. 18. Kraeling RR, Rampacek GB, Kiser TE. Corpus luteum function after indomethacin treatment during the estrous cycle and following hysterectomy in the gilt. Biol Reprod 2S:5 1 l-5 18, 1981. 19. Barb CR, Rampacek GB, Kraeling RR, Estienne MJ, Taras E, Estienne CE, Whisnant CS. Absence of brain opioid peptide modulation of luteinizing hormone secretion in the prepubertal gilt. Biol Reprod 39:603609,1988. 20. Byerley DJ, Bertrand JK, Berardinelli JG, Kiser TE. Testosterone and luteinizing hormone response to GnRH in yearling bulls of different libido. Theriogenology 34:1041-1049, 1990. 21. SAS, SAS User’s Guide, Gary, N.C.:Statistical Analysis Systems Institute, Inc., 1982. 22. Sesti LAC, Britt JH. Agonist-induced release of GnRH, LH and FSH and their associations with basal secretion of LH and FSH in lactating sows. Society for the Study of Reproduction 44 [Suppl. 1]:105, 1991. 23. Barb CR, Kraeling RR, Rampacek GB, Leshin LS. Gpioid modulation of follicle stimulating hormone (FSH) and prolactin (PRL) secretion in the postpartum sow. In: Mahesh VB, Dhindsa DS, Anderson E and Kalra SP (eds). Advances in Experimental Medicine and Biology, Plenum Press, New York, p. 647-652, 1987. 24. Pohl CR, Lee LR, Smith MS. Qualitative changes in luteinizing hormone and prolactin responses to nmethyl-aspartic acid during lactation in the rat. Endocrinology 124 (4):1905-1911, 1989. 25. Ondo JG, Wheeler DD, Dom RM. Hypothalamic site of action on N-methyl-D-aspartate (NMDA) on LH secretion. Life Sci 43:2283-2286, 1988. 26. Farah JM, Mick SJ, Michel J, Rao TS, Iyengar S. N-methyl-D,L-aspartic acid treatment increases circulating ACTH and immunoreactive B-endorphin in rats. Program of the 71st Annual Meeting of The Endocrine Society.Seatt1e.WA.p 180 1989. 27. Messi E, Zanisi M, Martini L. Excitatory amino acids stimulate LH release with a dual mechanism. Neuroendocrinology 52 [Suppl.l]:95:P2.93, 1990. 28. Barb CR, Kraeling RR, Rampacek GB. Opioid modulation of gonadotropin and prolactin secretion in domestic farm animals. Domest Anim Endocrinol8: 15-27, 199 1. 29. Fonda ES, Rampacek GB, Kraeling RR. The effect of adrenocorticotropin or hydrocortisone on serum luteinizing hormone concentrations after adrenalectomy and/or ovariectomy in the prepuberal gilt. Endocrinology 114:268-273, 1984. 30. Kraeling RR, Barb CR. Hypothalamic control of gonadotropin and prolactin secretion in pigs. J Reprod Fertil40:3-17, 1990. 3 1. Barb CR, Kraeling RR, Rampacek GB. Gpioid modulation of FSH, growth hormone and prolactin secretion in the prepuberal gilt. J Endocrinol 133:13-19, 1992. 32. Arslan M, Pohl CR, Smith MS, Plant TM. Studies of the role of the n-methyl-d-aspartate (NMDA) receptor in the hypothalamic control of prolactin secretion. Life Sci 50:295-300, 1992.