0013-7227/79/1054-0875$02.00/0 Endocrinology Copyright © 1979 by The Endocrine Society
Vol. 105, No. 4 Printed in U.S.A.
Circulating Luteinizing Hormone and Prolactin Concentrations in Intact or Castrated Male Rats Treated with 5-Hydroxytryptamine* NANCY S. PILOTTE AND JOHN C. PORTER Cecil H. and Ida Green Center for Reproductive Biology Sciences, the Departments of Obstetrics and Gynecology and Physiology, The University of Texas Health Science Center at Dallas, Southwestern Medical School, Dallas, Texas 75235
right atrium by way of the right jugular vein at the time of castration, and the animals were used 2 days later. Blood was withdrawn through the cannula before and after serotonin or the solvent vehicle was administered sc to these rats. Within 30 min after the injection of serotonin, the serum concentration of PRL was significantly increased and that of LH was significantly (P < 0.01) decreased relative to the preinjection levels. When bromocriptine was given to these acutely castrated rats 3 h before the administration of serotonin, PRL release was completely inhibited; however, LH release was still reduced significantly (P < 0.01) relative to the controls. These findings are in accord with the hypothesis that treatment with serotonin can lead to an increase in PRL release and, conversely, to an inhibition of LH release in acutely castrated male rats. Moreover, the effect of serotonin on LH release is not dependent on PRL
ABSTRACT. The effects of sc administered 5-hydroxytryptamine (serotonin) on LH and PRL release, as reflected by circulating concentrations of these hormones, were investigated in intact and castrated male rats. In the initial experiment, the animals were given serotonin or its solvent vehicle and were decapitated 30 min later. In all serotonin-treated animals (intact or castrated 2 or 30 days before use), the serum PRL concentration was increased markedly (at least 5 times) over that of the vehicle-treated animals, but the serum LH concentration was affected significantly only in rats that had been castrated for 2 days. In these animals, the serum LH concentration was 230 ± 21 ng/ml (mean ± SE) compared to 395 ± 39 ng/ml in the controls. However, in intact male rats or male rats that had been castrated for 30 days, the serum LH concentration was not significantly depressed in serotonin-treated animals. In subsequent experiments, an indwelling cannula was inserted into the
T
HE ADMINISTRATION of 5-hydroxytryptamine (serotonin) to rats (1, 2) or its precursor, 5-hydroxytryptophan, to humans (3) or rats (4) results in an augmented release of PRL. However, the relationship of serotonin to the release of LH is less clear. The administration of serotonin to female rats during the critical period of proestrus leads to inhibition of the subsequent preovulatory release of LH (5), and the intraventricular administration of serotonin to pentobarbital-treated proestrous rats results in inhibition of the release of LH after electrochemical stimulation of the medial preoptic area (6). Schneider and McCann (7) found that serotonin administered intraventricularly did not affect LH release during the estrous cycle but did appear to suppress LH release in castrated female rats. Other investigators (8) have found that serotonin is necessary for the cyclic
release. (Endocrinology 105: 875, 1979)
release of LH in castrated female rats treated with estradiol. These findings are supportive of the possibility that the effect of serotonin on LH release is influenced by gonadal steroids. To test this hypothesis, we evaluated the release of LH and PRL after sc administration of serotonin to intact and castrated male rats. Since hyperprolactinemia results in suppression of the postcastration rise in LH release in male rats (9) and since the administration of serotonin results in an increase in PRL release, we addressed the question of whether the effects of serotonin on the release of LH and PRL are conjoint events. Materials and Methods Animals Adult male rats of the Long-Evans strain (250-300 g) were used in this investigation. The animals were housed under conditions of controlled temperature (22-24 C) and lighting (lights on at 0500 h, off at 1900 h) and had free access to food and water. Intact as well as acutely or chronically orchiectomized rats were used in these experiments. Acutely and chronically castrated rats were animals that had been orchiectomized
Received March 9, 1979. Address all correspondence and requests for reprints to: Dr. Nancy S. Pilotte, Department of Obstetrics and Gynecology, The University of Texas Health Science Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235. * This work was supported by Research Grants AM-01237, AG00306, and HD-11149 from the NIH, Bethesda, MD. 875
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Endo 1979 Vol 105 , No 4
PILOTTE AND PORTER
876
2 and 30 days before use, respectively. All surgical procedures were performed under ether anesthesia.
Duncan's multiple range test were utilized in the statistical evaluation of the results (11). Results
Experimental procedures Serotonin creatinine sulfate obtained from Sigma Chemical Co. (St. Louis, MO) was dissolved in 0.01 M phosphate-0.14 M NaCl (PBS), pH 7.6, immediately before use. Bromocriptine (2a-bromoergocriptine), a gift from Sandoz Laboratories (Hanover, NJ), was dissolved in 0.3% tartaric acid immediately before use. All animals were given serotonin (11.5 mg free base/ kg BW, sc) or 0.5 ml PBS daily on 3 consecutive days before use. In some animals, bromocriptine (1 mg/kg BW, sc) was given 3 h before the administration of serotonin or PBS. Exp 1. Acutely orchiectomized rats were given the first injection of serotonin or PBS at the time of castration. The chronically castrated animals were given the first injection of serotonin or PBS 28 days after orchiectomy; the intact rats were given the first injection of serotonin or PBS 3 days before use. After decapitation, trunk blood from these animals was collected 30 min after the last, i.e. the third, injection of serotonin or PBS. After centrifugation of the blood, the serum was removed and stored at -20 C to await assay. Exp 2. The tip of an indwelling cannula was inserted into the right atrium by way of the right jugular vein of each rat at the time of castration, and serotonin or PBS was injected sc immediately after castration and daily for 2 more days. To minimize the effects of handling and withdrawal of blood on the release of PRL, the animals were habituated to the experimental procedures in the following manner. During the hour preceding the preinjection period, blood (0.4 ml) was withdrawn into a syringe and then reinjected through the cannula at 10min intervals. At 60, 50, 40, 30, 20, and 10 min before the injection of serotonin or PBS (preinjection period), blood (0.4 ml) was withdrawn from each rat and replaced with 0.4 ml 0.15 M NaCl containing heparin (300 U/ml). At time zero, the animals were given PBS or serotonin. During the following hour (postinjection period), blood (0.4 ml) was withdrawn every 10 min from each animal, and 0.4 ml 0.15 M NaCl containing heparin was infused to replace the blood loss, as in the preinjection period. After centrifugation of the blood, the plasma was removed and stored at -20 C to await assay.
Exp 1 In acutely castrated male rats, the serum concentration of LH was 230 ± 21 ng/ml (mean ± SE) in animals given serotonin and 395 ± 39 ng/ml in animals given the solvent vehicle (Fig. 1, left panel). These two means are significantly (P < 0.01) different. In contrast to these findings in the acutely castrated rats, the mean serum concentration of LH in intact or chronically castrated animals given serotonin was not significantly different from that in the control animals. The mean serum PRL concentration in intact or acutely castrated rats given serotonin was significantly (P < 0.01) greater than that in the vehicle-treated rats (Fig. 1, right panel). In the chronically castrated animals, the mean PRL concentration in the serotonin-treated rats (123 ± 37 ng/ml) was also significantly (P < 0.05) greater than that in the controls (24 ± 6 ng/ml), but the difference was less pronounced than that seen in intact or acutely castrated animals. Exp 2 Inasmuch as the serum LH concentrations were significantly lower whereas the serum PRL concentrations were significantly higher in acutely castrated rats treated with serotonin than in the controls, the question arose concerning the temporal relationship of the increase in PRL release to the reduction in LH release. As illustrated in Figs. 2 and 3, the plasma concentrations of LH and
Exp 3. The rats used in this experiment were treated identically to those used in Exp 2, except that bromocriptine was administered 3 h before the injection of serotonin or PBS. RIA of LH and PRL The serum or plasma was analyzed for LH and PRL by RIA. LH was measured according to the method of Niswender et al. (10). PRL was assayed according to the procedure outlined by the Rat Pituitary Program of the NIH (Bethesda, MD). The values for PRL and LH are expressed in terms of the NIAMDDRat PRL-RP-1 and the NIAMDD-Rat LH-RP-1 reference preparations, respectively. Statistical analysis Student's t test, repeated measures analysis of variance, and
Intact
2-Day Castrates
30-Day Castrates
Intact
2-Day
Castrates
3 0 - Day Castrates
FIG. 1. Serum concentrations of LH and PRL 30 min after the injection of serotonin or PBS into intact or castrated male rats. Serum concentrations of LH (left panel) and PRL (right panel) were measured in the same animals. D, Results obtained using rats treated with PBS (0.5 ml, sc). IS, Results obtained using rats treated with serotonin (5-HT). Column heights and vertical lines represent the means and magnitude of the SE, respectively. Ten rats were used in each group.
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CIRCULATING LH AND PRL AFTER SEROTONIN — © — VEHICLE (n=9) — A — 5-HT (n = 10)
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o
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After the injection of serotonin, the PRL concentration in plasma rose steadily for 40 min and then began to fall. In contrast, the plasma PRL concentration in the PBStreated animals changed little throughout the postinjection period. At 30, 40, 50, and 60 min after the injection, the plasma PRL levels in the serotonin-treated animals were significantly (P < 0.01) greater than those of the PBS-treated rats.
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FIG. 2. Plasma concentrations of LH before and after the administration of serotonin or PBS to acutely castrated male rats. The left half of the figure denotes the plasma concentrations of LH in serotonin- and vehicle-treated rats measured at 10-min intervals during the 60-min preinjection period. The preinjection plasma concentrations of LH of both groups were not significantly different and were combined. The right half of the figure denotes the plasma concentrations of LH in serotonin- and vehicle-treated animals measured at 10-min intervals for 60 min after the injection of serotonin or PBS. The symbols (0 and A) and the vertical lines represent the mean plasma LH concentrations and the magnitude of the SE, respectively. Significant differences (P < 0.01) between the groups occurred at 30, 40, 50, and 60 min after the administration of the test substances. »•••©••-•
_ © _
PRE-INJECTION (n=19)
Discussion
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— A — 5 - H T (n=10)
200
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p 100
To ascertain whether the release of PRL was causally related to the reduction in the release of LH after serotonin treatment, we evaluated the effect of serotonin on LH release in rats pretreated with bromocriptine. During the 60-min preinjection period, the plasma PRL concentrations of the bromocriptine-treated rats varied little from the mean of 13 ng/ml (Fig. 4). During the 60-min postinjection period after the administration of serotonin or PBS to the bromocriptine-treated animals, the mean plasma PRL concentrations were 16 and 13 ng/ml, respectively. In contrast to these findings in bromocriptinetreated rats given serotonin, the plasma concentrations of LH fell steadily throughout the 20- to 60-min postinjection period and were significantly (P < 0.01) less than those in bromocriptine-treated rats given PBS.
/ N
Although it is generally acknowledged that the administration of serotonin modulates the secretion of pituitary hormones, especially LH and PRL, the mechanism by which these actions of serotonin are mediated is obscure. Since hyperprolactinemia is often associated with deBROMOCRIPTINE (n = 20)
VEHICLE (n=10) 5-HT (n«10) INJECTION
©••--••.©..,
••©—0 -60
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FIG. 3. Plasma concentrations of PRL before and after the administration of serotonin or PBS to acutely castrated male rats. Significant (P < 0.01) differences between groups occurred at 30, 40, 50, and 60 min after the administration of the test substances. For further details, see Fig. 2.
PRL attained moderately steady levels of approximately 450 and 30 ng/ml, respectively, during the 60-min preinjection period. During the 10- to 60-min postinjection period, the plasma LH concentration steadily declined in the serotonin-treated rats but remained unchanged in the controls. The plasma LH concentrations at 30,40, 50, and 60 min after the administration of serotonin were significantly (P < 0.01) less than the plasma LH concentrations in rats given PBS.
30 z
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FIG. 4. Plasma concentrations of LH and PRL before and after the administration of serotonin or PBS to acutely castrated male rats pretreated with bromocriptine. The rats were treated with bromocriptine (1 mg/kg) 3 h before use. • — • , 0—0, and A—A, Plasma concentrations of LH. • • • • , © • . 0 , and A • • A, Plasma concentrations of PRL. Significant (P < 0.01) differences between the plasma concentrations of LH in the experimental and control groups occurred at 20, 30, 40, 50, and 60 min. For further details, see Fig. 2.
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878
PILOTTE AND PORTER
pressed plasma concentrations of gonadotropins in rats (9) as well as in man (3), it seems reasonable to suspect that the increase in PRL seen after the administration of serotonin is related to the concomitant decrease in circulating LH. In the present investigation, serotonin administration led to a significant release of PRL in each experimental model used, namely, intact male rats, acutely castrated male rats, and chronically castrated male rats. However, the effects of serotonin on the release of LH appear to be dependent on the animal model employed. Serotonin, administered sc, had little effect on LH release in intact male rats when the basal rate of LH release was low or in rats that had been orchiectomized for 30 days when the rate of LH release was high. It was only when LH release was examined 2 days after orchiectomy, a time when the rate of LHRH release is presumably undergoing rapid modification, that the administration of serotonin led to a significant reduction in the concentration of LH in plasma. For these reasons, the male rat which had been castrated for 2 days was employed as the model to address the question of whether the inhibitory effects of serotonin on LH release and the stimulatory effect of this indoleamine on PRL release were causally related events. To determine whether the inhibition of LH release was dependent on a marked augmentation of circulating PRL concentrations, bromocriptine was used to inhibit the serotonin-induced secretion of PRL. The plasma LH concentrations of bromocriptine-treated rats were markedly reduced in rats given serotonin compared to LH levels in vehicle-treated controls. Accordingly, the decreased release of LH induced by serotonin administration does not require a simultaneous enhancement of PRL release. Therefore, if not PRL, what is the mechanism of the inhibition of LH release by serotonin? It is unlikely that serotonin has a direct inhibitory effect on the gonadotropes, since incubation of rat pituitary glands in the presence of serotonin does not suppress the LHRH-induced release of LH (12). Serotonin may act as a neuromodulator which leads to suppression of LHRH secretion (6). It is also possible that serotonin may stimulate the secretion of an LH release-inhibiting factor. Lastly, what is the mechanism by which the administration of serotonin leads to the increased release of PRL? It is unlikely that serotonin stimulates PRL release by a direct action on the anterior pituitary gland (4). Perhaps serotonin acts as a neuromodulator substance to suppress the release of dopamine into hypophysial portal blood. It has been shown by Gudelsky and Porter (13) that a-methyltyrosine, a substance which inhibits the release of dopamine into portal blood, leads to the increased release of PRL. Hence, if serotonin were to
Endo 1979 Vol 105 « No 4
suppress dopamine release into portal blood, one could expect PRL secretion to increase. In preliminary experiments, it has been found that dopamine secretion into portal blood is depressed after serotonin administration (Pilotte, N. S., and J. C. Porter, unpublished observations). Serotonin may suppress dopamine synthesis in the tuberoinfundibular neurons, leading to decreased dopamine release into portal blood. Another possibility which cannot be excluded is that serotonin stimulates the secretion of a PRL-releasing factor. Acknowledgments The authors thank Sue Sherwin Martin, Linda Akers, and Jodie Roberts for technical assistance and lone CrandaLI and Judy Wagers for editorial assistance. The antiserum to rat PRL and the reference preparations used in the assay of PRL and LH were provided by the NIAMDD Rat Pituitary Hormone Program and Dr. A. Parlow. The antiserum to LH was a gift from Dr. G. D. Niswender. We thank Ms. K. D. Roskaz of Sandoz, Inc., for the gift of the bromocriptine.
References 1. Kamberi, I. A., R. S. Mical, and J. C. Porter, Effects of melatonin and serotonin on the release of FSH and prolactin, Endocrinology 88: 1288, 1971. 2. Lawson, D. M., and R. R. Gala, The influence of pharmacological manipulations of serotonergic and dopaminergic mechanisms on plasma prolactin in ovariectomized, estrogen-treated rats, Endocrinology 102: 973, 1978. 3. Maclndoe, J. H., and R. W. Turkington, Stimulation of human prolactin secretion by intravenous infusion of L-tryptophan, J Clin Invest 52: 1972, 1973. 4. Lamberts, S. W. J., and R. M. MacLeod, The interaction of the serotonergic and dopaminergic systems on prolactin secretion in the rat, Endocrinology 103: 287, 1978. 5. Wilson, C. A., and P. G. McDonald, Inhibitory effect of serotonin on ovulation in adult rats, J Endocrinol 60: 253, 1974. 6. Cramer, O. M., and C. A. Barraclough, The actions of serotonin, norepinephrine, and epinephrine on hypothalamic processes leading to adenohypophyseal luteinizing hormone release, Endocrinology 103: 694, 1978. 7. Schneider, H. P. G., and S. M. McCann, Dopaminergic pathways and gonadotropin releasing factors, In Bargmann, W., and B. Scharrer (eds.), Aspects of Neuroendocrinology, Springer-Verlag, Berlin, 1970, p. 177. 8. Hery, M., E. Laplante, and C. Kordon, Participation of serotonin in the phasic release of LH. I. Evidence from pharmacological experiments, Endocrinology 99: 496, 1976. 9. Winters, S. J., and D. L. Loriaux, Suppression of plasma luteinizing hormone by prolactin in the male rat, Endocrinology 102: 864, 1978. 10. Niswender, G. D., A. R. Midgley, Jr., S. E. Monroe, and L. E. Reichert, Jr., Radioimmunoassay for rat luteinizing hormone with antiovine LH serum and ovine LH-I3II, Proc Soc Exp Biol Med 128: 807, 1968. 11. Myers, J. L., Fundamentals of Experimental Design, Allyn and Bacon, Boston, 1966. 12. Martin, J. E., J. N. Engel, and D. C. Klein, Inhibition of the in vitro pituitary response to luteinizing hormone releasing hormone by melatonin, serotonin, and 5-methoxytryptamine, Endocrinology 100: 675, 1977. 13. Gudelsky, G. A., and J. C. Porter, Release of newly-synthesized dopamine into the hypophysial portal vasculature of the rat, Endocrinology 104: 583, 1979.
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Vol. 105, No. 4 Printed in U.S.A.
Circulating Luteinizing Hormone and Prolactin Concentrations in Intact or Castrated Male Rats Treated with 5-Hydroxytryptamine* NANCY S. PILOTTE AND JOHN C. PORTER Cecil H. and Ida Green Center for Reproductive Biology Sciences, the Departments of Obstetrics and Gynecology and Physiology, The University of Texas Health Science Center at Dallas, Southwestern Medical School, Dallas, Texas 75235
right atrium by way of the right jugular vein at the time of castration, and the animals were used 2 days later. Blood was withdrawn through the cannula before and after serotonin or the solvent vehicle was administered sc to these rats. Within 30 min after the injection of serotonin, the serum concentration of PRL was significantly increased and that of LH was significantly (P < 0.01) decreased relative to the preinjection levels. When bromocriptine was given to these acutely castrated rats 3 h before the administration of serotonin, PRL release was completely inhibited; however, LH release was still reduced significantly (P < 0.01) relative to the controls. These findings are in accord with the hypothesis that treatment with serotonin can lead to an increase in PRL release and, conversely, to an inhibition of LH release in acutely castrated male rats. Moreover, the effect of serotonin on LH release is not dependent on PRL
ABSTRACT. The effects of sc administered 5-hydroxytryptamine (serotonin) on LH and PRL release, as reflected by circulating concentrations of these hormones, were investigated in intact and castrated male rats. In the initial experiment, the animals were given serotonin or its solvent vehicle and were decapitated 30 min later. In all serotonin-treated animals (intact or castrated 2 or 30 days before use), the serum PRL concentration was increased markedly (at least 5 times) over that of the vehicle-treated animals, but the serum LH concentration was affected significantly only in rats that had been castrated for 2 days. In these animals, the serum LH concentration was 230 ± 21 ng/ml (mean ± SE) compared to 395 ± 39 ng/ml in the controls. However, in intact male rats or male rats that had been castrated for 30 days, the serum LH concentration was not significantly depressed in serotonin-treated animals. In subsequent experiments, an indwelling cannula was inserted into the
T
HE ADMINISTRATION of 5-hydroxytryptamine (serotonin) to rats (1, 2) or its precursor, 5-hydroxytryptophan, to humans (3) or rats (4) results in an augmented release of PRL. However, the relationship of serotonin to the release of LH is less clear. The administration of serotonin to female rats during the critical period of proestrus leads to inhibition of the subsequent preovulatory release of LH (5), and the intraventricular administration of serotonin to pentobarbital-treated proestrous rats results in inhibition of the release of LH after electrochemical stimulation of the medial preoptic area (6). Schneider and McCann (7) found that serotonin administered intraventricularly did not affect LH release during the estrous cycle but did appear to suppress LH release in castrated female rats. Other investigators (8) have found that serotonin is necessary for the cyclic
release. (Endocrinology 105: 875, 1979)
release of LH in castrated female rats treated with estradiol. These findings are supportive of the possibility that the effect of serotonin on LH release is influenced by gonadal steroids. To test this hypothesis, we evaluated the release of LH and PRL after sc administration of serotonin to intact and castrated male rats. Since hyperprolactinemia results in suppression of the postcastration rise in LH release in male rats (9) and since the administration of serotonin results in an increase in PRL release, we addressed the question of whether the effects of serotonin on the release of LH and PRL are conjoint events. Materials and Methods Animals Adult male rats of the Long-Evans strain (250-300 g) were used in this investigation. The animals were housed under conditions of controlled temperature (22-24 C) and lighting (lights on at 0500 h, off at 1900 h) and had free access to food and water. Intact as well as acutely or chronically orchiectomized rats were used in these experiments. Acutely and chronically castrated rats were animals that had been orchiectomized
Received March 9, 1979. Address all correspondence and requests for reprints to: Dr. Nancy S. Pilotte, Department of Obstetrics and Gynecology, The University of Texas Health Science Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235. * This work was supported by Research Grants AM-01237, AG00306, and HD-11149 from the NIH, Bethesda, MD. 875
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Endo 1979 Vol 105 , No 4
PILOTTE AND PORTER
876
2 and 30 days before use, respectively. All surgical procedures were performed under ether anesthesia.
Duncan's multiple range test were utilized in the statistical evaluation of the results (11). Results
Experimental procedures Serotonin creatinine sulfate obtained from Sigma Chemical Co. (St. Louis, MO) was dissolved in 0.01 M phosphate-0.14 M NaCl (PBS), pH 7.6, immediately before use. Bromocriptine (2a-bromoergocriptine), a gift from Sandoz Laboratories (Hanover, NJ), was dissolved in 0.3% tartaric acid immediately before use. All animals were given serotonin (11.5 mg free base/ kg BW, sc) or 0.5 ml PBS daily on 3 consecutive days before use. In some animals, bromocriptine (1 mg/kg BW, sc) was given 3 h before the administration of serotonin or PBS. Exp 1. Acutely orchiectomized rats were given the first injection of serotonin or PBS at the time of castration. The chronically castrated animals were given the first injection of serotonin or PBS 28 days after orchiectomy; the intact rats were given the first injection of serotonin or PBS 3 days before use. After decapitation, trunk blood from these animals was collected 30 min after the last, i.e. the third, injection of serotonin or PBS. After centrifugation of the blood, the serum was removed and stored at -20 C to await assay. Exp 2. The tip of an indwelling cannula was inserted into the right atrium by way of the right jugular vein of each rat at the time of castration, and serotonin or PBS was injected sc immediately after castration and daily for 2 more days. To minimize the effects of handling and withdrawal of blood on the release of PRL, the animals were habituated to the experimental procedures in the following manner. During the hour preceding the preinjection period, blood (0.4 ml) was withdrawn into a syringe and then reinjected through the cannula at 10min intervals. At 60, 50, 40, 30, 20, and 10 min before the injection of serotonin or PBS (preinjection period), blood (0.4 ml) was withdrawn from each rat and replaced with 0.4 ml 0.15 M NaCl containing heparin (300 U/ml). At time zero, the animals were given PBS or serotonin. During the following hour (postinjection period), blood (0.4 ml) was withdrawn every 10 min from each animal, and 0.4 ml 0.15 M NaCl containing heparin was infused to replace the blood loss, as in the preinjection period. After centrifugation of the blood, the plasma was removed and stored at -20 C to await assay.
Exp 1 In acutely castrated male rats, the serum concentration of LH was 230 ± 21 ng/ml (mean ± SE) in animals given serotonin and 395 ± 39 ng/ml in animals given the solvent vehicle (Fig. 1, left panel). These two means are significantly (P < 0.01) different. In contrast to these findings in the acutely castrated rats, the mean serum concentration of LH in intact or chronically castrated animals given serotonin was not significantly different from that in the control animals. The mean serum PRL concentration in intact or acutely castrated rats given serotonin was significantly (P < 0.01) greater than that in the vehicle-treated rats (Fig. 1, right panel). In the chronically castrated animals, the mean PRL concentration in the serotonin-treated rats (123 ± 37 ng/ml) was also significantly (P < 0.05) greater than that in the controls (24 ± 6 ng/ml), but the difference was less pronounced than that seen in intact or acutely castrated animals. Exp 2 Inasmuch as the serum LH concentrations were significantly lower whereas the serum PRL concentrations were significantly higher in acutely castrated rats treated with serotonin than in the controls, the question arose concerning the temporal relationship of the increase in PRL release to the reduction in LH release. As illustrated in Figs. 2 and 3, the plasma concentrations of LH and
Exp 3. The rats used in this experiment were treated identically to those used in Exp 2, except that bromocriptine was administered 3 h before the injection of serotonin or PBS. RIA of LH and PRL The serum or plasma was analyzed for LH and PRL by RIA. LH was measured according to the method of Niswender et al. (10). PRL was assayed according to the procedure outlined by the Rat Pituitary Program of the NIH (Bethesda, MD). The values for PRL and LH are expressed in terms of the NIAMDDRat PRL-RP-1 and the NIAMDD-Rat LH-RP-1 reference preparations, respectively. Statistical analysis Student's t test, repeated measures analysis of variance, and
Intact
2-Day Castrates
30-Day Castrates
Intact
2-Day
Castrates
3 0 - Day Castrates
FIG. 1. Serum concentrations of LH and PRL 30 min after the injection of serotonin or PBS into intact or castrated male rats. Serum concentrations of LH (left panel) and PRL (right panel) were measured in the same animals. D, Results obtained using rats treated with PBS (0.5 ml, sc). IS, Results obtained using rats treated with serotonin (5-HT). Column heights and vertical lines represent the means and magnitude of the SE, respectively. Ten rats were used in each group.
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CIRCULATING LH AND PRL AFTER SEROTONIN — © — VEHICLE (n=9) — A — 5-HT (n = 10)
••••••©••-• PRE-INJECTION (n=19)
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After the injection of serotonin, the PRL concentration in plasma rose steadily for 40 min and then began to fall. In contrast, the plasma PRL concentration in the PBStreated animals changed little throughout the postinjection period. At 30, 40, 50, and 60 min after the injection, the plasma PRL levels in the serotonin-treated animals were significantly (P < 0.01) greater than those of the PBS-treated rats.
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FIG. 2. Plasma concentrations of LH before and after the administration of serotonin or PBS to acutely castrated male rats. The left half of the figure denotes the plasma concentrations of LH in serotonin- and vehicle-treated rats measured at 10-min intervals during the 60-min preinjection period. The preinjection plasma concentrations of LH of both groups were not significantly different and were combined. The right half of the figure denotes the plasma concentrations of LH in serotonin- and vehicle-treated animals measured at 10-min intervals for 60 min after the injection of serotonin or PBS. The symbols (0 and A) and the vertical lines represent the mean plasma LH concentrations and the magnitude of the SE, respectively. Significant differences (P < 0.01) between the groups occurred at 30, 40, 50, and 60 min after the administration of the test substances. »•••©••-•
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PRE-INJECTION (n=19)
Discussion
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To ascertain whether the release of PRL was causally related to the reduction in the release of LH after serotonin treatment, we evaluated the effect of serotonin on LH release in rats pretreated with bromocriptine. During the 60-min preinjection period, the plasma PRL concentrations of the bromocriptine-treated rats varied little from the mean of 13 ng/ml (Fig. 4). During the 60-min postinjection period after the administration of serotonin or PBS to the bromocriptine-treated animals, the mean plasma PRL concentrations were 16 and 13 ng/ml, respectively. In contrast to these findings in bromocriptinetreated rats given serotonin, the plasma concentrations of LH fell steadily throughout the 20- to 60-min postinjection period and were significantly (P < 0.01) less than those in bromocriptine-treated rats given PBS.
/ N
Although it is generally acknowledged that the administration of serotonin modulates the secretion of pituitary hormones, especially LH and PRL, the mechanism by which these actions of serotonin are mediated is obscure. Since hyperprolactinemia is often associated with deBROMOCRIPTINE (n = 20)
VEHICLE (n=10) 5-HT (n«10) INJECTION
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FIG. 3. Plasma concentrations of PRL before and after the administration of serotonin or PBS to acutely castrated male rats. Significant (P < 0.01) differences between groups occurred at 30, 40, 50, and 60 min after the administration of the test substances. For further details, see Fig. 2.
PRL attained moderately steady levels of approximately 450 and 30 ng/ml, respectively, during the 60-min preinjection period. During the 10- to 60-min postinjection period, the plasma LH concentration steadily declined in the serotonin-treated rats but remained unchanged in the controls. The plasma LH concentrations at 30,40, 50, and 60 min after the administration of serotonin were significantly (P < 0.01) less than the plasma LH concentrations in rats given PBS.
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FIG. 4. Plasma concentrations of LH and PRL before and after the administration of serotonin or PBS to acutely castrated male rats pretreated with bromocriptine. The rats were treated with bromocriptine (1 mg/kg) 3 h before use. • — • , 0—0, and A—A, Plasma concentrations of LH. • • • • , © • . 0 , and A • • A, Plasma concentrations of PRL. Significant (P < 0.01) differences between the plasma concentrations of LH in the experimental and control groups occurred at 20, 30, 40, 50, and 60 min. For further details, see Fig. 2.
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878
PILOTTE AND PORTER
pressed plasma concentrations of gonadotropins in rats (9) as well as in man (3), it seems reasonable to suspect that the increase in PRL seen after the administration of serotonin is related to the concomitant decrease in circulating LH. In the present investigation, serotonin administration led to a significant release of PRL in each experimental model used, namely, intact male rats, acutely castrated male rats, and chronically castrated male rats. However, the effects of serotonin on the release of LH appear to be dependent on the animal model employed. Serotonin, administered sc, had little effect on LH release in intact male rats when the basal rate of LH release was low or in rats that had been orchiectomized for 30 days when the rate of LH release was high. It was only when LH release was examined 2 days after orchiectomy, a time when the rate of LHRH release is presumably undergoing rapid modification, that the administration of serotonin led to a significant reduction in the concentration of LH in plasma. For these reasons, the male rat which had been castrated for 2 days was employed as the model to address the question of whether the inhibitory effects of serotonin on LH release and the stimulatory effect of this indoleamine on PRL release were causally related events. To determine whether the inhibition of LH release was dependent on a marked augmentation of circulating PRL concentrations, bromocriptine was used to inhibit the serotonin-induced secretion of PRL. The plasma LH concentrations of bromocriptine-treated rats were markedly reduced in rats given serotonin compared to LH levels in vehicle-treated controls. Accordingly, the decreased release of LH induced by serotonin administration does not require a simultaneous enhancement of PRL release. Therefore, if not PRL, what is the mechanism of the inhibition of LH release by serotonin? It is unlikely that serotonin has a direct inhibitory effect on the gonadotropes, since incubation of rat pituitary glands in the presence of serotonin does not suppress the LHRH-induced release of LH (12). Serotonin may act as a neuromodulator which leads to suppression of LHRH secretion (6). It is also possible that serotonin may stimulate the secretion of an LH release-inhibiting factor. Lastly, what is the mechanism by which the administration of serotonin leads to the increased release of PRL? It is unlikely that serotonin stimulates PRL release by a direct action on the anterior pituitary gland (4). Perhaps serotonin acts as a neuromodulator substance to suppress the release of dopamine into hypophysial portal blood. It has been shown by Gudelsky and Porter (13) that a-methyltyrosine, a substance which inhibits the release of dopamine into portal blood, leads to the increased release of PRL. Hence, if serotonin were to
Endo 1979 Vol 105 « No 4
suppress dopamine release into portal blood, one could expect PRL secretion to increase. In preliminary experiments, it has been found that dopamine secretion into portal blood is depressed after serotonin administration (Pilotte, N. S., and J. C. Porter, unpublished observations). Serotonin may suppress dopamine synthesis in the tuberoinfundibular neurons, leading to decreased dopamine release into portal blood. Another possibility which cannot be excluded is that serotonin stimulates the secretion of a PRL-releasing factor. Acknowledgments The authors thank Sue Sherwin Martin, Linda Akers, and Jodie Roberts for technical assistance and lone CrandaLI and Judy Wagers for editorial assistance. The antiserum to rat PRL and the reference preparations used in the assay of PRL and LH were provided by the NIAMDD Rat Pituitary Hormone Program and Dr. A. Parlow. The antiserum to LH was a gift from Dr. G. D. Niswender. We thank Ms. K. D. Roskaz of Sandoz, Inc., for the gift of the bromocriptine.
References 1. Kamberi, I. A., R. S. Mical, and J. C. Porter, Effects of melatonin and serotonin on the release of FSH and prolactin, Endocrinology 88: 1288, 1971. 2. Lawson, D. M., and R. R. Gala, The influence of pharmacological manipulations of serotonergic and dopaminergic mechanisms on plasma prolactin in ovariectomized, estrogen-treated rats, Endocrinology 102: 973, 1978. 3. Maclndoe, J. H., and R. W. Turkington, Stimulation of human prolactin secretion by intravenous infusion of L-tryptophan, J Clin Invest 52: 1972, 1973. 4. Lamberts, S. W. J., and R. M. MacLeod, The interaction of the serotonergic and dopaminergic systems on prolactin secretion in the rat, Endocrinology 103: 287, 1978. 5. Wilson, C. A., and P. G. McDonald, Inhibitory effect of serotonin on ovulation in adult rats, J Endocrinol 60: 253, 1974. 6. Cramer, O. M., and C. A. Barraclough, The actions of serotonin, norepinephrine, and epinephrine on hypothalamic processes leading to adenohypophyseal luteinizing hormone release, Endocrinology 103: 694, 1978. 7. Schneider, H. P. G., and S. M. McCann, Dopaminergic pathways and gonadotropin releasing factors, In Bargmann, W., and B. Scharrer (eds.), Aspects of Neuroendocrinology, Springer-Verlag, Berlin, 1970, p. 177. 8. Hery, M., E. Laplante, and C. Kordon, Participation of serotonin in the phasic release of LH. I. Evidence from pharmacological experiments, Endocrinology 99: 496, 1976. 9. Winters, S. J., and D. L. Loriaux, Suppression of plasma luteinizing hormone by prolactin in the male rat, Endocrinology 102: 864, 1978. 10. Niswender, G. D., A. R. Midgley, Jr., S. E. Monroe, and L. E. Reichert, Jr., Radioimmunoassay for rat luteinizing hormone with antiovine LH serum and ovine LH-I3II, Proc Soc Exp Biol Med 128: 807, 1968. 11. Myers, J. L., Fundamentals of Experimental Design, Allyn and Bacon, Boston, 1966. 12. Martin, J. E., J. N. Engel, and D. C. Klein, Inhibition of the in vitro pituitary response to luteinizing hormone releasing hormone by melatonin, serotonin, and 5-methoxytryptamine, Endocrinology 100: 675, 1977. 13. Gudelsky, G. A., and J. C. Porter, Release of newly-synthesized dopamine into the hypophysial portal vasculature of the rat, Endocrinology 104: 583, 1979.
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