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Effect of estradiol benzoate and clomiphene on tyrosine hydroxylase activity and on luteinizing hormone and prolactin levels in ovariectomized rats
Life Sciences, Vol. 29, pp. 711-716 Printed in the U.S.A.
Pergamon Press
EFFECT OF ESTRADIOL BENZOATE AND CLOMIPHENE ON TYROSINE HYDROXYLASE ACTIVITY AND ON LUTEINIZING HORMONE AND PROLACTIN LEVELS IN OVARIECTOMIZED RATS H. Tobias, L. A. Carr and J. L. Voogt Department of Pharmacology and Toxicology, University of Louisville Health Sciences Center, Louisville, Kentucky 40292 and Department of Physiology, University of Kansas Medical Center, Kansas City, Kansas 66103 (Received in final form June 9, 1981) SUMMARY The effects of estradiol benzoate (EB) on tyrosine hydroxylase (TH) activity in the medial basal hypothalamus (MBH) and on plasma levels of luteinizing hormone (LH) and prolactin were studied in long-term ovariectomized rats. Administration of i0 ~g EB produced significant elevation of TH activity on Days 1 and 3 following injection. LH levels were significantly lower than controls throughout the three day treatment period, although there was a significant increase from Day 1 to Day 2. TH activity and LH levels were inversely related throughout the experimental period. Clomiphene (15 ~g/rat/day), a purported estrogen antagonist, was administered over a period of three days to control and EB-treated rats to determine whether the effect of EB on plasma LH levels was causally related to changes in TH activity. In rats receiving both EB and clomiphene, TH activity was lower and plasma LH was higher than after EB alone. The results support the hypothesis that the feedback effects of estradiol on LH release involve an action on the tuberoinfundibular dopaminergic (TIDA) neurons of the MBH and that clomiphene can oppose the inhibitory effect of estradiol on LH release by directly inhibiting TIDA neuron activity. Furthermore, EB-induced release of prolactin does not appear to involve detectable changes in the activity of TIDA neurons. Noradrenergic and dopaminergic neurons terminating in the hypothalamus are among the postulated sites for the feedback effects of estradiol on LH and prolactin release (i-3). Several parameters of catecholamine neuron activity, such as transmitter content (4,5), turnover (6,7) and synthesis enzyme activity (8), have been studied with a variety of experimental manipulations to determine the role of these neurons in the feedback effects of estradiol on the release of these hormones. However, not all of these studies have been in agreement. For example, Luine et al. (2) observed a decrease in tyrosine hydroxylase (TH) activity in the mediobasal hypothalamus (MBH) following administration of EB to ovariectomized rats whereas Beattie et al. (8) found a significant increase. Differences in dose, duration of treatment and areas of hypothalamus studied may account for the disparate observations (5). Furthermore, few attempts have been made to determine simultaneous changes in the activity of catecholamine synthesis enzymes and in plasma levels of LH and prolactin. The present investigation involved the effects of estradiol and an antiestrogen, clomiphene, on the activity of tyrosine hdyroxylase in the MBH and on 0024-3205/81/070711-06502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
712
Estradiol and Clomiphene on TH and LH
Vol. 29, No. 7, 1981
plasma levels of LH and prolactin in adult ovariectomized rats. MATERIALS AND METHODS Animals Mature female Sprague-Dawley rats weighing 180-200 g (Laboratory Supply Co., Indianapolis, Indiana) were housed 5 to a cage in a temperature-controlled room (25 + 2oc) with lights on from 5 am until 7 pm daily. Water and Purina Rat Chow were supplied ad libitum. They were ovariectomized under ether anesthesia and used 3-4 weeks after surgery. Experimental procedure Ovariectomized animals were injected subcutaneously with either i0 ~g EB (Sigma) in 0.i ml peanut oil or peanut oil alone at 9 am (Day 0). Groups of rats were killed by decapitation at 2, 4, and 6 pm on each of the following 3 days. Trunk blood was collected for radioimmunoassays of LH and prolactin. Following centrifugation, the plasma was frozen until analyzed. Immediately after decapitation, the brain was removed and dissected on a cold aluminum block under a dissecting microscope. The severed end of the pituitary stalk was held with a fine forceps and 2 parallel cuts were made in an anterior direction lateral to the portal vessels, using the lateral limits of the infundibular recess as a reference. The rostral border of the medial basal hypothalamus was taken as the area where the portal vessels were no longer visible and a cut was made there. This tissue includes all of the median eminence and part of the pituitary stalk and areuate nucleus. This method is similar to the one used by Chiocchio et al. (9). The superficial mediobasal hypothalamus (i0) (approx. avg. protein=50 ~g) was sonicated in 75 ~i of Tris-Triton buffer (pH 6.0) with a Kontes cell disrupter. The tissue sonicate was centrifuged at i0,000 X g for I0 minutes. Ten ~i of the supernatant was removed for protein determination (ii). In a second experiment, enclomiphene citrate (Merrell National Laboratories), the trans isomer of clomiphene, was dissolved in 0.9% saline and 10 ~g was injected subcutaneously in 0.i ml at 9 am on 3 successive days. Control animals received 0. i ml of the vehicle. The animals received either 10 ~g EB subcutaneously or the oil vehicle at 9 am on the second day of clomiphene or saline treatment. The animals were killed on the third day (first day after EB or oil) at 2 pm. Blood collection and tissue dissection were carried out as described above except that the sonicate volume was 80 ~i. Assay of tyrosine hydroxylase (TH) Tyrosine hydroxylase activity was determined by slight alteration of the method of Coyle (12) as modified by Saavedra et al. (13). To 25 ~i of tissue supernatant were added 85 ~I of incubation mixture containing i0 ~i bovine catalase (ii00 U, Sigma Chemical Co.), i0 ~I I0 mM ferrous ammonium sulfate, i0 ~i TPNH (i0 mM, Sigma Chemical Co.), i0 D1 sheep liver dihydropteridine reductase, i0 ~i 1 M potassium phosphate buffer (pH 5.5), 25 ~i Tris-Triton buffer (pH 6) and i0 ~i 1.8 mM tyrosine containing 1 ~Ci 2,6 3H tyrosine (Amersham, 37 Ci/mmole). The reaction was initiated by the addition of I0 ~I DL-6-methyl 5,6,7,8 tetrahydropterine (Calbiochem, 6.4 mM). After 45 min of incubation, 3H DOPA was isolated on alumina and counted by liquid scintillation spectrometry. Endogenous catecholamines were assumed to have no effect on the assay of enzyme activity due to dilution by the incubation medium. Assay of luteinizin$ hormone and prolactin Trunk blood was obtained following decapitation and the plasma separated from the cells and stored at -20oc for subsequent assay. Each plasma sample was assayed in duplicate or triplicate for prolactin and LH by the radioimmuno-
Vol. 29, No. 7, 1981
Estradiol and Clomiphene on TH and LH
713
assay methods of Niswender et al. (14). The antiovine LH serum for the LH assay (GND-15) was kindly provided by Dr. Gordon Niswender and the ovine LH for iodination (LER-1056 C2) was kindly provided by Dr. Leo Reichert. The remainder of the assay materials was provided by the NIAMDD Hormone Distribution Program. The reference preparations used were NIAMDD rat prolactin-RP-1 and NIAMDD rat LH-RP-I. Data analysis Enzyme activity data and hormone data were analyzed by one-way analysis of variance and by Student's t-test. RESULTS Effect of EB on TH~ LH and prolactin No significant differences among the three time periods were observed for TH, LH or prolactin on any day of the experiment. Thus, the data were combined within each day for further comparison. Plasma levels of LH were significantly decreased on each of the 3 days after treatment with EB (Table I). On Day 2, LH levels were significantly higher than on Day i, although they were still lower than oil-treated controls. Plasma levels of prolactin increased on Days i and 2 and remained elevated on Day 3. TH activity in the MBH was significantly elevated on Days I and 3 compared with oil-treated controls. Activity on Day 2 was significantly lower than on Day i. TABLE I Effect of estradiol benzoate on TH activity in the MBH and on plasma levels of LH and prolactin
Days following treatment
Control
TH (nmoles DOPA/mg protein/hr)
3.32+0.39
(26)
LH (ng/ml)
627+63
prolactin (ng/ml)
(39)
15+ i (31)
1
6.79+0.85a(18)
243+21 a (20)
52+ 8a(23)
2
3.14!0.68b(24)
418+_63ab(28)
181+32a(22)
3
5.33_+0.88a(21)
299!38 a (22)
143+39a(15)
Rats were administered I0 ~g EB at 9 am on Day 0. Enzyme activity and plasma hormone levels were determined in samples obtained during the afternoon of Days 1-3. Each value represents the mean + S.E. Numbers in parentheses refer to number of animals. aSignificantly different from control (p<.05) bSignificantly different from Day 1 (p<.05) Interaction of EB and clomiphene on TH~ LH and prolactin Another study was carried out with clomiphene citrate, a purported estradiol antagonist, to determine whether the increase in TH activity was involved in the EB-induced decrease of plasma LH. Clomiphene was administered to rats which were also treated with EB or oil. Clomiphene alone significantly decreased TH activity compared to oil-treated rats (Table II). Plasma levels in these animals were significantly higher than in controls (972 vs 621 ng/ml). When EB was administered to rats which also received clomiphene, TH activity was not different from controls. Neither the increase in TH activity previously observed with EB alone nor the decrease in TH activity caused by clomiphene
714
Estradiol and Clomiphene on TH and LH
Vol. 29, No. 7, 1981
alone was observed. Furthermore, plasma LH levels following combined treatment with EB and clomiphene were significantly higher than after EB alone and significantly lower than after clomiphene alone. Clomiphene did not alter the EB-induced increase in plasma prolactin. TABLE II Effects of clomiphene and estradiol benzoate on TH and activity in the MBH and on plasm~ LH and prolactin
Treatment
TH (nmoles DOPA/mg protein/hr)
LH (ng/ml)
Control
4.61+0.67
(8)
621+ 57
EB
7.18+1.38
(6)
197+ 21 a (16)
26+ 3 a ( 8 )
1.70+0.49 a (5)
972+i04 a ( 5 )
29+12
3.28+0.34bc(5)
320~ 4 2 b c ( 6 )
31+ 9 ( 6 )
Clomiphene Clomiphene + EB
(17)
prolactin (ng/ml)
15+ 1 (ii)
(4)
Rats were administered 15 Dg clomiphene or saline vehicle for 3 consecutive days at 9 am. EB or oil was given at 9 am on the second day of clomiphene treatment. The rats were killed at 2:00 pm on Day 3. The data for the control and EB-treated groups are from those animals in Table I which were killed at 2:00 pm. Each value represents the mean + 1 S . E . Numbers in parentheses refer to number of animals. aSignificantly different from control (p<.05) bSignificantly different from clomiphene (p<.05) CSignificantly different from EB (p<.05) DISCUSSION In agreement with several other investigators (15-17), EB suppressed plasma LH levels one day after administration of a single dose. Surges of LH have been shown to occur beginning in the afternoon of the first through third day in some, but not all, rats following administration of EB (15,17). In the present study, a modest elevation of LH levels did occur during the afternoon on Day 2 but not on Day 3. Throughout the three-day period following EB treatment there was a consistent inverse relationship between plasma LH levels and TH activity in the MBH. It has been demonstrated that TH activity in various areas of the central nervous system, including the hypothalamus, is closely linked to the rates of catecholamine utilization and release (18). Although the present study does not distinguish between TH localized in dopaminergic neurons and that in noradrenergic neurons, there is ample documented evidence that TH in this area is contained almost entirely in dopaminergic neurons (19) and that changes in TH activity in the median eminence parallel changes in dopamine turnover (20). In addition, it has been shown previously that changes in TH and dopamine-Bhydroxylase activities occur independently during the rat estrus cycle (21). These findings indicate that determination of TH activity in the MBH provides a useful index of dopamine synthesis and release in this region. This approach appears to have distinct advantages over methods used to measure dopamine turnover rates since it avoids the use of large doses of a-methyl-ptyrosine which can indirectly alter plasma hormone levels (20) with subsequent feedback effects on catecholamine neurons (22). Therefore, the increase in TH
Vol. 29, No. 7, 1981
Estradiol and Clomiphene on TH and LH
715
activity which occurred one day after administration of EB supports the hypotheses that dopaminergic neurons terminating in the median eminence inhibit LH release (23) and that EB stimulates neurotransmitter synthesis and utilization in these neurons (24,25). Such a mechanism is further supported by the present study when the results on Day I are compared to those of Day 2. LH levels were significantly increased on Day 2 compared to Day 1 whereas TH activity was decreased. Fuxe et al. (25) have proposed that the effect of estradiol on dopamine turnover is mediated by an estrogen receptor in the MBH which may be located on dopaminergic cell bodies. The lack of any effect of estrogen on dopamine turnover (26) or TH activity (2) in the nigral-striatal system suggests that the effect of estrogen on dopaminergic neurons may not be a generalized observation in brain tissue, but may be localized in theMBH. It has been suggested that the increase in dopaminergic turnover induced by estrogen may be mediated by increased release of prolactin (26). However, this mechanism is not supported by others (23,27) nor by the present study, since prolactin continued to increase over the first two days, whereas TH activity decreased from Day 1 to Day 2. There does not appear to be a correlation between TH activity and prolactin release during the three-day period. Thus, the stimulatory effect of EB on prolactin is apparently not related to dopaminergic neuron activity but may be mediated instead by norepinephrine (28). Schally et al. (29) have treated ovariectomized rats with both enclomiphene and zuclomiphene (trans and cis isomers, respectively) to determine their effects on LH release. Enclomiphene generally caused a further increase in plasma LH levels in these rats, whereas zuclomiphene decreased LH levels. These results and those of the present study suggest that enclomiphene, which is considered an estrogen antagonist, may actually stimulate LH release directly. Such an action would be distinct from its ability to block estrogen receptors (30,31). The latter mechanism would be unlikely in rats ovariectomized for 3-4 weeks since estrogen titers are presumably minimal. A direct action of clomiphene is also indicated by its effects on TH activity. Treatment with clomiphene alone caused a significant decrease in activity, in contrast to the effect seen with EB. These results further suggest that the actions of both EB and clomiphene on LH release may be mediated by dopaminergic neurons: EB-induced stimulation of neuronal activity (increased TH activity) leads to decreased LH release and clomiphene-induced inhibition of neuronal activity (decreased TH activity) allows increased LH release. The opposing actions of EB and clomiphene on TH activity indicate that they may be acting on separate, but possibly allosterically linked sites on dopaminergic neurons. Whether or not this is true, treatment with both drugs appeared to attenuate the effects of each drug alone on TH activity and on LH release. Further studies are required to determine whether luteinizing hormone releasing hormone (LHRH) is involved in this interaction. ACKNOWLEDGEMENTS This study was supported by Public Health Service Grant HD 11922. The authors wish to thank Ms. Yie-Jane Wu for her excellent technical assistance and Dr. W. L. Albrecht of Merrell National Laboratories for providing enclomiphene citrate. REFERENCES i. 2. 3. 4.
W. V. N. W.
LADOSKY and D. E. WANDSCHEER, J. Steroid Biochem. 6 1013-1020 (1975). N. LUINE, B. S. MCEWEN and I. B. BLACK, Brain Res. 120 188-192 (1977). I. MUNARO, Acta endocr., Copenh. 86 235-242 (1977). R. CROWLEY, T. L. O'DONOHUE and D. M. JACOBOWITZ, Brain Res. 147
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Estradiol and Clomiphene on TH and LH
Vol. 29, No. 7, 198]
30.
315-326 (1978). E. ENDROCZI and G. SZABO, Acta physiol, hung. 43 43-48 (1973). A. O. CONOSO, W. BISHOP, C. P. FAWCETT, L. KRULICH and S.M. MCCANN, Endocrinology 8 9 774-784 (1971). K. G. HOHN, P. WITTE and W. WUTTKE, Acta endocr., Copenh. Suppl. 215 103 (1978). C. W. BEATTIE, C. H. RODGERS and L. F. SOYKA, Endocrinology 9 1 276-279 (1972). S. R. CHIOCCHIO, M. A. CANNATA and J. H. TRAMEZZANI, Brain Res. Ii0 612-618 (1976). A. KAVANAGH and J. WEISZ, Neuroendocrinology 13 201-212 (1973/1974). O. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR and R. J. RANDALL, J. biol. Chem. 193 265-275 (1951). J. T. COYLE, Biochem. Pharmac. 21 1935-1944 (1972). J. M. SAAVEDRA, M. BROWNSTEIN, M. PALKOVITS, S. KIZER and J. AXELROD, J. Neurochem. 23 869-871 (1974). G. D. NISWENDER, A. R. MIDGELY, JR., S. E. MONROE and L. E. REICHERT, JR., Proc. Soc. exp. Biol. Med. 128 807-811 (1968). C. A. BLAKE, Endocrinology 101 1122-1129 (1977). L. CALIGARIS, J. J. ASTRADA and S. TALEISNIK, Endocrinology 88 810-815 (1971). S. J. LEGAN, V. L. GAY and A. R. MIDGELY, JR., Endocrinology 93 781-785 (1973). N. G. BACOPOULOS and R. K. BHATNAGAR, J. Neurochem. 29 639-643 (1977). J. S. KIZER, E. MUTH and D. M. JACOBOWITZ, Endocrinology 9 8 886-893 (1976). G. NICHOLSON, G. H. GREELEY, J. H UMM, W. W. YOUNGBLOOD and J. S. KIZER, Brain Res. 190 447-457 (1980). L. A. CARR and J. L. VOOGT, Brain Res. 196 437-445 (1980). G. A. GUDELSKY, J. SIMPKINS, G. P. MUELLER, J. MEITES and K. E. MOORE, Neuroendocrinology 22 206-215 (1976). K. FUXE, A. LOFSTROM--~ P. ENEROT~, J-~. GUSTAFSSON, P. SKETT, T. HOKFELT, F-A. WIESEL and L. AGNATI, Psychoneuroendocrinology 2 203-225 (1977). A. LOFSTROM, P. ENEROTH, J-~. GUSTAFSSON and P. SKETT, Endocrinology i01 1559-1569 (1977). F-A. WIESEL, K. FUXE, T. HOKFELT and L. F. AGNATI, Brain Res. 148 339411 (1978). D. C. EIKENBURG, A. J. RAVITZ, G. A. GUDELSKY and K. E. MOORE, J. Neural Transm. 40 235-244 (1977). J. S. KIZ~R, J. HUMM, G° NICHOLSON, G. GREELEY and W. YOUNGBLOOD, Brain Res. 146 95-107 (1978). L. A. CARR, P. M. CONWAY and J. L. VOOGT, Brain Res. 133 305-314 (1977). A. V. SCHALLY, W. H. CARTER, A. F. PARLOW, M. SAITO, A. ARIMURA, C. Y. BOWERS and D. E. HOLTKAMP, Am. J. Obstet. Gynec. 107 1156-1167 (1970). J. KATO, T. KOBAYASHI and C. A. VILLEE, Endocrinology 8 2 1049-1052
31.
(1968). R. MAURER a n d D. WOOLLEY, E n d o c r i n o l o g y
5. 6. 7. 8. 9. I0. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.
8_88 1 2 8 1 - 1 2 8 7
(1971).
Pergamon Press
EFFECT OF ESTRADIOL BENZOATE AND CLOMIPHENE ON TYROSINE HYDROXYLASE ACTIVITY AND ON LUTEINIZING HORMONE AND PROLACTIN LEVELS IN OVARIECTOMIZED RATS H. Tobias, L. A. Carr and J. L. Voogt Department of Pharmacology and Toxicology, University of Louisville Health Sciences Center, Louisville, Kentucky 40292 and Department of Physiology, University of Kansas Medical Center, Kansas City, Kansas 66103 (Received in final form June 9, 1981) SUMMARY The effects of estradiol benzoate (EB) on tyrosine hydroxylase (TH) activity in the medial basal hypothalamus (MBH) and on plasma levels of luteinizing hormone (LH) and prolactin were studied in long-term ovariectomized rats. Administration of i0 ~g EB produced significant elevation of TH activity on Days 1 and 3 following injection. LH levels were significantly lower than controls throughout the three day treatment period, although there was a significant increase from Day 1 to Day 2. TH activity and LH levels were inversely related throughout the experimental period. Clomiphene (15 ~g/rat/day), a purported estrogen antagonist, was administered over a period of three days to control and EB-treated rats to determine whether the effect of EB on plasma LH levels was causally related to changes in TH activity. In rats receiving both EB and clomiphene, TH activity was lower and plasma LH was higher than after EB alone. The results support the hypothesis that the feedback effects of estradiol on LH release involve an action on the tuberoinfundibular dopaminergic (TIDA) neurons of the MBH and that clomiphene can oppose the inhibitory effect of estradiol on LH release by directly inhibiting TIDA neuron activity. Furthermore, EB-induced release of prolactin does not appear to involve detectable changes in the activity of TIDA neurons. Noradrenergic and dopaminergic neurons terminating in the hypothalamus are among the postulated sites for the feedback effects of estradiol on LH and prolactin release (i-3). Several parameters of catecholamine neuron activity, such as transmitter content (4,5), turnover (6,7) and synthesis enzyme activity (8), have been studied with a variety of experimental manipulations to determine the role of these neurons in the feedback effects of estradiol on the release of these hormones. However, not all of these studies have been in agreement. For example, Luine et al. (2) observed a decrease in tyrosine hydroxylase (TH) activity in the mediobasal hypothalamus (MBH) following administration of EB to ovariectomized rats whereas Beattie et al. (8) found a significant increase. Differences in dose, duration of treatment and areas of hypothalamus studied may account for the disparate observations (5). Furthermore, few attempts have been made to determine simultaneous changes in the activity of catecholamine synthesis enzymes and in plasma levels of LH and prolactin. The present investigation involved the effects of estradiol and an antiestrogen, clomiphene, on the activity of tyrosine hdyroxylase in the MBH and on 0024-3205/81/070711-06502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
712
Estradiol and Clomiphene on TH and LH
Vol. 29, No. 7, 1981
plasma levels of LH and prolactin in adult ovariectomized rats. MATERIALS AND METHODS Animals Mature female Sprague-Dawley rats weighing 180-200 g (Laboratory Supply Co., Indianapolis, Indiana) were housed 5 to a cage in a temperature-controlled room (25 + 2oc) with lights on from 5 am until 7 pm daily. Water and Purina Rat Chow were supplied ad libitum. They were ovariectomized under ether anesthesia and used 3-4 weeks after surgery. Experimental procedure Ovariectomized animals were injected subcutaneously with either i0 ~g EB (Sigma) in 0.i ml peanut oil or peanut oil alone at 9 am (Day 0). Groups of rats were killed by decapitation at 2, 4, and 6 pm on each of the following 3 days. Trunk blood was collected for radioimmunoassays of LH and prolactin. Following centrifugation, the plasma was frozen until analyzed. Immediately after decapitation, the brain was removed and dissected on a cold aluminum block under a dissecting microscope. The severed end of the pituitary stalk was held with a fine forceps and 2 parallel cuts were made in an anterior direction lateral to the portal vessels, using the lateral limits of the infundibular recess as a reference. The rostral border of the medial basal hypothalamus was taken as the area where the portal vessels were no longer visible and a cut was made there. This tissue includes all of the median eminence and part of the pituitary stalk and areuate nucleus. This method is similar to the one used by Chiocchio et al. (9). The superficial mediobasal hypothalamus (i0) (approx. avg. protein=50 ~g) was sonicated in 75 ~i of Tris-Triton buffer (pH 6.0) with a Kontes cell disrupter. The tissue sonicate was centrifuged at i0,000 X g for I0 minutes. Ten ~i of the supernatant was removed for protein determination (ii). In a second experiment, enclomiphene citrate (Merrell National Laboratories), the trans isomer of clomiphene, was dissolved in 0.9% saline and 10 ~g was injected subcutaneously in 0.i ml at 9 am on 3 successive days. Control animals received 0. i ml of the vehicle. The animals received either 10 ~g EB subcutaneously or the oil vehicle at 9 am on the second day of clomiphene or saline treatment. The animals were killed on the third day (first day after EB or oil) at 2 pm. Blood collection and tissue dissection were carried out as described above except that the sonicate volume was 80 ~i. Assay of tyrosine hydroxylase (TH) Tyrosine hydroxylase activity was determined by slight alteration of the method of Coyle (12) as modified by Saavedra et al. (13). To 25 ~i of tissue supernatant were added 85 ~I of incubation mixture containing i0 ~i bovine catalase (ii00 U, Sigma Chemical Co.), i0 ~I I0 mM ferrous ammonium sulfate, i0 ~i TPNH (i0 mM, Sigma Chemical Co.), i0 D1 sheep liver dihydropteridine reductase, i0 ~i 1 M potassium phosphate buffer (pH 5.5), 25 ~i Tris-Triton buffer (pH 6) and i0 ~i 1.8 mM tyrosine containing 1 ~Ci 2,6 3H tyrosine (Amersham, 37 Ci/mmole). The reaction was initiated by the addition of I0 ~I DL-6-methyl 5,6,7,8 tetrahydropterine (Calbiochem, 6.4 mM). After 45 min of incubation, 3H DOPA was isolated on alumina and counted by liquid scintillation spectrometry. Endogenous catecholamines were assumed to have no effect on the assay of enzyme activity due to dilution by the incubation medium. Assay of luteinizin$ hormone and prolactin Trunk blood was obtained following decapitation and the plasma separated from the cells and stored at -20oc for subsequent assay. Each plasma sample was assayed in duplicate or triplicate for prolactin and LH by the radioimmuno-
Vol. 29, No. 7, 1981
Estradiol and Clomiphene on TH and LH
713
assay methods of Niswender et al. (14). The antiovine LH serum for the LH assay (GND-15) was kindly provided by Dr. Gordon Niswender and the ovine LH for iodination (LER-1056 C2) was kindly provided by Dr. Leo Reichert. The remainder of the assay materials was provided by the NIAMDD Hormone Distribution Program. The reference preparations used were NIAMDD rat prolactin-RP-1 and NIAMDD rat LH-RP-I. Data analysis Enzyme activity data and hormone data were analyzed by one-way analysis of variance and by Student's t-test. RESULTS Effect of EB on TH~ LH and prolactin No significant differences among the three time periods were observed for TH, LH or prolactin on any day of the experiment. Thus, the data were combined within each day for further comparison. Plasma levels of LH were significantly decreased on each of the 3 days after treatment with EB (Table I). On Day 2, LH levels were significantly higher than on Day i, although they were still lower than oil-treated controls. Plasma levels of prolactin increased on Days i and 2 and remained elevated on Day 3. TH activity in the MBH was significantly elevated on Days I and 3 compared with oil-treated controls. Activity on Day 2 was significantly lower than on Day i. TABLE I Effect of estradiol benzoate on TH activity in the MBH and on plasma levels of LH and prolactin
Days following treatment
Control
TH (nmoles DOPA/mg protein/hr)
3.32+0.39
(26)
LH (ng/ml)
627+63
prolactin (ng/ml)
(39)
15+ i (31)
1
6.79+0.85a(18)
243+21 a (20)
52+ 8a(23)
2
3.14!0.68b(24)
418+_63ab(28)
181+32a(22)
3
5.33_+0.88a(21)
299!38 a (22)
143+39a(15)
Rats were administered I0 ~g EB at 9 am on Day 0. Enzyme activity and plasma hormone levels were determined in samples obtained during the afternoon of Days 1-3. Each value represents the mean + S.E. Numbers in parentheses refer to number of animals. aSignificantly different from control (p<.05) bSignificantly different from Day 1 (p<.05) Interaction of EB and clomiphene on TH~ LH and prolactin Another study was carried out with clomiphene citrate, a purported estradiol antagonist, to determine whether the increase in TH activity was involved in the EB-induced decrease of plasma LH. Clomiphene was administered to rats which were also treated with EB or oil. Clomiphene alone significantly decreased TH activity compared to oil-treated rats (Table II). Plasma levels in these animals were significantly higher than in controls (972 vs 621 ng/ml). When EB was administered to rats which also received clomiphene, TH activity was not different from controls. Neither the increase in TH activity previously observed with EB alone nor the decrease in TH activity caused by clomiphene
714
Estradiol and Clomiphene on TH and LH
Vol. 29, No. 7, 1981
alone was observed. Furthermore, plasma LH levels following combined treatment with EB and clomiphene were significantly higher than after EB alone and significantly lower than after clomiphene alone. Clomiphene did not alter the EB-induced increase in plasma prolactin. TABLE II Effects of clomiphene and estradiol benzoate on TH and activity in the MBH and on plasm~ LH and prolactin
Treatment
TH (nmoles DOPA/mg protein/hr)
LH (ng/ml)
Control
4.61+0.67
(8)
621+ 57
EB
7.18+1.38
(6)
197+ 21 a (16)
26+ 3 a ( 8 )
1.70+0.49 a (5)
972+i04 a ( 5 )
29+12
3.28+0.34bc(5)
320~ 4 2 b c ( 6 )
31+ 9 ( 6 )
Clomiphene Clomiphene + EB
(17)
prolactin (ng/ml)
15+ 1 (ii)
(4)
Rats were administered 15 Dg clomiphene or saline vehicle for 3 consecutive days at 9 am. EB or oil was given at 9 am on the second day of clomiphene treatment. The rats were killed at 2:00 pm on Day 3. The data for the control and EB-treated groups are from those animals in Table I which were killed at 2:00 pm. Each value represents the mean + 1 S . E . Numbers in parentheses refer to number of animals. aSignificantly different from control (p<.05) bSignificantly different from clomiphene (p<.05) CSignificantly different from EB (p<.05) DISCUSSION In agreement with several other investigators (15-17), EB suppressed plasma LH levels one day after administration of a single dose. Surges of LH have been shown to occur beginning in the afternoon of the first through third day in some, but not all, rats following administration of EB (15,17). In the present study, a modest elevation of LH levels did occur during the afternoon on Day 2 but not on Day 3. Throughout the three-day period following EB treatment there was a consistent inverse relationship between plasma LH levels and TH activity in the MBH. It has been demonstrated that TH activity in various areas of the central nervous system, including the hypothalamus, is closely linked to the rates of catecholamine utilization and release (18). Although the present study does not distinguish between TH localized in dopaminergic neurons and that in noradrenergic neurons, there is ample documented evidence that TH in this area is contained almost entirely in dopaminergic neurons (19) and that changes in TH activity in the median eminence parallel changes in dopamine turnover (20). In addition, it has been shown previously that changes in TH and dopamine-Bhydroxylase activities occur independently during the rat estrus cycle (21). These findings indicate that determination of TH activity in the MBH provides a useful index of dopamine synthesis and release in this region. This approach appears to have distinct advantages over methods used to measure dopamine turnover rates since it avoids the use of large doses of a-methyl-ptyrosine which can indirectly alter plasma hormone levels (20) with subsequent feedback effects on catecholamine neurons (22). Therefore, the increase in TH
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activity which occurred one day after administration of EB supports the hypotheses that dopaminergic neurons terminating in the median eminence inhibit LH release (23) and that EB stimulates neurotransmitter synthesis and utilization in these neurons (24,25). Such a mechanism is further supported by the present study when the results on Day I are compared to those of Day 2. LH levels were significantly increased on Day 2 compared to Day 1 whereas TH activity was decreased. Fuxe et al. (25) have proposed that the effect of estradiol on dopamine turnover is mediated by an estrogen receptor in the MBH which may be located on dopaminergic cell bodies. The lack of any effect of estrogen on dopamine turnover (26) or TH activity (2) in the nigral-striatal system suggests that the effect of estrogen on dopaminergic neurons may not be a generalized observation in brain tissue, but may be localized in theMBH. It has been suggested that the increase in dopaminergic turnover induced by estrogen may be mediated by increased release of prolactin (26). However, this mechanism is not supported by others (23,27) nor by the present study, since prolactin continued to increase over the first two days, whereas TH activity decreased from Day 1 to Day 2. There does not appear to be a correlation between TH activity and prolactin release during the three-day period. Thus, the stimulatory effect of EB on prolactin is apparently not related to dopaminergic neuron activity but may be mediated instead by norepinephrine (28). Schally et al. (29) have treated ovariectomized rats with both enclomiphene and zuclomiphene (trans and cis isomers, respectively) to determine their effects on LH release. Enclomiphene generally caused a further increase in plasma LH levels in these rats, whereas zuclomiphene decreased LH levels. These results and those of the present study suggest that enclomiphene, which is considered an estrogen antagonist, may actually stimulate LH release directly. Such an action would be distinct from its ability to block estrogen receptors (30,31). The latter mechanism would be unlikely in rats ovariectomized for 3-4 weeks since estrogen titers are presumably minimal. A direct action of clomiphene is also indicated by its effects on TH activity. Treatment with clomiphene alone caused a significant decrease in activity, in contrast to the effect seen with EB. These results further suggest that the actions of both EB and clomiphene on LH release may be mediated by dopaminergic neurons: EB-induced stimulation of neuronal activity (increased TH activity) leads to decreased LH release and clomiphene-induced inhibition of neuronal activity (decreased TH activity) allows increased LH release. The opposing actions of EB and clomiphene on TH activity indicate that they may be acting on separate, but possibly allosterically linked sites on dopaminergic neurons. Whether or not this is true, treatment with both drugs appeared to attenuate the effects of each drug alone on TH activity and on LH release. Further studies are required to determine whether luteinizing hormone releasing hormone (LHRH) is involved in this interaction. ACKNOWLEDGEMENTS This study was supported by Public Health Service Grant HD 11922. The authors wish to thank Ms. Yie-Jane Wu for her excellent technical assistance and Dr. W. L. Albrecht of Merrell National Laboratories for providing enclomiphene citrate. REFERENCES i. 2. 3. 4.
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