Gonadotropin-Releasing Hormone-Associated Peptide Exerts a ...

0013-7227/88/1231-0390$02.00/0 Endocrinology Copyright © 1988 by The Endocrine Society

Vol. 123, No. 1 Printed in U.S.A.

Gonadotropin-Releasing Hormone-Associated Peptide Exerts a Prolactin-Inhibiting and Weak GonadotropinReleasing Activity in Vivo* WEN H. YU, PETER H. SEEBURG, KAROLY NIKOLICS, AND SAMUEL M. McCANN Department of Physiology, University of Texas Health Science Center (W.H. Y., S.M.M.), Dallas, Texas 75235; and Genentech, Inc. (P.H.S., KM), San Francisco, California 94080

ABSTRACT. The in vivo effects of GnRH-associated peptide (GAP) on PRL, LH, and FSH release have been examined by injecting this peptide iv into the following types of conscious rats: 1) ovariectomized steroid-blocked females, 2) ether-stressed males, and 3) lactating females. GAP (2.4 x 10"10 and 2.4 x 10~9 mol) suppressed plasma PRL release but did not affect the levels of plasma LH and FSH in ovariectomized steroid-blocked rats. Furthermore, with 1-min etherization, GAP (1.6 X 1(T10 and 8.0 x 10~10 mol) reduced the stress-induced rise of plasma PRL, but had no effect on the stress-induced decline of plasma gonadotropin levels in male rats. A single iv injection of GAP (8.0 X 10~10 mol) into lactating rats before the onset of nursing did not block the elevation of plasma PRL induced by suckling. However, a

T

HE STRUCTURE of the precursor for LHRH has been deduced from cloned cDNA sequences derived from mRNA from human placenta (1) and human (2) and rat hypothalamus (2). Nucleotide sequence analyses showed that the LHRH precursor proteins from human placenta and hypothalamus (prepro-LHRH) were identical (2). This precursor comprises the LHRH decapeptide preceded by a signal sequence of 23 amino acids and followed by an enzymatic processing site that separates LHRH from a 56-amino acid peptide termed GnRHassociated peptide (GAP). Recently, an enzyme that yielded a C-terminal extended form of LHRH and GAP directly from the precursor protein has been discovered (3). Immunocytochemical studies showed that antigenic determinants of GAP were found in LHRH neurons and coexisted with LHRH secretory granules in nerve terminals in the median eminence of the rat (4). Furthermore, GAP possessed potent PRL release-inhibiting ac-

second injection of GAP (1.6 x 10 10 mol) at 30 min after the onset of suckling partially lowered plasma PRL levels 15 min later. By contrast, plasma FSH levels were significantly elevated by the second injection of GAP, and plasma LH also rose after iv administration of GAP in the nursing rats. These results indicate that the activity of GAP to stimulate FSH and LH release is limited, since GAP stimulated the release of FSH and LH only when plasma gonadotropin levels were extremely low. However, the in vivo evidence that GAP inhibited PRL release in a variety of conditions reinforces the possibility that GAP could be the peptidic PRL-inhibiting factor {Endocrinology 123: 390-395, 1988)

tion and stimulated the release of gonadotropins in rat pituitary cell cultures (5). These results indicate that the central control of gonadotropins and PRL may be coupled by the synthesis of a common LHRH-PRL releaseinhibiting factor (PIF) precursor from hypothalamic neurons. Since it was reported that the ovariectomized (OVX) steroid-blocked rat was the most sensitive animal for in vivo assay of LH-releasing activity (6), it was used to assay the gonadotropin-releasing activity of GAP in vivo. Plasma PRL levels were also measured in these rats. To further evaluate the PIF activity of GAP under conditions in which PRL release is enhanced, the effects of GAP on ether stress (7, 8) and suckling-induced (9-11) release of PRL were also studied. The results have appeared in part in abstract form (12). Materials and Methods Human GAP was synthesized by a modification of the procedure described previously (5). In this process, GAP is expressed in Escherichia coli as part of a fusion protein of only 75 amino acids which after CNBr cleavage generates the 56amino acid peptide. This process gives significantly higher yields and simplifies the purification of the peptide to at least 95% purity. GAP was dissolved in 0.9% NaCl (saline) at the

Received September 14,1987. Address requests for reprints to: Dr. Samuel M. McCann, Department of Physiology, University of Texas Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235. * This work was supported by NIH Grants HD-09988 and AM10079.

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EFFECT OF GAP ON PRL AND GONADOTROPIN time of experimentation. Synthetic LHRH was purchased from Sigma (St. Louis, MO). All rats (Sprague-Dawley-derived; Holtzman, Madison, WI) were housed under controlled conditions of photoperiod (lights on, 0500-1900 h) and temperature (23-25 C) with ad libitum access to food (Purina rat chow, Ralston-Purina, St. Louis, MO) and water. Exp. 1: effects of GAP in OVX steroid-blocked rats Adult female rats (160-180 g) were OVX while lightly anesthetized with ether and used for experimentation 4 weeks later. Three days before the experiment, the animals were injected sc in separate injection sites with 50 fig estradiol benzoate dissolved in 0.1 ml sesame oil and 25 mg progesterone in 0.5 ml oil (6). A Silastic catheter (Dow-Corning, Midland, MI) was implanted in the external jugular vein 2 days before the experiment (13) to facilitate blood sampling and iv injection. On the morning of sampling, an extension tubing was connected to the jugular cannula on the dorsum of the neck. After at least 1 h of acclimatization in the laboratory, an initial blood sample (0.8 ml) was withdrawn from the conscious rats. GAP (0.15,1.5, or 15 /txg in 1 ml saline, doses equal to 2.4 x 10~n, 2.4 X 10~10, and 2.4 x 10~9 mol, respectively) or synthetic LHRH (30 ng in 1 ml saline) was injected iv into each animal through the jugular cannula. The controls received an equal volume of saline. Blood samples (0.8 ml) were removed 10,40, and 70 min after iv injection. Blood volume was replaced by injecting an equal volume of warmed (37 C), heparinized (25 U/ml) saline into the animal after each sampling. All samplings were conducted between 0900 and 1100 h. Exp 2: effects of GAP in ether-stressed male rats Adult male rats (300-350 g) were implanted with jugular cannulae as described above. On the following morning, two blood samples (0.5 ml each) were withdrawn from the jugular cannula to determine the initial values of hormones. Animals were injected iv with GAP (1 or 5 ng in 1 ml saline/rat, doses equal to 1.6 X 10"10 and 8.0 X 10~10 mol) or an equal volume of saline and placed in a glass container saturated with ether for 1 min. Then, the rats were removed and allowed to recover in individual cages. Blood samples (0.5 ml) were obtained 10, 20, 30, 45, 60, and 90 min after the onset of exposure to ether. Subsequent sampling volumes were replaced with saline as described above. Exp 3: effects of GAP in lactating rats Lactating female rats with pups were obtained 5-8 days after delivery, and the number of pups was adjusted to eight per litter upon arrival. The animals were housed in individual cages. Each mother was implanted with an intraatrial cannula through the right jugular vein 2 days before sampling. All experiments were done on days 13-15 of lactation. Mothers were separated from their pups for 8 h before experimentation. Before returning the pups to the mother, initial blood samples (0.5 ml) were withdrawn and synthetic GAP (1 or 5 ng in 1 ml saline/rat) or saline was injected iv into each mother. Suckling was defined as starting when at least four pups had attached to the nipples of the dam. The average time for commencing

391

suckling was within 1 min. Blood samples (0.5 ml) were obtained frequently during the suckling period. Heparinized saline was used to replace blood volume, as described in Exp 1. To determine whether GAP could affect the already elevated plasma PRL levels in suckling rats, a second injection of GAP (1 ng) was administered iv after 30 min of suckling into mothers that had been preinjected with a 1-ng dose of GAP (14). Since plasma PRL levels decline after the cessation of suckling, only mothers that maintained a 90-min suckling period were used in this experiment. RIAs All plasma samples were run in duplicate at a single dilution, which was in the measurable range of the RIA. In Exp 1, LH was estimated by the method of Niswender et al. (15), which uses antiovine LH 15 serum (supplied by Dr. G. D. Niswender, Colorado State University), and the results were expressed in terms of the NIH LH RP-2 standard. Since plasma LH levels were very low in intact male rats and lactating rats, another LH assay, with antirat LH S4 serum and LH RP-1 standard (NIDDK), which can measure lower concentrations of plasma LH (16), was used in Exp 2 and 3. FSH and PRL were measured with the RIA kits (supplied by NIDDK), and hormone values were expressed in terms of NIH RP-1 and NIH RP-3 standards, respectively. Statistics Kruskal-Wallis nonparametric multiple comparisons were used to examine the variation of plasma hormone among the multiple groups at each time point. The nonparametric MannWhitney two-tailed U test was used to determine the difference in hormone values between two groups.

Results Exp 1: effects of GAP in OVX steroid-blocked rats To eliminate the variation of baseline among animals, plasma PRL levels were expressed as percentages of initial values. GAP (2.4 X 10"10 and 2.4 X 10"9 mol) significantly lowered plasma PRL in OVX steroidblocked rats (Fig. la). In addition to the point to point comparisons, areas under each plasma PRL curve were calculated by a computerized program (HP-85, HewlettPackard, Palo Alto, CA). Again, GAP significantly lowered the areas under the plasma PRL curves at doses of 2.4 x 10"10 and 2.4 x 10"9 mol (Fig. lb). There was no significant decline of plasma PRL at a dose of 2.4 x 10~n mol. However, plasma gonadotropin levels were not significantly changed after iv injection of GAP at any of the doses tested (Figs. 2 and 3). On the other hand, synthetic LHRH stimulated the release of LH and FSH at a low dose of 2.5 X 1CT11 mol in OVX steroid-blocked rats (Figs. 2 and 3).

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EFFECT OF GAP ON PRL AND GONADOTROPIN

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+ 120 H IO.O3ug) •—• GAP - • - * CAP — O0AP (0.13MO)

+ 100 +80 +60 +40 + 20 0 -20

70 Time After Injection ( m i n )

10

20

30 40 TIME (min)

o O Q.

70

• LHRH (O.OSjig) •GAPdSuq) »GAPH5»l)

10,000 8,000

60

FIG. 2. Lack of effect of GAP on plasma LH (ALH) in OVX steroidprimed rats. The initial plasma LH concentrations in the groups (in descending order from LHRH to saline control group: 1.63 ± 0.30,1.56 ± 0.41, 2.11 ± 0.41, 1.26 ± 0.29, and 1.45 ± 0.23 ng/ml) were not significantly different. **, P < 0.01; *•*, P < 0.001 (us. saline controls). A, Change.

B o _) a: a.

50

1 1

6,000

**

4,000

X*•*

•o C

2,000 0

A

B

C

D

FIG. 1. GAP inhibited PRL release in 0VX estrogen-progesteroneblocked rats. Percentage of initial values of plasma PRL after iv injection of GAP. A, Saline (n = 7); B, 2.4 X 10" n mol GAP (n = 6); C, 2.4 X 10"10 mol GAP (n = 6): D, 2.4 X 1(T9 mol GAP (n = 6). b, Mean areas under plasma PRL curves. GAP suppressed PRL release with doses of 2.4 x 10~10 and 2.4 x 10"9 mol. Values are expressed as the mean ± SEM. *, P < 0.05; **, P < 0.01 (us. controls).

Exp 2: effects of GAP in ether-stressed male rats In normal male rats, ether anesthesia induced a rapid elevation of plasma PRL within 10 min after removal from the ether jar (Fig. 4). Then, PRL levels declined and returned to baseline about 30 min after ether stress. Administration of GAP (1 or 5 ng, equal to 1.6 X 10~10 and 8.0 X 10~10 mol) just before etherization significantly reduced the stress-induced rise of plasma PRL 10 min after etherization (Fig. 4). However, the responses of LH and FSH release to the 1-min etherization were different in normal male rats. Plasma LH was lowered and remained at low levels until 90 min after ether stress (Table 1), whereas plasma FSH

-1004

30

40

TIME (min)

FIG. 3. Lack of effect of GAP on plasma FSH (AFSH) in OVX steroidprimed rats. The initial plasma FSH levels (in descending order from LHRH to control group: 788 ± 39, 696 ± 44, 885 ± 41, 743 ± 76, and 764 ± 38 ng/ml) were not significantly different before iv injection. ***, P < 0.001 us. saline controls. A, Change.

only showed a slight (nonsignificant) decrease 10 min after etherization, and then rebounded to its initial levels (Table 2). Since blood samples were taken 10 min after etherization, the early (within 2 min) elevation of LH and FSH reported previously (18) was not clearly seen in this experiment. GAP had no effect on plasma levels of FSH or LH in ether-stressed male rats (Tables 1 and 2). Exp 3: effects of GAP in lactating rats Suckling resulted in a dramatic increase in plasma PRL in lactating rats (Fig. 5). Injection of GAP (5 /xg/ rat; 8.0 x 10~10 mol) just before suckling had no effect

EFFECT OF GAP ON PRL AND GONADOTROPIN 160

393

TABLE 2. GAP has no effect on plasma FSH release in ether-stressed male rats

140

Saline o—o Soline (n = 7) GAP (n = 8) GAP(n=7)

120

100

80 or Q_

10 20 30 45 60 90

o

60 40

/xg GAP

5 fig GAP

7

8

7

188 ± 11

190 ± 11

225 ± 17

166 ± 7 177 ± 6 191 ± 13 183 ± 14 179 ± 12 170 ± 12

172 ± 173 ± 181 ± 168 ± 174 ± 180 ±

208 ± 201 ± 200 ± 198 ± 199 ± 192 ±

No. of rats Baseline Min after etherization

Plasma FSH (ng/ml)

7 9 12 11 7 10

15 15 15 17 20 18

Plasma FSH levels are not different between GAP-treated and saline-treated groups. Values are the mean ± SEM.

20

800

0

L

-10 0 10 20 30 45 60

75 90

700

Time (min.) FIG. 4. GAP reduced the ether stressed-induced rise of plasma PRL in male rats. The time of etherization was 1 min. Values are expressed as the mean ± SEM. *,P< 0.05; **, < 0.01 (vs. controls).

600

500

TABLE 1. GAP has no effect on the stress-induced changes in plasma LH in male rats

400

Plasma LH (ng/ml) No. of rats Baseline Min after etherization 10 20 30 45 60 90

1 Mg GAP

5 tig GAP

7

8

7

99 ± 12.0

121 ± 15.1

116 ± 18.0

2OO Suckling 100

108 ± 14.0 82 ± 6.0 69 ± 4.3° 61 ± 3.3° 55 ± 2.6" 59 ± 5.1°

106 ± 11.5 84 ±8.5 69 ± 6.8° 62 ± 5.1" 55 ± 3.9" 54 ± 4.06

101 ± 91 ± 75 ± 66 ± 65 ± 55 ±

• — • 1/xq GAP (n=7) o—o Saline (nMO)

io 300

18.0 16.2 12.9 7.2° 6.9° 4.16

Plasma LH levels are decreased about 30-60 min after etherization. LH concentrations are expressed in terms of NIH LH RP-1 standard. Values are the mean ± SEM. ° P < 0.05 us. baseline. b P < 0.01 us. baseline.

on the suckling-induced elevation of plasma PRL in lactating rats (data not shown); however, the second injection of GAP (1 Mg/rat; 1.6 X 10~10 mol) in rats receiving 1 fig GAP just before suckling partially lowered plasma PRL levels (P < 0.05), and they gradually rebounded to the original high suckling levels (Fig. 5). The concentrations of plasma gonadotropins were measured in these suckling rats. The control animals showed a tendency for plasma LH to increase about 30 min after suckling (Fig. 6), but the plasma FSH levels were unchanged after the onset of suckling (Fig. 7). However,

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I===! -20-10 0 10 20 30 45

60

75 90

Time (min.)

FIG. 5. A second iv injection of GAP (1 ng; 1.6 x 10"10 mol) partially lowered the already elevated plasma PRL level in suckling rats. The rats were injected iv with 1 fig GAP just before the onset of suckling. Values are expressed as the mean ± SEM. *, P < 0.05 vs. controls.

plasma FSH was increased (P < 0.01) after the second injection of 1 /xg GAP, and plasma LH levels were elevated (P < 0.05) after each iv injection of GAP (Figs. 6 and 7).

Discussion Previous in vitro studies showed that GAP stimulated the release of gonadotropins. The minimum effective dose to stimulate FSH release was one fourth that needed for LH release (5). To test the hypothesis that GAP might be a FSH-releasing factor (FSHRF), we injected this peptide into the OVX steroid-blocked rats that were used in both the original (17) and latest (18) studies to

EFFECT OF GAP ON PRL AND GONADOTROPIN

394

460 r 140

120

80

o 60

• — • 1/Lig GAP (n=7) o—o Saline (n=10)

E 40

Suckling 20

0L

L

i

i

i

i

-20-10 0 10 20 30

45

60

75

90

Time (min.)

FlG. 6. Effect of GAP on plasma LH release in suckling rats. Plasma LH levels were expressed in terms of NIH LH RP-1 standard. *, P < 0.05 us. controls. 120 110

100

90

80

o

I 70

• — • 1/xg GAP (n=7) o—o Saline (n=10)

60 Suckling 10 -

0

L

-20-10

0

10 20 30

45

60

75

90

Time (min.)

FIG. 7. Effect of GAP on plasma FSH release in suckling rats. The second injection of GAP increased FSH release. **, P < 0.01 us. controls.

demonstrate the existence of FSHRF. However, iv injection of GAP (up to 2.4 X 10~9 mol) did not elevate plasma levels of FSH and LH, whereas a lower dose of LHRH (2.5 x 10~n mol) stimulated the release of LH and FSH in 0VX steroid-blocked rats. These data indicate that the activity of GAP as a releaser of FSH and LH was much weaker than that of LHRH. Similarly, GAP had

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no FSH- or LH-releasing activity in the ether-stressed rats. On the other hand, the initial dose of GAP increased LH but not FSH, and a second injection of GAP increased both plasma FSH and LH release in the suckling rats. These results suggest that pulsatile challenges of GAP may facilitate FSH release in vivo. This is of special significance in view of the pulsatile secretion pattern of both LHRH and GAP (19). The discrepancy between these in vivo and the previous in vitro results may be due to the following possibilities. 1) A long period of continuous exposure may be required for the action of GAP on gonadotropins. 2) The rate of degradation of GAP may be much more rapid in vivo than in vitro. 3) GAP may affect primarily the synthesis, but not the release, of gonadotropins. Furthermore, the possibility that a fragment of GAP may stimulate the release of FSH and LH in vivo is not excluded, since it was reported that a fragment of GAP (the first 13 amino acid residues from its N-terminal) was able to stimulate the release of FSH and LH in human and baboon pituitary cell cultures (20). Under in vivo conditions, GAP may have undergone processing to fragments. We have preliminary results that support this hypothesis (Yu, W. H., R. P. Millar, and S. M. McCann unpublished data). With regard to the control of PRL secretion, GAP (1.5 and 15 ng) suppressed PRL release in OVX steroidprimed rats. Preinjection of GAP (1 or 5 /tig) in intact male rats was able to reduce the ether-induced rise of plasma PRL. However, there was no dose dependence of the inhibition of PRL release by GAP. The doses of 5 and 15 fig GAP may be above the maximal inhibitory dose in both of these assay systems. Moreover, the second injection of GAP (1 /ug) partially lowered the already elevated plasma PRL levels in lactating rats. All of these results indicate that GAP or a fragment of GAP plays a role in inhibiting PRL release in vivo. The reason that preadministration of GAP did not reduce the sucklinginduced rise of plasma PRL is unknown. It is possible that GAP cannot overcome the stimulation by PRLreleasing factors (PRFs) (21, 22) during the initiation of suckling that increase PRL levels to 20-30 times the presuckling levels. The existence of a peptidic PIF in the hypothalamus had been reported, and the activity has been partially purified (23, 24). The peptidic PIF from ovine hypothalamic extract emerged from a Sephadex G-25 column in the same fractions as LHRH, a decapeptide (25). This is not consistent with the expected migration of a 56-amino acid peptide on such a column. Therefore, it is possible that the PIF identified in column fractions may be a biologically active fragment of GAP. If a peptidic PIF shares a precursor with LHRH, this may explain the inverse relationship between the plasma

EFFECT OF GAP ON PRL AND GONADOTROPIN levels of PRL and gonadotropins that exist under various physiological conditions (26-28). However, the central control of PRL secretion is extremely complex and involves other PIFs and PRFs (21). Dopamine (DA) is a potent inhibitor of PRL release (29-31), but it cannot be solely responsible for the hypothalamic control of PRL release (21, 32). On the other hand, many PRFs, such as TRH, vasoactive intestinal peptide, and oxytocin have been reported to stimulate PRL release in certain physiological or pharmacological conditions (21, 33). In summary, GAP inhibited PRL release in vivo and exerted a weak stimulatory activity on gonadotropin release. This evidence clearly reinforces the possibility that this peptide or a fragment of it could be a physiological PIF. However, the actually secreted form of this peptide in the hypothalamo-hypophysial portal system, the contribution of GAP to PRL synthesis, its secretion in relation to other PRFs and PIFs, and comparison of the relative potencies of human and rat GAP, which share a 70% identity in amino acid sequence (2), remain for further investigation.

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releasing hormone associated peptide (GAP) suppresses prolactin release in ovariectomized steroid-blocked rats. Fed Proc 46:1063 (Abstract) Harms PG, Ojeda SR1974 A rapid and simple procedure for chronic cannulation of the rat jugular vein. J Appl Physiol 36:391 Grosvenor CE, McCann SM, Nallar R 1965 Inhibition of nursinginduced and stress-induced fall in pituitary prolactin concentration in lactating rats by injection of acid extracts of bovine hypothalamus. Endocrinology 76:883 Niswender GD, Midgley Jr AR, Monroe SE, Reichert Jr LE 1968 Radioimmunoassay for rat LH with aniovine LH serum and ovine LH-1131. Proc Soc Exp Biol Med 128:807 Urbanski HF, Urbanski D, Ojeda SR 1984 An automated system for the study of pulsatile hormone secretion in the immature rat. Neuroendocrinology 38:403 Igarashi M, McCann SM 1964 A hypothalamic FSH-releasing factor. Endocrinology 74:446 Lumpkin MD, Moltz JH, Yu WH, Samson WK, McCann SM 1987 Purification of FSH-releasing factor: its dissimilarity from LHRH of mammalian, avian, and piscian origin. Brain Res Bull 18:175 Clarke IJ, Cummins JT, Karsch FJ, Seeburg PH, Nikolics K 1987 GnRH-associated peptide (GAP) is cosecreted with GnRH into the hypophysial portal blood of ovariectomiz,ed sheep. Biochem Biophys Res Commun 143:665 Millar RP, Wormald PJ, Milton RCL 1986 Stimulation of gonadotropin release by a non-GnRH peptide sequence of the GnRH precursor. Science 232:68 Leong DA, Frawley LS, Neill JD 1983 Neuroendocrine control of prolactin secretion. Annu Rev Physiol 45:109 Samson WK, Lumpkin MD, McCann SM 1986 Evidence for a physiological role for oxytocin in the control of prolactin secretion. Endocrinology 119:554 Dhariwal APS, Grosvenor CE, Antunes-Rodrigues J, McCann SM 1968 Studies on the purification of ovine prolactin-inhibiting factor. Endocrinology 82:1236 Kuhn E, Krulich L, Fawcett CP, McCann SM 1974 The ability of hypothalamic extracts to lower blood prolactin levels in lactating rats. Proc Soc Exp Biol Med 146:104 Mizunuma H, Khorram O, McCann SM 1985 Purification of a non-dopaminergic and non-gabaergic prolactin release-inhibiting factor (PIF) in sheep stalk-median eminence. Proc Soc Exp Biol Med 178:114 Minaguchi H, Meites J 1967 Effects of suckling on hypothalamic LH-releasing factor and prolactin inhibiting factor, and on pituitary LH and prolactin. Endocrinology 80:603 Winters SJ, Troen P 1984 Altered pulsatile secretion of luteinizing hormone in hypogonadal men with hyperprolactinaemia. Clin Endocrinol (Oxf) 21:257 Klibanski A, Beitlins IZ, Merriam GR, McArthur JW, Zervas NT, Ridgway EC 1984 Gonadotropin and prolactin pulsations in hyperprolactinemic women before and during bromocriptine therapy. J Clin Endocrinol Metab 58:1141 MacLeod RM, Fontham EH, Lehmeyer JE 1970 Prolactin and growth hormone production as influenced by catecholamines and agents that affect brain catecholamines. Neuroendocrinology 6:283 Ben-Jonathan N, Oliver C, Weiner HJ, Mical RS, Porter JC 1977 Dopamine in hypophysial portal plasma of the rat during the estrous cycle and throughout pregnancy. Endocrinology 100:452 Ben-Jonathan N 1985 Dopamine: a prolactin-inhibiting hormone. Endocr Rev 6:564 Demarest KT, Riegle GD, Moore KE 1984 Adenohypophysial dopamine content during physiological changes in prolactin secretion. Endocrinology 115:2091 McCann SM, Lumpkin MD, Mizunuma H, Khorram, O, Ottlecz A, Samson WK 1984 Peptidergic and dopaminergic control of prolactin release. Trends Neurosci 7:127