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Hypothalamic catecholamine biosynthesis and pituitary gonadotropin secretion in vitro: Effect of hyperprolactinemia
Molecular and Cellular Endocrinology, 30 ( 1983) 149- 160 Elsevier Scientific Publishers Ireland, Ltd.
149
HYPOTHALAMIC CATECHOLAMINE BIOSYNTHESIS AND PITUITARY GONADOTROPIN SECRETION IN VITRO: EFFECT OF HYPERPROLACTINEMIA * B.A. BENNETT
** and D.K. SUNDBERG
***
Department of Physiology and Pharmacology, Bowman Gray School of Medicine, Wake Forest Universrty, 300 S. Hawthorne Road, Winston -Salem, NC 27103 (U.S.A.) Received 1982
9 September
1982; revision
received
20 December
1982; accepted
20 December
The effect of hyperprolactinemia on central catecholamine biosynthesis and anterior pituitary hormone release was studied using an in vitro methodology. The incorporation of [ ‘Hltyrosine into hypothalamic and neurohypophyseal catecholamines was determined using a new method which combines high performance liquid chromatography (HPLC) with amperometric detection (LCEC). Elevated plasma prolactin levels, induced by pituitary transplants, resulted in increased in vitro biosynthesis of medial basal hypothalamic (MBH) dopamine (DA), but not norepinephrine (NE). Neurohypophyseal DA biosynthesis (including the intermediate lobe) was not affected. Plasma LH levels were depressed by hyperprolactinemia although the content of hypothalamic luteinizing hormone-releasing hormone (LHRH) was not changed. In parallel studies, the anterior pituitaries from these animals were incubated in vitro using a paired-half technique and LH and PRL release measured. While the basal release of prolactin was not altered by hyperprolactinemia, LH release was significantly decreased. Gonadotroph responsiveness to LHRH was significantly increased, while the inhibition of prolactin by dopamine was not altered. There was a decrease in pituitary prolactin content with normal LH levels. These experiments confirm several in vivo reports which show that hypothalamic dopaminergic but not noradrenergic activity is increased by prolactin. This action is specifically localized in the tuberoinfundibular dopaminergic neurons. Furthermore, these experiments suggest that these central changes result in alterations in both gonadotroph and mammotroph function. Keywords: norepinephrine; dopamine; amperometric detection; neurohypophyseal hormones and dopamine; luteinizing hormone-releasing hormone. * Supported by NIH Grants HD-10900 to D.K.S. and HL-22411 to Dr. Mariana Morris. A preliminary report of this work was reported at the 63rd Annual Meeting of the Endocrine Society, Cincinnati, Ohio, 1981. ** Current address: Department of Clinical Pharmacology: C237, University of Colorado Medical Center, 4200 East Ninth Avenue, Denver, CO 80262 (U.S.A.). *** To whom all correspondence and requests for reprints should be addressed.
0303-7207/83/0000-0000/$03.00
0 1983 Elsevier Scientific
Publishers
Ireland,
Ltd.
B.A. Bennett and D.K. Sundberg
150
It now seems well established that dopamine secreted into the hypothalamic-portal system tonically inhibits the secretion of anterior pituitary prolactin (Hokfelt and Fuxe, 1972; MacLeod, 1969; Meites and Clemens, 1972; Ben-Jonathan et al., 1977). Recent investigation has shown that elevated serum prolactin levels feedback at the hypothalamus (Clemens and Meites, 1968) and increase the turnover of tuberoinfundibular dopamine (Gudelsky et al., 1976; Hohn and Wuttke, 1978; Morgan and Herbert, 1980). These studies were performed by measuring the disappearance of hypothalamic catecholamines after inhibition of tyrosine hydroxylase. The activity of this enzyme, which is present in both dopaminergic and noradrenergic neurons, is also increased after transplant-induced hyperprolactinemia (Kreiger and Wuttke, 1980). Another study shows that pharmacologically induced increases of prolactin or prolactin-secreting tumors increase the in vitro biosynthesis of hypothalamic dopamine from [3H]tyrosine (Perkins et al., 1979). Both clinical (Quigley et al., 1980; Monroe et al., 1981) and experimental evidence (Gudelsky et al., 1976; Beck and Wuttke, 1977) show that hyperprolactinemia also results in a coincident decrease in circulating LH and altered gonadotroph responsiveness to LHRH (Monroe et al., 1981). The specific mechanism of this interaction remains elusive. While it has been suggested that hypothalamic dopamine inhibits the secretion of LHRH, several laboratories now concur that norepinephrine may be more important in stimulating its release (McCann et al., 1979). In order to elucidate some of the possible mechanisms associated with these alterations, we have investigated several neuroendocrine parameters using an in vitro methodology. Specifically we report here the effect of bilateral anterior pituitary transplants on (1) the in vitro biosynthesis of hypothalamic and neurohypophyseal catecholamines using a new technique employing reverse phase liquid chromatography with amperometric detection (Sundberg et al., 1980; Bennett and Sundberg, 1981; Morris and Sundberg, 1981) and (2) the ability of the anterior pituitary from these animals to release LH and prolactin and to respond to LHRH and dopamine in vitro.
MATERIALS
AND
METHODS
Animals and surgical procedures Adult male Sprague-Dawley rats, 125-150 g, ether and rat anterior pituitaries were bilaterally kidney capsule. Sham-operated control animals the kidneys exposed. All animals were maintained
were anesthetized with transplanted under the were anesthetized and on a 14 : 10 light : dark
Catecholamine biosynthesis in vitro and hyperprolactinemin
151
cycle with food and water ad libitum. After a minimum of 4 days post-surgery, experimental and age-matched controls were sacrificed by decapitation and the brains rapidly removed. Trunk blood was collected for LH, prolactin, vasopressin (AVP) and oxytocin (OT) measurement. Tissues studied were the medial basal hypothalamus, the anterior and the posterior pituitary. The MBH consisted of areas bounded by the postchiasmatic recess and the prernamillary recess with 1.5 mm cuts on either side of the median eminence crest. Average tissue weights were 9.8 f 0.6 mg. Anterior pituitaries were separated from the posterior lobes and hemisected. One half of each pituitary served as the control for the other experimental half. All tissues were pre-incubated for 30 min in 1 ml of Earle’s buffered salt solution, pH 7.3, at 37°C in an atmosphere of 95% 0,: 5% co,.
After preincubation, the MBH and the neurohypophysis were transferred to incubation media containing 5 PCi of [2,6-3H]L-tyrosine (30 Ci/mmole, NEN). The purity of this labeled precursor was routinely determined by reverse phase liquid chromatography. The tissues were incubated for 60 min, after which they were immediately transferred to 1 ml of 98% methanol : 0.02 N acetic acid containing 50 ng of the internal amine standard, dihydroxybenzylamine (DHBA). The tissues were weighed, homogenized and the supernatant aliquoted for measurement of tissue catecholamines (8/10) and neuropeptides (2/10). Catecholamines were alumina extracted (15 mg, BAS, Inc.) after adjustment of the pH to 8.6 with 0.7 ml of 0.5 M Tris buffer. The alumina was washed ( X 3) with distilled water and amines eluted with 50 ~1 of 0.1 N HCl. We have found that this modification of the technique of Anton and Sayres (1962) is useful for the concurrent measurement of biogenic amines and hypothalamic peptides (Sundberg et al., 1980; Morris et al., 1981). Separation of catecholamines was by reverse phase high performance liquid chromatography (C,, ~-Bondap~) with a mobile phase of 0.1 M phosphate buffer, 1 mM EDTA, pH 4.5, and 0.1 M heptane sulfonate (an ion-pairing agent). Catecholamines were quantitated by amperometric detection with an applied voltage of 0.7 V. The electrode was a TL-7A (BAS) glassy carbon which allows for sample collection of the eluant using a RE3 reference electrode (BAS). Fractions were collected after passage through the electrode and the amount of [ 3H]amine determined by liquid scintillation counting. Anterior pituitary incubation The anterior pituitaries from both the experimental
and control
rats
152
B.A. Bennert and D. K. Sundberg
were incubated using a paired-half technique. After the 30 min preincubation, the experimental halves were incubated in the presence of dopamine HCl (Sigma) or synthetic LHRH (Beckman) for 60 min. The media and tissues were then collected and processed for measurement of LH and PRL release and content. Radioimmunoassay LH, PRL, LHRH, AVP and OT were all determined by double-antibody radioimmunoassays. The LH assay used an anti-ovine LH serum obtained from Dr. G.D. Niswender (Niswender et al., 1968) and ovine LH for radiolabeling from Dr. L.E. Reichert. The assay buffer (PBS-BSA) contained 0.01 M phosphate, 0.15 M saline and 0.5% bovine serum albumin, pH 7.0. Prolactin assay reagents were obtained from the NIAMDD Pituitary Hormone Distribution Program. The PRL and LH assays contained their respective NIAMDD RP-1 standards and results are expressed as ng/ml of each standard. The potency of the standards, as measured by bioassay, was 0.03 X NIH-LH-Sl for LH and 11 IU/mg for PRL. Hypothalamic LHRH content was examined in the methanolic supernatant which was diluted and air-dried. LHRH was measured with an antiserum and a procedure previously described (Sorrentino and Sundberg, 1975). Synthetic LHRH (Bachem) was used for iodination and standards. AVP was measured with an antiserum supplied by Dr. Tj. van Wimersma Greidanus; its characteristics have been previously described (Dogterom et al., 1978; Morris et al., 1981). Oxytocin was measured with an antiserum developed and characterized in our laboratory (Morris et al., 1980). Synthetic AVP (Ferring AB) and oxytocin (Vega Biochemicals) were used for iodination and standards. Plasma levels of AVP and OT can only be measured following extraction to remove interfering substances (Robertson et al., 1973). Plasma samples (0.4 ml) were extracted with acetone according to this procedure, dried and stored at - 20°C until measurement by RIA. No correction is made for recovery which was 81.0 _+ 3.0% and 95.1 + 2.6% when 4 pg of AVP or OT, respectively, was added to pools of rat plasma. CaicuIation of data RIA results were calculated on a PDP-8 computer using a logit transformation of the raw data. Results are expressed as mean &SEM. Statistical significance of these results was obtained using several methods. Absolute changes in catecholamine biosynthesis, plasma hormone levels and responsiveness of the anterior pituitaries to DA or LHRH were determined using an unpaired Student’s r-test. The statistical signifi-
Catecholamine
biosynthesis
in vitro and hyperprolactinemia
153
cance of absolute changes in LH and PRL release induced by LHRH or DA between the control and hyperprolactinemic animals was determined by a two-way analysis of variance followed by a Scheffe’s multiple range test.
RESULTS Plasma LH and pro/a&n Bilateral transplants of rat anterior pituitaries to the kidney capsule resulted in a significant elevation of plasma PRL levels as early as 4 days after surgery (Fig. 1). This elevation persisted for up to 12 days although the magnitude was reduced. Circulating levels of LH were significantly depressed at 6 but not 4 days after transplantation. Hypothalamic catechoiamine biosynthesis Incubation of the MBH with [3H]tyrosine for 60 min resulted in significant inco~oration of this precursor into both NE and DA. In control animals, the absolute incorporation averaged 252 rt 42 and 1007 & 141 dpm/MBH for NE and DA, respectively. The incorporation was
Prolactin
Fig. 1. Plasma prolactin and LH levels at 4, 6, 8 and 12 days after anterior pituitary transplantation under the kidney capsule. Values are expressed as 5%of control + SEM and stippled area represents control + SEM. * P e 0.05.
154
B.A. Bennett and D.K. Sundberg
found to be directly related to amine content and tissue size. Therefore, the results are expressed as specific activity (fg [3H]amine/ng amine) corrected for the tissue uptake of labeled tyrosine. The MBH content of NE was not significantly different between control and hyperprolactinemic animals (2.21 + 0.40 vs. 2.06 f 0.07 ng/mg) at any of the times examined. However, 6 and 8 days after pituitary transplantation, the steady-state MBH content of DA was significantly depressed (0.88 5 0.10 vs. 0.64 f 0.01 ng/mg). The in vitro biosynthesis of NE and DA is shown in Table 1 and Fig. 2, respectively. While the incorporation of [ 3H]tyrosine into NE was never altered by pituitary transplantation, MBH dopamine synthesis was significantly elevated at all times investigated.
Hypothalamic LHRH content and release in vitro The MBH content of LHRH and the amount released into the media were measured by RIA. Although plasma LH concentration was significantly lower, neither the hypothalamic content (14.8 k 2.1 vs. 12.4 ~fr1.4 ng/MBH) nor the release during a 60 min incubation period (98 * 7.8 vs. 108 _+ 7.5 pg/MBH) was changed by these experimental elevations of circulating prolactin.
Neurohypophyseal dopamine biosynthesis Incubation of the neurohypophysis with [3H]tyrosine also resulted in its incorporation into dopamine (710 + 70 dpm/neurohypophysis); however, we were unable to show incorporation of label into NE. Table 2 shows the content and biosynthesis of neurohypophyseal DA in these experiments. Experimentally induced hyperprolactinemia had no significant effect on either parameter. In addition, plasma concentrations of the neurohypophyseal hormones vasopressin or oxytocin were not signifi-
Table
I
The in vitro biosynthesis of hypothalamic norepinephrine and those with anterior pituitary transplants Days a
Control
Hyperprolactinemic
4 6 8 12
0.18*0.05 0.28 f 0.06 0.40&0.14 0.51+0.10
0.41 0.32 0.61 0.50
Values are expressed a Days after anterior
as fg of NE synthesized pituitary transplants.
from
[ 3H]tyrosine
kO.13 + 0.03 +0.13 + 0.06 per ng of endogenous
NE.
in control
rats
155
Catecholamine biosynthesis in vitro and hyperprolactinemia
Days Post-transplant
Fig. 2. Medial basal days after pituitary ng total DA present groups. * P i 0.05,
hypothalamic DA biosynthesis was examined in vitro 4, 6, 8 and 12 transplantation. Values are expressed as fg newly synthesized DA per k SEM. Hatched bars represent hyperprolactinemic rats. n = 10 animal ** P i 0.01.
cantly altered (2.6 + 0.54 vs. 2.3 k 0.11 pg/ml 11 k 0.9 pg/ml OT).
AVP,
and
18 + 6.5 vs.
Anterior pituitary hormone release and content In order to evaluate the functional state of the anterior pituitary, glands were removed and incubated using a paired-half technique. In an
Table 2 Neurohypophyseal DA content and 12 days after transplantation Days
4 6 8 12
Dopamine
(ng/neurohypophysis) of anterior pituitaries
and biosynthesis
Dopamine
content
(dpm/ng)
biosynthesis
Control
Hyperprolactinemic
Control
Hyperprolactinemic
2.7 f 2.4 2.2kO.17 2.9 + 0.27 1.7*0.14
1.8 + 2.1 f 2.6 + 1.8 f
148+ 14 161k48 191*14 141+23
193f27 109+34 200 f 24 187+40
0.22 0.27 0.20 0.07
4, 6, 8
156
B.A. Bennerr and D. K. Sundberg
initial experiment using intact (unpaired) pituitaries we found that the basal release of LH was significantly depressed in hyperprolactinemic rats, while prolactin release was unaffected. In a reciprocal manner the anterior pituitary content of prolactin was significantly lower while LH was unchanged (Fig. 3). Although the basal release of LH was depressed by hyperprolactinemia, the percentage released when stimulated with 10 nM of LHRH was significantly greater. Fig. 4 shows the absolute release of LH (ng/ml) in control and LHRH-stimulated pituitary halves. The absolute magnitude of the response to LHRH (ng released/60 min) in both groups was not different; however, the percentage change over control was significantly increased when related to the lower basal LH release (4.0 + 0.92 vs. 12.3 + 2.1-fold increase over controls, P -c 0.01). Although the anterior pituitary content of prolactin was significantly
Prolactin 3.0 c
h
2.0
* * XI
1.0 / 0w
E 2000 -F
I
3 lOOO-
Days Post-transplant Fig. 3. Anterior pituitary content of prolactin and LH measured at 4 and 8 days after pituitary transplantation. Values are expressed as gg of prolactin or LH per anterior pituitary f SEM. Open bars are controls, hatched bars represent hyperprolactinetnic rats. * P < 0.01. Fig. 4. LH release from the anterior pituitaries of control and hyperprolactinemic (Hyp. Prl.) rats when incubated with 10 nM LHRH for 60 min. Values are expressed as pg LH released per anterior pituitary f SEM 6 days after pituitary transplantation. Stippled bars represent the LHRH-stimulated hemipituitary. Basal LH release (open bars) was significantly depressed by hyperprolactinemia (* P -z 0.05). LHRH significantly stimulated LH release (** P < 0.01) in both groups (ANOVA + Scheffe’s).
Catecholamine biosynthesis in vitro and hyperprolactinemia
157
decreased by hyperprolactinemia, neither the basal release nor the in vitro sensitivity to DA inhibition appeared to be affected. Six days after transplantation, a time when plasma LH and prolactin were significantly changed (Fig. l), the supramaximal dose of lop5 M DA caused an equal suppression of prolactin secretion in vitro (38.9 k 16.3% vs. 33.8 f 7.9% inhibition over control hemipituitaries). Since pituitary content of prolactin was also decreased, this data was calculated in terms of the DA inhibition of PRL release as percentage of content. Even in this instance DA was capable of inhibiting prolactin equally in controls and hyperprolactinemic rats.
DISCUSSION The results from these experiments confirm the hypothesis that prolactin can regulate its own release via a feedback mechanism that specifically stimulates tuberoinfundibular but not tuberohypophyseal dopaminergic activity. In this paper we describe the effects of bilateral pituitary transplants on plasma hormone levels, the pituitary responsiveness to LHRH and dopamine and the biosynthesis of NE and DA in both the MBH and neurohypophysis. These last studies were performed by a recently developed technique which utilizes high performance liquid chromatography with amperometric detection (Sundberg et al., 1980; Morris and Sundberg, 1981). These experiments demonstrate that a variety of neuroendocrine changes are induced by transplanting pituitaries under the kidney capsule. The resulting elevation of plasma prolactin levels in intact male rats coincides with a decline in circulating LH, which returns toward normal after several days. Using ovariectomized female rats, Beck and Wuttke (1977) demonstrated that hyperprolactinemia caused a similar transient inhibition of the post-castration rise of LH. Their results suggest that the gradual return of LH toward normal levels could be due to a central desensitization of DA receptors or to changes in noradrenergic activity in the preoptic area (Kreiger and Wuttke, 1980). Our experiments demonstrate that pituitary transplants consistently elevated the in vitro biosynthesis of hypothalamic DA, but not NE, at all time periods examined. Other groups, using the decline of central catecholamines after pharmacological inhibition of tyrosine hydroxylase, have reported that an increased turnover of DA is associated with elevated prolactin levels (Hokfelt and Fuxe, 1972; Gudelsky et al., 1976; Hohn and Wuttke, 1978; Morgan and Herbert, 1980). Using semiquantitative histofluorescent measurements, Wiesel et al. (1978) showed
158
B.A. Bennett and Q. K. Sundberg
that this decline was most pronounced in the lateral and medial palisade zone of the median eminence. These areas are closely associated with the hypothalamic portal capillaries and have been implicated in the control of LHRH and prolactin secretion. In fact, increased portal concentrations of DA have been reported after intraventricular injections of prolactin (Cramer et al., 1979; Gudelsky and Porter, 1980). A central site of action of elevated prolactin is suggested by other studies. Results from the laboratory of Moore and colleagues have shown that peripheral or central administration of prolactin results in an increased in vivo DA turnover after a latent period of 12-24 h (Gudelsky et al., 1976; Annunziato and Moore, 1978). They suggested that this was due to an increased synthesis of tyrosine hydroxylase, since it was obliterated by the protein synthesis inhibitor, cycloheximide (Johnston et al., 1980). Recently Kreiger and Wuttke (1980) confirmed that tyrosine hydroxylase, but not dopamine P-hydroxylase, was elevated in hyperprolactinemic rats. Since NE synthesis was not affected in our experiments, we conclude that the elevated DA synthesis in vitro represents a specific increase in tyrosine hydroxylase activity in the TIDA neurons. These studies also extend the finding of Perkins et al. (1979) who showed that pharmacological elevation of prolactin by trifluoroperazine (a DA blocker) increased hypothalamic DA synthesis in normal but not hypophysectomized rats. They were also able to show a small elevation of DA synthesis by implantation of pituitary tumors, which secrete microgram quantities of prolactin and other hormones. Their chromatographic system, however, did not allow them to examine NE biosynthesis. Early studies by Bjorklund et al. (1973) showed that the dopaminergic neurons from the anterior portion of the arcuate nucleus specifically innervate the neurointermediate lobe of the pituitary. These neurons are possibly involved in modifying neurohypophyseal or intermediate lobe hormone secretion. Because of the close association of these cell bodies with the tuberoinfundibular DA system, we examined the effects on this brain region. Elevated plasma prolactin did not alter circulating AVP or OT levels, or neurohypophyseal DA synthesis in vitro. That this system can be physiologically manipulated was shown by our earlier studies in which dehydration accelerated neurohypophyseal DA synthesis and also increased AVP and OT release (Morris and Sundberg, 1981). The present studies agree with in vivo reports which show that elevated prolactin does not effect the decline of neurohypophyseal DA after enzymatic inhibition (Morgan and Herbert, 1980; Johnston et al., 1980). Observations using other brain areas (Gudelsky et al., 1976) emphasize the marked specificity of prolactin’s action. Based on the rationale that the in vitro secretion of anterior pituitary
Catecholamine biosynthesis in vitro and hyperprolactinemia
159
hormones might reflect their prior in vivo environment, we also studied the basal and pharmacolo~cally induced release of LH and prolactin. Examination of the anterior pituitaries from the hype~rolactinemic rats showed that this manipulation Ieads to a significant reduction in proIactin content. Several studies have shown that the artificial elevation of serum prolactin leads to a decrease in content (Meites and Clemens, 1972; Cramer et al., 1979) and biosynthesis (MacLeod, 1974) of anterior pituitary prolactin. Cramer et al. (1979) suggested that this might be a direct effect of prolactin on mammotroph metabolism. Another possibib ity is that the continuous exposure of these pituitaries to DA results in a reduction in prolactin synthesis. Nonetheless, neither the basal release of prolactin nor the mammotroph’s sensitivity to DA were altered. A study of dopamine receptor sensitivity using a similar in vitro procedure revealed that an increased in~bition of prolactin release by DA is seen after 14 days in MBH-lesioned rats (Cheuug and Weiner, 1978). Dopamine receptor desensitization may require a longer time of exposure or a greater agonist concentration, On the other hand, while anterior pituitary content of LH was not changed, the basal release of LH was significantly depressed. The sensitivity to LHRH (when expressed as a percentage of control) was increased, as has been reported for h~e~rolactinemic women with decreased plasma LH levels (Spellacy et al., 1978; Monroe et al., 1981). These results suggest that the increase in TIDA activity is also coincident with an inhibition of hypothalamic LHRH secretion. In conclusion, elevation of circulating prolactin by anterior pituitary explants specifically activates tuberoinfundibular dop~ine neurons while not affecting the neurohypophyseal axis. That this perturbation also affects the h~othalamic-pituita~-~onadal axis is indicated by the decline in plasma LH and the fact that the anterior pituitary releases less LH and is more responsive to LHRH. Therefore, elevated serum prolactin levels appear to activate TIDA neurons which could result in a coincident in~bition of hypothal~ic LHRH reIease.
ACKNOWLEDGEMENTS We wish to thank Drs. Marina Morris and WK. C?‘Steen for their advice and editorial assistance. We are grateful to the NIAMDD Rat Pituitary Hormone Distribution Program for the rat PRL, to Dr. Gordon Niswender for antiserum to LH, to Dr. L.E. Reichert for LH for radiolabeling, and to Dr. Tj. B. van Wimersma Greidanus for AVP
160
antiserum. assistance
B.A. Bennett and D.K. Sundberg
The authors wish to thank Laurie Pittman and Rose Watson for her secretarial excellence.
for technical
REFERENCES Annunziato, L. and Moore, K.E. (1978) Life Sci. 22, 2037-2042. Anton, A.H. and Sayres, D.F. (1962) J. Pharmacol. Exp. Ther. 138, 360-375. Beck, W. and Wuttke, W. (1977) J. Endocrinol. 74, 67-74. Ben-Jonathan, N., Oliver, C., Weiner, H.J., Mical, R.S. and Porter. J.C. (1977) Endocrinology 100, 452-458. Bennett, B.A. and Sundberg, D.K. (1981) Life Sci. 28, 2811-2817. Bjorklund, A., Moore, R.Y., Nobin, A. and Stenevi, U. (1973) Brain Res. 5 1, 171- 191. Cheung, C.Y. and Weiner, R.I. (1978) Endocrinology 102, 1614-1620. Clemens, J.A. and Meites, J. (1968) Endocrinology 82, 878-881. Cramer, O.M., Parker, CR. and Porter, J.C. (1979) Endocrinology 105, 6366640. Dogterom, J., van Wimersma Greidanus, Tj.B. and DeWied, D. (1978) Am. J. Physiol. 234, E463-E467. Gudelsky, G.A. and Porter, J.C. (1980) Endocrinology 106, 526-529. Gudelsky, G.A., Simpkins, J., Mueller, G.P., Meites, J. and Moore, K.E. (1976) Neuroendocrinology 22, 206-2 15. Hohn, K.G. and Wuttke, W.O. (1978) Brain Res. 156, 241-252. Hokfelt, T. and Fuxe, K. (1972) Neuroendocrinology 9, 100-122. Johnston, CA., Demarest, K.T. and Moore, K.E. (1980) Brain Res. 195, 236-240. Kreiger, A. and Wuttke, W. (1980) Brain Res. 193, 173-180. MacLeod, R.M. (1969) Endocrinology 85, 916-923. MacLeod, R.M. (1974) In: Mammary Cancer and Neuroendocrine Therapy, Ed.: B.A. Stall (Butterworth, London) pp. 139-159. McCann, SM., Krulich, L. and Ojeda, S.R. (1979) In: Central Regulation of Endocrine Systems, Eds.: K. Fuxe, T. Hokfelt and R. Lufts (Plenum Press, New York) pp. 329-347. Meites, J. and Clemens, J.A. (1972) Vitam. Hormones 30, 165-221. Monroe, S.E., Levine, L., Chang, R.J., Keye, W.R. Yamamoto, M. and Jaffe R.B. (1981) J. Clin. Endocrinol. Metab. 52, 1171-l 178. Morgan, W.W. and Herbert, D.C. (1980) Neuroendocrinology 31. 215-221. Morris, M. and Sundberg, D.K. (1981) Clin. Exp. Hyper. 3, 1 l65- 1181. Morris, M., Stevens, S.W. and Adams, M.R. (1980) Biol. Reprod. 23, 782-787. Morris, M., Wren, J.A. and Sundberg, D.K. (1981) Peptides 2, 207-211. Niswender, G.D., Midgley, Jr., A.R., Monroe, S.E. and Reichert, L.E. (1968) Proc. Sot. Exp. Biol. Med. 128, 807-8 11. Perkins, N.A., Westfall. T.C.. Paul, C.V.. MacLeod, R.M. and Rogol, A.D. (1979) Brain Res. 160, 431-444. Quigley, M.E., Sheehan, K.L., Casper, R.F. and Yen, S.S.C. (1980) J. Clin. Endocrinol. Metab. 50, 427-430. Robertson, G.L., Mahr, E.A., Athar, S. and Sinha, T. (1973) J. Clin. Invest. 52, 2340-2352. Sorrentino, Jr., S. and Sundberg, D.K. (1975) Neuroendocrinology 17, 274-282. Sundberg, D.K., Bennett, B., Wendel, O.T. and Morris, M. (1980) Res. Commun. Chem. Pathol. Pharmacol. 29, 599-602. Wiesel, F.A., Fuxe, K., Hokfelt, T. and Agnati, L.F. (1978) Brain Res. 148. 399-411.
149
HYPOTHALAMIC CATECHOLAMINE BIOSYNTHESIS AND PITUITARY GONADOTROPIN SECRETION IN VITRO: EFFECT OF HYPERPROLACTINEMIA * B.A. BENNETT
** and D.K. SUNDBERG
***
Department of Physiology and Pharmacology, Bowman Gray School of Medicine, Wake Forest Universrty, 300 S. Hawthorne Road, Winston -Salem, NC 27103 (U.S.A.) Received 1982
9 September
1982; revision
received
20 December
1982; accepted
20 December
The effect of hyperprolactinemia on central catecholamine biosynthesis and anterior pituitary hormone release was studied using an in vitro methodology. The incorporation of [ ‘Hltyrosine into hypothalamic and neurohypophyseal catecholamines was determined using a new method which combines high performance liquid chromatography (HPLC) with amperometric detection (LCEC). Elevated plasma prolactin levels, induced by pituitary transplants, resulted in increased in vitro biosynthesis of medial basal hypothalamic (MBH) dopamine (DA), but not norepinephrine (NE). Neurohypophyseal DA biosynthesis (including the intermediate lobe) was not affected. Plasma LH levels were depressed by hyperprolactinemia although the content of hypothalamic luteinizing hormone-releasing hormone (LHRH) was not changed. In parallel studies, the anterior pituitaries from these animals were incubated in vitro using a paired-half technique and LH and PRL release measured. While the basal release of prolactin was not altered by hyperprolactinemia, LH release was significantly decreased. Gonadotroph responsiveness to LHRH was significantly increased, while the inhibition of prolactin by dopamine was not altered. There was a decrease in pituitary prolactin content with normal LH levels. These experiments confirm several in vivo reports which show that hypothalamic dopaminergic but not noradrenergic activity is increased by prolactin. This action is specifically localized in the tuberoinfundibular dopaminergic neurons. Furthermore, these experiments suggest that these central changes result in alterations in both gonadotroph and mammotroph function. Keywords: norepinephrine; dopamine; amperometric detection; neurohypophyseal hormones and dopamine; luteinizing hormone-releasing hormone. * Supported by NIH Grants HD-10900 to D.K.S. and HL-22411 to Dr. Mariana Morris. A preliminary report of this work was reported at the 63rd Annual Meeting of the Endocrine Society, Cincinnati, Ohio, 1981. ** Current address: Department of Clinical Pharmacology: C237, University of Colorado Medical Center, 4200 East Ninth Avenue, Denver, CO 80262 (U.S.A.). *** To whom all correspondence and requests for reprints should be addressed.
0303-7207/83/0000-0000/$03.00
0 1983 Elsevier Scientific
Publishers
Ireland,
Ltd.
B.A. Bennett and D.K. Sundberg
150
It now seems well established that dopamine secreted into the hypothalamic-portal system tonically inhibits the secretion of anterior pituitary prolactin (Hokfelt and Fuxe, 1972; MacLeod, 1969; Meites and Clemens, 1972; Ben-Jonathan et al., 1977). Recent investigation has shown that elevated serum prolactin levels feedback at the hypothalamus (Clemens and Meites, 1968) and increase the turnover of tuberoinfundibular dopamine (Gudelsky et al., 1976; Hohn and Wuttke, 1978; Morgan and Herbert, 1980). These studies were performed by measuring the disappearance of hypothalamic catecholamines after inhibition of tyrosine hydroxylase. The activity of this enzyme, which is present in both dopaminergic and noradrenergic neurons, is also increased after transplant-induced hyperprolactinemia (Kreiger and Wuttke, 1980). Another study shows that pharmacologically induced increases of prolactin or prolactin-secreting tumors increase the in vitro biosynthesis of hypothalamic dopamine from [3H]tyrosine (Perkins et al., 1979). Both clinical (Quigley et al., 1980; Monroe et al., 1981) and experimental evidence (Gudelsky et al., 1976; Beck and Wuttke, 1977) show that hyperprolactinemia also results in a coincident decrease in circulating LH and altered gonadotroph responsiveness to LHRH (Monroe et al., 1981). The specific mechanism of this interaction remains elusive. While it has been suggested that hypothalamic dopamine inhibits the secretion of LHRH, several laboratories now concur that norepinephrine may be more important in stimulating its release (McCann et al., 1979). In order to elucidate some of the possible mechanisms associated with these alterations, we have investigated several neuroendocrine parameters using an in vitro methodology. Specifically we report here the effect of bilateral anterior pituitary transplants on (1) the in vitro biosynthesis of hypothalamic and neurohypophyseal catecholamines using a new technique employing reverse phase liquid chromatography with amperometric detection (Sundberg et al., 1980; Bennett and Sundberg, 1981; Morris and Sundberg, 1981) and (2) the ability of the anterior pituitary from these animals to release LH and prolactin and to respond to LHRH and dopamine in vitro.
MATERIALS
AND
METHODS
Animals and surgical procedures Adult male Sprague-Dawley rats, 125-150 g, ether and rat anterior pituitaries were bilaterally kidney capsule. Sham-operated control animals the kidneys exposed. All animals were maintained
were anesthetized with transplanted under the were anesthetized and on a 14 : 10 light : dark
Catecholamine biosynthesis in vitro and hyperprolactinemin
151
cycle with food and water ad libitum. After a minimum of 4 days post-surgery, experimental and age-matched controls were sacrificed by decapitation and the brains rapidly removed. Trunk blood was collected for LH, prolactin, vasopressin (AVP) and oxytocin (OT) measurement. Tissues studied were the medial basal hypothalamus, the anterior and the posterior pituitary. The MBH consisted of areas bounded by the postchiasmatic recess and the prernamillary recess with 1.5 mm cuts on either side of the median eminence crest. Average tissue weights were 9.8 f 0.6 mg. Anterior pituitaries were separated from the posterior lobes and hemisected. One half of each pituitary served as the control for the other experimental half. All tissues were pre-incubated for 30 min in 1 ml of Earle’s buffered salt solution, pH 7.3, at 37°C in an atmosphere of 95% 0,: 5% co,.
After preincubation, the MBH and the neurohypophysis were transferred to incubation media containing 5 PCi of [2,6-3H]L-tyrosine (30 Ci/mmole, NEN). The purity of this labeled precursor was routinely determined by reverse phase liquid chromatography. The tissues were incubated for 60 min, after which they were immediately transferred to 1 ml of 98% methanol : 0.02 N acetic acid containing 50 ng of the internal amine standard, dihydroxybenzylamine (DHBA). The tissues were weighed, homogenized and the supernatant aliquoted for measurement of tissue catecholamines (8/10) and neuropeptides (2/10). Catecholamines were alumina extracted (15 mg, BAS, Inc.) after adjustment of the pH to 8.6 with 0.7 ml of 0.5 M Tris buffer. The alumina was washed ( X 3) with distilled water and amines eluted with 50 ~1 of 0.1 N HCl. We have found that this modification of the technique of Anton and Sayres (1962) is useful for the concurrent measurement of biogenic amines and hypothalamic peptides (Sundberg et al., 1980; Morris et al., 1981). Separation of catecholamines was by reverse phase high performance liquid chromatography (C,, ~-Bondap~) with a mobile phase of 0.1 M phosphate buffer, 1 mM EDTA, pH 4.5, and 0.1 M heptane sulfonate (an ion-pairing agent). Catecholamines were quantitated by amperometric detection with an applied voltage of 0.7 V. The electrode was a TL-7A (BAS) glassy carbon which allows for sample collection of the eluant using a RE3 reference electrode (BAS). Fractions were collected after passage through the electrode and the amount of [ 3H]amine determined by liquid scintillation counting. Anterior pituitary incubation The anterior pituitaries from both the experimental
and control
rats
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B.A. Bennert and D. K. Sundberg
were incubated using a paired-half technique. After the 30 min preincubation, the experimental halves were incubated in the presence of dopamine HCl (Sigma) or synthetic LHRH (Beckman) for 60 min. The media and tissues were then collected and processed for measurement of LH and PRL release and content. Radioimmunoassay LH, PRL, LHRH, AVP and OT were all determined by double-antibody radioimmunoassays. The LH assay used an anti-ovine LH serum obtained from Dr. G.D. Niswender (Niswender et al., 1968) and ovine LH for radiolabeling from Dr. L.E. Reichert. The assay buffer (PBS-BSA) contained 0.01 M phosphate, 0.15 M saline and 0.5% bovine serum albumin, pH 7.0. Prolactin assay reagents were obtained from the NIAMDD Pituitary Hormone Distribution Program. The PRL and LH assays contained their respective NIAMDD RP-1 standards and results are expressed as ng/ml of each standard. The potency of the standards, as measured by bioassay, was 0.03 X NIH-LH-Sl for LH and 11 IU/mg for PRL. Hypothalamic LHRH content was examined in the methanolic supernatant which was diluted and air-dried. LHRH was measured with an antiserum and a procedure previously described (Sorrentino and Sundberg, 1975). Synthetic LHRH (Bachem) was used for iodination and standards. AVP was measured with an antiserum supplied by Dr. Tj. van Wimersma Greidanus; its characteristics have been previously described (Dogterom et al., 1978; Morris et al., 1981). Oxytocin was measured with an antiserum developed and characterized in our laboratory (Morris et al., 1980). Synthetic AVP (Ferring AB) and oxytocin (Vega Biochemicals) were used for iodination and standards. Plasma levels of AVP and OT can only be measured following extraction to remove interfering substances (Robertson et al., 1973). Plasma samples (0.4 ml) were extracted with acetone according to this procedure, dried and stored at - 20°C until measurement by RIA. No correction is made for recovery which was 81.0 _+ 3.0% and 95.1 + 2.6% when 4 pg of AVP or OT, respectively, was added to pools of rat plasma. CaicuIation of data RIA results were calculated on a PDP-8 computer using a logit transformation of the raw data. Results are expressed as mean &SEM. Statistical significance of these results was obtained using several methods. Absolute changes in catecholamine biosynthesis, plasma hormone levels and responsiveness of the anterior pituitaries to DA or LHRH were determined using an unpaired Student’s r-test. The statistical signifi-
Catecholamine
biosynthesis
in vitro and hyperprolactinemia
153
cance of absolute changes in LH and PRL release induced by LHRH or DA between the control and hyperprolactinemic animals was determined by a two-way analysis of variance followed by a Scheffe’s multiple range test.
RESULTS Plasma LH and pro/a&n Bilateral transplants of rat anterior pituitaries to the kidney capsule resulted in a significant elevation of plasma PRL levels as early as 4 days after surgery (Fig. 1). This elevation persisted for up to 12 days although the magnitude was reduced. Circulating levels of LH were significantly depressed at 6 but not 4 days after transplantation. Hypothalamic catechoiamine biosynthesis Incubation of the MBH with [3H]tyrosine for 60 min resulted in significant inco~oration of this precursor into both NE and DA. In control animals, the absolute incorporation averaged 252 rt 42 and 1007 & 141 dpm/MBH for NE and DA, respectively. The incorporation was
Prolactin
Fig. 1. Plasma prolactin and LH levels at 4, 6, 8 and 12 days after anterior pituitary transplantation under the kidney capsule. Values are expressed as 5%of control + SEM and stippled area represents control + SEM. * P e 0.05.
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B.A. Bennett and D.K. Sundberg
found to be directly related to amine content and tissue size. Therefore, the results are expressed as specific activity (fg [3H]amine/ng amine) corrected for the tissue uptake of labeled tyrosine. The MBH content of NE was not significantly different between control and hyperprolactinemic animals (2.21 + 0.40 vs. 2.06 f 0.07 ng/mg) at any of the times examined. However, 6 and 8 days after pituitary transplantation, the steady-state MBH content of DA was significantly depressed (0.88 5 0.10 vs. 0.64 f 0.01 ng/mg). The in vitro biosynthesis of NE and DA is shown in Table 1 and Fig. 2, respectively. While the incorporation of [ 3H]tyrosine into NE was never altered by pituitary transplantation, MBH dopamine synthesis was significantly elevated at all times investigated.
Hypothalamic LHRH content and release in vitro The MBH content of LHRH and the amount released into the media were measured by RIA. Although plasma LH concentration was significantly lower, neither the hypothalamic content (14.8 k 2.1 vs. 12.4 ~fr1.4 ng/MBH) nor the release during a 60 min incubation period (98 * 7.8 vs. 108 _+ 7.5 pg/MBH) was changed by these experimental elevations of circulating prolactin.
Neurohypophyseal dopamine biosynthesis Incubation of the neurohypophysis with [3H]tyrosine also resulted in its incorporation into dopamine (710 + 70 dpm/neurohypophysis); however, we were unable to show incorporation of label into NE. Table 2 shows the content and biosynthesis of neurohypophyseal DA in these experiments. Experimentally induced hyperprolactinemia had no significant effect on either parameter. In addition, plasma concentrations of the neurohypophyseal hormones vasopressin or oxytocin were not signifi-
Table
I
The in vitro biosynthesis of hypothalamic norepinephrine and those with anterior pituitary transplants Days a
Control
Hyperprolactinemic
4 6 8 12
0.18*0.05 0.28 f 0.06 0.40&0.14 0.51+0.10
0.41 0.32 0.61 0.50
Values are expressed a Days after anterior
as fg of NE synthesized pituitary transplants.
from
[ 3H]tyrosine
kO.13 + 0.03 +0.13 + 0.06 per ng of endogenous
NE.
in control
rats
155
Catecholamine biosynthesis in vitro and hyperprolactinemia
Days Post-transplant
Fig. 2. Medial basal days after pituitary ng total DA present groups. * P i 0.05,
hypothalamic DA biosynthesis was examined in vitro 4, 6, 8 and 12 transplantation. Values are expressed as fg newly synthesized DA per k SEM. Hatched bars represent hyperprolactinemic rats. n = 10 animal ** P i 0.01.
cantly altered (2.6 + 0.54 vs. 2.3 k 0.11 pg/ml 11 k 0.9 pg/ml OT).
AVP,
and
18 + 6.5 vs.
Anterior pituitary hormone release and content In order to evaluate the functional state of the anterior pituitary, glands were removed and incubated using a paired-half technique. In an
Table 2 Neurohypophyseal DA content and 12 days after transplantation Days
4 6 8 12
Dopamine
(ng/neurohypophysis) of anterior pituitaries
and biosynthesis
Dopamine
content
(dpm/ng)
biosynthesis
Control
Hyperprolactinemic
Control
Hyperprolactinemic
2.7 f 2.4 2.2kO.17 2.9 + 0.27 1.7*0.14
1.8 + 2.1 f 2.6 + 1.8 f
148+ 14 161k48 191*14 141+23
193f27 109+34 200 f 24 187+40
0.22 0.27 0.20 0.07
4, 6, 8
156
B.A. Bennerr and D. K. Sundberg
initial experiment using intact (unpaired) pituitaries we found that the basal release of LH was significantly depressed in hyperprolactinemic rats, while prolactin release was unaffected. In a reciprocal manner the anterior pituitary content of prolactin was significantly lower while LH was unchanged (Fig. 3). Although the basal release of LH was depressed by hyperprolactinemia, the percentage released when stimulated with 10 nM of LHRH was significantly greater. Fig. 4 shows the absolute release of LH (ng/ml) in control and LHRH-stimulated pituitary halves. The absolute magnitude of the response to LHRH (ng released/60 min) in both groups was not different; however, the percentage change over control was significantly increased when related to the lower basal LH release (4.0 + 0.92 vs. 12.3 + 2.1-fold increase over controls, P -c 0.01). Although the anterior pituitary content of prolactin was significantly
Prolactin 3.0 c
h
2.0
* * XI
1.0 / 0w
E 2000 -F
I
3 lOOO-
Days Post-transplant Fig. 3. Anterior pituitary content of prolactin and LH measured at 4 and 8 days after pituitary transplantation. Values are expressed as gg of prolactin or LH per anterior pituitary f SEM. Open bars are controls, hatched bars represent hyperprolactinetnic rats. * P < 0.01. Fig. 4. LH release from the anterior pituitaries of control and hyperprolactinemic (Hyp. Prl.) rats when incubated with 10 nM LHRH for 60 min. Values are expressed as pg LH released per anterior pituitary f SEM 6 days after pituitary transplantation. Stippled bars represent the LHRH-stimulated hemipituitary. Basal LH release (open bars) was significantly depressed by hyperprolactinemia (* P -z 0.05). LHRH significantly stimulated LH release (** P < 0.01) in both groups (ANOVA + Scheffe’s).
Catecholamine biosynthesis in vitro and hyperprolactinemia
157
decreased by hyperprolactinemia, neither the basal release nor the in vitro sensitivity to DA inhibition appeared to be affected. Six days after transplantation, a time when plasma LH and prolactin were significantly changed (Fig. l), the supramaximal dose of lop5 M DA caused an equal suppression of prolactin secretion in vitro (38.9 k 16.3% vs. 33.8 f 7.9% inhibition over control hemipituitaries). Since pituitary content of prolactin was also decreased, this data was calculated in terms of the DA inhibition of PRL release as percentage of content. Even in this instance DA was capable of inhibiting prolactin equally in controls and hyperprolactinemic rats.
DISCUSSION The results from these experiments confirm the hypothesis that prolactin can regulate its own release via a feedback mechanism that specifically stimulates tuberoinfundibular but not tuberohypophyseal dopaminergic activity. In this paper we describe the effects of bilateral pituitary transplants on plasma hormone levels, the pituitary responsiveness to LHRH and dopamine and the biosynthesis of NE and DA in both the MBH and neurohypophysis. These last studies were performed by a recently developed technique which utilizes high performance liquid chromatography with amperometric detection (Sundberg et al., 1980; Morris and Sundberg, 1981). These experiments demonstrate that a variety of neuroendocrine changes are induced by transplanting pituitaries under the kidney capsule. The resulting elevation of plasma prolactin levels in intact male rats coincides with a decline in circulating LH, which returns toward normal after several days. Using ovariectomized female rats, Beck and Wuttke (1977) demonstrated that hyperprolactinemia caused a similar transient inhibition of the post-castration rise of LH. Their results suggest that the gradual return of LH toward normal levels could be due to a central desensitization of DA receptors or to changes in noradrenergic activity in the preoptic area (Kreiger and Wuttke, 1980). Our experiments demonstrate that pituitary transplants consistently elevated the in vitro biosynthesis of hypothalamic DA, but not NE, at all time periods examined. Other groups, using the decline of central catecholamines after pharmacological inhibition of tyrosine hydroxylase, have reported that an increased turnover of DA is associated with elevated prolactin levels (Hokfelt and Fuxe, 1972; Gudelsky et al., 1976; Hohn and Wuttke, 1978; Morgan and Herbert, 1980). Using semiquantitative histofluorescent measurements, Wiesel et al. (1978) showed
158
B.A. Bennett and Q. K. Sundberg
that this decline was most pronounced in the lateral and medial palisade zone of the median eminence. These areas are closely associated with the hypothalamic portal capillaries and have been implicated in the control of LHRH and prolactin secretion. In fact, increased portal concentrations of DA have been reported after intraventricular injections of prolactin (Cramer et al., 1979; Gudelsky and Porter, 1980). A central site of action of elevated prolactin is suggested by other studies. Results from the laboratory of Moore and colleagues have shown that peripheral or central administration of prolactin results in an increased in vivo DA turnover after a latent period of 12-24 h (Gudelsky et al., 1976; Annunziato and Moore, 1978). They suggested that this was due to an increased synthesis of tyrosine hydroxylase, since it was obliterated by the protein synthesis inhibitor, cycloheximide (Johnston et al., 1980). Recently Kreiger and Wuttke (1980) confirmed that tyrosine hydroxylase, but not dopamine P-hydroxylase, was elevated in hyperprolactinemic rats. Since NE synthesis was not affected in our experiments, we conclude that the elevated DA synthesis in vitro represents a specific increase in tyrosine hydroxylase activity in the TIDA neurons. These studies also extend the finding of Perkins et al. (1979) who showed that pharmacological elevation of prolactin by trifluoroperazine (a DA blocker) increased hypothalamic DA synthesis in normal but not hypophysectomized rats. They were also able to show a small elevation of DA synthesis by implantation of pituitary tumors, which secrete microgram quantities of prolactin and other hormones. Their chromatographic system, however, did not allow them to examine NE biosynthesis. Early studies by Bjorklund et al. (1973) showed that the dopaminergic neurons from the anterior portion of the arcuate nucleus specifically innervate the neurointermediate lobe of the pituitary. These neurons are possibly involved in modifying neurohypophyseal or intermediate lobe hormone secretion. Because of the close association of these cell bodies with the tuberoinfundibular DA system, we examined the effects on this brain region. Elevated plasma prolactin did not alter circulating AVP or OT levels, or neurohypophyseal DA synthesis in vitro. That this system can be physiologically manipulated was shown by our earlier studies in which dehydration accelerated neurohypophyseal DA synthesis and also increased AVP and OT release (Morris and Sundberg, 1981). The present studies agree with in vivo reports which show that elevated prolactin does not effect the decline of neurohypophyseal DA after enzymatic inhibition (Morgan and Herbert, 1980; Johnston et al., 1980). Observations using other brain areas (Gudelsky et al., 1976) emphasize the marked specificity of prolactin’s action. Based on the rationale that the in vitro secretion of anterior pituitary
Catecholamine biosynthesis in vitro and hyperprolactinemia
159
hormones might reflect their prior in vivo environment, we also studied the basal and pharmacolo~cally induced release of LH and prolactin. Examination of the anterior pituitaries from the hype~rolactinemic rats showed that this manipulation Ieads to a significant reduction in proIactin content. Several studies have shown that the artificial elevation of serum prolactin leads to a decrease in content (Meites and Clemens, 1972; Cramer et al., 1979) and biosynthesis (MacLeod, 1974) of anterior pituitary prolactin. Cramer et al. (1979) suggested that this might be a direct effect of prolactin on mammotroph metabolism. Another possibib ity is that the continuous exposure of these pituitaries to DA results in a reduction in prolactin synthesis. Nonetheless, neither the basal release of prolactin nor the mammotroph’s sensitivity to DA were altered. A study of dopamine receptor sensitivity using a similar in vitro procedure revealed that an increased in~bition of prolactin release by DA is seen after 14 days in MBH-lesioned rats (Cheuug and Weiner, 1978). Dopamine receptor desensitization may require a longer time of exposure or a greater agonist concentration, On the other hand, while anterior pituitary content of LH was not changed, the basal release of LH was significantly depressed. The sensitivity to LHRH (when expressed as a percentage of control) was increased, as has been reported for h~e~rolactinemic women with decreased plasma LH levels (Spellacy et al., 1978; Monroe et al., 1981). These results suggest that the increase in TIDA activity is also coincident with an inhibition of hypothalamic LHRH secretion. In conclusion, elevation of circulating prolactin by anterior pituitary explants specifically activates tuberoinfundibular dop~ine neurons while not affecting the neurohypophyseal axis. That this perturbation also affects the h~othalamic-pituita~-~onadal axis is indicated by the decline in plasma LH and the fact that the anterior pituitary releases less LH and is more responsive to LHRH. Therefore, elevated serum prolactin levels appear to activate TIDA neurons which could result in a coincident in~bition of hypothal~ic LHRH reIease.
ACKNOWLEDGEMENTS We wish to thank Drs. Marina Morris and WK. C?‘Steen for their advice and editorial assistance. We are grateful to the NIAMDD Rat Pituitary Hormone Distribution Program for the rat PRL, to Dr. Gordon Niswender for antiserum to LH, to Dr. L.E. Reichert for LH for radiolabeling, and to Dr. Tj. B. van Wimersma Greidanus for AVP
160
antiserum. assistance
B.A. Bennett and D.K. Sundberg
The authors wish to thank Laurie Pittman and Rose Watson for her secretarial excellence.
for technical
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