The effects of dopamine on prolactin mRNA levels in rat pituitary cells in culture.

Dopamine is known to be the prolactin-release inhibiting factor, but the effects of dopamine itself on regulation of prolactin messenger RNA have been little studied because of the instability of dopamine. We have compared the effects of dopamine and bromocriptine on the levels of prolactin mRNA and on the rates of synthesis, storage, and release of prolactin in primary cultured rat pituitary cells. The cells were incubated for 72 h with no secretagogue (control group) or in the presence of either dopamine (10 mumol/L) plus ascorbic acid (100 mumol/L) or bromocriptine (0.1 mumol/L). Prolactin mRNA was measured in cell extracts by means of slot blots, and newly synthesized prolactin was measured in similar incubations by the addition of [3H]leucine, followed by gel electrophoresis. The levels of total prolactin were measured by radioimmunoassay. Prolactin mRNA was reduced to 78 +/- 9% (mean +/- SEM) of control levels in bromocriptine-treated cells and to 59 +/- 7% in dopamine-treated cells, demonstrating that dopamine stabilized by ascorbic acid was able to reduce the levels of prolactin mRNA in rat pituitary cells in culture. Dopamine may act at sites in addition to the dopaminergic D2 receptor, since the level of prolactin mRNA was reduced more by a supramaximal dose of dopamine than by a supramaximal dose of bromocriptine. The results of the [3H]prolactin and prolactin measurements suggested that availability of mRNA was not a major factor in controlling the rate of prolactin synthesis.     

The effect of dopamine on the release of prolactin in sheep with lesions of the hypothetical centre producing prolactin inhibiting factor (PIF)

It has been demonstrated in our previous papers that in the anterior part of medial basal hypothalamus (AM BH) in sheep a stimulating, while in the caudal part of MBH (CMBH) an inhibiting centre of prolactin release are situated. These results suggested that CMBH might be the site of PIF production and prompted us to investigate the effect of dopamine (DA) on the concentration of prolactin in the peripheral blood (p.bl.) in animals in which CMBH had been previously lesioned and this concentration was very high. Microinfusion of L-dopamine into the third cerebral ventricle (c.v.) or into the internal maxillary artery in intact as well as in lesioned lactating ewes depressed distinctly the prolactin level in the p.bl. This action of DA suggests that in the CMBH exists dopaminergic system which itself plays an inhibitory role in the control of prolactin release without involvement of PIF.     

Restricted exposure of mice to primer pheromones coincident with prolactin surges blocks pregnancy by changing hypothalamic dopamine release

Exposure of recently mated female mice to strange male urine revealed that exposure for 8 h was sufficient to produce pregnancy block providing exposure is for two 4-h periods coincident with prolactin surges. Exposure for 8 h between prolactin surges or one 4-h exposure coincident with either the nocturnal or the diurnal prolactin surge was without effect. When bromocriptine, a dopamine agonist, was given coincident with the nocturnal and diurnal prolactin surges, it was equally effective, but the opiate antagonist (naltrexone) administered in a similar manner was without effect. This result indicates that pheromonal action is through excitation of the tuberoinfundibular neurones rather than by inhibition of beta-endorphin neurones. Further evidence for dopamine involvement in pregnancy block is demonstrated by showing DOPA accumulation in the medio-basal hypothalamus following exposure to male urinary pheromones after dihydroxybenzylhydrazine (DHBH) administration, which blocks the enzyme DOPA-decarboxylase. Taken together, this series of experiments provides convincing evidence for the dopamine inhibition of prolactin release being the final pathway for pheromone action in the context of pregnancy block.     

Effects of dopamine on the release of thyrotropin-releasing hormone from the rat retina in vitro.

The effects of dopamine on the release of thyrotropin-releasing hormone (TRH) from the rat retina in vitro were studied. The rat retina was incubated in the medium 199 (pH 7.4) with 1.0 mg/ml of bacitracin and 100 micrograms/ml of ascorbic acid. The amount of TRH release into the medium was measured by radioimmunoassay. The TRH release from the rat retina was inhibited significantly in a dose-related manner with the addition of dopamine, but not with pimozide. The inhibitory effects of dopamine on TRH release from the rat retina were blocked with an addition of pimozide to the medium. The elution profile of methanol-extracted rat retina on sephadex G-10 was identical to that of synthetic TRH. From these findings it is concluded that the dopaminergic system inhibits TRH release from the rat retina in vitro.     

Effect of atrial natriuretic peptide on the release of beta-endorphin from rat hypothalamo-neurohypophysial complex

The aim of this study was to determine whether atrial natriuretic peptide (ANP) alters beta-endorphin (beta-END) secretion from rat intermediate pituitary and whether this effect is a direct action on the intermediate pituitary or an indirect one mediated by hypothalamic factor(s). We studied the release of beta-END from rat neuro-intermediate lobes of the pituitary (NIL) and from the hypothalamo-neurohypophysial complex (HNC), which consists of the hypothalamus, pituitary stalk, intermediate and posterior lobes of the pituitary, by means of an in vitro perifusion system. NIL and HNC were prepared from male Wistar rats and individually perifused for 30 min with perifusion medium followed by 20 min perifusion with medium containing alpha-rat ANP and/or dopamine (DA). Samples of perifusion medium were collected every 5 min and subjected to RIA for beta-END. The basal release of beta-END from NIL was 180% of that from HNC (p less than 0.01), which provides further support for the presence of hypothalamic factors that inhibit beta-END release from the intermediate pituitary. The perifusion of HNC with ANP at 10(-7) and 10(-6) M increased the beta-END concentration by 25 and 50%, respectively (p less than 0.01). In contrast, ANP (10(-8) to 10(-6) M) had no effect on beta-END release from NIL. The inhibitory effect of DA (10(6) M) on beta-END release from NIL and HNC (51% and 50% of the basal release, respectively, p less than 0.01) was confirmed. However, this inhibitory effect was not reversed by ANP.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of estradiol on pituitary responsiveness to dopamine in vitro: a comparison of ovariectomized Fischer 344 and Holtzman rats.

The objective of this study was to determine if the effectiveness of dopamine as an inhibitor of prolactin is altered by estradiol in strains of rats which show marked differences in estrogen-induced pituitary hyperplasia. Groups of Fischer 344 and Holtzman Sprague-Dawley rats were ovariectomized and implanted with Silastic capsules of estradiol. Rats were sacrificed by rapid decapitation following a brief period of ether anesthesia at 2, 4, 6, 8 weeks (F-344) or at 2 and 8 weeks (Holtzman) of estradiol treatment. The pituitary was removed and cut into fragments which were either snap frozen for initial prolactin content measurements or incubated for 60 min in the presence or absence of dopamine (1 x 10(-6) M). Prolactin was measured in the plasma, in sonicates of the pituitary and in the incubation medium by double antibody radioimmunoassay. Pituitary weight and plasma levels of prolactin were significantly less in Holtzman rats compared to Fischer 344 females at 2 or 8 weeks of estradiol treatment but pituitary concentrations of prolactin were not different between the two strains. Pituitary fragments from Fischer 344 rats studied at 2 and 4 weeks of estradiol treatment did not respond to the removal of dopamine in vitro whereas pituitary fragments from Holtzman rats obtained at 2 weeks of estradiol treatment did release significantly more prolactin in the absence than in the presence of dopamine. Pituitary fragments taken from Fischer 344 rats at 6 and 8 weeks were responsive to dopamine whereas pituitary tissue from Holtzman rats was not responsive at 8 weeks. The data indicate that temporal differences in responsiveness to the inhibitory effects of dopamine occur in strains which are susceptible or resistant to the formation of pituitary tumors following prolonged estradiol treatment.     

Evidence that pimozide is not a partial agonist of dopamine receptors.

The dopamine receptor antagonist pimozide, at concentrations up to 10 nM, competitively antagonized the inhibitory action of a pomorphine on prolactin (PRL) secretion by cultured rat pituitary cells. At higher concentrations pimozide as well as the analogues clopimozide and penfluridol suppressed PRL secretion. The latter effect could not be reversed by dopamine antagonists devoid of intrinsic effects on PRL release. Suppression of PRL release was also observed with compounds which were devoid of dopamine receptor agonistic or antagonistic properties such as R 6694 and R 5052, structurally related to pimozide, and also with loperamide. The inhibitory action of pimozide on PRL release resembled that of the calcium antagonist flunarizine. Concentration effect curves showed parallel slopes and the effect of both compounds could be reversed by increasing the concentration of calcium ions (Ca²⁺). Both flunarizine and pimozide were also capable of inhibiting releasing factor-stimulated luteinizing hormone secretion, an effect not shared by apomorphine. Pimozide and the various structurally related compounds used, also antagonized Ca²⁺-induced smooth muscle contractions of the isolated caudal artery of the rat.The present findings indicate that pimozide is a competitive antagonist without partial agonistic activity on apomorphine-sensitive dopamine receptors in the pituitary and that its inhibitory effect on PRL release as well as on vascular smooth muscle contractions is due to interference with a Ca²⁺-dependent mechanism of the stimulus-effect coupling process.     

Both dopamine and the putative dopamine D3 receptor antagonist PNU-99194A induce a biphasic inhibition of phorbol ester-stimulated arachidonic acid release from CHO cells transfected with the dopamine D3 receptor

In Chinese hamster ovary (CHO) cells transfected with the cDNA for the dopamine D3 receptor, low concentrations of dopamine (IC50: 0.5 nM) counteracted the release of arachidonic acid (AA) induced by the protein kinase C activator TPA (maximal inhibition: 15% at 10 - 30 nM). The effect of dopamine -- which was antagonized by pretreatment with pertussis toxin (PTX) or by the dopamine receptor antagonist haloperidol -- was biphasic; thus, at increasing concentrations of dopamine (100 nM - 1 microM), AA levels approached baseline. The preferential dopamine D3 receptor ligand PNU-99194A displayed an effect similar to that of dopamine; thus, whereas low concentrations of PNU-99194A (IC50: 1.9 nM) reduced TPA-induced AA release (maximal inhibition: 15% at 30 - 100 nM), higher concentrations (> or =1 microM) were ineffective. When dopamine and PNU-99194A were administered together at concentrations yielding maximal inhibition of AA release, no additive effect was observed; moreover, a high concentration of dopamine counteracted the AA-reducing effect of a low concentration of PNU-99194A and vice versa. It is suggested that D3 receptors in transfected CHO cells may exert mainly an inhibitory, but also a stimulatory influence on TPA-induced AA release, and that PNU-99194A acts as an agonist in this system.     

Oral cadmium exposure throughout puberty does not inhibit secretion of prolactin, GH and ACTH through dopamine metabolism changes in male rat

This work analyzed possible dopamine-mediated cadmium effects on plasma prolactin, GH and ACTH levels and if these changes were related to metal accumulation. Male rats were treated from day 30 to 60 of life with 50 mg/L of CdCl₂ in the drinking water. Cadmium exposure decreased the dopamine (DA) metabolism (DOPAC/DA ratio) in all brain areas studied, and plasma levels of prolactin, GH and ACTH were diminished. The cadmium concentration did not increase nor in hypothalamus nor in the pituitary after the metal exposure. These results suggest that cadmium inhibits the secretion of these pituitary hormones and this inhibitory effect is not mediated by dopamine or the degree of metal accumulation.     

Purification of a non-dopaminergic and non-GABAergic prolactin release-inhibiting factor (PIF) in sheep stalk-median eminence

Prolactin release-inhibiting factor (PIF) extracted from 1200 sheep stalk-median eminences was purified by gel filtration on a Sephadex G-25 column (4.5 X 150 cm). PIF activity was determined by measuring the inhibition of prolactin release from dispersed anterior pituitary cells of adult male or estrogen-primed, ovariectomized rats. Using this system, PIF was detected in tube fractions 122-127 (volume = 20 ml/tube). These fractions also contained LHRH and somatostatin; however, these peptides had no prolactin-inhibiting activity in the quantities present. No dopamine or gamma-aminobutyric acid (GABA) was detected in the active fractions by radioenzymatic assay and fluorophotoenzymatic assay, respectively. Furthermore, receptor blockers for dopamine or GABA did not interfere with the PIF activity. These findings indicate that the PIF activity cannot be attributed to either dopamine or GABA, both of which are known to inhibit prolactin release, and provide evidence for the presence of a non-dopaminergic and non-GABAergic PIF within the hypothalamus.     

Effects of dopamine on prolactin secretion and cyclic AMP accumulation in the rat anterior pituitary gland

The effects of dopamine on pituitary prolactin secretion and pituitary cyclic AMP accumulation were studied by using anterior pituitary glands from adult female rats, incubated in vitro. During 2h incubations, significant inhibition of prolactin secretion was achieved at concentrations between 1 and 10nm-dopamine. However, 0.1–1μm-dopamine was required before a significant decrease in pituitary cyclic AMP content was observed. In the presence of 1μm-dopamine, pituitary cyclic AMP content decreased rapidly to reach about 75% of the control value within 20min and there was no further decrease for at least 2h. Incubation with the phosphodiesterase inhibitors theophylline (8mm) or isobutylmethylxanthine (2mm) increased pituitary cyclic AMP concentrations 3- and 6-fold respectively. Dopamine (1μm) had no effect on the cyclic AMP accumulation measured in the presence of theophylline, but inhibited the isobutylmethylxanthine-induced increase by 50%. The dopamine inhibition of prolactin secretion was not affected by either inhibitor. Two derivatives of cyclic AMP (dibutyryl cyclic AMP and 8-bromo cyclic AMP) were unable to block the dopamine (1μm) inhibition of prolactin secretion, although 8-bromo cyclic AMP (2mm) significantly stimulated prolactin secretion and both compounds increased somatotropin (growth hormone) release. Cholera toxin (3μg/ml for 4h) increased pituitary cyclic AMP concentrations 4–5-fold, but had no effect on prolactin secretion. The inhibition of prolactin secretion by dopamine was unaffected by cholera toxin, despite the fact that dopamine had no effect on the raised pituitary cyclic AMP concentration caused by this factor. Dopamine had no significant effect on either basal or stimulated somatotropin secretion under any of the conditions tested. We conclude that the inhibitory effects of dopamine on prolactin secretion are probably not mediated by lowering of cyclic AMP concentration, although modulation of the concentration of this nucleotide in some other circumstances may alter the secretion of the hormone.     

Effect of the GABAA agonist muscimol on prolactin secretion from human prolactin-secreting adenomas and GH3 rat pituitary tumour cells.

The effect of muscimol, a specific potent GABAA receptor agonist, on prolactin release from human prolactin-secreting tissue was investigated using a perifusion system. Perifusion studies on normal rat anterior pituitary tissue, which has identical GABA receptors to those found in normal human pituitary glands, show that muscimol has a specific biphasic effect on prolactin release. This is characterized by an initial transient stimulation (222.3 +/- 21.6% of basal) lasting for 5-10 min followed by a more prolonged inhibitory phase (63.9 +/- 3.1% inhibition of basal). Five human prolactin-secreting adenomas were studied, and in none of the tumours could a biphasic response be demonstrated. One of the prolactin-secreting adenomas had a blunted inhibitory response, but the other 4 showed no inhibitory effect of muscimol on prolactin release. Muscimol had no significant effect on basal or thyrotropin-releasing-hormone (TRH)-stimulated prolactin secretion from GH3 rat pituitary tumour cells. These studies suggest that the GABAergic effect on prolactin secretion is absent or altered in both rat and human prolactin-secreting tumour cells.     

Interaction between opiates and hypothalamic dopamine on prolactin release.

Opiate stimulation of prolactin (PRL) release appears to involve a hypothalamic mechanism(s). The present study utilized both central acting drugs and direct measurement of hypothalamic dopamine (DA) to investigate this problem. Administration of L-dopa, the precursor of DA; piribedil, a DA agonist; or amineptine, a DA reuptake inhibitor, each decreased serum PRL concentrations. Morphine sulfate (MS) and haloperidol (HAL) significantly increased serum PRL levels. L-dopa and piribedil reversed the stimulatory effect of MS on serum PRL concentrations by increasing dopamine activity. MS blocked the inhibitory effects of amineptine on serum PRL release, possibly by decreasing the concentration of DA available for reuptake. Injection of subeffective doses of HAL concurrently with a subeffective dose of MS increased serum PRL concentrations, by an additive inhibitory action on dopaminergic activity. β-endorphin, an endogenous opioid peptide, decreased the rate of DA turnover in the median eminence, and increased serum PRL levels approximately 10 - fold. These observations indicate that opiates stimulate PRL release by decreasing DA activity in the median eminence.     

Ascorbic acid and Mg-ATP co-regulate dopamine beta-monooxygenase activity in intact chromaffin granules

Ascorbic acid and Mg-ATP were found to regulate norepinephrine biosynthesis in intact secretory vesicles synergistically and specifically, using the model system of isolated bovine chromaffin granules. Dopamine uptake into chromaffin granules was shown to be unrelated to the presence of Mg-ATP and ascorbic acid at external dopamine concentrations of 7.5 and 10 mM. Under these conditions of dopamine uptake, norepinephrine biosynthesis was enhanced 5-6-fold by Mg-ATP and ascorbic acid compared to control experiments with dopamine only. Furthermore, norepinephrine formation was enhanced approximately 3-fold by ascorbic acid and Mg-ATP together compared to norepinephrine formation in granules incubated with either substance alone. The action of Mg-ATP and ascorbic acid together was synergistic and independent of dopamine content of chromaffin granules as well as of dopamine uptake. The apparent Km of norepinephrine formation for external ascorbic acid was 376 microM and for external Mg-ATP was 132 microM, consistent with the larger amounts of cytosolic ascorbic acid and ATP that are available to chromaffin granules. Other physiologic reducing agents were not able to increase norepinephrine biosynthesis in the presence or absence of Mg-ATP. In addition, maximum enhancement of norepinephrine biosynthesis occurred only with the nucleotide ATP and the cation magnesium. The mechanism of the effect of ascorbic acid and Mg-ATP on norepinephrine biosynthesis was investigated and appeared to be independent of a positive membrane potential. The effect was also not mediated by direct action of ADP, ATP, or magnesium on the activity of soluble or particulate dopamine beta-monooxygenase. These data indicate that Mg-ATP and ascorbic acid specifically and synergistically co-regulate dopamine beta-monooxygenase activity in intact chromaffin granules, independent of substrate uptake. Although the mechanism is not known, the data are consistent with the possibility that the chromaffin granule ATPase mediates these effects.     

In vitro release of prolactin by thyrotropin-releasing hormone: influence of dopamine, thyroxine, and cycloheximide.

An acute incubation procedure, using explanted normal rat hemipituitaries pretreated with fresh plasma obtained from pituitary donor animals, was employed to further investigate the in vitro stimulation of prolactin (PRL release by thyrotropin-releasing hormone (TRH). Pretreatment with dopamine (0.1 microgram/ml) caused a 30-50% decrease in the amount of PRL released into incubation media; the inhibitory effect of dopamine was not reversed by treatment with 0.5-6.0 ng. TRH, although these TRH concentrations consistently stimulated PRL release from pituitaries not exposed to dopamine. Treatment with thyroxine (10(-6) to 10(-5) M) showed a competitive inhibition of thyrotropin release by TRH (0.5 ng), but was without effect on TRH-stimulated PRL release. Cycloheximide (100 microgram/ml) blocked a net increase in PRL levels. TRH, nevertheless, significantly increased PRL release in the presence of cycloheximide. The results indicate that neither dopamine nor thyroxine compete with TRH in causing PRL release, and that the TRH stimulation of PRL release is unrelated to ongoing levels of hormone synthesis.     

Failure of 2 Hydroxyestradiol to interact with dopamine inhibition of human prolactin secretion in vitro and with dopamine receptors of prolactin-secreting adenomas

The ability of 2-Hydroxyestradiol, a catecholestrogen, and 17 beta Estradiol to interact with the dopamine inhibition of prolactin and with dopamine receptors has been tested on dispersed human prolactin-secreting cells obtained from ten pituitary adenomas. There is a 80% inhibition of prolactin secretion obtained by addition of dopamine in a superfusion system. This inhibition is not affected by preexposure to the steroids, or by their introduction into the perifusion medium. Moreover 2 Hydroxyestradiol and 17 beta Estradiol do not interact with the binding of 3H Domperidone to DA receptors.     

Catecholamine mechanisms in the feedback effects of estradiol benzoate on the release of LH and prolactin

The role of hypothalamic catecholamines and luteinizing hormone releasing hormone (LHRH) in the negative feedback effect of estradiol benzoate (EB) on luteinizing hormone (LH) release was studied in chronic ovariectomized rats. Administration of 10 micrograms EB decreased plasma LH levels and increased LHRH content in the medial basal hypothalamus (MBH) 1 day after injection. Inhibition of dopamine and norepinephrine synthesis with alpha-methyl-p-tyrosine (alpha-MT) reduced the LHRH content in the MBH in both oil- and EB-treated animals and partially reversed the decrease in plasma LH levels. Inhibition of norepinephrine synthesis with fusaric acid decreased LHRH content in both oil- and EB-treated rats but had no effect on plasma LH levels. The results suggest that at least a portion of the inhibitory effect of EB on LH release is due to the stimulation of an inhibitory dopaminergic mechanism which reduces LHRH release from the MBH. This feedback mechanism is apparently not susceptible to dopaminergic receptor blockade since administration of pimozide had no effect on LH levels. The stimulatory feedback effect of EB on prolactin release was studied in the same animals. alpha-MT and EB produced additive effects on plasma prolactin levels whereas fusaric acid blocked the EB-induced increase in plasma prolactin levels. Pimozide appeared to potentiate the effect of EB on prolactin release. The results reconfirm the possible role of noradrenergic neurons in the release of prolactin induced by EB and also suggest that EB stimulates a dopaminergic mechanism which is inhibitory to prolactin release but is normally masked by increased noradrenergic activity.     

Ascorbic acid enables reversible dopamine receptor 3H-agonist binding

The effects of ascorbic acid on dopaminergic ³H-agonist receptor binding were studied in membrane homogenates of bovine anterior pituitary and caudate, and rat striatum. In all tissues virtually no stereospecific binding (defined using luM (+)butaclamol) of the ³H-agonists N-propylnorapomorphine (NPA), apomorphine, or dopamine could be demonstrated in the absence of ascorbic acid. Although levels of total ³H-agonist binding were three to five times greater in the absence than in the presence of 0.1% ascorbic acid, the increased binding was entirely non-stereospecific. Greater amounts of dopamine-inhibitable ³H-NPA binding could be demonstrated in the absence of 0.1% ascorbic acid, but this measure of “specific binding” was demonstrated not to represent dopamine receptor binding since several other catecholamines and catechol were equipotent with dopamine and more potent than the dopamine agonist (±)amino-6,7-dihydroxy-1,2,3,4-tetrahydronapthalene (ADTN) in inhibiting this binding. High levels of dopamine-displaceable ³H-agonist binding were detected in fresh and boiled homogenates of cerebellum, an area of brain which receives no dopaminergic innervation, further demonstrating the non-specific nature of ³H-agonist binding in the absence of ascorbic acid. These studies emphasize that under typical assay conditions ascorbic acid is required in order to demonstrate reversible and specific ³H-agonist binding to dopamine receptors.     

Dopamine Release in Rat Striatum: Physiological Coupling to Tyrosine Supply

Intracerebral microdialysis was used to monitor dopamine release in rat striatal extracellular fluid following the intraperitoneal administration of dopamine's precursor amino acid, L-tyrosine. Dopamine concentrations in dialysates increased transiently after tyrosine (50-100 mg/kg) administration. Pretreatment with haloperidol or the partial lesioning of nigrostriatal neurons enhanced the effect of tyrosine on dopamine release, and haloperidol also prolonged this effect. These data suggest that nigrostriatal dopaminergic neurons are responsive to changes in precursor availability under basal conditions, but that receptor-mediated feedback mechanisms limit the magnitude and duration of this effect.