Opioid and dopamine involvement in prolactin release induced by arginine vasotocin and vasopressin in the male rat

The potential involvement of the endogenous opioid and dopamine (DA) systems in the mechanism(s) mediating arginine vasotocin (AVT)- and arginine vasopressin (AVP)-induced prolactin (PRL) release was investigated in vivo. The injection of AVT (5 micrograms) into unanesthetized male rats resulted in a 2-fold stimulation of PRL release 15 min later, followed by an inhibition of PRL release 30 min thereafter; both the stimulatory and inhibitory PRL responses to AVT were obviated by naloxone (NAL) (200 micrograms). Similarly, the administration of either AVT or AVP (5 micrograms) to urethane-anesthetized rats led to a 3- and 5-fold increase in plasma PRL levels, respectively, 10 min after injection. The PRL stimulatory response to both peptides was completely blocked by pretreating the animals with apomorphine (APO) (5 mg); however, the injection of APO by itself had no effect on PRL secretion in these animals. Both AVT and AVP were also effective in stimulating PRL release 10 min after injection in estrogen (50 micrograms)-progesterone (25 mg) (EP)-treated rats anesthetized with urethane. APO negated the PRL stimulatory response to these compounds in the EP-treated rat as well. Normal, urethane-treated rats experienced a 7- to 8-fold increase in PRL levels 20 min following the injection of methysergide (MET) (250 micrograms). Both AVT and AVP caused approximately a 2.5-fold greater PRL response in MET-treated animals than in AVT and AVP controls, respectively; however, only in the MET + AVT-treated rats was the PRL stimulatory response greater than in the MET controls. MET probably stimulated PRL through its DA antagonistic properties.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracellular transformation of prolactin following internalization into rat liver

We investigated prolactin (PRL) degradation in rat liver lysosomes both in vivo and in vitro. In previous studies we showed that, in addition to the Golgi apparatus, PRL is internalized towards lysosomes and light, lysosome-like vesicles which we identified as 'prelysosomes'. Injected [125I]oPRL that localized in lysosomes and prelysosomes at times varying from 0 to 45 min showed significant differences from fresh and plasma membrane- (PM) or Golgi-bound hormone. First, it was more easily dissociable by 3 M MgCl2 than Golgi- but less than PM-bound [125I]oPRL. Second, it was only in lysosomal fractions that, as time following injection increased, a significant part of dissociable radioactivity became non-TAC-precipitable. When MgCl2-extracted [125I]oPRL was subjected to gel filtration on a Sephadex G-75 fine column, some of the radioactivity, and especially that extracted from prelysosomal or lysosomal fractions, eluted as a high molecular weight (HMW) entity, most co-migrated with fresh [125I]oPRL, and a little was found in small fragments. Only the central peak had any rebinding activity, which was comparable to that of fresh hormone. In an in vitro study we incubated [125I]hGH with lysosomal fractions for 16 h at 25 degrees C. After centrifugation, an aliquot of supernatant hormone was assayed for its binding capacity to standard receptor preparations and the rest subjected to gel filtration. Peak fractions were also tested in binding assay. [125I]hGH that had been in contact with prelysosomes lost almost all of its ability to bind to standard receptors and totally migrated in the HMW peak, at the void volume of the column.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship between dopamine release into hypophysial portal blood and prolactin release after morphine treatment in rats

To address the issue of whether, after morphine treatment, the reduced release of dopamine (DA) into portal blood is entirely responsible for the increased prolactin (PRL) release, the following study was conducted. The concentration of DA in plasma from a single portal vessel of untreated, ovariectomized rats was 1.85 +/- 0.33 ng/ml (mean +/- SE). Treatment of ovariectomized rats with alpha-methyl-p-tyrosine (alpha MT) caused a 91% reduction in the concentration of DA in portal plasma. Infusion of DA (0.4 microgram/min/kg BW) into a jugular vein of rats pretreated with alpha MT restored the DA concentration in portal plasma to that seen in untreated rats. Injection of morphine sulfate elicited a marked increase in the concentration of PRL in plasma. Infusion of DA at rates of 0.4 and 0.8 microgram/min/kg BW suppressed by 52 and 75%, respectively, the secretion of PRL after morphine treatment. Infusion of DA had no effect on the release of PRL induced by intracerebroventricularly administered beta-endorphin. Although treatment of rats with alpha MT caused release of PRL that was similar to that seen in rats treated with morphine, infusions of DA at 0.4 and 0.8 microgram/min/kg BW into alpha MT-treated, ovariectomized rats suppressed the secretion of PRL by 89 and 96%, respectively. Thus, after morphine treatment, the decreased release of DA into portal blood is not in itself sufficient to account for the increase seen in the secretion of PRL. It is suggested that morphine and opiate-like peptides induce the release of a hypothalamic substance(s) that stimulates PRL release.     

Evidence for a role of endorphins in stress- and suckling-induced prolactin release in the rat

Injection of the opiate antagonist naloxone completely prevented the rise of serum prolactin induced by ether stress in intact male rats. Naloxone also led to a 50–95% inhibition of the marked elevation of plasma prolactin levels induced by suckling. These data suggest that endogenous opiates (endorphins) are involved in the stimulation of prolactin release induced by both stress and suckling in the rat.     

Changes in molecular variants during in vitro transformation and release of prolactin by the pituitary gland of the lactating rat.

PRL transformation involves a dopamine (DA)-controlled, thiol-mediated decrease in pituitary PRL detectability that precedes and may determine increased PRL release. The present studies were designed to determine 1) whether structural changes occur to adenohypophyseal (AP) PRL during in vitro transformation and may account for decreased PRL detectability; 2) whether such changes occur within AP PRL granules; 3) the type and proportion of secreted PRL variants; and 4) the influence of DA and/or thiol reducing agents upon AP PRL molecular changes. Quartered APs of 8 h nonsuckled lactating rats, injected or not with [3H]leucine (5 mCi/g body wt, 8 h before death) were incubated for 30-240 min. The effects of DA (50 microM) with or without reduced glutathione (10 mM), and the alkylant N-ethylmaleimide (1 mM) were compared to the changes in PRL molecular variants occurring in control incubated AP fragments. Polyacrylamide gel electrophoresis (PAGE) under native conditions and sodium dodecyl sulfate (SDS)-PAGE under reducing (R) or nonreducing (NR) conditions were used to resolve molecular variants of PRL and [3H]PRL, followed by Western blotting and densitometric/liquid scintillation techniques, respectively. Up to 5 and 10 major forms of immunoreactive PRL were resolved by PAGE and SDS-PAGE, respectively. The spontaneous transformation after 30 min incubation was correlated with a decrease of 23K PRL and 23K [3H]PRL bands and an increase in high mol wt (80 to greater than 100K) PRL forms. These changes were reversible upon reduction of the proteins before electrophoresis or after extraction of preparative gels. DA and N-ethylmaleimide prevented, whereas reduced glutathione induced these changes. Similar changes were observed in isolated granules from nonincubated and incubated pituitary fragments. Secreted PRL was also polymorphic, with a high predominance of 23K PRL and 23K [3H]PRL. In addition, a polymeric PRL variant, which in reducing gels showed an apparent mol wt of 27K, was secreted in high proportion, similar to that of 23K PRL. The nature of this polymeric variant of PRL is unclear at present. Altogether, these results suggest that a dynamic interconversion of PRL molecular variants occur during transformation. The decreased detectability associated with PRL transformation appears to involve the association of 23K PRL molecules into a 80 to greater than 100K disulfide-linked oligomer. This association is reversible upon reduction and takes place within secretory granules. DA appears to inhibit PRL transformation by preventing thiol-disulfide interchange.     

Interactions of dopamine and neurotensin on calcium fluxes and prolactin release in normal rat pituitary cells.

We studied the role of calcium in dopaminergic control of the neuroendocrine effects of neurotensin. In primary cultures of dispersed normal female rat anterior pituitary cells the interactions of dopamine and neurotensin were examined with reference to the rate of PRL release, the magnitude of 45Ca2+ uptake, the rate of fractional 45Ca2+ efflux, and the dynamic response of the intracellular calcium concentration (Cai) monitored with the fluorescent dye, Indo-1. Neurotensin stimulated calcium uptake and also mobilized a pool of intracellular calcium to increase Cai in a sustained plateau-like pattern. The response of PRL release and fractional efflux to neurotensin, however, each displayed typical spike and plateau profiles. In the presence of dopamine the stimulation of PRL release and calcium uptake due to neurotensin were abolished, and the rise in Cai was barely detectable, but neurotensin-stimulated fractional efflux persisted almost unchanged. These data suggest that dopamine may modulate Cai by inhibiting calcium uptake and possibly also by enhancing cellular calcium extrusion under stimulated conditions. Further, the increased inositol trisphosphate production reportedly stimulated by neurotensin apparently does not generate a spike-like response of intracellular calcium, and stimulated hormone release may display a spike and plateau pattern solely with a plateau Cai profile.     

Ascorbic acid potentiates the inhibitory effect of dopamine on prolactin release in primary cultured rat pituitary cells

The chemical structure of dopamine includes an ortho-catechol group which is labile to oxidizing agents. Ascorbic acid, a reducing agent, has in the past been added to the incubation medium in order to protect dopamine against oxidation. However, there has been no thorough examination of the biological effect of ascorbic acid on prolactin release. In this present study we have shown that ascorbic acid has neither a stimulatory nor an inhibitory effect on prolactin release but reduces by approximately two orders of magnitude the concentration of dopamine necessary to inhibit prolactin release from cultured anterior pituitary cells. The strong potentiation effect of ascorbic acid was reproduced using apomorphine. We compared the effect of ascorbic acid and isoascorbic acid on dopamine inhibition of prolactin release. Isoascorbic acid is an epimer of ascorbic acid, having the same reduction-oxidation potential as ascorbic acid, but is less biologically active. Isoascorbic acid was less effective in potentiating the dopaminergic effect than was ascorbic acid, which supports the notion that potentiation by ascorbic acid is not entirely due to its reducing property. In order to dissociate further the chemical protection of dopamine from the biological potentiation, the inhibitory effects of freshly made and 3-h-old dopamine solutions were compared. Neither one of the two solutions contained any ascorbic acid, yet the two solutions did not show any difference in their ability to inhibit prolactin release during the 3-h incubation period, indicating that no significant amount of dopamine was oxidized.(ABSTRACT TRUNCATED AT 250 WORDS)     

Suppression of prolactin release and mRNA accumulation by two novel dopamine agonist agents

Two novel dopamine agonist drugs, CV 205-502 and CQP 201-403, have been investigated to compare their effects on prolactin secretion and prolactin mRNA accumulation in cultured rat pituitary cells. Both drugs gave dose-dependent suppression of prolactin release over a 24 h incubation period: when each drug was used at 100 nmol/l CV 205-502 and CQP 201-403 induced suppression to 8.9 +/- 1.7 and 10.2 +/- 1.8% of control release, respectively, compared to 26.7 +/- 4.8% of control with 100 nmol/l bromocriptine. There was no consistent effect on growth hormone release. Cytoplasmic accumulation of prolactin mRNA was also inhibited by both drugs at this concentration, to 50.2 +/- 5.5% of control values by CV 205-502 and to 67.4 +/- 8% of control by CQP 201-403, and to a similar extent by 100 nmol/l bromocriptine (50.6 +/- 9.1% of control). None of the drugs had any significant effect on GH mRNA levels. These data suggest that the agents exert their effect at a pretranslational stage of prolactin synthesis, as well as at the level of hormone release.     

Ovine prolactin (PRL) and dopamine preferentially inhibit PRL release from the same subpopulation of rat mammotropes

Autoregulation of PRL release was studied at the single cell level by the use of a reverse hemolytic plaque assay. Monodispersed pituitary cells from adult male rats were first preincubated with test substances and then coincubated with antirat PRL antiserum before development of plaques with complement. At the conclusion of the assay, the percentage of all pituitary cells in culture that formed plaques was evaluated microscopically, and the rate of plaque development was used as an index for the rate of hormone release. In controls, the maximal percentage of pituitary cells formed PRL plaques within a 1.5-h antibody incubation period, and addition of TRH (1 X 10(-7) M) did not increase this proportion. Treatment with ovine PRL (oPRL, 100 ng/ml) or dopamine (1 X 10(-7) M), either alone or in combination, caused a comparable suppression of the rate of PRL plaque development, which was reversed by the presence of TRH. Pretreatment of cells with the lysosomotropic agent chloroquine (1 X 10(-5) M) overrode dopamine inhibition of PRL plaque development, but did not influence oPRL inhibition. Taken together, these results demonstrate that oPRL inhibits basal, but not TRH-induced, PRL release from rat pituitary cells and support the view that PRL can act at the pituitary level to inhibit its own secretion. Moreover, the equipotency and lack of additivity exhibited by oPRL and dopamine coupled with the differential effects of chloroquine suggest that these factors both act upon the same subpopulation of mammotropes to inhibit PRL release, but by separate intracellular mechanisms.     

Antipsychotic drugs inhibit prolactin release from rat anterior pituitary cells in culture by a mechanism not involving the dopamine receptor.

Bromocriptine, a dopamine agonist, inhibited secretion of PRL and did not affect GH release from rat anterior pituitary cells in culture. The reversal of this inhibition of PRL release by butaclamol, a dopamine antagonist, was stereospecific; 10 nM d-butaclamol completely reversed the inhibition caused by 10 nM bromocriptine, while l-butaclamol had no effect at concentrations up to 10 microM. However, both enantiomers at 10 microM inhibited PRL release to 30% and GH release to 91% of control values. Two other dopamine antagonists also inhibited hormone release. Haloperidol (10 microM) inhibited PRL release to 23% of control values and did not affect GH release; 3.3 microM pimozide inhibited PRL and GH release to 18% and 38% of control values, respectively. These data indicate that, the inhibition of PRL by antipsychotic drugs is not mediated through the dopamine receptor.     

The release of surfactant in rat lung by brief periods of hyperventilation

We investigated the release of surfactant-type phospholipids (S) using the isolated perfused rat lung (IPL). Following a 20 min equilibration period the lungs were hyperventilated for up to 15 min and then lavaged. Changing the peak inspired pressure (PIP) from 10 to 20 cm H2O rapidly increased the rate of release of S; this rate declined after 2 min. In contrast, doubling frequency of ventilation while maintaining the control tidal volume had no effect. The increase in alveolar S reflected release. rather than redistribution, and after 2 min amounted to about 8% of total S in lung tissue. Equivalent hyperventilation in an open-chested intact rat released significantly less S, suggesting possible tonic neurohumoral suppression in vivo. The release of S in the IPL was depressed by reducing temperature, but was not affected by hypoxia, 2,4 dinitrophenol, phenylephrine or dibutyrylguanosine 3',5'-cyclic monophosphate. We suggest that increasing tidal volume may directly distort the alveolar type II cell; each cell reacts to its own threshold distortion by releasing a pool of S in all-or-none fashion.     

Involvement of adenohypophysial dopamine in the regulation of prolactin release during suckling.

Dopamine (DA) was measured in the anterior lobe of the pituitary and median eminence from lactating rats. The effect of pup separation and suckling was studied in order to correlate changes in DA levels with changes in serum PRL. In lactating rats separated from their pups, low levels of circulating PRL were found at 2,4, and 8 h. DA levels in the median eminence showed a decline at 2h; at 4 and 8 h of separation, a significant increase was observed. In the pars distalis, the concentration of DA increased with the length of the nonsuckling interval. Suckling induced a rapid rise in serum PRL levels in rats that were separated from their pups 4 h earlier. Under these conditions, a significant decrease in DA levels in the median eminence and pars distalis was observed as early as 5 min after the onset of suckling; at 30 min, the DA levels were still low. In the situations studied (suckling and pup separation), a negative correlation between serum PRL and DA levels in both the median eminence and pars distalis was always found.     

Delayed puberty induced by chronic suppression of prolactin release in the female rat

To study the effect of PRL deficiency on the onset of puberty, PRL release was chronically inhibited by treating immature female rats with the dopaminergic receptor agonist, bromoergocriptine (CB-154). The resulting alterations in the time of puberty onset, and in other associated parameters, such as serum levels of pituitary hormones, ovarian responsiveness to gonadotropins and ovarian hCG receptor content were then evaluated. CB-154 was provided in the drinking water from day 22 onward at the concentration of 20 and 100 micrograms/ml. The treatment resulted in almost complete suppression of serum PRL levels throughout the entire period studied (day 22 to first diestrus). In contrast, serum GH, FSH, and LH levels were not depressed. Likewise, pulsatile release of FSH was not affected and only a subtle alteration in pulsatile LH release was apparent. The onset of puberty, as determined by the age at vaginal opening, and at first diestrus after the first estrus and by the presence of corpora lutea at sacrifice (first diestrus), was markedly delayed in the hypoprolactinemic (HPO) rats. This inhibitory effect of CB-154 was completely prevented by concomitant administration of PRL. Ovarian weight was significantly decreased in HPO rats at the three ages studied (27, 32, and 36 days of age). By day 36, 50% of the control animals had already ovulated, as compared with only 9% of the HPO rats. Microscopic examination of ovaries from HPO rats revealed a retarded follicular development. In vitro ovarian progesterone response to hCG studied at day 32 and 36 of age was reduced in the HPO rats. Uterine growth was also depressed in HPO rats, the ovaries of which, when incubated in vitro, failed to show the prepubertal increase in estrogen response to hCG seen in control rats between day 32 ad 36. Aromatase activity, as measured by the in vitro release of estradiol from ovaries incubated in the presence of an excess of androgen substrate, was depressed in HPO rats. hCG receptor content in the ovaries from HPO rats (counts per min [125]hCG bound per micrograms DNA) was also lower than that of control animals at day 32 and 34 but not at day 36. However, at this later time the hCG receptor content per milligram of ovary was still significantly reduced in HPO rats. The results support the view that PRL plays an important role in the process of ovarian development that leads to the onset of puberty in the female rat and that this effect is, at least in part, exerted through a positive influence of PRL on ovarian LH receptor content.     

Inhibition of gonadotropin and prolactin release by dopamine: effect of endogenous estradiol levels.

The influence of endogenous estradiol (E2) levels on gonadotropin and PRL sensitivity to dopamine (DA) infusion (4 micrograms/kg/min) was assessed at different stages of the follicular phase of the menstrual cycle. Basal LH and FSH levels were comparable in day 2 and day 12 subjects, and despite a 4-fold increase in E2 concentration, the inhibition of LH by DA was small and quantitatively similar and there was no discernible effect on FSH in either group. In marked contrast, day 14 subjects with an elevated basal LH level exhibited a dramatic increase in the sensitivity of LH and FSH to DA inhibition. Further, a remarkable rebound release for LH but not FSH occurred on the termination of DA infusion. There was a significant correlation between basal LH and response to DA (r = 0.979). This unique increase in response to DA at a time when hypothalamic LRF secretion is assumed to be elevated suggests that DA may exert its effect by inhibiting LRF release. The inhibition of PRL release by DA is correlated with endogenous E2 levels (r equal 0.685) as well as basal PRL levels (r = 0.878). Rebound release of PRL occurs in all three groups of women on termination of the DA infusion, but the magnitude was greatest in Day 14 subjects with the highest endogenous E2 levels. These data suggest that while E2 seems to augment the sensitivity of PRL inhibition by DA, its does not seem to directly influence gonadotropin sensitivity to DA inhibition. The selective hypersensitivity of both LH and FSH to DA observed on the day before midcycle LH peak is consistent with a reduction in LRF neuronal inhibition by tuberoinfundibular DA neurons at this time.     

Evidence for the involvement of hypothalamic dopamine and thyrotrophin-releasing hormone in suckling-induced release of prolactin

The interactions of several mammalian follicle-stimulating hormones and luteinizing hormones with specific gonadotrophin receptors in macropodid marsupial testicular homogenates were investigated with a view to developing radioreceptor assays for marsupial FSH and LH. Testes from Eastern grey kangaroos and tammar wallabies possessed high affinity (dissociation constant congruent to 10(-10) mol/l) saturable receptor sites which were highly specific for LH or FSH. Luteinizing hormone receptor sites bound only highly purified LH preparations (human, ovine and rat) but did not bind highly purified FSH, TSH or prolactin while FSH receptor sites were equally specific for highly purified FSH preparations. These sites demonstrated a degree of species specificity in that marsupial pituitary extracts were relatively more potent in these assays than in assays using gonadotrophin receptors from rat testes. Serum from hypophysectomized female tammar wallabies had little effect on the slope and position of the LH standard curve but significantly depressed the dose-response curve for FSH. For this reason it was not possible to develop a radioreceptor assay for serum FSH using marsupial testicular FSH receptors. However, gonadotrophin receptors from both rat and marsupial testes have been employed in the successful development of radioreceptor assays for marsupial pituitary LH and FSH and marsupial serum LH.     

Evidence that opioid peptides and dopamine participate in the suckling-induced release of prolactin in the ewe

A pharmacological approach was used to study the involvement of opioid peptides and dopamine in mediating the suckling-induced release of prolactin in the lactating ewe (10-20 days post partum). To promote reliable and predictable suckling activity lambs were fitted with elasticated masks to prevent sucking for 4.5 h. After a 1-hour control period of frequent blood sampling, ewes were treated (i.v. injections every 5 min) for a further 75 min with either saline vehicle, an opioid antagonist (naloxone; 4.17 mg/5 min), a dopamine antagonist (metoclopramide; 1.25 mg/5 min), a mixture of naloxone + metoclopramide or a dopamine agonist (apomorphine; 6.6 mg/5 min). Blood was withdrawn at 5-min intervals for determination of plasma prolactin and luteinizing hormone (LH) by radioimmunoassay. Plasma LH concentrations (less than or equal to 1 microgram/l) were not significantly affected by any of the drug treatments and there was no evidence for an acute fall in LH associated with suckling- or TRH-induced increases in prolactin secretion. Naloxone significantly (p less than 0.05) reduced the mean incremental change in prolactin concentration (delta PRL) in response to suckling (+7 +/- 18 micrograms/ml) compared with saline-infused controls (+79 +/- 26 micrograms/ml), an effect which was completely reversed by combined treatment with naloxone and metoclopramide (+146 +/- 56 micrograms/ml). Metoclopramide alone raised basal prolactin levels by 46% (p less than 0.01) but did not affect delta PRL in response to suckling (+115 +/- 52 micrograms/ml). Neither naloxone, metoclopramide nor a combination of the two drugs affected the subsequent prolactin to TRH (10 micrograms). Apomorphine, however, completely abolished both the suckling- and TRH-induced release of prolactin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of antiserum to neurotensin reveals a physiological role for the peptide in rat prolactin release.

Previous studies have indicated that the brain peptide neurotensin can stimulate prolactin release by direct action on the pituitary gland, whereas its action within the hypothalamus is inhibitory. The inhibitory action is mediated by the release of dopamine into the hypophyseal portal veins, which deliver the neurotransmitter to the anterior pituitary gland to inhibit prolactin release. Our experiments were done to evaluate the physiologic significance of these neurotensin actions by injecting the globulin fraction of highly specific neurotensin antiserum either intravenously or intraventricularly. Injection into the third ventricle of either 1 or 3 microliter of neurotensin antiserum significantly increased plasma prolactin concentrations in (i) ovariectomized and (ii) ovariectomized estrogen- and progesterone-primed rats within 1 hr of injection. The response was more pronounced in the ovariectomized than in the ovariectomized estrogen- and progesterone-treated animals and was dose related. Intraventricular injection of these doses of neurotensin antiserum also evoked elevations in plasma prolactin in intact males, which were significant but smaller in magnitude than those seen in female rats. To evaluate the effect of the antiserum on the pituitary directly, the antiserum was injected intravenously at a dose of 40 microliter, which was sufficient to block the blood pressure-lowering effect of neurotensin. After the intravenous injection of antiserum, a highly significant suppression of plasma prolactin occurred, detectable when first measured at 1 hr after injection in both ovariectomized and ovariectomized estrogen- and progesterone-treated animals; however, the intravenous injection of antiserum had no significant effect on the prolactin release in males. These data indicate the physiological significance of the hypothalamic inhibitory actions of neurotensin on prolactin release, which are probably mediated by its stimulation of dopamine release that in turn, inhibits prolactin secretion by the lactotropes. The direct stimulatory effect of the peptide on prolactin release after its presumed release into portal vessels also appears to be physiologically significant in female but not in male rats.