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.     

Reduced responses of prolactin and catecholamine to stress in the lactating rat

Prolactin, GH, TSH, adrenaline and noradrenaline responses to the stress of immobilization were compared between lactating and non-lactating dioestrous rats. The concentrations of GH in plasma were reduced to a similar degree by the immobilization of lactating and non-lactating rats, and TSH levels were unchanged in both groups. The increases in plasma concentrations of adrenaline and noradrenaline induced by stress were significantly smaller in lactating than in non-lactating rats. Immobilization caused a marked increase in prolactin levels in the plasma of non-lactating rats but no increase in lactating rats. These changes may help to save energy and maintain milk production during the period of lactation.     

Serotonergic mediation of the suckling-induced release of prolactin in the lactating rat

Lactating female rats were infused with the serotonergic neurotoxin, p-chloramphetamine (PCA), via stainless steel cannulae implanted into either the 3rd or the lateral ventricle. Those mothers receiving the drug in the 3rd ventricle sustained a more severe depletion of hypothalmic serotonin (43% vs 23%) and a significantly greater incidence of pup mortality (98% vs 48%) than those receiving the drug in the lateral ventricle. Coincident with the depletion of hypothalmic serotonin was a reduction in serum prolactin (Prl). Subcutaneous (s.c.) infections of Prl into each group of females sharply reduced the number of pup deaths. It was concluded that hypothalmic serotonergic neurons mediate the suckling-induced release of Prl necessary for the maintenance of lactation.     

The release of prolactin in the lactating rat: effect of chloroquine

The storage form of prolactin (PRL) was converted into the releasable form in the lactating rat pituitary gland within 10 min of suckling by 6 pups following 4-5 h of nonsuckling on postpartum day 13-14. The characteristics of the PRL discharge from the releasable pool into the circulation was then studied using a stimulus which is known to effectively release PRL (exposure of the mother to the exteroceptive stimuli emanating from 2 pups) but which is not of sufficient strength to influence the conversion of storage PRL. We found that the concentration of PRL could be repeatedly elevated to the same extent in the plasma with repetitive 10-min applications of this stimulus. With continuous 75 min of exposure to 2 pups, the plasma PRL concentration of the mother rose to maximum within 15 min which then was sustained for the remaining 60 min, suggesting a steady release of PRL into the circulation had occurred. These data indicate that, unlike the storage form of PRL, the discharge of the releasable form occurs in relation to the length of time the stimulus is applied and exhibits neither summation nor refractoriness. Subsequently it was noted that PRL could be released up to 8 h after the releasable pool had been formed and that the plasma concentration curves were not altered by injecting 5 mg of the lysosome inhibitor, chloroquine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship between the suckling-induced release of oxytocin and prolactin in the urethane-anaesthetized lactating rat

The effects of changes in the intensity of the suckling stimulus on the reflex release of oxytocin and prolactin were compared in urethane-anaesthetized lactating rats. Mothers which had previously suckled 12 pups (Group 1) showed a graded increase in the amount of oxytocin released during a 3 h suckling test when the number of pups applied to the nipples was increased from six to eight or ten. Mothers which had suckled six pups during their lactation (Group 2) appeared to show a maximum frequency of milk ejection whether six, eight or ten pups were applied to the nipples. The release of prolactin was not elicited from either Group 1 or Group 2 mothers when six pups were applied to their nipples. With eight pups suckling, the Group 1 mothers again showed no evidence of prolactin release. In contrast, the Group 2 mothers showed a significant increase in the level of prolactin in the plasma during the 3 h suckling test. With ten pups suckling the release of prolactin was evident in both groups of mothers, although the response was earlier and more pronounced in Group 2 than Group 1. These results suggest that in the urethane-anaesthetized rat, the threshold for the suckling-induced reflex release of oxytocin is distinct from the threshold for the release of prolactin and that these thresholds are, at least in part, set by the preceding suckling experience of the mothers. In those animals which showed both reflex milk ejection and prolactin release there was a linear relationship between the magnitude of the two endocrine responses.     

The endogenous stimulatory rhythm regulating prolactin secretion is present in the lactating rat

Prolactin (PRL) secretion in the female rat is regulated by an endogenous stimulatory rhythm (ESR), which is normally under the tonic inhibition of dopamine (DA). The ESR consists of a nocturnal (N) component which peaks at approximately 03.00 h, and a diurnal (D) component which peaks at approximately 17.00 h. This ESR has been shown to be present in ovariectomized and cervically stimulated rats. We have proposed that the ESR is continuously present in the female rat and that any suppression of the tonic inhibitory influence on PRL secretion can reveal its existence. In this study, the effects of the DA-lowering stimulus of suckling was investigated at different times of day in lactating rats. In addition, the pattern of PRL secretion in freely lactating rats throughout a 24-hour period was studied. Female rats were separated from their pups for 6 h prior to reunion at either 03.00 (coincident with the N component), 12.00 (control) or 17.00 h (coincident with the D component) at various stages of lactation. Blood samples were collected from intra-atrial cannulae immediately before separation of pups and dams, immediately before reunion of pups and dams (0 time), and 15, 30, 60 and 120 min following reunion of pups and dams. Four days following parturition, dams suckled at either 03.00 or 17.00 h secreted significantly greater PRL than rats suckled at 12.00 h. Peak levels of PRL were 60-, 90- and 25-fold greater than 0 time levels, at 03.00, 17.00 and 12.00 h, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thiol regulation of depletion-transformation and release of prolactin by the pituitary of the lactating rat

We investigated the possibility that thiol-disulfide interchange mechanisms are involved in depletion-transformation (loss of tissue PRL detectability) and release of PRL from adenohypophyses (AP) of lactating rats. The influence of pH, bicarbonate, and Triton X-100 as well as thiol-related compounds on these processes was assessed. Tissue PRL was depleted-transformed by the use of three different conditions: 1) 30 min of suckling after 8 h of nonsuckling; 2) in vitro incubation of APs for 2 h; or 3) in vivo cysteamine (CSH) treatment. Lactating rats nonsuckled for 8 h served as controls (no depletion-transformation). The depletion-transformation phenomenon was unchanged by extraction with Tris-HCl-0.1% Triton X-100 buffers but reversed either by extraction with bicarbonate buffer (pH 8.2 or 9.7) or by incubation of pH 8.2 homogenates for 3 h at 37 C. Reduced glutathione (GSH) added to these homogenates further enhanced PRL detectability. At pH 6.5, however, incubation with or without GSH had the opposite effect and decreased PRL detectability. In AP incubations, depletion was increased in a dose- and time-dependent fashion by the aminothiol CSH, and by GSH, dithiothreitol, or mercaptoethanol but not sodium ascorbate. These agents also inhibited PRL release. Similar results were obtained after injection of CSH (20-120 mg/kg BW) 4 h before death. Depletion and release of PRL in incubated APs were prevented by iodoacetamide and N-ethylmaleimide (0.1-5 mM); GSH or CSH counteracted these effects. In contrast to the alkylating agents, oxidized glutathione and 5,5'-dithio-2-nitrobenzoic acid inhibited PRL depletion but stimulated PRL release. Thus, thiols and aminothiols may preferentially lead to depletion-transformation of PRL, whereas disulfides may inhibit depletion and facilitate PRL release. Although in some experiments increased depletion was dissociated from increased release, nonetheless the data support the concept that shifts in PRL detectability during depletion-transformation, repletion, and release involve thiol-disulfide interchange mechanisms.     

Abolition of prolactin surge induced by ovarian steroid hormones in the lactating rat.

There are data indicating that stress-induced prolactin (PRL) release is blunted in the lactating rat like the release of other stress-associated hormones. In this experiment, the PRL release evoked by administration of estrogen, which is another principal stimulus for PRL release, was examined in ovariectomized lactating rats 8-15 days after delivery. Estradiol benzoate (EB, 20 micrograms) injections into ovariectomized nonlactating rats induced a PRL surge starting between 13:00 and 15:00 h with a peak at 17:00 h 2 days after the treatment, whereas the EB-induced PRL surge was absent in ovariectomized lactating rats separated from their pups at 09:00 h on the day or in mothers without separation from their pups. Injection of either thyrotropin-releasing hormone (TRH; 10 micrograms/kg) or pimozide (0.5 mg/kg) elevated serum PRL concentrations similarly in lactating and nonlactating rats when examined just before the beginning of the expected estrogen-induced PRL surge. Thus, the main cause for the reduced PRL response to estrogen in lactating rats seems not to be in the pituitary gland but in the brain. Progesterone, which is known to induce a PRL surge in ovariectomized estrogen-primed rats by acting on the mediobasal hypothalamus, also failed to evoke a PRL surge in lactating rats. Recovery from the inhibitory influence of suckling on PRL response to EB followed a time course similar to that observed in response to immobilization stress or to morphine injection; estrogen-induced PRL surge started to recover at 6 days and was almost fully recovered 8 days after weaning.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclosporine inhibits prolactin induction of ornithine decarboxylase in rat tissues

Cyclosporine (CyA), formerly cyclosporin A, significantly inhibited the ability of prolactin (PRL) to elevate ornithine decarboxylase (ODC) activity in a variety of rat tissues. Administration of PRL to hypophysectomized rats also resulted in an induction of ODC activity which was inhibited markedly in all tissues studied in the presence of CyA. Transglutaminase ( TGase ) activity was not affected in any significant manner by PRL or CyA in most tissues studied. However, it was elevated in the adrenal by 10(-8) M PRL. Bromocryptine, which selectively antagonizes pituitary PRL release, decreased the kidney ODC basal levels to 30% of vehicle control and serum PRL level to 4.3 +/- 1.4 compared to 28 +/- 10 in controls, suggestive of PRL maintenance of steady-state ODC activity in the kidney. CyA administration did not affect the action of glucagon, a known cyclic AMP-mediated hormone, or 8-bromo-cyclic AMP on kidney ODC activity. The elevation of rat kidney ODC activity by dexamethasone and triiodothyronine (T3), compounds which elevated serum prolactin levels in all cases, was also blocked by administration of CyA. Epidermal growth factor (EGF), which did not induce rat kidney ODC activity by itself, was capable of producing a small increment in ODC activity in the presence of CyA. The marked effect of CyA to selectively block ODC induction by PRL may be due to the ability of CyA to interact with receptor-required phospholipids in membranes and thus to antagonize hormone-receptor interaction.     

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)     

Effect of ovine prolactin on tubulin function in the anterior pituitary lobe of the lactating rat

Ovine PRL (oPRL) was employed to investigate the role of anterior pituitary lobe microtubules in suckling-induced PRL secretion in the lactating rat. Groups of primiparous rats on days 12-14 postpartum were isolated from their pups for 4-5 h, then suckled for 10 min, and killed, and the anterior pituitary lobes were dissected out. Each pituitary lobe was processed to obtain the two tubulin pools, viz. soluble and polymerized tubulin fractions. After 10 min of suckling the pituitary soluble tubulin levels were reduced by about 25% (P less than 0.05), and polymerized tubulin levels increased by about 40% (P less than 0.05). When 3 mg oPRL were injected 4 h before suckling, the suckling-induced rise in the polymerized tubulin levels in the anterior pituitary lobe as well as plasma PRL levels were significantly inhibited (P less than 0.05). In the suckled rats injected with oPRL a 25% reduction in the total tubulin levels (soluble and polymerized) was observed. In a second experiment, each anterior pituitary lobe obtained from groups of rats suckled for 10 min was processed to obtain the total tubulin fraction. Suckling for 10 min stimulated the in vitro assembly of total tubulin fraction into microtubules by about 150% (P less than 0.05); 3 mg oPRL injected 4 h before suckling inhibited the suckling-induced rise in tubulin assembly (P less than 0.05). In a third experiment, suckling for 10 min stimulated GTPase activity in the total tubulin fraction by about 60% (P less than 0.05). Administration of oPRL 4 h before suckling caused about an 80% increase in GTPase activity. At a 1:2000 dilution, antitubulin antibodies maximally inhibited GTPase activity by about 40%, suggesting that a significant proportion of the enzyme activity can be due to tubulin present in the pituitary. These results suggest that PRL secretion is coupled to pituitary microtubules and that in addition to tubulin, other GTP-binding proteins might be involved in PRL secretion.     

Annexin 5 inhibits thyrotropin releasing hormone (TRH) stimulated prolactin release in the primary culture of rat anterior pituitary cells

Annexin 5, a novel calcium-phospholipid binding protein, is thought to be involved in hormone secretion by the anterior pituitary gland. Gonadotropin releasing hormone stimulates annexin 5 synthesis, which, in turn, enhances gonadotoropin secretion. On the other hand, annexin 5 was shown to inhibit prolactin release in vitro. To understand the nature of the opposing effects of annexin 5 on these two major pituitary hormones, the present study examines the inhibitory effect of annexin 5 on prolactin release in relation to thyrotropin stimulating hormone (TRH) using primary cultures of anterior pituitary cells of adult female rats. While recombinant rat annexin 5 was found to have little effect on basal prolactin release, it significantly inhibited TRH-stimulated prolactin release. Addition of specific anti-annexin 5 serum to the culture increased basal prolactin release in a concentration dependent manner, and no further increase in prolactin release was observed following application of TRH in the presence of anti-annexin 5. The enhanced basal prolactin release induced by anti-annexin 5 was reversed by the simultaneous administration of indomethacin, an inhibitor of cyclooxygenase. These results demonstrate that endogenous pituitary annexin 5 exerts an inhibitory effect on prolactin release and suggest that this is attained by suppression of eicosanoid synthesis in vitro.     

Rat serum inhibits prolactin release from rat pituitary mammotrophs in culture

Rat pituitary cells maintained in culture for 3 days in α-MEM supplemented with 17% horse serum produce and release 4?8× the quantity of prolactin (PRL) originally contained in the cells at the time of plating. Addition of small (200 μl) quantities of rat serum repeatedly suppressed PRL release by $\text{\$}\text{?}$50%. Inhibition was related to dose of rat serum. Other studies showed (a) that the cells could recover from serum-induced inhibition, (b) that the effect was independent of the age of the pituitary and/or serum donor, (c) that GH release was unaffected by rat serum addition, (d) that the effect could not be attributed to proteases in the serum preparations and (e) that addition of T₃ to the culture medium prevented the inhibitory response. The inhibitory material(s) in rat serum is stable on freezing; is non-dialyzable; is heat-labile; is stable over 3 days incubation at 37°C; and is associated with molecules between 85 000 and 146 000 dalton. Inhibitory activity was generated upon incubating whole clotted blood at 37°C for 23 h. It is suggested that the inhibitory material might (a) be released from platelets during clotting or (b) be generated from an inactive precursor molecule cleaved by proteases released during the clotting process.     

Hysterectomy-induced alterations in prolactin secretion of lactating rats

The contribution of the uterus to the regulation of PRL secretion in lactating dams and cycling female rats was investigated. Lactating animals were hysterectomized or sham operated 2 days after parturition, and the number of pups was adjusted to eight. Blood samples for PRL RIA were obtained through intra-atrial cannulae implanted 2 days before experimentation. In order to study the PRL secretory profile in undisturbed freely lactating rats, blood samples were taken every 2 h for 24 h starting at 1400 h. During early lactation (days 7-8), hysterectomy did not alter the PRL secretory profile compared to that of sham-operated controls. On days 14-15 post partum, PRL secretion followed a characteristic bimodal pattern showing two PRL surges at 1800 h and 0600 h. After hysterectomy, the early morning PRL surge disappeared and PRL secretion showed an unimodal daily rhythm reaching its peak at 1800 h. The possible effect of the absence of the uterus on suckling-induced PRL release at various stages of lactation was studied. On days 7-8, suckling stimuli after 4 h of pup deprivation induced robust PRL release. Hysterectomy did not significantly alter PRL release at this earlier stage of lactation. In control groups, the suckling-induced PRL secretory response markedly declined as the postpartum period advanced. On the other hand, the hysterectomized animals retained significantly greater responsiveness to suckling during the second half of lactation. These data indicate an inhibitory influence of the uterus on PRL secretion. The onset of this uterine effect is considerably delayed, and its influence became prominent only at a later phase of lactation. The effect of length of pup deprivation preceding the suckling stimulus, in combination with hysterectomy, was also investigated. Hysterectomy significantly increased suckling-induced PRL release after 4 and 24 h separation compared to the sham-hysterectomized animals. When the separation was longer than 48 h, the inducibility of PRL release by suckling declined and was not influenced by hysterectomy. In order to study the possible influence of the uterus on PRL secretion during the estrous cycle, regularly cycling female rats were hysterectomized at diestrus 1. Twelve days later the animals were cannulated, and serial blood samples were taken during the subsequent proestrus. Hysterectomy did not alter the PRL surge which occurred on the afternoon of proestrus indicating that the uterus does not have a major function in regulating PRL secretion on proestrus. In conclusion, hysterectomy significantly delayed the extinction of suckling-induced PRL release revealing the active role of the uterus in the regulation of this neuroendocrine reflex.     

Vesicular release of prolactin from preformed prolactin granules is stimulated by soluble factor(s) from the anterior pituitary of lactating rats

This study demonstrates that conditioned media (CM) from the anterior pituitary gland (AP) of lactating rats contains soluble factors that promote in vitro prolactin (PRL) release from the pituitary glands of male rats. CM-induced PRL release was confirmed by polyacrylamide gel electrophoresis, ELISA and bioassay. In cultured AP cells challenged with CM, increased intracellular staining with the dye FM1-43 was observed, suggesting vesicular PRL release and subsequent endocytosis. The percentage and hormone content of PRL-containing cells but not of growth hormone-containing cells increased in cultured male AP cells when exposed to CM. When the release of PRL, prelabeled with [3H] leucine for 30 min to 24 h was examined, no stimulatory effect of CM was observed, suggesting that released PRL originates from hormone synthesized more than 24 h earlier. Accordingly, the PRL content of mature granules from male pituitary tissues decreased after CM treatment. These findings were confirmed by electron microscopy immunogold PRL labeling. Treatment with inhibitors of protein synthesis or vesicle trafficking between the endoplasmic reticulum and the Golgi complex did not prevent the stimulatory effect of CM on PRL release. However, blockage of traffic to the plasma membrane completely abolished the effect of CM. These results suggest that CM from the AP of lactators contains soluble factor(s) capable of inducing rapid vesicular release of PRL in the male AP, which originates from preformed, mature granules by mechanisms independent of protein synthesis.