Histidyl-proline-diketopiperazine (cyclo-his-pro) inhibits the suckling- and domperidone-induced transformation of prolactin in the lactating rat

The histidyl-proline-diketopiperazine (cyclo-his-pro) metabolite of TRH inhibited the transformation of prereleasable PRL within the anterior pituitary and as a consequence, the normal rise in plasma PRL levels when injected before suckling. The dose (400 ng) was administered five times at 1-min intervals. PRL suppression was similar to that resulting from dopamine injected at the same rate. Combined injections of cyclo-his-pro and dopamine, each at the 400 ng/min for 5 min rate, provoked a greater inhibition of PRL than did either alone. Neither cyclo-his-pro nor dopamine inhibited the suckling-induced release of PRL in rats whose PRL had previously been transformed by a short (10-min) period of suckling. The stimulatory effects of iv administered domperidone (0.005 or 0.01 mg/rat) and haloperidol (0.2 mg/rat) upon PRL transformation and release in the lactating rat were substantially reduced by cyclo-his-pro in a dose-related fashion over a range of doses from 200-800 ng/min for 5 min. These results demonstrate that cyclo-his-pro inhibits PRL secretion in the lactating rat after suckling, primarily through inhibition of the transformation phase, and that the inhibitory mechanism may involve an interaction with dopamine.     

Evidence that thyrotropin-releasing hormone is not a major prolactin-releasing factor during suckling in the rat

The purpose of this study was to investigate whether TRH could be an important PRL-releasing factor during suckling in the rat. Plasma PRL, TSH, beta-endorphin-like immunoreactivity, and GH responses in serial blood samples from unanesthetized suckled rats were determined. The resulting hormonal profile was compared with that obtained when TRH (500 ng/kg BW, iv) was injected at the onset of suckling. Suckling evoked a rise in plasma levels of PRL, beta-endorphin-like immunoreactivity, and GH, but not in TSH. In contrast, exogenous TRH caused a 9-fold increase in plasma TSH levels during suckling without further increasing the PRL response. Since plasma PRL responses are reportedly enhanced by previous suckling, we also determined plasma PRL and TSH levels when TRH (25 ng/rat, iv) was given 30 min after a brief suckling episode. TRH caused a 2.5-fold increase in plasma TSH, but did not significantly increase plasma PRL levels. Since suckling increases plasma PRL without increasing plasma TSH, and TRH increases TSH but not PRL levels, we conclude that TRH is not a major PRL-releasing factor during suckling.     

Ether releases large amounts of prolactin from rat pituitaries previously "depleted" by short term suckling.

The effect of ether inhalation upon plasma PRL levels was assessed in lactating rats whose pituitary stores of the hormone first had been depleted by a short term (10-min) period of suckling in comparison with those whose PRL stores had not been depleted by prior suckling. Ether, unlike suckling or handling-decapitation stress, did not deplete pituitary PRL and caused only a small increase (36 to 52 ng/ml) in the plasma concentration of the hormone. However, when ether was given 10, 60, or 120 min after the plasma concentration had subsided from a prior 10-min period of suckling, the plasma concentration of PRL in each instance rose within 5--10 min to 200--250 ng/ml and was sustained at that level for the remainder of the 30-min exposure period. The amplitude and profile of the plasma PRL concentration after ether inhalation were similar to those obtained when a second suckling, rather than ether, was administered after the short term suckling. These data support the hypothesis that releasable PRL derives from depleted PRL and also indicate that, once it is formed, releasable PRL remains available for discharge into the circulation for a relatively long period of time.     

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.     

Reversal by thiols of dopamine-, stalk-median eminence-, and zinc-induced inhibition of prolactin transformation in adenohypophyses of lactating rats

Depletion-transformation of PRL is a decrease in tissue PRL detectability which precedes and may often be required for increased PRL release. The present studies were designed to determine whether thiol-disulfide interchange mechanisms may be involved in PRL transformation and release by the pituitary of the lactating rat. Thirds of 8-h nonsuckled lactating rat adenohypophyses were incubated with or without thiols (reduced glutathione, the aminothiol cysteamine, or mercaptoethanol), in the presence of known inhibitors of transformation and release such as dopamine (DA), stalk median eminence (SME) extract, or Zn++. PRL concentrations in pre- and postincubated tissues, as well as the amount of released PRL, were determined by polyacrylamide gel electrophoresis and densitometry. In 30-min incubations without additions, 12-22% of the tissue PRL was depleted; however, in the presence of 17-50 microM DA, all doses of SME extracts tested (0.5-2.0 eq), or 0.1 mM Zn++, depletion was partially or totally prevented and PRL release was inhibited 25-60%. On the other hand, when thiols were added in addition to the above agents, a complete, dose-related, restoration of PRL depletion was obtained. In 120-min incubations, thiols similarly reversed the effects of 0.05 mM DA on depletion, but thiols did not reverse the inhibition of PRL release caused by DA, SME, or Zn++. Other data indicate that thiols alone may inhibit rat PRL release and also facilitate or induce PRL depletion; in bovine PRL granules, thiols reverse Zn++ inhibition of PRL release and detectability. These data suggest that thiol-disulfide interchange reactions may be importantly involved in both depletion-transformation and in secretion. The precise thiol sensitivity of the two processes does not appear identical, secretion being more sensitive to DA and less sensitive to thiols than depletion-transformation.     

Calcitonin inhibition of prolactin secretion in lactating rats: mechanism of action

The effects of intracerebroventricular (10 ng/rat) or intravenous (10 or 40 microg/15 min/rat) administration of salmon calcitonin (sCT) on the prolactin (PRL) response to suckling and the activity of tyrosine hydroxylase (TH) were examined in lactating rats. Plasma concentration of PRL increased dramatically in control rats after the onset of the suckling stimulus, while administration of sCT resulted in inhibition of PRL response to suckling. The action of sCT was much more effective with intracerebroventricular administration, which totally blocked PRL release, compared to intravenous administration. The intracerebroventricular administration of sCT increased TH activity of tuberoinfundibular dopamine neuron (TIDA) in the stalk-median eminence, as measured by DOPA accumulation, while completely suppressing the PRL response to suckling. Injection of alpha-methyl-p-tyrosine (alpha-MT; 50 mg/kg), an inhibitor of TH and thus dopamine synthesis, increased PRL levels, and suckling caused a further increase in plasma concentrations of PRL. Injection of sCT (intracerebroventricularly) did not inhibit the PRL response to suckling in the presence of a depletion of dopamine. These results suggest that sCT inhibition of PRL secretion in lactating rats is mediated mainly by TIDA neurons without involvement of other neuroendocrine mechanisms.     

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)     

PROLACTIN SECRETION IN GIRLS WITH ISOLATED GONADOTROPHIN DEFICIENCY

Plasma prolactin, basal levels and the response to an i.v. injection of TRH (100 microgram/m2) was determined in five girls with isolated gonadotrophin deficiency of hypothalamic origin before and after at least 3 months cyclic replacement therapy with conjugated oestrogens (1.25 mg/day). The basal plasma prolactin levels were similar during both tests, however, during oestrogen therapy the mean peak response to TRH almost doubled and the sum of all the values obtained (basal, +15, +30 and +60 min) was significantly higher. It is of note that even upon prolonged oestrogen deprivation the releasable prolactin response to TRH was adequate.     

Thyrotrophin-releasing hormone receptor 1 and prothyrotrophin-releasing hormone mRNA expression in the central nervous system are regulated by suckling in lactating rats

BACKGROUND: The accepted function of the hypothalamic peptide, thyrotrophin-releasing hormone (TRH), is to initiate release of thyrotrophin (TSH) from the pituitary. A physiological role for TRH in lactating rats has not yet been established. METHODS: Tissues were prepared from random-cycling and lactating rats and analysed using Northern blot, real time RT-PCR and quantitative in situ hybridisation. RESULTS: This study demonstrates that TRH receptor 1 (TRHR1) mRNA expression is up-regulated in the pituitary and in discrete nuclei of the hypothalamus in lactating rats, while proTRH mRNA expression levels are increased only in the hypothalamus. The results were corroborated by quantitative in situ analysis of proTRH and TRHR1. Bromocriptine, which reduced prolactin (PRL) concentrations in plasma of lactating and nursing rats, also counteracted the suckling-induced increase in TRHR1 mRNA expression in the hypothalamus, but had an opposite effect in the pituitary. These changes were confined to the hypothalamus and the amygdala in the brain. CONCLUSIONS: The present study shows that the mechanisms of suckling-induced lactation involve region-specific regulation of TRHR1 and proTRH mRNAs in the central nervous system notably at the hypothalamic level. The results demonstrate that continued suckling is critical to maintain plasma prolactin (PRL) levels as well as proTRH and TRHR1 mRNA expression in the hypothalamus. Increased plasma PRL levels may have a positive modulatory role on the proTRH/TRHR1 system during suckling.     

Vasoactive intestinal peptide is a physiological mediator of prolactin release in the rat

To determine whether VIP functions as a physiological PRL-releasing factor, the effects of immunoneutralization of endogenous vasoactive intestinal peptide (VIP) on the PRL secretory response to suckling and ether stress were assessed. Using a porcine VIP-thyroglobulin conjugate as antigen, a peptide-specific antiserum was generated in a rabbit which bound porcine VIP with a Kd of 5.1 X 10(-11) M and a maximum binding capacity of 1830 ng/ml. In a RIA, this antiserum demonstrated immunoreactive VIP in tissue extracts of various regions of the brain and gastrointestinal tract. IR VIP in extracts of cerebral cortex and hypothalamus coeluted with synthetic porcine VIP on Bio-Gel P-30 column chromatography. Using chronically implanted right atrial catheters for blood sampling to avoid effects of stress and anesthesia, PRL blood levels in normal controls began to rise almost immediately after initiation of suckling from basal values of 3.0 +/- 0.9 ng/ml to reach a plateau of 158.1 +/- 33.5 ng/ml after 40 min. When the VIP antiserum was administered immediately before initiation of suckling, the onset of the PRL response was delayed by 40 min, but PRL levels then rose at a slower rate to reach the plateau level of normal animals approximately 80 min later. When VIP antiserum was administered to rats who had been suckling for at least 1 h, PRL levels fell from a mean basal elevated level of 152.7 +/- 16.0 ng/ml to a nadir of 50.4 +/- 9.1 ng/ml 80 min after injection and then gradually returned to basal levels. The effect of VIP antiserum was studied in rats in whom PRL secretion was increased by exposure to ether, a stimulus that acts on the release phase of PRL secretion. In rats in whom the depletion-transformation of PRL was induced by a prior brief period of suckling, subsequent exposure to ether caused a rise in serum PRL levels. The response was completely blocked in rats given VIP antiserum, whereas animals given nonimmune serum showed a significant increase in serum PRL to 38.6 +/- 17.3 ng/ml. We conclude from these studies that VIP mediates the acute PRL response to suckling and is required for maintenance of PRL levels in continuously suckling animals but is not the only factor causing PRL elevation. Complete abolition by the VIP antiserum of the PRL response to ether indicates that the effect of the anesthetic is mediated entirely by the release of VIP. These findings are consistent with the view that VIP is a physiological PRL-releasing factor in the rat.     

Antagonist of pituitary adenylate cyclase activating polypeptide suppresses prolactin secretion without changing the activity of dopamine neurons in lactating rats

Pituitary adenylate cyclase activating polypeptide (PACAP) is a relatively new neuropeptide, and it has a potent stimulatory effect on adenylate cyclase activity in rat pituitary cells. However, the role of PACAP in the physiological control of prolactin (PRL) secretion is still unclear. In the present study, we investigated the physiological significance of endogenous PACAP on PRL secretion in lactating rats. On lactation days 7-8, pups were separated from their mother rats for 5 h before the onset of suckling and PACAP6-38 (16 microg), a receptor antagonist, was injected through the lateral ventricle cannula just after the removal of pups. The effects of PACAP6-38 on PRL and oxytocin secretion, and on the activity of tyrosine hydroxylase (TH), were examined after the onset of suckling. Administration of PACAP6-38 inhibited PRL levels in response to suckling, but it did not affect the activity of TH, as measured by DOPA accumulation at 15 min after administration of NSD 1015 (25.0 mg/kg), an L-aromatic amino acid decarboxylase inhibitor, or the plasma concentrations of oxytocin in lactating rats. Injection of alpha-methyl-p-tyrosine (alpha-MT; 50 mg/kg), an inhibitor of dopamine synthesis, increased PRL levels, and suckling caused a further increase in the plasma concentrations of PRL. An injection of PACAP6-38 (i.c.v.) also inhibited the PRL response to suckling under dopamine depletion. These results suggest that endogenous PACAP acts as a neurotransmitter or neuromodulator within the hypothalamus and plays an important role for PRL secretion in lactating rats. Endogenous PACAP may regulate PRL secretion, possibly mediated by PRL-releasing factors such as vasoactive intestinal polypeptide or vasopressin.     

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)     

A study on the prolactin releasing mechanism using an in vitro perfusion system with a cell column of cultured rat anterior pituitary cells

It is well-known that the hypothalamus predominantly exerts an inhibitory control on prolactin secretion and that dopamine (DA) is the main prolactin inhibiting factor (PIF). In addition, the hypothalamus contains prolactin-releasing factors (PRF). Thyrotropin-releasing hormone (TRH), vasoactive intestinal polypeptide (VIP) and peptide-histidine-isoleucine (PHI) are the components of PRF. However, the detailed mechanism by which the peptides release prolactin (PRL) at the pituitary level is still unknown. Therefore, in this paper, an in vitro perifusion system using the cell column of cultured rat pituitary cells attached on Cytodex beads was employed to investigate the mechanism of PRL release. The rat anterior pituitary cells were isolated using collagenase, and the dispersed pituitary cells were cultured with swollen Cytodex beads in Dulbecco's modified Eagle medium (DMEM) containing fetal calf serum at 37 degrees C in 5% CO2 and 95% air for 2--3 days. The cultured anterior pituitary cells attached on Cytodex beads were packed in a column and perifused with DMEM at a constant flow rate of 0.4 ml/min using a peristaltic pump. The following results were obtained. A five minute perifusion with 100 pg/ml to 100 ng/ml TRH caused a significant increase of PRL in a dose-related manner. A continuous perifusion with 2 ng/ml or 10 ng/ml DA inhibited PRL release in a dose-related manner. When TRH at a dose of 1 ng/ml, 10 ng/ml or 100 ng/ml was perifused for 120 min at a rate of 0.4 ml/min, a large amount of PRL was released during the early period of the TRH infusion, and then the PRL release gradually decreased to the basal levels in spite of the continuous TRH infusion. An additional TRH, of which the concentration was ten-fold higher than the TRH level in the continuous infusion, when added at the end of the continuous TRH infusion, had no effect on PRL release. On the other hand, a 5 minute TRH infusion given at 30 min after the end of the continuous TRH infusion caused a significant increase in PRL release. A continuous perifusion with 1 mM 8-bromo-cyclic AMP caused a small but continuous PRL release. An additional continuous 8-bromo-cyclic AMP infusion during the late period of a continuous TRH infusion caused a continuous PRL release similar to that induced by the continuous infusion of cyclic AMP only. A short period perifusion with 1 X 10(-9)M to 1 X 10(-7)M of vasoactive intestinal polypeptide (VIP) enhanced a significant increase of PRL release in a dose-related manner, but the amounts of PRL release induced by VIP were smaller than those induced by TRH.(ABSTRACT TRUNCATED AT 400 WORDS)     

Does bromocriptine block thyrotropin-releasing hormone-induced prolactin release during pregnancy?

PRL responses to 200 microgram of iv TRH were measured in 16 healthy women with normal early pregnancy before and at the endo of bromocriptine treatment of 5.0--7.5 mg daily for 1--2 weeks. Before the start of bromocriptine, TRH caused a PRL elevation from 19.1 +/- 2.2 to 95.2 +/- 12.6 ng/ml (mean +/- SE) after 20 min, with a mean maximal PRL increment of 71.7 +/- 11.6 ng/ml. Bromocriptine suppressed basal plasma PRL level to 3.6 +/- 0.8 ng/ml (P less than 0.001). TRH then caused a PRL rise to 18.8 +/- 1.8 ng/ml at 20 min, with a mean maximal PRL increment of 15.7 +/- 1.8 ng/ml. The absolute PRL response was significantly smaller (P less than 0.001) during bromocriptine intake than before, whereas the mean percent increments in PRL levels after TRH administration were similar in the presence and absence of bromocriptine. Fifteen of these women were restudied with TRH stimulation 4--6 weeks after legal abortion, and the PRL responses to TRH were normal. When 7 of these women were once again treated with bromocriptine and retested with TRH, no absolute or relative PRL response to TRH emerged. These results release differs between the pregnant and nonpregnant states.     

Control of prolactin release induced by suckling

In the present study, the role of dopamine and TRH in suckling-induced PRL release was investigated. Bupropion, a dopamine reuptake blocker, increased hypophysial stalk dopamine levels and inhibited suckling-induced PRL release. A short period of suckling, thought to induce a transient decrease in hypothalamic dopamine release, led to higher PRL levels following an iv injection of TRH than in rats which had not nursed their young for a short period after 4- to 6-h separation. These results, in combination with previous data, suggest that a decrease in hypothalamic dopamine release is important for suckling-induced PRL release. Increased PRL release may be in part due to an augmented hypothalamic release of TRH. Since serotonergic mechanisms seem involved in TRH release, lactating rats were treated with drugs acting on serotonergic pathways. Parachlorophenylalanine and pizotifen did not alter suckling-induced PRL release. Methysergide, a serotonin receptor blocker, prevented this PRL release when administered ip but not when injected into the lateral brain ventricle. Since methysergide is converted peripherally into metabolite(s) with dopamine agonistic activity, its effect on suckling-induced PRL release may be due to this action, rather than to its action on serotonin receptors. Thus, these data do not indicate that serotonergic mechanisms are important for suckling-induced PRL release. Passive immunization against TRH inhibited suckling-induced PRL release, indicating that TRH is a hypophysiotropic mediator of this PRL release.     

Suckling-induced changes of vasoactive intestinal peptide concentrations in hypothalamic areas implicated in the control of prolactin release

The present study was designed to investigate whether the vasoactive intestinal peptide (VIP) concentration in hypothalamic nuclei, dorsal raphe nucleus (DR) and pituitary lobes of lactating rats changes in physiological situations when prolactin (PRL) secretion is stimulated (suckling) or inhibited (pup separation). In addition VIP levels in blood plasma were determined in both situations. Acute suckling induced changes in VIP concentration only in the rostral part of the anterior hypothalamic (rAHN) and the paraventricular (PVN) nuclei of all the brain areas examined. VIP concentration in the rAHN increased at 5 min from 3.52 +/- 0.30 (mean +/- SEM) to 8.67 +/- 1.91 ng/mg protein (p less than 0.05) but fell to baseline values after 30 min suckling (p less than 0.05; 5 vs. 30 min). Although changes in VIP concentration in the suprachiasmatic nucleus (SCN) did not attain statistical significance, they followed the same trends as the changes of VIP in the rAHN. The opposite pattern of changes was observed in the PVN with a decrease in VIP concentration following 5 min suckling (p less than 0.01). At 30 min the VIP values showed a trend towards 0-min values. Pup removal did not affect VIP concentrations in the rAHN, PVN, SCN, median eminence, supraoptic nucleus and DR. VIP values were not detectable in the arcuate nucleus in any of the experimental situations examined. Lactation increased VIP concentration only in the rAHN and PVN when lactating rats with their pups were compared with virgin female diestrous rats. VIP concentration in the anterior lobe of the pituitary from lactating rats did not change with pup separation or suckling.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of exteroceptive pup stimuli on the responsiveness of prolactin release mechanisms to suckling stimuli in the lactating rat

In the lactating rat, the neural stimulus of suckling not only acutely releases PRL but also maintains the responsiveness of PRL regulatory mechanisms to subsequent nursing stimuli. Beginning near midlactation exteroceptive pup stimuli (ECS) can acutely release PRL. We have examined the capacity of this signal also to maintain the responsiveness of PRL release mechanisms to subsequent suckling stimuli. On day 14 postpartum lactating rats were either isolated from their young or exposed to ECS (without suckling) for approximately 24 h. When both groups were later nursed, plasma PRL of mothers earlier exposed to ECS rose significantly higher than that of subjects previously isolated from their young. Suckling produced a significant depletion in pituitary PRL and GH concentrations of ECS-exposed mothers; it did not produce a similar depletion in the pituitaries of the previously isolated group. When the pups were returned for suckling, ECS-exposed mothers began to nurse their pups substantially faster than did females of the isolated group. During the 6 h after nursing, the mammary glands of ECS-exposed mothers secreted milk at twice the rate of mammary glands of the isolated females. We conclude that ECS can maintain the capacity of neuroendocrine mechanisms to respond to galactopoetic hormone-releasing stimuli (consequently enhancing milk secretion) and support the maternal behavior pattern necessary for suckling to occur. As a result, ECS may become an important factor during later stages of lactation, compensating for the decline in suckling stimuli known to occur at that time.     

Prolactin-releasing and release-inhibiting factor activities in the bovine, rat, and human pineal gland: in vitro and in vivo studies.

The effects of crude extracts of bovine, rat, and human pineal glands on prolactin (PRL) release were studied using an in vitro system. In addition, the effects of a known pineal constituent, arginine vasotocin (AVT), and crude bovine pineal extract (bPE) on PRL secretion were studied in vivo. Normal male rat hemipituitaries (HP), incubated with bPE (13 mg tissue/HP)released 200%, 150%, and 285% more PRL into the medium than did their corresponding untreated control halves incubated in either Medium 199 alone, hypothalamic extract, or cerebral cortical extract, respectively. HP incubated with either rat (6 mg of tissue/HP) or human (25 mg of tissue/HP) pineal extract released 110% and 75% more PRL, respectively, than did their corresponding untreated control halves. HP exposed to 10 mg tissue eq of either bovine pineal fraction A1 or bovine pineal fraction A3 released 88% and 63%, respectively, less PRL than did their corresponding untreated control halves incubated in Krebs-Ringer Bicarbonate (KRB) medium. Quantitites of melatonin, thyrotropin-releasing hormone (TRH), or estrogen, comparable to those found in the pineal, had no significant effect on PRL secretion in vitro. The iv injection of either bPE (90 mg tissue/rat) or AVT (10 mug/rat) into estrogen and progesterone-treated male rats resulted in a 40% and 138% increase, respectively, in plasma PRL titers, 10 min after injection, over pre-injection control levels. The per cent of increase in plasma PRL levels in these animals was significantly greater than that observed in control rats receiving either saline or cortical extract. The results suggest that crude extracts of pineal glands of three different species contain prolactin-releasing factor (PRF) activity which is probably not due to any endogenous melatonin, TRH, or estrogen that may be present. Conversely, two bovine pineal fractions, A1 and A3, appeared to exhibit prolactin-inhibiting factor (PIF) activity. We have concluded that the pineal gland may serve as an alternate or supplemental source of PRF and/or PIF.     

Evidence for a physiological role for oxytocin in the control of prolactin secretion

The presence of oxytocin (OT) in neuronal elements of the external layer of the median eminence and in hypophysial portal plasma suggests a role for the peptide in the control of anterior pituitary function. We have reported previously that OT stimulates PRL release in vitro; therefore, we attempted to establish evidence for a physiological PRL-releasing role for OT. Plasma OT levels rose significantly just before the PRL surges occurring during a suckling stimulus in lactating rats (10 min after pup reinstatement vs. 15 min for PRL) and 48 h after estrogen injection in ovariectomized (OVX) rats (at 1200 h vs. 1300 h). Dispersed anterior pituitary cells harvested from lactating female rats and OVX estrogen-primed rats released PRL in a specific, significant, and dose-related fashion when perifused in vitro with incubation medium containing 10(-7)-10(-9) M OT, doses similar to levels found previously in hypophysial portal plasma. Infusion of antiserum specific for OT into lactating females before pup reinstatement and into estrogen-primed OVX rats 2 h before the expected release of endogenous OT delayed and significantly reduced subsequent PRL surges compared to levels in saline-or normal rabbit serum-infused rats; however, PRL release was not completely abolished. These data indicate that OT plays a physiological role in the hypothalamic control of PRL secretion and further suggest the importance of multiple factors in coordinated regulation of PRL release.     

Microtubules are mobilized in the lactating rat anterior pituitary gland during suckling

Microtubules in the lactating rat anterior pituitary gland were depolymerized into their constituent tubulin dimers by exposure of the pituitary to 4 C. The tubulin then was quantified with the [3H]colchicine binding assay, which was adapted for use with individual rat pituitary glands. The binding of [3H]colchicine to the tubulin fraction contained in high speed supernatants of lactating rat pituitary glands proved to be specific and saturable, and was pH, temperature, and time dependent. The amount of [3H]colchicine bound was linear over the range of protein concentrations tested (2-22 micrograms). To determine whether suckling affected the levels of microtubules in the anterior pituitary, tubulin levels were measured in groups of lactating rats after they were suckled (six pups per litter) for 0, 5, 10, 15, and 30 min following 4-5 h of nonsuckling. The tubulin concentration in the anterior pituitary progressively increased from 4 to 22 mumol during the 30 min of suckling; the increase was statistically significant (P less than 0.05) by the 10th min. Plasma PRL levels analyzed from trunk blood rose from 5 to 75 ng/ml during the 30-min suckling period. These results indicate that a mobilization of microtubules occurs in the anterior pituitary at the same time that PRL is being transformed and released into the circulation.