Mice deficient in the interferon type I receptor have reduced REM sleep and altered hypothalamic hypocretin, prolactin and 2',5'-oligoadenylate synthetase expression.

We report that mice with a targeted null mutation in the interferon type I receptor (IFN-RI), which cannot respond to such IFNs as IFNalpha and IFNbeta, have a 30% reduction in time spent in spontaneous rapid eye movement sleep (REMS) as a consequence of a reduced number of REMS episodes. Time spent in nonrapid eye movement sleep (NREMS) was essentially unaltered in IFN-RI knockouts (KOs) compared to 129 SvEv controls. Body temperature and locomotor activity were similar in both strains of mice. Hypothalamic expression of mRNAs for molecules previously linked to sleep-wake regulation and an IFN-inducible antiviral gene, 2',5'-oligoadenylate synthetase 1a (OAS), were determined by real-time reverse-transcriptase polymerase chain reaction (RT2-PCR). The level of hypocretin A mRNA was elevated in IFN-RI KO mice compared to 129 SvEv mice, while prolactin mRNA and OAS mRNA levels were suppressed. Vasoactive intestinal peptide (VIP) and corticotropin-releasing hormone (CRH) mRNA levels were unchanged relative to controls. Serum prolactin levels were similar in both strains. Results are consistent with the hypothesis that increased hypocretin and reduced prolactin in the hypothalamus of IFN-RI KO mice are responsible for their reduced REMS. In addition, the reduced OAS expression may result in modulation of prolactin receptor signaling and thus contribute to suppression of REMS.

Deficiency of growth hormone-releasing hormone signaling is associated with sleep alterations in the dwarf rat.

The somatotropic axis, and particularly growth hormone-releasing hormone (GHRH), is implicated in the regulation of sleep-wake activity. To evaluate sleep in chronic somatotropic deficiency, sleep-wake activity was studied in dwarf (dw/dw) rats that are known to have a defective GHRH signaling mechanism in the pituitary and in normal Lewis rats, the parental strain of the dw/dw rats. In addition, expression of GHRH receptor (GHRH-R) mRNA in the hypothalamus/preoptic region and in the pituitary was also determined by means of reverse transcription-PCR, and GHRH content of the hypothalamus was measured. Hypothalamic/preoptic and pituitary GHRH-R mRNA levels were decreased in the dw/dw rats, indicating deficits in the central GHRHergic transmission. Hypothalamic GHRH content in dw/dw rats was also less than that found in Lewis rats. The dw/dw rats had less spontaneous nonrapid eye movement sleep (NREMS) (light and dark period) and rapid eye movement sleep (REMS) (light period) than did the control Lewis rats. After 4 hr of sleep deprivation, rebound increases in NREMS and REMS were normal in the dw/dw rat. As determined by fast Fourier analysis of the electroencephalogram (EEG), the sleep deprivation-induced enhancements in EEG slow-wave activity in the dw/dw rats were only one-half of the response in the Lewis rats. The results are compared with sleep findings previously obtained in GHRH-deficient transgenic mice. The alterations in NREMS are attributed to the defect in GHRH signaling, whereas the decreases in REMS might result from the growth hormone deficiency in the dw/dw rat.

Antiserum to growth hormone decreases sleep in the rat.

To determine whether an acute withdrawal of growth hormone (GH) alters sleep, the effects of antiserum to GH (GH-AS) on sleep were studied in the rat. Sleep-wake activity and cortical brain temperature (Tc) were recorded for 2 days after systemic injection of physiological saline. Then, one group of rats (n = 6) received GH-AS whereas another group of rats was injected with normal rabbit serum (n = 6). The injections were given 1 h before light onset, and the rats' behaviors were recorded for 23 h during the subsequent 12-hour light and 12-hour dark period. Sleep and Tc were not altered after normal rabbit serum. The durations of both rapid eye movement sleep (REMS) and non-REMS (NREMS), and the EEG slow-wave activity during NREMS were significantly suppressed during the light period following the injection of GH-AS. Tc tended to decrease for 3 h and a small rise was observed thereafter during the light period, but these changes were not statistically significant. The assay of GH in plasma samples obtained at 30-min intervals for 5 h after injection of normal rabbit serum or GH-AS verified the decreases in plasma GH concentrations in response to GH-AS. It is suggested that GH may promote sleep possibly via some metabolic actions.

Sleep in rats rendered chronically hyperprolactinemic with anterior pituitary grafts.

A hyperprolactinemic rat model [rats bearing anterior pituitary grafts under the capsule of the kidney (AP-grafted rats)] was used to study sleep-wake activity and cortical brain temperature (T(crt)). Fisher 344 male rats (n = 24) were implanted with anterior pituitaries from rat pups; the control rats (n = 12) were sham-operated. Sleep-wake activity and T(crt) were recorded for 2 days between weeks 3 and 7 after surgery. The hyperprolactinemic state of the rats was confirmed by plasma prolactin (PRL) assays on week 7 and by determination of PRL mRNA levels in the anterior pituitary of the AP-grafted rats. Neither growth hormone plasma concentration nor pituitary mRNA levels were affected by the pituitary grafts. Duration of non-rapid eye movement sleep (NREMS) was slightly enhanced in the AP-grafted rats. A large increase in rapid eye movement sleep (REMS) during the 12-h light period was the major effect of the implantation of the extra pituitaries. Both the duration and the frequency of the REMS episodes increased and persisted for weeks 4-7 post-implantation. The nocturnal states of vigilance, T(crt), and intensity of NREMS (EEG slow wave activity) were not altered. The results clearly indicate that the enhancements in REMS persist during hyperprolactinemia, and support the hypothesis that PRL possesses REMS-promoting activity.

Prolactin-like biological activity in the pituitary glands of the marsupial Monodelphis domestica and of the amphibian Rana pipiens detected by a colorimetric Nb2 lymphoma cell proliferation assay.

An inexpensive and reliable colorimetric microplate version of the Nb2 lymphoma cell proliferation bioassay for prolactin (PRL) was developed and optimized. The useful range of the assay is between 0.1 and 12.8 ng/ml in terms of rat pituitary PRL. The assay can accommodate up to 20 microl sample/well. The physiological relevance of the assay was verified by measuring thyrotropin-releasing hormone (TRH)-induced secretion of PRL in pituitary cultures and in serum samples of neonatal rats. Through the use of the colorimetric Nb2 assay, PRL-like bioactivities were demonstrated in pituitary extracts of the marsupial, Monodelphis domestica (1.47 ng PRL/microg protein) and of the amphibian, Rana pipiens (1.86 ng PRL/microg protein). Marsupial and amphibian PRLs are predicted to have low specific activities in the Nb2 assay. Since the PRL values were calculated in terms of a rat PRL standard, they probably underestimate the amounts of PRL present. Parallel dose-response curves were obtained with these pituitary extracts and standard rat PRL over a wide range of dilutions. The Nb2 bioassay may serve as a tool for the purification of PRL from these species. The colorimetric version of the Nb2 bioassay may be a useful alternative to traditional Nb2 assays that rely on direct cell count or [3H]thymidine uptake.

Growth hormone release induced by growth hormone-releasing hexapeptide is not mediated by thyrotropin-releasing hormone in neonatal rats.

GH-releasing hexapeptide (GHRP-6) and nursing stimulate GH secretion in rat pups via GH-releasing factors (GRFs: distinct from GH-releasing hormone (GHRH). It was determined whether GH secretion induced by GHRP-6 or nursing was mediated by TSH-releasing hormone (TRH) in 2-d-old rats. In vitro. GHRP-6 and TRH stimulated GH secretion of neonatal pituitary glands. At their maximally effective doses, GHRP-6 and TRH evoked approximately equal GH responses. Treatment with a combination of the maximally effective doses of GHRP-6 and TRH resulted in a GH response comparable to that evoked by either treatment alone. GHRP-6 in vivo induced a greater GH response than did TRH. Treatment in vivo with a combination of the maximally effective doses of GHRP-6 and TRH synergistically increased serum GH levels. Unlike GHRP-6 TRH was an effective stimulus of prolactin secretion either in vitro or in vivo. Nursing was an effective stimulus for GH secretion, but only marginally increased serum prolactin levels. The effects of either of the peptides and nursing on GH secretion were additive. These results suggest that GHRP-6 stimulates GH secretion both by acting directly on the pituitary gland and indirectly via a hypothalamic GRF. The indirect effect appears to be greater. The alternative GRFs released by GHRP-6 or nursing are distinct from each other and from TRH. These findings suggest that alternative GRFs play a significant role in the regulation of GH secretion in neonatal rats.

Increase of prolactin mRNA in the rat hypothalamus after intracerebroventricular injection of VIP or PACAP.

Vasoactive intestinal peptide (VIP), the structurally homologous pituitary adenylate cyclase-activating peptide (PACAP) and the pituitary hormone, prolactin (PRL) enhance rapid eye movement sleep (REMS). VIP and PACAP are both inducers of PRL gene expression and release in the pituitary gland. Little is known about PRL regulation in the brain although it is hypothesized that the REMS-promoting activity of i.c.v. administered VIP may be mediated via the activation of cerebral PRL. To test whether VIP or PACAP in fact increase intracerebral mRNA, the peptides (VIP: 30 or 300 pmol; PACAP: 220 pmol) were injected i.c.v. into rats at dark onset. 1 h later, cDNA was synthesized from purified hypothalamic mRNA. Standardized amounts were analysed for PRL using the polymerase chain reaction followed by Southern blotting and hybridization. Compared with beta-actin mRNA levels, both VIP and PACAP increased PRL mRNA levels in a dose-dependent fashion though VIP was more effective on a molar basis. The previously reported alternatively spliced PRL mRNA (lacking exon 4) was not detected. The data support the hypothesis that the REMS-promoting activity of central VIP and PACAP might be mediated by cerebral PRL.

Involvement of prolactin in the REM sleep-promoting activity of systemic vasoactive intestinal peptide (VIP).

The involvement of pituitary prolactin (PRL) in systemic vasoactive intestinal peptide (VIP)-induced sleep was studied. Male rats were implanted with electrodes for EEG-recording, with brain thermistors to record cortical temperature (Tcrt) and with chronic intracardial catheters to obtain blood samples and to deliver substances. One group of rats (n = 8) received normal rabbit serum (NS)+physiological saline (SAL) on the baseline day and was injected with NS+VIP on the experimental day. In the other group of rats (n = 6), the baseline day was followed by administration of PRL-antiserum (PRL-AS) + VIP on the experimental day. The sera and VIP or SAL were injected 30 min before and at light onset, respectively. Sleep-wake activity was then recorded for the next 12-h light period. Systemic VIP-stimulated PRL secretion as measured by RIA in serial samples obtained hour 1 postinjection. VIP also elicited selective increases in REM sleep (REMS) in the rats pretreated with NS. Tcrt was not affected by VIP. Administration of PRL-AS blocked the increase in circulating levels of free (non-IgG-bound) PRL and prevented VIP-enhanced REMS. Comparisons of the sleep effects of PRL-AS+VIP with the previously reported changes in sleep after PRL-AS alone indicate that PRL has a major role in the mediation of the REMS-promoting activity of systemic VIP. The results suggest that an increased release of endogenous pituitary PRL modulates REMS.

Bioactive and immunoreactive variants of prolactin in milk and serum of lactating rats and their pups.

Prolactin (PRL)-like bioactivity (in Nb2 lymphoma assay) and immunoreactivity (in radioimmunoassay (RIA)) in rat milk, maternal and neonatal sera and in neonatal rat pituitary cultures were investigated. The PRL-like bioactivity in the water-soluble fraction of rat milk was high and exceeded its immunoreactivity 5.8-, 4.0- and 2.1-fold, on days 2, 12 and 22 of lactation respectively. The elevated bioactivity to immunoreactivity (B/I) ratio of PRL in milk was not due to the presence of interleukin-2 (IL-2) in milk, since the proliferation of the CTLL-2 murine T cells, which are not sensitive to PRL, was promoted by IL-2 but not by milk. Serum levels of immunoreactive PRL were low in sera of non-weaned rat pups on days 2, 12 and 22 postpartum. Similar to milk, the B/I ratio of PRL in sera of rat pups was high and decreased with time postpartum. Pituitary glands of pups obtained on days 2, 12 and 22 secreted progressively increasing amounts of PRL in vitro; the B/I ratio ranged between 1.2 and 2.1 without a significant change. The relative concentrations of size variants in milk were not proportional to those in serum of lactating rats on day 2 postpartum as assessed by Sepharcryl S-100 HR gel permeation chromatography and Nb2 bioassay or RIA. Size variants of biologically active PRL were abundant in early milk and gradually diminished as lactation progressed: a partially resolved peak representing monomeric to dimeric PRL variants (relative molecular weights ranging between 18 k and 42 k) became progressively narrower between days 2 and 22. Biologically active and immunoreactive PRLs displayed disparate elution profiles. The elution profile of PRL in sera of neonatal rats on day 2 post-partum was different from that of maternal serum or milk. The major immunological (and possibly biological) PRL-like activity eluted as two adjacent peaks at 2.2 k and 1.5 k, raising the possibility that fragments of milk-borne PRL were absorbed from the gut after partial proteolytic degradation. In contrast with PRL, GH (which is present in rat milk only in minute concentrations) did not show heterogeneity in sera of 2-day-old rat pups in gel permeation chromatography. The present results demonstrate that the concentrations of PRL-like activity in rat milk and newborn rat serum have been grossly underestimated because levels have been measured by RIA. The high B/I ratio of PRL in milk and neonatal sera is due to the presence of PRL-related compounds.(ABSTRACT TRUNCATED AT 250 WORDS)

Mammary epithelial cells of lactating rats express prolactin messenger ribonucleic acid.

The presence of prolactin (PRL) mRNA in the mammary gland, placenta, and pituitary gland of lactating and pregnant rats was investigated by polymerase chain reaction (PCR). Polyadenylated RNA was prepared from total RNA samples by oligo(dT)-cellulose chromatography, and complementary cDNAs were synthesized. A standardized amount of cDNA from each sample was used as the template in a Taq PCR under high-stringency conditions. PCR amplified a signal with the predicted size of approximately 375 bp in mammary and pituitary glands of lactating and pregnant rats, and in placentae of pregnant rats. This band specifically hybridized with a probe overlapping the entire sequence of the mature rat (r) PRL mRNA in Southern blot analysis. When the rPRL-specific primers were used, PCR revealed no signal in the liver or in lactating mammary gland explants cultured in vitro for 48 h, while the same cDNA preparations gave strong signals for beta-actin. The viability of the mammary gland explants was also suggested by their ability to secrete immunoreactive casein in vitro. PRL mRNA was localized in the epithelium of alveoli and ducts of the lactating mammary gland by in situ hybridization. These data provide evidence that the PRL gene is expressed in the mammary gland of pregnant and lactating rats, and suggest that the mammary gland might contribute to PRL in milk by de novo synthesis. Thus, while the placenta is an exogenous source of PRL-like activities for the fetus in utero, the mammary gland might take over this function after birth.

Interaction between the alpha 2-adrenergic system and nursing in the regulation of growth hormone secretion in the neonatal rat.

Separation of neonatal rats from their mothers decreases, while a subsequent period of suckling (nursing) increases, serum growth hormone (GH) levels in neonatal rats. Milk-borne (humoral) factors and neural factors inherent in mother-offspring interaction have been implicated in these phenomena. Conflicting reports have demonstrated the alpha 2-adrenergic agonist clonidine to increase and to decrease serum GH levels in 10-day-old rats. The present experiments were aimed at testing whether an interaction between the alpha 2-adrenergic system and the nursing-induced changes in GH secretion could account for the discrepancy. Rat pups were treated with clonidine (150 micrograms/kg) or the alpha 2-adrenergic antagonist yohimbine (10 mg/kg), and the drug treatment was combined with separation of the mothers and nursing. Yohimbine did not affect serum GH levels in separated two-day-old pups (i.e. basal levels of the hormone), but prevented the nursing-induced increase in serum GH concentration. In two-day-old pups, clonidine had no effect on basal GH levels but, like yohimbine, prevented the increase in serum GH normally associated with nursing. Both yohimbine and clonidine prevented active sucking behavior, i.e. the pups did not search for and/or attach to the nipples of their mothers. Moreover, the pups treated with yohimbine and clonidine were cooler to the touch than the littermate controls. In eight-day-old pups, yohimbine prevented the nursing-induced increase in serum GH and decreased GH levels below the saline-injected, separated control.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro control of prolactin (PRL) and growth hormone secretion of neonatal rat pituitary glands: effects of ovine PRL, salmon calcitonin, endothelin-3, angiotensin II, bromocryptine and somatostatin.

Bromocryptine potently decreased prolactin (PRL) secretion of pituitary glands of 2-day-old rats in vitro (up to 85% inhibition; ED50 between 0.1 and 1.0 nM) without altering the bioactivity to immunoreactivity (B/I) ratio. Bromocryptine tended to suppress growth hormone (GH) secretion although the effect did not reach statistical significance. Angiotensin-II (A-II; 1-1000 nM) stimulated PRL secretion in a dose-dependent manner without affecting secretion of GH. The B/I ratio of PRL secreted in response to A-II was increased. Somatostatin (SRIF) had no effect on PRL secretion but inhibited GH secretion in a dose-dependent manner; significant inhibition (50%) was observed at 100 nM. A 6-h exposure to ovine PRL (oPRL) in concentrations equipotent with 1.2-120 ng/ml rat PRL (rPRL) in the Nb2 bioassay had no effect on immunoreactive rPRL secretion. Salmon calcitonin (sCT) and endothelin-3 (ET-3; 0.1-100 nM) failed to inhibit secretion of PRL or GH. PRL secretion was slightly stimulated by sCT with no apparent dose-response relationship. The present findings suggest that neonatal pituitary glands do not display autoregulation of PRL secretion, and sCT and ET-3 (either endogenous or milk-derived) may not function as PRL inhibiting factors in 2-day-old pups. Thus, the receptors of PRL, sCT and ET-3 on lactotropes, or their functional coupling with inhibition of basal PRL secretion, occur at a later stage of development. The specificity of the PRL releasing factor (PRF) activity of A-II at this age is unique for established PRFs and might reflect a physiological function of PRL in osmoregulation. The increased B/I ratio of PRL secreted in response to A-II may be due to the release of specific PRL variants, and might be a sign of functional heterogeneity among lactotropes. The differential sensitivity of PRL and GH to the applied secretagogues suggests that the intracellular regulation of PRL and GH are compartmentalized in the mammosomatotrope cell.

Neuroendocrine control of immunoreactive growth hormone and bioactive prolactin secretion in neonatal rats: ontogeny and interactions between the serotonergic, cholinergic and alpha 2-adrenergic systems.

The effects of the alpha 2-agonist clonidine (CLO), the serotonin (5-HT) precursor 5-hydroxy-L-tryptophan (5-HTP), the 5-HT2/histamine (H1) antagonist cyproheptadine (CYPRO), the muscarinic cholinergic antagonist atropine (ATR), and an affinity-purified polyclonal anti-rat growth hormone-releasing hormone (rGHRH) immunoglobulin on serum concentrations of growth hormone (GH) and prolactin (PRL) were tested in 2- and 10-day-old litter-mate rat pups. Serum levels of GH and PRL were detected in RIA and Nb2 lymphoma bioassay, respectively. The effects of two different drugs either alone or in combination with each other were evaluated by two-factor analysis of variance. The data indicated that secretion of GH and PRL was regulated by alpha 2-adrenergic, serotonergic and cholinergic mechanisms; the pathways regulating the two hormones, however, were distinct. 5-HTP stimulated GH secretion as early as day 2 postpartum via cholinergic mechanisms not involving GHRH; this pathway was also present in 10-day-old pups. An additional serotonergic pathway was functional in 10-day-old pups which mediated CLO-induced release of GH, and did not include cholinergic transmission.(ABSTRACT TRUNCATED AT 250 WORDS)

Antiserum to prolactin decreases rapid eye movement sleep (REM sleep) in the male rat.

Previous reports suggest that blood-born prolactin (PRL) may selectively promote rapid eye movement sleep (REMS). To study the possible involvement of endogenous PRL in sleep regulation, rats were systemically injected with either antiserum to PRL or normal rabbit serum, and the sleep-wake activity was determined during the subsequent 12-h light cycle. The administration of normal rabbit serum in physiological saline did not alter sleep-wake activity compared to control recordings, whereas the PRL antiserum caused a modest and selective suppression in REMS. Immunoreactive PRL was eliminated from the serial plasma samples obtained between 6 to 11 h after the injection of the antiserum. Brain temperature was not affected by the antiserum. The results indicate that physiological pituitary PRL secretion has a slight REMS-promoting activity in the male rat. It is speculated that an increased release of pituitary PRL or the PRL-like substance previously demonstrated in the brain may significantly stimulate REMS.

Thyrotropin-releasing hormone mediates growth hormone release induced by milk and nursing in neonatal rats.

Previous evidence from this laboratory suggested that growth hormone (GH) release induced by milk in vitro and by nursing in vivo from neonatal rat pituitary glands is mediated by an alternative GH-releasing factor(s) (GRF) distinct from GH-releasing hormone (GHRH(1-43) ). In the present experiments we tested whether thyrotropin-releasing hormone (TRH) could fulfil the criteria of this alternative GRF in neonatal rats. The water-soluble fraction of rat milk (infranatant, prepared by ultracentrifugation) and its methanol/acetic acid extract (milk-borne peptides) stimulated GH release from perifused pituitary glands obtained from 2-day-old rats. Dialysis of the infranatant (mol wt cut-off: 2,000) against 500 volumes of culture medium at 4°C eliminated its GH-releasing activity in the perifusion system, while the infranatant retained its full GRF-like activity when incubated at 4°C without dialysis. The milk-borne GRF eluted as a single peak and coeluted with TRH in a combined gel permeation chromatography (Sephadex G-10) and perifusion set-up. Prolactin secretion was also stimulated simultaneously with the release of GH induced either by milk or by TRH. In a stepwise C(18) reversed-phase chromatography, milk-borne GRF was highly hydrophilic and coeluted with synthetic TRH. The in vitro GH-releasing bioactivities of synthetic TRH and a milk extract purified in C(18) reversed-phase chromatography were abolished by proline-specific endopeptidase. Thus, TRH and milk-borne GRF displayed similar molecular weights, hydropathic characteristics and proteolytic enzyme resistance. In vivo, nursing (which has been reported as a potent stimulus of GH secretion even in the absence of milk-intake) increased serum GH levels in 2-day-old pups. A supramaximal dose of TRH (10 ng/g intraperitoneally) stimulated GH release in 2-day-old pups separated from their mothers for 6 h to a similar extent as nursing. Nursing-induced levels of serum GH were not further elevated by TRH. This failure of TRH to further increase serum GH levels was not due to a maximal GH output by the neonatal pituitary gland, since the GH release induced by the serotonin precursor 5-hydroxy-L-tryptophan was augmented either by TRH or by nursing. These data provide evidence that the milk-borne GRF-like activity in vitro is indistinguishable from TRH, and suggest that TRH (probably of hypothalamic origin) might be the mediator of the nursing-induced release of GH in vivo as a physiological GRF in neonatal rats.

Rat milk inhibits the mitogenic response of Nb2 lymphoma cells to prolactin.

Prolactin-like bioactivity in the rat milk was observed using the lactogen specific Nb2 lymphoma assay. The water soluble fraction (infranatant) of pooled milk samples obtained on days 2, 12 or 22 postpartum stimulated Nb2 cell growth in the range of 0.08-2.5 microliters/well. Higher concentrations of day-12 and day-22 (but not day-2) milk infranatant, however, decreased Nb2 proliferation in a dose-dependent fashion. [3H]-thymidine incorporation used as an indicator of cell growth was decreased by 21%, 49% and 83% at the doses of 5, 10, 20 microliters/well concentrations of day-22 milk infranatant, respectively. Milk infranatant did not reduce cell viability as assessed by Erythrosin B exclusion test. Addition of exogenous rat PRL (NIH B-6) at concentrations of ED50-ED90 did not restore the Nb2 proliferation rate decreased by milk infranatant. Saturating doses of PRL (ED100-ED400) resulted in maximal cell growth, but failed to counteract the inhibitory effect of milk infranatant. The relative molecular weight of the putative Nb2 cell inhibitor of rat milk is between 10 kDa and 30 kDa as determined by ultrafiltration and dialysis. The inhibitory activity of milk infranatant is stable at physiological pH, but is destroyed upon acidification. Thirty min of incubation at 37 degrees C enhanced but 30 min of incubation at 100 degrees C only slightly decreased the calculated total inhibitory effect of milk infranatant. These initial results indicate the presence of a water-soluble antimitogenic factor in rat milk (rMAF) which inhibits the Nb2 lymphoma cell response to prolactin in a non-competitive manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of neonatal milk-prolactin deprivation on the ontogeny of the immune system of the rat.

A growing body of evidence suggests that prolactin (PRL) is involved in regulation of the immune system in the adult. PRL provided to the neonate in mother's milk also has been shown to be important in development of the neonatal neuroendocrine regulation of PRL secretion. Therefore, in this study we asked if deprivation of the neonate of milk-PRL on days 2-5 postpartum affected the ontogeny of the immune system. Two aspects, DNA synthesis (3H-thymidine incorporation) of neonatal lymphocytes in response to polyclonal mitogens in vitro and expression of lymphoid cell surface antigens, were examined. Splenocytes and thymocytes from neonates ranging in age from 5 to 28 days were taken from mothers treated with bromocriptine or saline on days 2-5 of lactation. Splenocytes from pups of vehicle-treated mothers showed a gradual increase in surface antigen expression by day 5 to 28. Thymocyte patterns and percentages of these surface proteins were at adult levels at the earliest times tested. Thymocytes from day 5 and 10 neonates were more responsive to Con A than were splenocytes, but both thymocytes and splenocytes showed an increase in mitogenic responsiveness until day 18, a sharp decline at day 21, and an increase again at day 28. The fact that day 21 is the time of intestinal closure (cessation of absorption of macromolecules from the gut) suggested that milk-borne material plays a role in immune cell maturation.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular heterogeneity of prolactin in lactating rats and their pups: biological and immunological activities in pituitary gland, serum and milk.

Prolactin (PRL)-like bioactivities (in Nb2 lymphoma assay), immunoreactivities (in RIA) and B/I ratios in rat milk, maternal and neonatal pituitary glands and sera were investigated. The PRL-like bioactivity in the water-soluble fraction of rat milk (infranatant prepared by ultracentrifugation) exceeded its immunoreactivity 3-7-fold. The elevated B/I ratio was in part due to the presence of a glycosalated PRL (G-rPRL)-like material, since 5-70% of the PRL-like bioactivity was recovered from the glycosylated fraction of rat milk infranatant prepared by concanavalin-A affinity chromatography. We were unable to detect PRL-like immunoreactivity in the glycosylated fraction of rat milk, and calculated that the maximal cross-reactivity of G-rPRL in the RIA is less than 3.8%. In day 12 milk, over 80% of the G-rPRL-like bioactivity eluted from a Sephadex G-100 column as a high apparent molecular weight (Mr) substance (approximately 50 kD), while the rest eluted as a monomeric G-rPRL (24-25 kD). The PRL-like bioactivity in the nonglycosylated fraction eluted in three peaks (Mr: 50, 24 and 16 kD), while two immunoreactive peaks occurred (Mr: 24 and 8 kD). The concentration of rPRL-like immunoreactivity in rat milk increased during the first days of lactation, remained high in midlactation, and declined by the end of lactation. The PRL-like bioactivity in the nonglycosylated fraction of rat milk displayed a similar timecourse. G-rPRL-like bioactivity in rat milk, however, changes inversely, i.e. decreased between days 2 and 18 postpartum then increased by day 22. The concentration of high Mr PRL-like bioactivity in rat milk was greatly reduced by day 22 from day 2 postpartum. No PRL-like bioactivity or immunoreactivity was recovered from the IgG fraction (prepared by protein A affinity chromatography) of rat milk. The B/I ratio in day 2 maternal pituitary glands was close to 1. In neonatal pituitaries and in maternal sera, however, the B/I ratio was slightly elevated (2-3). The B/I ratio in day 2 neonatal serum was between 6 and 22, while the B/I ratio of PRL secreted by day 2 neonatal pituitary glands in vitro was 1. The present results demonstrate that the concentrations of PRL in rat milk and neonatal serum have been grossly underestimated because levels were detected by RIA. The high B/I ratio reflects the presence of PRL variants. Milk appears to be the most likely source of PRL variants in the circulation of the neonate.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of Basal and nursing-induced secretion of growth hormone in the neonatal rat: the involvement of serotonergic, muscarinic cholinergic, alpha-adrenergic, somatostatin and growth hormone-releasing hormone systems.

Abstract Various neural factors are involved in the suckling-induced increase in serum growth hormone (GH) levels in neonatal rats, and, in the present study the serotonergic, cholinergic, somatostatin and GH-releasing hormone (GHRH) systems were investigated. The serotonin (5-HT) precursor 5-hydroxy-L-tryptophan (5-HTP) and the 5-HT receptor agonist quipazine maleate stimulated serum GH levels in 2-day-old rat pups separated from their mothers for 6 h. The increase in serum GH during suckling was further elevated by 5-HTP. The 5-HT antagonist cyproheptadine decreased serum GH levels in separated 2-day-old pups, and although it reduced the amplitude of the suckling-induced increase in serum GH concentration, it did not alter the increase in serum GH on a percentage basis. The effect of the cholinergic muscarinic antagonist atropine sulfate (ATR) was similar to that of cyproheptadine. Moreover, in separated pups, ATR prevented the increase in serum GH induced by 5-HTP. In contrast with 2-day-old pups, ATR completely eliminated the suckling-induced release of GH in 10-day-old rats. However, ATR failed to prevent GH release induced by the alpha(2)-adrenergic agonist clonidine HCI in 10-day-old male pups. While thyrotropin-releasing hormone increased serum GH levels, rat GHRH failed to alter serum GH levels either in separated or in suckled 2-day-old rat pups. Immunoneutralization for rat GHRH eliminated the increase in serum GH induced by clonidine HCI in 10-day-old pups, but (on a percentage basis) failed to prevent the GH-increasing effect of suckling in 2-day-old pups. While somatostatin failed to significantly decrease serum GH in separated 2-day-old pups, it effectively decreased serum GH levels in 2-day-old pups which were suckled. Cysteamine, which depletes hypothalamic somatostatin, increased serum GH in separated 2-day-old pups, and further increased the suckling-induced levels of serum GH. Cysteamine partially prevented the GH-decreasing effect of ATR. The present findings suggest that 1) the serotonergic and cholinergic systems are involved in the regulation of GH secretion as early as day 2 postpartum; 2) the serotonergic and cholinergic systems modulate the basal, and do not modulate the suckling-induced levels of serum GH; 3) the serotonergic system may exert its stimulatory influence on GH secretion only in the presence of a functional muscarinic cholinergic system; 4) the cholinergic system, at least in part, stimulates GH secretion via a cysteamine-sensitive system (probably by inhibiting somatostatin); 5) the cholinergic system is not functionally coupled with the alpha(2)-adrenergic system, which stimulates GH secretion via rat GHRH; 6) since in 10-day-old pups clonidine HCI was effective only in males, while suckling was effective in both sexes, the alpha(2)-adrenergic system is not involved in the suckling-induced increase of serum GH; and finally 7) neither somatostatin nor rat GHRH seem to be involved in the suckling-induced changes in serum GH. The findings are consistent with the hypothesis that the high circulating GH levels in the neonatal rat are due to alternative GH-releasing factors, perhaps thyrotropin-releasing hormone or gamma-aminobutyric acid. The neurohumoral mediator of the suckling-induced GH release in neonatal rats remains to be identified.

Fetal pituitary graft is capable of initiating hormone synthesis in median eminence removed adult rat.

Rathke's pouches of 12- and 13-day-old rat embryos were implanted beneath the kidney capsule of adult male rats subjected to the removal of median eminence or to sham-operation. Host animals were sacrificed 28 days after grafting and the implanted pituitaries were processed for immunohistological examination. ACTH, LH-beta, FSH-beta, TSH-beta, GH and PRL immunopositive cells could be observed in fetal grafts of all experimental groups. However, the number and staining intensity of different hormone containing cells largely varied and presumably depended on the hormonal state of host animals. The results indicate that undifferentiated fetal pituitary does not require hypothalamic hypophysiotrophic neurohormones for proliferation and cytodifferentiation and that its development might be modulated by circulating trophic hormones of host animals.