Increased hypothalamic somatostatin mRNA following dexamethasone administration in rats.

There is increasing evidence to suggest that supraphysiological doses of glucocorticoids suppress growth hormone secretion in vivo by augmenting somatostatin release from the hypothalamus; previously, we reported an increase in hypothalamic somatostatin content in dexamethasone-treated rats. To further examine whether the production of somatostatin really is augmented, hypothalamic somatostatin mRNA levels were determined by the Northern blot technique in female rats receiving 330 micrograms of dexamethasone daily for three days. In two series of experiments, hypothalamic somatostatin mRNA levels in dexamethasone-treated rats were significantly (p < 0.05) increased to 133 +/- 19 (mean +/- SD)% and 153 +/- 38% of the controls. In the dexamethasone-treated rats, plasma growth hormone levels were markedly suppressed compared with those of the controls. These results further support the hypothesis that pharmacological doses of glucocorticoids increase the production and release of somatostatin from the hypothalamus and thus inhibit growth hormone secretion, overriding the direct stimulatory effect of glucocorticoids on growth hormone production at the pituitary level.     

Reproductive actions of prolactin mediated through short and long receptor isoforms

Prolactin (PRL) is a polypeptide hormone with a wide range of physiological functions, and is critical for female reproduction. PRL exerts its action by binding to membrane bound receptor isoforms broadly classified as the long form and the short form receptors. Both receptor isoforms are highly expressed in the ovary as well as in the uterus. Although signaling through the long form is believed to be more predominant, it remains unclear whether activation of this isoform alone is sufficient to support reproductive functions or whether both types of receptor are required. The generation of transgenic mice selectively expressing either the short or the long form of PRL receptor has provided insight into the differential signaling mechanisms and physiological functions of these receptors. This review describes the essential finding that both long and short receptor isoforms are crucial for ovarian functions and female fertility, and highlights novel mechanisms of action for these receptors.     

Increased neuropeptide Y content in individual hypothalamic nuclei, but not neuropeptide Y mRNA, in diet-induced obesity in rats.

Neuropeptide Y (NPY) is the most powerful appetite stimulant known, and chronic administration leads to obesity. The hypothalamic content of NPY varies with nutritional status, suggesting that it is of physiological importance. We measured NPY in specific hypothalamic nuclei and NPY mRNA in the hypothalamus by Northern blotting in rats made obese by feeding a highly palatable diet compared with controls fed standard chow. In animals fed the palatable diet, NPY concentrations were increased in the paraventricular nucleus (mean +/- S.E.M.; 19.5 +/- 2.3 vs 11.1 +/- 1.1 fmol/micrograms protein, P less than 0.02), the arcuate nucleus (20.4 +/- 3.3 vs 9.3 +/- 0.6 fmol/micrograms protein, P less than 0.01), the medial preoptic area (9.1 +/- 0.9 vs 5.9 +/- 0.7 fmol/micrograms protein, P less than 0.02) and the anterior hypothalamus (2.7 +/- 0.2 vs 2.0 +/- 0.1 fmol/micrograms, P less than 0.02). Hypothalamic NPY mRNA measured by Northern blot analysis was, however, unchanged. These results suggest that the increase in NPY was due to decreased release rather than increased NPYergic activity. The findings are in accord with the neuroendocrine disturbance and increased thermogenesis observed in this model of obesity.     

The hypothalamic paraventricular nucleus has a pivotal role in regulation of prolactin release in lactating rats

The affect of paraventricular nucleus (PVN) lesions on PRL secretory response to suckling was studied in adult female rats. Basal levels of PRL were similar in the control and lesioned groups. Substantial decreases in PRL levels occurred after separation of pups from their mothers in the control as well as lesioned animals. When mothers and pups were reunited, the circulating PRL concentrations of the control groups rose immediately from basal values of 50-100 micrograms/liter to reach peaks of 450-550 micrograms/liter. PVN lesions significantly decreased the suckling-induced rise of PRL levels. Furthermore, PVN lesions abolished the high amplitude, episodic pattern of PRL release in continuously lactating rats. These findings are consistent with the view that PVN neurons produce PRL releasing factor(s), which is (are) required for normal secretory patterns of PRL in lactating rats.     

Effect of suckling and adrenergic stimulation on peripheral deiodination in lactating rats: differential expression of type 1 deiodinase mRNA forms.

Previous works led us to propose that peripheral iodothyronine deiodination is mainly regulated by the reciprocal interaction between the thyroid and the sympathetic nervous system (SNS). In this study, we analyzed the role suckling exerts, through SNS activation, upon deiodination of thyronines in liver, heart, brown adipose tissue and mammary gland during lactation. Our results showed that resuckling causes a concurrent stimulatory response on deiodinase type 1 (D1) in heart and mammary gland, but not in liver and brown adipose tissue. The stimulatory response was mimicked by norepinephrine and by the beta-adrenergic agonist isoproterenol, through the overexpression of the large form of D1 mRNA. These results suggested that, during lactation, peripheral thyronine deiodination is co-ordinated by the SNS, and suckling is a major modulatory influence.     

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.     

Increased expression of IL-12 receptor mRNA in active pulmonary tuberculosis and sarcoidosis.

Cytokines have been implicated in the pathophysiology and development of pulmonary diseases such as tuberculosis and sarcoidosis. In particular, the numbers of cells expressing Th1-type cytokines such as IFN-gamma and IL-12 are increased within the lungs of patients with these granulomatous diseases. As a factor promoting the commitment of naive lymphocytes to a Th1-type profile of cytokine expression, IL-12 may be pivotal in the cascade of proinflammatory events within the airways. In this study, we examined the expression of the IL-12 receptor (IL-12R) mRNA in bronchoalveolar lavage (BAL) fluid from patients with active pulmonary tuberculosis (n = 6) and active pulmonary sarcoidosis (n = 6), and from allergic asthmatics (n = 6) and normal control subjects (n = 6). Bronchoscopy with BAL was undertaken, and cell cytospins were examined using the technique of in situ hybridization. There was a significant increase in the numbers of cells expressing mRNA for both beta(1) and beta(2) subunits of the IL-12R in active pulmonary sarcoidosis (p < 0.02, p < 0.01, respectively) and active pulmonary tuberculosis (p < 0.01, p < 0.005, respectively) compared with normal control subjects. In contrast, the allergic asthmatic patients exhibited a significant decrease in the number of IL-12R mRNA-positive cells (both beta(1) and beta(2) subunits (p < 0.01, p < 0.005, respectively), compared with the normal control subjects. These patients did, however, exhibit a significant increase in IL-4R mRNA, which was not evident in those with either tuberculosis or sarcoidosis when compared with normal subjects (p < 0.05). Colocalization studies demonstrated that CD8+ve cells are a principal site for the expression of IL-12R in tuberculosis. In sarcoidosis, IL-12R was expressed both on CD4+ve and CD8+ve cells. The increased expression of receptors for IL-12 in granulomatous diseases such as pulmonary tuberculosis and sarcoidosis provides evidence supporting the commitment of lymphocytes to a Th1-type cytokine profile in vivo.     

H1 and H2 histamine receptor participation in the brain control of prolactin secretion in lactating rats.

The aim of the present research was to evaluate the histaminergic regulation of prolactin secretion in the lactating rat and the possible involvement of H1 and H2 histamine receptors in this control. Prolactin was measured by radioimmunoassay in blood samples withdrawn through an intrajugular silastic catheter from undisturbed lactating mothers 10 to 15 days after delivery. In some of those rats a stainless steel cannula was placed in the third ventricle. The tested drugs, H1 and H2 receptor agonists and antagonists, were injected either by the intrasilastic route or intraventricularly immediately before the onset of suckling and after a basal sample was taken. New samples were withdrawn 10, 20, 30 and 60 min thereafter. Suckling caused a 12- to 18-fold increase in serum prolactin by 10 min in control saline-injected mothers. In non-suckled mothers (NSM) injected with saline, prolactin levels were low at all times. Systemic or intraventricular diphenhydramine and mepyramine, H1 receptor antagonists, suppressed the increment in prolactin observed in suckled mothers (SM). Intraventricular metiamide, an H2 receptor antagonist, did not modify prolactin secretion in SM but drastically increased serum prolactin in NSM. A small but significant increase in prolactin titers was observed in NSM injected intraventricularly with histamine. 4-Methylhistamine, an H2 agonist, was ineffective when used intraventricularly in NSM, but clearly suppressed prolactin enhancement in SM. It is postulated that in lactating mothers, brain histamine has a dual control on prolactin secretion. H2 receptors mediate events related to inhibition of prolactin release, since the agonist 4-methylhistamine blocked the prolactin rise in SM, while the antagonist metiamide promoted release of the hormone in NSM. H1 receptors seem to be related to a facilitatory mechanism since classical antihistamines suppress the serum prolactin increase that follows the onset of suckling, while histamine itself is able to release prolactin in NSM.     

Increased abundance of alternatively spliced forms of D2 dopamine receptor mRNA after denervation.

The existence of two molecular forms of D2 dopamine receptors suggests that differences in the distribution or regulation of the two forms could be exploited for the pharmacological treatment of disease. Using probes selective for each alternatively spliced variant of D2 receptor mRNA, we determined that both variants were widely distributed in rat brain and pituitary but that the ratio of the forms varied among regions. mRNA for the 444-amino acid-long variant, D2(444), was the most abundant form in pituitary and neostriatum. Intermediate levels of both D2(444) mRNA and the short form, D2(415), were detected in midbrain, and low levels of D2(444) and D2(415) mRNAs were detected in all other regions examined, including hippocampus, cerebellum, and cortex. The D2(444)/D2(415) ratio was generally lower in the regions of low expression than in pituitary and neostriatum. Dopamine-depleting lesions increased the density of D2 receptors in the denervated neostriatum by 29% without altering the affinity of the receptors for [3H]spiperone. The proliferation of receptors appeared to be due to a lesion-induced increase of up to 120% in the abundance of both variants of mRNA in the neostriatum.     

The role of prolactin in the luteotrophic process of lactating rats.

Treatment of pseudopregnant rats with ergocryptine mesylate (ECR) depressed serum prolactin levels but had no apparent effect on LH secretion. Ovarian progesterone secretion was significantly, reduced 24 hr after ECR treatment on day 6 or 9 of pseudopregnancy and the secretion rate of 20alpha-OH-P remained constant. When lactating rats nursing 6 pups were treated with ECR on day 6 or 9 postpartum, progesterone secretion was significantly decreased by 48 hr after treatment and 20 alpha-OH-P secretion was not altered. Furthermore, ECR inhibited litter weight gains of these lactating dams. After treatment of normal pregnant and pregnant lactating rats with ECR on day 6 of pregnancy, gestation was terminated in all animals. If ECR was given on day 9 to normal pregnant rats, to pregnant lactating animals whose pups were removed on day 9 postpartum, or to pregnant lactating rats treated with LH, gestation was not terminated. However, treatment with ECR of day 9 pregnant lactating rats whose pups continued to suckle terminated pregnancy in 13 of 21 animals. The results of these studies suggest the elevated pituitary prolactin secretion is necessary for maintenance of luteal function in pseudopregnant and lactating rats, and in pregnant lactating rats elevated pituitary prolactin secretion is a component of the luteotrophic complex for a longer period of time than in normal pregnant rats.     

Effect of L-dopa on milk ejection and prolactin release in lactating rats.

The effect of L-DOPA on milk ejection and on prolactin release during 30 min of suckling was studied in lactating rats. Various doses of L-DOPA (1-25, 2-5, 5 and 10 mg/100 g body wt) were injected i.p. 30 min before the suckling period. Control rats were injected with 0-9% NaCl solution only. An inhibition of milk ejection proportional to the dose of drug administered was obtained. The dose of 10 mg completely blocked milk ejection but 1-25 mg had no effect. A normal milk-ejection response was obtained with a small dose of oxytocin injected immediately before nursing into mothers treated with 10 mg L-DOPA, indicating that the blocking effect was not due to a lack of mammary gland response. In control mothers, serum prolactin levels increased from 67-2 +/- 25-9 (S.E.M.) to 950-3 +/- 118-7 ng/ml after a 30 min suckling period. L-DOPA (5 and 10 mg) prevented the release of prolactin induced by suckling, but 1-25 and 2-5 mg L-DOPA had no effect. The results indicate that oxytocin and prolactin release induced by suckling in lactating rats is inhibited by an increase of catecholamines at the hypothalamic-hypophysial axis.     

Cloning and expression of a unique short form of the porcine prolactin receptor

Prolactin (PRL) is required not only for maintenance of gestation in pigs but also for mammary gland development and subsequent lactogenesis. The actions of PRL are modulated by both long and short isoforms of the PRL receptor (PRLR), where short isoforms can interfere with the essential signaling function of the long isoform. Using 3' RACE we have isolated a unique splice variant of the porcine PRLR (pPRLR) that contains a short intracellular domain of 38 aa that is encoded by splicing from exon 9 to a novel exon 11 located 17.5 kb downstream of exon 10 on chromosome 16. The short pPRLR was detected as a 42 kDa protein in membranes from porcine mammary glands. Functional analyses indicated that the short pPRLR functions as a dominant negative against the differentiative function of the long pPRLR and does not transduce a signal to the rat β-casein promoter. Differential abundance of long pPRLR and short pPRLR mRNA was established in a range of porcine tissues. The binding affinity of the short pPRLR for pPRL was lower (K(d) = 3.7 nM) than the affinity of the long pPRLR (K(d) = 1.6 nM) despite a fourfold higher level of binding sites for the short pPRLR. Our data raise the possibility that the short pPRLR in pigs may function independently from the long pPRLR, where the splicing strategy used to generate the short pPRLR likely evolved under different selection pressures to those acting on the long pPRLR.     

Catecholamines in individual hypothalamic nuclei of acutely and repeatedly stressed rats.

Norepinephrine (NE) and dopamine (DA) concentrations in 17 individual hypothalamic nuclei and 3 other brain regions were measured in rats, acutely or repeatedly stressed by immobilization, using a microdissection technique and a radioisotopic-enzymatic assay. Following the first 20 min immobilization (IMO) a significant NE decrease in the ventromedial (NVM) and supraoptic (NSO) nuclei and a DA decrease in the arcuate nucleus (NA) as well as NE and DA increase in the dorsomedial nucleus (NDM) were seen. Repeated IMO (40 times) produced a NE increase in the NVM, NDM, NSO paraventricular nucleus (NPV) and median eminence (ME), and a DA increase in the NDM and NPV. Changes of NE and DA concentration found in some individual hypothalamic nuclei under the influence of stress indicate that catecholamines (CAs), particularly in the medial basal hypothalamus, could be involved in the regulation of some neuroendocrine processes which are being activated during stress, especially ACTH release.     

Different forms of the prolactin receptor insights into the mechanism of prolactin action.

The primary structure of two forms (short [ approximately 300 amino acids] and long [ approximately 600 amino acids]) of prolactin (PRL) receptor have been deduced by cloning their cDNAs. PRL receptors belong to the superfamily of cytokine growth hormone-PRL receptors, based on conserved sequences in their extracellular domains. No clear second messenger of PRL has been identified, and because of the wide range of actions associated with PRL, it is doubtful that a single unifying mechanism will be found. Mutagenesis of extracellular and intracellular domains of the PRL receptor should help define subdomains involved in hormone binding, and coupled with a functional assay, delineate regions necessary for signal transduction.     

Mammary gland prolactin receptor and pituitary prolactin secretion in lactating mice with different lactational performance

SHN female mice, a high mammary tumour strain, are superior to SLN, a low mammary tumour strain, in lactational performance. Mammary gland prolactin receptor and pituitary prolactin secretion during lactation were compared between these strains. The binding activity, the number of receptor sites per mg tissue and the association constant were measured by the in vitro incubation of mammary gland slices with 125I-labelled bovine prolactin, and the pituitary and plasma levels of prolactin were assayed by homologous radioimmunoassay. There was only a slight difference between strains in any of the parameters for prolactin receptor and for prolactin secretion on either day 4 or day 9 of the first lactation. Almost all the correlation coefficients between each parameter for prolactin receptor and the pituitary or plasma level of prolactin were not statistically significant. These findings suggest that any parameter for prolactin examined in this study is not always directly indicative of lactational performance and further show that the individual variation in the pituitary prolactin secretion during lactation is not so great as to alter the prolactin receptor.     

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.