Immunoreactive dynorphin in pituitary and brain.

Distribution of the potent opioid peptide dynorphin has been determined in pituitary gland (pig, beef, rat), in the various regions of rat brain, and in rat spinal cord, by using a highly specific antiserum. By gel permeation chromatography in 4 M guanidine, the porcine pituitary immunoreactivity is found in a major peak of apparent molecular weight about 1700 and a minor peak of about 3400. Similar peaks are found in rat pituitary extracts, whereas rat brain contains, in addition, two peaks of larger apparent molecular weight. In the pituitary, immunoreactive dynorphin is found predominantly in pars nervosa. In the central nervous system, it is distributed widely, with highest concentrations in hypothalamus, medulla-pons, midbrain, and spinal cord. Although dynorphin contains leucine-enkephalin, the regional distribution of dynorphin is different from that of enkephalin or of any other known opioid peptide.     

Calcitonin expression in rat anterior pituitary gland is regulated by ovarian steroid hormones

Gonadotroph-derived calcitonin-like peptide (pit-CT) is a potent inhibitor of lactotroph function. We investigated the effect of ovarian hormones on pit-CT mRNA expression in the anterior pituitary (AP) gland of cycling female rats. Levels of mRNAs for pit-CT, CT receptor, prolactin (PRL), and beta-LH during 4-d estrous cycle were determined. In a second study, the effects of estrogens and progesterone on pit-CT and PRL mRNA levels were investigated. In a third group, the effect of estrogen or progesterone depletion on pit-CT mRNA expression was studied. In a fourth group, the effect of passive pit-CT immunization on PRL and LH mRNA expression was examined. Pit-CT mRNA levels varied during estrous cycle. They were highest in diestrus, but lowest in the evening of proestrus. CT-receptor mRNA levels displayed smaller fluctuations. Estrogen repletion caused a decline in pit-CT mRNA expression in ovariectomized rats, but progesterone produced a marked increase. ICI 182,780 prevented the decline of pit-CT mRNA levels during late proestrus-estrus, but RU 486 attenuated pit-CT mRNA levels. Passive CT immunization in diestrus altered PRL and LH mRNA expression, and advanced the estrus cycle. These results suggest that pit-CT mRNA expression is regulated by ovarian hormones, and depletion of pit-CT advances their estrous cycle.     

Chromogranin A messenger RNA expression in the rat anterior pituitary is permissively regulated by the adrenal gland

The influence of the adrenal gland on the expression of chromogranin A in the anterior pituitary was studied in the rat. Adrenalectomy caused a progressive and pronounced (20% of control levels at day 10 after adrenalectomy) decrease of Chromogranin A mRNA levels in anterior pituitary. Daily injection of dexamethasone (15 micrograms/animal, s.c.) fully reversed the postadrenalectomy decrement in chromogranin A mRNA levels. Chromogranin A protein content, however, was unchanged 10 days after adrenalectomy. In contrast, pro-opiomelanocortin mRNA levels were significantly elevated after adrenalectomy and restored to normal by dexamethasone, with a time course similar to the changes in chromogranin A mRNA levels. These data demonstrate that the adrenal gland permissively regulates chromogranin A expression in the anterior pituitary, at a pretranslational locus, and that this regulation is probably mediated by glucocorticoids.     

Dopamine D2-receptor messenger RNA is differentially regulated by dopaminergic agents in rat anterior and neurointermediate pituitary

Hypothalamic dopamine (DA), acting at DA D2-receptors (D2-R) on pituitary target cells, mediates peptide release and biosynthesis of rat pituitary anterior lobe (AL) prolactin, and neurointermediate lobe (NIL) pro-opiomelanocortin (POMC). We were interested in determining if dopamine agonists and antagonists were capable of modifying D2-R gene expression in these pituitary cells. Utilizing the recently published sequence of the rat D2-R, we isolated a rat D2-R cDNA clone by polymerase chain reaction, and have synthesized RNA probes to quantitate levels of D2-R mRNA by solution hybridization/nuclease protection assay. We report here that 5-day administration of the DA antagonist haloperidol led to significant increases in both D2-R mRNA and POMC mRNA in the NIL; the DA agonist bromocriptine caused a significant decrease in NIL POMC mRNA with no parallel change in D2-R mRNA. In contrast, no significant changes in D2-R mRNA in AL were observed following treatment with either the DA agonist or antagonist. These data provide evidence for tissue-specific regulation of D2-R mRNA in response to dopaminergic manipulation.     

Mineralo- and glucocorticoid receptor mrnas are differently regulated by corticosterone in the rat hippocampus and anterior pituitary

In most cell lines and animal tissues, glucocorticoid receptors undergo downregulation after exposure to corticosterone. However, corticosterone treatment has not shown a consistent effect on mineralocorticoid (MR) and glucocorticoid receptors (GR) in the hippocampus, and it has been rarely assessed in the anterior pituitary. In this study we investigated dose-dependent effects of corticosterone on MR and GR mRNAs in the hippocampus and anterior pituitary. Adrenalectomized rats substituted with corticosterone in drinking fluid were injected subcutaneously with vehicle or 1, 10, 50, 100, or 200 mg of corticosterone, and sacrificed 4 h later. In the hippocampus we found a progressive decrease in MR and GR mRNAs with increasing doses of corticosterone. This was significant with 50 and 100 mg corticosterone for MR mRNA and with 10-200 mg corticosterone for GR mRNA at plasma corticosterone levels above 30 microg/dl. The anterior pituitary did not show significant changes at any dose. A time-course with 2 mg of corticosterone (non-response dose range at 4 h) revealed a significant decrease in MR and GR mRNAs in the hippocampus 8 h after the subcutaneous injection. In the anterior pituitary both mRNAs showed an increase that was significant 24 h after injection for MR and from 8 to 24 h for GR. In the hippocampus, adrenalectomy (absence of corticosterone) induced a significant increase in MR and GR mRNAs on day 3, but not on days 1, 8 and 21 after adrenalectomy. In the anterior pituitary there were no significant changes at any time after adrenalectomy. In summary, we have found an in vivo corticosterone dose- and time-dependent downregulation of MR and GR mRNAs in the hippocampus, whereas anterior pituitary MRs and GRs seem relatively insensitive to the excess or the absence of corticosterone, suggesting the lack of an autoregulatory effect in this tissue. Significant mRNA changes appearing later in time could suggest a secondary response via a glucocorticoid-induced gene product. Corticosteroid receptor downregulation in the hippocampus could prevent overstimulation or tissue damage when plasma corticosterone is high, while increased corticosteroid receptors in the anterior pituitary could buffer the excessive brain drive on the pituitary during chronic stress or pathological conditions associated with increased plasma glucocorticoids, such as depression.     

alpha 2u-Globulin is present in the rat anterior pituitary.

The possibility that the pituitary gland may contain as yet undiscovered regulatory factors is intriguing. Recent reports have suggested the presence, in the anterior pituitary, or a number of proteins of extrapituitary origin. alpha 2u-Globulin, a rat serum and urinary protein, previously shown to be synthesized in the submaxillary gland and in the liver under anterior pituitary control, has now been localized by immunocytochemistry in the cytoplasm of some cells of the anterior pituitary. No alpha 2u-globulin could be detected in either the intermediate or posterior pituitary. The presence of alpha 2u-globulin was confirmed and quantitated by radioimmunoassay. Using RNA blot analysis and cloned alpha 2u-globulin cDNA probes, we could not detect alpha 2u-globulin mRNA sequences in pituitary RNA, indicating that alpha 2u-globulin is not synthesized therein. The presence of alpha 2u-globulin, presumably of circulatory origin, in certain anterior pituitary cells suggests that it may play a role in anterior pituitary function.     

Monooxygenase mediating catecholestrogen formation by rat anterior pituitary is an estrogen-4-hydroxylase

Microsomes from rat anterior pituitaries (AP) were incubated with (3H)estradiol under conditions previously shown to support catecholestrogen (CE) formation by placental microsomes via an NADPH- or an organic hydroperoxide-dependent, peroxidatic mechanism. Under conditions optimized for monooxygenase activity (pH 8.0, 5 mM NADPH), 4-hydroxylation predominated (apparent Vmax = 65 pmol and 13 pmol/mg protein/30 min for 4- and 2-hydroxy-E2, respectively). Under conditions optimized for peroxidatic activity (pH 6.0, 50 mM cumene hydroperoxide) 2- and 4-hydroxylated-E2 were produced in similar amounts. Thus in the AP, unlike in other target tissues studied, NADPH-dependent CE synthetase is a 4-hydroxylase and significant 2-hydroxylation occurs only via the peroxidatic mechanism. We propose that 4-hydroxylated CEs, which are both potent, long acting estrogens and catechols, serve as local mediators of actions of phenolic estrogens on the AP.     

The characteristic change in the distribution of S-100 immunoreactive folliculostellate cells in rat anterior pituitary upon long-term estrogen treatment is prevented by concomitant progesterone treatment

The presence of folliculostellate cells in the anterior pituitary was described 49 years ago. These cells give about 10% of the whole cell population and through their long processes they provide intrahypophyseal communication. The folliculostellate cells contain S-100 protein. Its immunostaining was used to identify these cells. It was previously found that the diethylstilbestrol treatment basically influences the morphology and function of the trophic hormone secreting as well as the folliculostellate cells. In the present experiment, we have studied whether a concomitant progesterone treatment can prevent or attenuate changes caused by diethylstilbestrol treatment in the distribution of folliculostellate, prolactin, and GH cells. Diethylstilbestrol alone induced the appearance of prolactinomas. Inside the prolactinomas, folliculostellate cells were scattered but outside the prolactinomas they formed a demarcation line. Inside the prolactinomas, there were only a few growth hormone immunoreactive cells but they surrounded the prolactinomas in a ring-like pattern. When diethylstilbestrol was implanted with progesterone, the changes being characteristic for diethylstilbestrol treatment, could not develop. Concomitant progesterone influence prevented morphological changes in the anterior pituitary. Progesterone alone had no effect. In accordance with the formation of prolactinomas, the plasma prolactin level was very high in diethylstilbestrol treated rats. Concomitant progesterone treatment prevented the effect of diethylstilbestrol. Progesterone alone did not influence the prolactin level. GH levels did not significantly differ in any groups.     

Immunoreactive gamma-melanotropin in rat pituitary and plasma: a partial characterization

A RIA for the gamma MSH region of proopiomelanocortin has been established and validated. The antiserum was raised to synthetic bovine gamma MSH, and it cross-reacts well with larger polypeptides, including gamma MSH and 16K fragment, which contain the gamma MSH sequence. Gel filtration chromatography of extracts prepared from the anterior and neurointermediate lobes of rat pituitary and from rat plasma reveal two heterogeneous immunoreactive (IR-) forms of gamma MSH in each venue which elute in molecular weight peaks of approximately 11,000 (11K) and 6,000 (6K). Addition experiments indicate that the smaller material is not an artifact generated from the larger form during preparation. By the criterion of retention on affinity columns of Concanavalin A-agarose, these peptides are glycosylated. The 6K form represents 8-17% of the total IR-gamma MSH in the anterior lobe and about 30% of that in the neurointermediate lobe. IR-gamma MSH are released by dispersed rat pituitary cells in response to several of the same secretagogues which modulate the secretion of other proopiomelanocortin-derived peptides. Changes in the plasma and anterior lobe content of IR-gamma MSHs and ACTH resulting from perturbation of the pituitary-adrenal axis are concordant. In particular, the plasma concentration of 6K IR-gamma MSH increases dramatically after imposed stress.     

Effect of estrogen on the expression of a cell-surface antigen associated with rat anterior pituitary somatotrophs

The long-term in vivo effect of diethylstilbestrol (DES) on the expression of a cell-surface antigen associated with the anterior pituitary somatotroph was studied in two strains of female rats using double immunofluorescence techniques. Mab WHC-1, a recently generated and characterized monoclonal antibody, was used to detect the antigen associated with somatotrophs, whereas rabbit anti-rat prolactin (rPRL) and anti-human growth hormone (hGH) antisera were used to identify mammotrophs and somatotrophs, respectively. In F344 rats, Mab WHC-1-positive cells increased from 13.8 +/- 0.5% of total pituitary cells in normal anterior pituitaries to 34.2 +/- 4.0% in DES-induced pituitary tumors. The number of mammotrophs also increased significantly from 58.0 +/- 3.2% in controls to 75.9 +/- 2.2% in tumors. On the other hand, somatotrophs decreased significantly in number following ovariectomy (OVX) and DES implantation (19.7 +/- 0.5% vs. 6.1 +/- 1.2%). Based on double immunofluorescence, the percentage of Mab WHC-1-positive cells, which were somatotrophs, decreased from 85.5 +/- 2.7% in normal controls to 6.7 +/- 1.5% in DES-induced tumors. On the other hand, the percentage of Mab WHC-1-positive cells which were mammotrophs increased significantly from 14.0 +/- 1.4% to 86.1 +/- 1.8% following OVX and DES implantation. A similar change was found in the number of somatotrophs and mammotrophs following the same treatment in Sprague-Dawley (SD) rats which did not develop pituitary tumors. In contrast to F344 rats, the number of Mab WHC-1-positive cells in SD rats decreased significantly from 32.4 +/- 2.8% in sham-operated controls to 19.3 +/- 2.9% in OVX + DES-implanted rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

The affinity of catechol estrogens for estrogen receptors in the pituitary and anterior hypothalamus of the rat.

The purpose of these experiments was to evaluate the potential for interaction between 2-hydroxyestrone and 2-hydroxyestradiol and estrogen receptors in rat pituitary and anterior hypothalamus. The 150,000 X g supernatant fractions of these tissues were prepared, the estrogen receptor-site concentration was measured, and the relative abilities of unlabelled estradiol, estrone, 2-hydroxyestradiol and 2-hydroxyestrone to compete with [3H]estradiol for estrogen binding sites was determined. From these results, and the previously determined association constant for [3H]estradiol, 10(10)M-1, the association constants of the other estrogens were calculated. The introduction of the 2-hydroxy group caused only a modest reduction in the affinity of these estrogens for the receptors. The association constants of the 2-hydroxy derivatives were within one order of magnitude of those of the parent compounds. These results demonstrate the potential for interaction between catechol estrogens and estrogen receptor in rat brain and pituitary of a magnitude which could be biologically significant.     

Galanin secretion from anterior pituitary cells in vitro is regulated by dopamine, somatostatin, and thyrotropin-releasing hormone.

Lactotrophs, somatotrophs, and thyrotrophs have been shown to contain immunoreactive galanin. Furthermore, estrogen stimulates galanin mRNA and peptide levels in the rat anterior pituitary, particularly within lactotrophs. To determine whether galanin is released from the anterior pituitary in a regulated manner, we used cultured pituitary cells from male and ovariectomized Fischer 344 rats implanted with estrogen-containing capsules. Anterior pituitary cells (5 x 10(5) cells/well) were challenged (0.5-3 h) with hypothalamic factors known to regulate anterior pituitary hormone secretion, and medium galanin levels were measured by RIA. In female pituitary cells, galanin secretion was inhibited by dopamine (10 and 100 nM) and stimulated by TRH (20 and 100 nM). Although galanin release was significantly lower in male pituitary cells, dopamine and TRH inhibited and stimulated galanin secretion, respectively. Medium galanin levels were also significantly reduced by somatostatin (5 nM) in both female and male cells. The pattern of PRL release in response to dopamine, TRH, and somatostatin was similar to that observed for galanin, regardless of the sex of the pituitary donor. Although galanin has been localized in somatotrophs, 5 nM GH-releasing hormone (GRF) failed to alter galanin release in male as well as female pituitary cells; GH secretion was significantly increased by GRF. LHRH (5 nM) and CRF (5 nM) failed to alter galanin release in vitro. We conclude that in estrogen-exposed pituitary cells obtained from male and ovariectomized Fischer 344 rats: 1) galanin secretion is inhibited by dopamine and somatostatin, and stimulated by TRH; 2) GRF, LHRH, and CRF do not regulate galanin release in these cells; and 3) the profile of the regulated pathway for galanin release suggests that the primary location of galanin is the lactotroph, probably within secretory granules.     

Immunoreactive alpha-MSH and ACTH levels in rat plasma and pituitary.

A radioimmunoassay is described for the measurement of alpha-melanocyte-stimulating hormone (alpha-MSH). The antibody was produced in rabbits by immunization with alpha-MSH coupled to bovine serum albumin with carbodiimide. The antibody did not react significantly with ACTH, beta-MSH, or 6 fragments of ACTH. The sensitivity and reliability of the assay were improved by employing a simple plasma extraction procedure. When applied to a 2 ml plasma sample, the detection limit of the radioimmunoassay was 6 pg/ml. ACTH was measured with a sensitive and specific radioimmunoassay previously described for humans and adapted for the rat. The anti-ACTH serum cross-reacted with the biologically active portion of alpha-p ACTH and not with alpha-MSH, beta-MSH or the alpha-p 17-39 and alpha-p 25-39 fragments of ACTH. The detection limit was 20 pg/ml. Plasma and pituitary alpha-MSH and ACTH had the same immunoreactivity as synthetic alpha-MSH and ACTH. alpha-MSH and ACTH contents of the rat neurointermediate lobe were 1398 +/- 360 (SE) ng and 28.2 +/- 2.9 ng, respectively, while in the anterior lobe they were 102 +/- 31 ng and 551 +/- 36 ng, respectively. The plasma alpha-MSH concentration at 8 AM in male rats was 64 +/- 8 pg/ml when the plasma ACTH concentration was 92 +/- 15 pg/ml. Over a 24-hour period two peaks of plasma alpha-MSH were observed, one at 4 AM (142 +/- 35 pg/ml) and the other at 4 PM (139 +/- 26 pg/ml). Plasma ACTH was higher at noon (151 +/- 43 pg/ml) and 4 PM (130 +/- 48 pg/ml). Short-term exposure to ether induced a transient increase in alpha-MSH level 5 min later and a rapid return to normal levels. Plasma ACTH increased significantly 2.5 min after the onset of ether stress and remained high for 30 min. Two hours' exposure to ether did not change plasma alpha-MSH, although a 3-fold increase in plasma ACTH was observed. Haloperidol injection was followed by a large increase in plasma alpha-MSH, whereas ACTH levels increased similarly after saline and Haloperidol injection. Corticoid administration reduced ACTH, but not alpha-MSH. Three weeks after adrenalectomy, alpha-MSH levels had not changed but ACTH levels had increased ten-fold. These data indicate that alpha-MSH is secreted in the rat, and that the regulation of its secretion is different from that of ACTH.     

Immunoreactive pit-1 protein in hyperplastic pars intermedia induced by calcitonin of the rat pituitary gland

To elucidate the effects of synthetic salmon calcitonin (sCT) on the cells in the rat pituitary gland, we histopathologically and immunohistochemically examined the early changes after 4 or 13 weeks treatment with sCT 120 IU/kg. Focal proliferative lesions of the anterior pituitary glands were consistently found after treatment with sCT for 13 weeks. Histologically, the cells with the focal proliferative lesions were classified into the following three groups: 1) enlarged basophilic cell focus, 2) vacuolated cell focus and 3) chromophobe cell focus. These focal proliferative lesions had positive staining only for the alpha-subunit and failed to show Pit-1 protein immunoreactivity. The sCT treatment also increased the thickness of the pars intermedia. Hypertrophy of the pars intermediate cells was characteristically seen. Furthermore, Pit-1 protein immunoreactivity was clearly detected in the nuclei of the hyperplastic pars intermediate cells. All pars intermediate cells were equally stained by alpha- or beta-MSH and beta-endorphin in both vehicle- and sCT-treatment. No difference was seen. These findings strongly suggest a very close relationship between Pit-1 protein immunoreactivity and cellular proliferation induced by sCT.     

Kallikrein gene expression in the rat anterior pituitary

The report of 'kallikrein-like' activity in the rat neuro-intermediate lobe (N-IL) and its possible involvement in pro-opiomelanocortin processing led us to explore the expression of the kallikrein gene(s) in the pituitary. Using 32P-labelled rat pancreatic kallikrein cDNA, we have shown positive hybridization for rat anterior pituitary poly(A)+ RNA, of identical size on Northern blots (approximately 1.0 kb) to rat kidney poly(A)+ RNA run in parallel. Prior adrenalectomy or ovariectomy decreased the level of kallikrein mRNA seen in the anterior pituitary; total RNA from rat N-IL showed no significant hybridization. On hybridization histochemistry the anterior pituitary was strongly positive, and the neural and intermediate lobes negative. The previously reported kallikrein-like activity in the N-IL is therefore probably due to a non-kallikrein kininogenase; in the anterior pituitary, kallikrein may have a physiological role in limited precursor proteolysis, but lack kininogen activity.     

Proenkephalin messenger RNA is expressed both in the rat anterior and posterior pituitary.

The presence of proenkephalin (PENK)-derived opioid peptides in the pituitary gland is well known. However, the cellular sources of their biosynthetic origin in all three pituitary lobes are less clear. In this study we identified the potential sites of synthesis by localizing the mRNA coding for PENK in the rat pituitary gland using in situ hybridization histochemistry. Numerous cells containing PENK mRNA were detected throughout the anterior lobe. Although suggested by previous reports, no mRNA signal could be detected in the intermediate lobe. Surprisingly, high levels of PENK mRNA were found in the posterior lobe. The cellular distribution in the neural lobe implies that pituicytes, a special class of glial cells, may express PENK mRNA.     

Galanin is an estrogen-inducible, secretory product of the rat anterior pituitary.

Galanin is a peptide widely distributed throughout vertebrate central and peripheral nervous systems. Although its precise physiologic role is unknown, it can stimulate the pituitary secretion of prolactin and growth hormone. We examined the control of rat galanin (rGal) gene expression in the anterior pituitary using RNA blot and in situ hybridization analyses and using specific RIA. Pituitaries of normal male and ovariectomized female rats contained little detectable rGal mRNA. Treatment of these animals with 17 beta-estradiol increased pituitary rGal mRNA up to 4000-fold. These increases depended on time and dose of estrogen administration and correlated with up to 50-fold increases in pituitary galanin-like immunoreactivity. Galanin-like immunoreactivity was detectable in the plasma of estrogen-treated animals. Pituitary levels of rGal mRNA in female rats varied greater than 30-fold during the estrous cycle, with a peak on estrus and a nadir on diestrus. Estrogen-induced rGal gene expression was also observed in transplantable MtTW15 prolactin- and growth hormone-containing tumors but not in neuronal tissues expressing this gene. These data demonstrate that rGal is a secreted product of rat anterior pituitary cells, where its gene expression is strongly affected by physiologic levels of circulating estrogen.     

Chronic estrogen treatment promotes a functional uncoupling of the D2 dopamine receptor in rat anterior pituitary gland

The effect of chronic estrogen treatment on the anterior pituitary D2 dopamine receptor was studied by treating rats with diethylstilbestrol (DES) over a 6-week period. DES treatment resulted in an increase in anterior pituitary weight and PRL content and serum PRL levels compared to those in sham-treated controls. The status of the anterior pituitary D2 dopamine receptor was evaluated using both radioligand binding and adenylate cyclase assays. [125I]N-(p-aminophenethyl)spiroperidol [( 125I]NAPS), a derivative of the D2-selective antagonist spiperone, was used to quantitate D2 receptors. Saturation analysis of [125I]NAPS binding indicated that DES treatment had no effect on the affinity or maximum binding capacity of the radioligand for the D2 receptor. Competition analysis with unlabeled D2 antagonists for [125I]NAPS binding also indicated that DES treatment did not affect antagonist interactions with the receptor. In contrast, the interactions of agonists with the D2 receptors from DES-treated rats were modified, as assessed through [125I]NAPS competition analysis. Using control tissue, agonist competition curves revealed both high and low affinity agonist binding states of the receptor. In the presence of guanine nucleotides, the high affinity agonist binding state is abolished, reflecting coupling of the receptor with a guanine nucleotide regulatory (G) protein. In DES-treated tissue, agonist competition curves indicated the presence of only low affinity agonist binding, with minimal effects of guanine nucleotides, suggesting uncoupling of receptor-G-protein interactions. The functionality of the D2 receptor was further assessed by examining dopaminergic inhibition of vasoactive intestinal peptide-stimulated adenylate cyclase activity. Although DES treatment resulted in a reduction of vasoactive intestinal peptide-stimulated enzyme activity itself, the ability of dopaminergic agonists to inhibit this activity was reduced by about 50%. These results suggest that estrogen is capable of attenuating the functional coupling of the D2 receptor with its biochemical effector system in the anterior pituitary gland.