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Cessation of lactation causes a massive loss of surplus lactotrophs in the rat pituitary gland. The factors and mechanisms involved in this phenomenon have not yet been elucidated. Besides its inhibitory control on prolactin secretion and lactotroph proliferation, evidence suggests that dopamine (DA) may be a proapoptotic factor for lactotrophs. We therefore tested the proapoptotic effect of DA on pituitary glands from virgin, lactating, and postlactating rats. By measuring mitochondrial membrane potential loss, caspase-3 activation, and nuclear fragmentation, we show that DA induces apoptosis specifically in lactotrophs from postlactating rats. We then determined that this effect was partly mediated by the DA transporter (DAT) rather than the D(2) receptor, as corroborated by the detection of DAT expression exclusively in lactotrophs from postlactating rats. We also observed tyrosine hydroxylase (TH) expression in postlactating lactotrophs that was accompanied by an increase in DA content in the anterior pituitary gland of postlactating compared with virgin rats. Finally, we observed that cells expressing TH coexpressed DAT and cleaved caspase-3. These findings show that DA may play a role in lactotroph regression during the postlactation period by inducing apoptosis. The fact that this process requires DAT and TH expression by lactotrophs themselves suggests that it may be "autocrine" in nature.

Previous studies showed that 17 beta-estradiol (17 beta-E2) regulates the nitric oxide (NO)/soluble guanylyl cyclase (sGC)/cGMP pathway in many tissues. Evidence from our laboratory indicates that 17 beta-E2 disrupts the inhibitory effect of NO on prolactin release, decreasing sGC activity and affecting the cGMP pathway in anterior pituitary gland of adult ovariectomized and estrogenized rats. To ascertain the mechanisms by which 17 beta-E2 affects sGC activity, we investigated the in vivo and in vitro effects of 17 beta-E2 on sGC protein and mRNA expression in anterior pituitary gland from immature female rats. In the present work, we showed that 17 beta-E2 acute treatment exerted opposite effects on the two sGC subunits, increasing alpha1 and decreasing beta1 subunit protein and mRNA expression. This action on sGC protein expression was maximal 6-9 h after 17 beta-E2 administration. 17beta-E2 also caused the same effect on mRNA expression at earlier times. Concomitantly, 17 beta-E2 dramatically decreased sGC activity 6 and 9 h after injection. These effects were specific of 17 beta-E2, because they were not observed with the administration of other steroids such as progesterone and 17 alpha-estradiol. This inhibitory action of 17beta-E2 on sGC also required the activation of estrogen receptor (ER), because treatment with the pure ER antagonist ICI 182,780 completely blocked 17 beta-E2 action. 17 beta-E2 acute treatment caused the same effects on pituitary cells in culture. These results suggest that 17 beta-E2 exerts an acute inhibitory effect on sGC in anterior pituitary gland by down-regulating sGC beta 1 subunit and sGC activity in a specific, ER-dependent manner.

To determine whether the type 2 iodothyronine deiodinase (D2), the principal central nervous system enzyme converting T(4) to biologically active T(3), is regulated in tanycytes by immune activation, D2 activity was measured in the mediobasal hypothalamus (MBH) 4, 12, and 24 h after administration of bacterial lipopolysaccharide (LPS) and compared with D2 levels in the cortex and anterior pituitary of rats. In contrast to D2 activity in the cortex and anterior pituitary that showed a steady linear increase over 24 h, which was coincident with a decline in thyroid hormone and TSH levels, D2 activity peaked in the MBH 12 h after LPS administration. By in situ hybridization, the increased D2 mRNA synthesis induced by LPS was specifically localized to tanycytes lining the third ventricle. In vitro assays in HC11 and HEK-293 cells demonstrated that the p65 subunit of nuclear factor-kappaB markedly increased both rat and human D2 genes (dio2) as analyzed by promoter assays. No activation of human dio2 was observed when an 83-bp minimal promoter was used. We propose that LPS or LPS-induced cytokines directly induce D2 mRNA in tanycytes. The ensuing MBH-specific D2-mediated local thyrotoxicosis may suppress the hypothalamus-pituitary-thyroid axis by local feedback inhibition of hypophysiotropic TRH and/or TSH and contribute to the mechanism of central hypothyroidism associated with infection.

Previous studies dealing with the mechanisms underlying the tissue-specific and regulated expression of the GnRH receptor (GnRH-R) gene led us to define several cis-acting regulatory sequences in the rat GnRH-R gene promoter. These include functional sites for steroidogenic factor 1, activator protein 1, and motifs related to GATA and LIM homeodomain response elements as demonstrated primarily in transient transfection assays in mouse gonadotrope-derived cell lines. To understand these mechanisms in more depth, we generated transgenic mice bearing the 3.3-kb rat GnRH-R promoter linked to the human placental alkaline phosphatase reporter gene. Here we show that the rat GnRH-R promoter drives the expression of the reporter gene in pituitary cells expressing the LHbeta and/or FSHbeta subunit but not in TSHbeta- or GH-positive cells. Furthermore, the spatial and temporal pattern of the transgene expression during the development of the pituitary was compatible with that characterizing the emergence of the gonadotrope lineage. In particular, transgene expression is colocalized with the expression of the glycoprotein hormone alpha-subunit at embryonic day 13.5 and with that of steroidogenic factor 1 at later stages of pituitary development. Transgene expression was also found in specific brain areas, such as the lateral septum and the hippocampus. A single promoter is thus capable of directing transcription in highly diverse tissues, raising the question of the different combinations of transcription factors that lead to such a multiple, but nevertheless cell-specific, expressions of the GnRH-R gene.

Objective: Thyrotropin-releasing hormone (TRH) is inactivated in the extracellular compartment by pyroglutamyl aminopeptidase II (PPII), a narrow specificity ectopeptidase present in the brain and in the lactotrophs of the adenohypophysis. TRH and various hypothalamic/paracrine agents regulate the activity of PPII on the surface of adenohypophyseal cells in primary culture. The activity of the hypothalamic-pituitary-thyroid axis presents circadian variations including an increase of serum thyrotropin levels in the early hours of the day. The purpose of this study was to determine whether adenohypophyseal PPII activity fluctuates during the daytime in the male rat and the role of TRH in these regulatory events in vivo., Results: Adenohypophyseal PPII specific activity and mRNA levels presented diurnal variations. A decrease in specific activity occurred with a minimum between 0930 and 1130 h, associated with increased serum thyrotropin levels. PPII mRNA levels were lowest at 0800 h. Intraperitoneal injection at 0800 or 1000 h of [3-Me-His(2)]-TRH, a potent agonist of the TRH receptor, reduced PPII specific activity at 30 min post-injection which was followed by a return to basal levels at 2 h. A second phase of decrease occurred between 4 and 8 h post-injection. Intravenous injection of a TRH-immune serum induced, at 2 h post-injection, an increase in adenohypophyseal PPII specific activity, which lasted up to 6 h., Conclusions: Adenohypophyseal PPII activity and mRNA levels fluctuate during the day; TRH down-regulates PPII activity in vivo, contributing to some of these variations. These new findings, and previous data, suggest that adenohypophyseal PPII activity varies in distinct physiological events, in response to endocrine and hypothalamic/paracrine factors, potentially modulating responses to TRH.

Objective: Local 5'-deiodination of l-thyroxine (T(4)) to active thyroid hormone 3,3',5-tri-iodothyronine (T(3)) catalyzed by the two 5'-deiodinase enzymes (D1 and D2) regulates various T(3)-dependent functions in the anterior pituitary and has been well studied in rodents. Only limited information about deiodinase expression and its cellular distribution in human anterior pituitaries is available., Design: We examined 5'-deiodinase enzyme activities in pituitary adenomas (18 non-functioning, seven TSH-producing, one GH- and TSH-producing, five GH-producing, eight prolactin (PRL)-producing, two adenomas each from patients with Cushing's disease and Nelson's syndrome) and three normal anterior pituitaries., Methods: Activities were measured as release of (125)I(-) from tyrosyl-ring labeled reverse T(3) with or without propylthiouracil, a potent inhibitor of D1 which does not influence D2 activities., Results: Most of the adenomas and normal tissues expressed both isoenzymes, with D2 activity higher than D1. In a few tissues D1 activity was higher than D2 and some tissues did not express D1 activity at all. Highest activities of both enzymes were found in TSH- and PRL-producing adenomas but absolute activities and the D1/D2 ratio were variable in the same kind of tumor in different patients., Conclusion: The finding that all examined tissues expressed 5'-deiodinase activity, most of them expressing both isoenzymes, implies that both enzymes are still active in tumors and that local deiodination is important for the function and feedback regulation of human anterior pituitary.

The matrix metalloproteinases (MMPs) are a family of zinc-containing endopeptidases that are able to degrade the extracellular matrix and allow angiogenesis and tumor invasion. The vast majority of pituitary tumors are benign and do not metastasize to distant sites, although they may invade locally. The aim of this study was to determine whether expression of the collagenase MMP-9 may play a role in allowing angiogenesis and invasion by different pituitary tumor types. Tumor expression of MMP-9 was investigated using a monoclonal antibody on a series of well-characterized paraffin-embedded sections of pituitary tumors. Invasive macroprolactinomas (n = 11) were significantly more likely to express MMP-9 than noninvasive macroprolactinomas (n = 8) (P = 0.003). Invasive macroprolactinomas showed higher-density MMP-9 staining than noninvasive tumors (P < 0.05). MMP-9 expression did not differ between noninvasive tumors and normal pituitary gland, or between different sized prolactinomas. MMP-9 expression was related to aggressive tumor behavior. It was higher in invasive macroprolactinomas (P = 0.003) when compared with noninvasive macroprolactinomas or the normal anterior pituitary gland. In addition, although there was no difference in whether MMP-9 was present or not when nonfunctioning adenomas that recurred were compared with those that did not, samples of recurrent tumor at the second presentation were more likely to express MMP-9 (P = 0.01). Pituitary carcinomas were significantly more likely to be MMP-9 positive compared with normal anterior pituitary gland (P = 0.05), but there was no difference from invasive adenomas. Angiogenesis assessed by vascular density was related to MMP-9 expression (P < 0.05). In summary, we have shown the presence of MMP-9 expression in some invasive and recurrent pituitary adenomas, and in the majority of pituitary carcinoma. The mechanisms whereby MMP-9 expression influences tumor recurrence and invasiveness, and its association with angiogenesis, remains to be elucidated. However, these observations suggest that a future potential therapeutic strategy for some pituitary tumors may be administration of a synthetic MMP-9 inhibitor.

Peptidylglycine alpha-amidating monooxygenase (PAM) is a bifunctional enzyme expressed in each major anterior pituitary cell type. We used primary cultures of adult male rat anterior pituitary to examine PAM expression, processing, and secretion in the different pituitary cell types and to compare these patterns to those observed in transfected AtT-20 corticotrope tumor cells. Immunostaining and subcellular fractionation identified PAM in pituitary secretory granules and additional vesicular compartments; in contrast, in AtT-20 cells, transfected PAM was primarily localized to the trans-Golgi network. PAM expression was highest in gonadotropes, with moderate levels in somatotropes and thyrotropes and lower levels in corticotropes and lactotropes. Under basal conditions, less than 1% of the cell content of monooxygenase activity was secreted per h, a rate comparable to the basal rate of release of individual pituitary hormones. General secretagogues stimulated PAM secretion 3- to 5-fold. Stimulation with specific hypothalamic releasing hormones demonstrated that different pituitary cell types secrete characteristic sets of PAM proteins. Gonadotropes and thyrotropes release primarily monofunctional monooxygenase. Somatotropes secrete primarily bifunctional PAM, whereas corticotropes secrete a mixture of mono- and bifunctional proteins. As observed in transfected AtT-20 cells, pituitary cells rapidly internalize the PAM/PAM-antibody complex from the cell surface. The distinctly different steady-state localizations of endogenous PAM in primary pituitary cells and transfected PAM in AtT-20 cell lines may simply reflect the increased storage capacity of primary pituitary cells.

GH-releasing hormone (GHRH) can induce proliferation of somatotroph cells. The pathway involving adenylyl cyclase/cAMP/protein kinase A pathway in its target cells seems to be important for this action, or at least it is deregulated in some somatotroph pituitary adenomas. We studied in this work whether GHRH can also stimulate mitogen-activated protein (MAP) kinase. GHRH can activate MAP kinase both in pituitary cells and in a cell line overexpressing the GHRH receptor. Although both protein kinase A and protein kinase C could activate MAP kinase in the CHO cell line studied, neither protein kinase A nor protein kinase C appears to be required for GHRH activation of MAP kinase in this system. However, sequestration of the betagamma-subunits of the G protein coupled to the receptor inhibits MAP kinase activation mediated by GHRH. This pathway also involves p21ras and a phosphatidylinositol 3-kinase, probably phosphatidylinositol 3-kinase-gamma. Despite the involvement of p21ras, the protein kinase Raf-1 is not hyperphosphorylated in response to GHRH, contrary to what usually occurs when the Ras-Raf-MAP kinase pathway is activated. In summary, this work describes for the first time the activation of MAP kinase by GHRH and outlines a path for this activation that is different from the cAMP-dependent mechanism that has been traditionally described as mediating the mitogenic actions of GHRH.

Peptidylglycine alpha-amidating monooxygenase (PAM) is a bifunctional enzyme that catalyzes the carboxyl-terminal amidation of glycine-extended peptides in a two-step reaction involving a monooxygenase and a lyase. Several forms of PAM messenger RNA result from alternative splicing of the single copy PAM gene. The presence of alternately spliced exon A between the two enzymatic domains allows endoproteolytic cleavage to occur in selected tissues, generating soluble monooxygenase and membrane lyase from integral membrane PAM. While using an exon A antiserum, we made the unexpected observation that Charles River Sprague Dawley rats expressed forms of PAM containing exon A in their pituitaries, whereas Harlan Sprague Dawley rats did not. Forms of PAM containing exon A were expressed in the atrium and hypothalamus of both types of Sprague Dawley rat, although in different proportions. PAM transmembrane domain splicing also differed between rat breeders, and full-length PAM-1 was not prevalent in the anterior pituitary of either type of rat. Despite striking differences in PAM splicing, no differences in levels of monooxygenase or lyase activity were observed in tissue or serum samples. The splicing patterns of other alternatively spliced genes, pituitary adenylate cyclase-activating polypeptide receptor type 1 and cardiac troponin T, did not vary with rat breeder. Strain-specific variations in the splicing of transcripts such as PAM must be taken into account in analyzing the resultant proteins, and knowledge of these differences should identify variations with functional significance.

Previous studies have shown that interaction of GnRH with its serpentine, G protein-coupled receptor results in activation of the extracellular signal regulated protein kinase (ERK) and the Jun N-terminal protein kinase (JNK) pathways in pituitary gonadotropes. In the present study, we examined GnRH-stimulated activation of an additional member of the mitogen-activated protein kinase (MAPK) superfamily, p38 MAPK GnRH treatment of alphaT3-1 cells resulted in tyrosine phosphorylation of several intracellular proteins. Separation of phosphorylated proteins by ion exchange chromatography suggested that GnRH receptor stimulation can activate the p38 MAPK pathway. Immunoprecipitation studies using a phospho-tyrosine antibody resulted in increased amounts of immunoprecipitable p38 MAPK from alphaT3-1 cells treated with GnRH. Immunoblot analysis of whole cell lysates using a phospho-specific antibody directed against dual phosphorylated p38 kinase revealed that GnRH-induced phosphorylation of p38 kinase was dose and time dependent and was correlated with increased p38 kinase activity in vitro. Activation of p38 kinase was blocked by chronic phorbol ester treatment, which depletes protein kinase C isozymes alpha and epsilon. Overexpression of p38 MAPK and an activated form of MAPK kinase 6 resulted in activation of c-jun and c-fos reporter genes, but did not alter the expression of the glycoprotein hormone alpha-subunit reporter. Inhibition of p38 activity with SB203580 resulted in attenuation of GnRH-induced c-fos reporter gene expression, but was not sufficient to reduce GnRH-induced c-jun or glycoprotein hormone alpha-subunit promoter activity. These studies provide evidence that the GnRH signaling pathway in alphaT3-1 cells includes protein kinase C-dependent activation of the p38 MAPK pathway. GnRH integration of c-fos promoter activity may include regulation by p38 MAPK.

To determine the site and mechanism of action of gonadal steroids on pituitary nitric oxide synthase type I (NOS I), present in both gonadotrophs and folliculo-stellate cells, the effects of castration and steroids were examined in male rats, in the presence of a GnRH antagonist (Antarelix). Western analysis showed a rapid and substantial increase with time, after orchidectomy, of NOS I protein, the concentration doubling in 24 h and reaching a maximal 4- to 5-fold increase after 3-7 days, followed by a progressive decline after 2 weeks. Testosterone or estradiol replacement, or administration of GnRH antagonist, totally abolished the effects of castration, demonstrating a mediation of the steroid effects via GnRH. In noncastrated rats, steroids and the GnRH antagonist also caused a reduction in the levels of NOS I (by 50-60%), consistent with inhibition of endogenous GnRH stimulation. In marked contrast, administration of a potent GnRH agonist (Triptorelin) to intact rats increased the levels of NOS I. A time-course study with a long-lasting formulation showed that rise in NOS I developed rapidly after a lag of approximately 5 h, with a 2-fold increase detectable after 8 h and a maximal 4.5-fold after 48 h. The level declined afterwards in a manner consistent with homologous desensitization that may occur in the continuous presence of GnRH; however, the profile was different and delayed compared with those of gonadotropin release. As observed for NOS I protein, NOS I messenger RNA concentration was increased by castration or GnRH agonist and reduced by steroids or GnRH antagonist. Taken together, these data demonstrate that steroids indirectly regulate NOS I messenger RNA and protein levels, through the hypothalamic modulation of GnRH, which represents the primary regulator of NOS I. No effect of steroids on NOS I was seen in the posterior lobe. NADPH-diaphorase histochemistry coupled to immuno-identification of the cells revealed that the treatments affecting the concentration of NOS I concomitantly altered the activity but exclusively in gonadotrophs and not in folliculo-stellate cells (which do not respond to GnRH), reinforcing the idea that GnRH played a major regulatory role. Expression in gonadotrophs of a GnRH-dependent NOS I and the ensuing production of nitric oxide represents a potentially novel signaling pathway for the neuropeptide in the anterior pituitary, consistent with the previously reported GnRH-induced cGMP production, the role of which remains to be evaluated.

TRH is inactivated by the TRH-degrading ectoenzyme, a TRH-specific metallopeptidase. At the pituitary level, this enzyme is stringently regulated by thyroid hormones. We describe here gender-related differences and the effect of estradiol (E2) on the expression of this enzyme in the anterior pituitary. Compared with male rats, only about one third of the enzymatic activities and the messenger RNA levels were found in the anterior pituitary of female rats, whereas the TRH receptor transcript levels were found inversely related. When male rats received a single injection of 0.5 microg E2/100 g BW, the enzymatic activity decreased to 65% of control values within 14 h, preceded by a decrease of the transcript levels to 25% of control within 6 h. Basal values were reached again 24-48 h after the injection. E2 had no effect on the expression of the enzyme in the brain. In vivo and with GH3 cells in vitro, E2 effectively counteracted the increase in enzymatic activity induced by T3, whereas neither testosterone nor progesterone, aldosterone, or dexamethasone showed any significant effects. Because the expression of the adenohypophyseal TRH-degrading ectoenzyme is tightly regulated by both T3 and E2 with adequate dynamics, we conclude that this peptidase serves integrative functions for the control of TRH-stimulated hormone secretion.

To identify the specific locations of type 2 deiodinase (D2) messenger RNA (mRNA) in the hypothalamus and pituitary gland and determine its regulation by thyroid hormone, we performed in situ hybridization histochemistry, Northern analysis, and quantitative RT-PCR in euthyroid, hypothyroid, and hyperthyroid rats. By in situ hybridization histochemistry, silver grains were concentrated over ependymal cells lining the floor and infralateral walls of the third ventricle extending from the rostral tip of the median eminence (ME) to the infundibular recess, surrounding blood vessels in the arcuate nucleus (ARC), and in the ME adjacent to the portal vessels and overlying the tuberoinfundibular sulci. Silver grains also accumulated over distinct cells in the midportion of the anterior pituitary. In hypothyroid animals, an increase in signal intensity was observed in the caudal hypothalamus, and a marked increase in the number of positive cells occurred in the anterior pituitary. Microdissection of the hypothalamus for Northern and PCR analysis established the authenticity of D2 mRNA in the caudal hypothalamus, and confirmed that the majority of D2 mRNA is concentrated in this region. The distribution of D2 mRNA suggests its expression in specialized ependymal cells, termed tanycytes, originating from the third ventricle. Thus, the tanycyte is the source of the high D2 activity previously found in the ARC-ME region of the hypothalamus. The results indicate that tanycytes may have a previously unrecognized integral role in feedback regulation of TSH secretion by T4.

Local deiodination of L-thyroxine (T4) to the active thyroid hormone T3 via two 5'deiodinase isoenzymes (5'DI and 5'DII) plays an important role for various T3-dependent functions of the anterior pituitary (AP). Recently, it was reported that 3,5-T2, the 5'deiodination product of T3, acts as a specific agonist in the feedback mechanism on TSH secretion at the pituitary level. We now examined the effects of 3,5-T2 on pituitary 5'deiodinase activities in vivo in male, adult rats and in vitro using rat AP reaggregate cultures and the somatomammotroph cell line GH3. 5'DI activity in the AP was transiently increased after a single injection of 3,5-T2. Serum TSH levels declined, and 24 h after 3,5-T2 application, betaTSH steady-state mRNA levels in the APs were markedly lower. In reaggregate cultures of the AP, 3,5-T2 stimulated 5'DI activity 24 h after application, dose-dependently. Compared with 5'DI activities, those of 5'DII were an order of magnitude lower, in vivo as well as in vitro, and were rapidly and transiently decreased by the higher dose of 3,5-T2. GH3 cells responded to 3,5-T2 and T3 by an 1.7-fold stimulation of 5'DI activity. Stimulation of DNA-binding was demonstrated in electrophoretic mobility shift assays for a specific RXR-containing protein complex with a DR+4 thyroid hormone response element of the human type 1 5'DI promoter using nuclear extracts from GH3 cells treated with 3,5-T2. In summary, 3,5-T2 and T3 exert direct thyromimetic effects on 5'DI activity and TSHbeta expression at the pituitary level. 5'DI is regulated by its substrate(s) and/or products and may serve an important function within the modulation of thyroid hormone-dependent gene expression in the AP.

The mechanism of mitogen-activated protein kinase (MAPK, ERK) stimulation by the GnRH analog [D-Trp6]GnRH (GnRH-a) was investigated in the gonadotroph-derived alphaT3-1 cell line. GnRH-a as well as the protein kinase C (PKC) activator 12-O-tetradecanoyl phorbol-13-acetate (TPA) stimulated a sustained response of MAPK activity, whereas epidermal growth factor (EGF) stimulated a transient response. MAPK kinase (MEK) is also activated by GnRH-a, but in a transient manner. GnRH-a and TPA apparently activated mainly the MAPK isoform ERK1, as revealed by Mono-Q fast protein liquid chromatography followed by Western blotting as well as by gel kinase assay. GnRH-a and TPA stimulated the tyrosine phosphorylation of several proteins, and this effect as well as the stimulation of MAPK activity were inhibited by the PKC inhibitor GF 109203X. Similarly, down-regulation of TPA-sensitive PKC subspecies nearly abolished the effect of GnRH-a and TPA on MAPK activity. Furthermore, the protein tyrosine kinase (PTK) inhibitor genistein inhibited protein tyrosine phosphorylation and reduced GnRH-a-stimulated MAPK activity by 50%, suggesting the participation of genistein-sensitive and insensitive pathways in GnRH-a action. Although Ca2+ ionophores have only a marginal stimulatory effect, the removal of Ca2+ markedly reduced MAPK activation by GnRH-a and TPA, but had no effect on GnRH-a and TPA stimulation of protein tyrosine phosphorylation. Interestingly, the removal of Ca2+ also partly inhibited the activation of MAPK by EGF and vanadate/H2O2. Thus, a calcium-dependent component(s) downstream of PKC and PTK might also participate in MAPK activation. Elevation of cAMP by forskolin exerted partial inhibition on EGF, but not on TPA or GnRH-a action, suggesting that MEK activators other than Raf-1 might be involved in GnRH action. We conclude that Ca2+, PTK, and PKC participate in the activation of MAPK by GnRH-a, with Ca2+ being necessary downstream to PKC and PTK.

The pituitary is a rich source of peptidylglycine alpha-amidating monooxygenase (PAM). This bifunctional protein contains peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase catalytic domains necessary for the two-step formation of alpha-amidated peptides from their COOH-terminal glycine extended precursors. Expression of PAM was evaluated in the anterior pituitary of intact cycling adult female rat and after experimental manipulation of estrogen status. PAM messenger RNA (mRNA) levels showed changes inversely related to the physiological variations of plasma estrogen levels during the estrous cycle. Chronic treatment of ovariectomized (OVX) rats with 17 beta-estradiol decreased PAM mRNA levels to values comparable with those found in intact rats at proestrus. In situ hybridization of anterior pituitary sections using 35S-labeled full length RNA antisense transcripts of rat PAM-1 complementary DNA showed that 17 beta-estradiol treatment induced an overall decrease of the hybridization signal, as compared with OVX rats. Progesterone treatment did not change PAM mRNA levels both in OVX or OVX + E2 rats. Based on Northern blot analysis and amplification of fragments derived from rat PAM-1 by RT-PCR, it was found that estrogen status does not affect the distribution of PAM mRNA among its various alternatively spliced forms. In OVX 17 beta-estradiol treated rats, the specific activity of PAM in the anterior pituitary decreased in both soluble and particulate fractions compared with OVX animals. Western blot analysis demonstrated a 105-kDa PAM protein in particulate fractions prepared from OVX and OVX-17 beta-estradiol treated animals. The soluble fraction from OVX animals contained major PAM proteins of 105, 95, 84, 75, and 45 kDa, and 17 beta-estradiol treatment caused a decrease in the prevalence of these proteins. These results indicate that estrogens are involved, either directly or indirectly, in regulating the expression of PAM in several cell types in the anterior pituitary gland.

Vasoactive intestinal polypeptide (VIP) and galanin were isolated in pure form from the stomach of the European green frog, Rana ridibunda. Frog VIP is identical to the previously characterized VIP from chicken and alligator. The primary structure of frog galanin contains only two amino acid substitutions (asparagine for histidine at position 23 and histidine for tyrosine at position 26) compared with porcine galanin. The data indicate that evolutionary pressure to conserve the amino acid sequence of both peptides during the evolution of amphibia to mammals has been strong. Synthetic frog VIP produced a dose-dependent increase in cAMP concentration in frog anterior pituitary fragments. The potency of the peptide (ED50 = 1.2 x 10(-6) M; mean +/- SE; n = 8) was comparable to that of porcine VIP (EC50 = 1.3 x 10(-6) M), but was approximately 10-fold less than that of frog pituitary adenylate cyclase-activating polypeptide [PACAP-(1-38); ED50 = 1.1 x 10(-7) M] in the same system. The increases in cAMP concentrations produced by maximal doses of PACAP (10(-5) M) and VIP (10(-5) M) were not additive. The data suggest that the effects of both peptides are mediated through a common PACAP-preferring receptor that is pharmacologically different from the mammalian PACAP type I receptor. Synthetic frog galanin also produced a dose-dependent increase in the concentration of cAMP in isolated frog anterior pituitary fragments (ED50 = 9.3 x 10(-8) M) consistent with a possible role for the peptide as a hypophysiotropic factor in amphibians.

PRL release from rat lactotrophs in response to TRH is Ca2+ dependent. TRH-induced PRL release is inhibited either after repeated pulses of TRH or in the presence of dopamine. TRH, however, generates increases in intracellular Ca2+ concentrations ([Ca2+]i) in both conditions. Calcium/calmodulin-dependent protein kinase-II (CaM kinase-II) is a ubiquitous enzyme implicated in secretion. To determine whether down-regulation of CaM kinase-II activity caused the lack of responsiveness to increases in [Ca2+]i, we measured the generation of calcium/calmodulin-independent kinase activity. Anterior pituitary cells contain a 50-kilodalton form of CaM kinase-II, determined by immunoblot, and the enzyme is in lactotrophs, determined by immunocytochemistry. TRH rapidly and transiently increased calcium/calmodulin-independent kinase activity; the increase was maximal by 15 sec and returned to basal by 2 min. When TRH pulses (1 microM, 15 sec) were applied every 10 min, each pulse caused an increase in calcium/calmodulin-independent kinase activity of similar magnitude, and the activity returned to basal values between pulses. Pretreatment of cells with dopamine (1 microM; 30 min) inhibited PRL release, but did not prevent the increase in calcium/calmodulin-independent kinase activity. These results indicate that TRH still activates CaM kinase-II when PRL release is inhibited. Dopamine and repeated pulses of TRH must inhibit PRL release at a site after the TRH-induced increase in [Ca2+]i and at a site other than CaM kinase-II.

Expression of the genes encoding the alpha- and beta-isoforms of the catalytic (C) and regulatory type I (RI) and type II (RII) subunits of cAMP-dependent protein kinases (PKA) in the anterior pituitary gland of rats was examined by hybridization of specific 32P-labeled cDNA probes to mRNA. C alpha, C beta, RI alpha, RI beta, RII alpha, and RII beta mRNAs were all present in the anterior pituitary gland and cultured cells, but in different proportions; C alpha was the most abundant, and RII alpha was the least. For C alpha, C beta, RI beta, and RII alpha, Northern blot analysis revealed single mRNAs with sizes of 2.4, 4.5, 2.8, and 6.0 kilobases (kb), respectively. For RI alpha, three distinct mRNAs (1.7, 3.2, and 3.7 kb) were identified, and for RII beta, two were found (1.8 and 3.5 kb). Compared to other tissues and species, including ovine and rat pituitary and testis, differences in sizes and relative abundance of mRNAs and/or mRNA subtypes were observed, demonstrating that great diversity exists, which may reflect tissue and species variation in posttranscriptional processing of mRNA transcripts. When rat anterior pituitary cells were cultured in the presence of 8-bromo-cAMP (8-Br-cAMP), RII beta mRNA levels increased in a dose- and time-dependent manner to a maximum of about 4- to 5-fold the basal values after 5 h in the presence of 2 mM 8-Br-cAMP. Under the same conditions, the C alpha mRNA, already at high levels, further increased, but to a lesser extent (1.5-2 times compared to basal abundancy). Cholera toxin (6 nM) reproduced the effects of 8-Br-cAMP on both C alpha and RII beta mRNAs. These effects were completely abolished in the presence of actinomycin-D, suggesting that cAMP enhances RII beta and C alpha gene transcription. On the other hand, cell exposure to the phorbol ester 12-O-tetradecanoyl 13-phorbol acetate, a potent activator of protein kinase-C (PKC), readily induced a dose-dependent actinomycin-D-inhibited increase in the RII beta mRNA content to levels about 2-fold those in control unstimulated cells, whereas it depressed levels of C alpha mRNA by 25%. In conclusion, we have found that the six genes coding for the alpha- and beta-isoforms of PKA subunits are all expressed in the anterior pituitary, and that the expression of two of those genes (RII beta and C alpha) is regulated by cAMP and phorbol ester in a similar or opposite manner.(ABSTRACT TRUNCATED AT 400 WORDS)

Glucocorticoids promote the development of many organ systems vital for extrauterine survival, and fetal cortisol provides the trigger for birth in sheep. The activity of glucocorticoids may be influenced at a cellular level by 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD), which is responsible for the interconversion of cortisol and cortisone. To examine 11 beta-HSD gene expression during fetal development, two overlapping clones which yield a 1.4 kilobase (kb) complementary DNA encoding sheep 11 beta-HSD from a liver library were isolated by using a rat 11 beta-HSD cDNA as the probe. This cDNA contains a 879 base pair open reading frame for a protein of 292 amino acids that has more than 70% sequence identity to rat and human 11 beta-HSDs. To define the tissue distribution of 11 beta-HSD messenger RNA in sheep, selected tissues were collected from one fetus at day 130 and term (approximately 145 days), and from a nonpregnant ewe. Cellular RNA was extracted and subjected to Northern blot analysis, and a single 1.8 kb transcript was detected in the fetal and adult liver, lung, hypothalamus, anterior pituitary, and placenta. This was undetectable in adrenals and kidneys, but a smaller (1.5 kb) transcript was present in fetal and adult kidney RNA. The relative abundance of 11 beta-HSD mRNA was greatest in fetal and adult livers, and it was much higher in adult liver, lung, and kidney than in the corresponding fetal tissues. To examine whether 11 beta-HSD gene expression is developmentally regulated in the fetal sheep, liver, lung, and kidney tissues were taken from fetuses at day 60-70, day 100-110, day 125-130, at term, and from newborn lambs (24-48 h old). In the lung and kidney, the relative abundance of 11 beta-HSD mRNA did not change from day 60 to term but increased in the lungs of newborn lambs. In contrast, 11 beta-HSD mRNA levels in the liver increased between day 125 and term and rose further in the newborn. Collectively, these results demonstrate that 11 beta-HSD gene expression in sheep is regulated in a tissue-specific and developmentally programmed manner.

A GTPase assay was employed to determine the relative proportions of the enzymatic activity in soluble and polymerized tubulin pools in the anterior pituitary lobe of the lactating rat. The GTPase activity in the tubulin fractions was estimated in 25-50 micrograms protein using [gamma-32P]GTP. The liberation of inorganic phosphate (Pi) was proportional to the protein concentration with either of the tubulin fractions. The enzymatic activity appeared to reach equilibrium by 1 min. Antitubulin antibodies inhibited the enzymatic activity in a concentration-dependent manner in both the tubulin fractions; at a final dilution of 1:2000 the antibody maximally inhibited the enzyme activity in both the tubulin fractions by 39-44%. After establishing the optimal conditions for the GTPase assay, the effect of suckling on pituitary GTPase activity was studied. Soluble and polymerized tubulin fractions were prepared from anterior pituitaries obtained from lactating rats killed after suckling for 30, 60, and 90 min; GTPase activity was assayed in both the tubulin fractions in the absence of antitubulin antibody. Compared to the nonsuckled control, suckling for 60 and 90 min stimulated the enzymatic activity in the soluble tubulin fraction by 80% and 44%, respectively (P less than 0.05). The enzymatic activity in the polymerized tubulin fraction increased by 30% at 60 min and decreased by about 20% at 90 min (P less than 0.05). The suckling-stimulated GTPase activity in the two pituitary fractions cannot be attributed to tubulin alone since there are other proteins also capable of hydrolyzing GTP. Therefore, GTPase activity was assayed in the pituitary tubulin fractions in the presence of antitubulin antibody (1:2000 dilution); tubulin-GTPase activity is the difference between the activity assayed in the absence of the antibody and that which was determined in the presence of the antibody. In the soluble tubulin fraction, tubulin-GTPase activity increased by 166% at 30 min suckling (P less than 0.05), decreased by 40% at 60 min (P less than 0.05), and again increased by 148% at 90 min (P less than 0.05). In the polymerized tubulin fraction, the enzyme activity decreased by 82% at 30 min (P less than 0.05), increased by 742% at 60 min (P less than 0.05), and again decreased by 95% at 90 min (P less than 0.05). Thus, an inverse relationship between tubulin-GTPase activities in the two pituitary fractions was observed and provides further evidence in support of our hypothesis that microtubules are recruited to transport PRL granules from the Golgi apparatus to the plasma membrane.

The ability of GnRH to modulate protein kinase-C (PKC) activity was examined in perifused rat pituitary cell cultures. Under these conditions, LH release and GnRH receptor number remained unchanged after repeated pulses of 1 nM GnRM, whereas PKC (measured both enzymatically and by radioligand assay) showed an initial increase in kinase activity after the first pulse of GnRH (approximately 2-fold), followed by down-regulation of PKC activity with subsequent pulses of the releasing hormone. It was also observed that the GnRH-stimulated down-regulation of PKC was dependent on the presence of extracellular calcium, which was not the case for the initial up-regulation of PKC. These findings are consistent with a modulating role of the GnRH receptor on PKC activity through a Ca2(+)-dependent process. This study also provides further evidence that GnRH-stimulated LH release and PKC activity can be uncoupled.

Using monolayer cultures from murine brain and reaggregate cell cultures of rat anterior pituitary we observed that TRH (pyroGlu-His-Pro-NH2) added to the culture medium was not taken up by these cells but hydrolyzed at the pyroGlu-His bond by an enzyme obviously located at the cell surface. This enzyme exhibited a high degree of substrate specificity and other characteristics of the membrane-bound TRH-degrading enzyme. Relatively high enzymatic activity was associated with cultured neuronal cells from embryonic rat brain while glial cells were almost devoid of this peptidase activity. Rather low, but significant activity was found on anterior pituitary cell aggregates. In agreement with previous in vivo studies we observed that the TRH-degrading ectoenzyme on adenohypophyseal cells was regulated by estradiol and stringently controlled by T3, but that the activity of the brain enzyme was not. When pituitary cells were separated according to their size and density and established in reaggregate cell culture, a close correlation was always observed between enzyme activity and the distribution of lactotrophic cells regardless of the animal models (eu- and hypothyroid adult male rats) used and the cell fractionation techniques (velocity sedimentation and sequential velocity/buoyant density sedimentation) employed. Such a close correlation was not observed with other cell types, such as the somatotrophic cells, the folliculo-stellate cells, the ACTH-producing AtT20 pituitary cells, or thyrotrophic cells. In conclusion, the high degree of substrate specificity, the tissue-specific regulation, and the very heterogeneous distribution of the TRH-degrading ectoenzyme on brain and pituitary cells strongly support the hypothesis that this enzyme serves very specialized functions in the transmission of TRH signals at specific target sites.

The activity of T4 5'-monodeiodinase (5'D) in the pituitary was measured in 12 patients with pituitary adenoma (3 patients with acromegaly, 2 with prolactinoma, 1 with Cushing's disease, 1 with TSH-producing tumor, and 5 with nonfunctioning tumor) and, as a control, in a patient who died of parotid cancer. The pituitaries, obtained at operation or autopsy, were homogenized in 0.1 mol/L potassium phosphate buffer, pH 7.0, and centrifuged at 800 x g. Supernatants were incubated with [125I]T4 and 20 mmol/L dithiothreitol (DTT) at 37C for 90 min. T4 5'-D was measured by the release of 125I- with the ion exchange method. The activity of T4 5'-D in the pituitaries from patients with prolactinoma and parotid cancer was dependent on protein concentration, incubation time, incubation temperature, and T4 concentration, and was labile to prior heating at 70 C for 30 min. T4 5'-D was not inhibited by 1 mmol/L propylthiouracil, but was inhibited 95% by 0.1 mmol/L iopanoic acid. The apparent Km and maximum velocity for T4 5'-D in homogenates of prolactinoma at 20 mmol/L DTT were 11 nmol/L and 1.54 pmol/mg protein.h, respectively. This reaction followed sequential-type reaction kinetics when the DTT concentration was varied. All other homogenates of pituitary tumors, except two nonfunctioning tumors, also demonstrated T4 5'-D activity. These results indicate that 1) the human pituitary express a low Km and PTU-insensitive T4 5'-D activity which is very similar to the type II enzyme activity in the rat pituitary; and 2) various types of pituitary tumor cells contain T4 5'-D activity.

In primary cultures of sheep pituitary cells extracellular nucleotides stimulated rapid increases in inositol tris- and bisphosphate, accompanied by intracellular Ca2+ mobilization. A similar stimulation of inositol phosphate production by extracellular nucleotides was observed in rat and baboon pituitary cells. The inositol phosphate response to nucleotides was greater than that elicited by any of the known hypothalamic releasing peptides. UTP, ATP, and ATP gamma S were the most potent agonists, with EC50 values for inositol phosphate production of 1.2, 2.6, and 2.7 microM. The relative potencies of a range of nucleotides indicates that the pharmacological specificity of the pituitary nucleotide receptor is different from that of the previously characterized P2X and P2Y purinoceptors present in other tissues. Increasing extracellular Mg2+ concentrations caused a shift to the right of the ATP dose-response curves, indicating that the predominantly active agonist species is not MgATP and may be ATP4-. In the absence of both Ca2+ and Mg2+ (1 mM EDTA) ATP stimulated inositol phosphate production with high potency (EC50 = 200 nM), indicating that an ectokinase or ecto-ATPase reaction is not involved in its mode of action. Phosphoinositidase-C activation by ATP was insensitive to pertussis toxin. The magnitude of the inositol phosphate and 45Ca2+ responses to extracellular nucleotides indicates that a substantial fraction of the cells in primary pituitary cultures bears nucleotide receptors. None of the major pituitary hormones appear to be released by extracellular nucleotides. The cell types in the pituitary that bear these nucleotide receptors are at present unidentified.

The capacity of the 5 alpha-dihydroprogesterone analog, 4-aza-4-methyl-5 alpha-pregnane-3,20-dione (AMPD), to inhibit progesterone 5 alpha-reductase and both 5 alpha-dihydroprogesterone 3 alpha-hydroxysteroid oxidoreductase activities (NADPH- and NADH-linked) from the female rat anterior pituitary has been investigated. Dose response studies demonstrate that AMPD is a powerful inhibitor of pituitary progesterone 5 alpha-reduction but is ineffective at inhibiting either of the 3 alpha-hydroxysteroid oxidoreductase activities, even at concentrations up to 10 microM. A kinetic analysis of the interaction of AMPD with progesterone 5 alpha-reductase indicates that it is a competitive inhibitor vs. progesterone [Kislope = 7.2 +/- 0.6 nM; apparent Michaelis-Menten constant (Km) (progesterone) = 193 +/- 18 nM] and an uncompetitive inhibitor vs. NADPH (Kiintercept = 17.9 +/- 1.4 nM). These inhibition patterns are consistent with the view that NADPH binding precedes that of either AMPD or progesterone. Furthermore, AMPD does not appear to be an irreversible inhibitor since preincubation of the enzyme (at 37 C) with AMPD and NADPH, for periods of time up to 60 min, does not lead to a time-dependent loss of activity. The inhibition can also be readily removed by dilution, even after a 60-min preincubation with the inhibitor and NADPH. It is postulated that the selective and potent inhibition of the 5 alpha-reduction of progesterone by AMPD may be due to the steroid functioning as a transition state analog. This inhibitor should prove useful in studying the properties of progesterone 5 alpha-reductase and the function of anterior pituitary progestin metabolism.

We have investigated the effects of SRIF and human pancreatic GH-releasing factor-44 (hpGRF-44) on adenylate cyclase (AC) activity of male rat anterior pituitaries (in which somatotrophs are present in large proportion) and of human GH-secreting pituitary adenomas (which are almost homogeneously constituted by somatotrophs). The adenoma's responsiveness to both agents in terms of secretion was previously demonstrated in in vitro experiments. SRIF inhibited in a dose-dependent fashion the GH release from monolayer cultures of the tumors. The inhibition ranged from 32-66% at the maximal effective concentration (10(-6) M). hpGRF-44 stimulated GH release in a dose-dependent fashion. The stimulation was 78-172% at 10(-7) M. SRIF and hpGRF-44 markedly affected AC activity in both systems. SRIF elicited a pronounced inhibition of the enzyme activity in a dose-dependent manner. The inhibition was about 40% in the rat and ranged from 16-49% in adenomas at the maximal effective concentration (10(-5) M SRIF). The inhibitory effect was GTP-dependent. hpGRF-44 markedly stimulated AC activity. The stimulation was dose dependent and GTP dependent. The stimulation was about 650% in the rat and 26-350% in adenomas at the maximal effective concentration (10(-6) M). These results suggest the presence of a dually regulated (by SRIF and hpGRF-44) AC in GH-secreting cells; an involvement of cAMP in the intracellular mechanisms transducing the signals of SRIF and hpGRF-44 in somatotrophs.

We found a nyctohemeral rhythm of type II iodothyronine 5'-deiodinase (5'-D-II) with a zenith at midnight in rat anterior pituitary, but not in brown adipose tissue. There was no nyctohemeral rhythm of 5'-D-I in anterior pituitary, liver, or kidney. Hypothyroidism abolished the nyctohemeral rhythmicity in anterior pituitary 5'-D-II. The rhythmicity of anterior pituitary 5'-D-II may play a role in setting the nyctohemeral rhythm of TSH secretion by regulating the degree of negative feedback by locally generated T3 in the thyrotroph.

The effect of free calcium (Ca2+) on adenylate cyclase (AC) activity of rat anterior pituitary gland have been investigated in order to shed some light on the interrelationships between the two second messengers (cAMP and calcium) which operate in pituitary cells. Anterior pituitary homogenates or crude membranes preparations (obtained using buffers free of divalent cation chelators) were assayed and the concentrations of Ca2+ in the assay mixture containing EGTA were calculated by a computer program for each addition of CaCl2. A wide range of Ca2+ concentrations (from 2 X 10(-9) to 6 X 10(-4)M) was spanned. Ca2+ was found to markedly inhibit pituitary AC and the mathematical analysis of data indicated the presence of two inhibition The two KiS were: 1.78 +/- 0.48 X 10(-7) M and 2.47 +/- 0.52 X 10(-4) M for the homogenates and 1.71 +/- 0.45 X 10(-7) M and 3.15 +/- 0.85 X 10(-4) M for the membrane preparations. No stimulation of the enzyme could be detected at any Ca2+ concentration tested. Furthermore, because of our experimental conditions it is unlikely that there was substantial loss of endogenous calmodulin, or other calcium binding protein(s) required to mediate AC stimulation by calcium. The lack of a calcium-calmodulin stimulation of pituitary AC was confirmed by experiments with anticalmodulin drugs (trifluoperazine and calmidazolium, R24571) and experiments with EGTA-washed membranes in the presence of exogenous calmodulin. At any Ca2+ concentration, the same AC activity was observed in the presence and in the absence of anticalmodulin drugs or added calmodulin. The mechanism of pituitary AC inhibition by Ca2+ was investigated focusing on a range of Ca2+ concentrations near the Ki for the high affinity calcium site and thus similar to the intracellular Ca2+ concentrations. Ca2+ was found to act as a competitive inhibitor of the Mg2+ activation of AC and as a noncompetitive inhibitor with respect to the MgATP2-, the substrate of the enzyme. The effects of Ca2+ on AC were also studied in cell populations and tissues extremely rich in PRL-secreting cells (cell fractions purified from rat anterior pituitaries and human prolactinomas). The pattern of Ca2+ action was found to be nearly superimposable on that observed in total pituitary.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Peptidase activity capable of inactivating luteinizing hormone (LHRH) may have a physiological role in partially determining hypothalamic LHRH levels as well as LHRH levels at the gonadotrope. In our previous work ( Lapp and O' Conner , 1984, companion paper), use of the synthetic substrate leucine-p-nitroanilide (Leu-p-NA) to assay LHRH-degradative activity was validated by several methods. The current studies were conducted in order to monitor peptidase activity in the hypothalamus and pituitary throughout the rat 4-day estrous cycle. Activity in both tissues was significantly decreased during proestrus and diestrus I. It seems possible that the proestrous reduction in peptidase activity represents a permissive period necessary for the induction of the LHRH and LH surges. The decreased degradative activity in the pituitary on diestrus I may be involved in inducing the pituitary LHRH receptors which are reportedly synthesized prior to proestrus. The peptidase exhibits positive cooperativity with Leu-p-NA, and the degree of this cooperativity also fluctuates during the estrous cycle. Estradiol and progesterone given alone or in combination to prepubertal castrate animals increased the activity of the hypothalamic peptidase in vitro. The degree of positive cooperativity with which the enzyme functioned was also apparently altered by these gonadal steroids.

Lysates from purified secretory granules of rat anterior pituitary glands were incubated with [3H]phenylalanine or [3H]arginine-labeled toad proopiocortin. The processed products formed were identified by immunoprecipitation with ACTH and beta-endorphin antisera, and by comigration with known markers on acid-urea polyacrylamide gels. Proopiocortin was cleaved by the secretory granule lysate primarily to 21,000 mol wt ACTH, 13,000 mol wt ACTH, 16,000 mol wt NH2-terminal glycopeptide, beta-lipotropin, a beta-endorphin-like peptide, and beta-endorphin. Characterization of the anterior pituitary proopicortin-converting activity shows that it: (1) cleaves specifically at the peptide bond on the carboxy side of the lysine-arginine residues of proopiocortin, (2) has a pH optimum in the acidic range, (3) is present in membrane and soluble fractions of the granule lysate, and (4) is inhibited by leupeptin, pepstatin A, and 2,2' dithiodipyridine, but not by p-chloromercuribenzoate, diisopropyl fluorophosphate, N alpha-p-tosyl-L-lysine chloromethyl ketone hydrochloride, chloroquine, L-1-tosylamide-2-phenylethyl-chloromethyl ketone, or EDTA.

Cerebrocortical and pituitary iodothyronine 5'-deiodinases produce 50% or more of the T3 found in these tissues and show rapid 3- to 5-fold changes in response to changes in the thyroid status. Since many T3 responses are initiated in the cell nucleus and require protein synthesis, we studied the influence of cycloheximide and actinomycin D on the acute T3 suppression of cerebrocortical and pituitary 5'-deiodinase activities in hypothyroid rats. Cycloheximide inhibited protein synthesis by more than 95%, but did not prevent the rapid (4-h) T3-mediated decreases in 5'-deiodinase activity. In the presence of cycloheximide, T3 decreased enzyme activity by more than 85%, with a t1/2 of approximately 70 min in the cerebral cortex and about 110 min in the pituitary. In vitro, neither T3 nor cycloheximide in large excess had any effect on enzyme activity. In cycloheximide-treated rats, cerebrocortical 5'-deiodinase activity decreased with a fractional turnover rate (k) of 1.2 h-1 in euthyroid and 0.07 h-1 in hypothyroid rats, respectively, with corresponding steady state enzyme levels of 18 +/- 3 and 145 +/- 9 U/mg protein. The resulting production rates of cerebrocortical 5'-deiodinase were 22 and 10 U/mg protein X h. Actinomycin D failed to alter either the cerebrocortical 5'-deiodinase in euthyroid rats in 4 h or the inhibitory effect of T3, but inhibited RNA synthesis by more than 70%. These data indicate that the T3-dependent fall in cerebrocortical 5'-deiodinase is mediated by a posttranscriptional mechanism that ultimately increases the rate of degradation/inactivation of the enzyme.

Incubation of cultured rat pituitary cell aggregates with [3H]choline ([3H]Chol) yielded a derivative that was identified as [3H]acetylcholine ([3H]ACh) by several criteria: 1) the [3H]Chol derivative with the highest retention time coeluted with a [14C]ACh standard in cation exchange and reverse phase HPLC; 2) cholinesterase treatment converted this derivative to a substance with the retention time of [3H]Chol; 3) two blockers of ACh production, hemicholinium and 4-[(1-naphthylvinyl)pyridinium], eliminated 3H-labeled material in the HPLC fractions with ACh retention time. Spontaneous [3H]ACh release was increased by depolarizing potassium concentrations, and both synthesis and release of ACh were increased by the glucocorticoid hormone dexamethasone. Double immunostaining of choline acetyltransferase (CAT) and, respectively, of ACTH, GH, PRL, TSH, S100, LH, and FSH in rat pituitary cells revealed that most of the CAT-immunoreactive cells were also ACTH immunoreactive. A small proportion (less than 10%) of the PRL-immunoreactive cells also showed CAT immunoreactivity, but all other cell types were negative. The immunocytochemical evidence for colocalization of CAT within the ACTH cell was strengthened by the finding of a significantly higher rate of [3H]ACh synthesis in a corticotroph-enriched cell population obtained by separating pituitary cells on a velocity sedimentation gradient. In addition, the mouse pituitary corticotropic cell line AtT20 contained CAT immunoreactivity, converted [3H]Chol to [3H]ACh, and released bioactive ACh-like material. In conclusion, the present data provide strong evidence that pituitary corticotrophs synthesize and release ACh, and that the activity of this intrapituitary cholinergic transmission system is under regulatory control.

The activities of a number of peptide-degrading enzymes were compared in homogenates of GH3 cells and rat anterior pituitaries. The enzymes studied were prolyl endopeptidase (EC 3.4.21.26), a soluble metalloendopeptidase, pyroglutamyl peptide hydrolase (EC 3.4.11.8), a multicatalytic protease complex, cathepsin B (EC 3.4.22.1), cathepsin D (EC 3.4.23.5), aminopeptidase (EC 3.4.11.2), and a membrane-bound neutral metalloendopeptidase (EC 3.4.24.11). Specific substrates were used to measure the activities, and active-site-directed inhibitors were used to verify the identities of the enzymes studied. Of the two lysosomal enzymes studied, cathepsin B, the enzyme with the highest activity in both preparations, had 5 times the activity in GH3 cell homogenates as in anterior pituitary homogenates. Cathespin D had a somewhat higher activity in the anterior pituitary homogenates than in the GH3 cell homogenates. Soluble metalloendopeptidase and prolyl endopeptidase, both cytoplasmic enzymes, had about twice the activity in GH3 cell homogenates as in anterior pituitary homogenates. Membrane-bound neutral metalloendopeptidase in the GH3 cell homogenates had 25% of the activity of the anterior pituitary homogenates. Of the two TRH-degrading enzymes, the activity of prolyl endopeptidase in GH3 cell homogenates was about 25 times higher than that of pyroglutamyl peptide hydrolase. Since the secretory function of the pituitary is in part controlled by neuropeptides, the knowledge of the enzyme profiles of the GH3 cells and the anterior pituitary should be of value in studying the metabolism of neuropeptides and peptide hormones in these systems.

5-Monodeiodination of T4 and T3 and 5'-monodeiodination of T4 and rT3 were studied in brain homogenates of male Sprague-Dawley rats, aged 1-60 days. Portions of the homogenates were incubated with the substrates at 37 C for 30 min. The reaction products were estimated by specific RIAs. All of the four reactions were dependent upon time, temperature, pH, and upon the concentrations of substrate, thiol, and tissue protein. Maximal reactions were obtained between 40 and 160 mM dithioerythritol. T4 5'-deiodination proceeded optimally at pH 7.4 and 0.4 microM substrate, the other reactions at pH 8.5 and 10 microM substrate. The four reactions were inactivated by heat (56 C, 30 min) and inhibited by 10(-5) M iopanoic acid. Only rT3 5'-deiodination was inhibited by 3 X 10(-4) M propylthiouracil (greater than 95%). In cerebellum, basal ganglia, brainstem, and hypothalamus both T4 and T3 5-deiodinase activity were very high in perinatal rats [up to 5.56 pmol/(min X mg protein) in hypothalamus], and decreased rapidly with age. In cortex and olfactory bulb these enzyme activities were low after birth, followed by an increase during the growth spurt [up to 632 fmol/(min X mg protein) in olfactory bulb]. T4 and rT3 5'-deiodinase activity in all brain regions studied were at their lowest in perinatal rats. During and after the growth spurt an increase was observed [up to 457 fmol/(min X mg protein) in cerebellum]. The reciprocal course of 5- and 5'-deiodination between birth and growth spurt in most of the brain regions studied might lead to a reduced intracellular thyromimetic activity during the perinatal period.

The degree of endocrine activity by rat pituitary lactotrophs was manipulated and the lysosomal involvement in the release and intracellular degradation of PRL was monitored by concomitant assays of acid phosphatase, beta-glucuronidase, dipeptidyl peptidases I and II, and nonspecific esterases in the anterior lobe. The in vitro release of PRL by cell cultures was inhibited by 0.5 or 1.5 microM bromocriptine during 24 or 72 h of incubation and by 0.75 microM for 4 h. Long term treatment caused a 40% reduction in the total PRL content of cells and media; however, after 4 h of bromocriptine treatment, no reduction in PRL content was found. TRH (10 ng/ml) in medium for 4 h increased PRL release, whereas it produced intracellular hormone depletion. In vitro treatment of anterior lobes of diestrous rats with 1 microM dopamine decreased PRL release by 50%. No changes in lysosomal enzyme activities were observed after the inhibition of release or stimulation of the in vitro secretion of PRL. Haloperidol (2.5 mg/kg BW) caused a 90% increase in serum PRL concentrations in diestrous rats 1 h after sc injection. alpha-Methyl-p-tyrosine (200 mg/kg BW, ip) stimulated in vivo PRL secretion, monitored 1, 4, and 24 h after drug administration. The administration of 100 micrograms/rat bromocriptine for 3 consecutive days, a single dose of 100 micrograms/rat polyestradiol phosphate, and their combination resulted in the expected changes in PRL production in female rats. These in vivo treatments failed to alter lysosomal enzyme activities in the anterior pituitary. These findings suggest that the release, intracellular accumulation, or depletion of PRL occurred without concomitant changes in lysosomal enzyme activity in the anterior pituitary. We suggest that the system of lysosome-dependent hormone degradation may involve more specific enzymes than those monitored to date.

Aging is characterized by a decreased secretion of thyroid hormones in rats associated with unchanged plasma TSH suggestive of impairments in the hypothalamo-pituitary-thyroid axis. Since it is known that pituitary T3 is more determinant on TSH secretion than plasma T3, we measured in young (4 months) and old (26 months) male rats the concentration of T3 in the anterior pituitary gland and found that it was similar in young and old animals despite the low circulating levels of thyroid hormones. This was suggestive of age-related differences in the intrapituitary T4 to T3 conversion. We therefore determined the activity of 5'-deiodinase (5'-D, type I and type II) in the adenohypophysis and investigated possible age-related changes in this enzyme activity in peripheral tissues by its determination in the thyroid gland and liver (type I) of young and old rats. Intrapituitary 5'-D activity was increased in old compared to young rats (type I 5'-D: 4.59 +/- 0.13 vs. 2.92 +/- 0.33 pmol rT3/h x mg protein; type II: 0.54 +/- 0.5 vs. 0.21 +/- 0.03 pmol rT3/h x mg protein; P less than 0.001). In contrast, in the thyroid gland and in the liver, type I 5'-D was reduced with age (4.7 +/- 0.6 vs. 7.4 +/- 0.8 and 3.1 +/- 0.4 vs. 5.6 +/- 0.5 nmol rT3/h x mg protein, respectively; P less than 0.01). These data are illustrative of age-related changes in the activity of 5'-D, different according to the tissues in agreement with the known tissue-specific regulation of the 5'-Ds. The reduced activity of 5'-D in the thyroid and liver of old rats is indicative of an impaired thyroid hormones disposal in peripheral tissues with age. In contrast, in the adenohypophysis of old rats, the increase in the activity of 5'-D is similar to that reported in hypothyroid animals and suggests the development with age of an adaptative mechanism in the presence of low circulating thyroid hormones; this mechanism leads to unchanged intrapituitary concentration of T3 and consequently to unaltered plasma levels of TSH in old rats.

Type II T4 5'-deiodinase (5'-D) activity was studied in pineal gland, frontal cortex, and anterior pituitary of male rats. Enzymatic activity was determined by the release of 125I using [3',5'-125I]T4 as substrate. Daytime levels of 5'-D activity were maximal for anterior pituitary (346.2 +/- 142.5 fmol 125I released/mg protein.h), followed by pineal gland (61.5 +/- 8.7 fmol 125I released/mg protein.h) and frontal cortex (3.1 +/- 0.5 fmol 125I released/mg protein.h). Twenty-four-hour fluctuations of 5'-D activity were apparent in the pineal gland and cortex. For pineal, peak 5'-D activity (43.4 +/- 11.4 fmol 125I released/gland.h) occurred at 0100 h, with values at this time being 8-fold greater than basal daytime levels. Superior cervical ganglionectomy (SCGX) or exposure to continuous light (LL) completely abolished the nocturnal rise of 5'-D activity. Treatment with iopanoic acid also inhibited 5'-D activity in pineal (32.1 +/- 3.5 vs. 18.8 +/- 1.7 fmol 125I released/gland.h) without influencing either the N-acetyltransferase activity or the melatonin content. In cortex, peak 5'-D activity occurred at 0500 h (5.3 +/- 0.4 fmol 125I released/mg protein.h), with values at this time being 1.5-fold greater than basal daytime levels. SCGX moderately depressed enzymatic activity, while LL exposure strikingly enhanced the nocturnal increase (11.7 +/- 1.1 vs. 19.3 +/- 2.4 fmol 125I released/mg protein.h at 0300 h). 5'-D activity exhibited no 24-h fluctuation in the anterior pituitary gland, and neither SCGX nor LL exposure affected enzyme levels in this tissue. Our data demonstrate the existence of a 24-h rhythm of type II T4 5'-D activity in pineal gland and cortex, with peak levels occurring at night; in pineal, the sympathetic neural input is indispensable for the rhythm, since SCGX prevented it. In cortex, the apparent paradoxical effect of LL and SCGX on 5'-D activity indicates that other mechanisms are involved in its regulation.

To study the effect of rT3 on the 5'-deiodinase type II (5'D-II) of rat brain cortex and anterior pituitary, daily infusions of rT3 at rates of 0.5, 1.5, 4.5, and 13.5 nmol/day X 100 g body weight were given to a total of 87 hypothyroid rats. rT3 inhibited the 5'D-II activity in the total homogenate and in the microsomal fraction in the brain cortex, inhibition becoming significant (28-33%) with the infusion of 4.5 nmol/day X 100 g body weight, at serum levels of 2.4 pmol rT3/ml. Infusion of 13.5 nmol/day X 100 g body weight produced 71-73% inhibition. In homogenates of the anterior pituitary, inhibition was significant with infusions of 13.5 nmol/day X 100 g body weight (30%). To examine how rT3 might inhibit 5'D-II activity, labeled rT3 (137 muCi/day) was infused into 4 hypothyroid rats, and the rT3 content in the homogenate and in the cell subfractions was measured by HPLC. No rT3 was detectable in the nuclei, and less than 1% was found in the microsomal fraction. The infused rT3 was thus no longer present in the microsome preparations and homogenates. The inhibitory effect of rT3 was also compared with that of T4. Serum T4 levels of 45 pmol/ml were required in order to inhibit 5'D-II to the same extent as with 7 pmol rT3/ml. The requirement of this high serum level of T4 excludes the potential role of T4 impurities in the infused rT3 preparation. We therefore conclude that rT3 inhibits 5'D-II in brain cortex per se independently of T4.

To assess the role in hormone release of the recently characterized anion-sensitive Mg+2-ATPase of pituitary secretory granules, three types of evidence were accumulated. First, granule suspensions were incubated with varying quantities of MgCl2 and ATP, and the effects on ATPase activity and release were measured. The main stimulatory influence on ATPase activity was the concentration of the complex between Mg+2 and ATP (MgATP), although very high concentrations of complex inhibited. In contrast, release of hormone was nearly totally independent of MgATP. Rather, release was primarily controlled by free Mg+2, which inhibited release at concentrations as low as 0.1 mM and reduced basal release by approximately 65-75% at concentrations approximating 2.0 mM or higher. Free ATP had small consistent inhibitory effects on ATPase activity, but stimulated protein release. Second, granules were incubated with other nucleotides and related compounds. Incubation with GTP, ITP, CTP, TTP, and UTP resulted in augmentation of hormone release duplicating that seen with ATP. Some increase was also seen with the nonhydrolyzable ATP derivative 5'-adenylylimido-diphosphate, whereas adenosine was inhibitory. Since the catalytic activity of the granule ATPase demonstrates purine nucleotide substrate specificity, these results provide additional evidence for the dissociation of ATPase activity from hormone release. Third, granules were incubated with several ATPase inhibitors. Though all inhibited ATPase activity to a comparable extent, only tri-n-butyltin inhibited hormone release; oligomycin, efrapeptin, and other tin compounds were inactive. Taken together, these data indicate that the conditions that influence ATPase differ strikingly from those affecting hormone release. Whatever the role of the anion-sensitive granule membrane ATPase, we suggest that it is not involved in the process of hormone release from isolated granules.

Pituitary GH secretion is regulated by Ca+2 and cAMP. We show that human pancreatic tumor GRF (hpGRF) stimulates anterior pituitary adenylate cyclase activity, cAMP accumulation, and GH release. The relationship between Ca+2 and the stimulating effects of the Ca+2 ionophore A23187 on cAMP accumulation and GH release in vitro was studied. To evaluate the role of the Ca+2-binding protein calmodulin in this system, we used the calmodulin antagonist W7, a naphthalene-sulfonamide derivative, and its less active analog W5. W7 inhibited hpGRF-stimulated adenylate cyclase activity, cAMP accumulation, and GH release, whereas W5 was either poorly effective or ineffective. Somatostatin (SRIF) also attenuated hpGRF stimulation of adenylate cyclase. These results suggest that the actions of Ca+2-calmodulin and cAMP are interrelated in modulating GH release. Calmodulin participates in hpGRF stimulation of adenylate cyclase, cAMP formation, and GH release. The attenuation of hpGRF-stimulated adenylate cyclase activity by SRIF may be one of the mechanisms for its GH inhibitory action.

An adenylate cyclase present in a PRL-producing tumor cell line, GH1, which is selectively stimulated by chlorpromazine, has been characterized with respect to several biochemical properties. The parameters studied include divalent metal ion specificity, guanyl nucleotide interaction, and sensitivity to sodium fluoride. The effects of calcium and the calcium chelator, EGTA, were also tested on the chlorpromazine response. The data reported herein establish the optimal conditions for the activation of adenylate cyclase by chlorpromazine in homogenates of GH1 cells. In addition, the results from this study identify a heat-stable protein in these cells which regulates cyclase activity. This protein, which can be released into the supernatant by the pretreatment of GH1 cells with EGTA, is an absolute requirement for chlorpromazine stimulation of adenylate cyclase activity in these cells. The activation of adenylate cyclase by chlorpromazine in homogenates of normal rat pituitaries was demonstrated as well as the presence of a protein factor which regulates this activity.

We have examined the influences of dopamine and the D2 receptor agonist bromocriptine on phosphoinositide metabolism in primary cultures of rat anterior pituitary cells, monitoring changes in the levels of phosphatidylinositol (PtdIns), phosphatidylinositol-4-phosphate [PtdIns(4)P], and phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2]. Basal incorporation of [3H]inositol ([3H]Ins) into phosphoinositides was progressive, and radioisotopic equilibrium was attained in all three species within 48 h. The inclusion of dopamine or bromocriptine in the incubation medium promoted concentration-dependent reductions in the rate, but not the magnitude, of phosphoinositide radiolabeling. The onset of this effect was rapid; inhibition of [3H]Ins incorporation by dopamine (500 nM) and bromocriptine (100 nM) could be detected within 2 h. This treatment also produced a comparable reduction in the incorporation of [32P]orthophosphate into PtdIns(4,5)P2. In extended time-course studies, bromocriptine dramatically retarded the radiolabeling of PtdIns(4)P and PtdIns(4,5)P2, and apparent equilibria in these species were attained only after 96 h. We also assessed the ability of dopamine to modify the concentration-response characteristics of [3H]Ins-labeled inositol phosphate ([3H]InsPx) production by TRH, angiotensin II (AII), neurotensin (NTS), bombesin (BBS), and vasoactive intestinal polypeptide (VIP). Neither dopamine nor bromocriptine altered the rate or magnitude of TRH-, AII-, NTS-, or BBS-related InsPx generation. VIP was completely ineffective in stimulating InsPx generation. PRL release was significantly reduced in all dopamine-treated groups. That the InsPx concentration-response relationships for each of these peptides remained unimpaired by exposure to dopamine or bromocriptine extends our previous observation that the phosphoinositide-specific phospholipase-C is insensitive to dopaminergic tone. Consistent with our earlier findings, these data indicate that activation of the D2 dopamine receptor attenuates the activity of mechanisms associated with the serial phosphorylations of PtdIns and PtdIns(4)P, reactions that give rise to PtdIns(4)P and PtdIns(4,5)P2, respectively. It is our conclusion that dopamine, in addition to its other actions, attenuates the phosphorylation, rather than the hydrolysis, of anterior pituitary phosphoinositide. This attenuation appears to be mediated by an inhibitory coupling of the D2 receptor with the phosphoryltransferase activities that catalyze PtdIns(4)P and PtdIns(4,5)P2 formation.(ABSTRACT TRUNCATED AT 400 WORDS)

The content of dopamine (DA) and the activity of beta-glucuronidase (a marker for lysosomal enzymes) in the anterior pituitary and the concentration of PRL in serum were determined in male rats that were treated with a variety of drugs that influence the release, storage, or actions of DA. Drugs that increase DA in hypophysial portal blood (amphetamine, methylphenidate and L-dopa) increased DA content, decreased PRL secretion, and had no effect on lysosomal enzyme activity. Drugs that activate DA receptors in the anterior pituitary (apomorphine and bromocriptine) decreased serum PRL but had no effect on DA content or lysosomal enzyme activity. Drugs that decrease DA in the hypophysial portal blood (alpha-methyltyrosine, gamma-butyrolactone, and reserpine) or block DA receptors (haloperidol) increased PRL secretion and decreased DA content and lysosomal enzyme activity. These results suggest that there is no obvious relationship between anterior pituitary DA content and PRL secretion. In addition, lysosomal enzyme activity is not stimulated by increasing the concentration of DA or activating DA receptors in the anterior pituitary, but lysosomal enzyme activity does appear to be tonically stimulated by DA in the control animals since decreasing DA concentrations or receptor activation in the anterior pituitary decreases beta-glucuronidase activity. This latter proposal was confirmed by the demonstration that apomorphine reduced the alpha-methyltyrosine-induced increase in serum PRL and prevented the decrease in anterior pituitary DA content and beta-glucuronidase activity. Furthermore, pretreatment with bromocriptine and L-dopa blocked both the increase in serum PRL concentration and the decrease in anterior pituitary DA content induced by alpha-methyltyrosine and gamma-butyrolactone. On the other hand, bromocriptine and L-dopa blocked the increase in serum PRL concentration but not the reduction in anterior pituitary DA content caused by reserpine, indicating that reserpine has a direct action on DA storage mechanisms in the anterior pituitary. These data suggest that the ability of DA to inhibit PRL secretion and the incorporation of DA into the anterior pituitary are not causally related.

We have detected tissue kallikrein and kallikrein mRNA in various brain regions with a kallikrein direct RIA and with nucleic acid hybridization using a kallikrein cDNA probe. In the direct RIA, rat urinary kallikrein-like activity was found in the pituitary and pineal glands, hypothalamus, cerebral cortex, cerebellum, and brain stem. Pituitary and pineal gland kallikrein concentrations were significantly higher than those in other regions. Only in pituitary was there a significant difference in tissue kallikrein concentration according to sex, with glands from female rats showing levels 4-fold higher than those from male rats. Kallikrein mRNAs were detected in all of the regions and were about 4-fold higher in female than in male pituitary gland. Northern blot analyses show sex dimorphism of pituitary kallikrein mRNA, similar in size to submandibular gland and kidney mRNA. In castrated male rats, whole pituitary kallikrein content was reduced to 50% of the control value and increased 1.7-fold with testosterone replacement and 18-fold with 17 beta-estradiol treatment. Neither T4 nor cortisol affected whole pituitary kallikrein levels in the castrated male rat, but testosterone decreased pituitary kallikrein in normal female rats by 35%. When anterior pituitary or neurointermediate lobe extracts were separately examined, immunoreactive kallikrein was 10.2- and 1.3-fold higher respectively, in female than in male rat lobes. Estradiol benzoate (30 micrograms/kg) administration increased kallikrein levels 90- and 22-fold, respectively, in the anterior pituitary of gonadectomized male and female rats, while it increased by only 40-50% kallikrein levels in the male and female neurointermediate lobe. In dot blot analysis, kallikrein mRNA levels were increased 5-fold by 17 beta-estradiol in the whole pituitary of castrated male rats. In the cytoplasmic dot hybridization analysis, estradiol benzoate treatment increased kallikrein mRNA levels 54-fold in the anterior pituitary of ovariectomized rats. The data show that a tissue kallikrein indistinguishable thus far from a urinary kallikrein is widely distributed in brain and pituitary and that levels of enzyme and mRNA are comparable in certain central sites. Kallikrein levels in the anterior and neurointermediate pituitaries are differentially regulated by estrogen.