GABA differentially regulates the gene expression of proopiomelanocortin in rat intermediate and anterior pituitary

The GABAergic regulation of proopiomelanocortin messenger RNA (POMC mRNA) levels in rat pituitary was investigated using molecular hybridization of DNA complementary to POMC mRNA. Endogenous GABA levels increased, in vivo, by inhibiting the GABA catabolic enzyme GABA-transaminase (GAT) with ethalonamine-O-sulfate (EOS) or with vinyl-GABA (VG). Rats were treated with VG (100 mg/kg or 800 mg/kg) or EOS (100 mg/kg), administered each second day. GABA levels in the neurointermediate lobe (NIL) and anterior lobe (AL) of the hypophysis and in the hypothalamus were significantly increased following 4 days of VG treatment (800 mg/kg). All treatments resulted in a 40-60% decrease in POMC mRNA levels after 4 days in the NIL but not in the AL. A similar decrease of about 60% in POMC mRNA levels in the NIL was seen when EOS was given in the drinking water (5 mg/ml). In this set of experiments the time course of alteration of POMC mRNA in the NIL and the concentration of alpha-MSH, a POMC-derived peptide, were analysed. After one day of EOS treatment, when POMC levels had already decreased by 40%, alpha-MSH levels were significantly elevated (34% above controls), possibly reflecting an inhibition of alpha-MSH secretion. However, after 4 and 8 days, POMC mRNA levels and tissue alpha-MSH levels had significantly decreased. When tested in vitro, on primary cultures of IL cells, GABA (10 microM) reduced POMC mRNA levels by 40% after 3 days of treatment. These results show that GABA exerts a direct inhibitory effect on POMC gene expression in the intermediate lobe.     

Binding site of salsolinol: its properties in different regions of the brain and the pituitary gland of the rat

It has been recently shown that salsolinol (SAL) is present in the hypothalamic neuroendocrine dopaminergic (NEDA) system and appears to be a selective and potent stimulator of prolactin (PRL) secretion in the rat. Furthermore, the lack of interference of SAL with 3H-spiperone binding in the striatum and the anterior lobe (AL) of the pituitary gland has been also demonstrated. These data clearly indicate that SAL does not act at the dopamine (DA) D(2) receptors, and suggest that SAL supposedly has a binding site through which the secretion of PRL may be affected. Therefore, binding of 3H-SAL to different regions of the central nervous system (CNS) has been investigated. Specific and saturable binding has been detected in the striatum, cortex, median eminence and in the hypothalamus as well as in the AL and the neuro-intermediate lobe (NIL) of the pituitary gland. K(D) values of the bindings were in the nanomolar range in all tissue tested. 3H-SAL displacing activity of several agonists and antagonists of known DA receptors have also been tested. It has been found that DA and in a lesser extent, apomorphine could displace 3H-SAL, but other DA receptor specific ligands have not been able to affect it. Furthermore, several pharmacologically active compounds, selected on the basis of their influence on DA synthesis, transport mechanisms and signal transduction, have also been tested. Neither mazindol (a selective DA transporter inhibitor) nor clonidine (an alpha(2)-adrenoreceptor agonist) could alter SAL binding. At the same time, L-dopa, carbidopa, benserazide and alpha-methyldopa were able to displace 3H-SAL. The possible changes in SAL binding due to physiological and pharmacological stimuli, like suckling stimulus and reserpine pretreatment (that blocks vesicular monoamine transport in DA terminals), respectively, have also been investigated. In the NIL of the pituitary gland and in the median eminence of the hypothalamus the binding decreased following 10 min of suckling stimulus compared to the binding detected in the same tissues obtained from mothers separated from their pups for 4h and not allowed to be suckled. At the same time, there were no changes in the binding at the AL and striatum. Following reserpine pretreatment that has completely prevented PRL releasing effect of SAL, the binding was significantly augmented. These results support our assumption that SAL should have specific binding sites through which it can affect PRL secretion. Furthermore, it clearly suggests that it may regulate DAergic neurotransmission of NEDA neurons by an altered intracellular or intraterminal synthesis and/or distribution of hypophysiotropic DA.     

Endogenous opioids inhibit the in vitro release of endogenous dopamine preferentially in the neural lobe of the rat neurointermediate lobe

The release of endogenous dopamine (DA) from the in vitro incubated combined neurointermediate lobe (NIL) or isolated neural lobe (NL) was studied. In the presence of the DA uptake inhibitor GBR 12921 (200 nM), electrical stimulation of the pituitary stalk caused an increase of the outflow of DA from the NIL in a frequency-dependent manner. Naloxone (1 microM) enhanced the DA release from the NIL evoked by electrical stimulation at 7 or 15 Hz by about 40%, but had no effect on DA release evoked by stimulation at 3 Hz. When the electrical stimulation was carried out at 15 Hz, the evoked DA release (expressed as fraction of the DA tissue content) from the NL amounted to only 15% of that from the combined NIL. Naloxone (1 microM) increased the evoked DA release from the isolated NL by 242%. Thus, the effect of naloxone on DA release from the combined NIL may be confined mainly to the NL. In conclusion, DA release from the NL is under inhibitory control of endogenous opioids released from the NL during stimulation at 7 or 15 Hz. Beta-Endorphin, known to be released spontaneously at a high rate from in vitro incubated NILs, appears to lack inhibitory effects on DA release from the NIL.     

Changes in the spontaneous action potentials from the whole, isolated, intact bovine pituitary gland following an injection of various bovine pituitary intraglandular colloid fractions

Bovine pituitary intraglandular colloid (IGC) of intermediate lobe (IL) origin with accessions from the anterior lobe (AL), can modify the spontaneous action potentials (SAP) from AL, IL and posterior lobe (PL) cells. It was discovered that intraglandular colloid contains peptides ACTH, alpha-MSH, and beta-LPH when subjected to a series of radioimmunoassays. These peptides are thought, in part, to be responsible for altering the SAP.     

An investigation of N-terminal pro-opiocortin peptides in the rat pituitary

Extracts of neurointermediate lobe (NIL) and anterior lobe (AL) of the rat pituitary, and material released from perfused rat pars distalis (PD) and pars intermedia (PI) cells were gel chromatographed and monitored using three antisera, each recognizing different regions of the non-corticotropin (ACTH)-lipotropin (LPH) portion of pro-opiocortin (POC). Two peaks (termed N-POC I) which emerged close to the elution position of rat beta-LPH were detected. The first peak was reduced significantly in the PI. Two smaller N-POC fragments which eluted near beta-endorphin were detected only in extracts and secretions of intermediate lobe tissue. One peak cross-reacted in the gamma 3-melanotropin (MSH) assay (N-POC III) whereas the other peak possessed amino (N)-terminal N-POC immunoreactivity (N-POC II). The results demonstrated differences in the distribution and nature of N-POC peptides released and extracted from the PD and PI of the rat pituitary, and suggest that the enzymic processing of N-POC is different in the two pituitary lobes.     

Subcellular localization of immunoreactive dynorphin and vasopressin in rat pituitary and hypothalamus

Dynorphin is found mainly in the particulate fraction of rat pituitary gland and hypothalamus homogenates. Dynorphin-like immunoreactivity (DYN-LI) from neurointermediate lobe (NIL) homogenates migrates at the same rate as vasopressin-like immunoreactivity (AVP-LI), in sucrose density gradients, whereas DYN-LI from the hypothalamus appears to migrate principally in a less dense region of the gradient. This suggests that dynorphin and vasopressin from pituitary are present in organelles of similar size and density, while the bulk of the dynorphin in the hypothalamus appears to be stored in a different subcellular organelle. Anterior lobe (AL) dynorphin appears to migrate in two separate bands on density gradients: the less dense band (slower) migrates at a similar rate to that of dynorphin and vasopressin from NIL. When alpha-neo-endorphin was measured in sucrose gradients of NIL and hypothalamus, it was found to co-migrate with DYN-LI.     

Release of beta-endorphin-immunoreactivity from rat pituitary and hypothalamus in vitro: effects of isoproterenol, dopamine, corticotropin-releasing factor and arginine8-vasopressin

The release of beta-endorphin-immunoreactivity (beta E-IR) from rat pituitary anterior lobe (AL) quarters, neurointermediate lobes (NILs), and hypothalamic fragments was investigated in vitro. The beta-adrenoceptor agonist isoproterenol (ISO) and the hypothalamic neurohormone corticotropin-releasing factor (CRF) concentration-dependently stimulated the release of beta E-IR from superfused AL quarters and NILs, but not from incubated hypothalamic fragments. Dopamine (DA) inhibited the release of beta E-IR from NILs and hypothalamic tissue in a concentration-dependent manner, whereas it did not affect the release from AL quarters. Arginine8-vasopressin (AVP) stimulated the release of beta E-IR from AL quarters and hypothalamic fragments, but did not affect the release from NILs. The data indicate that the release of beta E-IR from cells in the pituitary lobes and in the hypothalamus is differentially regulated, but that common principles are involved. In particular, the results provide first direct evidence for an action of vasopressin as a stimulator of the release of POMC-derived peptides in the hypothalamus.     

Post-translational processing of pro-opiomelanocortin (POMC)-derived peptides during fetal monkey pituitary development. I. Adrenocorticotropin (ACTH) and alpha-melanotropins (alpha-MSHs)

We have studied the post-translational processing of POMC-derived peptides during fetal monkey development using immunoassay and reverse-phase high-performance liquid chromatography (RP HPLC). Pituitary tissues obtained from fetal monkeys ranging from Gestational Day 50 to 155 were fractionated and analyzed for ACTH- and alpha-MSH-related peptides and compared to adult forms. Extracts of whole pituitary from Fetal Days 50 and 55 contained ACTH(1-39) and very small amounts of CLIP (corticotropin-like intermediate-lobe peptide; ACTH(18-39))-like immunoactivity. Acetylated alpha-MSHs were not detectable at Day 50. alpha-MSHs were barely detectable at Day 55. By Day 65, when pituitary lobes were separable, small amounts of des-, mono-, and diacetyl alpha-MSH were detectable in NIL extracts, but not in anterior lobe extracts. ACTH(1-39) levels were negligible when compared to increasing alpha-MSHs through Fetal Day 80 to 155 in the intermediate lobe. The CLIP immunoactivity was negligible in Day 80 and adult anterior lobe extracts. Thus, lobe-specific proteolytic processing of ACTH-related peptides was well established by midterm gestation. Marked increases of alpha-N- and alpha-N,O-acetylated forms of alpha-MSHs were detected during middle and late stage fetal development. Diacetyl alpha-MSH was the predominant form of alpha-MSH in adult NIL extracts. No acetylated alpha-MSHs were found in anterior lobe tissues, thus adult anterior lobe extracts contained almost exclusively ACTH(1-39). However adult NIL extracts contained two distinct forms of CLIP-related immunoactivity. Therefore changes in post-translational processing patterns of ACTH-related and alpha-MSH-related peptides continued to some extent, postnatally. These data indicate that marked changes in post-translational processing of POMC-derived ACTH-related products occur during the first half of monkey gestation.     

Glucocorticoid inhibition of immunoreactive beta-endorphin release from the anterior lobe of the rat pituitary: in vitro and in vivo studies

Glucocorticoid control of pituitary beta-endorphin (beta-END) release was investigated in vitro and in vivo. Cultured cells of both rat anterior (AL) and neurointermediate (NIL) lobe released beta-END-like immunoreactivity (beta-END-LI) in response to epinephrine (10(-7) M); however, only the response of AL cells was prevented by corticosterone (10(-8)-10(-6) M) or dexamethasone (10(-9)-10(-7) M). Gel chromatographic analysis (Sephadex G-50) revealed that the major forms of beta-END-LI released by AL cells corresponded to beta-END and beta-lipotropin (beta-LPH) in molecular size, whereas virtually all of the immunoreactivity released by NIL cells resembled beta-END. In vivo administration of dexamethasone attenuated the stress-induced release of beta-END-LI in a dose- and time-related fashion, having a more pronounced effect on plasma levels of beta-END-LI corresponding to beta-LPH in molecular size. Metyrapone (100 mg/kg), an inhibitor of glucocorticoid synthesis, evoked a rapid (20-40 min) four- to sixfold increase in total plasma beta-END-LI and 75% of this rise was due to immunoreactivity resembling beta-LPH in size. This response was diminished by coadministration of either dexamethasone (0.05-1.25 mg/kg) or corticosterone (0.05-1.25 mg/kg) and completely prevented by 4-hr pretreatment with dexamethasone (50 micrograms/kg). The briskness of the plasma beta-END-LI response to acute changes in glucocorticoid status suggests that a "rapid" feedback mechanism operates in the physiologic control of pituitary beta-END-LI secretion. Moreover, the ability of glucocorticoids to selectively inhibit AL release of beta-END-LI in vitro and their pronounced effect on plasma levels of beta-END-LI resembling beta-LPH, a marker of AL secretion, together indicate that glucocorticoids exert a selective influence over the secretion of AL corticotrophs in vivo. This demonstration of differential regulation of the AL versus IL secretion of beta-END-LI in vivo most likely reflects a phenomena having biologic importance related to the different physiologic actions of the several molecular forms of beta-END-LI secreted by the two tissues.     

Beta-endorphin concentrations in the hypothalamus, pituitary and plasma of streptozotocin-diabetic rats with and without insulin substitution therapy

The concentrations of beta-endorphin like immunoreactivity (beta-END) in the hypothalamus, pituitary and plasma were studied in rats of either sex, one month after induction of diabetes by single iv injection of streptozotocin. As controls, both normal and undernourished rats, weight-matched with diabetic rats, were used. Diabetic male and female rats had a marked depletion of beta-END stores in the hypothalamus and neurointermediate lobe (NIL) but not in the anterior pituitary. Depletion of beta-END was reversed to normal by insulin replacement therapy. Severe undernourishment was not as effective as diabetes to reduce beta-END stores in the hypothalamus and NIL. A significant reduction of beta-END was observed only in the NIL of undernourished female rats. Plasma beta-END and beta-lipotropin (beta-LPH) concentrations were not significantly altered in diabetic rats. These results indicate that the lack of insulin may affect beta-END synthesis in the hypothalamus and NIL.     

Post-translational processing of pro-opiomelanocortin (POMC)-derived peptides during fetal monkey pituitary development. II. beta-Lipotropin (beta-LPH)-related peptides

We have studied the post-translational processing of POMC-derived peptides during fetal monkey pituitary development using immunoassay and reverse-phase high-performance liquid chromatography (RP HPLC). Whole pituitary glands obtained from Day 50 and 55 fetal monkeys and separated lobes From Day 65 to 155 were extracted, fractionated, and analyzed for beta-melanotropin (beta-MSH), midportion beta-endorphin (beta-EP), and acetylated beta-EP immunoactivity. Separated adult pituitary lobes were analyzed for comparison. At Day 50, POMC-containing cells were located in both the anterior and intermediate pituitary lobes by immunofluorescence staining, the majority of these cells were localized in the anterior lobe. The Day 50 and 55 whole pituitaries contained predominantly beta-lipotropin (beta-LPH), gamma-lipotropin (gamma-LPH), beta-EP(1-31), and 2.2-kda beta-MSH. No acetylated products were found in Day 50 whole pituitary extracts. By Day 55, carboxy-shortened and acetylated beta-EPs were barely detectable in whole pituitary extracts. These forms were more apparent in the Day 65 separated neurointermediate lobe (NIL) extracts, and were similar to adult proportions by Day 80. The adult anterior lobe contained predominantly beta-LPH, beta-EP, and gamma-LPH. Adult NILs contained almost exclusively 2.2-kda beta-MSH, alpha-N-acetyl beta-EP(1-31) and alpha-N-acetyl beta-EP(1-27). The production of 2.2-kda beta-LPH in the monkey NIL indicates that monkey beta-LPH is different from rat beta-LPH in that it must contain the paired-basic cleavage site required for the formation of 2.2-kda beta-MSH that is known to be lacking in rat beta-LPH. Another finding was that monkey beta-EP contains a Tyr residue at position 27 as found in human beta-EP but appears to have the rat Gln substitution at position 31. The post-translational processing patterns characteristic of each lobe were well established by midterm fetal development (Day 80).     

Effects of adrenalectomy and hypercorticism on the ACTH content of the anterior and posterior pituitary in rats with inherited diabetes insipidus (Brattleboro strain).

The effects of adrenalectomy (Adx) and hypercorticism on the ACTH content in the anterior (AH) and the neurointermediate lobe (NIL) of the pituitary in Long Evans (+/+), heterozygous (+/DI) and homozygous (DI/DI) Brattleboro rats were determined using dispersed adrenal cells bioassay. Adx decreased the NIL-ACTH content in +/DI and DI/DI rats and left it unchanged in the +/+ rats. Adx increased the AH-ACTH content in the three groups. Hypercorticism had a delayed decreasing effect both in the AH and in the NIL in all rats, with one exception for the NIL in DI/DI rats. Conversely to what appeared in Wistar rats, in Long Evans and Brattleboro rats the corticosterone administered in drinking water was unable to reduce the increase in AH-ACTH activity. These data suggest that Brattleboro, and, to a lesser extent, Long Evans rats from which the former are derived present some particularities in the regulation of their corticotropic function at the AH and the NIL level. We also observed that NaCl (0.9%) added to drinking water and hypercorticism are two factors able to increase diabetes insipidus in homozygous rats without modifying the water intake in Long Evans and heterozygous rats.     

Independent differentiation of mammotropes and somatotropes in the chicken embryonic pituitary gland

It has been reported that mammotropes in a rodent pituitary gland are derived from somatotropes via somatomammotropes (SMTs), cells that produce both growth hormone (GH) and prolactin (Prl). However, no studies have been done on the transdifferentiation of somatotropes in the chicken pituitary gland. In this study, in order to determine the origin of mammotropes, we studied detail property of appearance of chicken somatotropes, mammotropes and pit-1 cells and then evaluated the existence of SMTs in the chicken embryonic pituitary gland. Immunohistochemical analysis revealed that GH-immunopositive (GH-ip) cells appeared on embryonic day (E) 14 and were mainly distributed in the caudal lobe, while Prl-immunopositive (Prl-ip) cells appeared in the cephalic lobe of the pituitary gland on E16. In situ hybridization (ISH) and RT-PCR analysis showed that expression of GH and Prl mRNA starts at E12 in the caudal lobe and at E14 in the cephalic lobe respectively. Pit-1 mRNA was first detected on E5 by RT-PCR, and pit-1 mRNA-expressing cells were found in the cephalic lobe on E8. Then with the ontogeny of the chicken, these cells spread into both lobes. Using a double staining method with ISH and immunohistochemistry, we could not detect the existence of SMTs in the chicken embryonic pituitary gland even in the marginal region of either lobe. These results suggest that chicken somatotropes and mammotropes independently appear in different lobes of pituitary gland and that transdifferentiation from somatotropes to mammotropes is not the central route for differentiation of mammotropes in the embryonic chicken pituitary gland.     

Cocaine influences beta-endorphin levels and release

Immunoreactive beta-endorphin (IR-BE) was measured in the plasma, anterior pituitary (AP), neurointermediate lobe of the pituitary (NIL) and hypothalamus of male rats treated chronically (once daily for ten days) with cocaine. Cocaine produced a consistent elevation in the concentration of IR-BE in the plasma, the AP and the NIL at doses of 2.5 - 20 mg/kg/ip. The release of IR-BE from the AP and the NIL was determined in vitro and was found to be increased by treatment with cocaine. Chronic administration of cocaine did not affect the concentration of IR-BE in the hypothalamus. Chromatographic analysis revealed that cocaine produced a slight decrease in the amount of beta-endorphin relative to beta-lipotropin in the AP. Beta-endorphin was the major form of IR-BE released by the AP and the sole constituent and secretory product of the NIL. These data indicate that chronic administration of cocaine stimulates the endogenous opiate system, elevating the levels of IR-BE in the pituitary and promoting beta-endorphin release.     

The effect of streptozotocin-induced diabetes on pituitary and hypothalamic endorphin equivalents

This study examines the effect of experimentally induced diabetes mellitus in rats on tissue concentrations of opioid peptides in the neurointermediate lobe (NIL), anterior pituitary (AP) and hypothalamus. Diabetic animals were found to have a marked increase in endorphin equivalents, measured by opiate receptor binding assay, in the NIL whereas no change was observed in beta endorphin-like immunoreactivity (beta ELI) or ACTH measured by RIA. These results may indicate the presence of a feedback mechanism and suggest the possibility that opioid peptides may be physiologically important in the maintenance of glucose homeostasis.     

Glucocorticoid regulation of proopiomelanocortin messenger ribonucleic acid content of rat hypothalamus

We have verified the possibility that the POMC gene of the rat hypothalamus might be subject to regulation by glucocorticoids. Adrenalectomy increased the concentration of POMC mRNA in anterior pituitary and in hypothalamus, but not in the neurointermediate lobe of the pituitary gland. Dexamethasone and, to a slightly lesser extent, corticosterone treatments reversed the adrenalectomy-induced increase in POMC mRNA concentrations in both anterior pituitary and hypothalamus. Dexamethasone caused a slight decrease of POMC mRNA levels in the neurointermediate lobe of the pituitary gland, while corticosterone had no effect. These results indicate that the POMC gene of the rat brain hypothalamus is also under negative control by glucocorticoids.     

Effect of Aging on 24-Hour Changes in Serotonin and Dopamine Turnover, and Somatostatin and Amino Acid Content, of the Rat Hypothalamus and Pituitary Gland

The effect of aging on neurotransmitter and peptide content in the hypothalamichypophysial unit has commonly been analyzed at single time points in the 24-h cycle. Since significant changes in circadian rhythmicity occur during aging, this study aimed to examine 24-h rhythmicity in hypothalamic and pituitary serotonin (5HT) and dopamine (DA) turnover and content, and somatostatin and amino acid content in 2 months-old and 18-20 months-old rats, killed at 6 different time intervals throughout the light-dark cycle. Aged rats showed suppressed or disrupted 24-h rhythms of 5HT and DA turnover and of somatostatin, glutamate, aspartate and taurine content (anterior hypothalamus), of 5HT and DA turnover and of somatostatin, glutamate, taurine and glycine content (medial hypothalamus) and of DA turnover and amino acid content (posterior hypothalamus). Twenty-four h variations in DA, somatostatin, aspartate, GABA and glycine content of the anterior hypophysis and in all parameters tested in the neurointermediate lobe became suppressed or disrupted in aged rats. Mean values generally decreased with age, except for DA content in the anterior pituitary lobe and aspartate content in the neurointermediate lobe. Conclusions: Examination of neurotransmitter and neuropeptide content at different times of the day is needed to analyze the effects of aging in the hypothalamic-hypophysial unit.     

Effect of atrial natriuretic peptide on the release of beta-endorphin from rat hypothalamo-neurohypophysial complex

The aim of this study was to determine whether atrial natriuretic peptide (ANP) alters beta-endorphin (beta-END) secretion from rat intermediate pituitary and whether this effect is a direct action on the intermediate pituitary or an indirect one mediated by hypothalamic factor(s). We studied the release of beta-END from rat neuro-intermediate lobes of the pituitary (NIL) and from the hypothalamo-neurohypophysial complex (HNC), which consists of the hypothalamus, pituitary stalk, intermediate and posterior lobes of the pituitary, by means of an in vitro perifusion system. NIL and HNC were prepared from male Wistar rats and individually perifused for 30 min with perifusion medium followed by 20 min perifusion with medium containing alpha-rat ANP and/or dopamine (DA). Samples of perifusion medium were collected every 5 min and subjected to RIA for beta-END. The basal release of beta-END from NIL was 180% of that from HNC (p less than 0.01), which provides further support for the presence of hypothalamic factors that inhibit beta-END release from the intermediate pituitary. The perifusion of HNC with ANP at 10(-7) and 10(-6) M increased the beta-END concentration by 25 and 50%, respectively (p less than 0.01). In contrast, ANP (10(-8) to 10(-6) M) had no effect on beta-END release from NIL. The inhibitory effect of DA (10(6) M) on beta-END release from NIL and HNC (51% and 50% of the basal release, respectively, p less than 0.01) was confirmed. However, this inhibitory effect was not reversed by ANP.(ABSTRACT TRUNCATED AT 250 WORDS)