Effect of dopaminergic drugs on hypothalamic and pituitary immunoreactive beta-endorphin concentrations in the rat

Beta-endorphin concentrations have been evaluated in the hypothalamus, pituitary lobes and plasma after 1-and 3-week treatment with 2-Br-alpha-ergocriptine or lisuride, two potent dopaminergic drugs. Hypothalamic beta-endorphin concentrations were significantly decreased after the administration of the dopaminergic agents for 1 or 3 weeks. Similarly, beta-endorphin concentrations decreased in the neurointermediate lobe and plasma. After gel chromatography, it appeared that in the anterior pituitary, beta-lipotropin concentrations were unchanged or lightly increased concomitantly with a decrease of beta-endorphin. Our data indicate that, both in the hypothalamus and the neurointermediate pituitary lobe, beta-endorphin is under an inhibitory dopaminergic tone. The latter may also play a role in inhibiting beta-endorphin cleavage from beta-lipotropin in the anterior pituitary.     

Effect of acute ethanol in vivo and in vitro on the beta-endorphin system in the rat

Acute ethanol treatment in vivo (i.p. injection of 3.5 g ethanol/Kg B. wt.) stimulated the release of beta-endorphin like peptides by the pituitary gland as was indicated by the increased content of beta-endorphin like immunoreactivity (beta-EPLIR) in the plasma. Furthermore, a significant decrease in the anterior lobe content of beta-EPLIR was observed, while the decrease in the neurointermediate lobe beta-EPLIR content at 45 min after the i.p. ethanol injection was not statistically significant. In vitro incubation of neurointermediate lobes, from animals injected with either ethanol or saline, in the presence of 3H phenylalanine indicated that the content of beta-EPLIR in the incubation medium was increased, the content of the newly biosynthesized 3H-phenylalanine labelled proteins in the neurointermediate lobe extract was decreased, while the content of 3H-phenylalanine labelled pro-opiomelanocortin, beta-lipotropin and beta-endorphin in the neurointermediate lobes extract were not significantly changed by the ethanol treatment, though a small increase was observed. When neurointermediate lobes from untreated control animals were incubated for 3 hrs with 3H-phenylalanine in the presence or absence of 300 mg ethanol per 100 ml incubation medium, there was no significant difference in the beta-EPLIR content in the incubation medium, or in the content of 3H-phenylalanine labelled proteins, pro-opiomelanocortin, beta-lipotropin and beta-endorphin in the neurointermediate lobe extract. These results suggest that ethanol has little or no direct effect on the beta-endorphin peptides in the pars intermedia cells.     

Eight weeks of streptozotocin-induced diabetes influences the effects of cold stress on immunoreactive beta-endorphin levels in female rats

Cold stress produced a significant reduction in the concentration of immunoreactive beta-endorphin (IR-BE) in the anterior pituitary of diabetic female rats. IR-BE levels in the anterior pituitary of non-diabetic female rats were not affected by exposure to the cold. The effects of cold stress on IR-BE levels in the neurointermediate lobe of the pituitary and the hypothalamus were attenuated in diabetic as compared to control animals. These data suggest that in female rats, eight weeks of diabetes produced alterations in the neuroendocrine mechanisms which modulate IR-BE levels in the pituitary and hypothalamus in response to cold stress.     

Corticotropin-releasing factor (CRF) receptors in intermediate lobe of the pituitary: biochemical characterization and autoradiographic localization

CRF receptors were characterized using radioligand binding and chemical affinity cross-linking techniques and localized using autoradiographic techniques in porcine, bovine and rat pituitaries. The binding of 125I-[Tyr0]-ovine CRF (125I-oCRF) to porcine anterior and neurointermediate lobe membranes was saturable and of high affinity with comparable KD values (200-600 pM) and receptor densities (100-200 fmoles/mg protein). The pharmacological rank order of potencies for various analogs and fragments of CRF in inhibiting 125I-oCRF binding in neurointermediate lobe was characteristic of the well-established CRF receptor in anterior pituitary. Furthermore, the binding of 125I-oCRF to both anterior and neurointermediate lobes of the pituitary was guanine nucleotide-sensitive. Affinity cross-linking studies revealed that the molecular weight of the CRF binding protein in rat intermediate lobe was identical to that in rat anterior lobe (Mr = 75,000). While the CRF binding protein in the anterior lobes of porcine and bovine pituitaries had identical molecular weights to CRF receptors in rat pituitary (Mr = 75,000), the molecular weight of the CRF binding protein in porcine and bovine intermediate lobe was slightly higher (Mr = 78,000). Pituitary autoradiograms from the three species showed specific binding sites for 125I-oCRF in anterior and intermediate lobes, with none being apparent in the posterior pituitary. The identification of CRF receptors in the intermediate lobe with comparable characteristics to those previously identified in the anterior pituitary substantiate further the physiological role of CRF in regulating intermediate lobe hormone secretion.     

Effects of food deprivation and high fat diet on immunoreactive beta-endorphin levels in brain regions of Zucker rats

The levels of immunoreactive beta-endorphin (ir-beta-EP) were measured in the brain and pituitary of lean Zucker rats subjected to food deprivation for 72 h and to a high fat diet, and in fatty Zucker rats after food deprivation for 72 h. Ir-beta-EP was increased in the neurointermediate (NI-) pituitary lobe but reduced in the medulla-pons of fatty rats when compared to lean littermates fed ad libitum. Food deprivation decreased ir-beta-EP in the cortex and medulla-pons of lean rats and in the cortex, midbrain and NI-pituitary of fatty rats. In contrast, ir-beta-EP was increased in the anterior pituitary of lean rats and in the striatum of fatty rats after deprivation. The high fat diet produced a decrease in ir-beta-EP in the cortex, midbrain and NI-pituitary with an increase in the striatum and hypothalamus of lean rats. These results suggest that the ir-beta-EP concentration could be differentially affected in different brain regions of Zucker rats by changes in the energy balance.     

Derivatization of progesterone to a neurally active steroid by pituitary neurointermediate lobe

The melanotrophs of the neurointermediate lobe and peptidergic terminals of the neural lobe are regulated by gamma-aminobutyric acid (GABA) via GABA-A receptors and therefore, may be important sites for the modulatory actions of neurally active steroids. These steroid compounds might be produced peripherally, synthesized de novo in the pituitary, or derivatized from circulating steroids, each pathway having different physiological implications. In the present study, we show that neurointermediate lobe tissue can derivatize progesterone to the neurally active steroid 3α-hydroxy-5α-pregnan-20-one. The neurointermediate lobe was found to be four times as active as anterior pituitary and mediobasal hypothalamus in conversion of progesterone to 3α-hydroxy-5α-pregnan-20-one; mediobasal hypothalamus was relatively more active in the production of the intermediate 5α-pregnan-3,20-dione. The identity of the compounds was confirmed by the method of serial isotopic dilution. We observed rates of synthesis in the neurointermediate lobe consistent with the production of physiologically relevant quantities of 3α-hydroxy-5α-pregnan-20-one from concentrations of progesterone which can occur naturally. In support of these findings, we demonstrate the presence of 3α-hydroxysteroid oxidoreductase in neurointermediate lobe by immunocytochemistry.     

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.     

Development of the sex difference in glandular kallikrein and prolactin levels in the anterior pituitary of the rat

Glandular kallikrein is a major estrogen-induced and dopamine-repressed protein of the rat anterior pituitary that appears to originate from lactotrophs. This study examined the development of glandular kallikrein levels in the anterior pituitary in both female and male rats and compared it to anterior pituitary prolactin. In addition, the development of glandular kallikrein levels in the neurointermediate lobe of the pituitary and the kidney were also examined. During puberty, a dramatic surge in glandular kallikrein occurred in female anterior pituitaries (16- to 20-fold increase) and levels remained elevated thereafter. The dynamics of the increase were biphasic--glandular kallikrein increased between Day 30 and 45, plateaued between Days 45 and 55, and then increased again between Days 55 and 65. Female anterior pituitary prolactin increased 7- to 8-fold during puberty. The rise during puberty was biphasic and was generally synchronized with increases in glandular kallikrein. However, the initial rise was proportionately less than that of glandular kallikrein, and the secondary surge was more dramatic. In contrast to females, anterior pituitary glandular kallikrein remained at low levels in male rats; prolactin levels also remained unchanged through puberty and increased moderately thereafter. Glandular kallikrein in the female neurointermediate lobe remained unchanged through Day 55, almost doubled on Day 60, and returned to prepubertal levels by Day 65; males did not exhibit the transient surge in neurointermediate lobe levels. Starting at age 60 days, renal glandular kallikrein was found to be slightly higher (15-20%) in females than in males.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inbred strains of mice with variable sensitivity to ethanol exhibit differences in the content and processing of beta-endorphin

The content of beta-endorphin-like immunoreactivity (beta-EPLIR) in the anterior and neurointermediate lobe of the pituitary gland, the hypothalamus and the serum of the c57BL/6, BALB/C and DBA/2 inbred strains of mice was estimated at the resting state as well as 45 min after i.p. injection of either ethanol solution (3.0 g/kg.b.wt.) or saline. At the resting state, the neurointermediate lobe and the serum of the c57BL/6 mice showed the highest content of beta-EPLIR, while no statistically significant difference was noticed in the total beta-EPLIR content in the anterior lobe and hypothalamus. At 45 min post-ethanol treatment the beta-EPLIR content was increased in the serum of all three strains of mice studied and was decreased in the hypothalamus of the c57BL/6 mice only. Further analysis of the beta-endorphin peptides using sephadex G-75 chromatography and reverse phase high performance liquid chromatography indicated strain differences in the relative proportions of the various forms of beta-endorphin in the anterior lobe, neurointermediate lobe and the hypothalamus. These strain specific differences in the content and post-translational processing of beta-endorphin may be involved in some of the genetically determined differences in responses to ethanol by these inbred strains of mice.     

Antagonism of diazepam-induced feeding in rats by antisera to opioid peptides

Diazepam-induced feeding in rats is antagonized not only by the opiate antagonist naloxone but also intraventricular administration of specific antisera to the endogenous opioid peptides met-enkephalin or beta-endorphin. Pituitary beta-endorphin is probably not implicated in the diazepam effect since blockade with the glucocorticoid dexamethasone of the release of beta-endorphin from the anterior pituitary does not modify the diazepam-induced feeding, which is however prevented by TRH, a suggested physiological antagonist of some of the effects of opioid peptides. The possible central participation of both beta-endorphin and met-enkephalin in the ingestive behavior induced by diazepam gives further support to the postulated physiological role of endogenous opioids in appetite regulation.     

Administration of gonadal steroids to neonatal rats affects beta-endorphin levels in the adult

Administration of gonadal steroids to neonatal rats has a profound effect on the function of the neuroendocrine system in the adult animal. Considering that gonadal steroids modulate hypothalamic and pituitary levels of beta-endorphin (BE) in adult male and female rats, the effects of neonatal gonadal steroid treatment on BE levels in the adult animal were investigated. Neonatal male rats were administered testosterone and neonatal female rats were treated with estrogen. Matched control littermates received vehicle. All animals were sacrificed at 90 days of age. Neonatal gonadal steroid treatment did not affect the level of immunoreactive beta-endorphin (IR-BE) in the anterior pituitary (AP) of male rats but did result in a significant increase in IR-BE in the AP of female rats. Neonatal administration of gonadal steroids produced a significant decrease in IR-BE in the neurointermediate lobe of the pituitary (NIL) of both male and female rats, with the magnitude of the decrease being greater in the NIL of the female rats. IR-BE levels in the hypothalamus of male or female rats were not altered by the treatments. Column chromatography indicated that the increase in IR-BE in the AP represented a proportional increase in BE and beta-lipotropin, while the reduction in IR-BE in the NIL of the treated rats represented a reduction in BE. These findings suggest that gonadal steroids may influence the development of the neurotransmitter systems which regulate BE levels in the adult pituitary, the development of the biosynthetic mechanisms of the adult pituitary, or both.     

Molecular and Pharmacological Characterization of GABAA Receptors in the Rat Pituitary

Abstract: Levels of mRNA for the major subunits of the GABA_A receptor were assayed in the rat pituitary anterior and neurointermediate lobes by ribonuclease protection assay. α1, β1, β2, β3, and γ2s were found to be the predominant subunits in the anterior lobe, whereas α2, α3, β1, β3, γ2s, and γ1 were the predominant subunits expressed in the neurointermediate lobe. α5, α6, and δ subunits were not detectable. Hill and Scatchard analysis of [³H]muscimol binding to anterior and neurointermediate lobe membranes showed high-affinity binding sites with dissociation constants of 5.6 and 4.5 n M , respectively, and Hill coefficients near 1. Muscimol sites were present at a maximum of 126 fmol/mg in the anterior lobe and 138 fmol/mg in the neurointermediate lobe. The central-type benzodiazepine antagonist [³H]Ro 15-1788 bound to a high-affinity site with a dissociation constant of 1.5 n M in both tissues, at a maximum of 60 fmol/mg in anterior pituitary and 72 fmol/mg in neurointermediate lobe. A Hill coefficient of 1 was measured for this site in both tissues. Assays of CL 218 872 displacement of Ro 15-1788 were consistent with a pure type I benzodiazepine site in the anterior lobe and a pure type II site in the intermediate lobe. These results are consistent with both tissue-specific expression of particular GABA_A receptor subunits and receptor heterogeneity within individual cells in the pituitary.     

Age-related and diurnal changes in Met5-Enk-Arg6-Phe7 and Met5-enkephalin contents of pituitary and rat brain structures

The beta-endorphin, met5-enkephalin-arg6-phe7 (MEAP) and met5-enkephalin (ME) changes related to age and diurnal rhythms were studied in various regions of rat brain and in the pituitary by specific radioimmunoassays. The contents of MEAP, met5-enkephalin and beta-endorphin were higher in the pituitary of old rats (18 months old) than that of young rats (23 days old) while the content of these opioid peptides was higher in the hypothalamus of young rats than in that of old rats. Beta-endorphin was also higher in the striatum of 23 days old rats, but no age-associated changes were observed in the hippocampus, brain stem or cortex. In the diurnal rhythm study, it was found that in the hypothalamus and striatum of the adult rat (2-3 months old), both MEAP and ME contents were higher at mid-dark than at mid-light and that in the intermediate posterior lobe of the pituitary, the ME content was also higher at mid-dark.     

Secretin in the rat hypothalamo-pituitary system: Localization,identification and characterization

Secretin-like immunoreactivity (SLI) has been identified and characterized in the pituitary of the rat. The concentration in the neurointermediate lobe is about 45 fold higher than the concentration of SLI observed in the anterior lobe. Transections of the pituitary stalk of the rat caused a significant depletion of SLI in the neurointermediate lobe without affecting the content in the anterior lobe. In view of the relatively high concentration of SLI reported to occur in the hypothalamus, it appears that there may be a secretinergic pathway between the brain and the neurointermediate lobe of the pituitary.     

Dopamine receptor blockade increases glandular kallikrein-like activity in the neurointermediate lobe of the rat pituitary

The effect of dopamine receptor blockers on glandular kallikrein-like activity in the neurointermediate lobe of the rat pituitary was examined. Male rats were given daily injections of haloperidol (2.5 mg/kg), perphenazine (5 mg/kg) or sulpiride (60 mg/kg) for 7 days. Homogenates of the neurointermediate lobe were prepared. Latent proteases were activated with trypsin and proteolytic activity was measured at 37 degrees C, pH 8.0 using chromogenic peptide substrates. All three dopamine receptor blockers produced about a 100% increase in glandular kallikrein-like activity. The results suggest that glandular kallikrein-like activity in the neurointermediate lobe is under inhibitory control by dopaminergic mechanisms.     

Met-enkephalin-like immunoreactivity in neurointermediate pituitary is decreased by DA receptor stimulation

The effect of dopamine receptor stimulation by administration of the dopamine analogue bromocriptine on Met-enkephalin-LI was examined in rat hypothalamus, and neurointermediate and anterior lobes of pituitary. Bromocriptine treatment resulted in a dramatic decline of Met-enkephalin-LI in neurointermediate pituitary which was significant by 3 days of treatment. Maximal reduction of Met-enkephalin-LI ranged between 60-70% of pretreatment values and was maintained as long as bromocriptine was administered (4 weeks), with no evidence of desensitization or "escape." The effects of bromocriptine on neurointermediate lobe were of long duration and persisted for at least 4 days after discontinuation of treatment. No significant effects of bromocriptine were detected on Met-enkephalin-LI in hypothalamus or anterior pituitary. Whether these differences represent truly different regional regulation of Met-enkephalin-LI or whether the changes are more sensitively reflected in an area such as neurointermediate lobe that largely consists of nerve terminals, remains to be shown.     

Pro-opiomelanocortin-derived peptides in the pig pituitary: alpha- and gamma 1-melanocyte-stimulating hormones and their glycine-extended forms

Pro-opiomelanocortin (POMC)-related peptides in extracts of anterior and neurointermediate pituitary lobes from pigs were characterized by gel chromatography, reversed-phase chromatography and radioimmunoassays. The peptide content was ca. 3-fold greater in the anterior lobe compared to the neurointermediate lobe (19.8 nmol POMC/anterior lobe vs 7.0 nmol/neurointermediate lobe). In the neurointermediate lobe 93% of POMC was processed to alpha-melanocyte-stimulating hormone (alpha-MSH) and analogs exclusively of low molecular weight. Most of the remaining adrenocorticotropic hormone (ACTH)-related material consisted of the glycine-extended intermediate ACTH-(1-14) and analogs. In contrast only one fourth to one third of the N-terminal part of POMC (N-POMC) was processed to amidated gamma-MSH and its C-terminal glycine-extended precursor. The relative amount of amidated gamma-MSH was the same as alpha-MSH and analogs (94%). However, more than 95% of these peptides were of high molecular weight. In the anterior lobe 2.3% of N-POMC was processed and 94% was amidated gamma-MSH of only high molecular weight. These results show that gamma-MSH and alpha-MSH are amidated to the same extent and that gamma 1-MSH and gamma 2-MSH immunoreactivity are present in both the anterior lobe and the neurointermediate lobe. The results suggest that the production of amidated peptides is not regulated by the amidation process itself but at an earlier step (e.g. at the proteolytic cleavage).     

Immunoreactive beta-endorphin in the plasma, pituitary and hypothalamus of young female rats on the day of estrus and intact and chronically castrated old constant estrous female rats

Immunoreactive beta-endorphin (IR-beta-ENDO) was compared in the plasma, pituitary and hypothalamus of young female rats on the day of estrus and old constant estrous (CE) female rats, and in intact and chronically castrated old CE female rats. The concentration of IR-beta-ENDO in the plasma and the content and concentration of IR-beta-ENDO in the neurointermediate lobe of the pituitary were significantly greater in the old CE female rats than in the young female rats on the day of estrus. The content and concentration of IR-beta-ENDO in the anterior pituitary and hypothalamus were similar in the two age groups. To determine if estrogen contributed to the increase in plasma and pituitary levels of IR-beta-ENDO observed in the old animals, a group of old CE female rats were castrated and compared to sham operated control CE rats. Thirty days after castration, levels of plasma, pituitary and hypothalamic IR-beta-ENDO were comparable in the intact and the chronically castrated old female rats. These data indicate that in old CE female rats, plasma and pituitary IR-beta-ENDO are significantly increased in comparison to young female rats on the day of estrus, and that these increased levels of IR-beta-ENDO observed in old female rats do not appear to be influenced by gonadal estrogen.     

Membrane-associated forms of peptidylglycine alpha-amidating monooxygenase activity in rat pituitary. Tissue specificity

Membrane-associated peptidylglycine alpha-amidating monooxygenase (PAM) activity was investigated in rat anterior and neurointermediate pituitary tissues and in pituitary AtT-20/D-16v and GH3 cell lines. A substantial fraction of total pituitary PAM activity was found to be membrane-associated. Triton X-100, N-octyl-beta-D-glucopyranoside, and Zwittergent were effective in solubilizing PAM activity from crude pituitary membranes. The distribution of enzyme activity between soluble and membrane-associated forms was tissue-specific. In the anterior pituitary lobe and pituitary cell lines, 40-60% of total PAM activity was membrane-associated while only 10% of the alpha-amidating activity in the neurointermediate lobe was membrane-associated. Soluble and membrane-associated forms of PAM shared nearly identical characteristics with respect to copper and ascorbate requirements, pH optima, and Km values. Upon subcellular fractionation of anterior and neurointermediate pituitary lobe homogenates on Percoll gradients, 12-18% of total PAM activity was found in the rough endoplasmic reticulum/Golgi fractions and 42-60% was localized to secretory granule fractions. For both tissues, membrane-associated PAM activity was enriched in the rough endoplasmic reticulum/Golgi pool, whereas most of the secretory granule-associated enzyme activity was soluble.