Participation of membrane progesterone receptor α in the inhibitory effect of progesterone on prolactin secretion

The membrane progesterone receptors (mPRα, mPRβ, mPRγ, mPRδ and mPRε) are known to mediate rapid nongenomic progesterone functions in different cell types. However, the functions of these receptors in the pituitary have not been reported to date. In the present study, we show that the expression of mPRα was the highest among the mPRs in the rat anterior pituitary gland. Immunostaining of mPRα was detected in somatotrophs, gonadotrophs and lactotrophs. Interestingly, 63% of mPRα‐positive cells within the pituitary were lactotrophs, suggesting that mPRα is involved in controlling prolactin (PRL) secretion in the pituitary. To test this hypothesis, rat pituitaries were incubated (1 hour) with either progesterone (P4) or the mPRα‐specific agonist Org OD 02‐0. PRL secretion was then measured by radioimmunoassay. The results of this experiment revealed that both P4 and Org OD 02‐0 decreased PRL secretion. Moreover, the results from the GH3 cell line (CCL‐82.1) showed that P4 and Org OD 02‐0 inhibited PRL release, although the nuclear PR agonist R5020 was ineffective. Our investigation of the cellular mechanisms behind mPRα activity indicated that both P4 and Org OD 02‐0 decreased cAMP accumulation, whereas R5020 was ineffective. In addition, the Org OD 02‐0‐effect on PRL release was blocked by pretreatment with pertussis toxin, an inhibitor of Go/Gi proteins. Because transforming growth factor (TGF)β1 is a potent inhibitor of PRL secretion in lactotrophs, we lastly evaluated whether TGFβ1 was activated by progesterone and whether this effect was mediated by mPRα. Our results showed that P4 and Org OD 02‐0, but not R5020, increased active TGFβ1 levels. This effect was not observed when cells were transfected with mPRα‐small interfering RNA. Taken together, these data provide new evidence suggesting that mPRα mediates the progesterone inhibitory effect on PRL secretion through both decreases in cAMP levels and activation of TGFβ1 in the lactotroph population.     

A guanine nucleotide-binding protein mediates the inhibition of voltage-dependent calcium currents by dopamine in rat lactotrophs

The present study examines the effect of dopamine (DA), known to inhibit prolactin (PRL) release, on voltage-activated calcium currents in identified rat lactotrophs. Two types of voltage-dependent Ca2+ currents were recorded using the whole-cell mode of the patch-clamp technique. Both were reversibly inhibited by DA application. The inhibitory action of DA was reduced by (i) sulpiride (D2 antagonist), (ii) preincubation of the cells with pertussis toxin (PTX), and (iii) inclusion of guanosine 5'-O-(2-thiodiphosphate) (GDP-beta-S) in the pipette solution, whereas it was potentiated by guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S). This DA-induced response could not be overcome by changing the adenosine 3',5'-cyclic monophosphate level. These findings suggest that DA can inhibit Ca2+ entry through voltage-activated Ca2+ channels via a PTX-sensitive G protein(s) pathway thereby affecting PRL release from rat lactotrophs.     

Endocrine cells in pancreatic islets of Langerhans are immunoreactive to antibody against guanine nucleotide-binding protein (Go) purified from rat brain

Guanine nucleotide-binding proteins (G proteins) are usually classified into four subclasses (Gs, Gi, Go and Gt or transducin). We localized the anti-Go immunoreactivities in islets of Langerhans of the rat pancreas by using affinity-purified antibody against the alpha-subunit of Go purified from rat brain. Endocrine cells of the islets of Langerhans were stained with this Go antibody, but the acinar cells in the exocrine portion of the pancreas were immunonegative to this antibody. These findings strongly suggest that Go protein functions as intermediaries in the transmembrane signalling pathway in the endocrine cells of the islets of Langerhans.     

Thyrotrophin-releasing hormone, vasoactive intestinal peptide, prolactin-releasing peptide and dopamine regulation of prolactin secretion by different lactotroph morphological subtypes in the rat

In the male rat anterior pituitary, three morphological subtypes of cells secreting primarily prolactin (PRL) (lactotrophs) have been described. Type I contain predominantly large irregularly shaped granules, whereas type II and type III lactotrophs contain smaller spherical granules. We have previously shown that oestradiol and testosterone exert a rapid stimulatory effect selectively on type II lactotrophs but it is not known how the lactotroph subtypes respond to peptide secretagogues. We have therefore examined which cell subtype(s) release PRL in response to vasoactive intestinal peptide (VIP), thyrotrophin-releasing hormone (TRH) and prolactin-releasing peptide (PrRP-31). Pituitary segments were incubated in medium containing tannic acid (to capture exocytosis of secretory granules), either alone or with secretagogue peptide. VIP (1-10 nM), TRH (10 nM) and PrRP-31 (10 nM) all caused a significant increase (P < 0.05) in the amount of PRL granule exocytosis from type II and III lactotrophs, but had no effect on PRL exocytosis from type I. Dopamine (100 nM) inhibited basal exocytosis of immunoreactive (ir)-PRL from type I, II and III lactotrophs and PrRP-31-stimulated ir-PRL granule exocytosis from II and III lactotrophs. Treatment of lactating female rats with the dopamine D(2) receptor antagonist sulpiride (40 microg/kg) produced a significant increase (P < 0.05) in PRL granule exocytosis from type I and type III lactotrophs and a significant increase (P < 0.05) in the proportion of type I and II cells undergoing exocytosis of PRL. In conclusion, VIP, TRH and PrRP-31 selectively stimulate exocytosis from type II and III lactotrophs in the male rat, whereas all three lactotroph types are sensitive to dopamine inhibition of exocytosis in male and female rats.     

Changes in the local metabolism of dopamine in the anterior and neural lobes but not in the intermediate lobe of the pituitary gland during nursing

Dopamine (DA), produced by tubero-infundibular dopaminergic (TIDA) neurons of the arcuate nucleus (ARN) is the established inhibitor of the secretion of prolactin (PRL). Changes in dopaminergic (DAergic) neuronal activity in the median eminence–long portal vessels (ME–LPV) and/or the concentration of DA in the anterior lobe (AL) are inversely related to the secretion of PRL. However, conflicting reports concerning DAergic neuronal activity during the suckling-induced release of PRL persist. In addition to TIDA neurons, PeVN-hypophysial DAergic (PHDA) and tubero-hypophysial DAergic (THDA) neurons which, respectively, innervate the intermediate lobe (IL) and the IL/neural lobe (NL) also have a significant role. We measured the concentrations of DA and its main metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), in the median eminence and the three pituitary lobes of lactating mothers. Concentrations of DA and DOPAC from tissues and the concentration of PRL in plasma were measured by HPLC-EC and RIA, respectively. There were no changes in the concentration of DA and DOPAC of the IL due to the suckling stimulus. In the NL, a decrease in the concentration of DOPAC was detected due to the suckling stimulus. In addition, there were no changes of DA or DOPAC in the outer zone of the AL (AL-OZ) due to suckling. However, a decrease in the concentrations of DA and DOPAC was detected in the inner zone of the AL (AL-IZ). These data suggest lactotrophs from the AL-IZ are responsible for the changes in the concentration of plasma PRL in response to the suckling stimulus. In addition TIDA and THDA neurons, but not PHDA neurons, regulate the control of the secretion of PRL in response to suckling.     

Interleukin-1 and tumor necrosis factor-mediated regulation of C3 gene expression in human astroglioma cells

In this report, we show that in the human astroglioma cell line D54-MG, both interleukin-1 (IL-1β) and tumor necrosis factor-alpha (TNF-α) enhance C3 gene expression in a time- and dose-dependent manner. Kinetic analysis demonstrates that after 96 h, C3 mRNA levels increase approximately 30-fold and 20-fold in response to IL-1β or TNF-α, respectively. C3 protein production increases proportionally, reaching levels 36-fold and 18-fold higher than untreated controls upon exposure to IL-1β or TNF-α, respectively. D54-MG cells require a minimal 1 h exposure to IL-1β in order to enhance C3 gene expression significantly, while 4 to 8 h are required for TNF-α. Simultaneous treatment of D54-MG cells with IL-1β and interferon-gamma (IFN-γ) resulted in an additive increase in both C3 mRNA and protein expression, a finding not seen with the combination of TNF-α and IFN-γ. Primary rat astrocytes also express increased C3 mRNA levels after 48 h in response to IL-1β (5.3-fold increase) and TNF-α (7-fold increase), while an additive effect was observed upon simultaneous treatment with both IL-1β and IFN-γ. In the central nervous system (CNS), endogenous complement and cytokine production by astrocytes, and enhancement by IFN-γ, a product of activated T cells often seen in the CNS in neural autoimmune disease, may contribute to the pathogenesis of inflammatory demyelinating diseases such as multiple sclerosis.     

Regulation of Basal and Stimulated Prolactin Release in Prolactin-Secreting Rat Pituitary Tumors*

To better understand the mechanisms of the inhibitory effects of dopamine on pituitary prolactin release, we have utilized an estrone-induced, benign and dopamine-sensitive rat pituitary adenoma and two malignant, transplantable and dopamine-resistant rat pituitary tumors, 7315a and MITW15. Enzymatically dispersed and Percoll purified cells obtained from the three tissues were incubated for 30 min in media with or without Na⁺ and in the presence or the absence of dopamine and/or various prolactin releasers for evaluating the secretion of prolactin under baseline and experimental conditions. In some experiments, the cells were pretreated for 16 h with pertussis toxin to evaluate the eventual presence and role of pertussis toxin-sensitive G proteins. Dopamine inhibited baseline prolactin release by adenomatous lactotrophs in a Na⁺-dependent manner, but was totally inactive with 7315a and MtTW15 cells. The Ca²⁺ channel agonist BAY K 8644 stimulated prolactin release with all three preparations and its effects were enhanced by a Na⁺-free medium. Dopamine antagonized the stimulatory effects of BAY K 8644 with adenomatous and 7315a cells only, even in the absence of Na⁺. Pertussis toxin pretreatment significantly increased baseline prolactin release by adenomatous and MtTW15 cells and abolished dopamine inhibition of adenomatous lactotrophs baseline hormone release. BAY K 8644, TRH and vasoactive intestinal peptide, stimulated prolactin release by adenomatous cells and this effect was antagonized by dopamine in a pertussis toxin-sensitive manner. All prolactin releasers, except TRH, were effective also with 7315a cells, and its actions were not blocked by pertussin toxin. The stimulatory effects of BAY K 8644 and vasoactive intestinal peptide on 7315a cells were enhanced by pertussis toxin pretreatment. The results obtained with an almost pure preparation of adenomatous lactotrophs confirm the existence of a dual mechanism of dopamine inhibitory action on prolactin release: 1) a Na⁺-dependent action exerted on baseline, and 2) a Na⁺-independent action exerted on stimulated prolactin release. They also indicate that both actions are exerted through pertussis toxin-sensitive G proteins. Furthermore, our results show the presence in transplantable pituitary tumors 7315a and MtTW15 of multiple and diverse anomalies in the regulation of prolactin release probably due, at least partly, to anomalies of one or more G proteins and/or neurotransmitter receptors.     

Mitogen-Activated Protein Kinase Pathways are Involved in the Upregulation of Calcitonin Gene-Related Peptide of Rat Trigeminal Ganglion After Organ Culture

The trigeminal ganglion (TG) can express and release calcitonin gene-related peptide (CGRP), an important neuropeptide that plays a crucial role in migraine attack and cluster headache. Activation of rat TG increases CGRP expression. However, the regulatory mechanism of CGRP expression in TG neurons remains to be explored. This study aims to evaluate the involvement of mitogen-activated protein kinase (MAPK) pathways in CGRP upregulation after rat TG organ culture. Rat TG was cultured alone for 24 h or cultured in combination with MAPK inhibitors, tumor necrosis factor α (TNF-α), or interleukin 1β (IL-1β) for 24 h. CGRP protein was determined using immunohistochemistry. The mRNA levels of CGRP, TNF-α, and IL-1β were analyzed through real-time quantitative polymerase chain reaction. MAPK phosphorylation was detected via western blot. After rat TG organ culture, the expressions of CGRP, TNF-α, and IL-1β were upregulated at 24 h. The phosphorylation of extracellular signal-regulated kinases (ERK1/2), P38, and c-jun N-terminal kinases (JNK) significantly increased at 30 min compared with fresh rat TG. In addition, both CGRP expression and phosphorylation of ERK1/2, P38, and JNK were enhanced obviously after rat TG treatment with TNF-α or IL-1β compared with fresh rat TG. However, they decreased markedly after rat TG pretreatment with PD98059 (ERK1/2 inhibitor), SB203580 (P38 inhibitor), or SP600125 (JNK inhibitor) compared with rat TG co-culture with TNF-α or IL-1β. In conclusion, the elevated CGRP expression after rat TG organ culture can be regulated via MAPK pathways. The findings provide insight into the molecular mechanisms and experimental evidence for therapeutic targets of migraine.     

Stimulation of group II phospholipase A2 mRNA expression and release in an immortalized astrocyte cell line (DITNC) by LPS,TNFα, and IL-1β

Astrocytes are immunoactive cells in brain and have been implicated in the defense mechanism in response to external injury. Previous studies using cultured glial cells indicated the ability of astrocytes to respond to bacteria endotoxin and cytokines, resulting in the release of phospholipase A₂. In this study, we examined the interactive effects of lipopolysaccharides (LPS), interleukin 1β (IL-1β) and tumor necrosis factor (TNFα) to stimulate phospholipase A₂ (PLA₂) in an immortalized astrocyte cell line (DITNC) with many properties of type I astrocytes. Northern blot analysis using oligonucleotide probes derived from the cDNA encoding the rat spleen group II PLA₂ indicated the ability of DITNC cells to respond to all three factors in the induction of gene expression and the release of PLA₂. After an initial lag time of 2 h, PLA₂ release was proportional to time, reaching a plateau by 12 h. This event occurred at a time period preceding any signs of cell death. Cycloheximide at 1.25 μM completely inhibited cytokine-induced PLA₂ release. When suboptimal amounts of TNFα were added to the DITNC culture together with IL-1β or LPS, a synergistic increase in the induction of PLA₂ release could be observed. On the other hand, combination of IL-1β and LPS resulted only in an additive increase in PLA₂ release. Antibodies to IL-1β and TNFα completely neutralized the effects of these two agents on PLA₂ release. However, neither antibody was able to inhibit the PLA₂ release induced by LPS, suggesting that the effect of LPS was not complicated by the release of IL-1β or TNFα. Taken together, results show that the immortalized astrocyte cell line (DITNC) can be used for studies to elucidate the molecular mechanism underlying the cytokine signaling cascade and subsequent induction of PLA₂ synthesis.     

Signal mechanisms underlying low-dose endothelial monocyte-activating polypeptide-II-induced opening of the blood-tumor barrier

Our previous studies have demonstrated that both the RhoA/Rho kinase and the protein kinase C (PKC) signaling pathways are involved in the low-dose endothelial monocyte-activating polypeptide-II (EMAP-II)-induced blood-tumor barrier (BTB) opening. In the present study, an in vitro BTB model was used to investigate which isoforms of PKC were involved in this process as well as the interactions between the RhoA/Rho kinase and the PKC signaling pathways. Our results showed that EMAP-II-activated PKC-α, β, and ζ and induced translocations of them from the cytosolic to the membrane fractions of rat brain microvascular endothelial cells. The EMAP-II-induced alterations in BTB permeability and tight junction (TJ) protein expression were partially blocked by G?6976, the inhibitor of PKC-α/β, and PKC-ζ pseudosubstrate inhibitor (PKC-ζ-PI). Meanwhile, we observed that G?6976 partly inhibited the EMAP-II-induced rearrangement of actin cytoskeleton as well as phosphorylation of myosin light chain and cofilin, whereas PKC-ζ-PI had no effect on these above-mentioned changes induced by EMAP-II. Also, our data revealed that inhibition of RhoA or inhibition of Rho kinase significantly diminished the activities and the translocations of PKC-α and PKC-β induced by EMAP-II, whereas PKC-ζ was unaffected. However, inhibition of PKC-α/β or inhibition of PKC-ζ did not cause any changes in the RhoA and Rho kinase activities. The effects of EMAP-II on BTB permeability and TJ proteins expression were completely blocked by inhibition of both RhoA and PKC-ζ, whereas inhibition of both RhoA and PKC-α/β had an effect similar to that of inhibition of RhoA alone. In summary, this study demonstrates for the first time that three PKC isoforms, PKC-α, β, and ζ, are involved in the EMAP-II-induced BTB opening. It is PKC-α/β, but not PKC-ζ, which serves as the downstream target for RhoA and Rho kinase, suggesting that EMAP-II induces BTB opening via the RhoA/Rho kinase/PKC-α/β signaling pathways. However, PKC-ζ is involved in this process by other mechanisms.     

G protein mRNA expression in immunohistochemically identified dopaminergic and noradrenergic neurons in the rat brain

A family of guanine nucleotide binding proteins (G proteins) is involved in the transduction of information from receptors on the cell surface into cellular responses. Two G proteins, Gi and Gs, were initially defined by their inhibitory or stimulatory actions on adenylyl cyclase, respectively. In addition, brain contains high levels of another G protein, Go. cDNAs for the alpha subunits for these G proteins have been cloned and sequenced. This allowed us to examine the distributions of the mRNAs for the alpha subunits for Gi, Go and Gs in the rat brain using in situ hybridization with radio-labelled, synthetic oligonucleotide probes. Various regions known to contain catecholamine cell groups displayed high levels of G protein mRNA. There is good physiological evidence supporting a role for G proteins in signal transduction in dopaminergic and noradrenergic neurons. Therefore, further experiments were undertaken using in situ hybridization combined with immunohistochemistry to examine G proteins expression in identified catecholamine neurons. The results indicate that the dopaminergic neurons of the substantia nigra and the noradrenergic neurons of the locus ceruleus express the mRNA for the alpha subunits of all three of these G proteins. These data provide evidence for the coexpression of multiple G proteins within identified catecholamine neurons in the brain.     

Propentofylline, a CNS glial modulator does not decrease pain in post-herpetic neuralgia patients: in vitro evidence for differential responses in human and rodent microglia and macrophages

There is a growing body of preclinical evidence for the potential involvement of glial cells in neuropathic pain conditions. Several glial-targeted agents are in development for the treatment of pain conditions. Here we report the failure of a glial modulating agent, propentofylline, to decrease pain reported in association with post-herpetic neuralgia. We offer new evidence to help explain why propentofylline failed in patients by describing in vitro functional differences between rodent and human microglia and macrophages. We directly compared the proinflammatory response induced by lipopolysaccharide (LPS) with or without propentofylline using rat postnatal microglia, rat peritoneal macrophages, human fetal microglia, human peripheral macrophages and human immortalized THP-1 cells. We measured tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β) and nitrite release (as an indicator of nitric oxide (NO)) as downstream indicators. We found that LPS treatment did not induce nitrite in human microglia, macrophages or THP-1 cells; however LPS treatment did induce nitrite release in rat microglia and macrophages. Following LPS exposure, propentofylline blocked TNF-α release in rodent microglia with all the doses tested (1-100 μM), and dose-dependently decreased TNF-α release in rodent macrophages. Propentofylline partially decreased TNF-α (35%) at 100 μM in human microglia, macrophages and THP-1 macrophages. Propentofylline blocked nitrite release from LPS stimulated rat microglia and inhibited nitrite in LPS-stimulated rat macrophages. IL-1β was decreased in LPS-stimulated human microglia following propentofylline at 100 μM. Overall, human microglia were less responsive to LPS stimulation and propentofylline treatment than the other cell types. Our data demonstrate significant functional differences between cell types and species following propentofylline treatment and LPS stimulation. These results may help explain the differential behavioral effects of propentofylline observed between rodent models of pain and the human clinical trial.     

Effects of Neonatal Exposure to Estradiol on Prolactin Secretion and Activity of the Tubero-lnfundibular Dopamine System in Young Adulthood: Comparison with Neonatal Prolactin Deficiency*

Previous results from this laboratory indicate that female rats who consume milk deficient in prolactin (PRL) during the neonatal period subsequently display hyperprolactinemia, associated with decreased activity in the tubero-infundibular dopamine (DA) system and decreased lactotrope responsiveness to DA receptor stimulation. The present studies tested whether these neuroendocrine consequences of neonatal PRL deficiency can be mimicked by exposure of neonatal rats to estradiol. Female rats were injected sc with 1 mUg estradiol benzoate or oil vehicle on postpartum Days one to 3, while in other experiments, females were made neonatally deficient in PRL through treatment of their mothers with the DA agonist bromocriptine, a treatment that reduces the levels of PRL in milk. Females treated neonatally with estradiol benzoate, as well as offspring of the bromocriptine-treated mothers, displayed hyperprolactinemia as young adults, as compared to their respective vehicle-matched controls, and in both cases, this was abolished by ovariectomy, indicating dependence upon ovarian secretions. As reported previously in neonatal PRL-deficient females, neonatal estradiol benzoate-treated animals also exhibited reduced steady state levels and decreased turnover rates of DA in the median eminence when 35 days of age. DA levels and turnover rates in this region were still significantly reduced on postpartum Day 60. The DA agonist bromocriptine suppressed PRL release to a similar extent in cultured anterior pituitary cells from neonatal estrogen-treated and control rats, suggesting normal responsiveness of DA receptors on lactotrope cells in both groups. The present results confirm the ability of estradiol treatment or induction of a PRL deficiency during the early neonatal period to induce subsequent hyperprolactinemia in female rats, and further indicate that the hyperprolactinemic conditions resulting from either neonatal manipulation are dependent on the ovary and are associated with decreased levels and turnover of DA in the median eminence during the prepubertal period. Although these findings suggest that increased exposure to estradiol during the neonatal period may underlie the similar effects of neonatal PRL deficiency, the further observations in neonatal estrogen-treated rats that 1) decreased DA turnover in the median eminence persists at Day 60, and 2) lactotrope responsiveness to DA is normal, differ from results obtained previously in PRL-deficient rats. Thus, enhanced exposure to estrogen during the neonatal period does not appear to account for all of the neuroendocrine consequences of neonatal PRL deficiency.     

Interleukin 1-β- and tumor necrosis factor-α-mediated regulation of ICAM-1 gene expression in astrocytes requires protein kinase C activity

Several adhesion molecules including intracellular adhesion molecule-1 (ICAM-1) are expressed by astrocytes, the predominant glial cell of the central nervous system (CNS). Such molecules are important in the trafficking of leukocytes to sites of inflammation, and in lymphocyte activation. ICAM-1 is constitutively expressed by neonatal rat astrocytes, and expression is enhanced by the proinflammatory cytokines interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ), with IL-1β and TNF-α being the strongest inducers. In this study, we have examined the nature of the second messengers involved in ICAM-1 gene expression induced by the cytokines IL-1β and TNF-α. Our results indicate that stimuli related to protein kinase C (PKC) such as the phorbol ester phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187 increase ICAM-1 mRNA expression, whereas cyclic nucleotide analogs and PKA agonists have no effect. Pharmacologic inhibitors of PKC such as H7, H8, and calphostin C inhibit ICAM-1 gene expression inducible by IL-1β and TNF-α. Prolonged treatment of astrocytes with PMA results in a time-dependent downregulating of the PKC isoforms α, δ, and ?, and a concomitant diminution of ICAM-1 mRNA expression induced by IL-1β, TNF-α, and PMA itself at specific time points post-PMA treatment. These data, collectively, demonstrate a role for various PKC isoforms in IL-1β and TNF-α enhancement of ICAM-1 gene expression in rat astrocytes. © 1995 Wiley-Liss, Inc.     

A guanine nucleotide-binding protein in human astrocytoma

We compared, by immunoblotting and immunohistochemical staining, the distribution of the a subunit of Go in human normal brain tissue with that in human brain tumours of neuroectodermal origin. The ten tumour samples included seven high-grade astrocytomas (grades III and IV) and three low-grade astrocytomas (grade II). Im mu noreactivity of high-grade astrocytomas with the α subunit of Go (Goα) antibody was lower than that of low-grade astrocytomas and normal white matter. In the immunohistochemical study, neoplastic cells in the astrocytomas were found to contain little or no significant Goα, although nonlesional white matter was strongly stained with Goα antibody. This study suggests that Goα antibody may assist in defining the boundary of an astrocytoma, and the difference in Go content may affect the alteration in the physical properties of the membrane, which exhibit, in part, the character of the transformation.     

Functional Lactotroph Heterogeneity in Lactating Rats and in vitro Modification by 17Beta-Estradiol

Lactotrophs from lactating rats were separated by unit gravity sedimentation on a continuous density gradient of bovine serum albumin and were identified in two populations located in the light fractions (fractions 3–5) and in the heavy fractions (fractions 7–9) of the gradient. After 7 days in vitro, the effects on prolactin release of thyrotropin-releasing hormone (TRH) and dopamine before and after pretreatment with 17β-estradiol were studied by a continuous perifusion system and reverse hemolytic plaque assay. Light fraction lactotrophs spontaneously released large quantities of prolactin (22 ng/ml/2 min/10⁶ cells) and this basal release was markedly elevated (51 ng/ml/2 min/10⁶ cells) by pretreatment with 17β-estradiol (10^?8 M, 48 h), while the amount of intracellular prolactin remained stable. Mean hemolytic plaque area was increased in the same manner by 17β-estradiol pretreatment but the number of cells and the percentage of plaque-forming cells were not changed. Perifusion of dopamine-containing medium (10^?7 M) almost completely blocked basal prolactin release from light fraction cells and this inhibition was markedly reduced by 17β-estradiol pretreatment. TRH-containing medium (10^?7 M) weakly stimulated basal prolactin release (about 190% from basal) and this response was significantly enhanced (to about 300% of basal release) by 17β-estradiol pretreatment. Both dopaminergic inhibition and TRH-stimulatory effects were dose-dependent and their half maximal effect values were not changed by 17β-estradiol pretreatment. Secretion of prolactin evaluated at the single cell level by the reverse hemolytic plaque assay corroborated the results obtained from perifusion experiments. Lactotrophs from heavy fractions released small amounts of prolactin (12 ng/ml/2 min/10⁶ cells) and neither this basal release nor the amount of intracellular prolactin were markedly modified by 17β-estradiol pretreatment. As opposed to the light fraction cells, lactotrophs found in heavy fractions were very sensitive to TRH (10^?7 M) stimulation with maximal stimulation reaching ten times basal release, but were less sensitive to dopamine (10^?7 M), with an inhibition of only 40% basal prolactin liberation. Pretreatment of heavy fraction lactotrophs with 17β-estradiol induced similar effects to those observed after pretreatment of light fraction cells: the stimulation by TRH was increased (from 11 times to 16 times) whereas the inhibition by dopamine was diminished (from 35% to 60%), but cell number and the percentage of prolactin-secreting cells remained unchanged. From the above results, we suggest that: 1) lactotrophs in the lactating rat pituitary can be divided into two major subpopulations with regard to cellular size and density, prolactin production and responsiveness to TRH and dopamine; 2) 17β-estradiol pretreatment increases basal prolactin release from light fraction cells but does not affect basal prolactin release from heavy fraction cells in this way; 3) pretreatment with 17β-estradiol enhances TRH stimulation and reduces dopaminergic inhibition of prolactin release from lactotrophs.     

Brain protein phosphatase 2A: Developmental regulation and distinct cellular and subcellular localization by B subunits

Protein phosphatase 2A (PP2A) is a heterotrimeric enzyme consisting of a catalytic subunit (C), a structural subunit (A), and a variable regulatory subunit (B). We have investigated the spatial and temporal expression patterns of three members of the B subunit family, Bα, Bβ, and Bγ, both at the message level by using ribonuclease protection analysis and at the protein level by using specific antibodies. Although A, Bα, and C protein are expressed in many tissues, Bβ and Bγ were detectable only in brain. Bα, Bβ, and Bγ are components of the brain PP2A heterotrimer, because they copurified with A and C subunits on immobilized microcystin. Whereas Bα and Bβ are mainly cytosolic, Bγ is enriched in the cytoskeletal fraction. In contrast to A, C, and Bα, which are expressed at constant levels, Bβ and Bγ RNA and protein are developmentally regulated, with Bβ levels decreasing and Bγ levels increasing sharply after birth. RNA and immunoblot analyses of subdissected brain regions as well as immunohistochemistry demonstrated that B subunits are expressed in distinct but overlapping neuronal populations and cellular domains. These data indicate that B subunits confer tissue and cell specificity, subcellular localization, and developmental regulation to the PP2A holoenzyme. The Bα-containing heterotrimer may be important in general neuronal functions that involve its partially nuclear localization. Holoenzymes containing Bβ likely function in early brain development as well as in somata and processes of subsets of mature neurons. Bγ may target PP2A to cytoskeletal substrates that are important in the establishment and maintenance of neuronal connections. J. Comp. Neurol. 392:515–527, 1998. © 1998 Wiley-Liss, Inc.     

Development of beta-adrenergic receptor and G protein messenger RNA in rat brain

The ontogeny of rat brain beta 1- and beta 2-adrenergic receptor (beta-AR) and G protein messenger RNA (mRNA) was examined by Northern blot analysis using nick-translated cDNA clones for probes. The level of beta 1-AR and beta 2-AR mRNA followed a pattern of development which paralleled that for the receptor binding sites; the levels of mRNA and binding sites were low at day 1 and increased gradually to adult levels by postnatal days 16-25. In contrast, the level of G protein mRNA, including that for Gs alpha, Gi1 alpha, Gi2 alpha, Go alpha and G beta, on postnatal day 1 was equal to or greater than adult levels, increased 40-80% between day 1 and 7 and then decreased to adult values by day 14-25. This developmental time course approximates that reported for the expression of Gs and Gi but not Go and G beta protein levels determined by immunolabeling and toxin catalyzed ADP-ribosylation. The level of beta-actin mRNA was also greater than adult levels on day 1 and then gradually decreased to adult levels by day 14-25. The results indicate that the ontogeny of beta-ARs and G proteins and the relationship between the amount of mRNA and protein are qualitatively different.     

Anterior pituitary gland synthesises dopamine from l‐3,4‐dihydroxyphenylalanine (l‐dopa)

Prolactin (PRL) is a hormone principally secreted by lactotrophs of the anterior pituitary gland. Although the synthesis and exocytosis of this hormone are mainly under the regulation of hypothalamic dopamine (DA), the possibility that the anterior pituitary synthesises this catecholamine remains unclear. The present study aimed to determine if the anterior pituitary produces DA from the precursor l ‐3,4‐dihydroxyphenylalanine (l ‐dopa). Accordingly, we investigated the expression of aromatic l ‐amino acid decarboxylase (AADC) enzyme and the transporter vesicular monoamine transporter 2 (VMAT2) in the anterior pituitary, AtT20 and GH3 cells by immunofluorescence and western blotting. Moreover, we investigated the production of DA from l ‐dopa and its release in vitro. Then, we explored the effects of l ‐dopa with respect to the secretion of PRL from anterior pituitary fragments. We observed that the anterior pituitary, AtT20 and GH3 cells express both AADC and VMAT2. Next, we detected an increase in DA content after anterior pituitary fragments were incubated with l ‐dopa. Also, the presence of l ‐dopa increased DA levels in incubation media and reduced PRL secretion. Likewise, the content of cellular DA increased after AtT20 cells were incubated with l ‐dopa. In addition, l ‐dopa reduced corticotrophin‐releasing hormone‐stimulated adrenocorticotrophic hormone release from these cells after AADC activity was inhibited by NSD‐1015. Moreover, DA formation from l ‐dopa increased apoptosis and decreased proliferation. However, in the presence of NSD‐1015, l ‐dopa decreased apoptosis and increased proliferation rates. These results suggest that the anterior pituitary synthesises DA from l ‐dopa by AADC and this catecholamine can be released from this gland contributing to the control of PRL secretion. In addition, our results suggest that l ‐dopa exerts direct actions independently from its metabolisation to DA.