Growth hormone-releasing hormone and glucocorticoids determine the balance between luteinising hormone (LH) beta- and LH beta/follicle-stimulating hormone beta-positive gonadotrophs and somatotrophs in the 14-day-old rat pituitary tissue in aggregate cell culture.

Fourteen-day-old rat pituitary tissue represents an attractive model for studying cell population dynamics, particularly of gonadotrophs. Prolonged three-dimensional culture in serum- and hormone-free medium causes a striking decline in somatotroph abundance but a several-fold rise in monohormonal LH beta-positive cell number, whereas bihormonal gonadotrophs almost disappear. In the present study, we investigated whether these changes are inter-related by examining the effects of growth hormone-releasing hormone (GHRH) and glucocorticoids, two protagonist regulators of somatotrophs. Cells were identified by single cell reverse transcriptase-polymerase chain reaction (RT-PCR) and immunofluorescence. Supplementation of the cultures for 2 weeks with GHRH (1 nm) did not augment the proportion of somatotrophs, but expanded the nonhormonal cell population. GHRH reduced the proportion of monohormonal luteinising hormone (LH)beta mRNA positive cells to approximately 50% of control, although the effect was not seen when these cells were visualised by immunostaining. Supplementation of the cultures with dexamethasone (4 nM) for 3 weeks partially rescued LH beta/follicle-stimulating hormone beta cells and fully rescued the GH mRNA cells in parallel with a decline in nonhormonal cell abundance, but strongly reduced bromodeoxyuridine labelling of GH-immunoreactive cells. As studied by patch-clamp single cell RT-PCR at the start of culture, GHRH caused an acute rise in intracellular [Ca(2+)] in some monohormonal GH cells, but at a higher incidence in cells expressing LH beta mRNA, alone or in combination with GH mRNA and/or pro-opiomelanocortin (POMC) mRNA. The present data suggest that, in the 14-day-old rat pituitary, the majority of GHRH target cells are cells expressing LH beta mRNA alone or in combination with GH and/or POMC mRNA. The data show co-regulation of gonadotroph and somatotroph population sizes by glucocorticoids and GHRH, with the former preserving bihormonal gonadotrophs and the latter repressing LH beta-only cell abundance. GHRH may not expand the somatotroph population unless glucocorticoid hormone is present to maintain terminal differentiation.

Paracrinicity: the story of 30 years of cellular pituitary crosstalk.

Living organisms represent, in essence, dynamic interactions of high complexity between membrane-separated compartments that cannot exist on their own, but reach behaviour in co-ordination. In multicellular organisms, there must be communication and co-ordination between individual cells and cell groups to achieve appropriate behaviour of the system. Depending on the mode of signal transportation and the target, intercellular communication is neuronal, hormonal, paracrine or juxtacrine. Cell signalling can also be self-targeting or autocrine. Although the notion of paracrine and autocrine signalling was already suggested more than 100 years ago, it is only during the last 30 years that these mechanisms have been characterised. In the anterior pituitary, paracrine communication and autocrine loops that operate during fetal and postnatal development in mammals and lower vertebrates have been shown in all hormonal cell types and in folliculo-stellate cells. More than 100 compounds have been identified that have, or may have, paracrine or autocrine actions. They include the neurotransmitters acetylcholine and gamma-aminobutyric acid, peptides such as vasoactive intestinal peptide, galanin, endothelins, calcitonin, neuromedin B and melanocortins, growth factors of the epidermal growth factor, fibroblast growth factor, nerve growth factor and transforming growth factor-beta families, cytokines, tissue factors such as annexin-1 and follistatin, hormones, nitric oxide, purines, retinoids and fatty acid derivatives. In addition, connective tissue cells, endothelial cells and vascular pericytes may influence paracrinicity by delivering growth factors, cytokines, heparan sulphate proteoglycans and proteases. Basement membranes may influence paracrine signalling through the binding of signalling molecules to heparan sulphate proteoglycans. Paracrine/autocrine actions are highly context-dependent. They are turned on/off when hormonal outputs need to be adapted to changing demands of the organism, such as during reproduction, stress, inflammation, starvation and circadian rhythms. Specificity and selectivity in autocrine/paracrine interactions may rely on microanatomical specialisations, functional compartmentalisation in receptor-ligand distribution and the non-equilibrium dynamics of the receptor-ligand interactions in the loops.

Stimulation of growth hormone release by 5-hydroxytryptamine (5-HT) in cultured rat anterior pituitary cell aggregates: evidence for mediation by 5-HT2B, 5-HT7, 5-HT1B, and ketanserin-sensitive receptors.

5-Hydroxytryptamine (5-HT) promotes the release of GH by a hypothalamic site of action. The present study explores a putative pituitary action in a perifused rat anterior pituitary aggregate cell culture system. In aggregates cultured with 1 nM estradiol for expression of the 5-HT4, -5, and -6 receptor (R), 5-HT promptly stimulated GH secretion with a dose dependency between 1 and 10 nM. The effect of 5-HT was partially blocked by methiothepin and methysergide; by SB-206553, a 5-HTR2B/C antagonist; SB-269970, a 5-HTR7/5A antagonist; and SB-224289, a 5-HTR1B antagonist. The GH response was fully blocked by combined administration of SB-206553+SB-269970 and SB-206553+ketanserin but not by SB-206553+spiperone. Culturing the aggregates without estradiol diminished the magnitude of the GH response to 5-HT as well as the impact of 5-HTR7/5 blockade on the response. Basal GH release was stimulated by the 5-HTR2 agonists 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane, m-chlorophenyl piperazine, and alpha-methyl 5-HT; 5-carboxytryptamine (agonist at 5-HTR1, -5, and -7); tryptamine (preferential 5-HTR7 agonist); and the selective 5-HTR1B agonist CP93129 but not the 5-HTR1A agonists 7-(dipropylamino)tetralin-1-ol-8-hydroxy-2-(di-n-propylamino)tetralin and the 5-HTR1B/D agonist sumatriptan. The selective 5-HTR2B agonist BW 723C86 stimulated GH release, and the selective 5-HTR2B antagonist SB-204741 attenuated the GH response to 5-HT. The present data suggest that 5-HT may release GH through a pituitary site of action, and that the 5-HTR2B, 5-HTR7 and 5-HTR1B mediate this response, with possibly an inhibitory component of the 5-HTR1D. The relative contribution of these receptors may be modulated by estrogen.

Estradiol induces expression of 5-hydroxytryptamine (5-HT) 4, 5-HT5, and 5-HT6 receptor messenger ribonucleic acid in rat anterior pituitary cell aggregates and allows prolactin release via the 5-HT4 receptor.

Serotonin [5-hydroxytryptamine (5-HT)] is known to control prolactin (PRL) release at a hypothalamic level, but a pituitary site of action remains poorly studied. The present study explores the acute effect of 5-HT on PRL release in rat anterior pituitary aggregate cell cultures, the influence of steroid and thyroid hormones, and the 5-HT receptor (5-HTR) subtype(s) involved. 5-HT elicited a prompt increase in basal PRL release, an effect strongly potentiated by estradiol (E(2)) in the culture medium (dose response 1-100 nm). In E(2) condition, the PRL response was not affected by the nonselective 5-HTR antagonists methysergide and methiothepin nor by 5-HTR1, 5-HTR2, 5-HTR3, 5-HTR6, and 5-HTR7/5 antagonists, but was fully blocked by the 5-HTR4 antagonist GR 113808. Among various agonist analogs, only the 5-HTR4 agonist cisapride and the 5-HTR2 agonist alpha-methyl-5-HT evoked PRL release. The effect of alpha-methyl-5-HT also required E(2) during culture and was abolished by GR 113808 but not by combined 5-HTR2A, B, and C blockade. In E(2)-treated aggregates, 5-HT caused a 5-fold increase in cAMP levels. The intact anterior pituitary expressed mRNA of all known members of the 5-HTR family. In aggregates, 5-HTR4, 5-HTR5, and 5-HTR6 mRNA expression required E(2) during culture. The effect of 5-HT on PRL release was not affected by blocking the serotonin transporter or the vesicular monoamine transporter. The present data suggest a widespread expression of 5-HTRs in the rat anterior pituitary, several of which are up-regulated by estrogen, and that, in the presence of estrogen, one of these, the 5-HTR4, mediates acute PRL release.

A Pituitary cell type coexpressing messenger ribonucleic acid of proopiomelanocortin and the glycoprotein hormone alpha-subunit in neonatal rat and chicken: rapid decline with age and reappearance in vitro under regulatory pressure of corticotropin-releasing hormone in the rat.

Promiscuous hormone mRNA expression in the pituitary remains poorly understood. We examined by means of RT-PCR and immunostaining whether glycoprotein hormone alpha-subunit (alphaGSU) could be coexpressed with proopiomelanocortin (POMC) in vivo and under pressure of CRH in vitro. Cells coexpressing alphaGSU and POMC mRNA amounted to 2.6% of the cells in ex vivo rat pituitary at birth [postnatal d 1 (P1)], fell to much lower level at P14, and were undetectable in adulthood. In cultured pituitary aggregates of P14 rats, alphaGSU/POMC cells remained scarce but represented up to 6.6% after chronic treatment with CRH but not leukemia inhibitory factor. CRH was less effective in aggregates from P1 and adult rats. The total alphaGSU population ex vivo at P1 was two times smaller than at P14, but in culture it expanded 2.5 times, concomitantly with a reciprocal change in POMC cell abundance. Tpit transcripts were detected in POMC-only and alphaGSU/POMC cells but not in alphaGSU-only cells. Cells coexpressing alphaGSU and POMC mRNA were relatively abundant in P14 chicken pituitary and aggregate cultures, but occurrence was not affected by CRH. Immunostaining showed alphaGSU and POMC colocalization in sporadic cells in intact rat pituitary and CRH-treated cultures at P1 but not at P14 and adult age. The data demonstrate the occurrence of cells coexpressing alphaGSU and POMC in rat and chicken pituitary. The developmental dynamics of this cell population and its response to CRH in vitro in the rat suggest a relationship of these cells with the embryonic branching of the POMC and alphaGSU cell lineages and their mutually opposite developmental course during early postnatal life.

Triiodothyronine expands the lactotroph and maintains the lactosomatotroph population, whereas thyrotrophin-releasing hormone augments thyrotroph abundance in aggregate cell cultures of postnatal rat pituitary gland.

In the present study, we used a three-dimensional pituitary cell culture system from early postnatal rats to examine the in vitro developmental potential of triiodothyronine (T3) and thyrotrophin-releasing hormone (TRH). Cell types were identified at the hormone mRNA level by single-cell reverse transcription-polymerase chain reaction and any change in abundance was further examined by immunofluorescence staining of the corresponding hormone protein. In aggregates from 14-day-old rats, long-term (12-16 days) treatment with T3 (0.5 nM) increased the abundance of cells expressing prolactin mRNA (PRLmRNA cells) by 2.5-fold and lowered that of nonhormonal cells and thyroid-stimulating hormone beta (TSHbeta)mRNA cells. The abundance of growth hormone (GH)mRNA cells decreased during culture compared to that in the freshly dispersed pituitary gland and T3 did not significantly affect this cell population. Cells coexpressing PRL mRNA and GH mRNA virtually disappeared during culture but reappeared in the presence of T3. T3 increased the abundance of PRL-immunoreactive (ir) cells in aggregates from 14-day-old rats, as well as in aggregates from newborn and 1-week-old rats. As estimated by bromodeoxyuridine (BrdU) labelling, a 3-day treatment with T3 enhanced the number of PRL-ir cells that had incorporated BrdU, but did not yet expand the total population of PRL-ir cells. Long-term treatment with TRH (100 nM) did not affect the proportion of PRLmRNA or GHmRNA cells, but consistently increased the proportional number of TSHbeta(mRNA) and TSHbeta-ir cells. The present data confirm the findings obtained in recent in vivo loss of function genetic studies suggesting that T3 plays a prominent role in postnatal expansion of the lactotroph population and that TRH is important for thyrotroph development. The data suggest that the effect of T3 is brought about by a direct action on the pituitary gland through a cell proliferation mechanism. T3 also appears to support the lactosomatotroph population. In view of the established theory that lactotrophs develop from GH-expressing progenitor cells and that this is a post mitotic event, we propose that T3 is mitogenic for GHmRNA cells that lack GH-ir material and that transdifferentiate into PRL-ir cells, but that a pathway of PRL cell development from mitotic nonhormonal cell progenitors may also be involved.

The chicken pituitary-specific transcription factor PIT-1 is involved in the hypothalamic regulation of pituitary hormones.

Pit-1 is a pituitary-specific POU-domain DNA binding factor, which binds to and trans-activates promoters of growth hormone- (GH), prolactin- (PRL) and thyroid stimulating hormone-beta- (TSHbeta) encoding genes. Thyrotropin-releasing hormone (TRH) is located in the hypothalamus and stimulates TSH, GH and PRL release from the pituitary gland. In the present study, we successfully used the cell aggregate culture system for chicken pituitary cells to study the effect of TRH administration on the ggPit-l* (chicken Pit-1), GH and TSHbeta mRNA expression in vitro. In pituitary cell aggregates of 11-day-old male broiler chicks the ggPit-l * mRNA expression was significantly increased following TRH administration, indicating that the stimulatory effects of TRH on several pituitary hormones are mediated via its effect on the ggPit-l* gene expression. Therefore, a semiquantitative RT-PCR method was used to detect possible changes in GH and TSHbeta mRNA levels. TRH affected both the GH and TSHbeta mRNA levels. The results of this in vitro study reveal that ggPit-1 * has a role in mediating the stimulatory effects of TRH on pituitary hormones like GH and TSHbeta in the chicken pituitary.

Stimulation and inhibition of prolactin release by prolactin-releasing Peptide in rat anterior pituitary cell aggregates.

Although the G-protein coupled receptor GPR10 is highly expressed in the anterior pituitary, the action of its ligand prolactin-releasing peptide-31 (PrRP) in this tissue is controversial. The present study examined the acute effect of this peptide on prolactin secretion in perifused rat pituitary reaggregate cell cultures from adult male rats. PrRP readily and dose-dependently stimulated prolactin release at concentrations of 10 and 100 nM, although with a magnitude several times lower than that of thyrotropin-releasing hormone. Surprisingly, PrRP inhibited prolactin release at 0.1 and 1 nm in a pertussis toxin-sensitive manner. Inhibition was markedly favoured by long-term culture. Stimulation and inhibition were differentially affected by the presence of hormones during culture: dexamethasone favoured the inhibitory effect and decreased the magnitude of the stimulatory effect, while oestradiol and triiodothyronine strongly reduced stimulation, as well as inhibition. PrRP, even at 1 nm, counteracted the inhibition of prolactin release by dopamine. There was no effect of PrRP on growth hormone release in aggregates cultured either in the absence or presence of hormones. The present results confirm the prolactin-releasing capacity of PrRP at nanomolar doses and reveal a hitherto unrecognized inhibitory activity of this peptide. Furthermore, dopamine inhibition of prolactin release is antagonized by PrRP, irrespective of the PrRP dose.

Combinatorial expression of phenotypes of different cell lineages in the rat and mouse pituitary.

As studied by single cell RT-PCR of pituitary hormones, we demonstrated that the pituitaries of rats and mice contain a subpopulation of cells that express two or more hormone phenotypes typically belonging to lineages that are branched separately early during embryonic development, such as glycoprotein hormone alpha-subunit (alphaGSU) mRNA + PRL mRNA, alphaGSU mRNA + POMC mRNA, and POMC mRNA + GH or PRL mRNA. GnRH in vitro selectively expands the population of cells coexpressing alphaGSU mRNA + PRL mRNA, and CRH selectively increases the proportion of cells coexpressing alphaGSU mRNA + POMC mRNA. Colocalization of alphaGSU + PRL or alphaGSU + POMC could not be detected by double immunofluorescence. This lineage promiscuity was also observed in the pituitary in vivo.

Melanocortin peptides stimulate prolactin gene expression and prolactin accumulation in rat pituitary aggregate cell cultures.

Treatment for 40 h of reaggregate pituitary cell cultures from 14-day-old female rats with nanomolar concentrations of gamma3-melanocyte-stimulating hormone (MSH) increased prolactin mRNA but not growth hormone (GH) mRNA expression levels as measured by quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR). During the 40 h incubation, gamma3-MSH stimulated prolactin accumulation in the culture medium. alpha-MSH, a potent agonist of the rat melanocortin-3 receptor (MC3R) and Ala(8)-gamma2-MSH, a very weak agonist of the MC3R, increased prolactin mRNA expression at a similar concentration range as gamma3-MSH. The effect of gamma3-MSH on prolactin mRNA expression was abolished when aggregates were cultured in the presence of thyroid or glucocorticoid hormones, but not of oestradiol. By contrast, oestradiol abolished the stimulatory effect of Ala(8)-gamma2-MSH on prolactin mRNA expression. In GH3 cells stably transfected with the enhanced green fluorescent protein (eGFP) gene under control of a 3-kb prolactin promoter fragment, a dose as low as 1 nMgamma3-MSH, added for 24 h, significantly increased eGFP fluorescence. Agouti-related protein (AgRP(83-132)), a known endogenous MC3R and MC4R antagonist, did not reduce the stimulation of prolactin mRNA expression by gamma3-MSH or Ala(8)-gamma2-MSH. On its own, AgRP(83-132) significantly increased prolactin mRNA expression level and prolactin accumulation. Both gamma2-MSH and Ala(8)-gamma2-MSH increased [S(35)]GTPgammaS binding in membrane preparations of 14-day-old rat pituitaries and of GH3 cells. Whereas MC3R and MC5R mRNA were detectable by RT-PCR in normal pituitary, these receptor mRNAs were undetectable in GH3 cells using various oligonucleotide primer sets. The present findings indicate that melanocortin peptides stimulate prolactin gene expression and production and that, at least in part, a receptor different from the classic MCR is involved. AgRP appears to have other actions than its known antagonistic activity on the MC3R and MC4R.

Gamma-MSH peptides in the pituitary: effects, target cells, and receptors.

The melanocortin (MC) gamma3-MSH is believed to signal through the MC3 receptor. We showed that it induces a sustained increase in intracellular free calcium levels ([Ca(2+)](i)) in a subpopulation of pituitary cells. Most of the cells responding to gamma3-MSH express more than one pituitary hormone mRNA. The effect of gamma3-MSH is blocked by SHU9119, a MC3R and MC4R antagonist, in only 50% of the responsive cells, suggesting that in half of these cells the mediating receptor is not the MC3R. Low picomolar doses of gamma3-MSH increase [Ca(2+)](i) in the growth hormone (GH)- and prolactin (PRL)-secreting GH3 cell line. gamma2-MSH and alpha-MSH display a similar effect. SHU9119 does not affect the gamma3-MSH-induced [Ca(2+)](i) response. MTII, a potent synthetic agonist of the MC3R, MC4R, and MC5R, also shows no or low potency in increasing [Ca(2+)](i). By means of RT-PCR, the mRNA of the MC2R, MC3R, and MC4R receptors is undetectable. Experiments testing gamma2-MSH analogues with single alanine replacements show that, unlike the classic MCRs, the His(5)-Phe(6)-Arg(7)-Trp(8) sequence in gamma2-MSH is not a core sequence for activating the gamma-MSH receptor in GH3 cells, whereas Met(3) is essential. Low nanomolar doses of gamma-MSH increase intracellular cAMP levels. Blockade of protein kinase A abolishes the [Ca(2+)](i) responses to gamma3-MSH. gamma2-MSH increases binding of [S(35)]GTPgammaS to membrane preparations of GH3 cells. The pharmacological characteristics of gamma-MSH peptides and analogues on [Ca(2+)](i) and the signal-transduction pathways present strong evidence for the expression of a hitherto uncharacterized gamma-MSH receptor in GH3 cells, belonging to the G protein-coupled receptor family.

Stimulation of combinatorial expression of prolactin and glycoprotein hormone alpha-subunit genes by gonadotropin-releasing hormone and estradiol-17beta in single rat pituitary cells during aggregate cell culture.

Previously we showed the existence of rat and mouse anterior pituitary cells coexpressing mRNA from two or more hormone genes in which production and/or storage of the corresponding hormones were not detectable. To substantiate a putative function for these cells, we investigated whether these phenotypes were retained during long-term reaggregate cell culture and whether protagonist regulatory factors could expand cell populations expressing particular hormone mRNA combinations. After 4-wk culture and treatments, aggregates were trypsinized and single cells collected by means of a fluo-rescence-activated cell sorter. Hormone mRNAs were detected by single-cell RT-PCR. Combinatorial hormone mRNA expression was retained in culture. Both estradiol (E2) and GnRH (1 nM) markedly augmented the proportion of cells expressing prolactin (PRL) mRNA together with other hormone mRNAs and cells expressing glycoprotein subunit (GSU)-alpha mRNA together with other hormone mRNAs. GnRH strongly increased the proportion of cells containing alphaGSU mRNA alone, but E2 did not. GnRH and (E2) affected the expansion of a population (approximately 20% of all cells) coexpressing PRL and alphaGSU mRNA without betaGSUs. Immunostaining of stored hormone on tissue sections revealed colocalization of PRL and alphaGSU in the E2- but not in the GnRH-treated cells. The present findings suggest that cells coexpressing different pituitary hormone mRNAs form a distinct population that survives without extrapituitary factors. Their occurrence can be markedly modified by regulatory factors. Certain hormone regimens favor unique coexpressions distinctly at mRNA and protein level. These peculiar characteristics support the notion that combinatorial expression of hormone genes in the pituitary serves a biological role.

Isolation and structure-bioactivity characterization of glycosylated N-pro-opiomelanocortin isoforms.

The N-terminal fragment of mouse pro-opiomelanocortin (N-POMC) was isolated from AtT-20 cell-conditioned medium on the basis of immunoreactivity to an anti-POMC1-50 monoclonal antibody by a concentration step, a cation exchange step, reversed phase high-performance liquid chromatography (HPLC) and size exclusion HPLC. Two groups of N-POMC isoforms with a molecular weight (MW) of approximately 11 kDa and 13 kDa, respectively, were identified by mass spectrometry and N-terminal amino acid sequencing. C-terminal sequencing indicated that 11 kDa isoforms correspond to POMC1-74 and 13 kDa isoforms to POMC1-95. Isoforms from both groups enhanced the prolactin mRNA content (measured by means of TaqMan real-time reverse transcription-polymerase chain reaction) in cultured rat pituitary cell aggregates in a dose-dependent manner, but not all of them showed this activity. POMC1-74 compounds were significantly more potent than POMC1-95 isoforms. The observed effects were abolished by coincubation with the monoclonal anti-POMC1-50 antibody, showing the specificity of this biological action. Incorporation of bromodeoxyuridine into DNA of immunostained lactotrophs was enhanced by only a minor part of the isoforms. Some of these had no effect on prolactin mRNA expression. The N-POMC isoforms appeared to be N- and at least in part O-glycosylated. After enzymatic N-deglycosylation of selected N-POMC isoforms, the stimulatory effect on the prolactin mRNA level was depressed (in case of the POMC1-95 isoforms) or totally abolished (in case of the POMC1-74 isoforms). The present findings show that N-POMC is a mixture of differentially glycosylated isoforms, that the isoforms of POMC1-74 are the biologically more effective forms and that different isoforms induce different biological responses in the same cell population. The data also show the essential role of N-glycosylation in the biological response.

Ontogeny of plurihormonal cells in the anterior pituitary of the mouse, as studied by means of hormone mRNA detection in single cells.

The expression of mRNA of growth hormone (GH), prolactin (PRL), pro-opiomelanocortin (POMC) and the common glycoprotein hormone alpha-subunit (alphaGSU) was studied by means of single cell reverse transcriptase-polymerase chain reaction in male mouse pituitary cells at key time points of fetal and postnatal development: embryonic day 16 (E16); postnatal day 1 (P1) and young-adult age (P38). At E16, the hormone mRNAs examined were detectable, although only in 44% of total cells. Most of the hormone-positive cells expressed only one of the tested hormone mRNAs (monohormonal) but 14% of them contained more than one hormone mRNA (plurihormonal cells). Combinations of GH mRNA with PRL mRNA, of alphaGSU mRNA with GH and/or PRL mRNA and of POMC mRNA with GH and/or PRL mRNA or alphaGSU mRNA were found. As expected, the proportion of hormone-positive cells rose as the mouse aged. The proportions of plurihormonal cells followed a developmental pattern independent of that of monohormonal cells and characteristic for each hormone mRNA examined. Cells coexpressing POMC mRNA with GH or PRL mRNA significantly rose in proportion between E16 and P1, while the proportion of cells coexpressing GH and PRL mRNA markedly increased between P1 and P38. The occurrence of cells displaying combined expression of alphaGSU mRNA with GH and/or PRL mRNA did not significantly change during development. Remarkably, the population of cells expressing PRL mRNA only, was larger at E16 than at P1 and expanded again thereafter. In conclusion, the normal mouse pituitary develops a cell population that is capable of expressing multiple hormone mRNAs, thereby combining typical phenotypes of different cell lineages. These plurihormonal cells are already present during embryonic life. This population is of potential physiological relevance because development-related factors appear to determine which hormone mRNAs are preferentially coexpressed. Coexpression of multiple hormone mRNAs may represent a mechanism to respond to temporally increased endocrine demands. The data also suggest that the control of combined hormone expression is different from that of single hormone expression, raising questions about the current view on pituitary cell lineage specifications.

Effects of melanocortins on cardiovascular regulation in rats.

1. Of the melanocortin peptides, gamma(2)-melanocyte-stimulating hormone (MSH) has been attributed a cardiovascular effect, inducing an increase in blood pressure and heart rate. Although still controversial, this effect, based on pharmacological blockade experiments, is supposed to be mediated through sympathetic activation. 2. The aims of the present study were to identify the N-terminal pro-opiomelanocortin (N-POMC) fragments and melanocortins that influence blood pressure and heart rate and to investigate the real-time changes in baroreflex sensitivity and in sympathetic and vagal modulation underlying cardiovascular effects in conscious rats without the use of pharmacological blockade. 3. Intracerebroventricular administration of different melanocortins and N-POMC induced a long-lasting dose- dependent pressor response from 1 nmol onwards, with only a small initial bradycardic response with the highest dose. 4. Coinciding with this pressor response, an elicitation of the low-frequency (LF) component was observed in spectral analysis of both blood pressure variability (BPV) and heart rate variability (HRV), followed by the high-frequency (HF) component in at least BPV. Baroreflex sensitivity remained unchanged. 5. After intravenous administration, gamma(2)-MSH produced a short-lasting dose-dependent pressor and cardioaccelerator response with very rapid onset with concentrations from 1 nmol onwards. 6. Continuous infusion of gamma(2)-MSH depressed baroreflex sensitivity and simultaneously increased both components of BPV, with a radical reduction of the LF component and a preserved vagal HF component in HRV. 7. Of all the intravenously administered melanocortins, only gamma(2)-MSH was active. The central effect is likely to depend on an increase of (alpha-)sympathetic outflow. 8. For the peripheral effect, gamma(2)-MSH appeared to act as a baroreceptor reflex-blocking agent, being compatible with a role in the acute stress response.

Combined expression of different hormone genes in single cells of normal rat and mouse pituitary.

Cells displaying combined expression of different pituitary hormone genes (further referred to as 'multi-hormone mRNA cells') were identified in normal rat and mouse pituitary by single cell RT-PCR. These cells do not seem to produce or store all the respective hormones the mRNAs encode for. The cells are already developed at day 16 of embryonic life (E16) in the mouse. Different peptides, such as gamma3-melanocyte-stimulating hormone (gamma3-MSH) and gonadotropin-releasing hormone (GnRH), affect different subsets of these cells. In culture, estrogen and GnRH increase the number of 'multi-hormone mRNA cells' that contain prolactin (PRL) mRNA or mRNA of the alpha-subunit of the glycoprotein hormones (alpha-GSU) but not the number of 'multi-hormone mRNA cells' not containing PRL or alpha-GSU mRNA. 'Multi-hormone mRNA cells' may function as 'reserve cells' in which a particular hormone mRNA may be translated under a particular physiological condition demanding a rapid increase of that hormone.

Alpha B-crystallin expression in human and rat hepatic stellate cells.

BACKGROUND/AIMS: We searched for factors implicated in early hepatic stellate cell (HSC) activation in diseased liver, by means of suppression subtractive hybridization (SSH). METHODS: SSH was performed between messenger RNA (mRNA) from normal rat HSC and mRNA from HSC, isolated from rats with acute D-galactosamine (Gal)-induced hepatitis. RESULTS: One of the potentially upregulated factors which we found was alpha B-crystallin (ABCRYS), a small heat-shock protein and a chaperone known to protect the cell against protein degradation in conditions of cellular stress and known to associate with various types of intermediate filaments. Upregulation of ABCRYS mRNA in HSC, following Gal-intoxication (3.5-fold) as well as by culturing the HSC on plastic (20-30-fold), was confirmed by quantitative real-time reverse transcription polymerase chain reaction. The expression of ABCRYS protein in human and rat HSC was demonstrated by immunohistochemistry, in vitro and in vivo, in normal and diseased liver. Double-staining co-localized ABCRYS immunoreactivity with alpha-smooth muscle actin immunoreactivity in human liver and with desmin immunoreactivity in rat liver. In vivo upregulation of ABCRYS protein following Gal-intoxication was also shown, by comparison with desmin expression. CONCLUSIONS: Human and rat HSC express ABCRYS mRNA and protein. Both are rapidly upregulated following HSC activation.

Growth and differentiation factors derived from the N-terminal domain of pro-opiomelanocortin.

1. We describe a novel paracrine control system in the pituitary gland, consisting of peptides derived from the N-terminal fragment of pro-opiomelanocortin (N-POMC), for example POMC(1-74) and gamma3-melanocyte-stimulating hormone (MSH). 2. By searching the target cells of these N-POMC fragments, using the rise of intracellular free calcium as a response system and single cell reverse transcription-polymerase chain reaction of hormone mRNA as a cell type identification method, we found that a considerable number of cells in normal rat pituitary display combinatorial expression of different pituitary hormone genes (further referred to as 'multihormone mRNA cells'), without indication that all these cells also produce or store the respective hormones translatable from these mRNA. The N-POMC fragments POMC(1-74) and gamma3-MSH preferentially target particular subsets of these multihormone mRNA cells. 3. We discovered a potentially novel receptor for gamma3-MSH on these cells; more precisely, on cells coexpressing growth hormone and prolactin. The putative novel receptor displays properties highly divergent from those of the known gamma3-MSH receptor (i.e. the melanocortin-3 receptor) and even of all other melanocortin receptors cloned today.

Stimulation of intracellular free calcium in GH3 cells by gamma3-melanocyte-stimulating hormone. Involvement of a novel melanocortin receptor?

The melanocortin (MC) gamma3MSH is a peptide that can be generated from the N-terminal domain of POMC and is believed to signal through the MC3 receptor. We recently showed that it induces a sustained rise in intracellular free calcium levels ([Ca(2+)](i)) in a subpopulation of pituitary cells, particularly in the lactosomatotroph lineage. In the present study we report that gamma3MSH and some analogs increase [Ca(2+)](i) in the GH- and PRL-secreting GH3 cell line and evaluate on the basis of pharmacological experiments and gene expression studies which MC receptor may be involved. A dose as low as 1 pM gamma3MSH induced an oscillating [Ca(2+)](i) increase in a significant percentage of GH3 cells. Increasing the dose recruited an increasing number of responding cells; a maximum was reached at 0.1 nM. gamma2MSH, alphaMSH, and NDP-alphaMSH displayed a similar effect. SHU9119, an MC3 and MC4 receptor antagonist, and an MC5 receptor agonist, did not affect the number of cells showing a [Ca(2+)](i) rise in response to gamma3MSH. SHU9119 had also no effect when added alone. MTII, a potent synthetic agonist of the MC3, MC4, and MC5 receptor as well as an N-terminally extended recombinant analog of gamma3MSH showed low potency in increasing [Ca(2+)](i) in GH3 cells, but high potency in stimulating cAMP accumulation in HEK 293 cells stably transfected with the MC3 receptor. In contrast, a peptide corresponding to the gamma2MSH sequence of POMC-A of Acipenser transmontanus increased [Ca(2+)](i) in GH3 cells, but was about 50 times less potent than gamma2- or gamma3MSH in stimulating cAMP accumulation in the MC3 receptor expressing HEK 293 cells. By means of RT-PCR performed on a RNA extract from GH3 cells, the messenger RNA of the MC2, MC3, and MC4 receptor was undetectable, but messenger RNA of the MC5 receptor was clearly present. These data suggest that the GH3 cell line does not mediate the effect of gamma3MSH through the MC3 receptor. The involvement of the MC5 receptor is unlikely, but cannot definitely be excluded. The findings animate the hypothesis that there exists a second, hitherto unidentified, MC receptor that displays high affinity for gamma3MSH.

Human and rat hepatic stellate cells express neurotrophins and neurotrophin receptors.

The expression of neurotrophins and neurotrophin receptors in non-neural tissue is related to tissue remodeling, differentiation, proliferation and migration of target cells. The literature yields contradictory results on neurotrophin and neurotrophin receptor expression in the liver. We show immunoreactivity to antibodies to nerve growth factor (NGF), brain-derived neurotrophin (BDNF), neurotrophin 3 (NT-3), neurotrophin 4/5 (NT-4/5), the low-affinity nerve growth factor receptor p75 and the high-affinity tyrosine kinase receptors (Trk) B and C in hepatic stellate cells and weak reactivity for BDNF, NT-3, and NT-4/5 in hepatocytes, in cryosections of human and rat liver, in normal and varying pathologic conditions. Immunoreactivity is unequivocally localized to hepatic stellate cells by double staining with alpha-smooth muscle actin (alpha-SMA) and desmin, studied by confocal laser scanning microscopy. Finally, the presence of mRNA transcripts for the different neurotrophins and neurotrophin receptors, with the exception of Trk-B, is shown by reverse transcription polymerase chain reaction (RT-PCR) on RNA extracted from freshly isolated rat hepatic stellate cells, compared with hepatocyte RNA. Hepatocyte RNA was found to contain BDNF, NT-3, NT-4/5 mRNA (which is compatible with the immunohistochemical findings) and Trk-A mRNA. In conclusion, hepatic stellate cells are a source of several neurotrophins in the liver and they express neurotrophin receptors. These findings correspond with the known involvement of hepatic stellate cells in tissue remodeling, their production of extracellular matrix components and their proliferation in acute necrotizing liver pathology. In analogy with findings in other organs and systems, neurotrophins are hypothesized to play a role in the pathophysiology of liver disease.