Leukemia inhibitory factor induces differentiation of pituitary corticotroph function: an immuno-neuroendocrine phenotypic switch.

Leukemia inhibitory factor (LIF) promotes differentiated cell function in several systems. We recently reported LIF and LIF receptor expression in human fetal pituitary corticotrophs in vivo and demonstrated LIF stimulation of adrenocorticotrophin (ACTH) transcription in vitro, suggesting a role for LIF in corticotroph development. We therefore assessed the action of LIF on proliferating murine corticotroph cells (AtT20). LIF impairs proliferation of AtT20 cells (25% reduction versus control, P < 0.03), while simultaneously enhancing ACTH secretion (2-fold, P < 0.001) and augmenting ACTH responsiveness to corticotrophin-releasing hormone (CRH) action (4-fold, P < 0.001). This attenuation of cell growth is due to a block of cell cycle progression from G1 into S phase, as measured by flow cytometric analysis (24 +/- 0.8 versus 11.57 +/- 1.5, P < 0.001). Using bromodeoxyuridine incorporation assays, loss of cells in S phase was confirmed (25 +/- 0.08 to 9.4 +/- 1.4, P < 0.008). In contrast, CRH induced the G2/M phase (3.6 +/- 0.2 to 15.4 +/- 3, P < 0.001). This effect was blunted by LIF (P < 0.001 versus CRH alone). Cyclin A mRNA levels, which decline in S phase, were stimulated 3.5-fold by LIF and markedly suppressed by CRH. These results indicate a LIF-induced cell cycle block occurring at G1/S in corticotroph cells. Thus, LIF reduces proliferation, enhances ACTH secretion, and potentiates effects of CRH on ACTH secretion while blocking effects of CRH on the cell cycle. Responses of these three markers of differentiated corticotroph function indicate LIF to be a differentiation factor for pituitary corticotroph cells by preferential phenotypic switching from proliferative to synthetic.

Quantal release at a neuronal nicotinic synapse from rat adrenal gland.

The neuronal nicotinic synapse in tissue slices of the adrenal medulla was studied with whole-cell patch-clamp. Excitatory postsynaptic currents (EPSCs) were evoked by local field stimulation or occurred spontaneously especially when external [K+] was increased. EPSCs were carried by channels sharing biophysical and pharmacological properties of neuronal-type nicotinic receptors (nAChRs). A single-channel conductance (gamma) of 43-45 pS was found from nonstationary variance analysis of EPSCs. Spontaneous EPSCs were tetrodotoxin-insensitive and Ca(2+)-dependent and occurred in burst-like clusters. Quantal analysis of spontaneous EPSCs gave a quantal size of 20 pA and amplitude histograms were well described by binomial models with low values of quantal content, consistent with a small number of spontaneously active release sites. However, rare large amplitude EPSCs suggest that the total number of sites is higher and that extrajunctional receptors are involved. Our estimates of quantal content and size at the chromaffin cell neuronal nicotinic synapse may be useful in characterizing central neuronal-type nicotinic receptor-mediated cholinergic synaptic transmission.

Overview of the most prevalent hypothalamus-specific mRNAs, as identified by directional tag PCR subtraction.

We applied the directional tag PCR subtractive hybridization method to construct a rat hypothalamic cDNA library from which cerebellar and hippocampal sequences had been depleted, enriching 20-30-fold for sequences expressed selectively in the hypothalamus. We studied a sample of 94 clones selected for enrichment in the subtracted library. These clones corresponded to 43 distinct mRNA species, about half of which were novel. Thirty-eight of these 43 mRNAs (corresponding to 85 of the clones in the sample) exhibited enrichment in the hypothalamus; 23 were highly enriched. In situ hybridization studies revealed that one novel species was restricted to cells in a small bilaterally symmetric area of the paraventricular hypothalamus. Other novel mRNAs showed substantial enrichment in basal diencephalic structures, particularly the hypothalamus, without restriction to single hypothalamic nuclei. The data suggest that the hypothalamus utilizes at least two distinct strategies for employing its selectively expressed proteins. Secretory neuropeptides utilized for intercellular communication are produced by functionally discrete nuclei, while several other proteins are shared by structures that are unrelated in their physiological roles but may share biochemical systems.

P-OTX: a PIT-1-interacting homeodomain factor expressed during anterior pituitary gland development.

A novel OTX-related homeodomain transcription factor has been identified on the basis of its ability to interact with the transactivation domain of the pituitary-specific POU domain protein, Pit-1. This factor, referred to as P-OTX (pituitary OTX-related factor), is expressed in primordial Rathke's pouch, oral epithelium, first bronchial arch, duodenum, and hindlimb. In the developing anterior pituitary, it is expressed in all regions from which cells with distinct phenotypes will emerge in the mature gland. P-OTX is able to independently activate and to synergize with Pit-1 on pituitary-specific target gene promoters. Therefore, P-OTX may subserve functions in generating both precursor and specific cell phenotypes in the anterior pituitary gland and in several other organs.

Differential distribution of two ATP-gated channels (P2X receptors) determined by immunocytochemistry.

Several P2X receptor subunits were recently cloned; of these, one was cloned from the rat vas deferens (P2X1) and another from pheochromocytoma (PC12) cells differentiated with nerve growth factor (P2X2). Peptides corresponding to the C-terminal portions of the predicted receptor proteins (P2X1 391-399 and P2X2 460-472) were used to generate antisera in rabbits. The specificities of antisera were determined by staining human embryonic kidney cells stably transfected with either P2X1 or P2X2 receptors and by absorption controls with the cognate peptides. In the vas deferens and the ileal submucosa, P2X1 immunoreactivity (ir) was restricted to smooth muscle, whereas P2X2-ir was restricted to neurons and their processes. Chromaffin cells of the adrenal medulla and PC12 cells contained both P2X1- and P2X2-ir. P2X1-ir was also found in smooth muscle cells of the bladder, cardiac myocytes, and nerve fibers and terminals in the superficial dorsal horn of the spinal cord. In contrast, P2X2-ir was observed in scattered cells of the anterior pituitary, neurons in the hypothalamic arcuate and paraventricular nuclei, and catecholaminergic neurons in the olfactory bulb, the substantia nigra, ventral tegmental area, and locus coeruleus. A plexus of nerve fibers and terminals in the nucleus of the solitary tract contained P2X2-ir. This staining disappeared after nodose ganglionectomy, consistent with a presynaptic function. The location of the P2X1 subunit in smooth muscle is consistent with its role as a postjunctional receptor in autonomic transmission, while in neurons, these receptors appear in both postsynaptic and presynaptic locations.

Crystal structure at 2.6-A resolution of human macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF) was the first cytokine to be described, but for 30 years its role in the immune response remained enigmatic. In recent studies, MIF has been found to be a novel pituitary hormone and the first protein identified to be released from immune cells on glucocorticoid stimulation. Once secreted, MIF counterregulates the immunosuppressive effects of steroids and thus acts as a critical component of the immune system to control both local and systemic immune responses. We report herein the x-ray crystal structure of human MIF to 2.6 angstrom resolution. The protein is a trimer of identical subunits. Each monomer contains two antiparallel alpha-helices that pack against a four-stranded beta-sheet. The monomer has an additional two beta-strands that interact with the beta-sheets of adjacent subunits to form the interface between monomers. The three beta-sheets are arranged to form a barrel containing a solvent-accessible channel that runs through the center of the protein along a molecular 3-fold axis. Electrostatic potential maps reveal that the channel has a positive potential, suggesting that it binds negatively charged molecules. The elucidated structure for MIF is unique among cytokines or hormonal mediators, and suggests that this counterregulator of glucocorticoid action participates in novel ligand-receptor interactions.

Nerve growth factor in the anterior pituitary: localization in mammotroph cells and cosecretion with prolactin by a dopamine-regulated mechanism.

Nerve growth factor (NGF) is well characterized for its neurotrophic actions on peripheral sensory and sympathetic neurons and on central cholinergic neurons of the basal forebrain. Recent evidence, however, has shown high levels of NGF to be present in a variety of biological fluids after inflammatory and autoimmune responses, suggesting that NGF is a mediator of immune interactions. Increased NGF serum levels have been reported in both humans and experimental animal models of psychological and physical stress, thus implicating NGF in neuroendocrine interactions as well. The possible source(s) and the regulatory mechanisms involved in the control of serum NGF levels, however, still remain to be elucidated. We now report the presence of both NGF gene transcripts and protein in the anterior pituitary. Immunofluorescence analysis indicated that hypophysial NGF is selectively localized in mammotroph cells and stored in secretory granules. NGF is cosecreted with prolactin from mammotroph cells by a neurotransmitter-dependent mechanism that can be pharmacologically regulated. Activation of the dopamine D2 receptor subtype, which physiologically controls prolactin release, resulted in a complete inhibition of vasoactive intestinal peptide-stimulated NGF secretion in vitro, whereas the specific D2 antagonist (-)-sulpiride stimulated NGF secretion in vivo, suggesting that the anterior pituitary is a possible source of circulating NGF. Given the increased NGF serum levels in stressful conditions and the newly recognized immunoregulatory function of this protein, NGF, together with prolactin, may thus be envisaged as an immunological alerting signal under neuronal control.

Down-regulation of pituitary receptors for luteinizing hormone-releasing hormone (LH-RH) in rats by LH-RH antagonist Cetrorelix.

Antagonists of luteinizing hormone-releasing hormone (LH-RH), unlike the LH-RH agonists, suppress gonadotropins and sex steroid secretion immediately after administration, without initial stimulatory effects. [Ac-D-Nal(2)1,D-Ph(4Cl)2,D-Pal(3)3,D-Cit6,D-Ala10]LH-R H (SB-75; Cetrorelix) is a modern, potent antagonistic analog of LH-RH. In this study, the binding characteristics of receptors for LH-RH in membrane fractions from rat anterior pituitaries were investigated after a single injection of Cetrorelix at a dose of 100 microg per rat. To determine whether the treatment with Cetrorelix can affect the concentration of measurable LH-RH binding sites, we applied an in vitro method to desaturate LH-RH receptors by chaotropic agents such as manganous chloride (MnCl2) and ammonium thiocyanate (NH4SCN). Our results show that the percentages of occupied LH-RH receptors at 1, 3, and 6 h after administration of Cetrorelix were approximately 28%, 14%, and 10%, respectively, of total receptors. At later time intervals, we could not detect occupied LH-RH binding sites. Ligand competition assays, following in vitro desaturation, demonstrated that rat pituitary LH-RH receptors were significantly (P < 0.01) down-regulated for at least 72 h after administration of Cetrorelix. The lowest receptor concentration was found 3-6 h after Cetrorelix treatment and a recovery in receptor number began within approximately 24 h. The down-regulation of LH-RH binding sites induced by Cetrorelix was accompanied by serum LH and testosterone suppression. Higher LH-RH receptor concentrations coincided with elevated serum hormone levels at later time intervals. Our results indicate that administration of LH-RH antagonist Cetrorelix produces a marked down-regulation of pituitary receptors for LH-RH and not merely an occupancy of binding sites.

Nitric oxide inhibits the release of norepinephrine and dopamine from the medial basal hypothalamus of the rat.

Previous research indicates that norepinephrine and dopamine stimulate release of luteinizing hormone (LH)-releasing hormone (LHRH), which then reaches the adenohypophysis via the hypophyseal portal vessels to release LH. Norepinephrine exerts its effect via alpha 1-adrenergic receptors, which stimulate the release of nitric oxide (NO) from nitricoxidergic (NOergic) neurons in the medial basal hypothalamus (MBH). The NO activates guanylate cyclase and cyclooxygenase, thereby inducing release of LHRH into the hypophyseal portal vessels. We tested the hypothesis that these two catecholamines modulate NO release by local feedback. MBH explants were incubated in the presence of sodium nitroprusside (NP), a releaser of NO, and the effect on release of catecholamines was determined. NP inhibited release of norepinephrine. Basal release was increased by incubation of the tissue with the NO scavenger hemoglobin (20 micrograms/ml). Hemoglobin also blocked the inhibitory effect of NP. In the presence of high-potassium (40 mM) medium to depolarize cell membranes, norepinephrine release was increased by a factor of 3, and this was significantly inhibited by NP. Hemoglobin again produced a further increase in norepinephrine release and also blocked the action of NP. When constitutive NO synthase was inhibited by the competitive inhibitor NG-monomethyl-L-arginine (NMMA) at 300 microM, basal release of norepinephrine was increased, as was potassium-evoked release, and this was associated in the latter instance with a decrease in tissue concentration, presumably because synthesis did not keep up with the increased release in the presence of NMMA. The results were very similar with dopamine, except that reduction of potassium-evoked dopamine release by NP was not significant. However, the increase following incubation with hemoglobin was significant, and hemoglobin, when incubated with NP, caused a significant elevation in dopamine release above that with NP alone. In this case, NP increased tissue concentration of dopamine along with inhibiting release, suggesting that synthesis continued, thereby raising the tissue concentration in the face of diminished release. When the tissue was incubated with NP plus hemoglobin, which caused an increase in release above that obtained with NP alone, the tissue concentration decreased significantly compared with that in the absence of hemoglobin, indicating that, with increased release, release exceeded synthesis, causing a fall in tissue concentration. When NO synthase was blocked by NMMA, the release of dopamine, under either basal or potassium-evoked conditions, was increased. Again, in the latter instance the tissue concentration declined significantly, presumably because synthesis did not match release. Therefore, the results were very similar with both catecholamines and indicate that NO acts to suppress release of both amines. Since both catecholamines activate the release of LHRH, the inhibition of their release by NO serves as an ultra-short-loop negative feedback by which NO inhibits the release of the catecholamines, thereby reducing the activation of the NOergic neurons and decreasing the release of LHRH. This may be an important means for terminating the pulses of release of LHRH, which generate the pulsatile release of LH that stimulates gonadal function in both male and female mammals.

Localization of vasopressin mRNA and immunoreactivity in pituicytes of pituitary stalk-transected rats after osmotic stimulation.

The presence of [arginine] vasopressin (AVP) mRNA and AVP immunoreactivity in pituicytes of the neural lobe (NL) of intact and pituitary stalk-transected rats, with and without osmotic stimulation, was examined. AVP mRNA was analyzed by Northern blotting, as well as by in situ hybridization in combination with immunocytochemistry using anti-glial fibrillary acidic protein (GFAP) as a marker for pituicytes. In intact rats, a poly(A) tail-truncated 0.62-kb AVP mRNA was detected in the NL and was found to increase 10-fold with 7 days of continuous salt loading. Morphological analysis of the NL of 7-day salt-loaded rats revealed the presence of AVP mRNA in a significant number of GFAP-positive pituicytes in the NL and in areas most probably containing nerve fibers. Eight days after pituitary stalk transection the NL AVP mRNA diminished in animals given water to drink, whereas in those given 2% saline for 18 h followed by 6 h of water, a treatment repeated on 6 successive days beginning 2 days after surgery, the 0.62-kb AVP mRNA was present. The AVP mRNA in the pituitary stalk-transected, salt-loaded rats showed an exclusive cellular distribution in the NL, indicative of localization in pituicytes. Immunoelectron microscopy showed the presence of AVP immunoreactivity in a subpopulation of pituicytes 7 and 10 days after pituitary stalk transection in salt-loaded animals, when almost all AVP fibers had disappeared from the NL. These data show that a subset of pituicytes in the NL is activated to synthesize AVP mRNA and AVP in response to osmotic stimulation.

Pituitary follicle-stimulating hormone (FSH) induces CREM gene expression in Sertoli cells: involvement in long-term desensitization of the FSH receptor.

Transcription factor CREM (cAMP-responsive element modulator) plays a pivotal role in the nuclear response to cAMP in neuroendocrine cells. We have previously shown that follicle-stimulating hormone (FSH) directs CREM expression in male germ cells. The physiological importance of FSH in Sertoli cell function prompted us to analyze its effect on CREM expression in these cells. We observed a dramatic and specific increase in the CREM isoform ICER (inducible cAMP early repressor) expression, with a peak 4 h after FSH treatment of primary Sertoli cells. Interestingly, induced levels of ICER protein persist for a considerably longer time. Induction of the repressor ICER accompanies early down-regulation of the FSH receptor transcript, which leads to long-term desensitization. Here we show that ICER represses FSH receptor expression by binding to a CRE-like sequence in the regulatory region of the gene. Our results confirm the crucial role played by CREM in hormonal control and suggest its role in the long-term desensitization phenomenon of peptide membrane receptors.

The rotated hypoblast of the chicken embryo does not initiate an ectopic axis in the epiblast.

In the amniotes, two unique layers of cells, the epiblast and the hypoblast, constitute the embryo at the blastula stage. All the tissues of the adult will derive from the epiblast, whereas hypoblast cells will form extraembryonic yolk sac endoderm. During gastrulation, the endoderm and the mesoderm of the embryo arise from the primitive streak, which is an epiblast structure through which cells enter the interior. Previous investigations by others have led to the conclusion that the avian hypoblast, when rotated with regard to the epiblast, has inductive properties that can change the fate of competent cells in the epiblast to form an ectopic embryonic axis. Thus, it has been suggested that the hypoblast normally induces the epiblast to form a primitive streak at a specific locus. In the work reported here, an attempt was made to reexamine the issue of induction. In contrast to previous reports, it was found that the rotated hypoblast of the chicken embryo does not initiate formation of an ectopic axis in the epiblast. The embryonic axis always initiates and develops according to the basic polarity of the epiblast layer. These results provoke a reinterpretation of the issues of mesoderm induction and primitive streak initiation in the avian embryo.

Thyroid-stimulating hormone (TSH)-directed induction of the CREM gene in the thyroid gland participates in the long-term desensitization of the TSH receptor.

Thyroid gland function is regulated by the hypothalamic-pituitary axis via the secretion of TSH, according to environmental, developmental, and circadian stimuli. TSH modulates both the secretion of thyroid hormone and gland trophism through interaction with a specific guanine nucleotide-binding protein-coupled receptor (TSH receptor; TSH-R), which elicits the activation of the cAMP-dependent signaling pathway. After TSH stimulation, the levels of TSH-R RNA are known to decrease dramatically within a few hours. This phenomenon ultimately leads to homologous long-term desensitization of the TSH-R. Here we show that TSH drives the induction of the inducible cAMP early repressor (ICER) isoform of the cAMP response element (CRE) modulator gene both in rat thyroid gland and in the differentiated thyroid cell line FRTL-5. The kinetics of ICER protein induction mirrors the down-regulation of TSH-R mRNA. ICER binds to a CRE-like sequence in the TSH-R promoter and represses its expression. Thus, ICER induction by TSH in the thyroid gland represents a paradigm of the molecular mechanism by which pituitary hormones elicit homologous long-term desensitization.

Rapid endocytosis coupled to exocytosis in adrenal chromaffin cells involves Ca2+, GTP, and dynamin but not clathrin.

Rapid endocytosis (RE) occurs immediately after an exocytotic burst in adrenal chromaffin cells. Capacitance measurements of endoocytosis reveal that recovery of membrane is a biphasic process that is complete within 20 sec. The ultimate extent of membrane retrieval is precisely controlled and capacitance invariably returns to its prestimulation value. The mechanism of RE specifically requires intracellular Ca2+; Sr2+ and Ba2+ do not substitute, although all three cations support secretion. Thus the divalent cation receptors for RE and exocytosis must be distinct molecules. RE is dependent on GTP hydrolysis; it is blocked by GTP removal or replacement with guanosine 5'-[gamma-thio]triphosphate. In the presence of GTP, multiple rounds of secretion followed by RE could be elicited from the same cell. RE requires participation of dynamin, a guanine nucleotide binding protein, as revealed by intracellular immunological antagonism of this protein. Intact microtubules may be essential, as nocodazole also blocked RE. Whereas anti-dynamin antibodies blocked RE, anti-clathrin antibodies did not, suggesting that clathrin-coated vesicles are not involved in this form of endocytosis. RE may represent the initial step in the rapid recycling of secretory granules in the chromaffin cell.

Three gonadotropin-releasing hormone genes in one organism suggest novel roles for an ancient peptide.

Gonadotropin-releasing hormone (GnRH) is known and named for its essential role in vertebrate reproduction. Release of this decapeptide from neurons in the hypothalamus controls pituitary gonadotropin levels which, in turn, regulate gonadal state. The importance of GnRH is underscored by its widespread expression and conservation across vertebrate taxa: five amino acids are invariant in all nine known forms, whereas two others show only conservative changes. In most eutherian mammals, only one form, expressed in the hypothalamus, is thought to exist, although in a recent report, antibody staining in developing primates suggests an additional form. In contrast, multiple GnRH forms and expression loci have been reported in many non-mammalian vertebrates. However, evidence based on immunological discrimination does not always agree with analysis of gene expression, since GnRH forms encoded by different genes may not be reliably distinguished by antibodies. Here we report the expression of three distinct GnRH genes in a teleost fish brain, including the sequence encoding a novel GnRH preprohormone. Using in situ hybridization, we show that this form is found only in neurons that project to the pituitary and exhibit changes in soma size depending on social and reproductive state. The other two GnRH genes are expressed in other, distinct cell populations. All three genes share the motif of encoding a polypeptide consisting of GnRH and a GnRH-associated peptide. Whereas the GnRH moiety is highly conserved, the GnRH-associated peptides are not, reflecting differential selective pressure on different parts of the gene. GnRH forms expressed in nonhypothalamic regions may serve to coordinate reproductive activities of the animal.