Regulation of myoglobin in hypertrophied rat cardiomyocytes in experimental pulmonary hypertension.

A major problem in chronic heart failure is the inability of hypertrophied cardiomyocytes to maintain the required power output. A Hill-type oxygen diffusion model predicts that oxygen supply is limiting in hypertrophied cardiomyocytes at maximal rates of oxygen consumption and that this limitation can be reduced by increasing the myoglobin (Mb) concentration. We explored how cardiac hypertrophy, oxidative capacity, and Mb expression in right ventricular cardiomyocytes are regulated at the transcriptional and translational levels in an early stage of experimental pulmonary hypertension, in order to identify targets to improve the oxygen supply/demand ratio. Male Wistar rats were injected with monocrotaline to induce pulmonary hypertension (PH) and right ventricular heart failure. The messenger RNA (mRNA) expression levels per nucleus of growth factors insulin-like growth factor-1Ea (IGF-1Ea) and mechano growth factor (MGF) were higher in PH than in healthy controls, consistent with a doubling in cardiomyocyte cross-sectional area (CSA). Succinate dehydrogenase (SDH) activity was unaltered, indicating that oxidative capacity per cell increased. Although the Mb protein concentration was unchanged, Mb mRNA concentration was reduced. However, total RNA per nucleus was about threefold higher in PH rats versus controls, and Mb mRNA content expressed per nucleus was similar in the two groups. The increase in oxidative capacity without an increase in oxygen supply via Mb-facilitated diffusion caused a doubling of the critical extracellular oxygen tension required to prevent hypoxia (PO2crit). We conclude that Mb mRNA expression is not increased during pressure overload-induced right ventricular hypertrophy and that the increase in myoglobin content per myocyte is likely due to increased translation. We conclude that increasing Mb mRNA expression may be beneficial in the treatment of experimental PH.

Monolayer cultures of adult pancreatic islet cells on osmotically disrupted fibroblasts.

A simple method that permits the attachment of fully dissociated adult pancreatic islet cells to culture dishes, using osmotically disrupted fibroblasts, is described. Using this system, monolayer cultures consisting of either isolated or clustered adult endocrine islet cells can be readily established in standard culture media.

Discrepancy between prolactin (PRL) messenger ribonucleic acid and PRL content in rat fetal pituitary cells: possible role of dopamine.

Data are controversial concerning the time when PRL-synthesizing cells are detected for the first time in the rat pituitary. Using a very sensitive immunocytochemical technique, we could visualize only a few PRL cells before day 10 after birth. At that time, pituitary PRL was still 200 times less abundant than in the adult (on a tissue weight basis) whereas PRL mRNA per mg total RNA was only 80 times lower than in the adult. However, by in situ hybridization, we could demonstrate the presence of PRL mRNA in cells from fetal day 18 on. We have also followed the expression of GH gene in rat pituitary cells during development. In contrast to results obtained with PRL cells, quantitative analysis of cDNA probe hybridization to GH mRNA correlated well with measurements of immunostained cells. We found that PRL was released in the blood from fetal day 19 onwards. Thus, at that time PRL is synthesized and secreted but not stored. We therefore measured brain dopamine levels, and the data support the idea that the rise in dopamine levels after birth contributes to PRL storage. We confirmed in vitro that newborn pituitary cells can store PRL when cultured in the presence of dopamine.

Prolactin-induced expression of cytokine-inducible SH2 signaling inhibitors in human hematopoietic progenitors.

OBJECTIVE: The prolactin (PRL) receptor (PRLR) utilizes the JAK2/STAT-5 pathway and induces expression of cytokine-inducible SH2 (CIS)/JAK2 binding (JAB) signaling inhibitors. We and others recently showed that CIS-3 and JAB abolish PRLR-mediated JAK2 activation and STAT-5 activity, whereas CIS-1, CIS-2, and CIS-4 had a negligible effect. Human CD34(+) hematopoietic progenitors express PRLRs and respond to PRL in vitro by enhanced cytokine-induced colony formation. To assess the signaling mechanism(s) involved in PRL-mediated enhancement of hematopoiesis and to identify further the CIS/JAB targets for PRL-mediated cellular responses, we assayed the effect of PRL, alone or in the presence of interleukin-3 (IL-3), on activation of STAT-5 and expression of CIS/JAB RNA in human cord blood (CB) CD34(+) cells. MATERIALS AND METHODS: Isolated CB CD34(+) cells were incubated in serum-free cultures in the absence or presence of recombinant human (rh)PRL, rhIL-3, or both. Cell lysates were subjected to Western blot analysis with anti-STAT-5 and anti-phospho-STAT-5 antibodies. Isolated RNA was subjected to semiquantitative reverse transcriptase polymerase chain reaction analysis of CIS/JAB expression. RESULTS: STAT-5 tyrosine phosphorylation was similarly induced by PRL and IL-3, with an additive effect detected in the presence of both stimuli. Both PRL and IL-3, alone or combined, failed to induce CIS-3 or JAB RNA expression in CD34(+) cells. Interferon-gamma had no effect on CIS-3/JAB induction in these cells. However, CIS-1 was induced by PRL < IL-3 < PRL+IL-3, whereas CIS-2 expression was induced by PRL = IL-3 < PRL+IL-3. CONCLUSIONS: Our findings show that PRL induces activation of STAT-5 and expression of similar CIS/JAB family members as IL-3 does in human CB CD34(+) cells. Because CIS-1 abolishes STAT-5 activation via the IL-3 but not the PRL receptor, the hematopoietic growth-promoting effects of PRL may involve its capacity to provide sustained STAT-5-mediated stimulatory signals to the cells.

Antibody-induced modulation of rhabdovirus infection of neurons in vitro.

Dissociated neuron cultures of mice were inoculated with vesicular stomatitis virus (VSV) and subsequently fed with medium containing sufficient antiviral antibody (AB) to neutralize all free virus. In contrast to control acute neuronal infection, which lasts one to two days, AB-treated neuron cultures were maintained as long as two weeks. This protection was not obtained in infected monkey kidney cells treated with AB. Protected neuron cultures were examined with immunolabeling and EM techniques. During the first days of AB treatment, viral buds and surface antigens were often grouped in clusters instead of being diffuse on the neuronal surface. In addition, phagocytic cells often in contact with viruses released by neurons progressively engulfed aggregates of these viruses in their lysosomal systems. Dendritic processes in AB-treated cultures contained more viral antigen and ribonucleoproteins, but less assembly sites than in acutely-infected neurons. However, budding sites were frequent on the side of the post-synaptic density and some viruses seemed to enter directly into the lateral side of the presynaptic terminal to which the productive dendrite was connected. Removal of AB at this stage resulted in reactivation of viral infection in more neurons than those originally infected, but complete viral maturation and release occurred only 2 to 3 days after removal of AB. Chance of reactivation decreased with increasing length of AB treatment, and no viral antigens or budding sites were detected during the second week of AB treatment. Continuous treatment of VSV-infected neuron cultures with antiviral AB first induces (1) activation of non-neuronal cells to phagocytize clusters of viruses forming at the neuron surface, and (2) restriction of virus maturation sites mostly to the postsynaptic area where virus spreading to presynaptic endings seems favored. Prolonged treatment with AB later results in the apparent curing of the infection.

Growth hormone expression in murine bone marrow cells is independent of the pituitary transcription factor Pit-1.

GH has been shown to promote the development and function of leukocytes. The expression of both GH and GH-receptors in lymphoid cells has led to the hypothesis that GH acts in an autocrine or paracrine fashion. The described effects of GH on hematopoiesis and B cell development, led us to investigate GH expression in bone marrow cells. By immunocytochemistry, we show that bone marrow-derived granulocytes and macrophages contain immunoreactive GH. We found that 65 +/- 24% of the granulocytes were stained with anti-GH, whereas 5.8 +/- 1.5% of the granulocytes contained detectable amounts of GH mRNA as assessed by in situ hybridization. To address a possible alternative regulation mechanism in bone marrow and to establish whether locally derived GH might still play a role in pituitary-deficient dwarf mice, we also addressed GH expression in bone marrow from hypopituitary Snell dwarf mice. These mice have a mutated gene for the pituitary transcription factor Pit-1 that is deficient in DNA binding. Our finding that GH expression (immunoreactive protein and mRNA) in bone marrow cells from dwarf mice is similar to that in normal mice points to a Pit-1 independent regulation of GH in mouse bone marrow.

Human neutrophils express GH-N gene transcripts and the pituitary transcription factor Pit-1b.

Since GH stimulates the development and function of granulocytes, we investigated the expression of GH in granulocyte subsets. By immunocytochemistry, 25 +/- 7% of the human neutrophils were shown to express immunoreactive GH, whereas eosinophils were negative. Reversed transcription (RT)-PCR analysis demonstrated GH mRNA in neutrophils. Restriction analysis revealed that neutrophils express the GH-N gene but not the GH-V gene. Furthermore, we demonstrated by western blot analysis that neutrophils express an alternatively spliced variant of the pituitary transcription factor Pit-1, designated Pit-1b.

Pancreatic hormone receptors on islet cells.

To assess whether islet cells are equipped with recognition units which allow an intra-islet regulation via released hormones, the presence of insulin and glucagon receptors is investigated on purified pancreatic A and B cells. Mono-[125I]glucagon is shown to bind specifically to islet B cells, with similar binding characteristics as in isolated hepatocytes but involving less receptors per cell (2.10(4) per B cell vs. 8.10(5) per liver cell). Binding is half-maximally displaced by 5.10(-9) M glucagon, a concentration known to induce half-maximal biological effects in isolated B cells. These results are compatible with a regulatory role of glucagon in the insulin release process. No specific binding of [125I]tyr-A14-insulin is detected on pancreatic A cells. In order to increase receptor assay sensitivity, [123I]tyr-A14-insulin is prepared with at least 5-fold higher specific activity. Its validity for in vitro receptor analysis is demonstrated in IM-9 lymphocytes, where insulin binding is detectable down to 10(4) cells/ml. However, no insulin-binding sites are identified on pancreatic A cells, even at 10(6) cells/ml. If isolated A cells contain high affinity insulin receptors, their number should be inferior to 400 per cell, which is 50- to 500-fold lower than in classical insulin target cells. These findings explain the insensitivity of the glucagon release process to acute exposure to insulin.

Interplay of nutrients and hormones in the regulation of insulin release.

Single pancreatic B cells are purified by autofluorescence-activated cell sorting, and their secretory activity is measured after overnight culture. Compared to intact islets, the isolated cells release 2-fold more insulin under basal conditions and 5-fold less during nutrient stimulation. Their secretory activity can be induced by glucose, leucine, or arginine, but only 0.3-1.7% of their hormone content is liberated at 20 mM nutrient concentrations. This poor nutrient-induced insulin release from purified B cells is attributed to their low cAMP levels and is markedly increased after addition of (Bu)2cAMP, of glucagon, or of pancreatic A cells. These results strongly support the concept that the potent in vivo insulin-releasing action of glucose and leucine is not only dependent on their fuel capacity in pancreatic B cells but also on the concurrent cAMP levels in these cells. In isolated islets, endogenously released glucagon apparently determines the cAMP production in B cells and thus participates in the nutrient-induced secretory process. Somatostatin and epinephrine were shown to exert their suppressive effects via the glucagon-dependent messenger system. It is concluded that nutrients and hormones interact with two different messenger systems which amplify each others' stimulatory effect upon insulin release. cAMP might represent the hormone-induced messenger which sets the B cell's sensitivity and secretory capacity for nutrient stimuli such as glucose. The higher insulin secretory response observed after reaggregation of single B cells could not be attributed to an altered activity in the nutrient or hormonal regulatory units, raising the possibility that the aggregated state of the cells is rather responsible for a better organization or cooperation of the secretory effector unit.

Prolactin cell subpopulations separated on discontinuous Percoll gradient: an immunocytochemical, biochemical, and physiological characterization.

A single-step procedure was devised to separate PRL cells from the rat anterior pituitary gland. After dissociation, cells were centrifuged on a Percoll gradient. Three layers were recovered. The composition of the different layers was evaluated using immunocytochemistry (with antisera to the six pituitary hormones), and in situ hybridization [with DNA complementary to PRL or to GH messenger RNA (mRNA)]. Both methods yielded identical values. PRL cells were recovered in the lower density layer (layer 1) with a good yield (that is 81% of the total PRL cells of the initial cell suspension) and in addition, markedly enriched (indeed 85% of the cells in layer 1 stained for PRL). A second layer (layer 2: intermediate density) contained most of the remaining PRL cells which were, however, heavily contaminated mainly by GH cells and cells that did not stain for any of the known pituitary hormones. A third layer (layer 3: higher density) was enriched in GH cells to 93% (representing, however, only 10% of the initial pituitary GH cells). In addition, PRL and GH were measured by RIA in culture medium and in cell lysates. Hormone biosynthesis was monitored by polyacrylamide gel electrophoresis and autoradiography after culture in the presence of [35S]methionine. These experiments confirmed that layer 1 was enriched in cells containing, and producing, PRL and depleted from GH cells. Cells in layer 2 contained and produced more GH than PRL. PRL cells from layer 1 responded to dopamine and to vasoactive intestinal polypeptide in the same way as PRL cells in the unseparated pituitary cell population. In contrast PRL cells in layer 2 had a lower basal secretion rate but a higher response to vasoactive intestinal polypeptide. Unless this represents a paracrine effect of non-PRL cells, PRL cells in layer 2 exhibit different properties and may therefore form a distinct subpopulation of PRL cells.

Differential actions of the dopamine agonist bromocriptine on growth of SMtTW tumors exhibiting a prolactin and/or a somatotroph cell phenotype: relation to dopamine D2 receptor expression.

Dopamine (Da) and Da agonists are known to inhibit secretion and proliferation of normal and tumoral PRL cells, through receptors of D2 subtype. Because of the lack of an experimental model, the relationship between bromocriptine (BR) sensitivity and D2 receptor expression is poorly documented. Such a relationship was analyzed using five lineages of spontaneous transplantable rat pituitary tumors (SMtTW) exhibiting different PRL/GH phenotypes. From plasma PRL and GH concentrations of rats bearing the tumors and tumor messenger RNA contents, tumors were classified as PRL (SMtTW2), somatotroph (SMtTW10), or somatomammotroph (SMtTW5) tumors. Two lineages (SMtTW3 and SMtTW4) represented variants producing PRL and GH but with a high predominance of PRL. With the exception of SMtTW4 tumors, which were malignant, all the tumors were benign and differed in their growth rate. Hormone production and growth of tumors with a PRL or a somatomammotroph phenotype were reduced by about 90% under BR treatment, whereas somatotroph tumors and the PRL malignant tumors were totally insensitive to BR. D2 receptor messenger RNA was present in all BR-sensitive tumors and was not detected in BR-resistant tumors. In conclusion, using five lineages of SMtTW tumors that are representative of the most frequent tumors encountered in human pituitary pathology, we found a full concordance between tumor responses to BR and the expression of D2 receptor by the tumors. The identification of a tumor lineage with a malignant phenotype, secreting high amounts of PRL and presenting a resistance to BR, supports the idea that Da-resistant prolactinomas are aggressive tumors.

Receptors for pituitary adenylate cyclase activating peptides in human pituitary adenomas.

The presence of pituitary adenylate cyclase activating polypeptide (PACAP) receptors coupled to adenylate cyclase was investigated in four types of human pituitary adenomas: three null adenomas and five gonadotropin-, three ACTH-, four GH-, and four PRL-producing adenomas. In all samples, except in prolactinomas, PACAP(1-27) and PACAP(1-38) stimulated adenylate cyclase activity equally well and potently (K(act) around 3 nmol). Vasoactive intestinal polypeptide (VIP) was systematically 100- to 300-fold less potent than both PACAPs. In prolactinomas, PACAP(1-27), PACAP(1-38), and VIP were inactive despite a response of the enzyme to guanosine 5'-triphosphate, Gpp(NH)p, forskolin, and fluoride. [125I-AcHis1]PACAP(1-27) binding was detected in all samples except in prolactinomas. In addition, a detailed analysis of receptors was feasible in all five gonadotropin- and in two ACTH-producing adenomas, confirming the existence of selective PACAP receptors that recognized PACAP(1-27) and PACAP(1-38) with similar high affinity (IC50 0.8-1.5 nmol) and VIP with a low affinity (IC50 100 nmol/L).

A novel pituitary transcription factor is produced by alternative splicing of the human GHF-1/PIT-1 gene.

An alternative splice acceptor site in intron 1 of the human GHF-1/PIT-1 gene was sequenced. The use of this splice site is responsible for a 78-bp in-frame insertion upstream from exon 2 and leads to the hGHF-2/PIT-2 cDNA detected in normal human pituitary.

Pit-1/GHF-1 expression in pituitary adenomas: further analogy between human adenomas and rat SMtTW tumours.

Adenomas can develop from each cell type of the anterior pituitary. In the normal pituitary, three of these cells types, the GH-, prolactin- and TSH-secreting cells, express the transcription factor Pit-1/GHF-1 which is responsible for prolactin and GH (and probably TSH) cell commitment, differentiation, probably proliferation and gene expression. We have analysed the expression of Pit-1/GHF-1 in a panel of human pituitary adenomas. All GH-, prolactin- and TSH-expressing adenomas studied expressed the Pit-1/GHF-1 factor, as demonstrated by in-situ hybridization and immunocytochemistry. The expression was higher in adenomas than in normal human pituitary. In contrast, ACTH- and LH-FSH-secreting and non-secreting adenomas were negative. Seven transplants of the spontaneous rat prolactinoma SMtTW were also investigated and all were found to be positive. This further stresses the analogy between these tumours and human prolactinomas. Taken together, the data confirm that Pit-1/GHF-1 expression is restricted to GH-, prolactin- and TSH-expressing cells, and the increased expression in adenomas is compatible with a role of Pit-1/GHF-1 in cell proliferation.

Prolactin and growth hormone mRNA and protein characterization in SMtTW rat pituitary tumours.

We have combined different techniques to analyse passages of five different rat spontaneous pituitary tumours (SMtTW) that were transplanted under the kidney capsule. These tumours were secreting prolactin (PRL), GH or both hormones. RIA, immunocytochemistry (ICC) and Western blot analysis were applied to characterize the hormone(s) stored (ICC and Western blot) and secreted (RIA). mRNA content was analysed by PCR, Northern blot analysis and in situ hybridization. The data point not only to the reliability of the techniques used at both protein and RNA levels for each tumour studied but also to the complementarity of some techniques. For example, whereas Northern blot analysis demonstrates the presence and size of hormone mRNA, in situ hybridization indicates the percentage of cells expressing a given hormone mRNA and allows the presence of one population (or more) of cells in a given tumour to be identified. Moreover, the tumours were compared with normal rat pituitary. Although the PRL and GH mRNAs were identical in size, the amount of mRNA was lower in the tumours. At the protein level, the PRL and GH variants exhibited a different pattern of expression in tumours compared with the normal rat pituitary. The biological significance of these differences is discussed.

The Thy-1 glycoprotein on nerve cells in culture: electron-microscopic analysis and biochemical characterization.

The distribution and some biochemical properties of the Thy-1 glycoprotein, an antigen common to brain and thymus-derived lymphocytes, were analyzed in primary nerve cell cultures from mouse embryos. The Thy-1 antigen was detected on the plasma membrane of most neurons and fibroblast-like cells and on some astrocytes. The labelling pattern was homogeneous, except on some neuronal cell bodies where the distribution varied from a few patches to a homogeneous labelling. The Thy-l molecules immunoprecipitated from either nerve cell cultures or from fibroblast-like cells from the meninges had similar molecular weights and isoelectric points. In contrast, Thy-1 from thymocytes was more heterogeneous in molecular weight and in isoelectric point range.

Myeloid leukemic cells express and secrete bioactive pituitary-sized 23 kDa prolactin.

Prolactin (PRL) is a 23 kDa polypeptide hormone of pituitary origin which is of major importance for reproduction. In addition, PRL has immunomodulatory effects and can be produced in small quantities in nonpituitary tissues. To address possible autocrine or paracrine functions of PRL in leukemia, we characterized immunoreactive PRL from the culture medium of leukemic cells. The myeloid cell line Eol-1 expresses the long extrapituitary type mRNA for PRL and synthesizes immunoreactive PRL with a molecular weight of 23 kDa. The biological activity in Eol-1 culture medium was determined using the Nb2 bioassay. This activity co-eluted with recombinant human (rh) PRL on an S-200 Sephacryl gel filtration column and could be blocked by anti-PRL antiserum. Western blot analysis and Nb2 bioassays also suggest that acute myelogenous leukemic blasts secrete bioactive 23 kDa PRL in one out of three tested patients.

Cytokine-like effects of prolactin in human mononuclear and polymorphonuclear leukocytes.

Some biochemical events following the binding of prolactin (PRL) to its receptor in normal human leukocytes were investigated. PRL enhanced JAK2 phosphorylation in peripheral blood mononuclear cells (PBMC) but not in granulocytes. PRL also induced phosphorylation of Stat-5 in PBMC and Stat-1 in granulocytes. Subsequent binding of Stat-5- and of Stat-1-like molecules to a GAS responsive element from the beta-casein promoter was detected by EMSA. p38 MAPK (but not p42/p44 MAPK) was activated by PRL in both leukocyte populations. PRL induced iNOS and CIS mRNA expression in granulocytes. Increased expression of IRF-1 and SOCS-2 was observed in granulocytes and of SOCS-3 and iNOS in PBMC. Similar effects were obtained with ovine and human PRL. Antiserum to PRL reduced iNOS and IRF-1 expression induced by PRL in granulocytes and reduced iNOS expression in PBMC. Also, pretreatment of granulocytes with a p38 MAPK inhibitor (SB 203580) prevented in part PRL-induced iNOS and IRF-1 expression. In PBMC, the p38 inhibitor decreased PRL-induced iNOS gene expression. These results indicate that PRL-induced gene regulation in leukocytes requires the activation of at least two different pathways: the Stat and the MAP kinase pathways. Moreover, although PRL activates Stat in both leukocyte types, signal transduction is different in granulocytes and in PBMC. Most importantly, PRL modulates the expression of genes crucial to leukocyte function. The present findings reinforce the concept that PRL has "cytokine-like" activity in human leukocytes.

Expression of SOCS genes in normal and leukemic human leukocytes stimulated by prolactin, growth hormone and cytokines.

To evaluate the possible role of the recently described family of suppressors of cytokine signaling (SOCS) factors in the human lympho-hemopoietic system, we have monitored SOCS factor expression, both constitutive and induced by either cytokines, prolactin (PRL) or growth hormone (GH), using polymerase chain reaction in normal and leukemic cells. CIS (cytokine-inducible SH2-containing protein), SOCS-2 and SOCS-3 were constitutively expressed in peripheral blood mononuclear leukocytes. SOCS-3 expression was enhanced by PRL or by IFN-gamma. In bone marrow cells and granulocytes, CIS expression was induced and SOCS-2 enhanced by IFN-gamma and by PRL. In tonsillar cells, CIS expression was increased and SOCS-2 was induced by IL-1beta, IL-6, PRL and GH. SOCS-3 expression was enhanced by IL-1beta. The expression of SOCS-7 was increased by IL-6, PRL and GH. In Raji B-lymphoma cells, the expression of SOCS-2 and SOCS-7 was enhanced by IL-1beta. In THP-1 myeloid leukemia cells pretreated with TPA (to induce receptors for IFN-gamma), IFN-gamma induced SOCS-2. Jurkat cells expressed more SOCS-2 when exposed to PRL. Original observations in this work include the first report on SOCS-7 induction by cytokines. Also our data shed new light on the possible involvement of PRL and GH in the cytokine network. These hormones could modulate the transduction of signals originating from receptors for various cytokines.

Expression of IL-6 mRNA in normal rat and human pituitaries and in human pituitary adenomas.

Cells expressing IL-6 mRNA were detected by in situ hybridization in normal pituitaries. In normal untreated rat pituitary the expression was very low. Within hours after IP administration of liposaccharide, IL-6 mRNA accumulated in the anterior lobe of the pituitary. Production of IL-6 was monitored after dissociation and culture of pituitary cells. High levels (8000 pg/ml) were recovered after 72 hr in culture. In normal human pituitaries, less than 1% of cells expressed IL-6 mRNA or IL-6 receptor mRNA (IL-6-R mRNA). In gonadotropinomas, prolactinomas, and non-functioning adenomas, only rare, scattered positive cells were found for either IL-6 or IL-6-R. In contrast, both genes were highly expressed in ACTH- and GH-secreting tumors at the junction of adenoma and infiltrating fibrous tissue and around blood vessels. The combined expression of IL-6 and IL-6-R suggests that IL-6 acts in an autocrine or in a paracrine way in ACTH and GH adenomas.