ABSTRACT
INTRODUCTION: Chagas disease, the most important parasitic infection in Latin America, is caused by the intracellular protozoan Trypanosoma cruzi. To treat this disease, only two nitroheterocyclic compounds with toxic side effects exist and frequent treatment failures are reported. Hence there is an urgent need to develop new drugs. Recently, metabolomics has become an efficient and cost-effective strategy for dissecting drug mode of action, which has been applied to bacteria as well as parasites, such as different Trypanosome species and forms. OBJECTIVES: We assessed if the metabolomics approach can be applied to study drug action of the intracellular amastigote form of T. cruzi in a parasite-host cell system. METHODS: We applied a metabolic fingerprinting approach (DI-MS and NMR) to evaluate metabolic changes induced by six different (candidate) drugs in a parasite-host cell system. In a second part of our study, we analyzed the impact of two drugs on polar metabolites, lipid and proteins to evaluate if affected pathways can be identified. RESULTS: Metabolic signatures, obtained by the fingerprinting approach, resulted in three different clusters. Two can be explained by already known of mode actions, whereas the three experimental drugs formed a separate cluster. Significant changes induced by drug action were observed in all the three metabolic fractions (polar metabolites, lipids and proteins). We identified a general impact on the TCA cycle, but no specific pathways could be attributed to drug action, which might be caused by a high percentage of common metabolome between a eukaryotic host cell and a eukaryotic parasite. Additionally, ion suppression effects due to differences in abundance between host cells and parasites may have occurred. CONCLUSION: We validated the metabolic fingerprinting approach to a complex host-cell parasite system. This technique can potentially be applied in the early stage of drug discovery and could help to prioritize early leads or reconfirmed hits for further development.
Subject(s)
Host-Parasite Interactions , Metabolomics/methods , Myoblasts/parasitology , Proteomics/methods , Trypanocidal Agents/pharmacology , Trypanosoma cruzi/drug effects , Animals , Cell Line , Lipid Metabolism , Magnetic Resonance Spectroscopy/methods , Mass Spectrometry/methods , Metabolome , Myoblasts/metabolism , Proteome/chemistry , Rats , Trypanosoma cruzi/metabolism , Trypanosoma cruzi/pathogenicityABSTRACT
Major depressive disorder (MDD) affects around 350 million people worldwide; however, the underlying genetic basis remains largely unknown. In this study, we took into account that MDD is a gene-environment disorder, in which stress is a critical component, and used whole-genome screening of functional variants to investigate the 'missing heritability' in MDD. Genome-wide association studies (GWAS) using single- and multi-locus linear mixed-effect models were performed in a Los Angeles Mexican-American cohort (196 controls, 203 MDD) and in a replication European-ancestry cohort (499 controls, 473 MDD). Our analyses took into consideration the stress levels in the control populations. The Mexican-American controls, comprised primarily of recent immigrants, had high levels of stress due to acculturation issues and the European-ancestry controls with high stress levels were given higher weights in our analysis. We identified 44 common and rare functional variants associated with mild to moderate MDD in the Mexican-American cohort (genome-wide false discovery rate, FDR, <0.05), and their pathway analysis revealed that the three top overrepresented Gene Ontology (GO) processes were innate immune response, glutamate receptor signaling and detection of chemical stimulus in smell sensory perception. Rare variant analysis replicated the association of the PHF21B gene in the ethnically unrelated European-ancestry cohort. The TRPM2 gene, previously implicated in mood disorders, may also be considered replicated by our analyses. Whole-genome sequencing analyses of a subset of the cohorts revealed that European-ancestry individuals have a significantly reduced (50%) number of single nucleotide variants compared with Mexican-American individuals, and for this reason the role of rare variants may vary across populations. PHF21b variants contribute significantly to differences in the levels of expression of this gene in several brain areas, including the hippocampus. Furthermore, using an animal model of stress, we found that Phf21b hippocampal gene expression is significantly decreased in animals resilient to chronic restraint stress when compared with non-chronically stressed animals. Together, our results reveal that including stress level data enables the identification of novel rare functional variants associated with MDD.
Subject(s)
Depressive Disorder, Major/genetics , Adult , Case-Control Studies , Female , Gene Expression , Genetic Predisposition to Disease/genetics , Genome-Wide Association Study , Humans , Los Angeles , Male , Mexican Americans/genetics , Middle Aged , Polymorphism, Single Nucleotide/genetics , Risk Factors , Stress, Psychological , White People/geneticsABSTRACT
The inflammasome is hypothesized to be a key mediator of the response to physiological and psychological stressors, and its dysregulation may be implicated in major depressive disorder. Inflammasome activation causes the maturation of caspase-1 and activation of interleukin (IL)-1ß and IL-18, two proinflammatory cytokines involved in neuroimmunomodulation, neuroinflammation and neurodegeneration. In this study, C57BL/6 mice with genetic deficiency or pharmacological inhibition of caspase-1 were screened for anxiety- and depressive-like behaviors, and locomotion at baseline and after chronic stress. We found that genetic deficiency of caspase-1 decreased depressive- and anxiety-like behaviors, and conversely increased locomotor activity and skills. Caspase-1 deficiency also prevented the exacerbation of depressive-like behaviors following chronic stress. Furthermore, pharmacological caspase-1 antagonism with minocycline ameliorated stress-induced depressive-like behavior in wild-type mice. Interestingly, chronic stress or pharmacological inhibition of caspase-1 per se altered the fecal microbiome in a very similar manner. When stressed mice were treated with minocycline, the observed gut microbiota changes included increase in relative abundance of Akkermansia spp. and Blautia spp., which are compatible with beneficial effects of attenuated inflammation and rebalance of gut microbiota, respectively, and the increment in Lachnospiracea abundance was consistent with microbiota changes of caspase-1 deficiency. Our results suggest that the protective effect of caspase-1 inhibition involves the modulation of the relationship between stress and gut microbiota composition, and establishes the basis for a gut microbiota-inflammasome-brain axis, whereby the gut microbiota via inflammasome signaling modulate pathways that will alter brain function, and affect depressive- and anxiety-like behaviors. Our data also suggest that further elucidation of the gut microbiota-inflammasome-brain axis may offer novel therapeutic targets for psychiatric disorders.
Subject(s)
Anxiety/metabolism , Depression/metabolism , Gastrointestinal Microbiome/physiology , Inflammasomes/metabolism , Animals , Anxiety Disorders/complications , Behavior, Animal/physiology , Brain/metabolism , Caspase 1 , Cytokines/metabolism , Depressive Disorder, Major/metabolism , Gastrointestinal Microbiome/immunology , Inflammasomes/physiology , Inflammation/metabolism , Male , Mice , Mice, Inbred C57BL , Microbiota , Neuroimmunomodulation/physiology , Signal Transduction , Stress, Psychological/microbiologyABSTRACT
Women with breast cancer (BC) and antithyroid peroxidase (TPO) autoantibodies (TPOAb) have a better prognosis than women lacking TPOAb. Sera from women with TPOAb displayed immunoreactivity to BC tissue by immunofluorescence that was not apparent in women without TPOAb. We hypothesize a BC/thyroid shared antigen that provides a target for humoral or cell-mediated immune activity; candidates include the sodium/iodide symporter (expressed in thyroid and BC), cross-reacting epitopes in TPO and lactoperoxidase (LPO) or TPO itself. As the association is with TPOAb, we investigated TPO expression in BC, breast peritumoral tissue (PT), other tissues (tumoral and not) and thyroid as positive control. Transcripts for known and novel TPO isoforms were detected in BC (n = 8) and PT (n = 8) but at approximately 10(4) -fold lower than in thyroid while in non-BC tumors (n = 5) they were at the limit of detection. TPO was expressed also in adipose tissue (n = 17), 10(3) -fold lower than in thyroid. Full length TPO (Mr 105-110 kDa) was detected in Western blots in the majority of examined tissues; preabsorption of the TPO antibody with recombinant TPO (but not LPO) reduced the signal, indicating specificity. The same occurred with some lower molecular weight bands, which could correspond to smaller TPO transcript isoforms, present in all samples. In conclusion, TPO is weakly expressed in BC and other tissues; this could partly explain the high frequency and protective role of TPOAb in BC patients. Further studies will investigate tissue specificity, function and immunogenicity of the novel TPO variants (some BC-specific) identified.
Subject(s)
Antigens, Neoplasm/immunology , Breast Neoplasms/immunology , Iodide Peroxidase/immunology , Thyroid Gland/immunology , Adipose Tissue/enzymology , Adipose Tissue/immunology , Autoantibodies/immunology , Autoimmunity/immunology , Breast Neoplasms/enzymology , Cross Reactions/immunology , Epitopes/immunology , Female , Humans , Symporters/immunology , Thyroid Gland/enzymologyABSTRACT
Trypanosoma cruzi is the protozoan agent of Chagas disease, and the most important parasitic disease in Latin America. Protozoa of the genus Leishmania are global agents of visceral and cutaneous leishmaniasis, fatal and disfiguring diseases. In the 1970s multilocus enzyme electrophoresis demonstrated that T. cruzi is a heterogeneous complex. Six zymodemes were described, corresponding with currently recognized lineages, TcI and TcIIa-e--now defined by multiple genetic markers. Molecular epidemiology has substantially resolved the phylogeography and ecological niches of the T. cruzi lineages. Genetic hybridization has fundamentally influenced T. cruzi evolution and epidemiology of Chagas disease. Genetic exchange of T. cruzi in vitro involves fusion of diploids and genome erosion, producing aneuploid hybrids. Transgenic fluorescent clones are new tools to elucidate molecular genetics and phenotypic variation. We speculate that pericardial sequestration plays a role in pathogenesis. Multilocus sequence typing, microsatellites and, ultimately, comparative genomics are improving understanding of T. cruzi population genetics. Similarly, in Leishmania, genetic groups have been defined, including epidemiologically important hybrids; genetic exchange can occur in the sand fly vector. We describe the profound impact of this parallel research on genetic diversity of T. cruzi and Leishmania, in the context of epidemiology, taxonomy and disease control.
Subject(s)
Chagas Disease/epidemiology , Leishmania , Leishmaniasis/epidemiology , Molecular Epidemiology , Phylogeny , Trypanosoma cruzi , Animals , Chagas Disease/parasitology , Chagas Disease/transmission , Ecosystem , Leishmania/classification , Leishmania/genetics , Leishmaniasis/parasitology , Leishmaniasis/transmission , South America/epidemiology , Trypanosoma cruzi/classification , Trypanosoma cruzi/geneticsABSTRACT
Antidepressants are among the most-prescribed class of drugs in the world and though weight gain is a common outcome of antidepressant treatment, that effect is not well understood. We employed an animal model comprised of 2 weeks of chronic restraint stress with antidepressant treatment, followed by diet-induced obesity. We showed that short-term antidepressant treatment had long-lasting effects, not only leading to weight gain, but also enhancing trabecular and cortical bone features in rats; therefore, weight gain in this model was different from that of the classic diet-induced obesity. Late in the post-restraint recovery period, antidepressant-treated animals were significantly heavier and had better bone features than saline-treated controls, when assessed in the distal femoral metaphysis. The propensity to gain weight might have influenced the rate of catch-up growth and bone allometry, as heavier animals treated with fluoxetine also had enhanced bone features when compared to non-stressed animals. Therefore, short-term antidepressant treatment ameliorated the long-term effects of stress on body growth and bone. Growth and bone structural features were associated with leptin levels, and the interaction between leptin levels and antidepressant was significant for bone mineral content, suggesting that short-term antidepressants in the context of long-term diet-induced obesity modified the role of leptin in bone formation. To our knowledge this is the first study reporting that short-term antidepressant treatment has long-lasting effects in restoring the effects of chronic stress in body weight and bone formation. Our findings may be relevant to the understanding and treatment of osteoporosis, a condition of increasing prevalence due to the aging population.
Subject(s)
Antidepressive Agents/pharmacology , Bone Density/drug effects , Stress, Psychological/drug therapy , Weight Gain/drug effects , Animals , Behavior, Animal/drug effects , Disease Models, Animal , Fluoxetine/pharmacology , Leptin/metabolism , Male , Obesity/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
In addition to causing widespread cell death and loss of brain function, cerebral ischaemia also induces extensive neuroplasticity. In humans, stroke is often accompanied by severe cognitive and psychiatric changes that are thought to arise as a consequence of this infarct-induced remodelling. A candidate for producing these post-stroke neuropsychiatric changes is Npas4, an activity-dependent transcription factor involved in synaptic plasticity whose expression is aberrantly up-regulated following ischaemic injury. In this study we investigated the role of Npas4 in modulating these stroke-induced neuropsychiatric responses by comparing the performance of wildtype and Npas4-/- mice in various cognitive and behavioural tasks in a photochemical model of focal cortical stroke. We show that this stroke model results in impaired spatial recognition memory and a reduction in despair-like behaviour that affect both genotypes to a similar degree. Moreover, mice lacking Npas4 also show differences in some aspects of post-stroke sociability and anxiety. Specifically, we show that while stroke had no effect on anxiety levels in wildtype mice, Npas4-/- mice became significantly more anxious following stroke. In addition, Npas4-/- mice retained a greater level of sociability in the acute post-stroke period in comparison to their wildtype littermates. Thus, our findings suggest that Npas4 may be involved in post-stroke psychiatric changes related to anxiety and sociability.
Subject(s)
Anxiety/etiology , Basic Helix-Loop-Helix Transcription Factors/deficiency , Social Behavior , Stroke/complications , Analysis of Variance , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Exploratory Behavior/physiology , Light/adverse effects , Maze Learning , Memory Disorders/etiology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Photochemistry , Rose Bengal/adverse effects , Saccharin/administration & dosage , Stroke/etiology , Stroke/psychology , Time FactorsABSTRACT
ras oncogenes are present in several types of cancers but are most frequently described in colon and pancreatic carcinomas. Consequently, ras is being targeted for drug development as a means to develop therapies for these types of cancer. The ras protein is posttranslationally modified by the addition of a farnesyl group, followed by cleavage of the COOH-terminal 3 amino acids and methylation of the prenylated cysteine. Because the posttranslational addition of farnesyl is obligatory not only for the remaining modifications to take place but also for ras control of cell growth, inhibitors of farnesylation are being developed as potential antitumor agents. In this report, a new peptidomimetic inhibitor of farnesyl transferase is described. This compound, B956, and its methyl ester B1086, inhibit the formation of colonies in soft agar of 14 human tumor cell lines expressing different ras oncogenes at concentrations between 0.2 and 60 microM. Higher concentrations of B956 (10-80 microM) were required to inhibit colony formation by 5 tumor cell lines without ras mutations. B956/B1086 at 100 mg/kg also inhibited tumor growth by EJ-1 human bladder carcinoma, HT1080 human fibrosarcoma, and to a lesser extent by HCT116 human colon carcinoma xenografts in nude mice. Furthermore, inhibition of tumor growth by B956 is shown to be correlated with inhibition of ras posttranslational processing in the tumor. Thus, peptidomimetic inhibitors of ras farnesylation have the potential to be developed as therapy for ras-dependent tumors.
Subject(s)
Alkyl and Aryl Transferases , Antineoplastic Agents/therapeutic use , Enzyme Inhibitors/therapeutic use , Methionine/analogs & derivatives , Neoplasms, Experimental/drug therapy , Transferases/antagonists & inhibitors , Animals , Female , Genes, ras , Humans , Methionine/therapeutic use , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasm Transplantation , Neoplasms, Experimental/pathology , Transplantation, Heterologous , Tumor Cells, CulturedABSTRACT
Epidemiological studies have revealed a significantly higher incidence of toxic adenoma (TA) and toxic multi-nodular goitre (TMNG) in regions of iodine deficiency. Fifty to eighty percent of TA and TMNG are caused by activation of the cAMP pathway, mostly by mutations in the thyrotrophin receptor (TSHR). We aimed to investigate whether iodide could modulate the biological effects of activating TSHR mutations. We have applied an in vitro model of TA comprising FRTL-5 cells stably expressing activating TSHR. We have mimicked the in vivo situation by examining the effects of prolonged exposure to iodide on the proliferation and signal transduction etc. of these cells. We observed an iodide-induced 'inhibition of proliferation' which was significant from 10 mM in the presence of serum but from 1 mM in its absence. The inhibition of proliferation was significantly higher in the activating mutant expressing FRTL-5 compared with control Neo or wild-type TSHR, indicating that the effect was mediated via the cAMP cascade. The effect was neither due to hyper-tonicity nor was it the result of an increase in cell death either by apoptosis or necrosis. Prolonged exposure to iodide produces an increase in cells in the G2 and post-G2 phases, indicating that G2/M blockade contributes to the mechanism of inhibition. The mutant expressing FRTL-5 cells have increased proliferation when chronically exposed to TSH, and this is associated with a reduction in phosphorylated (p) CREB levels. This contrasts with the effect of iodide in which inhibition of proliferation is accompanied by an increase in pCREB. In conclusion, our studies indicate that the biological effects of activating TSHR mutations vary with the ambient iodide supply and could be masked in regions of high iodine intake.
Subject(s)
Iodides/metabolism , Receptors, Thyrotropin/genetics , Adenylyl Cyclases/metabolism , Animals , Apoptosis/physiology , Cell Cycle/physiology , Cells, Cultured , Cyclic AMP/metabolism , Cyclic AMP Response Element-Binding Protein/metabolism , Mutation , Necrosis/metabolism , Osmotic Pressure , Phosphorylation , Rats , Receptors, Thyrotropin/metabolism , Thyroid Gland/metabolism , Thyrotropin/metabolismABSTRACT
Activating TSH receptor (TSHR) mutations are a major cause of toxic thyroid adenoma and familial hyperthyroidism, and more than 37 such mutations have been described. Previously their functional activity had been assessed in terms of cAMP and inositol phosphate production and predominantly in transiently transfected COS-7 (monkey embryonic kidney cells), a model that does not reflect effects on thyrocyte proliferation and function. Here we have performed a systematic comparison of wild-type and seven gain-of-function TSHR mutants, introduced into rat FRTL-5 and human thyrocytes, using retroviral vectors. Our results show that 1) biological potency of TSHR mutants in thyroid cells does not correlate with their cAMP levels in transfected COS cells, highlighting the importance of cellular context and level of expression when assessing biological effects of oncogenic mutations; 2) dissociation between stimulation of function and growth occurs with thyrocyte differentiated functions more readily stimulated than growth; 3) TSHR mutants show a similar order of potency in FRTL-5 cells and human thyrocytes; 4) mutants inducing the highest stimulation of adenylyl cyclase may paradoxically fail to induce proliferation; and 5) biological effects of cAMP activating TSHR mutants are attenuated by complex counterregulatory mechanisms at least at the level of phosphodiesterases and cAMP regulatory element modulator isoforms.
Subject(s)
Point Mutation , Receptors, Thyrotropin/genetics , Receptors, Thyrotropin/metabolism , Signal Transduction/genetics , Amino Acid Sequence , Animals , COS Cells , Cell Differentiation/physiology , Cell Division/physiology , Cyclic AMP/metabolism , Humans , Molecular Sequence Data , Retroviridae/genetics , Thyroid Gland/cytology , Transduction, Genetic , TransfectionABSTRACT
A(1) and A(2) adenosine receptors have been identified in the pituitary gland, but the cell type(s) on which they are located and their effects on pituitary cell growth are not known. Therefore, we analyzed the expression of A(1) and A(2) receptors in primary rat anterior pituitary cells, two pituitary folliculostellate (TtT/GF and Tpit/F1) and two pituitary endocrine (GH(3) and AtT20) cell lines, and compared their effects on cell proliferation. In anterior pituitary and folliculostellate cells, adenosine and adenosine receptor agonists (5'-N-ethylcarboxamidoadenosine, a universal agonist, and CGS 21680, an A(2A) receptor agonist) stimulated cAMP levels with a rank order of potency that indicates the presence of functional A(2B) receptors. This stimulation, however, was not observed in either GH(3) or AtT20 cells, where adenosine and the A(1) receptor agonist 2-chloro-N(6)-cyclopentyladenosine inhibited VIP/forskolin-stimulated cAMP production. Expression of A(2B) and A(1) receptors in the folliculostellate cells and that of the A(1) receptor in the endocrine cells were confirmed by RT-PCR, immunocytochemistry, and ligand binding. Adenosine and 5'-N-ethylcarboxamidoadenosine dose-dependently (10 nM to 10 microM) stimulated growth in the folliculostellate, but not in the endocrine, cells, whereas in the latter, 100 microM adenosine and 2-chloro-N(6)-cyclopentyladenosine inhibited cell proliferation by slowing cell cycle progression. These data highlight the differential expression of A(1) and A(2B) adenosine receptors in pituitary cells and provide evidence for opposing effects of adenosine on pituitary folliculostellate and endocrine cell growth.
Subject(s)
Adenosine/physiology , Pituitary Gland/cytology , Receptors, Purinergic P1/physiology , Animals , Binding, Competitive/drug effects , Cell Cycle/physiology , Cell Division/physiology , Cell Line , Cyclic AMP/physiology , Immunohistochemistry , Ligands , Male , Pituitary Gland/drug effects , Pituitary Gland/metabolism , Protein Biosynthesis , Protein Conformation , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Rats , Rats, Wistar , Receptor, Adenosine A2B , Receptors, Purinergic P1/drug effects , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The mitogenic activity of extracts of human non-functional pituitary tumours has been studied. Previously we have reported that the tumour extracts could be resolved into both high (> 13,000) and low (< 3,000) molecular weight fractions using Sephadex G-50 chromatography. Mitogenic activity was assayed by looking at trichloroacetic acid precipitable 3H-thymidine incorporation into GH3 cells. We have now purified the major component of the low molecular weight mitogenic fraction using reversed phase HPLC: the material was identified and found to be 5'-adenosine monophosphate (5'-AMP) using electron spray mass spectrometry. The mitogenic activity of 5'-AMP and the purified tumour extract was confirmed as both produced an increase in GH3 cell number after 4 days of treatment. In conclusion our results show that the major component of the low molecular weight mitogenic activity in human non-functional pituitary tumour extracts is 5'-AMP.
Subject(s)
Adenosine Monophosphate/metabolism , Mitogens/metabolism , Pituitary Neoplasms/metabolism , Adenosine Monophosphate/chemical synthesis , Adenosine Monophosphate/pharmacology , Animals , Cell Line , Chromatography, Gel , Chromatography, High Pressure Liquid , Dose-Response Relationship, Drug , Humans , Molecular Weight , Pituitary Neoplasms/chemistry , Pituitary Neoplasms/pathology , Rats , Thymidine/metabolism , Tissue Extracts/pharmacologyABSTRACT
A major problem in the study of human pituitary cells is their lack of proliferative capacity in vitro. To address this issue, we have infected normal human, postmortem pituitary cells in monolayer culture with a temperature-sensitive (tsA58) mutant of SV40 large T antigen. Several epithelial-like colonies were isolated; and one, designated CHP2, has been studied in detail to identify its functional characteristics. CHP2 cells have undergone more than 150 culture passages and retain an epithelial morphology. They exhibit tight temperature-dependent growth, in the presence and absence of serum, with cell division at 33 C and growth inhibition at 39 C. CHP2 cells, at both temperatures, showed diffuse immunostaining for human alpha-subunit and focal staining for TSH beta. Gene expression was confirmed by RT-PCR and sequencing. TRH and GnRH receptors were not detectable, and their absence was confirmed by their lack of effects on intracellular calcium and inositol phospholipids. Cytogenetic analysis showed that the cells had a modal peak in the diploid range and a smaller peak in the tetraploid range. There was also a consistent loss of chromosome 22 and a normal chromosome 2 homologue, the latter being replaced by one of two chromosome 2 markers, M2A or M2B. In conclusion, we have immortalized human pituitary cells using SV40 tsT, from which we have cloned a cell line expressing alpha-subunit and TSH beta.
Subject(s)
Gene Expression , Glycoprotein Hormones, alpha Subunit/genetics , Pituitary Gland/metabolism , Thyrotropin/genetics , Antigens, Polyomavirus Transforming/genetics , Cell Division , Cell Line, Transformed , Epithelial Cells/metabolism , Glycoprotein Hormones, alpha Subunit/analysis , Humans , Immunohistochemistry , Karyotyping , Keratins/analysis , Mutation , Pituitary Gland/chemistry , Pituitary Gland/virology , Pituitary Hormones, Anterior/analysis , Polymerase Chain Reaction , RNA, Messenger/analysis , RNA-Directed DNA Polymerase , Temperature , Thyrotropin/analysis , TransfectionABSTRACT
The effects of free fatty acids on somatostatin secretion, content and mRNA levels in fetal rat hypothalamic and cortical cell cultures were investigated. Somatostatin secretion and content were quantified by radioimmunoassay. Somatostatin mRNA levels were measured by Northern blot hybridization using a cRNA probe. Treatment with either caprylic acid (5 x 10(-3) M) or oleic acid (5 x 10(-5) M) for 90 min inhibited basal somatostatin secretion in both hypothalamic and cortical cell cultures. In addition, the increase in somatostatin secretion induced by incubation with veratridine (10(-4) M) or carbachol (10(-4) M) for 90 min was significantly reduced by the addition of caprylic acid, but somatostatin release stimulated by 5.6 x 10(-2) M KCl was not affected. Furthermore, treatment with these free fatty acids for 90 min markedly decreased somatostatin mRNA levels in both types of neurone culture. These inhibitory effects were transient, being observed after 90 min, but not after 5 h. These results support the probability that there is a role for free fatty acids in the regulation of somatostatin mRNA levels and somatostatin secretion in both hypothalamic and cortical cell cultures.
Subject(s)
Cerebral Cortex/metabolism , Fatty Acids, Nonesterified/physiology , Hypothalamus/metabolism , Neurons/metabolism , RNA, Messenger/metabolism , Somatostatin/metabolism , Animals , Caprylates/pharmacology , Cells, Cultured , Cerebral Cortex/cytology , Cerebral Cortex/embryology , Hypothalamus/cytology , Hypothalamus/embryology , Neurons/cytology , Oleic Acid , Oleic Acids/pharmacology , Rats , Somatostatin/genetics , Time FactorsABSTRACT
We have developed a fetal rat hypothalamic cell culture system for the study of factors controlling the acute release of TRH. Release of TRH by the cells has been characterized by reversed-phase high pressure liquid chromatography and about 86% of the total immunoreactivity in the medium co-eluted with synthetic TRH. Release of TRH by the cells in response to 56 mmol K+/l increased between days 5 and 9 of culture but reached a plateau thereafter. Cell contents of TRH did not change significantly between days 5 and 14 of culture. Release of TRH from the cells was stimulated by K+ (56 mmol/l), veratridine (100 mumol/l) and ouabain (100 mumol/l) to 550, 480 and 335% of basal release respectively over a 1-h period. Release of TRH was dependent upon calcium in that it was absent when calcium-free medium was used and could be blocked by verapamil (20 mumol/l); however it could not be blocked by nifedipine (50 mumol/l). The calcium ionophore blocked by nifedipine (50 mumol/l). The calcium ionophore A23187 (1 mumol/l) stimulated TRH release to 340% of basal release. Tetrodotoxin (1 mumol/l) completely abolished the release in response to veratridine but had no effect on the release stimulated by K+ (56 mmol/l). The calmodulin antagonists trifluoperazine and triflupromazine (50 mumol/l) inhibited veratridine-stimulated TRH release. This was at a site after calcium influx as they also inhibited A23187-stimulated TRH release. The highly specific calmodulin antagonist W7 (10 mumol/l) also inhibited both veratridine and A23187-stimulated TRH release whereas, at the same concentration, its inactive analogue W5 did not significantly inhibit TRH release in response to either stimulus.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Calcium/physiology , Calmodulin/physiology , Hypothalamus/metabolism , Thyrotropin-Releasing Hormone/metabolism , Animals , Cells, Cultured , Rats , Rats, Inbred StrainsABSTRACT
We have studied the effect of dopamine together with agonist and antagonist drugs of different specificities on the release of TRH from the perfused, intact hypothalamus of the adult rat in vitro. Dopamine produced a dose-related stimulatory effect on TRH release with maximal effect being achieved at 1 mumol/l (increase over basal, 118 +/- 16.5 (S.E.M.) fmol TRH; P less than 0.001 vs basal). This effect was mimicked by the specific D2-agonist drugs bromocriptine (0.1 mumol/l) and LY 171555 (0.1 mumol/l) (increase over basal values, 137.5 +/- 13.75 fmol and 158.6 +/- 10.7 fmol respectively; P less than 0.001 vs basal), but not by the D1-agonist SKF 38393A. The stimulatory effect of dopamine (1 mumol/l) was blocked in a stereospecific manner by the active (D) but not by the inactive (L) isomers of the dopamine antagonist butaclamol. Similar blockade was achieved with the specific D2-antagonist domperidone (0.01 mumol/l) whereas the D1-antagonist SCH 23390 was only effective when used at a concentration 100 times greater. Lower concentrations (0.01 mumol/l) of this D1-antagonist did not block the stimulatory effect of dopamine. High-performance liquid chromatography characterization of the material secreted within the hypothalamus showed one single peak of immunoreactive material which coeluted with synthetic TRH. These data suggest that dopamine exerts a stimulatory role in the control of hypothalamic TRH release by acting at specific D2-receptors.
Subject(s)
Dopamine/pharmacology , Hypothalamus/drug effects , Receptors, Dopamine/drug effects , Thyrotropin-Releasing Hormone/metabolism , Animals , Hypothalamus/metabolism , Male , Perfusion , Rats , Rats, Inbred Strains , Receptors, Dopamine/physiology , Receptors, Dopamine D2ABSTRACT
Lysosomes degrade a wide range of macromolecules to yield monomer products which are exported out of the lysosome by a series of transporters. In addition, lysosomes perform a range of other functions which are cell or tissue specific. In order to gain insight into the tissue specific role of lysosomes, carrier-ampholyte two-dimensional electrophoresis (2-DE) was used in combination with N-terminal sequencing to identify the major proteins present in both the membrane and luminal space of placental lysosomes. From the 45 N-terminal peptide sequences generated, 14 luminal and five membrane proteins were identified while three other sequences were novel. The sequenced proteins were a mixture of lysosomal and non-lysosomal proteins. The lysosomal proteins consisted of gamma-interferon-inducible protein (IP-30), Saposin D, cathepsins B and D, beta-hexosaminidase, palmitoyl protein thioesterase, alpha-glucosidase, and LAMP-1. The non-lysosomal proteins were serum albumin, serotransferrin, haemoglobin gamma G chain, alpha-1-antitrypsin, placental lactogen, endoplasmin, peptide binding protein 74, p60 lymphocyte protein, p450 side chain cleavage enzyme and placental alkaline phosphatase. The 2-DE maps obtained in this study are the first to identify the major proteins in both the lumen and membrane of placental lysosomes through sequence analysis, and thus provide the basis upon which to build a complete 2-DE database of the lysosome. Furthermore, the identities of the proteins sequenced from the placental lysosomes suggest a role for lysosomes in the transport of nutrients across the trophoblastic layer.
Subject(s)
Lysosomes/chemistry , Membrane Proteins/analysis , Placenta/metabolism , Pregnancy Proteins/analysis , Pregnancy/metabolism , Adult , Amino Acid Sequence , Cell Fractionation , Electrophoresis, Gel, Two-Dimensional , Female , Humans , Intracellular Membranes/chemistry , Molecular Sequence Data , Peptide MappingABSTRACT
We have demonstrated that high doses of adenosine (0.1 mM) inhibit the growth of the rat pituitary GH(3) cell line. This effect was not mediated via cell surface adenosine receptors as the adenosine agonists N(6)-(2-phenylisopropyl)adenosine (PIA, 0.1 mM) and 5'-N- ethylcarboxamidoadenosine (NECA, 0.1 mM) were unable to reproduce the growth inhibitory effect of adenosine. The adenosine uptake inhibitor dipyridamole completely blocked the growth inhibitory effect of adenosine suggesting that its site of action is intracellular. Epidermal growth factor (EGF) was able to completely reverse the growth inhibitory effect, restoring the growth rate of cultures treated with adenosine and EGF to that of control cultures. The ratio of cells in G(0)/G(1):S:G(2)/M phases of the cell cycle was unaltered in adenosine treated compared with control cultures suggesting that there was no change in the rate of cell division, however the degree of apoptosis was markedly increased in adenosine treated cultures. EGF was able to reduce the adenosine induced apoptosis almost to levels seen in the control cultures. Thus the mechanism of the growth inhibitory effect of adenosine does not appear to be by reducing the rate of cell division but rather by increasing the rate of cell death and EGF restores the growth rate of adenosine treated cells to that of untreated cells by preventing adenosine induced apoptosis.
Subject(s)
Adenosine/pharmacology , Epidermal Growth Factor/pharmacology , Pituitary Gland/cytology , Adenosine/analogs & derivatives , Adenosine/metabolism , Adenosine-5'-(N-ethylcarboxamide) , Animals , Biological Transport/drug effects , Cell Cycle , Cell Division/drug effects , Cell Line , DNA Fragmentation/physiology , Dipyridamole/pharmacology , Phenylisopropyladenosine/pharmacology , Purinergic P1 Receptor Agonists , RatsABSTRACT
The proopiomelanocortin (POMC) gene is highly expressed in the pituitary gland where the resulting mRNA of 1200 base pairs (bp) gives rise to a full-length protein sequence. In peripheral tissues however both shorter and longer POMC variants have been described, these include for example placental tissue which contain 800 (truncated at the 5' end) and 1500 as well as the 1200 bp transcripts. The importance of the 800 bp transcript is unclear as the lack of a signal sequence renders the molecule to be non-functional. This transcript has not been previously demonstrated in the pituitary gland. In this report we show evidence of a 5' truncated POMC gene in human pituitary corticotroph macroadenoma cells (JE) maintained in primary culture for >1 year. The original tumour tissue and the derived cells during early passage (up to passage 4-5) immunostained for ACTH and in situ hybridisation confirmed the presence of the POMC gene in the cultured cells. These cells also secreted 15-40 pg/10(5) cells/24 h ACTH. In addition, as expected RT-PCR demonstrated the presence of all three POMC gene exons and is thus indicative of a full-length POMC gene. In late culture passages (passages 8-15) JE cells ceased to express ACTH and cell growth became very slow due presumably to cells reaching their Hayflick limit. ACTH immunostaining in these cells was undetectable and ACTH secretion was also at the detection limits of the assay and no greater than 10 pg/10(5) cells/24 h. ACTH precursor molecules were also undetectable. RT-PCR for the POMC gene in these late passage cells showed that only exon 3 was detectable, in contrast to early passage cells where all three exons were present. In summary we isolated in culture, human pituitary cells that possessed initially all three exons of the POMC gene and immunostained for ACTH. On further passaging these cells showed a loss of exons 1 and 2 in the POMC gene and a loss of ACTH immunostaining and secretion. We would like to suggest that the loss of ACTH peptide expression in these late passage cells is in part due to the loss of the POMC signal sequence. An alternative explanation for our findings is that there were originally two populations of corticotrophs in the cultures, one of which possessed the full-length POMC gene and the other only the 5' truncated POMC transcript and it is these latter cells which survived in culture. In either scenario this is the first report of the 5' truncated POMC gene occurring in pituitary cells.
Subject(s)
Adrenocorticotropic Hormone/metabolism , Pituitary Gland/cytology , Pro-Opiomelanocortin/genetics , Protein Sorting Signals , Adenoma , Adrenocorticotropic Hormone/genetics , Cells, Cultured , Exons/genetics , Humans , In Situ Hybridization , Pituitary Neoplasms , Pro-Opiomelanocortin/chemistry , Pro-Opiomelanocortin/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
Abstract We describe the development and validation of a two-site immunochemiluminometric assay for rat growth hormone-releasing hormone (GHRH) based on the affinity purification of polyclonal rabbit antisera to rat GHRH using a human 1-29 GHRH affinity column. Assay sensitivity is 3.2 pg/ml using 100 mul of unextracted sample and the working range for the assay within 15% confidence limits is 64 to 5,000 pg/ml. Rat hypothalamic extract and secreted material demonstrated a single large peak of immunoreactive material coeluting with synthetic rat GHRH on high-performance liquid chromatography with a smaller, earlier peak which probably represents methionine sulphoxide [Met(O)(27)] GHRH. Extracted material diluted in parallel to the standard curve. Incubated rat hypothalami readily released measurable amounts of rat GHRH which responded appropriately to depolarization with 60 mM K(+) in a Ca(2+)-dependent manner.