ABSTRACT
We evaluated the impact of early embryonic deletion of huntingtin (htt) from pyramidal neurons on cortical development, cortical neuron survival and motor behavior, using a cre-loxP strategy to inactivate the mouse htt gene (Hdh) in emx1-expressing cell lineages. Western blot confirmed substantial htt reduction in cerebral cortex of these Emx-httKO mice, with residual cortical htt in all likelihood restricted to cortical interneurons of the subpallial lineage and/or vascular endothelial cells. Despite the loss of htt early in development, cortical lamination was normal, as revealed by layer-specific markers. Cortical volume and neuron abundance were, however, significantly less than normal, and cortical neurons showed reduced brain-derived neurotrophic factor (BDNF) expression and reduced activation of BDNF signaling pathways. Nonetheless, cortical volume and neuron abundance did not show progressive age-related decline in Emx-httKO mice out to 24months. Although striatal neurochemistry was normal, reductions in striatal volume and neuron abundance were seen in Emx-httKO mice, which were again not progressive. Weight maintenance was normal in Emx-httKO mice, but a slight rotarod deficit and persistent hyperactivity were observed throughout the lifespan. Our results show that embryonic deletion of htt from developing pallium does not substantially alter migration of cortical neurons to their correct laminar destinations, but does yield reduced cortical and striatal size and neuron numbers. The Emx-httKO mice were persistently hyperactive, possibly due to defects in corticostriatal development. Importantly, deletion of htt from cortical pyramidal neurons did not yield age-related progressive cortical or striatal pathology.
Subject(s)
Cerebral Cortex/growth & development , Cerebral Cortex/metabolism , Corpus Striatum/growth & development , Corpus Striatum/metabolism , Huntingtin Protein/deficiency , Pyramidal Cells/metabolism , Animals , Brain-Derived Neurotrophic Factor/metabolism , Cell Count , Cell Survival/physiology , Cerebral Cortex/pathology , Corpus Striatum/pathology , Female , Huntingtin Protein/genetics , Male , Mice, Knockout , Motor Activity/physiology , Pyramidal Cells/pathologyABSTRACT
Exercise reduces the risk of developing a number of neurological disorders and increases the efficiency of cellular energy production. However, overly strenuous exercise produces oxidative stress. Proper oxygenation is crucial for the health of all tissues, and tight regulation of cellular oxygen is critical to balance O2 levels and redox homeostasis in the brain. Hypoxia Inducible Factor (HIF)1α and HIF2α are transcription factors regulated by cellular oxygen concentration that initiate gene regulation of vascular development, redox homeostasis, and cell cycle control. HIF1α and HIF2α contribute to important adaptive mechanisms that occur when oxygen and ROS homeostasis become unbalanced. It has been shown that preconditioning by exposure to a stressor prior to a hypoxic event reduces damage that would otherwise occur. Previously we reported that 3 months of exercise protects SNpc dopaminergic (DA) neurons from toxicity caused by Complex I inhibition. Here, we identify the cells in the SNpc that express HIF1α and HIF2α and show that running exercise produces hypoxia in SNpc DA neurons, and alters the expression of HIF1α and HIF2α. In mice carrying a conditional knockout of Hif1α in postnatal neurons we observe that exercise alone produces SNpc TH+ DA neuron loss. Loss of HIF1α also abolishes exercise-induced neuroprotection. In mice lacking Hif2α in postnatal neurons, the number of TH+ DA neurons in the adult SNpc is diminished, but 3months of exercise rescues this loss. We conclude that HIF1α is necessary for exercise-induced neuroprotection and both HIF1α and HIF2α are necessary for the survival and function of adult SNpc DA neurons.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Dopaminergic Neurons/physiology , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , MPTP Poisoning/rehabilitation , Pars Compacta/cytology , Physical Conditioning, Animal/methods , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Cell Survival/genetics , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , Glial Fibrillary Acidic Protein/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , MPTP Poisoning/chemically induced , MPTP Poisoning/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , RNA, Messenger/metabolism , RNA, Untranslated/genetics , RNA, Untranslated/metabolism , Tyrosine 3-Monooxygenase/metabolism , beta-Galactosidase/metabolismABSTRACT
Integrins are heterodimeric transmembrane cell adhesion receptors that are essential for a wide range of biological functions via cell-matrix and cell-cell interactions. Recent studies have provided evidence that some of the subunits in the integrin family are involved in synaptic and behavioral plasticity. To further understand the role of integrins in the mammalian central nervous system, we generated a postnatal forebrain and excitatory neuron-specific knockout of alpha8-integrin in the mouse. Behavioral studies showed that the mutant mice are normal in multiple hippocampal-dependent learning tasks, including a T-maze, non-match-to-place working memory task for which other integrin subunits like alpha3- and beta1-integrin are required. In contrast, mice mutant for alpha8-integrin exhibited a specific impairment of long-term potentiation (LTP) at Schaffer collateral-CA1 synapses, whereas basal synaptic transmission, paired-pulse facilitation and long-term depression (LTD) remained unaffected. Because LTP is also impaired in the absence of alpha3-integrin, our results indicate that multiple integrin molecules are required for the normal expression of LTP, and different integrins display distinct roles in behavioral and neurophysiological processes like synaptic plasticity.
Subject(s)
CA1 Region, Hippocampal/physiology , Integrins/physiology , Learning/physiology , Long-Term Potentiation/genetics , Animals , CA1 Region, Hippocampal/growth & development , Female , Integrins/deficiency , Integrins/genetics , Learning Disabilities/genetics , Learning Disabilities/metabolism , Learning Disabilities/physiopathology , Male , Maze Learning/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Protein Subunits/deficiency , Protein Subunits/genetics , Protein Subunits/physiology , Synaptic Transmission/geneticsABSTRACT
With spontaneous elongation of the CAG repeat in the R6/2 transgene to > or =335, resulting in a transgene protein too large for passive entry into nuclei via the nuclear pore, we observed an abrupt increase in lifespan to >20 weeks, compared to the 12 weeks common in R6/2 mice with 150 repeats. In the > or =335 CAG mice, large ubiquitinated aggregates of mutant protein were common in neuronal dendrites and perikaryal cytoplasm, but intranuclear aggregates were small and infrequent. Message and protein for the > or =335 CAG transgene were reduced to one-third that in 150 CAG R6/2 mice. Neurological and neurochemical abnormalities were delayed in onset and less severe than in 150 CAG R6/2 mice. These findings suggest that polyQ length and pathogenicity in Huntington's disease may not be linearly related, and pathogenicity may be less severe with extreme repeats. Both diminished mutant protein and reduced nuclear entry may contribute to phenotype attenuation.
Subject(s)
Huntington Disease/genetics , Longevity/genetics , Nerve Tissue Proteins/genetics , Nuclear Proteins/genetics , Trinucleotide Repeat Expansion , Aging , Animals , Base Sequence , Brain/metabolism , Brain/pathology , DNA Mutational Analysis , Disease Models, Animal , Gene Expression , Huntingtin Protein , Huntington Disease/mortality , Mice , Mice, Transgenic , Molecular Sequence Data , Neurons/metabolism , Neurons/ultrastructure , Peptides/physiology , Phenotype , RNA, Messenger/metabolism , Survival RateABSTRACT
To determine whether neurons lacking huntingtin can participate in development and survive in postnatal brain, we used two approaches in an effort to create mice consisting of wild-type cells and cells without huntingtin. In one approach, chimeras were created by aggregating the 4-8 cell embryos from matings of Hdh (+/-) mice with wild-type 4-8 cell embryos. No chimeric offspring that possessed homozygous Hdh (-/-) cells were obtained thereby, although statistical considerations suggest that such chimeras should have been created. By contrast, Hdh (-/-) ES cells injected into blastocysts yielded offspring that were born and in adulthood were found to have Hdh (-/-) neurons throughout brain. The Hdh (-/-) cells were, however, 5-10 times more common in hypothalamus, midbrain, and hindbrain than in telencephalon and thalamus. Chimeric animals tended to be smaller than wild-type littermates, and chimeric mice rich in Hdh (-/-) cells tended to show motor abnormalities. Nonetheless, no brain malformations or pathologies were evident. The apparent failure of aggregation chimeras possessing Hdh (-/-) cells to survive to birth is likely attributable to the previously demonstrated critical role of huntingtin in extraembryonic membranes. That Hdh (-/-) cells in chimeric mice created by blastocyst injection are under-represented in adult telencephalon and thalamus implies a role for huntingtin in the development of these regions, whereas the neurological dysfunction in brains enriched in Hdh (-/-) cells suggests a role for huntingtin in adult brain. Nonetheless, the lengthy survival of Hdh (-/-) cells in adult chimeric mice indicates that individual neurons in many brain regions do not require huntingtin to participate in normal brain development and to survive.
Subject(s)
Brain/pathology , Chimera/genetics , Huntington Disease/genetics , Nerve Tissue Proteins/deficiency , Neurons/metabolism , Nuclear Proteins/deficiency , Animals , Behavior, Animal , Brain/embryology , Brain/metabolism , Cell Differentiation , Cell Movement , Cell Survival , Genes, Reporter , Genotype , Huntingtin Protein , Hypothalamus/metabolism , Hypothalamus/pathology , Mesencephalon/metabolism , Mesencephalon/pathology , Mice , Motor Activity/genetics , Nerve Tissue Proteins/biosynthesis , Nerve Tissue Proteins/genetics , Nervous System Diseases/diagnosis , Nervous System Diseases/genetics , Neurons/pathology , Nuclear Proteins/biosynthesis , Nuclear Proteins/genetics , Organ Specificity/genetics , Rhombencephalon/metabolism , Rhombencephalon/pathology , Stem Cells , Survival Rate , Telencephalon/metabolism , Telencephalon/pathology , Thalamus/metabolism , Thalamus/pathologyABSTRACT
Conditional gene repair mutations in the mouse can assist in cell lineage analyses and provide a valuable complement to conditional gene inactivation strategies. We present a method for the generation of conditional gene repair mutations that employs a loxP-flanked (floxed) selectable marker and transcriptional/translational stop cassette (neostop) located within the first intron of a target gene. In the absence of Cre recombinase, expression of the targeted allele is suppressed generating a null allele, while in the presence of Cre, excision of neostop restores expression to wild-type levels. To test this strategy, we have generated a conditional gene repair allele of the mouse Huntington's disease gene homolog (HDH:). Insertion of neostop within the HDH: intron 1 generated a null allele and mice homozygous for this allele resembled nullizygous HDH: mutants and died after embryonic day 8.5. In the presence of a cre transgene expressed ubiquitously early in development, excision of neostop restored HDH: expression and rescued the early embryonic lethality. A simple modification of this strategy that permits the generation of conventional gene knockout, conditional gene knockout and conditional gene repair alleles using one targeting construct is discussed.
Subject(s)
Gene Silencing , Mutagenesis/genetics , Viral Proteins , Alleles , Animals , Blotting, Northern , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Female , Gene Expression Regulation, Developmental , Genetic Engineering/methods , Genotype , Huntingtin Protein , Integrases/genetics , Integrases/metabolism , Male , Mice , Mice, Knockout , Mutation , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , RNA/genetics , RNA/metabolism , Recombination, Genetic , TransfectionABSTRACT
Inactivation of the mouse homologue of the Huntington disease gene (Hdh) results in early embryonic lethality. To investigate the normal function of Hdh in the adult and to evaluate current models for Huntington disease (HD), we have used the Cre/loxP site-specific recombination strategy to inactivate Hdh expression in the forebrain and testis, resulting in a progressive degenerative neuronal phenotype and sterility. On the basis of these results, we propose that huntingtin is required for neuronal function and survival in the brain and that a loss-of-function mechanism may contribute to HD pathogenesis.
Subject(s)
Brain/metabolism , Gene Targeting , Infertility, Male/etiology , Nerve Tissue Proteins/antagonists & inhibitors , Neurodegenerative Diseases/etiology , Nuclear Proteins/antagonists & inhibitors , Testis/metabolism , Viral Proteins , Alleles , Animals , Brain/pathology , Female , Glial Fibrillary Acidic Protein/analysis , Huntingtin Protein , Infertility, Male/genetics , Infertility, Male/metabolism , Integrases/genetics , Integrases/metabolism , Longevity/genetics , Male , Mice , Microtubule-Associated Proteins/analysis , Molecular Sequence Data , Movement Disorders/etiology , Movement Disorders/genetics , Movement Disorders/metabolism , Nerve Tissue Proteins/physiology , Neurodegenerative Diseases/genetics , Neurodegenerative Diseases/metabolism , Nuclear Proteins/physiology , Phenotype , Recombinant Fusion Proteins/metabolism , Recombination, Genetic , Regulatory Sequences, Nucleic Acid , Spermatogenesis/genetics , Testis/pathologyABSTRACT
Huntingtin-associated protein 1 (HAP1) interacts with the product of the Huntington's disease gene. To investigate the function of Hap1 in development and in the adult mouse, we have examined the expression of Hap1 by northern analysis and in situ hybridization histochemistry. Hap1 expression is first detected in the embryonic day 8.5 (E8.5) neuroepithelium. Expression persists throughout development, predominantly in the brain and spinal cord, and to a lesser extent in enteric neurons and abdominal sympathetic ganglia. In the adult, Hap1 expression is detected not only in the brain but also in the ovary, testis, and the intermediate lobe of the pituitary.
Subject(s)
Nerve Tissue Proteins/metabolism , Animals , Blotting, Northern , Embryo, Mammalian/anatomy & histology , Embryo, Mammalian/metabolism , Embryonic and Fetal Development , In Situ Hybridization , Mice , Mice, Inbred C57BL , Organ SpecificityABSTRACT
To explore the potential of a simple and rapid approach for ubiquitous conditional gene disruption, we have generated Cre-producer mouse transgenic lines (Hs-cre1, 6 and 7) expressing a recombinase transgene (cre) from a heat shock gene promoter and tested their performance in Cre-mediated excision of target DNA in crosses with Cre-responder strains carrying loxP-modified alleles of the genes encoding the Huntington's disease gene homolog (Hdh), the epidermal growth factor receptor (Egfr), and the type 1 insulin-like growth factor receptor (Igf1r). Analyses of progeny possessing various transgene/reporter combinations showed that cre expression can occur without heat shock in early embryos, but this constitutive transcription is stochastic and transgene dependent. Thus, Hs-cre1 behaves predominantly as a "deleter" strain, since the majority of progeny (approximately 70-85%) exhibit complete recombination, regardless of reporter locus. Lines Hs-cre6 and Hs-cre7, however, function successfully as "mosaicking" strains because, in addition to two extreme classes of progeny with 0% or 100% recombination, they generate an intermediate class of mosaics exhibiting various degrees of partial DNA excision. Notably, the frequency of offspring in each class varies between reporters, but mosaic embryos are consistently obtained in adequate numbers (approximately 30-60%). The Hs-cre6 transgene is also inducible and can be used to introduce mosaicism into adult tissues at preselected developmental times by heat shock treatment of mice with 0% recombination in tail DNA. By bypassing the lethality resulting from some gene knockouts, mosaic embryos and mice make particular mutational analyses possible and are also very useful for the identification of cell lineage-specific gene functions.
Subject(s)
Heat-Shock Proteins/genetics , Integrases/genetics , Promoter Regions, Genetic/genetics , Transgenes/genetics , Viral Proteins , Animals , Embryo, Mammalian/metabolism , Female , Gene Expression Regulation , Gene Expression Regulation, Developmental , Genes, Reporter/genetics , HSP70 Heat-Shock Proteins/genetics , HSP70 Heat-Shock Proteins/metabolism , Heat-Shock Proteins/metabolism , Hot Temperature , Male , Mice , Mice, Transgenic , Mutagenesis, Site-Directed , Pregnancy , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Recombination, Genetic , Tissue DistributionABSTRACT
Huntington's disease (HD) is a neurodegenerative disease associated with polyglutamine expansion in huntingtin, a widely expressed protein. The function of huntingtin is unknown although huntingtin plays a fundamental role in development since gene targeted HD (-) (/-)mouse embryos die shortly after gastrulation. Expression of huntingtin is detected in spleen and thymus but its role in hematopoiesis has not been examined. To determine the function of huntingtin and to provide insight into potential pathologic mechanisms in HD, we analyzed the role of huntingtin in hematopoietic development. Expression of huntingtin was analyzed in a variety of hematopoietic cell types, and in vitro hematopoiesis was assessed using an HD ( +/-)and several HD( -) (/-)embryonic stem (ES) cell lines. Although wild-type, HD ( +/-)and HD( -) (/-)ES cell lines formed primary embryoid bodies (EBs) with similar efficiency, the numbers of hematopoietic progenitors detected at various stages of the in vitro differentiation were reduced in HD ( +/-)and HD( -/-)() ()ES cell lines examined. Expression analyses of hematopoietic markers within the EBs revealed that primitive and definitive hematopoiesis occurs in the absence of huntingtin. However, further analysis using a suspension culture in the presence of hematopoietic cytokines demonstrated a highly significant gene dosage-dependent decrease in proliferation and/or survival of HD ( +/-)and HD( -) (/-)cells. Enrichment for the CD34(+)cells within the EB confirmed that the impairment is intrinsic to the hematopoietic cells. These obser- vations suggest that huntingtin expression is required for the generation and expansion of hematopoietic cells and provides an alternative system in which to assess the function of huntingtin.
Subject(s)
Hematopoiesis/physiology , Nerve Tissue Proteins/metabolism , Nuclear Proteins/metabolism , Stem Cells/metabolism , Animals , Blotting, Southern , Blotting, Western , Cell Line , Cells, Cultured , Chimera , Embryo, Mammalian , Humans , Huntingtin Protein , Mice , Mice, Inbred C57BL , Mice, Inbred ICR , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Mouse embryos nullizygous for a targeted disruption of the Huntington's disease gene homologue (Hdh), which encodes a protein (huntingtin) of unknown biochemical function, become developmentally retarded and disorganized, and die early in development. Using chimeric analysis, we demonstrate that extensively chimeric embryos derived by injection of Hdh null ES cells into wild-type host blastocysts are rescued from lethality. In contrast, when wild-type ES cells are injected into Hdh null blastocysts, the chimeric embryos are morphologically indistinguishable from Hdh null mutants derived from natural matings, and die shortly after gastrulation. Therefore, the primary defect in the absence of huntingtin lies in extraembryonic tissues, whereas the epiblast and its derivatives are affected secondarily. It is likely that the mutation results in impairment of the nutritive functions of the visceral endoderm, which otherwise appears to differentiate normally, as evidenced by the expression of several specific marker genes. Consistent with preliminary histochemical analysis indicating that at least the transport of ferric ions is defective in Hdh mutants and in conjunction with the known localization of huntingtin in the membranes of vesicles associated with microtubules, we hypothesize that this protein is involved in the intracellular trafficking of nutrients in early embryos.
Subject(s)
Embryo, Mammalian/physiology , Embryonic and Fetal Development/genetics , Nerve Tissue Proteins/deficiency , Nerve Tissue Proteins/genetics , Nuclear Proteins/deficiency , Nuclear Proteins/genetics , Animals , Blastocyst/physiology , Chimera , Crosses, Genetic , DNA Primers , Endoderm/physiology , Female , Fetal Death , Gene Expression Regulation, Developmental , Genetic Markers , Heterozygote , Huntingtin Protein , Huntington Disease/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Mice, Knockout , Nerve Tissue Proteins/biosynthesis , Nuclear Proteins/biosynthesis , Polymerase Chain Reaction , Recombinant Proteins/biosynthesis , beta-Galactosidase/biosynthesisABSTRACT
A large body of evidence implicates the second and third intracellular loops and the carboxyl-terminal portion of many G protein-coupled receptors as sites responsible for the interaction to G proteins. We synthesized a number of peptides from selected sites of the murine delta-opioid receptor and measured their ability to modify ligand-stimulated G protein activation and 3H agonist binding to the receptor. In membranes from Rat-1 fibroblasts transfected to express the murine delta-opioid receptor stably (clone D2 cells), the delta-opioid agonist [D-Ser2-Leu5-Thr6]enkephalin (DSLET) stimulated high affinity GTPase activity, which was inhibited by peptides that are derived from the proximal (i3.1) and the distal portions (i3.3) of the third intracellular loop with IC50 values of 15 +/- 5 and 50 +/- 4 microM, respectively. Peptides i3.1 and i3.3 inhibited DSLET-stimulated [35S]guanosine 5'-O-thiotriphosphate binding in the same membranes. However, a peptide designated i4, which was derived from a juxtamembranous region of the carboxyl-terminal tail of the delta-opioid receptor, failed to alter agonist-mediated high affinity GTPase activity or agonist-driven [35S]guanosine 5'-O-thiotriphosphate binding. Specific binding of [3H]DSLET to membrane preparations from clone D2 was reduced by peptides i3.1 and i4. Combinations of these peptides abolished detectable [3H]DSLET binding in the same membranes. Peptides i3.1 and i3.3 also destabilized the high affinity state of the receptor as assessed in 3H agonist binding on membranes from neuroblastoma X glioma (NG108-15) hybrid cells, which express the delta-opioid receptor endogenously; furthermore, delta-opioid receptor-stimulated GTPase activity in the same membranes was inhibited by peptides i3.1 and i3.3 but i4 was inactive. In contrast, peptides derived from the second intracellular loop (i2.1 and i2.2), an intermediate portion of the third intracellular loop (i3.2), and the extreme amino-terminal region of the receptor were without effect in these assays. These observations indicate that although peptides i3.1, i3.3, and i4 act via different mechanisms, they provide evidence that at least two sites of the third intracellular loop and part of the carboxyl-terminal tail of the delta-opioid receptor are important in the interaction between this receptor and cellular G proteins. Collectively, these results provide novel information about regions of the delta-opioid receptor that are involved in G protein coupling and high affinity agonist binding.
Subject(s)
GTP-Binding Proteins/metabolism , Peptide Fragments/pharmacology , Protein Structure, Tertiary , Receptors, Opioid, delta/metabolism , Amino Acid Sequence , Analgesics/metabolism , Analgesics/pharmacology , Animals , Binding Sites , Enkephalin, Leucine/analogs & derivatives , Enkephalin, Leucine/metabolism , Enkephalin, Leucine/pharmacology , Fibroblasts/metabolism , GTP Phosphohydrolases/metabolism , GTP-Binding Proteins/agonists , Glioma , Hybrid Cells , Kinetics , Mice , Molecular Sequence Data , Neuroblastoma , Peptide Fragments/chemical synthesis , Peptide Fragments/metabolism , Rats , Receptors, Opioid, delta/agonists , Structure-Activity Relationship , Substrate Specificity , Transfection , Tritium , Tumor Cells, CulturedABSTRACT
1. In in vitro studies with adult male rats we have recently shown that the delta-opioid agonist DTLET inhibits the release of the Gonadotropin-Releasing Hormone (GnRH) from hypothalamic fragments containing the arcuate nucleus and the median eminence. This effect is receptor mediated and eicosanoid dependent (Gerozissis et al., 1993). 2. In the present study we report that the delta-opioid antagonists with negative intrinsic activity, Diallyl-G and ICI 174864, applied under the same experimental conditions (30 min static incubations at 37 degrees C, in a potassium rich milieu), in the absence of the agonist DTLET, also exert a similar to the agonist inhibitory effect on the release of GnRH. 3. The dose-dependent inhibitory effect of Diallyl-G on GnRH release is reversed by increasing concentrations of DTLET. The mu and delta opioid antagonist, naloxone is without effect in the absence of DTLET. However, naloxone acts as an antagonist on the Diallyl-G-induced inhibition of GnRH release. 4. Diallyl-G also inhibits the release of prostaglandin E2 (PGE2). In the presence of indomethacin or nordihydroguaiaretic acid, Diallyl-G is ineffective to further inhibit the release of GnRH. These latter observations taken together with the results of eicosanoid estimation suggest that PGE2 but not leukotrienes participate in the agonist-independent effects of Diallyl-G on GnRH release. 5. Therefore these results support the hypothesis that delta-opioid antagonists with negative intrinsic activity exert agonist-independent biological responses similar to those of the agonists.
Subject(s)
Gonadotropin-Releasing Hormone/metabolism , Receptors, Opioid, delta/antagonists & inhibitors , Animals , Arachidonic Acid/metabolism , Arcuate Nucleus of Hypothalamus/metabolism , Dinoprostone/antagonists & inhibitors , Dinoprostone/metabolism , Dinoprostone/physiology , Eicosanoids/antagonists & inhibitors , Eicosanoids/biosynthesis , Gonadotropin-Releasing Hormone/antagonists & inhibitors , Leukotrienes/biosynthesis , Male , Median Eminence/metabolism , Naloxone/pharmacology , Narcotic Antagonists/pharmacology , Oligopeptides/pharmacology , Rats , Rats, Wistar , Receptors, Opioid, delta/agonistsABSTRACT
It is currently accepted that opioids modify the secretion of LH by affecting the release of GnRH in the hypothalamus. A direct action of opioids at the pituitary level is not yet fully established. To this end, we tested the effects of opioids on the release of LH by the entire pituitary in adult male rats. Opioid agonists with mu (DAGO), delta (DS-LET) and kappa (U-50488H) specificity were tested at 0.01 to 10 microM in static incubations. DAGO inhibited dose-dependently the spontaneous and GnRH-induced release of LH. DSLET inhibited only the GnRH-induced release of LH. On the other hand, U-50488H increased spontaneous LH release dose-dependently. The opioid antagonists naloxone, diallyl-G (delta antagonist) and MR 2266 (kappa antagonist) blocked the effects induced by DAGO, DSLET or U-50488H respectively, implying an opioid receptor-mediated effect. The above results showed that opioids with mu, delta and kappa specificity act on the entire pituitary and modify differentially the release of LH. In this study we also compared spontaneous and GnRH-induced LH release by anterior and entire pituitaries and found that the amount of LH released by the anterior lobe was twofold higher, suggesting that inhibitory factors present in the neurointermediate part may affect the release of LH.
Subject(s)
Luteinizing Hormone/metabolism , Narcotics/metabolism , Pituitary Gland/metabolism , 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer , Analgesics/pharmacology , Animals , Benzomorphans/pharmacology , Dose-Response Relationship, Drug , Enkephalin, Ala(2)-MePhe(4)-Gly(5)- , Enkephalin, Leucine/analogs & derivatives , Enkephalin, Leucine/pharmacology , Enkephalins/pharmacology , Gonadotropin-Releasing Hormone/pharmacology , Male , Naloxone/pharmacology , Oligopeptides/pharmacology , Organ Culture Techniques , Pituitary Gland/drug effects , Pyrrolidines/pharmacology , Rats , Rats, Wistar , Receptors, Opioid, delta/agonists , Receptors, Opioid, delta/antagonists & inhibitors , Receptors, Opioid, kappa/agonists , Receptors, Opioid, kappa/antagonists & inhibitors , Receptors, Opioid, mu/agonistsABSTRACT
In static incubations, the K+ induced release of gonadotropin-releasing hormone (GnRH) and of prostaglandin (PG) E2, was 2-3 times higher in the isolated median eminence (ME) compared to the hypothalamic area containing the arcuate nucleus (ARN) plus the ME. The delta-opioid agonist DTLET, induced a parallel, dose-dependent reduction of GnRH and PGE2 release in the ARN plus ME. Both effects of DTLET were blocked by the delta-opioid antagonist Diallyl-G. In the isolated ME, DTLET reduced the secretion of PGE2 but enhanced the release of GnRH. In this area Diallyl-G had no effect on the PGE2 release but blocked the GnRH secretion. When the PGE2 production was blocked by indomethacin in the ARN plus ME preparation, DTLET increased the release of GnRH and induced the production of leukotrienes (LTs). On the other hand, DTLET decreased the release of both GnRH and PGE2 in the presence of nordihydroguaiaretic acid (NDGA), an inhibitor of the production of LTs. The above results suggest that: (a) the delta-opioid agonist DTLET modulates GnRH release differentially in the hypothalamic areas examined; and (b) the arachidonic acid metabolites are involved in the mode of action of DTLET on the release of GnRH in the ARN plus ME hypothalamic fragment.