ABSTRACT
Serotonin (5-hydroxytryptamine; 5-HT) is abundantly present throughout the gastrointestinal tract and stored mostly in enterochromaffin (EC) cells, which are located on the mucosal surface. 5-HT released from EC cells stimulate both intrinsic and extrinsic nerves, which results in various physiological and pathophysiological responses, such as gastrointestinal contractions. EC cells are believed to have the ability to respond to the chemical composition of the luminal contents of the gut; however, the underlying molecular and cellular mechanisms have not been identified. Here, we demonstrate that the transient receptor potential (TRP) cation channel TRPA1, which is activated by pungent compounds or cold temperature, is highly expressed in EC cells. We also found that TRPA1 agonists, including allyl isothiocyanate and cinnamaldehyde, stimulate EC cell functions, such as increasing intracellular Ca(2+) levels and 5-HT release, by using highly concentrated EC cell fractions and a model of EC cell function, the RIN14B cell line. Furthermore, we showed that allyl isothiocyanate promotes the contraction of isolated guinea pig ileum via the 5-HT(3) receptor. Taken together, our results indicate that TRPA1 acts as a sensor molecule for EC cells and may regulate gastrointestinal function.
Subject(s)
Calcium Channels/metabolism , Enterochromaffin Cells/metabolism , Gastrointestinal Motility , Nerve Tissue Proteins/metabolism , Serotonin/metabolism , Transient Receptor Potential Channels/metabolism , Animals , Ankyrins , Calcium/metabolism , Calcium Channels/genetics , Cell Line , Gene Expression Regulation/genetics , Guinea Pigs , Humans , Male , Mice , Nerve Tissue Proteins/genetics , Rats , TRPA1 Cation Channel , TRPC Cation Channels , Transient Receptor Potential Channels/geneticsABSTRACT
The G protein-coupled receptor (GPCR) family is highly diversified and involved in many forms of information processing. SREB2 (GPR85) is the most conserved GPCR throughout vertebrate evolution and is expressed abundantly in brain structures exhibiting high levels of plasticity, e.g., the hippocampal dentate gyrus. Here, we show that SREB2 is involved in determining brain size, modulating diverse behaviors, and potentially in vulnerability to schizophrenia. Mild overexpression of SREB2 caused significant brain weight reduction and ventricular enlargement in transgenic (Tg) mice as well as behavioral abnormalities mirroring psychiatric disorders, e.g., decreased social interaction, abnormal sensorimotor gating, and impaired memory. SREB2 KO mice showed a reciprocal phenotype, a significant increase in brain weight accompanying a trend toward enhanced memory without apparent other behavioral abnormalities. In both Tg and KO mice, no gross malformation of brain structures was observed. Because of phenotypic overlap between SREB2 Tg mice and schizophrenia, we sought a possible link between the two. Minor alleles of two SREB2 SNPs, located in intron 2 and in the 3' UTR, were overtransmitted to schizophrenia patients in a family-based sample and showed an allele load association with reduced hippocampal gray matter volume in patients. Our data implicate SREB2 as a potential risk factor for psychiatric disorders and its pathway as a target for psychiatric therapy.
Subject(s)
Brain/pathology , Genetic Predisposition to Disease/genetics , Nerve Tissue Proteins/genetics , Receptors, G-Protein-Coupled/genetics , Schizophrenia/genetics , Schizophrenia/pathology , Alleles , Amino Acid Sequence , Animals , Behavior, Animal , Evolution, Molecular , Humans , Magnetic Resonance Imaging , Male , Mice , Mice, Knockout , Molecular Sequence Data , Organ Size/genetics , Polymorphism, Single Nucleotide , Schizophrenic PsychologyABSTRACT
Histone deacetylase (HDAC) inhibitors have been shown to have antitumor activity in vitro and in vivo. Various studies related to their antitumor activity and mechanism of action have been reported for HDAC inhibitors, but the relationship of their antitumor effects to their pharmacodynamic and pharmacokinetic properties in vivo has not ever fully characterized. We report here the discovery of a novel cyclic-peptide-based HDAC inhibitor, YM753. YM753 is a bacteria-derived natural product containing a disulfide bond. It potently inhibited HDAC enzyme with an IC50 of 2.0 nM in the presence of dithiothreitol. YM753 was rapidly converted to a reduced form in tumor cells, and then induced accumulation of acetylated histones, followed by p21WAF1/Cip1 expression, tumor cell growth inhibition and tumor-selective cell death. In an in vitro washout study, YM753 showed prolonged accumulation of acetylated histones in WiDr human colon carcinoma cells. In vivo YM753 dosing of mice harboring WiDr colon tumor xenografts significantly inhibited the tumor growth via sustained accumulation of acetylated histones in the tumor tissue. In a pharmacokinetic study, YM753 rapidly disappeared from the plasma, but its reduced form remained in the tumor tissue. Moreover, the accumulation of acetylated histones induced by YM753 was tumor tissue selective compared to several normal tissues. This study provides evidence that YM753 has antitumor activity that is the result of selective, sustained accumulation of acetylated histones in tumor tissues despite rapid disappearance of the drug from the plasma. These results suggest that the novel HDAC inhibitor, YM753 has attractive pharmacodynamic and pharmacokinetic properties giving it potential as an antitumor agent.
Subject(s)
Colonic Neoplasms/drug therapy , Histone Deacetylase Inhibitors , Histones/metabolism , Peptides, Cyclic/therapeutic use , Acetylation/drug effects , Animals , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , HL-60 Cells , Humans , K562 Cells , Male , Mice , Mice, Nude , Models, Biological , Models, Molecular , Peptides, Cyclic/metabolism , Peptides, Cyclic/pharmacology , Prodrugs/metabolism , Substrate Specificity , Xenograft Model Antitumor AssaysABSTRACT
When the homozygous active form of porcine TGF-beta1 transgene (Tgf/Tgf) (under control of the rat glucagon promoter) is introduced into the nonobese diabetic mouse (NOD) genetic background, the mice develop endocrine and exocrine pancreatic hypoplasia, low serum insulin concentrations, and impaired glucose tolerance. To identify genetic modifiers of the diabetic phenotypes, we crossed hemizygous NOD-Tgf with DBA/2J mice (D2) or C3H/HeJ mice (C3H) and used the "transgenic mice" for quantitative trait loci (QTL) analysis. Genome-wide scans of F(2)-D Tgf/Tgf (D2 x NOD) and F(2)-C Tgf/Tgf (C3H x NOD), homozygous for the TGF-beta1 transgene, identified six statistically significant modifier QTLs: one QTL (Tdn1) in F(2)-D Tgf/Tgf, and five QTLs (Tcn1 to Tcn5) in F(2)-C Tgf/Tgf. Tdn1 (Chr 13, LOD = 4.39), and Tcn3 (Chr 2, LOD = 4.94) showed linkage to body weight at 8 weeks of age. Tcn2 (Chr 7, LOD = 4.38) and Tcn4 (Chr 14, LOD = 3.99 and 3.78) showed linkage to blood glucose (BG) concentrations in ipGTT at 30, 0, and 120 min, respectively. Tcn1 (Chr 1, LOD = 4.41) and Tcn5 (Chr 18, LOD = 4.99) showed linkage to serum insulin concentrations in ipGTT at 30 min. Tcn2 includes the candidate gene, uncoupling protein 2 (Ucp2), and shows linkage to Ucp2 mRNA levels in the soleus muscle (LOD = 4.90). Identification of six QTLs for diabetes-related traits in F(2)-D Tgf/Tgf and F(2)-C Tgf/Tgf raises the possibility of identifying candidate susceptibility genes and new targets for drug development for human type 2 diabetes.
Subject(s)
Crosses, Genetic , Diabetes Mellitus/genetics , Homozygote , Quantitative Trait Loci/genetics , Transforming Growth Factor beta/genetics , Transgenes/genetics , Animals , Blood Glucose , Body Weight , Chromosomes, Mammalian , Female , Food Deprivation , Genome , Insulin/blood , Lod Score , Male , Mice , Quantitative Trait, Heritable , Sex Characteristics , SwineABSTRACT
Prokineticins, multifunctional secreted proteins, activate two endogenous G protein-coupled receptors PKR1 and PKR2. From in situ analysis of the mouse brain, we discovered that PKR2 is predominantly expressed in the olfactory bulb (OB). To examine the role of PKR2 in the OB, we created PKR1- and PKR2-gene-disrupted mice (Pkr1(-/-) and Pkr2(-/-), respectively). Phenotypic analysis indicated that not Pkr1(-/-)but Pkr2(-/-)mice exhibited hypoplasia of the OB. This abnormality was observed in the early developmental stages of fetal OB in the Pkr2(-/-) mice. In addition, the Pkr2(-/-) mice showed severe atrophy of the reproductive system, including the testis, ovary, uterus, vagina, and mammary gland. In the Pkr2(-/-) mice, the plasma levels of testosterone and follicle-stimulating hormone were decreased, and the mRNA transcription levels of gonadotropin-releasing hormone in the hypothalamus and luteinizing hormone and follicle-stimulating hormone in the pituitary were also significantly reduced. Immunohistochemical analysis revealed that gonadotropin-releasing hormone neurons were absent in the hypothalamus in the Pkr2(-/-) mice. The phenotype of the Pkr2(-/-) mice showed similarity to the clinical features of Kallmann syndrome, a human disease characterized by association of hypogonadotropic hypogonadism and anosmia. Our current findings demonstrated that physiological activation of PKR2 is essential for normal development of the OB and sexual maturation.
Subject(s)
Gastrointestinal Hormones/metabolism , Genitalia/abnormalities , Neuropeptides/metabolism , Olfactory Bulb/abnormalities , Receptors, G-Protein-Coupled/deficiency , Receptors, Peptide/deficiency , Animals , Base Sequence , Female , Genitalia/metabolism , Gonadotropin-Releasing Hormone/genetics , Gonadotropin-Releasing Hormone/metabolism , Humans , Kallmann Syndrome/etiology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Neurons/metabolism , Olfactory Bulb/metabolism , Pregnancy , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, G-Protein-Coupled/genetics , Receptors, Peptide/genetics , Sexual Maturation/genetics , Sexual Maturation/physiologyABSTRACT
A lysophospholipid series, such as lysophosphatidic acid, lysophosphatidylserine, and lysophosphatidylcholine (LPC), is a bioactive lipid mediator with diverse physiological and pathological functions. LPC has been reported to induce insulin secretion from pancreatic beta-cells, however, the precise mechanism has remained elusive to date. Here we show that an orphan G-protein-coupled receptor GPR119 plays a pivotal role in this event. LPC potently enhances insulin secretion in response to high concentrations of glucose in the perfused rat pancreas via stimulation of adenylate cyclase, and dose-dependently induces intracellular cAMP accumulation and insulin secretion in a mouse pancreatic beta-cell line, NIT-1 cells. The Gs-protein-coupled receptor for LPC was identified as GPR119, which is predominantly expressed in the pancreas. GPR119-specific siRNA significantly blocked LPC-induced insulin secretion from NIT-1 cells. Our findings suggest that GPR119, which is a novel endogenous receptor for LPC, is involved in insulin secretion from beta-cells, and is a potential target for anti-diabetic drug development.
Subject(s)
Glucose/metabolism , Insulin/metabolism , Islets of Langerhans/drug effects , Islets of Langerhans/metabolism , Lysophosphatidylcholines/pharmacology , Receptors, G-Protein-Coupled/metabolism , Animals , Cell Line , Dose-Response Relationship, Drug , Hormones/metabolism , Humans , In Vitro Techniques , Insulin Secretion , Male , Organ Specificity , Pancreas , Rats , Rats, Wistar , Tissue DistributionABSTRACT
To find a novel human ion channel gene we have executed an extensive search by using a human genome draft sequencing data base. Here we report a novel two-pore domain K+ channel, TRESK (TWIK-related spinal cord K+ channel). TRESK is coded by 385 amino acids and shows low homology (19%) with previously characterized two-pore domain K+ channels. However, the most similar channel is TREK-2 (two-pore domain K+ channel), and TRESK also has two pore-forming domains and four transmembrane domains that are evolutionarily conserved in the two-pore domain K+ channel family. Moreover, we confirmed that TRESK is expressed in the spinal cord. Electrophysiological analysis demonstrated that TRESK induced outward rectification and functioned as a background K+ channel. Pharmacological analysis showed TRESK to be inhibited by previously reported K+ channel inhibitors Ba2+, propafenone, glyburide, lidocaine, quinine, quinidine, and triethanolamine. Functional analysis demonstrated TRESK to be inhibited by unsaturated free fatty acids such as arachidonic acid and docosahexaenoic acid. TRESK is also sensitive to extreme changes in extracellular and intracellular pH. These results indicate that TRESK is a novel two-pore domain K+ channel that may set the resting membrane potential of cells in the spinal cord.
Subject(s)
Potassium Channels/biosynthesis , Potassium Channels/physiology , Amino Acid Sequence , Analgesics, Non-Narcotic/pharmacology , Animals , Anti-Arrhythmia Agents/pharmacology , Arachidonic Acid/pharmacology , Barium/pharmacology , Cell Line , Cloning, Molecular , Docosahexaenoic Acids/pharmacology , Electrophysiology , Ethanolamines/pharmacology , Fatty Acids/metabolism , Fatty Acids, Nonesterified/metabolism , Glyburide/pharmacology , Humans , Hydrogen-Ion Concentration , Lidocaine/pharmacology , Mice , Models, Biological , Molecular Sequence Data , Patch-Clamp Techniques , Phylogeny , Potassium Channels/chemistry , Propafenone/pharmacology , Protein Structure, Tertiary , Quinidine/pharmacology , Quinine/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Spinal Cord/metabolism , Tissue Distribution , TransfectionABSTRACT
Nicotinic acid and its derivative, Acipimox, have been widely used in the treatment of hyperlipidemia. Pharmacological studies have demonstrated that they exert the beneficial effect through the activation of a Gi-protein-coupled receptor on adipocyte, which has remained elusive to date. Here we show that a novel GPCR, designated HM74b because of its high similarity to HM74, is a receptor for nicotinic acid. HM74b mRNA is found in human, murine, and rat adipose tissues. Nicotinic acid and Acipimox inhibit forskolin-stimulated intracellular cAMP accumulation in human HM74b-expressing cells and activate GTP gamma S binding in a dose-dependent manner. [3H]Nicotinic acid specifically binds to HM74b-expressing membrane and its binding is replaced by Acipimox. This finding will open a new phase of research on the physiological role of nicotinic acid and will be a clue to develop novel antihyperlipidemic drugs.
Subject(s)
Pyrazines/chemistry , Receptors, Nicotinic/genetics , Adipocytes/metabolism , Amino Acid Sequence , Animals , Cell Line , Cloning, Molecular , Colforsin/pharmacology , Cyclic AMP/metabolism , DNA, Complementary/metabolism , Dose-Response Relationship, Drug , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Humans , Mice , Molecular Sequence Data , Niacin/pharmacology , Open Reading Frames , Poly A , RNA, Messenger/metabolism , Radioligand Assay , Rats , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Tissue DistributionABSTRACT
Recent studies have identified two novel biofunctional proteins, termed prokineticin 1/EG-VEGF and prokineticin 2, which were mammalian homologues of mamba MIT1 and frog Bv8. Prokineticins have been demonstrated to exert their physiological functions through G-protein coupled receptors (GPCRs). In this study, we report the molecular identification of two endogenous prokineticin receptors, designated PK-R1 and PK-R2, through a search of the human genomic DNA database. PK-R1, locating in chromosome 2, and PK-R2, locating in chromosome 20p13, shared 87% homology, which was an extremely high value among known GPCRs. In functional assays, mammalian cells expressing PK-Rs responded to prokineticins in a concentration-dependent manner. Tissue distribution analysis revealed that expression of PK-R1 was observed in the testis, medulla oblongata, skeletal muscle and skin, while that of PK-R2 showed preferential expression in the central nervous system. The tissue distribution of PK-Rs reported in this paper suggests that the prokineticins play multifunctional roles in vivo.
Subject(s)
Neuropeptides , Receptors, Cell Surface/genetics , Receptors, G-Protein-Coupled , Receptors, Peptide/genetics , Amino Acid Sequence , Brain/metabolism , Cloning, Molecular , DNA, Complementary/biosynthesis , DNA, Complementary/chemistry , Gastrointestinal Hormones/biosynthesis , Gastrointestinal Hormones/metabolism , Genes, Reporter , Humans , Luciferases/genetics , Male , Molecular Sequence Data , Polymerase Chain Reaction , RNA, Messenger/analysis , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/metabolism , Receptors, Peptide/chemistry , Receptors, Peptide/metabolism , Testis/metabolismABSTRACT
The cDNA encoding histamine H4 receptor was cloned from the porcine spleen cDNA library. Porcine H4 receptor, which shares 72% homology with its human counterpart, bound to histamine in receptor-expressing mammalian cells. Isolation of the porcine H4 receptor, which is important for understanding of the pharmacology, will aid in better interpretation of physiological role of this subtype of histamine receptor.
Subject(s)
Receptors, G-Protein-Coupled , Receptors, Histamine/genetics , Amino Acid Sequence , Animals , CHO Cells , Cloning, Molecular , Cricetinae , DNA, Complementary/genetics , Molecular Sequence Data , Organ Specificity , Receptors, Histamine/metabolism , Sequence Alignment , SwineABSTRACT
We report identification and characterization of Kv6.3, a novel member of the voltage-gated K(+) channel. Reverse transcriptase-polymerase chain reaction analysis indicated that Kv6.3 was highly expressed in the brain. Electrophysiological studies indicated that homomultimeric Kv6.3 did not yield a functional voltage-gated ion channel. When Kv6.3 and Kv2.1 were co-expressed, the heteromultimeric channels displayed the decreased rate of deactivation compared to the homomultimeric Kv2.1 channels. Immunoprecipitation studies indicated that Kv6.3 bound with Kv2.1 in co-transfected cells. These results indicate that Kv6.3 is a novel member of the voltage-gated K(+) channel which functions as a modulatory subunit.