Rat brain myo-inositol 3-phosphate synthase is a phosphoprotein.

The therapeutic effects of lithium in bipolar disorder are poorly understood. Lithium decreases free inositol levels by inhibiting inositol monophosphatase 1 and myo-inositol 3-phosphate synthase (IPS). In this study, we demonstrate for the first time that IPS can be phosphorylated. This was evident when purified rat IPS was dephosphorylated by lambda protein phosphatase and analyzed by phospho-specific ProQ-Diamond staining and Western blot analysis. These techniques demonstrated a mobility shift consistent with IPS being phosphorylated. Mass spectral analysis revealed that Serine-524 (S524), which resides in the hinge region derived from exon 11 of the gene, is the site for phosphorylation. Further, an antibody generated against a synthetic peptide of IPS containing monophosphorylated-S524, was able to discriminate the phosphorylated and non-phosphorylated forms of IPS. The phosphoprotein is found in the brain and testis, but not in the intestine. The intestinal IPS isoform lacks the peptide bearing S524, and hence, cannot be phosphorylated. Evidences suggest that IPS is monophosphorylated at S524 and that the removal of this phosphate does not alter its enzymatic activity. These observations suggest a novel function for IPS in brain and other tissues. Future studies should resolve the functional role of phospho-IPS in brain inositol signaling.

Beta-cell sparing in transplanted islets by vascular endothelial growth factor.

We have reported that vascular endothelial growth factor (VEGF) promotes the revascularization of transplanted islets, thereby reducing the initial number required to prevent diabetes. The present study was undertaken to assess other mechanisms of beta-cell sparing by VEGF. For in vitro studies, islets were cultured for 14 days with versus without 20 ng/mL VEGF. Viability, necrosis, and apoptosis were examined by specific staining (Alcein AM, propidium iodide, and annexin/phosphatidylserine). The effects of VEGF on islets were also examined in a proteomic study. In vivo streptozotocin-treated diabetic Lewis rats received 1000 Lewis or Sprague-Dawley islets beneath the renal capsule. Oxygen levels at the transplant site were monitored by a Clark-type oxygen electrode. Fasting blood glucose served as an indicator of islet survival and function. VEGF enhanced oxygen levels at the transplant site. Syngeneic recipients were euglycemic for over 6 months, whereas control islets failed within 30 to 60 days. VEGF prevented allograft rejection for over 14 days, whereas controls were rejected within 6 to 7 days. Immunostaining suggested that VEGF inhibited the presentation of MHC II antigen and promoted islet survival by the inhibition of necrosis and apoptosis. Our proteomic study suggested VEGF preserved systems required for cellular preservation (heat shock proteins) and insulin secretion. VEGF promotes the preservation of isolated and transplanted islets by a variety of mechanisms, including enhanced oxygenation and inhibition of immune rejection, necrosis, and apoptosis. The provision of exogenous VEGF may be a useful adjunct to islet transplantation.

Human chromosomal localization of a gene for inositol monophosphatase by fluorescence in situ hybridization.

Inhibition of the enzyme inositol monophosphatase (IMPase) (E.C. 3.1.3.25) has been linked to the therapeutic action of lithium in the treatment of manic-depression (bipolar) disorder. Because of the link between bipolar and IMPase, we felt it would be of considerable importance to determine the human chromosomal localization of the IMPase gene. Fluorescence in situ hybridization analysis using a human cDNA clone, which included the 5'-UTR and the complete coding region, mapped the human IMPase gene to chromosome 8q21.2-21.3. No gene locus for manic-depressive disorder has yet been identified. Further studies on this IMPase gene, and other potential gene variants and mutations, should help to determine if specific subgroups of patients with manic-depressive disorder can be determined on a molecular basis, with regard to the IMPase gene.

Promising Psychotherapeutic Effects of the Natural Sugar: Myo-Inositol.

Myo-inositol is a common six-carbon sugar with unique biochemical and psychotherapeutic properties. It is involved in neuronal signaling and osmoregulation, and has been shown to be therapeutic in initial studies of depression, panic disorder, and obsessive-compulsive disorder. The inositol signaling system is a post-receptor second messenger system found in many cells, and is similar to the cAMP system. Myo-inositol exists in the free form, or as a component of membrane inositol phospholipids which are present largely on the inner leaflet of the plasma membrane. Inositol phospholipids, particularly phosphatidylinositol 4,5-bis-phosphate (PIP2), are linked to a number of brain receptor signaling systems including serotonergic, muscarinic, adrenergic, histaminergic, cholecystokinin, tachykinins, metabotropic, neurotensin, platelet activating factor, and others. With receptor stimulation, the signal is transmitted through a series of other proteins. Activation of a GTP-binding protein (Gq), in turn activates plasma membrane phospholipase C releasing the second messenger, myo-inositol 1,4,5-trisphosphate (InsP3), into the cytosol. InsP3 then causes release of free calcium from endoplasmic reticulum into the cytosol, which then activates a number of calcium-sensitive enzymes and receptors. Myo-inositol is made available to the brain through three sources: (i) receptor stimulation (salvage pathway), (ii) de novo production, and (iii) dietary intake. Initial clinical studies have shown that myo-inositol has psychoactive effects, and is effective in the treatment of specific mood and anxiety disorders. Recent preliminary clinical studies have suggested the fascinating possibility that myo-inositol has psychoactive effects, and may be effective in the treatment specific mood and anxiety disorders. Further clinical studies are required using larger groups of patients before definitive conclusions can be drawn upon the use of myo-inositol as a potential psychoactive compound. Myo-inositol as a natural medication increases interest in this newly emerging area of nutritional neuroscience.

The Use of Ginseng as an Adjunct in Treatment-Resistant Depression.

The treatment of depression has evolved over the past several years since the evolution of tricyclic antidepressants (TCAs) to the serotonin selective reuptake inhibitors (SSRIs). However, some patients are resistant to various medications, and various adjunctive medications have been added to the original medication, to promote a therapeutic response. This case report describes a woman, with a long history of treatment-resistant depression, who was treated with a combination of an SSRI and ginseng.

Molecular characterization of coding and untranslated regions of rat cortex lithium-sensitive myo-inositol monophosphatase cDNA.

Lithium sensitive myo-inositol monophosphatase (IMPase) is a pivotal enzyme which controls the levels of brain inositol within the inositol-based signaling system. Its capacity to release free myo-inositol from inositol monophosphates generated from receptor-linked and de novo pathways is crucial to the maintenance of appropriate amounts of intracellular myo-inositol, which is essential for both inositol-based cell signaling and cell volume control. We present here the full length cDNA encompassing the coding and untranslated regions (5'- and 3'-UTRs) of rat brain IMPase. This cDNA was derived from rat cortex mRNA by the RT-PCR technique. Analysis of this cDNA revealed several interesting features which include a short 5'-untranslated region (5'-UTR) of 68 nucleotides followed by coding region of approximately 0.8 kb and a long 3'-untranslated region (3'-UTR) of 1.2 kb. Both 5'-rapid amplification of cDNA ends (5'-RACE) and 3'-RACE techniques were carried out to isolate both UTRs and double stranded sequencing was carried out to its entirety (approximately 2.1 kb) by 'gene walking' using several oligonucleotide primers. All nucleotides were sequenced unambiguously using the sense and antisense strands of DNA. PCR analysis for the coding region and the deduced amino acid sequence demonstrated a DNA fragment of 831 bp and 277 amino acids, respectively, which are strikingly similar to human hippocampal IMPase. The 5'-UTR demonstrated distinct CpG doublets, characteristic of 'housekeeping' genes. The sequence around the initiator methionine, AAGATGG, conforms well to the Kozak consensus sequence for mammalian protein biosynthesis and the 3'-UTR demonstrated three canonical (AATAAT, AATTAA, AATACA) and one unusual polyadenylation signals (ATTAAA) followed by a 31 base poly(A) tail. The presence of a CCTGTG in the 3'-UTR (putative carbohydrate response element) links IMPase mRNA to brain carbohydrate metabolic pathways. Computer analyses demonstrated several unique features of this mRNA, including the potential formation of hairpin loops which might be important in its intracellular regulation and turn-over. In summary, this lithium-sensitive brain IMPase mRNA has the following characteristics: a 5'-CpG-rich short untranslated segment, a highly conserved coding region, and a long 3'-untranslated region with several polyadenylation signals.

Brain inositol monophosphatase identified as a galactose 1-phosphatase.

During the course of our analysis of myo-inositol monophosphatase (IMPase), a key enzyme of brain inositol signaling, we found it also hydrolyzes galactose 1-phosphate (Gal 1-P), an intermediate of galactose metabolism. Electrophoretically homogeneous IMPase was prepared from three different sources: (i) bovine brain, (ii) rat brain, and (iii) human brain (recombinant), which demonstrated similar ability to hydrolyze inositol monophosphates and galactose 1-phosphate. The ability of IMPase to use both inositol 1-phosphates and galactose 1-phosphate equally as substrates is of considerable importance in determining lithium's mechanism of action. Our current results suggest that during lithium therapy, both galactose and inositol metabolic pathways can be simultaneously modulated through lithium inhibition of IMPase. Enzyme studies with Mg2+ ions as activators and with Li+, Ca2+, Mn2+, Ba2+ ions as inhibitors demonstrate that IMPase is a single enzyme possessing the ability to hydrolyze both inositol monophosphates and Gal-1-P with equal efficiency. In addition, gel-filtration chromatographic analysis demonstrated that IMPase and galactose 1-phosphatase activities co-purify in our electrophoretically homogeneous enzyme preparations. Our results indicate that lithium inhibition of IMPases at clinically relevant concentrations, may modulate both inositol and galactose metabolism, and identifies yet another carbohydrate pathway utilizing IMPase.

Biochemical and molecular properties of lithium-sensitive myo-inositol monophosphatase.

Myo-inositol monophosphatase is a pivotal enzyme of the inositol second messenger system which is specifically inhibited by therapeutic levels of lithium salts, implicating inhibition of this enzyme as a potential site of its action in bipolar disease. This enzyme has a native molecular weight of 59,000, and has traditionally been found in the cytosolic fraction, although a membrane-bound form has also been identified. Possessing two identical subunits, this enzyme hydrolyzes those monophosphates which are equatorially located within the inositol ring, and several nucleoside monophosphates phosphorylated at the 2-position. Each subunit of the native enzyme contains an active site with unusually large caverns as revealed by crystallographic studies, which may explain the accommodation of these structurally unrelated substrates. We have suggested that the uncompetitive inhibition of this phosphatase by lithium ions may prevent the formation of an enzyme-bound non-isomeric (meso) intermediate, Mg(2+)-inositol 1,3 or 4,6 cyclic monophosphate when this enzyme hydrolyzes its respective isomeric substrates.

myo-inositol monophosphatase from rat testes: purification and properties.

myo-Inositol monophosphatase (EC 3.1.3.25) has been purified to homogeneity from the high-speed supernatant of rat testes and its properties were investigated. By means of ammonium sulfate precipitation, followed by heating, anion exchange, and gel filtration high-pressure liquid chromatographic techniques, polylysine agarose and phenyl-Sepharose column chromatographic methods, this phosphatase was purified 2563-fold to a specific activity of 7972 mU/mg protein. It showed an apparent native molecular weight of 58,000 as determined by gel filtration chromatography and was composed of two identical subunits of molecular weight of 29,000 as determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Among several divalent cations tested for activation of the enzyme, Mg2+ was most effective and optimally active at pH 7.8. The Km values for D- and L-myo-inositol 1-phosphate (which were equal) and 2'-AMP were 0.12 +/- 0.02 and 0.17 +/- 0.03 mM, respectively. Lithium ions inhibited this phosphatase specifically and kinetic studies demonstrated uncompetitive inhibition. Preparations of polyclonal antibodies against the homogeneous enzyme in rabbits cross-reacted with the partially purified enzyme preparations from liver, kidney, heart, and brain show immunological identity. Western blot analysis after SDS-polyacrylamide gel electrophoresis confirmed a major band corresponding to a subunit molecular weight of 29,000. A sensitive enzyme staining method was also developed to localize the site of myo-inositol monophosphatase activity on polyacrylamide gels which helped to differentiate this phosphatase from nonspecific contaminating phosphatases. To explain the unusual stereospecificity of this enzyme on its isomeric substrates, a working model was suggested involving the production of a myo-inositol 1,3-cyclic phosphate intermediate during the course of its reaction.

The identification of a membrane-bound myo-inositol 1-phosphatase in rat brain, liver, and testes.

A membrane-bound myo-inositol 1-phosphatase has been solubilized and partially purified from rat tissues. This particulate enzyme was detected in brain, liver and testis and certain physicochemical and enzymological properties were examined. Previously this major enzyme of the inositol signaling system was considered strictly cytosolic. The ratio of activity in the membrane form was approximately one-eighth of the activity found with the cytosolic fraction. The molecular weight of this phosphatase was found to be 59,000 by gel filtration chromatography and a subunit molecular weight of 29,000 by Western blot analysis, values comparable to the cytosolic form. This phosphatase cleaves both D- and L- myo-inositol 1-phosphates which originate from two different cellular pathways and is inhibited by lithium ions. Polyclonal antibodies were raised against homogeneous testicular cytosolic myo-inositol 1-phosphatase and cross-reacted with this membrane form as determined by western blot analysis showing immunological identity.

The effects of lithium isotopes on the myo-inositol 1-phosphatase reaction in rat brain, liver, and testes.

Enzyme inhibition studies were performed with several lithium isotopes in order to more precisely define how lithium inhibits the enzyme myo-inositol 1-phosphatase. This lithium-induced inhibition is thought to be central to the therapeutic effects of lithium in the treatment of manic-depressive disorder. Naturally occurring lithium (NLi) exists as a combination of isotopes: 6Li and 7Li. Lethality studies were performed comparing 6LiCl, 7LiCl, and NLiCl, did not demonstrate a differential effect as previous studies had suggested. Enzyme inhibition studies were performed with these individual lithium isotopes, and compared to the effects of the naturally occurring combination (NLi) on the inhibition of myo-inositol 1-phosphatase using a partially purified enzyme preparation from rat brain, liver and testes. Identical inhibition was observed with all lithium isotopes and their combinations. In addition, both D- and L-myo-inositol 1-phosphates were used as enzyme substrates and found to be equivalent. These experiments, along with previous work demonstrating lithium acting as an uncompetitive inhibitor in the reaction, and the lack of lithium binding sites on the enzyme, suggests the hypothesis that lithium is possibly inhibiting this reaction by interfering with the formation of a transition cyclic intermediate, myo-inositol 1,3-cyclic phosphate, which may be formed from either the D- or L-substrates. This proposal is in contrast to previous suggestions regarding the inhibitory mechanism of action of lithium on the myo-inositol 1-phosphatase reaction.