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.

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.

Ginkgo biloba and Memory: An Overview.

Ginkgo biloba extract has been increasingly popular for the treatment of memory problems. However, it is not commonly understood that this extract is composed of numerous chemicals, including flavonoid glycosides, terpene lactones, biflavones, and other miscellaneous components. It remains to be established exactly which components are biologically helpful. The extracts come from the leaves of the Ginkgo biloba tree which is cultivated extensively for this purpose. Our aging population will consist of approximately 79 million people 65 y.o. or older in the year 2050. Since memory disorders increase dramatically with age, this poses a major challenge to both the pharmaceutical and nutritional industries to provide products which improve or prevent problems with memory. Our culture is based on the ability to recall information, therefore problems with memory are fundamental to our entire social system. Dementias are disorders that affect memory and intellectual functioning, and are caused primarily by Alzheimer's disease and vascular disorders (multi-infarct dementia). New drug therapies have been developed to improve cognition, through stimulation of the cholinergic system. In recent decades, an extract of the leaves of the tree Ginkgo biloba L. has been used to improve memory in these disorders. The European experience with Ginkgo extract is much greater than that of the U.S. Clinical studies to date have indicated a probable therapeutic benefit of Ginkgo biloba extract. Further human studies are needed to identify which clinical population is most responsive to Ginkgo treatment. In addition, it would be very useful to identify which chemical compound or compounds provide therapeutic effects in memory disorders. These bioactive components may be further concentrated for increased benefit in increasing cognitive memory capabilities. In addition, pharmaceutical companies might be able to modify memory-enhancing Ginkgo-derived molecules to increase potency and effectiveness, leading to the next generation of memory-enhancing drugs.

Inositol and Ginkgo biloba as Adjuncts in Chronic Depression: Case Report.

The treatment of resistant depression has involved the use of various adjuncts in addition to the antidepressant, such as lithium or liothyronine (T3). This case report describes a woman with a history of chronic depression (dysthymic disorder), unresponsive to a number of traditional antidepressants, who was treated with the antidepressant bupropion and the nutritional and herbal supplements inositol and ginkgo. Her bupropion was able to be reduced, and she appeared to respond well to the added supplements, reducing her Ham-D score from 17 to 11. In this case, it was hypothesized that the inositol was the probable active adjunct with regard to the antidepressant effect, with the ginkgo helping with cognition. The inositol theory is reviewed with reference to mania and depression.

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.

Myo-inositol monophosphatase: diverse effects of lithium, carbamazepine, and valproate.

The therapeutic molecular sites of action for the mood-stabilizing medications are unknown. Myo-inositol monophosphatase (E.C. 3.1.3.25) is a major enzyme of the inositol signaling system that has previously been shown to be inhibited by clinically relevant concentrations of lithium, implicating this enzyme as a potential therapeutic site of action in manic-depressive disorder. Inhibition of myo-inositol monophosphatase (IMPase), which converts myo-inositol monophosphates to myo-inositol, results in increased levels of myo-inositol monophosphates and decreased myo-inositol available for the resynthesis of inositol phospholipids. In addition to lithium, carbamazepine and valproate are also used medically to treat manic-depressive disorder. It is of considerable interest to determine whether inhibition of IMPase activity is a common unifying mechanism for mood-stabilizing medications. Using a partially purified myo-inositol monophosphatase preparation derived from bovine brain, we examined the effects of lithium, carbamazepine, and valproate on the IMPase reaction. These results demonstrate that (1) lithium inhibited IMPase activity in the low millimolar range, (2) carbamazepine stimulated the IMPase reaction beginning in the low-micromolar range, and (3) valproate did not demonstrate any stimulation or inhibition of IMPase. We conclude that inhibition of IMPase is not a common neurochemical mechanism for mood-stabilizing medications.

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.

The identification of a novel inositol lipid, phosphatidylinositol trisphosphate (PIP3), in rat cerebrum using in vivo techniques.

Rats received intraventricular injections of 20 uCi of [3H]-myo-inositol, and were sacrificed 24 hrs later by high-power head-focused microwave fixation. Two inositol lipid extraction methods were compared: The Hauser and Eichberg method yielded higher recovery of inositol lipids, but a lower inositol phosphate content. The Schacht method yielded reduced radiolabel in the lipid fractions, but increased water soluble phosphates. Both methods extracted a novel inositol lipid (PIP3) which contained inositol tetrakisphosphate (IP4) as its polar head group. This was determined by alkaline hydrolysis and analyzed by high performance liquid chromatography with authentic IP4 standard. Furthermore, preliminary studies of the fatty acid composition indicated a similarity with other inositol lipids. The radiolabel ratio of PIP2:PIP3 was 5:1. In summary, we have isolated a novel inositol phospholipid in rat brain, PIP3, the parent compound for inositol tetrakisphosphate (IP4).

Carbamazepine inhibits electroconvulsive shock-induced inositol trisphosphate (IP3) accumulation in rat cerebral cortex and hippocampus.

Carbamazepine is used to treat manic-depressive disorder, and is also an anticonvulsant. Rats were injected with this drug 90 min prior to this experiment, when mild inhibition of convulsions took place. Intraventricular injections of 14 muCi [3H]myoinositol were made 20-24 hrs prior to the experiment. Ninety min after intraperitoneal injection of carbamazepine or vehicle, rats were given electroconvulsive shock or sham procedure and sacrificed 30 sec later. Incorporation of radiolabel into inositol lipids and inositol phosphates was analyzed in cerebral cortex and hippocampus. Carbamazepine's effects on the brain inositol lipid cycle, studied here for the first time, showed 1) enhanced labeling in the polyphosphoinositides (carbamazepine-ECS groups showed increases of about 40% in PIP2); 2) decreased [H]IP1 levels; and 3) inhibition of ECS-induced [3H]-IP3 accumulation.

Electroconvulsive shock stimulates polyphosphoinositide degradation and inositol trisphosphate accumulation in rat cerebrum: lithium pretreatment does not potentiate these changes.

Using an in vivo model, we explored the acute effects of electroconvulsive shock (ECS) and lithium on rat cerebral polyphosphoinositides and inositol phosphates. ECS was shown to increase the [3H]inositol trisphosphate ([3H]IP3) by 75%, decrease the endogenous mass of phosphatidylinositol 4,5-bisphosphate (PIP2) by 23%, and enhance [3H]myo-inositol labeling into the polyphosphoinositides. In contrast, lithium pretreatment 20-24 h prior to ECS appeared to attenuate the ECS-induced [3H]IP3 increase and the decrease in mass of PIP2; [3H]inositol monophosphate ([3H]IP1) levels demonstrated no differences between the lithium ECS and lithium-alone groups. These results indicate that ECS stimulates the inositol lipid cycle in brain possibly due to neurotransmitter release. Moreover, the effects of lithium suggest other possible sites of action of this cation on inositol lipid metabolism in addition to an inhibition of inositol-1-phosphatase.

Enhanced inositide turnover in brain during bicuculline-induced status epilepticus.

Because brain inositides are enriched in the 1-stearoyl-2-arachidonoyl species, they form a likely source for the tetraenoic free fatty acids (FFA) and diacylglycerols (DG) that are accumulated during seizures. To study inositide turnover during bicuculline-induced seizures, rats were injected intraventricularly and bilaterally with 10-20 microCi 32P, mechanically ventilated and sacrificed by 6.5 KW head-focused microwave irradiation. Seizure activity was recorded by electroencephalography. Bicuculline-induced seizure activity resulted in: a) almost 50% increase in 32P labeling of phosphatidic acid (PA); phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP2) also increased (24% and 36%, respectively); b) no change in other lipids; and c) water-soluble phosphodiesteratic degradation products, analyzed by high voltage paper electrophoresis, increased 24% in the amount of radiotracer recovered as inositol 1,4-bisphosphate (IP2) and by 44% in the amount recovered as inositol 1,4,5-trisphosphate (IP3). These data indicate that during experimental status epilepticus the cerebral inositide cycle is accelerated: PIP2----(IP3----IP2----IP----I) + DG----PA----PI----PIP----PIP2.