SMIT2 mediates all myo-inositol uptake in apical membranes of rat small intestine

This study presents the characterization of myo-inositol (MI) uptake in rat intestine as evaluated by use of purified membrane preparations. Three secondary active MI cotransporters have been identified; two are Na(+) coupled (SMIT1 and SMIT2) and one is H(+) coupled (HMIT). Through inhibition studies using selective substrates such as d-chiro-inositol (DCI, specific for SMIT2) and l-fucose (specific for SMIT1), we show that SMIT2 is exclusively responsible for apical MI transport in rat intestine; rabbit intestine appears to lack apical transport of MI. Other sugar transport systems known to be present in apical membranes, such as SGLT1 or GLUT5, lacked any significant contribution to MI uptake. Functional analysis of rat SMIT2 activity, via electrophysiological studies in Xenopus oocytes, demonstrated similarities to the activities of SMIT2 from other species (rabbit and human) displaying high affinities for MI (0.150 +/- 0.040 mM), DCI (0.31 +/- 0.06 mM), and phlorizin (Pz; 0.016 +/- 0.007 mM); low affinity for glucose (36 +/- 7 mM); and no affinity for l-fucose. Although these functional characteristics essentially confirmed those found in rat intestinal apical membranes, a unique discrepancy was seen between the two systems studied in that the affinity constant for glucose was approximately 40-fold lower in vesicles (K(i) = 0.94 +/- 0.35 mM) than in oocytes. Finally, the transport system responsible for the basolateral efflux transporter of glucose in intestine, GLUT2, did not mediate any significant radiolabeled MI uptake in oocytes, indicating that this transport system does not participate in the basolateral exit of MI from small intestine.     

Dephosphorylation of myo-inositol 1,4,5-trisphosphate and myo-inositol 1,3,4-triphosphate.

We have augmented our previous studies [Storey, Shears, Kirk & Michell (1984) Nature (London) 312, 374-376] on the subcellular location and properties of Ins(1,4,5)P3 (inositol 1,4,5-trisphosphate) phosphatases in rat liver and human erythrocytes. We also investigate Ins(1,3,4)P3 (inositol 1,3,4-trisphosphate) metabolism by rat liver. Membrane-bound and cytosolic Ins(1,4,5)P3 phosphatases both attack the 5-phosphate. The membrane-bound enzyme is located on the inner face of the plasma membrane, and there is little or no activity associated with Golgi apparatus. Cytosolic Ins(1,4,5)P3 5-phosphatase (Mr 77,000) was separated by gel filtration from Ins(1,4)P2 (inositol 1,4-bisphosphate) and inositol 1-phosphate phosphatases (Mr 54,000). Ins(1,4,5)P3 5-phosphatase activity in hepatocytes was unaffected by treatment of the cells with insulin, vasopressin, glucagon or dibutyryl cyclic AMP. Ins(1,4,5)P3 5-phosphatase activity in cell homogenates was unaffected by changes in [Ca2+] from 0.1 to 2 microM. After centrifugation of a liver homogenate at 100,000 g, Ins(1,3,4)P3 phosphatase activity was largely confined to the supernatant. The sum of the activities in the supernatant and the pellet exceeded that in the original homogenate. When these fractions were recombined, Ins(1,3,4)P3 phosphatase activity was restored to that observed in unfractionated homogenate. Ins(1,3,4)P3 was produced from Ins(1,3,4,5)P4 (inositol 1,3,4,5-tetrakisphosphate) and was metabolized to a novel InsP2 that was the 3,4-isomer. Ins(1,3,4)P3 phosphatase activity was not changed by 50 mM-Li+ or 0.07 mM-Ins(1,4)P2 alone, but when added together these agents inhibited Ins(1,3,4)P3 metabolism. In Li+-treated and vasopressin-stimulated hepatocytes, Ins(1,4)P2 may reach concentrations sufficient to inhibit Ins(1,3,4)P3 metabolism, with little effect on Ins(1,4,5)P3 hydrolysis.     

Expression and functionality of the Na+/myo-inositol cotransporter SMIT2 in rabbit kidney

Myo-inositol (MI) is involved in several important aspects of cell physiology including cell signaling and the control of intracellular osmolarity i.e. by serving as a "compatible osmolyte". Currently, three MI cotransporters have been identified: two are Na(+)-dependent (SMIT1 and SMIT2) and one is H(+)-dependent (HMIT) and predominantly expressed in the brain. The goal of this study was to characterize the expression of SMIT2 in rabbit kidney and to compare it to SMIT1. First, we quantified mRNA levels for both transporters using quantitative real-time PCR and found that SMIT1 was predominantly expressed in the medulla while SMIT2 was mainly in the cortex. This distribution of SMIT2 was confirmed on Western blots where an antibody raised against a SMIT2 epitope specifically detected a 75 kDa protein in both tissues. Characterization of MI transport in brush-border membrane vesicles (BBMV), in the presence of d-chiro-inositol and l-fucose to separately identify SMIT1 and SMIT2 activities, showed that only SMIT2 is expressed at the luminal side of proximal convoluted tubules. We thus conclude that, in the rabbit kidney, SMIT2 is predominantly expressed in the cortex where it is probably responsible for the apical transport of MI into the proximal tubule.     

Sodium-dependent myo-inositol transporter 1 is a cellular receptor for Mus cervicolor M813 murine leukemia virus

Retrovirus infection is initiated by binding of the surface (SU) portion of the viral envelope glycoprotein (Env) to specific receptors on cells. This binding triggers conformational changes in the transmembrane portion of Env, leading to membrane fusion and cell entry, and is thus a major determinant of retrovirus tissue and species tropism. The M813 murine leukemia virus (MuLV) is a highly fusogenic gammaretrovirus, isolated from Mus cervicolor, whose host range is limited to mouse cells. To delineate the molecular mechanisms of its restricted host range and its high fusogenic potential, we initiated studies to characterize the cell surface protein that mediates M813 infection. Screening of the T31 mouse-hamster radiation hybrid panel for M813 infectivity localized the receptor gene to the distal end of mouse chromosome 16. Expression of one of the likely candidate genes (slc5a3) within this region in human cells conferred susceptibility to both M813 infection and M813-induced fusogenicity. slc5a3 encodes sodium myo-inositol transporter 1 (SMIT1), thus adding another sodium-dependent transporter to the growing list of proteins used by MuLVs for cell entry. Characterization of SMIT1 orthologues in different species identified several amino acid variations within two extracellular loops that may restrict susceptibility to M813 infection.     

Identification of a novel Na+/myo-inositol cotransporter

rkST1, an orphan cDNA of the SLC5 family (43% identical in sequence to the sodium myo-inositol cotransporter SMIT), was expressed in Xenopus laevis oocytes that were subsequently voltage-clamped and exposed to likely substrates. Whereas superfusion with glucose and other sugars produced a small inward current, the largest current was observed with myo-inositol. The expressed protein, which we have named SMIT2, cotransports myo-inositol with a K(m) of 120 microm and displays a current-voltage relationship similar to that seen with SMIT (now called SMIT1). The transport is Na(+)-dependent, with a K(m) of 13 mm. SMIT2 exhibits phlorizin-inhibitable presteady-state currents and substrate-independent "Na(+) leak" currents similar to those of related cotransporters. The steady-state cotransport current is also phlorizin-inhibitable with a K(i) of 76 microm. SMIT2 exhibits stereospecific cotransport of both d-glucose and d-xylose but does not transport fucose. In addition, SMIT2 (but not SMIT1) transports d-chiro-inositol. Based on previous publications, the tissue distribution of SMIT2 is different from that of SMIT1, and the existence of this second cotransporter may explain much of the heterogeneity that has been reported for inositol transport.     

Sodium/myo-Inositol transporters: substrate transport requirements and regional brain expression in the TgCRND8 mouse model of amyloid pathology

Inositol stereoisomers, myo- and scyllo-inositol, are known to enter the brain and are significantly elevated following oral administration. Elevations in brain inositol levels occur across a concentration gradient as a result of active transport from the periphery. There are two sodium/myo-inositol transporters (SMIT1, SMIT2) that may be responsible for regulating brain inositol levels. The goals of this study were to determine the effects of aging and Alzheimer''s disease (AD)-like amyloid pathology on transporter expression, to compare regional expression and to analyze substrate requirements of the inositol transporters. QPCR was used to examine expression of the two transporters in the cortex, hippocampus and cerebellum of TgCRND8 mice, a mouse model of amyloid pathology, in comparison to non-transgenic littermates. In addition, we examined the structural features of inositol required for active transport, utilizing a cell-based competitive uptake assay. Disease pathology did not alter transporter expression in the cortex or hippocampus (p>0.005), with only minimal effects of aging observed in the cerebellum (SMIT1: F_2,26 = 12.62; p = 0.0002; SMIT2: F_2,26 = 8.71; p = 0.0015). Overall, brain SMIT1 levels were higher than SMIT2, however, regional differences were observed. For SMIT1, at 4 and 6 months cerebellar SMIT1 levels were significantly higher than cortical and hippocampal levels (p<0.05). For SMIT2, at all three ages both cortical and cerebellar SMIT2 levels were significantly higher than hippocampal levels (p<0.05) and at 4 and 6 months of age, cerebellar SMIT2 levels were also significantly higher than cortical levels (p<0.05). Inositol transporter levels are stably expressed as a function of age, and expression is unaltered with disease pathology in the TgCRND8 mouse. Given the fact that scyllo-inositol is currently in clinical trials for the treatment of AD, the stable expression of inositol transporters regardless of disease pathology is an important finding.     

Contributions in astrocytes of SMIT1/2 and HMIT to myo-inositol uptake at different concentrations and pH

myo-Inositol is important for cell signaling both in cytoplasm and in intracellular organelles. It is required in the plasma membrane and cytoplasm for maintained synthesis of the second messengers, inositoltrisphosphate (IP(3)) and diacylglycerol (DAG) from phosphatidylinositol bisphosphate (PIP(2)), and in organelles as precursor for synthesis of complex signaling phospholipids and inositolphosphates from IP(3) and PIP(2). myo-Inositol must be taken up into the cell where its is used, because neither neurons nor astrocytes synthesize it. It is also an osmolyte, taken up in response to surrounding hyperosmolarity and released during hypo-osmolarity. There are three myo-inositol transporters, the Na(+)-dependent SMIT1 and SMIT2, and HMIT, which co-transports myo-inositol with H(+). Their relative expressions in astrocytes and neurons are unknown. Uptake kinetics for myo-inositol in astrocytes has repeatedly been determined, but always on the assumption of only one component, leaving kinetics for the individual transporters unknown. This paper demonstrates that astrocytes obtained directly from the brain express SMIT1 and HMIT, but little SMIT2, and that all three transporters are expressed in neurons. Cultured mouse astrocytes show a high-affinity/low-capacity myo-inositol uptake (V(max): 60.0 ± 3.0 pmol/min per mg protein; K(m): 16.7 ± 2.6 μM), mediated by SMIT1 and perhaps partly by SMIT2. It was determined in cells pre-treated with HMIT-siRNA and confirmed by specific inhibition of SMIT. However at physiologically relevant myo-inositol concentrations most uptake is by a lower-affinity/higher-capacity uptake, mediated by HMIT (V(max): 358 ± 60 pmol/min per mg protein; K(m): 143 ± 36 μM) and determined by subtraction of SMIT-mediated from total uptake. At high myo-inositol concentrations, its uptake is inhibited by incubation in medium with increased pH, and increased during intracellular acidification with NH(4)Cl. This is in agreement with literature data for HMIT alone. At low concentration, where SMIT1/2 activity gains importance, myo-inositol uptake is reduced by ammonia-induced intracellular acidification, consistent with the transporter's pH sensitivity reported in the literature.     

ABCA3 inactivation in mice causes respiratory failure, loss of pulmonary surfactant, and depletion of lung phosphatidylglycerol

The highly branched mammalian lung relies on surfactant, a mixture of phospholipids, cholesterol, and hydrophobic proteins, to reduce intraalveolar surface tension and prevent lung collapse. Human mutations in the ABCA3 transporter have been associated with childhood respiratory disease of variable severity and onset. Here, we report the generation of Abca3 null mice, which became lethargic and cyanotic and died within 1 h of birth. Tissue blots found ABCA3 expression was highest in lung but was also detectable in other tissues, including the kidney. Gross development of kidney and lung was normal in neonatal Abca3(-/-) pups, but the mice failed to inflate their lungs, leading to death from atelectatic respiratory failure. Ultrastructural analysis of the Abca3(-/-) lungs revealed an absence of surfactant from the alveolar space and a profound loss of mature lamellar bodies, the intracellular storage organelle for surfactant. Mass spectrometry measurement of >300 phospholipids in lung tissue taken from Abca3(-/-) mice showed a dramatic reduction of phosphatidylglycerol (PG) levels as well as selective reductions in phosphatidylcholine species containing short acyl chains. These results establish a requirement of ABCA3 for lamellar body formation and pulmonary surfactant secretion and suggest a unique and critical role for the transporter in the metabolism of pulmonary PG. They also demonstrate the utility of the Abca3 null mouse as a model for a devastating human disease.     

Measurement of myo-inositol turnover in phosphatidylinositol: description of a model and mass spectrometric method for cultured cortical neurons

Rates of myo-inositol (Ins) incorporation and turnover in phosphatidylinositol (PtdIns) were determined in cultured mouse cortical neurons. Cells were incubated with deuterium-labeled myo-inositol (Ins) in culture medium free of unlabeled Ins. The time-dependent changes in the specific activity of cytosolic Ins and membrane PtdIns were measured by mass spectrometry. PtdIns turnover was modeled incorporating values for Ins flux, cytosolic dilution, PtdIns concentration, and rate of incorporation into PtdIns. Recycled Ins diluted the labeled precursor pool, and a time course was obtained for this cytosolic process. The specific activity of the precursor pool at the plateau of the time-course curve was 0.43 +/- 0.04 (mean +/- SD). The incorporation of the tracer into PtdIns was linear between 4 and 10 h incubation of the neurons. After factoring in the extent of dilution of the tracer in the precursor pool, the rate of Ins incorporation into PtdIns was found to be 315 +/- 51 nmol (g of protein)(-1) x h(-1). The half-life of Ins in PtdIns was calculated for each point on the linear incorporation curve and then corrected for the tracer reincorporation. The half-life of Ins in PtdIns was 6.7 +/- 0.2 h, which translates into a basal turnover rate of 10.3%/h in this in vitro system. The mathematical model and the stable isotope method described here should allow assessment of the dynamics of PtdIns signaling altered in certain diseases or by agents.     

Pulmonary abnormalities due to ABCA1 deficiency in mice

Mice gene targeted for ATP-binding cassette transporter A1 (ABCA1; Abca1(-/-)) have been shown to have low-serum high-density lipoprotein and abnormal lung morphology. We examined alterations in the structure and function of lungs from -/- mice (DBA1/J). Electron microscopy of the diseased mouse lung revealed areas of focal disease confirming previous results (47). Lipid analysis of the lung tissue of -/- mice showed a 1.2- and 1.4-fold elevation in total phospholipid (PL) and saturated phosphatidylcholine, respectively, and a marked 50% enrichment in total cholesterol content predominantly due to a 17.5-fold increase in cholesteryl ester compared with wild type (WT). Lung surfactant in the -/- mice was characterized by alveolar proteinosis (161%), a slight increase in total PL (124%), and a marked increase in free cholesterol (155%) compared with WT. Alveolar macrophages were enriched in cholesterol (4.8-fold) due to elevations in free cholesterol (2.4-fold) and in cholesteryl ester (14.8-fold) compared with WT macrophages. More PL mass was cleared from the alveolar space of -/- mice lungs, measured using intratracheal installation of (3)H-PL liposomes. Compared with WT mice, the Abca1(-/-) mice demonstrated respiratory distress with rapid, shallow breathing. Thus the lungs of mice lacking ABCA1 protein demonstrated abnormal morphology and physiology, with alveolar proteinosis and cholesterol enrichment of tissue, surfactant, and macrophages. The results indicate that the activity of ABCA1 is important for the maintenance of normal lung lipid composition, structure, and function.     

Antagonists of myo-inositol 3,4,5,6-tetrakisphosphate allow repeated epithelial chloride secretion

Cystic fibrosis (CF) patients suffer from a defect in hydration of mucosal membranes due to mutations in the cystic fibrosis transmembrane regulator (CFTR), an apical chloride channel in mucosal epithelia. Disease expression in CF knockout mice is organ specific, varying with the level of expression of calcium activated Cl(-) channels (CLCA). Therefore, restoring transepithelial Cl(-) secretion by augmenting alternate Cl(-) channels, such as CLCA, could be beneficial. However, CLCA-mediated Cl(-) secretion is transient, due in part to the inhibitory effects of myo-inositol 3,4,5,6-tetrakisphosphate [Ins(3,4,5,6)P(4)]. This suggests that antagonists of Ins(3,4,5,6)P(4) could be useful in treatment of CF. We have, therefore, synthesized a series of membrane-permeant Ins(3,4,5,6)P(4) derivatives, carrying alkyl substituents on the hydroxyl groups and screened them for effects on Cl(-) secretion in a human colonic epithelial cell line, T(84). While membrane-permeant Ins(3,4,5,6)P(4) derivatives had no direct effects on carbachol-stimulated Cl(-) secretion, Ins(3,4,5,6)P(4) derivatives, but not enantiomeric Ins(1,4,5,6)P(4) derivatives, reversed the inhibitory effect of Ins(3,4,5,6)P(4) on subsequent thapsigargin activation of Cl(-) secretion. The extent of the antagonistic effect of the Ins(3,4,5,6)P(4) derivatives varied with the position of the alkyl substituents. Derivatives with a cyclohexylidene ketal or a butyl-chain at the 1-position reversed the Ins(3,4,5,6)P(4)-mediated inhibition of Cl(-) secretion by up to 96 and 85%, respectively, whereas butylation of the 1- and 2-position generated a reversal effect of only 65%. Derivatives carrying the butyl chain only at the 2-position showed no antagonistic effect. These data: (1) Support the hypothesis that Ins(3,4,5,6)P(4) stereospecifically inhibits Ca(2+) activated Cl(-) secretion and that Ins(3,4,5,6)P(4) mediates most, if not all of the cholinergic-mediated inhibition of chloride secretion in T(84) cells; (2) Demonstrate Ins(3,4,5,6)P(4)-mediated inhibition can be completely reversed with rationally designed membrane-permeant Ins(3,4,5,6)P(4) antagonists; (3) Demonstrate that a SAR for membrane-permeant Ins(3,4,5,6) P(4) antagonists can be generated and screened in a physiologically relevant cell-based assay; (4) Indicate that Ins(3,4,5,6)P(4) derivatives could serve as a starting point for the development of therapeutics to treat cystic fibrosis.     

Neural function: metabolism and actions of inositol metabolites in mammalian brain

In the nervous system, a variety of cell types respond to external stimuli through the inositol lipid signalling pathways. The stimulus-coupled sequence of intracellular events has been investigated in a homogeneous model system, the cloned mammalian neural cell line NG115-401L. The neural peptide bradykinin stimulates a rapid production of identified inositol phosphate isomers and an intracellular Ca2+ discharge followed by a persistent plasma membrane influx. The temporal sequence suggests that Ins(1,4,5)P3 or Ins(1,3,4,5)P4 or both may coordinate these events in a neuronal cell, as has been suggested in other cell types. Thapsigargin, an irritant and tumour-promoting plant product, produces calcium transients in the absence of inositol phosphate production, and may provide a new tool for investigating the interactions between inositol phosphates and changes in cellular calcium homeostasis. In the 401L line, high levels of radiolabelled InsP5 and InsP6 have been detected, which has led to the evaluation of their possible occurrence and actions in normal brain. Both InsP5 and InsP6 are produced from a radiolabelled myo-inositol precursor in intact mature brain in a region-specific manner. This suggests that both inositol polyphosphates may be end products of regionally regulated biosynthetic pathways. When microinjected into a nucleus of the brainstem, or iontophoretically applied to the dorsal horn of the spinal cord, both InsP5 and InsP6, but not Ins(1,3,4,5)P4 isomers, appear to be potent neural stimulants. These results suggest that the inositol lipid signalling pathways may generate both intracellular and extracellular signals in brain.     

The pathway of myo-inositol 1,3,4-trisphosphate phosphorylation in liver. Identification of myo-inositol 1,3,4-trisphosphate 6-kinase, myo-inositol 1,3,4-trisphosphate 5-kinase, and myo-inositol 1,3,4,6-tetrakisphosphate 5-kinase

Inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) metabolism has been studied in liver homogenates and in 100,000 x g supernatant and particulate fractions. When liver homogenates were incubated in an "intracellular" medium containing 5 mM MgATP, equal proportions of Ins(1,3,4)P3 were dephosphorylated and phosphorylated. Two inositol tetrakisphosphate (InsP4) products and an inositol pentakisphosphate (InsP5) were detected. The InsP4 isomers were unequivocally identified as inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) and inositol 1,3,4,6-tetrakisphosphate (Ins(1,3,4,6)P4) by high performance liquid chromatography separation of inositol phosphates, periodate oxidation, alkaline hydrolysis, and stereo-specific polyol dehydrogenase. Ins(1,3,4)P3 5-kinase is a novel enzyme activity and accounted for 16% of the total Ins(1,3,4)P3 phosphorylation. Ins(1,3,4,6)P4 was also shown to be further phosphorylated to inositol 1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P5) by a kinase not previously known to occur in liver. About 75% of Ins(1,3,4)P3 kinase activities were soluble and were partly purified by anion-exchange fast protein liquid chromatography. The two Ins(1,3,4)P3 kinase activities eluted as a single peak that was well resolved from Ins(1,3,4)P3 phosphatase, Ins(1,3,4,6)P4 5-kinase, and Ins(1,3,4,5)P4 5-phosphatase activities. A further novel observation was that 10 microM Ins(1,3,4,5)P4 inhibited Ins(1,3,4)P3 kinase activities by 60%.     

myo-inositol 3,4,5,6-tetrakisphosphate inhibits an apical calcium-activated chloride conductance in polarized monolayers of a cystic fibrosis cell line

Does inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P(4)) inhibit apical Ca(2+)-activated Cl(-) conductance (CaCC)? We studied this question using human CFPAC-1 pancreatoma cells grown in polarized monolayers. Cellular Ins(3,4,5,6)P(4) levels were acutely sensitive to purinergic receptor activation, rising 3-fold within 1 min of agonist addition. Intracellular Ins(3,4,5,6)P(4) levels were therefore specifically elevated, independently of receptor activation, by incubating cells with a cell-permeant bioactivable analogue, 1,2-di-O-butyl-myo-inositol 3,4,5,6-tetrakisphosphate octakis(acetoxymethyl)ester (Bt(2)Ins (3,4,5,6)P(4)/AM). The latter inhibited Ca(2+)-activated Cl(-) secretion by 60%. We next used nystatin to selectively permeabilize the basolateral membrane to monovalent anions and cations, thereby preventing this membrane from electrochemically dominating ion movements through the apical membrane. Thus, we studied autonomous regulation of apical Cl(-) channels in situ. The properties of Cl(-) flux across the apical membrane were those expected of CaCC: niflumic acid sensitivity, outward rectification, and 2-fold greater permeability of I(-) over Cl(-). Following nystatin-treatment, we elevated intracellular levels of Ins(3,4,5,6)P(4) with either purinergic agonists or with Bt(2)Ins(3,4,5,6)P(4)/AM. Both protocols inhibited Ca(2+)-activated Cl(-) secretion (up to 70%). These studies provide the first demonstration that, in a physiologically relevant context of a polarized monolayer, there is an apical, Ins(3,4,5,6)P(4)-inhibited CaCC.     

BMCP1: a mitochondrial uncoupling protein in neurons which regulates mitochondrial function and oxidant production

Outside the nervous system, members of the mitochondrial uncoupling protein (UCP) family have been proposed to contribute to control of body temperature and energy metabolism, and regulation of mitochondrial production of reactive oxygen species (ROS). However, the function of brain mitochondrial carrier protein 1 (BMCP1), which is highly expressed in brain, remains to be determined. To study BMCP1 expression and function in the nervous system, a high-affinity antibody to BMCP1 was generated and used to analyze tissue expression of BMCP1 protein in mouse. BMCP1 protein was highly expressed in heart and kidney, but not liver or lung. In the nervous system, BMCP1 was present in cortex, basal ganglia, substantia nigra, cerebellum, and spinal cord. Both BMCP1 mRNA and protein expression was almost exclusively neuronal. To study the effect of BMCP1 expression on mitochondrial function, neuronal (GT1-1) cell lines with stable overexpression of BMCP1 were generated. Transfected cells had higher State 4 respiration and lower mitochondrial membrane potential (psi(m)), consistent with greater mitochondrial uncoupling. BMCP1 expression also decreased mitochondrial production of ROS. These data suggest that BMCP1 can modify mitochondrial respiratory efficiency and mitochondrial oxidant production, and raise the possibility that BMCP1 might alter the vulnerability of brain to both acute injury and to neurodegenerative conditions.     

Properties of inositol polyphosphate 1-phosphatase

We recently described inositol polyphosphate 1-phosphatase, an enzyme which cleaves the 1-phosphate from inositol 1,4-bisphosphate (Ins(1,4)P2) and inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) (Inhorn, R. C., and Majerus, P. W. (1987) J. Biol. Chem. 262, 15946-15952). We have now purified the enzyme to homogeneity from calf brain. The enzyme hydrolyzes 50.3 mumol of Ins(1,4)P2/min/mg protein. The enzyme has an apparent mass of 44,000 daltons as determined both by gel filtration chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that it is monomeric. Lithium ions inhibit Ins(1,3,4)P3 hydrolysis uncompetitively with an apparent Ki of approximately 0.3 mM LiCl. Calcium inhibits hydrolysis of Ins(1,4)P2 and Ins(1,3,4)P3 equally, with approximately 40% inhibition occurring at 1 microM free Ca2+. Rabbit polyclonal antiserum against purified inositol polyphosphate 1-phosphatase was prepared which immunoprecipitates approximately 0.3 milliunits of activity/microliter serum (1 unit = 1 mumol of Ins(1,4)P2 hydrolyzed per min). This antiserum was used to determine the enzyme content in several bovine tissues, all of which had a similar intrinsic specific activity (i.e. approximately 0.3 milliunits/microliter antiserum). Tissues studied included brain, heart, kidney, liver, lung, parotid, spleen, testis, and thymus. Approximately 10-15% of the total inositol polyphosphate 1-phosphatase activity in calf brain homogenates remains in a particulate fraction; antiserum also binds 0.3 milliunits of membrane-associated activity/microliter antiserum. Thus, a single enzyme can account for Ins(1,4)P2 hydrolytic activity in the bovine tissues. Ins(1,3,4)P3 metabolism was also investigated in bovine tissue homogenates. Inositol polyphosphate 1-phosphatase accounts for greater than 80% of the hydrolytic activity in all tissues studied except brain, where inositol polyphosphate 4-phosphatase is the major enzyme that hydrolyzes Ins(1,3,4)P3. The apparent Km of inositol polyphosphate 1-phosphatase for Ins(1,3,4)P3 varies approximately 3-4-fold among the bovine tissues.     

Evidence for a single pool of myo-inositol in hormone-responsive WRK-1 cells

Previous reports have suggested the existence of at least two pools of cellular myo-inositol (Ins); it has been further hypothesized that only one of these pools is utilized during hormone-activated, cyclic phosphatidylinositol (PtdIns) resynthesis. In an effort to investigate this possibility, we have undertaken kinetic studies of Ins metabolism in WRK-1 cells. Our results indicate that a single pool of Ins is involved in both basal and activated PtdIns synthesis. Ins generated by the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP₂) mixes with the existing pool of free Ins and is not used exclusively for resynthesis of PtdIns. © 1995 Wiley-Liss, Inc.     

Wortmannin and LY294002 inhibit myo-inositol accumulation by cultured bovine aorta endothelial cells and murine 3T3-L1 adipocytes

We have previously reported that myo-inositol uptake and metabolism is reduced in human fibroblasts derived from patients with ataxia telangiectasia (AT). Treating normal fibroblasts with 10-100 microM wortmannin duplicates some of the phenotypic properties of AT fibroblasts including the decrease in myo-inositol accumulation. In the present study we examined whether treatment of other types of mammalian cells with wortmannin or LY294002 altered myo-inositol uptake. Cultured bovine aorta endothelial cells or 3T3-L1 adipocytes were incubated with either wortmannin or LY294002, and afterwards, myo-inositol uptake and SMIT mRNA levels were determined. Incubating cultured bovine aorta endothelial cells and 3T3-L1 adipocytes with either wortmannin or LY294002 caused a time- and concentration-dependent decrease in myo-inositol accumulation that was independent of changes in SMIT mRNA levels. The effect of wortmannin and LY294002 on myo-inositol accumulation was not due to an increase in myo-inositol secretion. The effect of LY294002 on myo-inositol accumulation was reversible. Furthermore, the LY294002-induced decrease in myo-inositol accumulation was specific since the uptake of serine or choline by cultured bovine aorta endothelial cells and 3T3-L1 adipocytes treated with LY294002 was not significantly decreased. Co-incubation of cultured bovine aorta endothelial cells and 3T3-L1 adipocytes with either wortmannin or LY294002 and hyperosmotic medium caused a significant decrease in the induction of myo-inositol accumulation by hyperosmolarity without significantly affecting the hyperosmotic-induced increase in SMIT mRNA levels. These data suggest that myo-inositol accumulation is regulated post-translationally by wortmannin and LY294002.     

Expression and functionality of the Na/myo-inositol cotransporter SMIT2 in rabbit kidney

Myo-inositol (MI) is involved in several important aspects of cell physiology including cell signaling and the control of intracellular osmolarity i.e. by serving as a “compatible osmolyte”. Currently, three MI cotransporters have been identified: two are Na⁺-dependent (SMIT1 and SMIT2) and one is H⁺-dependent (HMIT) and predominantly expressed in the brain. The goal of this study was to characterize the expression of SMIT2 in rabbit kidney and to compare it to SMIT1. First, we quantified mRNA levels for both transporters using quantitative real-time PCR and found that SMIT1 was predominantly expressed in the medulla while SMIT2 was mainly in the cortex. This distribution of SMIT2 was confirmed on Western blots where an antibody raised against a SMIT2 epitope specifically detected a 75 kDa protein in both tissues. Characterization of MI transport in brush-border membrane vesicles (BBMV), in the presence of d-chiro-inositol and l-fucose to separately identify SMIT1 and SMIT2 activities, showed that only SMIT2 is expressed at the luminal side of proximal convoluted tubules. We thus conclude that, in the rabbit kidney, SMIT2 is predominantly expressed in the cortex where it is probably responsible for the apical transport of MI into the proximal tubule.     

Sequence analysis,structure and binding site prediction of Sigma 1 receptor protein by in silico method

Sigma 1 Receptor is a subtype of opioid receptor that participates in membrane remodeling and cellular differentiation in the nervous system. Sigma1 Receptor protein with amino acid length ranging from 229 is widely distributed in the liver and moderately in the intestine, kidney, white pulp of the spleen, adrenal gland, brain, placenta and the lung. In this study, the three dimensional structure for sigma 1 receptor protein has been developed by in- silico analysis based on evolutionary trace analysis of 37 sigma proteins from different sources. The present work focus on identification of functionally important residues and its interaction with antipsychotic drugs reported in literature.