Characteristics of a phosphatidylinositol exchange activity of soybean microsomes

Microsome fractions from hypocotyls of dark-grown soybean (Glycine max [L.] Merrill) seedlings incorporated myo-inositol into phosphatidylinositol by an exchange reaction stimulated by Mn²⁺ (optimum at 10 mm) and cytidine nucleotides (CMP = CDP CTP) but not by Mg²⁺ or nucleotides other than cytidine nucleotides. The activity was membrane associated, with an optimum pH of 8, stimulated by auxin, and inhibited by certain thiol reagents or by heating above 40°C. With radioactive inositol, phosphatidylinositol was the only radioactive product. That turnover was by myo-inositol exchange was verified from experiments where unlabeled inositol replaced already incorporated inositol with approximately the same kinetics as for the incorporation of label. Both the incorporation and the displacement reactions were stimulated by Mn²⁺ and CMP and both were responsive to auxin with comparable dose dependency. Corresponding exchange activities with choline or ethanolamine were not observed. The phosphatidylinositol-myo-inositol exchange activity was low or absent from plasma membrane, tonoplast, and mitochondria enriched fractions. The activity co-localized on free-flow electrophoresis and aqueous two-phase partition with NADPH cytochrome c reductase and latent IDPase, markers for endoplasmic reticulum and Golgi apparatus, respectively. With microsomes incubated with both ATP and inositol, polyphosphoinositides were unlabeled demonstrating separate locations for the inositol exchange and phosphatidylinositol kinase reactions. Thus, the auxin-responsive inositol turnover activity of soybean membranes is distinct from the usual de novo biosynthetic pathway. It is not the result of a traditional D-type phospholipase and appears not to involve plasma membrane-associated polyphosphoinositide metabolism. It most closely resembles previously described phosphatidylinositol-myo-inositol exchange activities of plant and animal endoplasmic reticulum.     

Solubilization of microsomal-associated phosphatidylinositol synthase from germinating soybeans

CDP-1,2-diacyl-sn-glycerol (CDP-diacylglycerol):myo-inositol phosphatidyltransferase (EC 2.7.8.11, phosphatidylinositol synthase) catalyzes the final step in the de novo synthesis of phosphatidylinositol in the endoplasmic reticulum fraction of germinating soybeans (Glycine max L. var Cutler 71). A variety of solubilization agents were examined for their ability to release phosphatidylinositol synthase activity from the microsome fraction. The most effective agent to solubilize the enzyme was the nonionic detergent Brij W-1. A 2.1-fold increase in specific activity was achieved using 1% Brij W-1 with 69% activity solubilized.     

Phosphatidylinositol synthesis by a mn-dependent exchange enzyme in castor bean endosperm

myo-Inositol is incorporated into phosphatidylinositol by an exchange reaction associated with the endoplasmic reticulum fraction isolated from post-germination castor bean endosperm. The reaction requires Mn²⁺, has a pH optimum of 8.0, an apparent K_m for myo-inositol of 26 micromolar, and is stimulated about 15-fold by certain cytidine derivatives. The cytidine derivatives appear to be converted to CMP, which may be the only active stimulator. These optimal exchange reaction conditions, both with and without CMP, differ from those for cytidine-5′ -diphosphodiglyceride: myo-inositol transferase (EC 2.7.8), so the exchange does not appear to be a reversal of the transferase. This conclusion is augmented by the low rates of CDP-diglyceride formation from cytidine derivatives when compared to the high rate of myo-inositol incorporation into phosphatidylinositol in the presence of the same cytidine derivatives and identical reaction conditions.     

Phosphatidylinositol synthesis in castor bean endosperm: cytidine diphosphate diglyceride:inositol transferase

CDP-diglyceride:inositol transferase in endoplasmic reticulum fractions from castor bean (Ricinus communis) endosperm was partially characterized. The enzyme had a pH optimum of 8.5 and required Mn²⁺ for activity. Maximal activity was at 1.5 millimolar MnCl₂. A K_m of 0.30 mM was calculated for myo-inositol and 1.35 millimolar was estimated for CDP-dipalmitoylglyceride. Concentrations of CDP-dipalmitoylglyceride above 1.2 millimolar inhibited the enzyme. A deoxycholate concentration of 0.1% (w/v) stimulated the reaction slightly while Triton X-100 inhibited at all concentrations tested. Some incorporation of myo-inositol into phosphatidylinositol occurred in the absence of CDP-diglyceride.     

myo-Inositol binding and transport in brush border membranes of rat kidney

Using hypotonically treated brush border membranes, binding and transport of myo-inositol were examined.By hypotonic treatment, both total and non-specific uptake decreased significantly, but specific uptake was not affected.myo-Inositol release from membranes preloaded by incubation for 2 min was very rapid and about 98% of preloaded myo-inositol was released in 5 min of incubation. However, myo-inositol release from membranes preloaded by incubation for 20 min was fairly slow and 50% of myo-inositol remained in the membranes even after 10 min of incubation.Uptake of myo-inositol decreased by the increase of osmolarity in the medium. However, effect of osmolarity on the uptake was less significant when myo-inositol concentration was lower.Under conditions in which mainly binding occurred, myo-inositol binding to the membranes was measured. Two binding systems were demonstrated and high affinity site could bind 22 pmol/mg protein at most and the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value was 8.3 μM.Both binding and transport processes were dependent on Na⁺ and enhanced by Na⁺-gradient.     

Reduced Na+/K+ ATPase transport activity,resting membrane potential,and bradykinin-stimulated phosphatidylinositol synthesis by polyol accumulation in cultured neuroblastoma cells

In these studies we examined the effect of polyol accumulation on neural cellmyo-inositol metabolism and properties. Neuroblastoma cells were cultured for two weeks in media containing 30 mM glucose, fructose, galactose or mannose with or without 0.4 mM sorbinil or 250 Mmyo-inositol. Chronic exposure of neuroblastoma cells to media containing 30 mM glucose, galactose, or mannose caused a decrease inmyo- inositol content and myo-[2-³H]inositol accumulation and incorporation into phosphoinositides compared to cells cultured in unsupplemented medium or medium containing 30 mM fructose as an osmotic control. These monosaccharides each caused an increase in intracellular polyol levels with galactitol > sorbitol = mannitol accumulation. Chronic exposure of neuroblastoma cells to media containing 30 mM glucose, galactose, or mannose caused a significant decrease in Na⁺/K⁺ ATPase transport activity, resting membrane potential, and bradykinin-stimulated³²P incorporation into phosphatidylinositol compared to cells cultured in medium containing 30 mM fructose. In contrast, basal incorporation of³²P into phosphatidylinositol or basal and bradykinin-stimulated³²P incorporation into phosphatidylinositol 4,5-bisphosphate were not effected. Each of these cellular functions as well asmyo-inositol metabolism and content and polyol levels remained near control values when 0.4 mM sorbinil, an aldose reductase inhibitor, was added to the glucose, galactose, or mannose supplemented media.myo-Inositol metabolism and content and bradykinin-stimulated phosphatidylinositol synthesis were also maintained when media containing 30 mM glucose, galactose, or mannose was supplemented with 250 Mmyo-inositol. The results suggest that polyol accumulation induces defects in neural cellmyo-inositol metabolism and certain cell functions which could, if they occurred in vivo, contribute to the pathological defects observed in diabetic neuropathy.     

RADIOAUTOGRAPHIC LOCALIZATION OF THE INCREASED SYNTHESIS OF PHOSPHATIDYLINOSITOL IN RESPONSE TO PANCREOZYMIN OR ACETYLCHOLINE IN GUINEA PIG PANCREAS SLICES

A technique is described for measuring the incorporation of myo-inositol-2-³H into the lipid of various regions of the guinea pig pancreatic acinar cell by radioautography. Stimulation of enzyme secretion with either pancreozymin or acetylcholine was associated with increased graining in both the basophilic cytoplasm and the nonbasophilic cytoplasm. Kinetic studies suggested that the incorporation of myo-inositol-2-³H was stimulated independently in the two regions. Most of the increment in graining due to stimulation with pancreozymin or acetylcholine plus eserine was abolished if the tissue was extracted with 2:1 chloroform-methanol before radioautography. On chromatography of lipid extracts of pancreas, the only lipid showing a detectable increment in radioactivity on stimulation with pancreozymin was phosphatidylinositol. Thus, essentially all of the increment in graining is likely to be due to increased incorporation of tritium into phosphatidylinositol. These studies, coupled with earlier studies employing differential centrifugation, indicate that on stimulation of enzyme secretion there is increased synthesis of phosphatidylinositol in the rough-surfaced endoplasmic reticulum and in the smooth-surfaced Golgi membranes. The significance of these observations is discussed in connection with membrane circulation presumed to occur in the pancreatic acinar cell on stimulation of protein secretion. It is suggested that the increased synthesis of phosphatidylinositol may be concerned with the formation of new endoplasmic reticulum and possibly Golgi membrane to replace that which is presumably converted to membrane of the zymogen granules during intracellular protein transport.     

myo-Inositol homeostasis in foetal rabbit lung

In several species, lung maturation is accompanied by a decline in the phosphatidylinositol content of lung surfactant and a concomitant increase in its phosphatidylglycerol content. To examine the possibility that this developmental change is influenced by the availability of myo-inositol, potential sources of myo-inositol for the developing rabbit lung were investigated. On day 28 of gestation the myo-inositol content of foetal rabbit lung tissue (2.3±0.5μmol/g of tissue) was not significantly different from that of adult lung tissue but the activity of d-glucose 6-phosphate:1l-myo-inositol 1-phosphate cyclase (cyclase) in foetal lung tissue (81.0±9.0nmol·h⁻¹·g of tissue⁻¹) was higher than that found in adult lung tissue (23.2±1.0nmol·h⁻¹·g of tissue⁻¹). Day 28 foetal rabbit lung tissue was found also to take up myo-inositol by a specific, energy-dependent, Na⁺-requiring mechanism. Half-maximal uptake of myo-inositol by foetal rabbit lung slices was observed when the concentration of myo-inositol in the incubation medium was 85μm. When the myo-inositol concentration was 1mm (but not 100μm) the addition of glucose (5.5mm) stimulated myo-inositol uptake. myo-Inositol uptake was observed also in adult rabbit lung and was found to be sub-maximal at the concentration of myo-inositol found in adult rabbit serum. The concentration of myo-inositol in the serum of pregnant adult rabbits (47.5±5.5μm) was significantly lower than that of non-pregnant adult female rabbits (77.9±9.2μm). On day 28 of gestation the concentration of myo-inositol in foetal serum (175.1±12.0μm) was much less than on day 25, but more than that found on day 30. A transient post-partum increase in the concentration of myo-inositol in serum was followed by a rapid decline. Much of the myo-inositol in foetal rabbit serum probably originates from the placenta, where on day 28 of gestation a high cyclase activity (527±64nmol·h⁻¹·g of tissue⁻¹) was measured. The gestational decline in serum myo-inositol concentration, together with the decreasing cyclase activity of the lungs, is consistent with the view that maturation of the lungs is accompanied by decreased availability of myo-inositol to this tissue.     

Membrane lipid biosynthesis in Chlamydomonas reinhardtii. Partial characterization of CDP-diacylglycerol: myo-inositol 3-phosphatidyltransferase

Myo-inositol may be incorporated in the formation of phosphatidylinositol by two mechanisms. One reaction utilizes CDP-diacylglycerol and is catalyzed by phosphatidylinositol (PtdIns) synthase (CDP-diacylglycerol: myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11). The second reaction is the phosphatidylinositol: myo-inositol exchange reaction, in which a free inositol is exchanged for an existing inositol headgroup. This characterization of inositol incorporation into phosphatidylinositol in the green alga Chlamydomonas reinhardtii provides evidence for the presence of both reactions. The transferase reaction required a divalent cation and exhibited its maximum activity at 2.0 mM Mn²⁺. The optimal pH for this reaction was 8.5–9.0. The best substrate concentrations were 0.5 mM CDP-diacylglycerol and 1.2 mM myo-inositol, with an estimated K_m for myo-inositol of 0.2 mM. The exchange reaction also required Mn²⁺ for activity, but became saturated at 0.5 mM Mn²⁺. The optimal pH of the exchange reaction was 8.0, the optimal myo-inositol concentration was 0.3 mM, and the estimated K_m for myo-inositol in this reaction was 0.015 mM. Measurement of the transferase reaction in cell fractions of C. reinhardtii indicated that the activity occurred primarily in the microsomal fraction, with little or no activity in the plastids.     

Characterisation of the FAM69 family of cysteine-rich endoplasmic reticulum proteins

Myo-inositol is one of the major organic osmolytes in the brain and the kidney. The accumulation of intracellular organic osmolytes allows cells to regulate intracellular osmolality without altering cytoplasmic ionic strength and to adapt to hyperosmotic conditions. Two types of myo-inositol transporters, sodium/myo-inositol transporter and H⁺/myo-inositol transporter (HMIT), have been identified. Sodium/myo-inositol transporters are induced by osmotic stress and might be involved in the intracellular accumulation of myo-inositol in mammals. The role of HMIT, however, remains unknown. In the present study, we characterized three Caenorhabditis elegansHMIT genes, hmit-1.1, hmit-1.2, and hmit-1.3. hmit-1.1 was expressed in the intestine, and hmit-1.2 was expressed in the glia and the excretory canal, which is an osmotic regulatory organ that is functionally analogous to the kidney. hmit-1.3 was expressed in the intestine and the glia. The expression of hmit-1.1 and hmit-1.2 but not hmit-1.3, was markedly induced under hyperosmotic conditions. Animals with mutant hmit-1.1 and hmit-1.2 were hypersensitive to osmotic stress. The defects of hmit-1.1 and hmit-1.2 mutants were rescued by hmit-1.1 and hmit-1.2 transgenes, respectively, and by modified human HMIT. In human cell lines, HMIT expression was induced in hyperosmotic conditions. These findings indicate that the C. elegans HMIT family has a crucial role in the osmoprotective response.     

A sensitive radioisotopic assay of myo-inositol: Its application to rat pancreatic islets

A radioisotopic procedure for the assay of myo-inositol is presented. It is based on the generation of NADH from NAD⁺ in the reaction catalyzed by myo-inositol dehydrogenase and the subsequent NADH-dependent conversion of 2-[U-¹⁴C]ketoglutarate to ¹⁴C-labeled l-glutamate in the reaction catalyzed by glutamate dehydrogenase. This method was applied to the measurement of myo-inositol in rat pancreatic islets. The myo-inositol islet content was decreased when the animals were fed a diet deprived of myo-inositol. When incubated in the absence of exogenous d-glucose, pancreatic islets, like parotid cells, released myo-inositol in the incubation medium. Over 90 min of incubation, a rise in extracellular d-glucose concentration increased the myo-inositol islet content, which was decreased, however, after incubation in the presence of carbamylcholine. These findings indicate that the myo-inositol content of islets is affected by nutritional and other environmental factors.     

Modulatory roles of NHERF1 and NHERF2 in cell surface expression of the glutamate transporter GLAST

The PDZ (PSD-95/Drosophila discs-large protein/zonula occludens protein) domain-containing proteins Na⁺/H⁺ exchanger regulatory factor 1 (NHERF1) and NHERF2 interact with the glutamate transporter GLAST. To characterize the roles of these NHERF proteins in the plasma membrane targeting of GLAST, we examined the interaction of green fluorescent protein (EGFP)-tagged GLAST with epitope-tagged NHERF proteins in human embryonic kidney (HEK) 293T cells. Co-expression of either NHERF protein increased the cell surface expression of EGFP-GLAST. Deletion of the C-terminal PDZ domain-binding motif caused an increase in EGFP-GLAST with immature endoglycosidase H-sensitive N-linked oligosaccharides, suggesting impaired exit of EGFP-GLAST from the endoplasmic reticulum (ER). Immunoprecipitation experiments revealed that NHERF1 predominantly bound EGFP-GLAST containing immature N-glycans, whereas NHERF2 co-precipitated EGFP-GLAST with mature N-glycans. Expression of a dominant-negative mutant of the GTPase Sar1 increased the interaction of EGFP-GLAST with NHERF1 in the ER. By contrast, immunofluorescence microscopy showed that NHERF2 co-localized with EGFP-GLAST in ER–Golgi intermediate compartments (ERGICs), at the plasma membrane and in early endosomes, but not in the ER. These results suggest that NHERF1 interacts with GLAST during ER export, while NHERF2 interacts with GLAST in the secretory pathway from the ERGIC to the plasma membrane, thereby modulating the cell surface expression of GLAST.     

Effect of Osmolality and myo-Inositol Deprivation on the Transport Properties of myo-Inositol in Primary Astrocyte Cultures

myo-Inositol uptake measured in primary astrocyte cultures was saturable in the presence of Na⁺ with a Km of 13–18 M and a Vmax of 9.4 nmoles/mg protein/hour in myo-inositol-fed cells, indicating a high affinity transport system. In myo-inositol-deprived cells, Km was about 53 M with a Vmax of 13.2 nmoles/mg protein/hour. Decreasing osmolality decreased the Vmax to about 1.9 nmoles/mg protein/hour whereas increasing osmolality increased Vmax about 5-fold, while Kms were essentially unchanged in myo-inositol fed cells. In cells deprived of myo-inositol, Vmax decreased in hypotonic medium and increased in hypertonic medium almost 10-fold, but with more than a doubling of the Km regardless of the osmolality. Glucose (25 mM) inhibited myo-inositol uptake 51% whereas the other hexoses used inhibited uptake much less. Our findings indicate that myo-inositol uptake in astrocytes occurs through an efficient carrier-mediated Na⁺-dependent co-transport system that is different from that of glucose and its kinetic properties are affected by myo-inositol availability and osmotic stress.     

myo-Inositol Synthesis from [1-H]Glucose in Phaseolus vulgaris L. during Early Stages of Germination

Radiolabeled d-[1-³H]glucose was fed by imbibition under sterile conditions to bean (Phaseolus vulgaris L.) seeds. After 72 and 96 hours of feeding, the ³H was located in uronic acid and pentose residues as well as hexose residues of cell wall polysaccharides in growing hypocotyl and root. Free myo-inositol present in cotyledons, hypocotyl, and root also contained ³H, showing that de novo synthesis of myo-inositol from [1-³H]glucose did occur during the first 72 hours of germination. More than 90% of the labeled, free myo-inositol was present in the cotyledons. The ³H percentage in trifluoroacetic acid-soluble arabinose residues of cell wall polysaccharides from 72-hour-old bean hypocotyls was only half of their mole percentage. On the other hand, ³H percentages in hexose residues were higher than their mole percentages. The results suggest that myo-inositol is synthesized from reserve sugars during the very early stages of germination, and that the newly synthesized myo-inositol, as well as that stored in cotyledons, can be used for the construction of new hypocotyl and root cell wall polysaccharides after conversion into uronic acids and pentoses via the myo-inositol oxidation pathway.     

D-chiro-inositol affects accumulation of raffinose family oligosaccharides in developing embryos of Pisum sativum

Developing garden pea embryos are able to take up exogenously applied cyclitols: myo-inositol, which naturally occurs in pea, and two cyclitols absent in pea plants: d-chiro-inositol and d-pinitol. The competition in the uptake of cyclitols by pea embryo, insensitivity to glucose and sucrose, and susceptibility to inhibitor(s) of H⁺-symporters (e.g. CCCP and antimycin A) suggest that a common cyclitol transporter is involved. Both d-chiro-inositol and d-pinitol can be translocated through the pea plant to developing embryos. During seed maturation drying, they are used for synthesis of mainly mono-galactosides, such as fagopyritol B1 and galactosyl pinitol A. Accumulation of d-chiro-inositol (and formation of fagopyritols), but not d-pinitol, strongly reduces accumulation of verbascose, the main raffinose oligosaccharide in pea seeds. The reasons for the observed changes are discussed.     

Phosphatidylinositol(4,5)bisphosphate and Phosphatidylinositol(4)phosphate in Plant Tissues

Pea (Pisum sativum) leaf discs or swimming suspensions of Chlamydomonas eugametos were radiolabeled with [³H]myo-inositol or [³²P]Pi and the lipids were extracted, deacylated, and their glycerol moieties removed. The resulting inositol trisphosphate and bisphosphate fractions were examined by periodate degradation, reduction and dephosphorylation, or by incubation with human red cell membranes. Their likely structures were identified as d-myo-inositol(1,4,5)trisphosphate and d-myo-inositol(1,4,)-bisphosphate. It is concluded that plants contain phosphatidylinositol(4)phosphate and phosphatidylinositol(4,5)bisphosphate; no other polyphosphoinositides were detected.     

Unimpaired formation of hormone-stimulated inositol triphosphate in human mesangial cells under hyperglycemic conditions

The relationship between bulk cellular myo-inositol content and phosphatidylinositol metabolism was evaluated in a human mesangial cell line under euglycemic and hyperglycemic conditions. Mesangial cells maintained in high glucose medium displayed a concentration-dependent fall in myo-inositol as measured by gas-liquid chromatography. Measurements of phosphatidylinositol, phosphatidylinositol 4-monophosphate and phosphatidylinositol 4,5-biphosphate mass revealed slight but statistically insignificant increases in cells exposed to high glucose containing medium. CDP-diacylglycerol: myo-inositol 3-phosphatidylinositol transferase activity, measured in plasma membranes from mesangial cells grwon under control and hyperglycemic conditions, was kinetically similar with Michaelis constants (K_m values) for myo-inositol of 2.9 and 2.1 mM, respectively. Finally hormone-stimulated intracellular calcium mobilization and myo-inositol 1,4,5-triphosphate mass was measured from mesangial cells grown under normal and hyperglycemic conditions. Both intracellular calcium and inositol triphosphate formation were unchanged in cells previously exposed to high glucose conditions (400 mg/dl) compared to cells grown under normal glucose concentration (100 mg/dl). These data indicate that bulk changes in myo-inositol induced by hyperglycemia are neither associated with alterations in basal levels of inositol containing glycerolipids nor with changes in hormone-stimulated calcium mobilization and inositol trisphosphate formation under conditions of short term changes in extracellular glucose.     

The distribution of phosphatidylinositol in microsomal membranes from rat liver after biosynthesis de novo. Evidence for the existence of different pools of microsomal phosphatidylinositol by the use of phosphatidylinositol-exchange protein

1. The phosphatidylinositol-exchange protein from bovine brain was used to determine to what extent phosphatidylinositol in rat liver microsomal membranes is available for transfer. 2. The microsomal membranes used in the transfer reaction contained either phosphatidyl[2-³H]inositol or ³²P-labelled phospholipid. The ³²P-labelled microsomal membranes were isolated from rat liver after an intraperitoneal injection of [³²P]P_i. The ³H-labelled microsomal membranes and rough- and smooth-endoplasmic-reticulum membranes were prepared in vitro by the incorporation of myo-[2-³H]inositol into phosphatidylinositol by either exchange in the presence of Mn²⁺ or biosynthesis de novo in the presence of CTP and Mg²⁺. 3. Tryptic or chymotryptic treatment of the microsomes impaired the biosynthesis de novo of phosphatidylinositol. It was therefore concluded that the biosynthesis of phosphatidylinositol and/or its immediate precursor CDP-diacylglycerol takes place on the cytoplasmic surface of the microsomal membrane. 4. Under the conditions of incubation 42% of the microsomal phosphatidyl[2-³H]inositol was transferred with an estimated half-life of 5min; 38% was transferred with an estimated half-life of about 1h; the remaining 20% was not transferable. Identical results were obtained irrespective of the method of myo-[2-³H]inositol incorporation. 5. Both measurement of phosphatidylinositol phosphorus in the microsomes after transfer and the transfer of microsomal [³²P]phosphatidylinositol indicate that phosphatidyl[2-³H]-inositol formed by exchange or biosynthesis de novo was homogeneously distributed throughout the microsomal phosphatidylinositol. 6. We present evidence that the slowly transferable pool of phosphatidylinositol does not represent the luminal side of the microsomal membrane; hence we suggest that this phosphatidylinositol is bound to membrane proteins.