Inositol phospholipid

The [3H]inositol-labeled phospholipids are separated from other phospholipids on thin-layer chromatography (TLC) plates. After elution, the radioactive inositol phospholipids are cochromatographed with standards and visualized by exposure to iodine vapors, or by spraying with 10% CUSO4 in 8% H3PO4, then scanned for radioactivity (Gamier et al., 1994 Baron et al., 1989). 

PI is synthesized from myo-inositol and DAG. First DAG is phosphorylated to phosphatidic acid (PA), then PA and CTP react to form CDP-diacylglycerol. The synthesis of PI (from CDP-diacylglycerol and inositol) occurs in the endoplasmic reticulum (ER) and it is transported from there to other membranes by a PI-specific transfer protein. Once at the plasma membrane, PI can be sequentially phosphorylated by PI 

4- kinase and PI(4)P 5-kinase to generate PI(4)P and PI(4,5)P2 (Majerus, 1992). PI 4-kinase is membrane associated in most tissues, and has been purified to homogeneity from bovine uterus it has a molecular mass of 55 kDa and is specific for the 4 position (Carsten and Miller, 1990). PI(4)P 5-kinase has been found in both the soluble and particulate fractions of cell homogenates. A 53-kDa form of this enzyme has been purified from human erythrocyte membranes. The activities of PI 4- and PI(4)P 5-kinases are stimulated by Mg + and inhibited by Ca + (Abdel-Latif, 1986). 

IP3 3-kinase is a widely distributed soluble enzyme that converts I(1,4,5)P3 to I(1,3,4,5)P4 in the presence of Mg2+ and ATP (Majerus, 1992). The 3-kinase has a low for IP3 (0.2 to 1.5 jjlM), thus the kinase can compete effectively with the inositol polyphosphate 

5- phosphatases for IP3, since the for the latter enzymes are higher (7-25 jcM). The native enzyme is composed of two catalytic subunits, a 53-kDa protein and calmodulin (CaM). Physiological concentrations of Ca + stimulate IP3 3-kinase activity via the calmodulin subunit. The enzyme has been purified from porcine aortic smooth muscle (Yamaguchi et al., 1987), the cytosolic fraction of which contains enough CaM so that the enzyme is fully active without the addition 

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Other metabolites of inositol phospholipids, e.g., inositol (1,3,4,5)-tetrakisphos-phate (IP4) may have additional signal transduction roles, particularly in accelerating the uptake of extracellular Ca ion into the SR following a contraction. 

Triacylglycerols and some phosphoglycerols are synthesized by progressive acylation of glycerol 3-phosphate. The pathway bifurcates at phosphatidate, forming inositol phospholipids and cardiolipin on the one hand and triacylglycerol and choline and ethanolamine phospholipids on the other. 

Eberhard, D. A., Cooper, C. L., Low, M. G. and Holz R. W. Evidence that the inositol phospholipids are necessary for exocytosis loss of inositol phospholipids and inhibition of secretion in permeabilized cells caused by a bacterial phospholipase C and removal of ATR Biochem. J. 268 15-25, 1990. 

Michell, R. H. (1975). Inositol phospholipids in cell surface receptor function. Biochim. Biophys. Acta 415, 81-147. 

Figure 14-3. Signaling through protein kinase C (PKC). Activated phospholipase C cleaves the inositol phospholipid PIP2 to form both soluble (IP3) and membrane-associated (DAG) second messengers. DAG recruits PKC to the membrane, where binding of calcium ions to PKC fully activates it. To accomplish this, IP3 promotes a transient increase of intracellular concentration by binding to a receptor on the endoplasmic reticulum, which opens a channel allowing release of stored calcium ions. PIP2, phosphatidylinositol 4,5-bisphosphate DAG, diacylglycerol PLC, phospholipase C IP3, inositol trisphosphate.

Platelet activation occurs in large part via G protein-coupled agonist receptors and intracellular signaling events that involve activation of phospholipase C (PLC). PLC catalyzes the breakdown of plasma membrane inositol phospholipids, resulting in generation of 1,2-diacylglycerol (DAG) and 1,4,5-inositol triphosphate (IP3). DAG activates protein kinase C, and IP3 induces mobilization of calcium from intracellular stores (10). 

Cartwright RD Rapid eye movement sleep characteristics during and after mood disturbing events. Arch Gen Psychiatry 40 197-201, 1983 Gasas M, Alvarez E, Duro P, et al Antiandrogenic treatment of obsessive-compulsive neurosis. Acta Psychiatr Scand 73 221-222, 1986 Gasebolt TL, Jope RS Long-term lithium treatment selectively reduces receptor-coupled inositol phospholipid hydrolysis in rat brain. Biol Psychiatry 25 329-340, 1989... 

Kendall DA, Nahorski SR Acute and chronic lithium treatments influence agonist-and depolarization-stimulated inositol phospholipid hydrolysis in rat cerebral cortex. J Pharmacol Exp Ther 241 1023-1027, 1987... 

Whitworth P, Kendall DA Effects of lithium on inositol phospholipid hydrolysis and inhibition of dopamine D, receptor-mediated cyclic AMP formation by carbachol in rat brain slices. J Neurochem 53 536-541, 1989 Whybrow PC The therapeutic use of triiodothyronine and high dose thyroxine in psychiatric disorder. Acta Med Austriaca 21 44-47, 1994 Whybrow PC Update on thyroid axis approaches to treatment of rapid cycling bipolar disorder. Paper presented at the annual meeting of the New Clinical Drug Evaluations Unit (NCDEU), Boca Raton, EL, May 30, 1996... 

Lynch M. A. and Voss K. L. (1990). Arachidonic acid increases inositol phospholipid metabolism and glutamate release in synaptosomes prepared from hippocampal tissue. J. Neurochem. 55 215-221. 

Shasby, D.M., M. Winter, and S. Shasby. 1988. Oxidants and conductance of cultured epithelial cell monolayers Inositol phospholipid hydrolysis. Am J Physiol 255 781. 

DAG species are derived from three main routes (1) PLC-mediated hydrolysis of phospholipids (2) phosphatase-mediated hydrolysis of phosphatidic acid (PA) and (3) lipase-mediated hydrolysis of triacylglycerol (TAG) species (Fig. 2). Targeted lipidomic analyses show that the fatty acid compositions of the DAGs formed by these various routes reflect the composition of the parent lipid (Fig. 5). In particular, those derived from inositol phospholipids are highly enriched in... 

Dickenson JM, Hill SJ. Human 5-HT1B receptor stimulated inositol phospholipid hydrolysis in CHO cells synergy with Gq-coupled receptors. Eur J Pharmacol 1998 348 279-285. 

Griendling, K. K., Rittenhouse, S. E., Brock, T. A., et al. 1986. Sustained diacylglycerol formation from inositol phospholipids in angiotensin II-stimulated vascular smooth muscle cells. J Biol Chem 261 5901-5906. 

Rather than being reincorporated into inositol phospholipids, DAG can be broken down to release some arachidonate which is the precursor of prostaglandin E which, in turn, can react with plasma membrane receptors linked to adenyl cyclase and cAMP production. IL-3 does not activate phospholipase C but does promote phosphorylation of the glucose transporter by phosphokinase C (Dexter and Spooner, 1987). 

An excellent, well-written review (really a tome) on the stereochemistry of the inositol phospholipids is one by Parthasarathy and Eisenberg (1986). It should be on the must read list of investigators laboring in this ever-expanding field of the biochemistry of the phosphatidylinositols and also for others laboring to understand what is happening in this area. 

Perhaps the best approach to extraction of the phosphatidylinositols and their various phosphorylated derivatives from a cellular preparation is through use of the Bligh-Dyer technique or some modification of it. However, it is of paramount importance that the chloroform-methanol-water (1 2 0.8, v/v) mixture, as an example, contain an acid, usually 1 N HC1 as the water component. Otherwise, there will be a decreased recovery of the inositol phospholipids in the final chloroform extract. This is directly attributable to the fact that these inositol-containing phospholipids, as already mentioned above, are found naturally as the Ca2+, Mg2+, K1, and/or Na+ salts. If the solvent is not acidic, these salts essentially will remain in a water-rich frac-... 

Perhaps the major take-home message here is that the fatty acid composition of the inositol phospholipids is quite different from that found in phosphoglycerides such as phosphatidylcholine or phosphatidylethanolamine. The uniqueness rests on the narrow spectrum of fatty acid chain lengths present. 

A Summation. It is possible at this point in time to isolate, separate, and identify glycerophosphoinositol and inositol monophosphate by sophisticated HPLC on anion exchange resins. This approach, using well-defined synthetic compounds (available from commercial suppliers) for standards, has made a significant impact on identification of inositol phospholipid metabolites in stimulated cell preparations. Certainly these latter techniques, together with the methodologies described earlier, have made life much easier for scientists in this field. 

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