Neuroprotocols最新文献

1-[6-[[17β-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]-hexyl]1H-pyrrole-2,5-dione (U-73122) is an aminosteroid that was identified initially as a potent inhibitor of platelet activation by receptor-specific agonists. U-73122 inhibits receptor-coupled generation of inositol 1,4,5-trisphosphate (but not cyclic AMP) and intracellular mobilization of Ca ²⁺ in a variety of cell types. U-73122 inhibits phosphoinositide-specific phospholipase C (PI-PLC) activity in cell-free systems, but exhibits little or no direct inhibition of phospholipases A₂ and D. Structure-activity analysis revealed that the maleimide group of U-73122 is essential, but not sufficient, for inhibitory activity. The succinimide analog of U-73122 (U-73343) has negligible inhibitory activity and is a useful control compound. On the basis of information derived from the use of U-73122 in a variety of cell types, procedures for storing, dissolving, and presenting U-73122 to cells are recommended. While knowledge of the mechanism of action of U-73122 would extend the utility of this compound, U-73122 has already been employed successfully to examine PI-PLC involvement in a variety of cellular processes. The application of U-73122 in an investigation of muscarinic receptor sequestration in SK-N-SH neuroblastoma cells is illustrated.

Phosphoinositide hydrolysis plays an important role in cellular signaling because it results in increased levels of calcium and diacylglycerols (DGs), which in turn activate protein kinase C (PKC). Agonist-induced hydrolysis of phosphatidylcholine (PtdCho) has been demonstrated, which also results in DG formation. However, it has not been clearly established whether PtdCho-derived DGs activate PKC in intact cells. We addressed this question directly, using α-thrombin stimulation of IIC9 fibroblasts as a model system. We show that DG produced from phosphoinositide, but not PtdCho hydrolysis, is associated with the activation of PKC. In addition, the methods used to quantify and chemically analyze agonist-induced changes in lipid levels, as well as PKC activation, are reviewed in detail.

A method for assessing stimulated phosphoinositide turnover by measurement of the liponucleotide CDP-diacylglycerol is presented. The phosphoinositide signal transduction pathway consists of a sequence of reactions in which the second messengers Inositol 1,4,5-triphosphate and diacylglycerol are recycled back to phosphatidylinositol (PtdIns), which then serves to replenish the initial hydrolyzed substrate, phosphatidylinositol 4,5-bis-phosphate. Receptor-stimulated inositol lipid turnover is most commonly assessed by measurement of the accumulation of [³H]inositol-labeled inositol phosphates in the presence of Li⁺. The latter blocks Inositol monophosphatase and thus can lead to a depletion of intracellular inositol. Because inositol is required for resynthesis of PtdIns, the immediate precursor of PtdIns, CDP-diacylglycerol, also accumulates in the presence of agonist and Li⁺. Measurement of radiolabeling of this liponucleotide following Incorporation of [³H]cytidine thus forms the basis for an alternative assay for Inositol lipid turnover. The general applicability of this method may be limited, since, In brain slices, not all receptors exhibit CDP-diacylglycerol responses that are consistent with their inositol phosphate responses. In addition, in cultured neural cells, growth in inositol-free, chemically defined medium is required to maximize the Li⁺ -dependent CDP-diacylglycerol response. A major advantage of this method may be its ability to provide insight Into the regulation of phosphoinositide turnover since this method uniquely reflects slowing of the regenerative cycle. Such in vitro studies may have relevance to the in vivo action of Li⁺ as a psychotherapeutic agent.

Phospholipase D is a commonly encountered but poorly understood member of the phospholipase family of cell signaling enzymes. Until recently, its study was inhibited by the lack of a simple and adaptable assay in intact cells that is not complicated by the presence of phospholipase C activity. Here, we review the various methods used to measure phospholipase D in whole cells in culture and with disrupted neuronal preparations, and we introduce the use of transphosphatidylation as a method of measuring the activity of phospholipase D in the presence of millimolar concentrations of alcohol. We then describe in detail the use of transphosphatidylation by butanol with ³²P-labeled neuron-like cells in culture. Alternative radiolabeling procedures, using [³H]glycerol and ³H-labeled fatty acids, with these cells are discussed. Finally, the application of procedures such as these to brain preparations, in particular, to intact synaptosomal preparations, is described.

Methods for the synthesis, purification, and use of an inhibitor of glucosylceramide synthesis (PDMP or D-1-phenyl-2-decanoylamino-3-morpholino-1-propanol) are given. The inhibitor is effective with a variety of cells and animals in producing a depletion of glucosylceramide. Because this cerebroside is the precursor of hundreds of other glycolipids, depletion of all of these compounds also takes place as each one is degraded by hydrolases. Use of PDMP causes accumulation of ceramide, the lipoidal precursor of glucosylceramide. Some of this simple sphingolipid is diverted to the synthesis of sphingomyelin and some, via hydrolysis, to formation of sphingols (sphingoid bases). The above changes in the biosynthesis of glycolipids result in pronounced effects on cells: slowing of growth, increased activity of the phospholipase C that catalyzes phosphatidylinositol bisphosphate hydrolysis, accumulation of N,N-dimethylsphingosine (an inhibitor of protein kinase C), accumulation of diacyiglycerol (an activator of PKC), and reduction of the ability to bind to extracellular matrix proteins. PDMP is rapidly absorbed and released by cells. In mice, it is metabolized by a microsomal monooxygenase and the products are excreted. The degradative process can be blocked by inhibitors of cytochrome P-450, such as piperonyl butoxide, cimetidine, and fluconazole. Understanding these properties permits the use of PDMP in both in vitro and in vivo studies in which glycolipids may exhibit important biological effects. Two recent examples of the in vitro and in vivo use of PDMP are provided.