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.