Lithium Enhances Muscarinic Receptor–Stimulated CDP-Diacylglycerol Formation in Inositol-Depleted SK-N-SH Neuroblastoma Cells

The psychotherapeutic action of Li + in brain has been proposed to result from the depletion of cellular inositol secondary to its block of inositol monophosphatase. This action is thought to slow phosphoinositide resynthesis, thereby attenuating stimulated phosphoinositidase-mediated signal transduction in affected cells. In the present study, the effect of Li + on muscarinic receptor–stimulated formation of the immediate precursor of phosphatidylinositol, CDP-diacylglycerol (CDP-DAG), has been examined in human SK-N-SH neuroblastoma cells that have been cultured under conditions that alter the cellular content of myo -inositol. Resting neuroblastoma cells, like brain cells in vivo, were found to concentrate inositol from the culture medium, achieving an intracellular level of 60.0 ± 4 nmol/mg of protein. The addition of carbachol to [ 3 H]cytidine-prelabeled cells elicited a four- to fivefold increase in the accumulation of labeled CDP-DAG. This stimulated formation of [ 3 H]CDP-DAG was completely blocked by the addition of 10 Μ M atropine, was not dependent on the presence of Li + , nor was it affected by co-incubation with myo -inositol. This result was in sharp contrast to findings in rat brain slices, in which carbachol-stimulated formation of [ 3 H]CDP-DAG was potentiated ∼ 10-fold by Li + and substantially reduced by coincubation with inositol. The formation of [ 3 H]CDP-DAG in labeled SK-N-SH cells by carbachol was both concentration and time dependent. The order of efficacy of muscarinic ligands in stimulating [ 3 H]-CDP-DAG accumulation paralleled that established in these cells for inositol phosphate accumulation, i.e., carbachol ≥ oxotremorine-M > bethanecol ≥ arecoline > oxotremorine > pilocarpine. Extended culture of the SK-N-SH cells in an inositol-free chemically defined growth medium progressively reduced the intracellular inositol content to <5 nmol/mg of protein, a level comparable with that seen in cortical slices. In these inositol-depleted cells, Li + potentiated carbachol-stimulated [ 3 H]CDP-DAG formation, and this effect was completely reversed by coincubation with inositol (EC 50 0.2 m M ). The present study thus demonstrates, in the same cultured cell line, the effects of normal and reduced intracellular inositol levels on the ability of Li + to attenuate phosphoinositide resynthesis, as inferred from [ 3 H]CDP-DAG accumulation. The results indicate that Li + can lead to a slowing of stimulated phosphoinositide turnover in neuroblastoma cells, provided that the intracellular inositol content has been significantly reduced.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65723/1/j.1471-4159.1993.tb03289.x.pd

Developmental and Regional Studies of the Metabolism of Inositol 1,4,5-Trisphosphate in Rat Brain

Coupling of CNS receptors to phosphoinositide turnover has previously been found to vary with both age and brain region. To determine whether the metabolism of the second messenger inositol 1,4,5-trisphosphate also displays such variations, activities of inositol 1,4,5-trisphosphate 5′-phosphatase and 3′-kinase were measured in developing rat cerebral cortex and adult rat brain regions. The 5′-phosphatase activity was relatively high at birth (∼50% of adult values) and increased to adult levels by 2 weeks postnatal. In contrast, the 3′-kinase activity was low at birth and reached ∼50% of adult levels by 2 weeks postnatal. In the adult rat, activities of the 3′-kinase were comparable in the cerebral cortex, hippocampus, and cerebellum, whereas much lower activities were found in hypothalamus and pons/medulla. The 5′-phosphatase activities were similar in cerebral cortex, hippocampus, hypothalamus, and pons/medulla, whereas 5-to 10-fold higher activity was present in the cerebellum. The cerebellum is estimated to contain 50–60% of the total inositol 1,4,5-trisphosphate 5′-phosphatase activity present in whole adult rat brain. The localization of the enriched 5′-phosphatase activity within the cerebellum was examined. Application of a histochemical lead-trapping technique for phosphatase indicated a concentration of inositol 1,4,5-trisphosphate 5′-phosphatase activity in the cerebellar molecular layer. Further support for this conclusion was obtained from studies of Purkinje cell-deficient mutant mice, in which a marked decrement of cerebellar 5′-phosphatase was observed. These results suggest that the metabolic fate of inositol 1,4,5-trisphosphate depends on both brain region and stage of development.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65545/1/j.1471-4159.1990.tb01976.x.pd

Properties of a CDP-Diglyceride Hydrolase from Guinea Pig Brain

Enzymatic hydrolysis of the pyrophosphate bond of CDP-diglyceride (CDP-DG), previously shown to occur in bacteria, is demonstrable in mammalian tissues. Activity was enriched in a lysosomal fraction obtained from guinea pig cerebral cortex and was purified 92-fold relative to the homogenate by a combination of XM-300 ultrafiltration and DEAE-cellulose column chromatography. When incubated with CDP-dipalmitin, the purified enzyme produced stoichiometric amounts of CMP and phosphatidate. dCDP-DG served as a substrate, while ADP-DG was an inhibitor, as were 5′-AMP and 5′-dAMP. CDP-DG hydrolysis was not affected by the presence of excess amounts of CDP-choline, CDP-glycerol, sodium pyrophosphate, or cyclic 3′,5′-AMP.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65407/1/j.1471-4159.1981.tb01691.x.pd

Isolation and separation of inositol phosphates from hydrolysates of rat tissues

1. 1.An electrophoretic method for the rapid separation of the phosphate esters of inositol is presented.2. 2.It is used, together with ion-exchange chromatography, to establish the presence of inositol di- and triphosphates in hydrolysates of trichloroacetic acid residues of rat brain, liver, heart, kidney, and lung.3. 3.With the aid of a 32P marker, inositol di- and triphosphates were also found in hydrolysates of rat erythrocyte stroma.4. 4.Glycerol diphosphate was found in hydrolysates of rat liver.5. 5.Inositol tetraphosphate was not found in the hydrolysates of any of the tissues studied.6. 6.Residues remaining after extraction of rat brain by established lipid extraction procedures retained substantial amounts of bound inositol di- and triphosphates.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/32019/1/0000061.pd

Cholesterol Synthesis and Nerve Regeneration

In this report, we examine the requirement of cholesterol biosynthesis and its axonal transport for goldfish optic nerve regeneration. Cholesterol, labeled by intraocular injection of [ 3 H]mevalonolactone. exhibited a delayed appearance in the optic tectum. Squalene and other minor components were labeled but not transported. Following optic nerve crush, the amount of labeled cholesterol transport was elevated, while retinal labeling was not altered relative to control fish. A requirement for cholesterol biosynthesis is inferred from the inhibition of neurite outgrowth in retinal explants caused by the cholesterol synthesis inhibitor, 20, 25-diazacholes-terol. The inhibition of growth could be overcome by addition of mevalonolactone, but not cholesterol, to the medium. Intraperitoneal administration of 200 nmol of dia-zacholesterol resulted in 92-98% inhibition of retinal cholesterol synthesis and accumulation of labeled des-mosterol and other lipids in fish retina and brain which persisted for 2 weeks. Diazacholesterol-treated fish showed no reduction in the amount of lipid-soluble radioactivity transported following intraocular injection of [ 3 H]mevalonolactone, but there were alterations in the chromatographic pattern of the transported labeled lipids. In contrast to its effects on neurite outgrowth in vitro , diazacholesterol did not inhibit optic nerve regeneration in vivo , as measured both by arrival of labeled rapidly transported protein at the tectum and by time required for the return of visual function.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65710/1/j.1471-4159.1984.tb12701.x.pd

EFFECTS OF PUROMYCIN, ACETOXYCYCLOHEXIMIDE AND ACTINOMYCIN D ON PROTEIN SYNTHESIS IN GOLDFISH BRAIN *

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65427/1/j.1471-4159.1966.tb10284.x.pd

Measurement of Receptor-Activated Phosphoinositide Turnover in Rat Brain: Nonequivalence of Inositol Phosphate and CDP-Diacylglycerol Formation

Two methods for the measurement of receptor-activated phosphoinositide turnover were evaluated for their degree of correspondence in slices of rat brain; they involved the Li + -dependent accumulations of either [ 3 H]-inositol-labeled inositol phosphates or [ 3 H]cytidine-labeled CDP-diacylglycerol. In contrast to the expectation that the ratio of these two responses would remain approximately constant, varying degrees of correspondence were obtained. The two extremes are exemplified by carbachol, which elicited large increases in both inositol phosphate and CDP-diacylglycerol labeling, and endothelin, which gave a robust inositol phosphate response with little or no accumulation of 3 H-CDP-diacylglycerol. No instance of the presence of the latter response in the absence of 3 H-inositol phosphate accumulation was observed. Measurement of 3 H-CDP-diacylglycerol accumulation thus may add additional insight into the regulation of phosphoinositide turnover and the complex actions of Li + .Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66135/1/j.1471-4159.1993.tb03258.x.pd

Uridine Metabolism in the Goldfish Retina During Optic Nerve Regeneration: Cell-Free Preparations

The activities of uridine kinase (EC 2.7.1.48), uridine monophosphate (UMP) kinase (EC 2.7.1.3.14), and uridine diphosphate (UDP) kinase (EC 2.7.4.6) were measured in retinal high-speed supernatant fractions following unilateral optic nerve crush in the goldfish. The enzyme activities followed a similar time course, with initial increases 2-3 days following nerve crush, peak activity at 4 days, and a gradual return to basal levels by day 21. The magnitude of the stimulation on day 4 was about 35% in each case. Activities of two enzymes of intermediary metabolism, pyruvate kinase (EC 2.7.1.40) and lactic dehydrogenase (EC 1.1.1.27), were not altered, indicating that the coordinate increases in nucleoside and nucleotide kinase activities were specific responses to the nerve injury. The increased labeling could not be explained by altered phosphohydrolytic activities. The nature of the enhancement was further studied in UDP kinase, the most active of the kinases examined. Neither low-molecular-weight components nor substrate availability could account for the observed increase in UDP kinase in the 4 day post-crush retinas. The K m , for UDP was unaltered, and a mixing experiment did not support the possibility that stimulatory or inhibitory factors played a role. The enhancement of UDP kinase activity was blocked by injection of actinomycin D following nerve crush. The results suggest that the observed increases in enzymes of uridine metabolism result from their increased formation following nerve crush.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65504/1/j.1471-4159.1981.tb01714.x.pd

Political boundary spanning:politicians at the interface between collaborative governance and representative democracy

Research finds that productive interfaces between collaborative and bureaucratic forms of governance hinges on the extent t

DISTRIBUTION AND PROPERTIES OF CDP-DIGLYCERIDE:INOSITOL TRANSFERASE FROM BRAIN 1

CDP-diglyceride is converted to phosphatidyl inositol by several particulate subcellular fractions of guinea pig brain, with highest specific activity in the microsomal fraction. Optimal conditions with respect to pH, metal ion concentration, and substrate concentrations have been determined. The reaction was stimulated by the addition of bovine serum albumin and by Tween 80. Of several dl-CDP-diglycerides synthesized and used as substrates in a spectrophoto-metric assay for the enzyme, dl-CDP-didecanoin was the most active. The enzyme showed a high selectivity for myo-inositol. Of a number of compounds tested, only scyllo -inosose and epi -inosose served as substrates. Three inositol isomers and three myo -inositol monophosphates inhibited the reaction slightly. The most potent inhibitor found was galactinol, a myo -inositol galactoside.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66197/1/j.1471-4159.1969.tb06850.x.pd