Journal of Neurochemistry

Ammonium trichloro(dioxoethylene-0,0') tellurate (AS101) is a non-toxic organotellurium compound with pleiotropic activities. It was recently shown to induce production of the neurotrophic factor glial cell line-derived neurotrophic factor and to rescue neuronal-like PC-12 cells from neurotrophic factor deprivation-induced apoptosis. In this study, we show that AS101 improves functional outcome and reduces brain damage in a mouse model of focal ischemic stroke. Both pre-stroke and post-stroke intraperitoneal treatments with AS101 reduced infarct size and edema and improved the neurological function of the animals. AS101 treatments reduced both apoptotic and inflammatory caspase activities, and also inhibited protein tyrosine nitration suggesting that AS101 suppresses oxidative stress. Studies of cultured neurons showed that AS101 confers protection against apoptosis induced by either glucose deprivation or the lipid peroxidation product 4-hydroxynonenal. Moreover, AS101 treatment reduced glutamate-induced intracellular calcium elevation, a major contributor to neuronal death in stroke. As AS101 has an excellent safety profile in humans, our pre-clinical data suggest a potential therapeutic benefit of AS101 in patients suffering from stroke and other neurodegenerative conditions.

Although members of the multiple vertebrate/mammalian dopamine D1 receptor gene family can be selectively classified on the basis of their molecular/phylogenetic, structural, and tissue distribution profiles, no subtype-specific discriminating agents have yet been identified that can functionally differentiate these receptors. To define distinct pharmacological/functional attributes of multiple D1-like receptors, we analyzed the ligand binding profiles, affinity, and functional activity of 12 novel NNC compounds at mammalian/vertebrate D1/D1A and D5/D1B, as well as vertebrate D1C/D1D, dopamine receptors transiently expressed in COS-7 cells. Of all the compounds tested, only NNC 01-0012 displayed preferential selectivity for vertebrate D1C receptors, inhibiting [3H]SCH-23390 binding with an estimated affinity (approximately 0.6 nM) 20-fold higher than either mammalian/vertebrate D1/D1A or D5/D1B receptors or the D1D receptor. Functionally, NNC 01-0012 is a potent antagonist at D1C receptors, inhibiting to basal levels dopamine (10 microM)-stimulated adenylyl cyclase activity. In contrast, NNC 01-0012 (10 microM) exhibits weak antagonist activity at D1A receptors, inhibiting only 60% of maximal cyclic AMP production by dopamine, while acting as a partial agonist at vertebrate D1B and D1D receptors, stimulating adenylyl cyclase activity by approximately 33% relative to the full agonist dopamine (10 microM), an effect that was blocked by the selective D1 receptor antagonist NNC 22-0010. These data clearly suggest that the benzazepine NNC 01-0012, despite lacking the N-methyl residue in the R3 position, is a selective and potent D1C receptor antagonist. Moreover, the differential signal transduction properties exhibited by NNC 01-0012 at these receptor subtypes provide further evidence, at least in vertebrates, for the classification of the D1C receptor as a distinct D1 receptor subtype.

In this study we evaluated UCN-01, a small molecule that inhibits protein kinases by interacting with the ATP-binding site, as a potential anti-cancer agent for neuroblastoma. UCN-01 was effective at inducing apoptosis in six neuroblastoma cell lines with diverse cellular and genetic phenotypes. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) assays, detection of active caspase-3 and cleaved poly ADP-ribose polymerase (PARP) confirmed that UCN-01 induced apoptosis. Cell cycle analysis determined that the UCN-01 treated cells accumulated in S phase by 16 h. Unlike vinblastine and docetaxel that increased survivin expression, UCN-01 treatment did not increase X-linked inhibitor of apoptosis protein (XIAP) and survivin levels. Analysis of specific phosphoepitopes on chk1/2, Akt, and GSK3beta following UCN-01 treatment determined that there was no significant change in phospho-chk1/2. However, there was decreased immunoreactivity at Ser473 and Thr308 of Akt and Ser9 of GSK3beta by 4 h indicating that the Akt survival pathway and downstream signalling was compromised. Thus, UCN-01 was effective at inducing apoptosis in neuroblastoma cell lines.

Using primary cultures of bovine adrenal chromaffin cells labelled with 32Pi, we show that stimulation with bradykinin, nicotine, or a depolarising concentration of potassium stimulates the accumulation of [32P]phosphatidic acid. The effects of nicotine and potassium are smaller than the effect of bradykinin, and are dependent entirely on extracellular calcium. The diacylglycerol kinase inhibitor R 59 022 attenuates the formation of phosphatidic acid by nicotine and depolarising concentrations of potassium. This inhibitor also blocks the nicotine and potassium stimulation of noradrenaline release from chromaffin cells. Using 45Ca2+ influx studies, we show that the nicotine-evoked calcium influx is also attenuated by R 59 022. These observations contrast with those in another report in which we showed that bradykinin stimulation of either [32P]phosphatidic acid accumulation or noradrenaline release is not affected by R 59 022. It is likely that the calcium influx produced by nicotine and depolarising potassium is blocked by R 59 022 by a mechanism that is independent of its ability to block diacylglycerol kinase. The nicotine- and potassium-stimulated [32P]phosphatidic acid accumulation is a consequence of this calcium influx and presumably reflects calcium activation of either phospholipase C or phospholipase D.

Feedback inhibition of tyrosine hydroxylase by catechols was evaluated using in situ and in vitro enzyme assays. The three catechol compounds used were norepinephrine, 2-hydroxyestradiol, and 3'4'-dihydroxy-2-methylpropiophenone (U-0521, Upjohn); representing endogenous catecholamines, catechol estrogens, and a synthetic catechol, respectively. The in situ experiments were performed with dissociated retinal cells from rats and with stationary phase adrenergic-like neuroblastoma cells (N1E-115). The catechol estrogen, 2-hydroxyestradiol, resembled the endogenous catecholamine in its potency to inhibit in vitro and in situ tyrosine hydroxylations with IC50 values of 10 microM in vitro and 100 microM in situ. The drug U-0521, which has been used as an inhibitor of catechol-O-methyltransferase (COMT), was also found to be an inhibitor of tyrosine hydroxylase. Further, it was shown to be more potent than the natural catechols, both in vitro and in situ, with IC50 values of 30--600 nM.

The effects of the selective dopamine D2 receptor antagonists YM-09151-2 and l-sulpiride on the in vivo release of dopamine (DA), L-3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) in rat striatum were investigated. The drugs were injected into the striatum through a microinjection needle attached to a dialysis probe. YM-09151-2 (0.1 or 1.0 microgram/0.5 microliter) injected into the striatum produced a dramatic rapid-onset transient increase in striatal DA release in a dose-dependent manner. However, the DA increase induced by l-sulpiride (15 or 75 ng/0.5 microliter) was small and of slower onset. An increase of DOPAC levels by YM-09151-2 was biphasic: The first peak occurred at 40 min, followed by a delayed-onset gradual increase. Slower-onset gradual increases were also found in DOPAC levels after l-sulpiride injection and in HVA levels after injections of both YM-09151-2 and l-sulpiride. The infusion of tetrodotoxin (TTX; 2 microM) revealed two different types of DA release mechanisms: The rapid-onset transient DA release induced by YM-09151-2 was TTX insensitive, whereas the slower-onset DA release induced by l-sulpiride was TTX sensitive. Moreover, the rapid-onset transient DA release was Ca2+ independent and was not affected by pretreatment with l-sulpiride or nomifensine. Therefore, it is concluded that YM-09151-2 injected into the striatum produced a transient striatal DA release that is independent of D2 receptors and the action potential.

The addition of nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) to PC12 cells prelabeled with [3H]inositol and preincubated for 15 min in the presence of 10 mM LiCl stimulated the production of inositol phosphates with maximal increases of 120-180% in inositol monophosphate (IP), 130-200% in inositol bisphosphate (IP2), and 45-50% in inositol trisphosphate (IP3) within 30 min. The majority of the overall increase (approximately 85%) was in IP; the remainder was recovered as IP2 and IP3 (approximately 10% as IP2 and 5% as IP3). Under similar conditions, carbachol (0.5 mM) stimulated about a 10-fold increase in IP, a sixfold increase in IP2, and a fourfold increase in IP3. The mass level of 1,2-diacylglycerol (DG) in PC12 cells was found to be dependent on the incubation conditions; in growth medium [Dulbecco's modified Eagle's medium (DME) plus serum], it was around 6.2 mol %, in DME without serum, 2.5 mol %, and after a 15-min incubation in Dulbecco's phosphate-buffered saline, 0.62 mol %. The addition of NGF and bFGF induced an increase in the mass level of DG of about twofold within 1-2 min, often rising to two- to threefold by 15 min, and then decreasing slightly by 30 min. This increase was dependent on the presence of extracellular Ca2+, and was inhibited by both phenylarsine oxide (25 microM) and 5'-deoxy-5'-methylthioadenosine (3 mM). Under similar conditions, 0.5 mM carbachol stimulated the production of DG to the same extent as 200 ng/ml NGF and 50 ng/ml bFGF. Because carbachol is much more effective in stimulating the production of inositol phosphates, the results suggest that both NGF and bFGF stimulate the production of DG primarily from phospholipids other than the phosphoinositides.

The uptake of [14C]ethylenediamine into slices of rat brain and its subsequent evoked release have been studied. An active uptake process was demonstrated by comparing uptake at 37 and 4 degrees C. This uptake showed a Km of 1.36 mM, was partly sodium-dependent and was reduced by nipecotic acid. Release could be readily evoked by 30 mM potassium, and by electrical stimulation, the release in both cases being calcium-dependent. In view of these findings and the reported interactions of ethylenediamine with gamma-aminobutyric acid-related mechanisms, it might be of interest to determine whether this simple diamine occurs endogenously in the mammalian brain.

The axonal outgrowth of cells of Neuro2a, a mouse neuroblastoma cell line, was suppressed on expression of the beta-galactoside alpha 1,2-fucosyltransferase (alpha 1,2-FT) gene. We recently cloned two types of rabbit alpha 1,2-FT, RFT-I and RFT-II. RFT-I exhibits comparable kinetic properties and structural homology with human H gene alpha 1,2-FT, and RFT-II shows comparable kinetic parameters with human Se gene alpha 1,2-FT. Neuro2a cells expressing RFT-I (N2A-RFT-I) contained a large amount of fucosyl GM1 instead of GM1 and GD1a, major gangliosides in the parent Neuro2a cells, whereas Neuro2a cells expressing RFT-II (N2A-RFT-II) showed a subtle change in the ganglioside pattern. N2A-RFT-II and parent Neuro2a cells showed axonal outgrowth in serum-free medium on the exogenous addition of GM1, whereas N2A-RFT-I cells exhibited multiple neurite sprouts but not axonal outgrowth. This phenotype was fully recovered by N2A-RFT-I cells on the addition of D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol and alpha-L-fucosidase to the culture medium, which resulted in pronounced reduction of fucosyl GM1 expression. These results suggested that expression of H-type alpha1,2-FT, and subsequent incorporation of fucose into glycolipids and glycoproteins, especially the formation of fucosyl GM1, modifies the response of neuronal cells to stimuli that induce axonal extension.

The relative suitability of different molecular species of 1,2-diacyl-sn-glycerols as substrates for the diacylglycerol kinase (ATP: 1,2-diacyl-sn-glycerol phosphotransferase) in rat brain microsomes was investigated. The diacylglycerols tested were a mixture of the 1-[3H]palmitoyl and 1-[14C]stearoyl homologues of either the 2-oleoyl (monoenoic), 2-linoleoyl (dienoic), 2-arachidonoyl (tetraenoic), or 2-docosahexaenoyl (hexaenoic) diacylglycerols with an isotope ratio (3H/14C) approximately equal to 1.00. At substrate concentrations of 0.125 mM and 0.60 mM, only a modest preference of the kinase for total (1-palmitoyl plus 1-stearoyl homologues) monoenoic over total hexaenoic species was indicated. The tetraenoic diacylglycerols gave reaction rates which were not significantly different from the monoenes, dienes, or hexaenes when the data were analyzed statistically. No significant enzyme selectivity for either the 1-palmitoyl or 1-stearoyl homologues of the various 1-saturated 2-unsaturated diacylglycerols was apparent. The present results, together with data on the composition of free 1,2-diacylglycerols in brain, which reveal a preponderance of tetraenoic molecular species, suggest that the tetraenoic phosphatidic acids (mainly as 1-stearoyl 2-arachidonoyl species) are quite possibly the major products of diacylglycerol kinase activity in rat brain under physiological conditions.

Highly purified rat brain myelin isolated by two different procedures showed appreciable activity for CDP-ethanolamine: 1,2-diacyl-sn-glycerol ethanolaminephosphotransferase (EC 2.7.8.1). Specific activity was close to that of total homogenate and approximately 12-16% that of brain microsomes. Three other lipid-synthesizing enzymes, cerebroside sulfotransferase, lactosylceramide sialyltransferase, and serine phospholipid exchange enzyme, were found to have less than 0.5% the specific activity in myelin compared with microsomes. Washing the myelin with buffered salt or taurocholate did not remove the phosphotransferase, but activity was lost from both myelin and microsomes by treatment with Triton X-100. It resembled the microsomal enzyme in having a pH optimum of 8.5 and a requirement for Mn2+ and detergent, but differed in showing no enhancement with EGTA. The diolein Km was similar for the two membranes (2.5-4 x 10(-4) M), but the CDP-ethanolamine Km was lower for myelin (3-4 x 10(-5) M) than for microsomes (11 - 13 x 10(-5 M). Evidence is reviewed that this enzyme is able to utilize substrate from the axon in situ.

N-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) treatment, both in vitro and in vivo, results in an irreversible blockade of cortical S2 5-hydroxytryptamine (serotonin) receptors. Incubation of rat cortical homogenates with EEDQ in vitro results in a concentration-dependent (EC50 approximately 5 microM) and time-dependent decrease in the Bmax of [3H]ketanserin-labeled S2 serotonin receptors. Extensive washing of the homogenate following in vitro or in vivo EEDQ treatment does not result in an increase in the amount of [3H]ketanserin binding, indicating that EEDQ acts to modify irreversibly cortical S2 serotonin receptors. That the modification of S2 receptor binding by EEDQ occurs at the recognition site of the receptor is indicated by the finding that coincubation with the S2 receptor antagonist ketanserin, but not the D2 3,4-dihydroxyphenylethylamine (dopamine) receptor antagonist domperidone, selectively protects against the irreversible blockade of S2 serotonin receptors. Peripheral administration of EEDQ results in a dose-dependent reduction in cortical S2 serotonin receptors with maximal effects (approximately 90% reduction) observed following 10 mg/kg (i.p.). Seven days following peripheral administration of EEDQ there is a recovery of S2 serotonin receptors back to 74% of the original receptor population. These data demonstrate that EEDQ in vitro and in vivo acts as an irreversible antagonist of S2 serotonin receptors and that it can be used to investigate the recovery rate of these receptors.

The content of 1,2-diacylglycerol (DAG) was determined in sciatic nerves from normal and streptozotocin-induced diabetic rats. In nerves frozen in situ, DAG content was reduced 22% in the proximal region and 77% in the distal region of diabetic nerve, principally because of the loss of associated fat. DAG levels in freshly dissected and desheathed diabetic nerve were decreased from 23 to 30% as compared with normal nerve. Determination of DAG molecular species distribution in desheathed normal nerve indicated that 18:0/20:4 accounted for 34%, 16:0/18:1 for 17%, and several other polyunsaturated fatty acid-containing species for 17% of the total. In diabetic nerve, the quantity of the 18:0/20:4 DAG, species was reduced by 37%, and this drop was 62% of the reduction in all molecular species. The content of the minor species, 16:0/20:4 DAG, was decreased by 48%. Our results suggest that nerve DAG arises in large part from phosphoinositide degradation. Moreover, these results provide support for the hypothesis that reduced Na+,K(+)-ATPase activity in diabetic nerve is a consequence of decreased phosphoinositide turnover, which thereby generates insufficient DAG to maintain a protein kinase C-mediated step necessary for activation of Na+,K(+)-ATPase.

1,2-Diacyl-sn-glycerol (DAG) is a product of cell activation that has emerged as an important intracellular messenger whose primary function appears to be the activation of protein kinase C. They originate by the activation of phospholipases, which hydrolyze different phospholipids depending on the external stimulus and the nature of the cells, leading to the production of different molecular species. In the present study the quantitative changes in the total mass and the molecular species of DAG formed on phorbol ester (12-O-tetradecanoyl-phorbol 13-acetate) stimulation were investigated in proliferating and retinoic acid (RA)-differentiated human LA-N-1 cells. The TPA treatment of both cell types elicited an increase in the total amount of DAG. The increase was biphasic; i.e., an initial peak at 2-5 min was followed by a sustained increase that persisted for > 30 min. The analysis of the molecular species of DAG and phospholipids showed that in proliferating LA-N-1 cells, the DAG increase corresponds to the production of mainly saturated/monounsaturated (16:0-18:1, 18:0-18:1) and saturated/saturated (16:0-16:0, 16:0-18:0) species, suggesting that they originate essentially from the hydrolysis of phosphatidylcholine. In contrast, RA-differentiated cells responded to TPA treatment by increasing the level of saturated/polyunsaturated (16:1-22:6, 18:0-22:6, 16:0-20:4, 18:0-20:4) and monounsaturated/monounsaturated (18:1-18:1) species, suggesting mainly a phosphatidylethanolamine origin. These findings indicate that the treatment of LA-N-1 cells with TPA generates different molecular species of DAG depending on their physiological state. These observations suggest in turn that different phospholipases are activated by TPA in proliferating and RA-differentiated cells.

Specific radioactivities of molecular species of phosphatidyl choline(PC), phosphatidyl ethanolamine(PE) and 1,2-diacylglycerol were determined in rabbit brain 15 and 30 min after intraventricular injection of 10OpCi of either [U-14C]glucose or [U-14C]glycerol. The rate of de nouo synthesis of glycerophospholipids and their molecular species could be determined after glycerol labelling, since 94.0–99.7% of 14C activity was recovered in glyceryl moieties of brain lipids. After injection of glucose radioactivity was measured in both glyccrol and acyl residues of lipids. High incorporation rates were measured in species of PC, PE and 1,2-diacylglycerol with oleic acid in position 2 and with palmitic, stearic or oleic acids in position 1. The conclusion may therefore be drawn that these molecular species were preferably synthesized de novo by selective acylation of glycerol 3-phosphate. The lowest specific activities were observed for 1,2-dipalmitoyl- and l-stearoyl-2- arachidonoyl-glycerol, -PC and -PE. These turnover rates point to incorporation of arachidonate, and probably also of palmitate in dipalmitoyl-PC, amounting to 20% of total PC, via deacylation-acylation- cycle.

The irreversible protein-modifying reagent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was used to investigate binding site characteristics on the gamma-aminobutyric acidA (GABAA) receptor complex. In vitro, preincubation with EEDQ led to a concentration-dependent decrease in receptor number for benzodiazepine, t-butylbicyclophosphorothionate (TBPS), and GABA binding sites in cerebral cortex. The effect was maximal at the highest concentration of EEDQ used (10(-4) M) and was greatest for the benzodiazepine site. Pretreatment of membranes with the benzodiazepine antagonist Ro 15-1788, 1 or 10 microM, or the agonist lorazepam, 10 microM, largely prevented the effects of EEDQ. Scatchard analysis indicated no effect of EEDQ, 10(-4) M, on apparent affinity, but a decrease in receptor density for each site. Administration of EEDQ to mice, 12.5 mg/kg i.p., led to a substantial (55-65%) decrease in number of benzodiazepine binding sites in cortex after 4 h. Slightly smaller changes were observed for TBPS and GABA binding. No changes were observed in apparent affinity at any site. Prior administration of Ro 15-1788, 5 mg/kg, prevented the effect of EEDQ on benzodiazepine binding. Density of benzodiazepine binding sites gradually recovered over time, and receptor density returned to control values by 96 h after EEDQ injection. Number of binding sites in cortex for TBPS and GABA also increased over time after EEDQ. Benzodiazepine sites in cerebellum were decreased proportionally to cortex after EEDQ, and increased over a similar time course. Function of the GABAA receptor in chloride uptake in cortex was markedly reduced (65%) by EEDQ.(ABSTRACT TRUNCATED AT 250 WORDS)

1,2-Diacyl-, 1-alk-1'-enyl-2-acyl- and 1-alkyl-2-acyl-sn-glycero-3- phosphorylcholine specifically labelled with different acids at the 2 positions, were prepared enzymically using the acyltransferase system of rabbit sarcoplasmic reticulum. The substrates were submitted to hydrolysis by phospholipase A2 (phospholipid acyl-hydrolase, EC 3.1.1.4) obtained from normal and brain tissue affected with subacute sclerosing panencephalitis. In the diseased tissue an increase of phospholipase A2 activity ranging from 46 to 54% could be observed in comparison to the control brain for all substrates investigated. Among the investigated substrates, phospholipase A2 had the highest affinity for the 1,2 diacylcompound, whereas alkenylacyl- and alkylacyl-sn-glycero-3-phosphorylcholine were cleaved at almost similar rates. The hydrolysis rate of choline plasmalogen and the corresponding diacyl compound by the enzyme was greatly influenced by the fatty acid moiety located at the 2 position of the substrates.

The neurons of dorsal root ganglia (DRG) mediate several sensation modalities. The carbohydrate antigens on DRG neurons differ with the sensation modalities that subsets of neurons convey. Despite the important roles of gangliosides and glycoproteins in neuronal differentiation and neuritogenesis of the mammalian nervous system, little is known about the mechanisms underlying the regulation of glycosylation. We previously demonstrated the expression of H-blood type antigens (Fuc alpha1, 2Gal beta) on rabbit DRG neurons of small diameter and dramatic changes in H antigens during the perinatal period. To investigate the possible biological roles and regulatory mechanisms of H antigens, we recently cloned three types of rabbit alpha1,2-fucosyltransferase gene that catalyze the biosynthesis of H antigens. Here, we analyze the expression of these genes, RFT-I, II, and III, in rabbit DRG. The H-type alpha1,2-fucosyltransferase gene, RFT-I, was expressed in DRG in late embryos to adult rabbits, as detected on northern blotting. The other two secretor-type alpha1,2-fucosyltransferase genes, RFT-II and III, were observed to be expressed in late embryonic DRG on RT-PCR analysis but were not detectable on northern blotting. The expression of the H-type alpha1,2-fucosyltransferase gene was analyzed by in situ hybridization and was found to be abundant in small-diameter DRG neurons. These results indicate that the H-type alpha1,2-fucosyltransferase gene plays a major role in the regulation of the H antigen expression in DRG during the perinatal period.

Peroxiredoxins are an important family of cysteine-based antioxidant enzymes that exert a neuroprotective effect in several models of neurodegeneration. However, under oxidative stress they are vulnerable to inactivation through hyperoxidation of their active site cysteine residues. We show that in cortical neurons, the chemopreventive inducer 3H-1,2-dithiole-3-thione (D3T), that activates the transcription factor Nuclear factor erythroid 2-related factor (Nrf2), inhibits the formation of inactivated, hyperoxidized peroxiredoxins following oxidative trauma, and protects neurons against oxidative stress. In both neurons and glia, Nrf2 expression and treatment with chemopreventive Nrf2 activators, including D3T and sulforaphane, up-regulates sulfiredoxin, an enzyme responsible for reducing hyperoxidized peroxiredoxins. Induction of sulfiredoxin expression is mediated by Nrf2, acting via a cis-acting antioxidant response element (ARE) in its promoter. The ARE element in Srxn1 contains an embedded activator protein-1 (AP-1) site which directs induction of Srxn1 by synaptic activity. Thus, raising Nrf2 activity in neurons prevents peroxiredoxin hyperoxidation and induces a new member of the ARE-gene family, whose enzymatic function of reducing hyperoxidized peroxiredoxins may contribute to the neuroprotective effects of Nrf2 activators.

We have applied the 19F-nuclear magnetic resonance (NMR) calcium indicator 1,2-bis(2-amino-5-fluoro-phenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBAPTA) to the measurement of the free intracellular calcium concentration [( Ca2+]i) in superfused brain slices. A mean +/- SD control value of 380 +/- 71 nM (n = 18) was obtained at 37 degrees C using 2.4 mM extracellular Ca2+. Subcellular fractionation studies using [3H]5FBAPTA showed that after loading of its tetraacetoxymethyl ester, approximately 55% was de-esterified, with the other 45% remaining as the tetraester bound to membranes. Of the de-esterified 5FBAPTA, greater than 90% was in the cytosolic fractions, with less than 1% in the mitochondria or microsomes. The NMR-visible de-esterified 5FBAPTA slowly disappeared from the tissue with a t1/2 of 4 h. A time course after loading confirmed that the calculated [Ca2+]i was constant over a 5-h period, although the scatter of individual results was +/- 20%. The [Ca2+]i was increased by a high extracellular K+ concentration ([K+]e), by a low extracellular concentration of Na+, and by the calcium ionophore A23187. On recovery from high [K+]e, the [Ca2+]i "overshot" to values lower than the original control value. The [Ca2+]i was surpisingly resistant to changes in extracellular Ca2+ concentration.

The alkylating agent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) is a peptide-coupling agent that is being used to inactivate irreversibly alpha 2-adrenoceptors and other receptors. The aim of the present study was to assess the in vitro and in vivo effects of EEDQ on the newly discovered brain I2-imidazoline sites, located mainly in mitochondria. Preincubation of rat cortical membranes with EEDQ (10(-8)-10(-5) M) markedly decreased (20-90%) the specific binding of the selective antagonist [3H]RX821002 to alpha 2-adrenoceptors without affecting that of [3H]idazoxan (in the presence of adrenaline) to I2-imidazoline sites. In EEDQ-pretreated membranes (10(-5) M, 30 min at 25 degrees C), the density of I2-imidazoline sites (Bmax = 80 +/- 4 fmol/mg of protein) was not different from that determined in untreated membranes in the presence of (10(-6) M (-)-adrenaline (Bmax = 83 +/- 4 fmol/mg of protein), and both densities were lower (24%, p < 0.05) than the total native density of [3H]idazoxan binding sites (Bmax = 107 +/- 6 fmol/mg of protein) (I2-imidazoline sites plus alpha 2-adrenoceptors). Treatment of rats with an optimal dose of EEDQ (1.6 mg/kg, i.p., for 2 h to 30 days) reduced maximally at 6 h (by 95 +/- 1%) the specific binding of [3H]-RX821002 to alpha 2-adrenoceptors, but also the binding of [3H]idazoxan to I2-imidazoline sites (by 44 +/- 5%). Pretreatment with yohimbine (10 mg/kg, i.p.) fully protected against EEDQ-induced alpha 2-adrenoceptor inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)

The object of the present study was to determine if a reduced amount of lipid in the Quaking mouse brain were accompanied by decreased activities of any or all of the enzymes in the de novo synthesis of phosphatidylcholine via the CDP-choline pathway. In contrast to expectations, the results show a significant (P<0.005) increase in the cytosolic activity of the cholinephosphate cytidylyltransferase. In addition, this is the first report of these enzyme activities in mouse brain.