Adenosine inhibits glutamate stimulated [3H]d-aspartate release from cerebellar granule cells

The release of [³H]d-aspartate from cultured cerebellar granule cells (a glutamatergic neuron) was studied. It was found that the l-glutamate induced stimulation of [³H]d-aspartate release from the neurons could be decreased 30–40% by adenosine, cyclohexyladenosine and (−)N⁶-(2-phenylisopropyl)-adenosine all in the dose range 1–10 μM. The effects of adenosine agonists could be blocked and even reversed by the adenosine antagonist theophyllin indicating a possible endogenous adenosine tonus on the neurons. The results add further evidence to the notion that adenosine A₁ receptors present on parallel fiber terminals in the cerebellar cortex are involved in the regulation of glutamate transmitter release from the nerve terminals.     

Cyclic-AMP inhibits neither A23187-stimulated [14C]-arachidonic acid release from prelabelled lipids nor phospholipase A2 activity in resident rat peritoneal macrophages

8-Bromo cyclic AMP inhibited A23187-stimulated PGE2 production in adherent resident rat peritoneal macrophages by 50% when this was assessed by radioimmunoassay. In contrast, neither exogenous 8-bromo cyclic AMP nor elevation of endogenous cyclic AMP with cholera toxin inhibited 14C-arachidonic acid release or labelled prostaglandin formation by [1-14C]-arachidonic acid-prelabelled macrophages stimulated with either A23187 or melittin. Inhibition by cyclic AMP appears to be confined to PGE2 originating from a pool of endogenous phosphoglyceride that does not readily exchange with isotopically-labelled arachidonic acid. Phospholipase A2 activity, assessed as calcium-dependent [1-14C]-arachidonic acid release from exogenous 1-stearoyl, 2-[1-14C]-arachidonyl phosphatidyl choline at pH 8.6, was activated by melittin but not by A23187 in 1000 x g supernates from sonicated cells. Neither melittin nor calcium activation of phospholipase A2 was inhibited by preincubation of the cells prior to breakage with 8-bromo-cyclic AMP, nor by inclusion of either 8-bromo cyclic AMP or the catalytic subunit of cyclic AMP-dependent kinase in the assay. The results are inconsistent with the hypothesis that inhibition of A23187-stimulated PGE2 production by cyclic AMP in peritoneal macrophages is due to inhibition of a calcium-stimulated phospholipase A2.     

Vasopressin decreases total free fatty acids but enhances release of radioactivity from isolated hepatocytes labelled with [3H]arachidonic acid

The short-term effects of vasopressin on free fatty acids and lysophospholipids were investigated in hepatocytes isolated from fed rats. Over the time period 0.25 to 10 min vasopressin decreased the steady-state concentrations of palmitic, stearic and oleic acids measured by gas liquid chromatography in extracts of cells incubated at 0.1 mM extracellular Ca2+. The concentrations of arachidonic and linoleic acids did not change. In hepatocytes labelled with [3H]arachidonic acid and incubated at 1.3 mM extracellular Ca2+ vasopressin or the Ca2+-selective ionophore A23187 increased the rate of accumulation of radioactivity in the incubation medium by 40%. The action of A23187 was dependent on extracellular Ca2+. When hepatocytes labelled with 32Pi were treated with vasopressin, no change in the amounts of [32P]lysophosphatidylethanolamine or [32P]lysophosphatidylcholine was observed. It is concluded that the action of vasopressin on hepatocytes is associated with the release of arachidonic acid or metabolites of arachidonic acid but is not accompanied by a general increase in the steady-state concentrations of free fatty acids and lysophospholipids.     

Bordetella heat-labile toxin causes release of radioactivity from smooth muscle cells labeled with [14C]arachidonic acid

The effect of Bordetella heat-labile toxin (HLT) on cultured vascular or tracheal smooth cells labeled with [14C]arachidonic acid for 3 h was examined. At 37 degrees C, in the presence of Ca2+, HLT induced the release of radioactive substances from the cells in a dose-dependent manner but HLT had no effect on release of radioactivity from cells at 0 degrees C or in the absence of Ca2+. After cells were exposed to HLT, a 2-h lag period occurred before release of radioactivity was detected. The substances released from cells by HLT were identified as arachidonic acid and phosphatidylcholine.     

Regional effects of neurotensin on the electrically stimulated release of [3H]dopamine and [14C]acetylcholine in the rat nucleus accumbens

This study has shown that neurotensin (NT) increases the electrically stimulated release of [³H]DA to a similar extent in all but the extreme caudolateral area of the rat nucleus accumbens and appears to modulate DA release equally in the medial and lateral zones of this brain area. The simultaneous release of ACh was not significantly affected by NT.     

Efflux of sphingolipids metabolically labeled with [1-3H]sphingosine, L-[3-3H]serine and [9,10-3H]palmitic acid from normal cells in culture

The membrane complex lipids of human fibroblasts and differentiated rat cerebellar granule cells in culture were metabolically radiolabeled with [1-³H]sphingosine, L-[3-³H]serine and [9,10-³H]palmitic acid. A relevant efflux of radioactive sphingolipids and phosphatidylcholine was observed from cells to the culture medium in the presence of fetal calf serum. This event was independent of the concentration and structure of the metabolic precursor administered to cells, and it was linearly time-dependent. The radioactive lipid patterns present in the medium were different from those present in the cells. Radioactive sphingomyelin and ganglioside GM3 containing short acyl chains were the main species present in the medium from human fibroblasts, while sphingomyelin and GD3 ganglioside in that from neuronal cells. In the absence of proteins in the culture medium, the efflux of complex lipids was much lower than in the presence of serum, and the patterns of released molecules were again different from those of cells. This work was supported by COFIN-PRIN, Consiglio Nazionale delle Ricerche (PF Biotechnology), Italy.     

Phenylarsine oxide inhibits alpha-latrotoxin-stimulated [3H]GABA release from rat brain synaptosomes

Phosphatidylinositol 4,5-biphosphate has been implicated in a variety of membrane-trafficking processes, including exocytosis of neurotransmitters. However, there are contradictory findings concerned ability of phenylarsine oxide (PAO), an inhibitor of phosphatidylinositol 4-kinase, to affect exocytotic release of different types of neurotransmitters. We bent our efforts to a detailed analysis of action of PAO on Ca(2+)-dependent and Ca(2+)-independent [3H]GABA release produced by exposure of rat brain synaptosomes to different concentrations of alpha-latrotoxin. We also compared PAO action on alpha-latrotoxin- and 4-aminopyridine (4-AP)-evoked [3H]GABA release. The experiments have shown that release of [3H]GABA evoked by the depolarization with 4-AP was decreased by 80% as a result of action of 3 microM PAO and the complete inhibition of release was observed with 10 microM PAO. When alpha-latrotoxin as a stimulant was applied, release of [3H]GABA was increased as toxin concentration used was elevated from 0.5 to 3.0 nM, however, concomitantly, the response of the toxin-induced [3H]GABA release to PAO became attenuated: 10 microM PAO led to almost complete inhibition of the effect of 0.5 nM alpha-latrotoxin and only partly decreased (by 40%) the response to 3.0 nM alpha-latrotoxin. To test whether the efficacy of PAO depended on the toxin-induced outflow of cytosolic [3H]GABA, synaptosomes with depleted cytosolic [3H]GABA pool were also exploited. Depletion was performed by means of heteroexchange of cytosolic [3H]GABA with nipecotic acid. The experiments have shown that treatment of loaded synaptosomes with nipecotic acid resulted in some increase of [3H]GABA release evoked by 0.5 nM alpha-latrotoxin, but in the two-fold decrease of the response to 3.0 nM alpha-latrotoxin. PAO essentially inhibited [3H]GABA release from depleted synaptosomes irrespective of alpha-latrotoxin concentration used. Therefore, the amount of [3H]GABA released from cytosolic pool determined, in considerable degree, the insensitivity of alpha-latrotoxin action to PAO. Thus, our data show that subnanomolar concentrations of alpha-latrotoxin may be used for stimulation of exocytotic release of [3H]GABA. Exposure of synaptosomes with nanomolar toxin concentrations leads not only to stimulation of exocytosis, but also to leakage of [3H]GABA from cytosolic pool. PAO potently inhibits exocytotic release of [3H]GABA and its inhibitory effectiveness is diminished as far as the outflow of [3H]GABA is elevated.     

Halothane inhibits the neurotoxin stimulated [14C]guanidinium influx through 'silent' sodium channels in rat glioma C6 cells

We have investigated the effect of pharmacological agents on [14C]guanidinium ion influx through sodium channels in C6 rat glioma and N18 mouse neuroblastoma cells. The sodium channels of the N18 cells can be activated by aconitine alone, indicating that they are voltage-dependent channels. In contrast, sodium channels in the C6 cells require the synergistic action of aconitine and scorpion toxin for activation and are therefore characterized as so-called silent channels. The general anesthetic halothane used at clinical concentrations, specifically inhibited the ion flux through the silent sodium channel of C6 rat glioma cells. The voltage-dependent channels of the N18 cells were insensitive to halothane at the concentrations tested.     

Measles-virus-persistent infection in BGM cells. Modification of the incorporation of [3H]arachidonic acid and [14C]stearic acid into lipids.

In BGM cells chronically infected with measles virus, although the composition of the phospholipids is unaltered, the fatty acid composition is modified. Uninfected, lytic and persistently infected cells were labelled with [3H]arachidonic acid and [14C]stearic acid and their metabolic fate analysed. No difference in the total incorporation was observed in the different systems. However, the [14C]stearic acid and [3H]arachidonic acid were incorporated up to 2-fold and 13-fold respectively greater into the neutral lipid of persistently infected compared with that of uninfected cells. Both radioactive fatty acids were specifically accumulated in the triacylglycerol and non-esterified fatty acids fractions. Lytically infected cells were similar to uninfected cells. Although there was no significant difference in the incorporation of radioactivity into the total phospholipid in either system, there was a large decrease in [3H]arachidonic acid incorporated into phosphatidylethanolamine and to a lesser extent phosphatidylcholine and phosphatidylinositol in persistently infected cells. [14C]Stearic acid incorporation was also reduced in phosphatidylcholine and phosphatidylethanolamine fractions of persistently infected cells.     

Phosphatidylinositol 4,5-bisphosphate promotes both [3H]-noradrenaline and [14C]-glutamate exocytosis from nerve endings

Inhibition of phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-bisphosphate (PI4,5P(2)) synthesis by phenylarsine oxide (PAO) inhibits both [3H]-noradrenaline ([3H]-NA) and [14C]-glutamate ([14C]-glu) exocytosis from streptolysin-O (SLO)-perforated synaptosomes. When PI4,5P(2) is blocked by an antibody or chelated by neomycin, neurotransmitter exocytosis again is inhibited. Also, when phosphoinositide (PI) synthesis is indirectly decreased by shunting phosphatidic acid (PA) synthesis into phosphatidylbutanol production, both [14C]-glutamate and [3H]-noradrenaline exocytosis are inhibited. All of these results indicate that PI4,5P(2) is necessary for exocytosis of both synaptic vesicles (SVs) and dense core vesicles (DCVs).     

Protein kinase C involved in zymosan-induced release of arachidonic acid and superoxide but not in calcium ionophore-elicited arachidonic acid release or formation of prostaglandin E2 from added arachidonate.

Zymosan and phorbol ester induced in liver macrophages the release of arachidonic acid, prostaglandin E2, and superoxide; the calcium ionophore A 23187 elicited a release of arachidonic acid and prostaglandin E2 but not of superoxide, and exogenously added arachidonic acid led to the formation of prostaglandin E2 only. The zymosan- and phorbol-ester-induced release of arachidonic acid, prostaglandin E2, and superoxide was dose-dependently inhibited by staurosporine and K252a, two inhibitors of protein kinase C, and by pretreatment of the cells with phorbol ester which desensitized protein kinase C. The release of arachidonic acid or prostaglandin E2 following the addition of A 23187 or arachidonic acid was not affected by these treatments. Zymosan and phorbol ester but not A 23187 or arachidonic acid induced a translocation of protein kinase C from the cytosol to membranes in intact cells. These results demonstrate an involvement of protein kinase C in the zymosan- and phorbol-ester-induced release of arachidonic acid, prostaglandin E2, and superoxide; the release of arachidonic acid and prostaglandin E2 elicited by A 23187 and the formation of prostaglandin E2 from exogenously added arachidonic acid, however, is independent of an activation of protein kinase C.     

Incorporation of dietary [14C]arachidonic acid and [3H]eicosapentaenoic acid into tissue lipids during absorption of a fish oil emulsion.

A preferential incorporation of dietary arachidonic acid (20:4, n-6) into chyle lipoprotein phospholipids, a relative resistance of 20:4 esters of chyle triacylglycerol (TG) to hydrolysis by lipoprotein lipase, a preferential utilization of 20:4 for phospholipid acylation, and a low rate of oxidation of 20:4 are factors that may contribute to the differences seen in the incorporation into tissue lipids between absorbed 20:4 and the predominant dietary 16-18 carbon fatty acids. In this study we fed [14C]20:4 and [3H]eicosapentaenoic acid (20:5, n-3) as free fatty acids in a fish oil emulsion to rats and analyzed the radioactivity in different tissue lipids after 1, 2, and 4 h. The purpose was to examine the degree of similarity in the fate of the two major eicosanoid precursors during the absorption of a fish oil meal. The recovery after 2 and 4 h of 14C exceeded that of 3H in lipids of small intestine, serum, liver, heart, kidneys, and spleen. The differences increased with time, e.g., the liver contained 9.7 (+/- 0.7)% 3H and 17.9 (+/- 1.4)% of the 14C (P less than 0.001), and the upper half of the small intestine 10.0 (+/- 0.8)% of the 3H and 22.8 (+/- 1.1)% of the 14C (P less than 0.001) after 4 h. The 14C and 3H radioactivity per g tissue after 4 h ranked as follows: liver and brown adipose tissue greater than kidneys greater than heart, lungs, spleen, and serum greater than colon greater than white adipose tissue and testes, the differences between tissues being up to 50-fold. There were up to fourfold variations in the 14C/3H ratios between tissues after 4 h, the highest value being observed in the heart and the lowest in white adipose tissue. Of the radioactivity retained in liver and intestine, more 14C and 3H was in phospholipids and less in triacylglycerol (TG), the differences being largest in the liver, e.g., after 4 h 57.6 (+/- 0.8)% of the 14C and 29.9 (+/- 0.9)% of the 3H (P less than 0.001) in the liver was in phosphatidylcholine (PC). In both intestine and liver the highest 14C/3H ratios were found in phosphatidylinositiol (PI). Also phosphatidylethanolamine (PE) contained more 14C than 3H but the quantitative differences were relatively small after 4 h. In heart the proportions of 3H and 14C found in PE and PI did not differ, whereas more of the 14C was in PC and more of the 3H was in cardiolipin and phosphatidylserine.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evaluation of radiation dose resulting from the ingestion of [3H]- and [14C]thymidine in the rat

Average doses to rat tissues from the ingestion of 2-[14C]thymidine were compared with those from methyl-[3H]thymidine or 6-[3H]thymidine. Among the three precursors, [14C]thymidine gave the highest dose to spleen and small intestine. The doses to other tissues from [14C]thymidine were almost the same or lower as compared with those from [3H]thymidine, irrespective of the 9 times higher beta-ray energy of 14C than that of 3H. In the case of [14C]thymidine, most of the dose was given by radioactivity incorporated into the organic tissue constituents (non-volatile radioactivity). In the case of [3H]thymidine, however, the dose contributions by non-volatile radioactivity were very small and the major contributions were rather from volatile radioactivity (3HHO), formed by degradation of [3H]thymidine. No significant difference in their total doses was found between the two [3H]precursors, but the dose from non-volatile radioactivity alone was 2-3 times higher with methyl-[3H]thymidine than with 6-[3H]thymidine. Estimates of the dose to cell nuclei in various tissues after the ingestion of [3H]thymidine were also made in order to predict more precisely possible radiation hazards.     

DNA strand breakage in Chinese hamster V79 cells caused by low levels of incorporated [3H] and [14C]thymidine

A sensitive alkali-unwinding assay was used to measure DNA strand breakage in Chinese hamster V79 cells caused by low-level incorporation of methyl-labelled [3H] and [14C] thymidine, and to estimate the effective dose per disintegration relative to low doses of gamma-irradiation. Damage equivalent to 0.0035 +/- 0.0006 and 0.0014 +/- 0.0005 Gy was observed for each 3H and 14C disintegration respectively. These values agree well with those expected from the estimated nuclear radiation dose delivered by the beta particles if a relative biological effect (r.b.e.) of 1.0 is assumed, and suggest that strand-breakage produced by these isotopes is determined by the nuclear radiation dose delivered by the beta particles.