Metabolic co-operation in HGPRT+ and HGPRT− embryonal carcinoma cells

HGPRT⁺ and HGPRT^? clones have been isolated from the mouse embryonal carcinoma tissue culture line PC13. Upon inoculation into isogeneic mice, all clones form tumors showing the same pattern of differentiation as that given by PC13, namely, a predominance of neural differentiation with the presence of smaller amounts of other tissues. The karyotype of one HGPRT clone, PC13TG8, has been compared with that of PC13 by G-banding. Both lines possess a marker isochromosome and show a substantial degree of chromosome imbalance. By two techniques, (a) autoradiography after [³H]hypoxanthine labelling and (b) co-culture in toxic purine analogues, PC13 and PC13TG8 cells have been shown to participate in metabolic co-operation with each other and with the Chinese hamster cell line Don and its derivatives. Like other tissue culture lines, however, they show little or no metabolic co-operation with derivatives of mouse L cells.     

Incidence of gap junctions and microvilli in variant cell lines with altered capacity for metabolic co-operation

The metabolic cooperation-defective embryonal carcinoma cell variant R5/3 has a reduced area of gap junctions per unit cell volume and an increased surface area of microvilli per unit volume compared with its parent PC13TG8. Both changes are reversed in the cooperation-competent revertant H2T12. This provides strong evidence in support of the view that metabolic cooperation is mediated by gap junctions, and suggests that the altered properties of R5/3 may be due to a cytoskeletal defect.     

Metabolic coupling between cultured pancreatic B-cells

The transfer of [³H]uridine nucleotides from donor (uridine-loaded) to recipient (thymidine-prelabelled) pancreatic endocrine islet cells in monolayer culture was qualitatively and quantitatively assessed by light and electron microscope autoradiography. Recipient cells showed a labelled cytoplasm only when they were in contact with donor cells or positive recipients. Controls indicated that the labelling was not due to the incorporation of [³H]uridine, ³H nucleotides or nucleic acids lost by donor cells in the medium. Quantitation showed that cytoplasmic labelling of positive recipient cells was higher than cellular background, but lower than the cytoplasmic labelling of donor cells. These data indicate that label in recipient cells was derived from donors by direct intercellular transfer of ³H nucleotides. Differentiated insulin-producing cells (B cells) were involved in the exchange.     

Studies on ether-phospholipids of vascular smooth muscle cells. Identification of a rapid Ca2+-dependent hydrolysis of alkyl-phosphatidylethanolamine promoted by endothelin-1

We have investigated the metabolism of 1-O-[³H]octadecyl-sn-glycero-3-phosphocholine ([³H]lyso PAF) and [³H]myristic acid in secondary cultures of aortic smooth muscle cells (SMC) to characterize the origin of second messengers generated upon stimulation with endothelin-1 (ET-1). When cells were labelled with [³H]lyso PAF, we observed a transfer of the label from phosphatidylcholine (PC) to phosphatidylethanolamine (PE). In contrast, incubation with [³H]myristate labelled mainly PC. Both precursors were incorporated into all PC and PE subclasses. However, [³H]lyso PAF labelled mainly alkyl-subclasses while [³H]myristate was associated with diacyl-subclasses. Using these specific labelling procedures, we have shown that ET-1 induced a strong hydrolysis of PE. This hydrolysis was specific for alkyl-PE with a maximum after 5 s of stimulation. We have also observed an extracellular Ca²⁺-dependent increase in diglyceride (DG), phosphatidic acid (PA) and mainly triglyceride (TG) concomitant to alkyl-PE hydrolysis. Thus, alkyl-DG generated from alkyl-PE appears to be a major product in ET-1 stimulation of SMC. These results suggest a new level of complexity in the signal transduction cascade involving a specificity for phospholipid subclasses.     

Impact of endothelial lipase on cellular lipid composition

Using mass spectrometry (MS), we examined the impact of endothelial lipase (EL) overexpression on the cellular phospholipid (PL) and triglyceride (TG) content of human aortic endothelial cells (HAEC) and of mouse plasma and liver tissue. In HAEC incubated with the major EL substrate, HDL, adenovirus (Ad)-mediated EL overexpression resulted in the generation of various lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) species in cell culture supernatants. While the cellular phosphatidylethanolamine (PE) content remained unaltered, cellular phosphatidylcholine (PC)-, LPC- and TG-contents were significantly increased upon EL overexpression. Importantly, cellular lipid composition was not altered when EL was overexpressed in the absence of HDL. [¹⁴C]-LPC accumulated in EL overexpressing, but not LacZ-control cells, incubated with [¹⁴C]-PC labeled HDL, indicating EL-mediated LPC supply. Exogenously added [¹⁴C]-LPC accumulated in HAEC as well. Its conversion to [¹⁴C]-PC was sensitive to a lysophospholipid acyltransferase (LPLAT) inhibitor, thimerosal. Incorporation of [³H]-Choline into cellular PC was 56% lower in EL compared with LacZ cells, indicating decreased endogenous PC synthesis. In mice, adenovirus mediated EL overexpression decreased plasma PC, PE and LPC and increased liver LPC, LPE and TG content. Based on our results, we conclude that EL not only supplies cells with FFA as found previously, but also with HDL-derived LPC and LPE species resulting in increased cellular TG and PC content as well as decreased endogenous PC synthesis.     

Histone H10 mRNA and protein accumulate early during retinoic acid induced differentiation of synchronized embryonal carcinoma cells

The very lysine-rich replacement histone variant H1⁰ is found to be present in different murine (C1003, PC13, P19) and human (Tera-2) embryonal carcinoma cell lines. The proportion of H1⁰ increases upon induction of differentiation of the different cell lines by various treatments. In undifferentiated PC13 EC cells H1⁰ mRNA is present at a low level. During retinoic acid induced differentiation of mitotically synchronized PC13 EC cells, accumulation of H1⁰ mRNA starts in the first cell cycle. The H1⁰ protein level starts to increase in the second synchronous cycle preceding changes in the cycle parameters that become apparent in the third cycle. The results provide further support for an important role of H1⁰ in the control of cellular differentiation in early mammalian development.Abbreviations EC embryonal carcinoma - RA retinoic acid - DAPT 4-6-diamino-2-phenylindole - SDS sodium dodecylsulphate - DMSO dimethyl sulfoxide - TCA trichloro acetic acid     

The lesion in a metabolic cooperation-defective embryonal carcinoma variant is of broad specificity with regard to metabolite and to contiguous cell type

Homotypic and heterotypic permeable junction formation between a metabolic cooperationdefective embryonal carcinoma variant R5/3 and its parent PC13TG8 have been studied by uridine prelabelling. The results indicate that the lesion results not in a modified specificity of cell recognition but in a deficiency in forming permeable junctions irrespective of contiguous cell type. New techniques have been developed for the study of intercellular transfer of sodium ions and of amino acids of the urea cycle. These have been used to demonstrate that the R5/3 lesion reduces the capacity of the cells to participate in intercellular transfer of three unrelated categories of small molecule.     

Ethanol inhibits phosphatidylcholine and phosphatidylethanolamine biosynthesis in human leukemic monocyte-like U937 cells

The effect of ethanol (ETOH) on the incorporation of [¹⁴C]oleic acid (18:1) into lipid in human monocyte-like U937 cells was investigated. With increasing time of exposure to ETOH, the percentage of the label distributed into neutral lipid (NL) declined from 35 per cent (3 h) to 10 per cent (24 h) accompanied by increased incorporation into phospholipid (PL). [¹⁴C] 18 : 1 was preferentially incorporated into triglyceride (TG) and phosphatidylcholine (PC), comprising over 65 per cent and 50 per cent of the label associated with NL and PL, respectively. Low concentrations of ETOH (⩽ 1·0 per cent; v/v) had no effect. At concentrations greater than 1·5 per cent, there was enhanced incorporation into TG and diacylglycerol (DAG) in a 24-h incubation period, while at 16 h the label in phosphatidylethanolamine (PE) was decreased. The effect of ETOH on the CDP-choline or ethanolamine pathway was examined by monitoring the incorporation of [³H]choline or [¹⁴C]ethanolamine into PC or PE, respectively. At low concentrations ETOH had no effect on either choline uptake or the incorporation into PC. Higher concentrations (≥ 1·5 per cent) for 3 and 6 h resulted in a slightly decreased choline uptake, and the reduction (40–50 per cent) of incorporation into PC suggests that the CDP-choline pathway was inhibited. There was a similar inhibition of the incorporation of [¹⁴C]ethanolamine into PE. When the cells were incubated for 3 h in the presence of 2 per cent ETOH and with labelled 18 : 1 and PL-base, the ratios of incorporation (base/18 : 1) into PC and PE fractions decreased, indicating that the major inhibition lay in blockage of the availability of the base moiety for PL formation. Analysis of the distribution of the label into metabolites revealed that ETOH inhibited the conversion of [¹⁴C] ethanolamine into [¹⁴C]phosphorylethanolamine. The reduction in incorporation was not due to the enhanced breakdown of base-labelled PL. Our results indicate that ETOH has an inhibitory effect on the CDP-choline or ethanolamine pathway.     

Characterization of an Adenylate Cyclase-Linked Serotonin (5-HT1) Receptor in a Neuroblastoma × Brain Explant Hybrid Cell Line (NCB-20)

Clonal cell line NCB-20 (a hybrid of mouse neuroblastoma N18TG2 and Chinese hamster 18-day embryonic brain expiant) expressed both high- (K_D 180 nM) and low-affinity (>3000 nM) binding sites for [³H]serotonin (5-HT) which were absent from the parent neuroblastoma. The low-affinity binding site was eliminated by 1 μM spiperone. The order of drug potency for inhibition of high-affinity [³H]5-HT binding was consistent with a 5-HT₁ receptor (5,6 - dihydroxytryptamine = 5-HT = methysergide = 5-methoxytryptamine > cyproheptadine = clozapine = mianserin > spiperone > dopamine = morphine = ketanserin = norepinephrine). [³H]5-HT binding was inhibited by guanine nucleotides (e.g., GTP and Gpp(NH)p), whereas antagonist binding was not; as-corbate was also inhibitory. A 30-min exposure of cells to 1—2 μM 5-HT or other agonists produced a three- to fivefold stimulation of cyclic AMP levels. The order of potency for 5-HT agonist stimulation of basal cyclic AMP levels and 5-HT antagonist reversal of agonist-stimulated levels was the same as the order of drug potency for inhibition of high-affinity [³H]5-HT binding, suggesting linkage of the 5-HT₁ receptor to adenylate cyclase in NCB-20 cells.     

Characterization of 5-HT transporter and receptor system in HeLaS3 cells by [H]8-OH-DPAT and other serotonergic ligands

[³H]8-OH-DPAT is a selective ligand for labeling 5-HT_1A receptor sites. In competition binding experiments, we found that classic biogenic amine transporter inhibitors displaced [³H]8-OH-DPAT binding at its high-affinity binding sites in HeLaS3 cells. [¹²⁵I]RTI-55 and [³H]paroxetine are known to specifically label amine transporter sites, and this was observed in our cells. Displacement studies showed that 8-OH-DPAT displayed affinity in a dose-dependent manner for the labeled amine transporter sites. These data suggest that [³H]8-OH-DPAT binds to amine uptake sites in HeLaS3 cells. A variety of drugs targeting different classes of receptors did not significantly affect [³H]8-OH-DPAT binding. Moreover, we determined the specific binding effects of various serotonergic ligands (i.e. [¹²⁵I]cyanopindolol, [³H]ketanserin/[³H]mesulergine, [³H]GR-65630, [³H]GR-113808 and [³H]LSD) that specifically labeled 5-HT₁, 5-HT₂, 5-HT₃, 5-HT₄ and 5-HT_5–7 receptors, respectively. It is suggested that HeLaS3 cells contain distinct types of the related to 5-HT receptor recognition binding sites. These observations could help elucidate the relevant characteristics of different types of 5-HT receptors and 5-HT membrane transporters in tumor cells and their role in tumorigenesis.     

5,8-Disubstituted indolizidines: A new class of noncompetitive blockers for nicotinic receptor-channels

A series of 8-methyl-5-substituted indolizidines inhibit binding of the noncompetitive blocking agent [³H]perhydrohistrionicotoxin to muscle-type nicotinic acetylcholine receptor-channels in membranes fromTorpedo electroplax. The K_i values range from 0.16 to 1.12 M, making these alkaloids among the most potent ligands for this site. Unlike most noncompetitive blockers, the potencies of the 8-methyl-5-substituted indolizidines arereduced in the presence of carbamylcholine. Indolizidine 205A (8-methyl-5-(4-pentynyl)indolizidine) is unique in enhancing binding of [³H]perhydrohistrionicotoxin by 1.5-fold. The enhancement is at a maximum at 0.01 to 0.1 M, followed by progressive inhibition with an IC₅₀ of about 20 M. In the presence of carbamylcholine, which itself enhances binding of [³H]perhydrohistrionicotoxin, indolizidine 205A causes only an inhibition of binding with an IC₅₀ of about 10 M. Indolizidines with a hydroxy substituent on the 8-methyl group have very low activity. None of the indolizidines affect binding of [¹²⁵I]-bungarotoxin to acetylcholine recognition sites. In pheochromocytoma PC12 cells, indolizidine 205A has no agonist activity, but only inhibits carbamylcholine-elicited²²Na⁺ influx. The profile of potencies for the 8-methyl-5-substituted indolizidines is similar in electroplax membranes and PC12 cells. Indolizidines 205A and 209B (8-methyl-5-pentylindolizidine) have no apparent effect on desensitization of receptors in PC12 cells. The 5,8-disubstituted indolizidines appear to represent an atypical and potent class of noncompetitive blockers for muscle-type and ganglionic nicotinic receptor-channels.     

Internode length in Zea mays L.

[¹³C, ³H]Gibberellin A₂₀ (GA₂₀) has been fed to seedlings of normal (tall) and dwarf-5 and dwarf-1 mutants of maize (Zea mays L.). The metabolites from these feeds were identified by combined gas chromatography-mass spectrometry. [¹³C, ³H]Gibberellin A₂₀ was metabolized to [¹³C, ³H]GA₂₉-catabolite and [¹³C, ³H]GA₁ by the normal, and to [¹³C, ³H]GA₂₉ and [¹³C, ³H]GA₁ by the dwarf-5 mutant. In the dwarf-1 mutant, [¹³C, ³H]GA₂₀ was metabolized to [¹³C, ³H]GA₂₉ and [¹³C, ³H]GA₂₉-catabolite; no evidence was found for the metabolism of [¹³C, ³H]GA₂₀ to [¹³C, ³H]GA₁. [¹³C, ³H]Gibberellin A₈ was not found in any of the feeds. In all feeds no dilution of ¹³C in recovered [¹³C, ³H]GA₂₀ was observed. Also in the dwarf-5 mutant, the [¹³C]label in the metabolites was apparently undiluted by endogenous [¹³C]GAs. However, dilution of the [¹³C]label in metabolites from [¹³C, ³H]GA₂₀ was observed in normal and dwarf-1 seedlings. The results from the feeding studies provide evidence that the dwarf-1 mutation of maize blocks the conversion of GA₂₀ to GA₁.Abbreviations GA_n gibberellin A_n - GC-MS combined gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - RP reverse phase     

Reserpine-induced reduction in norepinephrine transporter function requires catecholamine storage vesicles

Treatment of rats with reserpine, an inhibitor of the vesicular monoamine transporter (VMAT), depletes norepinephrine (NE) and regulates NE transporter (NET) expression. The present study examined the molecular mechanisms involved in regulation of the NET by reserpine using cultured cells. Exposure of rat PC12 cells to reserpine for a period as short as 5 min decreased [³H]NE uptake capacity, an effect characterized by a robust decrease in the V_max of the transport of [³H]NE. As expected, reserpine did not displace the binding of [³H]nisoxetine from the NET in membrane homogenates. The potency of reserpine for reducing [³H]NE uptake was dramatically lower in SK-N-SH cells that have reduced storage capacity for catecholamines. Reserpine had no effect on [³H]NE uptake in HEK-293 cells transfected with the rat NET (293-hNET), cells that lack catecholamine storage vesicles. NET regulation by reserpine was independent of trafficking of the NET from the cell surface. Pre-exposure of cells to inhibitors of several intracellular signaling cascades known to regulate the NET, including Ca²⁺/Ca²⁺–calmodulin dependent kinase and protein kinases A, C and G, did not affect the ability of reserpine to reduce [³H]NE uptake. Treatment of PC12 cells with the catecholamine depleting agent, α-methyl-p-tyrosine, increased [³H]NE uptake and eliminated the inhibitory effects of reserpine on [³H]NE uptake. Reserpine non-competitively inhibits NET activity through a Ca²⁺-independent process that requires catecholamine storage vesicles, revealing a novel pharmacological method to modify NET function. Further characterization of the molecular nature of reserpine's action could lead to the development of alternative therapeutic strategies for treating disorders known to be benefitted by treatment with traditional competitive NET inhibitors.     

Water stress provokes a generalized increase in phosphatidylcholine turnover in barley leaves

Water and salt stress promote betaine accumulation in leaves of barley (Hordeum vulgare L.) by accelerating the de-novo synthesis of betaine, via choline. Previous radiotracer kinetic studies have implicated stress-enhanced turnover of the choline moiety of phosphatidylcholine (PC) as a major source of choline for betaine synthesis. Two approaches have therefore been followed to show whether stress-induced PC turnover is a cellor organelle-specific phenomenon, or a generalized one. In the first approach, [³H]ethanolamine of high specific activity was supplied to second leaves of unstressed and water-stressed barley plants; after 1 h, paired sections of tissue were excised from each leaf, one for extraction and analysis of [³H]metabolites and the other for autoradiography. The³H-activity remaining in the leaf tissue after washing out the water-soluble³H-metabolites during preparation for autoradiography was taken to be mainly in phospholipids. In unstressed leaves, [³H]phosphatidylethanolamine (PE) was the major labeled phospholipid, whereas there were approximately equal amounts of [³H]PE and [³H]PC in stressed leaves. At the light-microscope level, silver grains were associated with all living cells in both unstressed and stressed leaves; grains were concentrated in the cytoplasmic regions of highly vacuolate mesophyll cells, and were distributed throughout densely cytoplasmic vascular parenchyma. At the electron-microscope level, silver grains were not confined to any particular types of membranes in unstressed or stressed leaves. In the second approach, second leaves of stressed plants received a 1-h pulse of [¹⁴C]ethanolamine, and were then homogenized. The brei was subjected to sucrose density gradient centrifugation. The specific radioactivity of [¹⁴C]PC was quite similar in the gradient fractions, whether they contained microsomes or mitochondrial plus chloroplast membranes. We infer that stress does not enhance the turnover of any structurally discrete class of PC, but rather stimulates PC turnover in several or all classes of membranes in most cells of the leaf.Abbreviations and symbols PE phosphatidylethanolamine - PC phosphatidylcholine - PMME phosphatidylmonomethylethanolamine - PDME phosphatidyldimethylethanolamine - TLC thin-layer chromatography - _leaf leaf water potential     

Metabolism and fate of neutral lipids of fetal lung fibroblast origin

Fetal rat lung fibroblasts characteristically increase their triacylglycerol (TG) stores during development. Both fibroblasts and alveolar type II (TII) cells can synthesize TG de novo, but only fibroblasts can absorb TG from culture medium, and retain the TG in a stable state. When fibroblasts pre-labelled with [³H]triolein are recombined with TII cells in organotypic culture the radiolabel appears in TII cell disaturated phosphatidylcholine (disatPC). When fibroblasts are preloaded with increasing amounts of TG there is a commensurate increase in TII cell disatPC following organotypic culture. Comparison of [³H]triacylglycerol and [¹⁴C]glucose incorporation into type II cell phospholipids revealed preferential use of TG for the surface-active phospholipids disatPC (10-fold greater) and phosphatidylglycerol (23-fold greater). These in vitro data suggest that fibroblasts provide lipid substrate for TII cell surfactant phospholipid synthesis.     

Differentiation of pre- and postsynaptic high affinity serotonin receptor binding sites using physico-chemical parameters and modifying agents

The two³H-labeled agonists [³H]8-hydroxy-2-(di-n-propylamino) tetralin ([³H]8-OH-DPAT) and [³H]serotonin ([³H]5-HT) have been used to examine the effects of physico-chemical parameters and modulatory agents on the high affinity 5-HT receptor binding sites in various regions of the rat central nervous system. Sites labeled by [³H]8-OH-DPAT and [³H]5-HT were differentially sensitive to changes in incubation demperature and pH, such that the optimal interaction of [³H]8-OH-DPAT with specific sites in the striatum was at 30°C and pH 7.4, whereas [³H]5-HT sites in the same region were most easily labeled at 2–23°C and pH 8.2. Micromolar concentrations of Mn²⁺ enhanced [³H]5-HT binding but inhibited markedly [³H]8-OH-DPAT binding to striatal membranes. In contrast, both [³H]5-HT and [³H]8-OH-DPAT binding were incerased by the cation in hippocampal membranes. Conversely, GTP reduced the binding of either ligand in the hippocampus but affected only [³H]5-HT binding in the striatum. Furthermore, N-ethylmaleimide inhibited equally [³H]5-HT and [³H]8-OH-DPAT binding to hippocampal membranes, but was markedly less potent against [³H]8-OH-DPAT binding to striatal     

Nerve Growth Factor Stimulates the Production of Inositol 1,3,4-and 1,4,5-Trisphosphate and Inositol 1,3,4,5-Tetrakisphosphate in PC 12 Cells

Abstract: In PC12 cells, preincubated with [³H]inositol, nerve growth factor (NGF) stimulated an ～ 100% increase in the levels of [³H]inositol 1,3,4-trisphosphate {[³H]-Ins(1,3,4)P₃}, [³H]inositol 1,4,5-trisphosphate {[³H]lns(1,4,5)P₃}, and [³H]inositol 1,3,4,5-tetrakisphosphate {[³H]-Ins(1,3,4,5)P₄} as early as 5–15 s after addition of NGF. This NGF-mediated response was apparent only when the cells had been cultured in the absence of fetal bovine serum (FBS). PC12 cells cultured in FBS-containing medium did not display NGF-mediated increases in [³H]-Ins(1,3,4)P₃, [³H]-Ins(1,4,5)P₃, and [³H]-Ins(1,3,4,5)P₄ levels. Using cells cultured in the absence of FBS, epidermal growth factor (EGF) and fibroblast growth factor also stimulated production of [³H]lns(1,3,4)P₃, [³H]-Ins(1,4,5)P₃, and [³H]lns(1,3,4,5)P₄. Lavendustin A, a tyrosine kinase inhibitor, inhibited both the EGF-and NGF-stimulated increases in the levels of these tritiated inositol phosphates. These results suggest that NGF stimulates the production of lns(1,3,4)P₃, lns(1,4,5)P₃, and lns(1,3,4,5)P₄ and that this response is dependent on tyrosine kinase activity. Furthermore, although the production of lns(1,3,4)P₃, lns(1,4,5)P₃, and lns(1,3,4,5)P₄ may be a common response to factors stimulating neuronal differentiation, it is not sufficient for stimulation of neuronal differentiation.     

Automated NMR resonance assignment strategy for RNA via the phosphodiester backbone based on high-dimensional through-bond APSY experiments

A fast, robust and reliable strategy for automated sequential resonance assignment for uniformly [¹³C, ¹⁵N]-labeled RNA via its phosphodiester backbone is presented. It is based on a series of high-dimensional through-bond APSY experiments: a 5D HCP-CCH COSY, a 4D H1′C1′CH TOCSY for ribose resonances, a 5D HCNCH for ribose-to-base connection, a 4D H6C6C5H5 TOCSY for pyrimidine resonances, and a 4D H8C8(C)C2H2 TOCSY for adenine resonances. The utilized pulse sequences are partially novel, and optimized to enable long evolution times in all dimensions. The highly precise APSY peak lists derived with these experiments could be used directly for reliable automated resonance assignment with the FLYA algorithm. This approach resulted in 98 % assignment completeness for all ¹³C–¹H, ¹⁵N1/9 and ³¹P resonances of a stem-loop with 14 nucleotides.     

Intracellular formation of analogues of cyclic AMP: Studies with brain slices labeled with radioactive derivatives of adenine and adenosine

A variety of radioactive analogs of adenine and adenosine were incubated with guinea pig cerebral cortical slices. Neither 1,N⁶-ethano[¹⁴C]adenosine nor 1,N⁶-ethanol[¹⁴C]adenine were significantly incorporated into intracellular nucleotides. 2-chloro[8-³H]adenine was incorporated, but at a very low rate and conclusive evidence for the formation of intracellular radioactive 2-chlorocyclic AMP was not obtained. N⁶-Benzyl[¹⁴C]adenosine was converted only to intracellular monophosphates and significant formation of radioactive N⁶-benzylcyclic AMP was not detected during a subsequent incubation. 2′-Deoxy-[8-¹⁴C] adenosine was converted to both intracellular radioactive 2′-deoxyadenine nucleotides and radioactive adenine nucleotides. Stimulation of these labeled slices with a variety of agents resulted in formation of both radioactive 2′-deoxycyclic AMP and cyclic AMP. Investigation of the effect of various other compounds on uptake of adenine or adenosine suggested that certain other adenosine analogs might serve as precursors of abnormal cyclic nucleotides in intact cells.     

Association of glucocerebroside homolog biosynthesis with Schwann cell proliferation

The biosynthesis of myelin-associated glycolipids was studied in quiescent secondary cultures of Schwann cells and in a rapidly proliferating population of transfected Schwann cells (TSC) by in vitro incorporation of [³H]galactose. The TSC demonstrated a marked increase (>10-fold) in [³H]galactose incorporation when compared to quiescent Schwann cells. The level (or amount) of [³H]galactose incorporation into lipids is dependent upon the number of TSC in culture. The majority of³H-labeled lipids were oligohexosylceramides (GL-2, GL-3, and GL-4). Substrates that inhibit TSC proliferation, collagen type I and Matrigel, an artificial basement membrane, decrease the [³H]galactose incorporation by 25% and 80%, respectively. Our results indicate that the synthesis of glucocerebroside and its homologs is associated with Schwann cell proliferation.Abbreviations HPTLC high-performance thin-layer chromatography - TL total lipids - NL non-polar lipids - GL glycolipids - PL phospholipids - MGDG monogalactosyl diacylglycerol - GalCe galactocerebroside - GalCe-OH galacto hydroxycerebroside - GlcCe glucocerebroside - Su sulfatide - Su-OH hydroxysulfatide - GL-2 lactosylceramide - GL-3 trihexosylceramide - GL-4 tetrahexosylceramide - PE phosphatidylethanolamine - PC phosphatidylcholine - PS phosphatidylserine - PI phosphatidylinositol - TSC transfected Schwann cells A preliminary report of this work was presented at the 22nd Annual Meeting of the American Society for Neurochemistry, Charleston, South Carolina, March 13, 1991.