Metabolism of palmitate in cultured rat Sertoli cells

Isolated rat Sertoli cells were incubated in the presence of [1-14C]palmitate at a cell concentration of 1.54 +/- 0.31 mg protein/flask (n = 7). The oxidation of palmitate was concentration dependent and maximal oxidation was obtained at 0.35 mM-palmitate. At a saturating concentration of palmitate the oxidation was linear for at least 6 h. About 65% of the total amount of palmitate oxidized during 5 h at 0.52 mM-palmitate (109 +/- 44 nmol/flask, n = 5) was recovered as CO2 and the rest as acid-soluble compounds. Almost all radioactive acid-soluble compounds which were secreted by the Sertoli cells were shown to be 3-hydroxybutyrate and acetoacetate. The palmitate recovery in cellular lipids and triacylglycerols was 9.4 +/- 5.1 nmol/flask (n = 5) and 3.5 +/- 2.8 nmol/flask (n = 5) respectively. Addition of glucose had no significant effect on palmitate oxidation but caused a 9-fold increase in esterification of palmitate into triacylglycerols. We conclude that cultured rat Sertoli cells can oxidize palmitate to CO2 and ketone bodies and that fatty acids appear to be a major energy substrate for these cells.     

Lipoprotein lipase activity is stimulated by insulin and dexamethasone in cardiomyocytes from diabetic rats.

Type 1 diabetes mellitus reduces lipoprotein lipase (LPL) activity in the heart. The diabetic phenotype of decreased LPL activity in freshly isolated cardiomyocytes persisted after overnight culture (16 h). Total cellular LPL activity was 311+/-56 nmol oleate released x h(-1) x mg(-1) cell protein in diabetic cultured cardiomyocytes compared with 661+/-81 nmol oleate released x h(-1) x mg(-1) cell protein for control cultured cells. Diabetes also resulted in lower heparin-releasable (HR) LPL activity compared with control cells (111+/-25 vs. 432+/-63 nmol x h(-1) x mg(-1) cell protein). In kinetic experiments, the reduction in total cellular LPL and HR-LPL activities in cultured cells from diabetic hearts was due to a decrease in maximal velocity, with no change in apparent Km for substrate (triolein). LPL activity in primary cultures of cardiomyocytes from control rats is stimulated by the combination of insulin (Ins) and dexamethasone (Dex). Overnight treatment of cultured cardiomyocytes from diabetic rats with Ins+Dex elicited an 84% increase in cellular LPL activity (to 572+/-65 nmol x h(-1) x mg(-1) cell protein) and a 194% increase in HR-LPL activity (to 326+/-46 nmol x h(-1) x mg(-1) cell protein). This stimulation occurred at subnanomolar concentrations of the hormones, but neither hormone was effective alone. The amount of immunoreactive LPL protein mass in cultured cardiomyocytes from diabetic hearts was unchanged by Ins+Dex treatment. Addition of oleic acid (60 microM) to the overnight culture medium inhibited the already reduced HR-LPL activity in diabetic cultured cells by 73% (to 30+/-4 nmol x h(-1) x mg(-1) cell protein). The presence of oleic acid also reduced hormone-stimulated HR-LPL activity. Increasing the glucose concentration in the culture medium to 26 mM had no effect on total cellular LPL or HR-LPL activities.     

Membrane potentials, electrolyte contents, cell pH, and some enzyme activities of fibroblasts

The resting membrane potential of the cultured fibroblasts derived from rabbit subcutaneous tissues was -10.2 +/- 0.20 mV (n = 390). This potential was affected by the potassium concentration in the culture medium, but not by other chemical or hormonal preparations, such as dibutyryladenosine 3',5'-cyclic monophosphate (0.5 to 5.0 mmol/l), sodium fluoride (10(-5) to 10(-4) M), hydrocortisone (10(-7) to 10(-6) M), parathyroid extract (0.5 to 1.0 U/ml), or thyrotrophin (5 to 10 mU/ml). The Na+, K+, and Cl- concentrations of the cultured fibroblasts were 35.4, 85.7, and 22.6 mmol/l cell water, respectively. The water and protein contents of these cells were 82.1 and 9.18 g/100-g cells, respectively. The intracellular pH of fibroblasts as determined by [14C] dimethyloxazolidine-2, 4-dione, and 3H2O ranged between 6.9 and 7.1 when the pH of the culture medium was maintained at 7.4. The activities of Na+, K+-, HCO3(-)-, and Ca++, Mg++-ATPases in these cultured cells were 19.0 +/- 2.1, 13.6 +/- 2.1, and 6.6 +/- 1.2 nmol pi/mg protein per minute, respectively, and the carbonic anhydrase activity was 0.054 U/mg protein. Calculations based on the values for the membrane potential and the electrolyte concentrations observed in this study indicate that Na+, K+, Cl-, and H+ are not distributed according to their electrochemical gradients across the cell membrane. Na+, Cl-, and H+ are actively transported out of the cells and K+ into the cells.     

Rates of utilization of glucose, glutamine and oleate and formation of end-products by mouse peritoneal macrophages in culture.

1. The metabolism of mouse thioglycollate-elicited peritoneal macrophages was studied in culture for up to 96 h. 2. The rates of glycolysis, lactate formation and glutamine utilization were approximately linear with time for at least 80 h of culture. 3. The rates of glucose and glutamine utilization by cultured macrophages were approx. 500 and 90 nmol/h per mg of protein respectively. This rate of glucose utilization is at least 50% greater than that previously reported for macrophages during 60 min incubation in a shaking flask; and it is now increased by addition of glutamine to the culture medium. The rate of glutamine utilization in culture is similar to that previously reported for macrophages during 60 min incubation. The major end-product of glucose metabolism is lactate, and those of glutamine metabolism are CO2, glutamate, ammonia and alanine. 4. Oleate was utilized by these cells: 14C from [14C]oleate was incorporated into CO2 and cellular lipid. The highest rate of oleate utilization was observed when both glucose and glutamine were present in the culture medium. The presence of oleate in the culture medium did not affect the rates of utilization of either glucose or glutamine. Of the [14C]oleate incorporated into lipid, approx. 80% was incorporated into triacylglycerol and only 18% into phospholipid. 5. The turnover rate for the total ATP content of the macrophage in culture is about 10 times per minute: the value for the perfused isolated maximally working rat heart is 22. This indicates a high metabolic rate for macrophages, and consequently emphasizes the importance of the provision of fuels for their function in an immune response.     

Developmental changes of gamma-aminobutyric acid (GABA) and putative amino acid neurotransmitters in the organotypic culture of newborn mouse cerebellum

The quantitative changes and metabolism of GABA and putative amino acid neurotransmitters during early developmental stages in the organotypic culture of newborn mouse cerebellum were examined by using the high-performance liquid chromatograph (HPLC) technique. D-[U-14C]Glucose was used as a precursor of amino acids. To analyze amino acid neurotransmitters, explants were incubated for 4 weeks under standard conditions. The amount of GABA linearly increased from 8.7 +/- 1.3 nmol/mg protein (2 days in vitro, 2 DIV) to 26.5 +/- 6.1 nmol/mg protein (15 DIV) and was saturated after that (24.0 +/- 3.6 nmol/mg protein at 30 DIV). During the period of GABA increase, the capability for GABA synthesis from [14C]glucose increased rapidly from 3.03 +/- 0.67 nCi/mg protein (2 DIV, 3 h incubation) to 9.32 +/- 1.34 nCi/mg protein (15 DIV, 3 h incubation). In the case of glutamic acid, a putative neurotransmitter of granule cell parallel fibers in the cerebellum, the amount in explants was nearly constant during incubation, in contrast with the fact that the amount in vivo gradually increased. However, the capability for glutamic acid synthesis from [14C]glucose increased from 10.80 +/- 3.01 nCi/mg protein (2 DIV, 1 h incubation) to 27.62 +/- 4.71 nCi/mg protein (22 DIV, 1 h incubation). In the case of taurine, found in abundance in fetal brain and supposed to play a specific role in the development and maturation of the central nervous system, the amount in explants decreased from 139.8 +/- 4.0 nmol/mg protein (2 DIV) to 54.0 +/- 0.8 nmol/mg protein (30 DIV).(ABSTRACT TRUNCATED AT 250 WORDS)     

Lactate production is the major metabolic fate of glucose in splenocytes and is altered in spontaneously diabetic BB rats.

Enhanced glucose metabolism is necessary to support the activation and proliferation of lymphocytes. To define further quantitatively the metabolic fates of glucose and assess glucose utilization both in normal cells and in an autoimmune disease with abnormal lymphocytes, [U-14C]glucose conversion into 14CO2 and the production of lactate and pyruvate were measured in splenocytes. Cells from non-diabetes-prone (BBn) and spontaneously diabetic (BBd) rats were studied both freshly isolated 'resting' and cultured for 96 h with and without concanavalin A (Con A) stimulation. (1) Lactate was confirmed to be the major end product in both freshly isolated (53% of utilized glucose) and unstimulated cultured (62% of utilized glucose) cells from BBn animals studied at (2-8) x 10(6) cells/ml concentration. The use of concentrations from 10 x 10(6) to 300 x 10(6) cells/ml resulted in progressively less lactate production per 10(6) splenocytes. (2) Cells from BBd animals after stimulation with Con A incorporated less [3H]thymidine and produced significantly less lactate (155 +/- 14 versus 305 +/- 24 nmol/2 h per 10(6) cells) than did BBn cells (P less than 0.05). (3) However, more lactate (101 +/- 8 versus 78 +/- 6 nmol/5 h per 10(6) cells) was produced by 'resting' cells from BBd animals compared with BBn (P less than 0.03), and this difference was sustained after 4 days in culture. (4) Significantly greater amounts of pyruvate were produced by BBd than by BBn cells, particularly when stimulated with Con A, suggesting an alteration in the availability of reducing equivalents in BBd cells. (5) These results are consistent with prior metabolic as well as immunological 'activation' of cells in vivo in the BB diabetic animals.     

Presence of ectonucleotidases in cultured chromaffin cells: hydrolysis of extracellular adenine nucleotides

The granular ATP released from chromaffin cells during the secretory response can be hydrolyzed by ectonucleotidases that are present in the plasma membrane of these cells. The ecto-ATPase activity showed a Km for ATP of 250 +/- 18 microM and a VMAX value of 167 +/- 25 nmol/10(6) cells x min (1.67 mumol/mg protein x min) for cultured chromaffin cells, while the ecto-ADPase activity showed a Km value for ADP of 375 +/- 40 microM and a VMAX of 125 +/- 20 nmol/10(6) cells x min (1.25 mumol/mg protein x min). The ecto 5'-nucleotidase activity of cultured chromaffin cells was more specific for the purine nucleotides, AMP and IMP, than for the pirimidine nucleotides, CMP and TMP. The Km for AMP was 55 +/- 5 microM and the VMAX value was 4.3 +/- 0.8 nmol/10(6) cells x min (43 nmol/mg protein x min). The nonhydrolyzable analogs of ADP and ATP, alpha, beta-methylene-adenosine 5'-diphosphate and adenylyl-(beta, gamma-methylene)-diphosphonate were good inhibitors of ecto 5'-nucleotidase activity, the KI values being 73.3 +/- 3.5 nM and 193 +/- 29 nM, respectively. The phosphatidylinositol-specific phospholipase C released the ecto-5'-nucleotidase from the chromaffin cells in culture, thus suggesting an anchorage through phosphatidylinositol to plasma membranes. The presence of ectonucleotidases in chromaffin cells may permit the recycling of the extracellular ATP exocytotically released from these neural cells.     

Effect of age and endurance training on the capacity for epinephrine-stimulated gluconeogenesis in rat hepatocytes.

The effects of endurance training on hepatic glucose production (HGP) from lactate were examined in 24-h-fasted young (4 mo) and old (24 mo) male Fischer 344 rats by using the isolated-hepatocyte technique. The liver cells were incubated for 30 min with 5 mM lactate ([U-14C]lactate; 25000 dpm/ml) and nine different concentrations of epinephrine (Epi). Basal HGP (with lactate only and no Epi) was significantly greater for young trained (T) (99.6 +/- 6.2 nmol/mg protein) compared with young controls (C) (78.2 +/- 6.0 nmol/mg protein). The basal HGP was also significantly greater for old T (97.3 +/- 5.9 nmol/mg protein) compared with old C (72.2 +/- 3.9 nmol/mg protein). After the incubation with the various concentrations of Epi, Hanes-Woolf plots were generated to determine kinetic constants (Vmax and EC50). Maximal Epi-stimulated hepatic glucose production (Vmax) was significantly greater for young T (142.5 +/- 6.5 nmol/mg protein) compared with young C (110.9 +/- 4.8 nmol/mg protein). Similarly, the Vmax was significantly greater for old T (138.2 +/- 5.0 nmol/mg protein) compared with old C (103.9 +/- 2.5 nmol/mg protein). Finally, there was an increase in the EC50 from the hepatocytes of old T (56.2 +/- 6.2 nM) compared with young T (32.6 +/- 4.9 nM). In like manner, there was an increase in the EC50 from the hepatocytes of old C (59.7 +/- 5.8 nM) compared with young C (33.1 +/- 2.7 nM). The results suggest that training elevates HGP in the basal and maximally Epi-stimulated condition, but with age there is a decline in EC50 that is independent of training status.     

Sialic acid, galactose and fucose on the surface of Ehrlich ascites tumor cells grown in glucose-free medium in the presence of uridine

1) The content and accessibility of terminal sialic acid and galactose residues as well as the incorporation of [3H]fucose into glycoconjugates were determined in 48-h cultures of Ehrlich ascites tumor cells in a glucose-free medium supplemented with uridine, a compound which can fulfil the necessary functions of glucose. 2) Sialic-acid residues accessible to sialidase cleavage were reduced from 695 +/- 80 nmol/10(9) cells (controls) to 284 +/- 22 nmol/10(9) cells (43% of controls). In situ labeling using periodate oxidation followed by sodium borotritiide reduction revealed a tritium incorporation of 47 +/- 11% that of controls (= 4.1 x 10(5) cpm/mg protein). 3) Labeling of galactose residues of 80-90% of that of controls was achieved after treatment of the cells with galactose oxidase/sodium borotritiide. A nearly six-fold enhancement of tritium incorporation into galactose of control cells was observed after sialidase/galactose oxidase treatment and sodium borotritiide reduction (1.5----8.8 x 10(5) cpm/mg protein); only a 3.6-fold increase (1.2 x 10(5)----4.3 x 10(5) cpm/mg protein) was found with glucose-free cultured cells. It is concluded that the galactose content of the cell surface is reduced to about 50% of controls. 4) The incorporation of tritium into acid-insoluble precipitate after 24 h incubation with [3H]fucose and the activity of the acid-soluble fraction were enhanced by about 85% as compared to controls. The pattern of inhibition by tunicamycin of [3H]fucose uptake and incorporation was the same in glucose-containing standard medium and in glucose-free uridine medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin regulation of glucose metabolism in HT29 colonic adenocarcinoma cells: activation of glycolysis without augmentation of glucose transport

The effects of insulin on glucose transport and metabolism were examined in cultured HT29 human colonic adenocarcinoma cells. The presence of glucose transporters was verified by D-glucose displaceable [3H]cytochalasin B binding. The Kd and Bmax values from cytochalasin B binding studies were 190 +/- 30 nM and 8.4 +/- 1.4 pmol/mg protein, respectively. Glucose transport determined with 3-O-methylglucose showed saturable kinetics with a Km of 5.8 +/- 0.4 mM and a Vmax of 0.047 +/- 0.003 mumol/mg protein per min at 25 degrees C. Moreover, in HT29 cells, two classes of insulin binding sites were detected in radioligand binding experiments. Although insulin failed to stimulate glucose transport, it was found to activate glycolysis in HT29 cells. Glucose consumption increased from 0.33 +/- 0.03 mumol/mg protein per h to 0.49 +/- 0.05 mumol/mg protein per h and lactate production was augmented from 0.67 +/- 0.04 mumol/mg protein per h to 0.87 +/- 0.06 mumol/mg protein per h in response to 10(-7) to 10(-5) M insulin. Insulin also enhanced mannose metabolism. Apart from these two hexoses, HT29 cells exhibited a surprisingly narrow substrate specificity. With the possible exception of glyceraldehyde, little lactate was produced from alternative substrates, including adenosine, inosine, ribose, deoxyribose, dihydroxyacetone, galactose and fructose either with or without insulin. Despite its limited utilization by the glycolytic pathway, adenosine was readily salvaged for de novo synthesis of adenine nucleotides. These findings suggest that insulin directly influences substrate utilization through the glycolytic pathway in HT29 cells without activating the glucose transport pathway.     

Carnitine stimulation of glucose oxidation in the fatty acid perfused isolated working rat heart.

The effects of L-carnitine on myocardial glycolysis, glucose oxidation, and palmitate oxidation were determined in isolated working rat hearts. Hearts were perfused under aerobic conditions with perfusate containing either 11 mM [2-3H/U-14C]glucose in the presence or absence of 1.2 mM palmitate or 11 mM glucose and 1.2 mM [1-14C]palmitate. Myocardial carnitine levels were elevated by perfusing hearts with 10 mM L-carnitine. A 60-min perfusion period resulted in significant increases in total myocardial carnitine from 4376 +/- 211 to 9496 +/- 473 nmol/g dry weight. Glycolysis (measured as 3H2O production) was unchanged in carnitine-treated hearts perfused in the absence of fatty acids (4418 +/- 300 versus 4547 +/- 600 nmol glucose/g dry weight.min). If 1.2 mM palmitate was present in the perfusate, glycolysis decreased almost 2-fold compared with hearts perfused in the absence of fatty acids. In carnitine-treated hearts this drop in glycolysis did not occur (glycolytic rates were 2911 +/- 231 to 4629 +/- 460 nmol glucose/g dry weight.min, in control and carnitine-treated hearts, respectively. Compared with control hearts, glucose oxidation rates (measured as 14CO2 production from [U-14C]glucose) were unaltered in carnitine-treated hearts perfused in the absence of fatty acids (1819 +/- 169 versus 2026 +/- 171 nmol glucose/g dry weight.min, respectively). In the presence of 1.2 mM palmitate, glucose oxidation decreased dramatically in control hearts (11-fold). In carnitine-treated hearts, however, glucose oxidation was significantly greater than control hearts under these conditions (158 +/- 21 to 454 +/- 85 nmol glucose/g dry weight.min, in control and carnitine-treated hearts, respectively). Palmitate oxidation rates (measured as 14CO2 production from [1-14C]palmitate) decreased in the carnitine-treated hearts from 728 +/- 61 to 572 +/- 111 nmol palmitate/g dry weight.min. This probably occurred secondary to an increase in overall ATP production from glucose oxidation (from 5.4 to 14.5% of steady state myocardial ATP production). The results reported in this study provide direct evidence that carnitine can stimulate glucose oxidation in the intact fatty acid perfused heart. This probably occurs secondary to facilitating the intramitochondrial transfer of acetyl groups from acetyl-CoA to acetylcarnitine, thereby relieving inhibition of the pyruvate dehydrogenase complex.     

Contraction of human airways by oxidative stress protection by N-acetylcysteine.

We examined the in vitro effects of tert-butylhydroperoxide (tBu-OOH) in human bronchial muscle. tert-Butylhydroperoxide produced concentration-dependent contractions of bronchial rings (maximum effect was 56.5 +/- 9.6% of contraction by 1 mM acetylcholine; effective concentration 50% was approximately 100 microM). tert-Butylhydroperoxide (0.5 mM)-induced contraction was enhanced by epithelial removal but abolished by indomethacin (cyclooxygenase inhibitor) and zileuton (lipoxygenase inhibitor). tert-Butylhydroperoxide produced a transient rise in intracellular calcium in human cultured airway smooth muscle cells (HCASMC). The bronchial reactivity to acetylcholine and histamine was not altered by tBu-OOH. In HCASMC, tBu-OOH (0.5 mM, 30 min) increased malondialdehyde levels (MDA; from 7.80 +/- 0.83 to 26.82 +/- 1.49 nmol mg(-1) protein), accompanied by a decrease of reduced glutathione (GSH; from 16.7 +/- 2.6 to 6.9 +/- 1.9 nmol mg(-1) protein) and an increase of oxidized glutathione (from 0.09 +/- 0.03 to 0.18 +/- 0.03 nmol mg(-1) protein). N-acetylcysteine (0.3 mM) inhibited by approximately 60% the bronchial contraction resulting from tBu-OOH (0.5 mM) and protected cultured cells exposed to tBu-OOH (MDA was lowered to 19.51 +/- 1.19 nmol mg(-1) protein, and GSH content was replenished). In summary, tBu-OOH caused contraction of human bronchial muscle mediated by release of cyclo-oxygenase and lipoxygenase products without producing airways hyperreactivity. N-acetylcysteine decreases tBu-OOH-induced contraction and protects human cultured airway smooth muscle cells exposed to tBu-OOH.     

Comparison of the oxidation of glutamine,glucose, ketone bodies and fatty acids by human diploid fibroblasts

The contribution of glutamine, glucose, ketone bodies and fatty acids to the oxidative energy metabolism of human diploid fibroblasts was studied. The rate of glutamine oxidation by fibroblasts was 98 nmol/h per mg cell protein compared to 2 nmol/h per mg cell protein or less for glucose, acetoacetate, d-3-hydroxybutyrate, octanoic acid and palmitic acid. Glucose inhibited glutamine oxidation by 85%, while the other substrates had no effect. Therefore, these cells meet their energy requirement almost solely by anaerobic glycolysis and glutamine oxidation.     

Thromboxane A2 receptors are influenced by cell density in cultured rat aortic smooth muscle cells.

The influence of cell density on the binding characteristics of thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors in rat aortic vascular smooth muscle cells in culture were determined using [1S- (1 alpha, 2 beta (5Z), 3a (1E, 3R*), 4 alpha)]- 7 -[3- (3-hydroxy -4- (4'-iodophenoxy)-1-butenyl)-7-oxabicyclo-[2.2.1]heptan- 2yl]-5-heptenoic acid (125I-BOP). The Bmax for 125I-BOP was 5,430 +/- 139 sites/cell (26.9 +/- 5.7 fmoles/mg protein) for cells cultured in 1% fetal calf serum and 2809 +/- 830 sites/cell (13.1 +/- 2.2 fmoles/mg protein) for cells cultured in 10% fetal calf serum. Cells were allowed to grow to varying densities and then harvested for assay. There was a negative correlation between the Bmax and the cell density per flask. The Kd for I-BOP did not significantly vary in any of the studies. The results demonstrate that cell density plays an important role in influencing the expression of vascular TXA2/PGH2 receptors.     

Neuronal and glial enzyme studies in cell culture

Summary Newborn BALB/c mouse brain was cultured as disaggregated cells after serial trypsin dissociations. The ontogeny of the cultures was followed by assays of cell number, deoxyribonucleic acid, and protein content and by the activities of three enzymes considered to be markers of neuronal differentiation. Aliquots of the freshly dissociated cells were assayed for choline acetylase, acetylcholinesterase, and glutamic acid decarboxylase activities and compared with intact brain. The percentages of recovery of activities, expressed as¹⁴C product formed per mg of protein per 10 min, at pH 6.8 and 37°C, were 37% for choline acetylase, 54% for acetylcholinesterase, and 24% for glutamic acid decarboxylase. The remainder of the freshly dissociated cells were placed into culture; enzyme assays were performed as the cells multiplied and then when the cultures became static. Choline acetylase activity increased as the cells rapidly divided, and glutamic acid decarboxylase activity increased only after the cultures became confluent. Under the culture conditions, acetylcholinesterase was not induced, despite active synthesis of acetylcholine. Neuroblastoma clone N18, C1300 cell line, was grown in cell culture, and the activity of acetylcholinesterase was measured as the cells multiplied and came to confluency. The specific activity of mouse neuroblastoma acetylcholinesterase increased 25-fold when the rate of cell division was restricted. The rate of cell division could be regulated by adjusting the serum concentration. By removing fetal calf serum during the growth period, cell division ceased, and acetylcholinesterase activity was significantly and rapidly induced. Choline-O-acetyltransferase specific activity was measured in rapidly dividing and in static cultures. Its specific activity was highest in nondividing cultures, compared to cultures containing actively dividing cells (6-fold), and the specific activity of thymidylate synthetase was increased 2.5-fold in actively dividing cultures, compared to static cultures. Glioblastoma cells obtained from the rat astrocytoma, clone C6, were grown in culture, and glucose metabolism was measured in control cultures, and in cultures containing norepinephrine (0.017 mg per ml). Norepinephrine produced a 50% inhibition in the incorporation ofd-[¹⁴C]glucose. Cells incubated for 2 hr in the presence ofd-[¹⁴C]glucose, washed and then incubated in control medium or in medium containing norepinephrine, resulted in the release of greater than 50% of radioactive metabolites in the norepinephrine treated plates. Norepinephrine caused a 50% increase in¹⁴CO₂ production in glioblastoma cells incubated withd-[1-¹⁴C]glucose. Norepinephrine, under similar conditions, did not affect the metabolism of glucose in clone C46, C1300 mouse neuroblastoma cells. Portions of this work were supported by a research grant (6-444946-58605) from the American Cancer Society.     

Halothane-Induced Alterations of Glucose and Pyruvate Metabolism in Rat Cerebra Synaptosomes

Synaptosomes isolated from rat cerebra were used to study the effects of the inhalational anesthetic, halothane, on cholinergic processes. To identify possible mechanisms responsible for the depression of acetylcholine synthesis, we examined the effects of halothane on precursor metabolite metabolism involved with supplying the cytosol with acetyl-CoA for acetylcholine synthesis. Three percent halothane/air (vol/vol) depressed 14CO2 evolution from labeled pyruvate and glucose. Steady-state 14CO2 evolution from [1-14C]glucose was depressed 84% by halothane, while 14CO2 evolution from [6-14C]glucose and [3,4-14C]glucose was decreased 67 and 52%, respectively, when compared with control conditions. Halothane inhibited the activities of both pyruvate dehydrogenase (14% depression) and ATP-citrate lyase (32% depression). Total synaptosomal acetyl-CoA concentrations were unaffected by halothane. Three percent halothane/air (vol/vol) caused a 77% increase in medium glucose depletion rate from 1.38 nmol (mg protein)-1 min-1 to 2.44 nmol (mg protein)-1 min-1. Production of lactate by the synaptosomes in the presence of halothane increased by 231% from a control rate of 1.44 nmol (mg protein)-1 min-1 to 4.77 nmol (mg protein)-1 min-1. Lactate production rate from pyruvate was also enhanced by 56% in the presence of halothane. These data lend support to the concept that the NAD+/NADH potential may be involved in the halothane-induced depression of acetylcholine synthesis.     

Rapid isolation of neuroblastoma plasma membranes on Percoll gradients. Characterization and lipid composition

A purified plasma membrane fraction was isolated from cultured neuroblastoma (N1E-115) cells on a discontinuous gradient of 5, 25 and 35% Percoll within 1 h of cell disruption by nitrogen cavitation. Yield of plasma membrane, banding in the 25% Percoll (d = 1.051), was high as judged by the recoveries of the marker enzymes, 5'-nucleotidase (58.0 +/- 5.4%, n = 5), alkaline phosphatase (46.0 +/- 3.0%, n = 4) and Mg2+-stimulated neutral sphingomyelinase (48.0 +/- 4.2%, n = 3); enrichment of specific activities of these enzymes relative to total cell homogenate (lysate) were 10.9 +/- 1.0-, 9.1 +/- 1.0- and 9.6 +/- 0.4-fold, respectively. Levels of marker enzymes for other organelles were less than 3% of total activity, except for microsomes (less than 9%). The plasma membrane fraction was further characterized by 2-, 5- and 6-fold higher content (nmol/mg protein) of total phospholipids, free cholesterol and sphingomyelin, respectively, compared to lysate. Ratios of free cholesterol to phospholipids and of sphingomyelin to phosphatidylcholine in the plasma membrane fraction were about 2-fold greater than that of lysate. The cholesterol ester content of plasma membrane (36 +/- 8 nmol/mg protein) was 2-3-fold higher than that of lysate. Sphingomyelin of the plasma membrane fraction had a higher concentration of long-chain fatty acids (more than 18 carbon atoms) relative to lysate or microsomes. Significant differences also were observed in the fatty acyl composition of diphosphatidylglycerol, cholesterol esters and triacylglycerol of plasma membrane. Thus, we have devised a rapid and reliable method for isolation of highly purified plasma membranes of cultured neuroblastoma cells that is suitable for comparison of metabolic relationships between the plasma membrane and other cellular organelles.     

Uptake of the cephalosporin, cephalexin, by a dipeptide transport carrier in the human intestinal cell line, Caco-2

The transport of the orally absorbed cephalosporin, cephalexin, was examined in the human epithelial cell line, Caco-2 that possesses intestinal enterocyte-like properties when cultured. In sodium-free buffer, the cells accumulated 1 mM D-[9-14C]cephalexin against a concentration gradient and obtained a distribution ratio of 3.5 within 180 min. Drug uptake was maximal when the extracellular pH was 6.0. Uptake was reduced by metabolic inhibitors and by protonophores indicating that uptake was energy- and proton-dependent. Kinetic analysis of the concentration dependence of the rate of cephalexin uptake showed that a non-saturable component (Kd of 0.18 +/- 0.01 nmol/min per mg protein per mM) and a transport system with a Km of 7.5 +/- 2.8 mM and a Vmax of 6.5 +/- 0.9 nmol/min per mg protein were responsible for drug uptake. Uptake was competitively inhibited by dipeptides. The transport carrier exhibited stereospecificity for the L-isomer of cephalexin. Drug uptake was not affected by the presence of amino acids, organic anions, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid or 4,4'-diisothiocyano-2,2'-disulfonic stilbene. Therefore, Caco-2 cells take up cephalexin by a proton-dependent dipeptide transport carrier that closely resembles the transporter present in the intestine. Caco-2 cells represent a cellular model for future studies of the dipeptide transporter.     

Forskolin induction of S-100 protein in glioma and hybrid cells

The S-100 protein level in mouse neuroblastoma (N18TG-2 and NIE-115), rat glioma (C6, C6BU-1, and C6V-1), and hybrid (NG108-15, 140-3, 141-B, NBr10A, NBr20A, NCB20, and NX3IT) cells was determined with a sensitive enzyme immunoassay system that uses a rabbit antibody to bovine brain S-100 protein. S-100 protein was detected in glioma but not in neuroblastoma cells. All seven hybrid cells derived from neuroblastoma and glioma or other types of cells were found to possess a very little or undetectable S-100 protein. The induction of S-100 protein level in prestationary phase cultures of glioma C6BU-1 cells was examined by forskolin, which was a highly specific activator of adenylate cyclase of the cells and produced morphological differentiation. After incubation with 10 microM forskolin for 48 hr, the S-100 protein level increased 2-2.5-fold in C6BU-1 glioma cells whose mean control level was 60 +/- 26 ng/mg protein (+/- SD). The forskolin induction of S-100 protein in the cells was dose dependent, and the concentration of forskolin required for 50% activation of S-100 protein was about 0.6 microM. The increase by forskolin was initiated from 10-15 hr after incubation with it and was inhibited with cycloheximide and actinomycin D. In NG108-15 hybrid cells the induction of S-100 protein was also observed by forskolin as well as prostaglandin (PG) E1 plus theophylline which are known to activate adenylate cyclase of the cells. The results indicate that S-100 protein biosynthesis is genetically controlled in these clonal cells, and that S-100 protein can be regulated in a cAMP-dependent fashion in prestationary cultures.