A human-mouse somatic hybrid line selected for human deoxycytidine deaminase.

A new selective medium has been developed for cells containing the enzyme deoxycytidine deaminase. This medium contains hypoxanthine, aminopterin, and 5-methyldeoxycytidine (HAM medium). To survive in the presence of the aminopterin, the cells must utilize deoxycytidine deaminase to convert the 5-methyldeoxycytidine to thymidine. The cells must also have thymidine kinase and hypoxanthine phosphoribosyltransferase. A mouse cell line deficient in deoxycytidine deaminase has been isolated from a deoxycytidine kinase-deficient line, using 5-bromodeoxycytidine as the selective agent. A hybrid line between this double mutant and a human diploid fibroblast was isolated in HAM medium. The hybrid line contains the chromosomes expected of a human-mouse hybrid. The deoxycytidine deaminase isozyme patterns on cellogel show that the human-mouse hybrid cell line produces an enzyme with an electrophoretic mobility intermediate between that of the human and that of the mouse.     

Bromodeoxyuridine induction of deoxycytidine deaminase activity in a hamster cell line

The Syrian hamster cell line, RPMI 3460, was found to express barely detectable levels of the enzyme deoxycytidine deaminase. In contrast, the cell lines B4 and HAB, which are derived from 3460 cells and have approx. 60 and 100% bromodeoxyuridine substitution in DNA, respectively, show an approx. 50-fold higher enzyme activity. Deoxycytidine deaminase activity can be "induced" in 3460 cells by growth in 10(-5) M bromodeoxyuridine, as well as by the other halogenated pyrimidines, iododeoxyuridine and chlorodeoxy-uridine. The time required for maximal enzyme activity to accrue (approx. 8 days) suggests that new genetic expression is required for enhanced deoxycytidine deaminase activity and inhibition of induction in the presence of Ara. C shows that bromodeoxyuridine must be incorporated into DNA. In addition, the extent of enhanced deoxycytidine deaminase activity is directly related to the level of bromodeoxyuridine substitution in DNA. Another hamster cell line, BHK21/C13, which shows no detectable deoxycytidine deaminase activity, cannot be induced by bromodeoxyuridine. These results are discussed with respect to a mechanism by which bromodeoxyuridine may alter gene expression due to an altered binding of both positive and negative regulatory proteins to DNA.     

Characterization of Na(+)-dependent, active nucleoside transport in rat and mouse peritoneal macrophages, a mouse macrophage cell line and normal rat kidney cells

Peritoneal rat macrophages expressed solely an Na(+)-dependent, concentrative nucleoside transporter, which possesses a single Na(+)-binding site and transports purine nucleosides and uridine but not thymidine or deoxycytidine. The Michaelis-Menten constants for formycin B and Na+ were about 6 microns and 14 mM, respectively, and the estimated Na+:formycin B stoichiometry was 1:1. Rat macrophages accumulated 5 microM formycin B to a steady-state level exceeding that in the medium by about 500-fold during 60 min of incubation at 37 degrees C. Concentrative formycin B transport was resistant to inhibition by nitrobenzylthioinosine, lidoflazine, dilazep and nifedipine, but was slightly inhibited by high concentrations of dipyridamole (greater than 10 microM) and probenecid (greater than 100 microM). Mouse peritoneal macrophages and lines of mouse macrophages and normal rat kidney cells expressed Na(+)-dependent, active nucleoside transport but in addition significant Na(+)-independent, facilitated nucleoside transport. Facilitated nucleoside transport in these cells was sensitive to inhibition by nitrobenzylthioinosine, dilazep and dipyridamole. The presence of these inhibitors greatly enhanced the concentrative accumulation of formycin B by these cells by inhibiting the efflux via the facilitated transporter of the formycin B actively transported into the cells. Whereas rat macrophages lacked high-affinity nitrobenzylthioinosine-binding sites, mouse macrophages and normal rat kidney cells possessed about 10,000 such sites/cell. Rat and mouse erythrocytes, rat lymphocytes, and lines of Novikoff rat hepatoma cells, Chinese hamster ovary cells, Mus dunni cells and embryonic monkey kidney cells expressed only facilitated nucleoside transport.     

Human immunodeficiency virus type 1 induces 1-beta-D-arabinofuranosylcytosine resistance in human H9 cell line.

We have found that chronically HIV-1(IIIB)-infected H9 cells showed 21-fold resistance to 1-beta-D-arabinofuranosylcytosine (ARA-C) compared with uninfected H9 cells. In the infected H9 cells, a 37% increase of dCTP pool and a 34% increase of dATP were observed, and no alteration of dTTP and dGTP was observed, compared with the uninfected H9 cells. A marked decrease of ARA-CTP generation was observed in the infected H9 cells after 3-h incubation with 0.1-10 microM ARA-C. The level of deoxycytidine kinase activity with ARA-C as substrate was similar in both the infected and the uninfected cells; however, a 37-fold increase of cytidine deaminase activity was observed in the infected H9 cells. These results indicate that the induction of cytidine deaminase activity by HIV-1(IIIB) infection conferred ARA-C resistance to H9 cells. This conclusion was supported by the observation that a marked reversal of ARA-C resistance in the infected H9 cells occurred after treatment with the inhibitor of cytidine deaminase, 3,4,5,6-tetrahydrouridine. The understanding of these cellular alterations in drug sensitivity may facilitate the development of effective therapeutic strategies against HIV-1-infected cells.     

Tetrahydrouridine specifically facilitates deoxycytidine incorporation into herpes simplex virus DNA.

As reported by Jamieson and Subak-Sharpe (J. Gen. Virol. 31:303-313, 1976), exogenous deoxycytidine is very poorly incorporated into herpes simplex virus DNA. Here it is shown that this incorporation was dramatically increased in the presence of tetrahydrouridine (THU), a specific inhibitor of cytidine-deoxycytidine deaminase. Thus, the exclusion of deoxycytidine from herpes simplex virus DNA probably results from massive degradation by the deaminase, which is consistent with the observation that in the absence of THU, most of the nucleotides formed from exogenous deoxycytidine are dUMP. The effect of tHU upon deoxycytidine incorporation was specific for herpes simplex virus-infected cells; THU did not increase deoxycytidine incorporation into DNA of uninfected cells. Therefore, one might expect THU to enhance the antiviral activity of 1-beta-D-arabinofuranasylcytosine since this analog is also readily deaminated. However, THU increased both the antiviral activity and the cell toxicity only slightly and to about the same extent. Therefore, the metabolism of 1-beta-D-arabnofuranosylcytosine is different from that of deoxycytidine in herpes simplex virus-infected cells.     

In vitro metabolism of deoxycoformycin in human T lymphoblastoid cells. Phosphorylation of deoxycoformycin and incorporation into cellular DNA

The biochemical and metabolic effects of deoxycoformycin, a potent inhibitor of adenosine deaminase, were investigated using two human T lymphoblastoid cell lines. A dose-response analysis demonstrated that the concentration of deoxycoformycin at which there was 50% inhibition of growth was greater than 1 X 10(-3) M in lymphoblastoid cells. Uptake of deoxycoformycin was biphasic and occurred much more slowly than for natural nucleosides, and lower saturation levels were reached. The intracellular concentration of deoxycoformycin achieved was 0.4 to 0.5 microM when the extracellular concentration was 1 microM. At 10 microM extracellular concentration, the intracellular concentration was 3-4 microM. Although deoxycoformycin at very low concentrations (1 or 10 microM) did not have any detectable effects on the growth of these cells, the nucleoside was found to be metabolized, and was phosphorylated to give the mono-, di-, and triphosphate derivatives. The triphosphate derivative was incorporated into cellular DNA with little incorporation into cellular RNA. Metabolism of deoxycoformycin in several mutant lymphoblastoid cells deficient in adenosine kinase and/or deoxycytidine kinase was found to be unchanged from wild-type cells, indicating that these major nucleoside kinases do not play a significant role in the phosphorylation of deoxycoformycin. These results may account, at least in part, for the differences that are observed between the pharmacologic inhibition of adenosine deaminase, and the inherited deficiency of adenosine deaminase.     

Isolation and localization of phosphoprotein pp 135 in the nucleoli of various cell lines

Phosphoprotein pp 135 is one of the dominant proteins endogenously phosphorylated in cellular sonicates during short-time exposure to [gamma-32P]ATP. Mouse cells growing exponentially show the highest pp 135 level as determined by endogenous phosphorylation and immunobinding assays. Disruption of cells in the absence of calcium at low magnesium concentration renders more than 90% pp 135 into the cytosolic fraction. A five-step purification yields greater than 95% pure pp 135. The cellular location of pp 135 was determined with a rat anti-(mouse pp 135) serum by immunofluorescence in mouse cell lines and cryostat sections of normal mouse tissue. We observed fluorescence predominantly of nucleolar structures, confirmed by studies of isolated nuclei and nucleoli. Cross-reacting nucleolar phosphoproteins were identified in cell lines of other species with molecular masses of 128 kDa (human), 135 kDa (hamster) and 118 kDa (Drosophila). Endogenous phosphorylation of pp 135 investigated with purified mouse nucleoli showed optimal activity at isotonicity, pH 7.3, in the presence of 10 mM magnesium ions.     

Counter-regulation of insulin-stimulated glucose transport by catecholamines in the isolated rat adipose cell

The interaction between catecholamines and insulin in regulating glucose transport in isolated rat adipose cells has been evaluated. In the absence of insulin, 1 microM isoproterenol stimulates 3-O-methylglucose transport approximately 2-fold. However, isoproterenol in combination with adenosine deaminase inhibits glucose transport activity approximately 60%. N6-Phenylisopropyladenosine, a nonmetabolizable adenosine analogue, substantially reverses this inhibitory effect and actually stimulates glucose transport activity approximately 2-fold in the absence of isoproterenol. Dibutyryl cAMP inhibits glucose transport activity approximately 75% regardless of adenosine deaminase. While none of these agents significantly influences the basal concentration of plasma membrane glucose transporters, as assessed by specific D-glucose-inhibitable cytochalasin B binding, isoproterenol or dibutyryl cAMP in combination with adenosine deaminase reduces that in the low density microsomes 19 and 58%, respectively. In the presence of insulin, both isoproterenol and adenosine deaminase alone inhibit glucose transport activity approximately 25%. However, only the latter is accompanied by a corresponding decrease in the insulin-stimulated concentration of plasma membrane glucose transporters. Together, isoproterenol and adenosine deaminase inhibit insulin-stimulated glucose transport activity approximately 75%, even in the presence of 5 mM glucose to maintain cellular ATP levels. A similar inhibition is observed with dibutyryl cAMP. However, these agents decrease the insulin-stimulated concentration of plasma membrane glucose transporters only approximately 45%. Nevertheless, all of these inhibitory effects occur through decreases in the transport Vmax. In addition, N6-phenylisopropyladenosine partially reverses the inhibitory effects induced by the presence of adenosine deaminase. These results suggest that catecholamines counter-regulate basal and insulin-stimulated glucose transport in rat adipose cells through a cAMP-mediated mechanism, but only in part by modulating the translocation of glucose transporters.     

Picric acid methods greatly overestimate serum creatinine in mice: more accurate results with high-performance liquid chromatography

The creatinine levels of blood and urine from humans, rats, and mice were measured by high-performance liquid chromatography. These were compared to the alkaline picrate analysis of creatinine performed by standard colorimetric, kinetic, and AutoAnalyzer techniques. For human serum and urine the values obtained using the HPLC technique gave good agreement with four out of five alkaline picrate techniques. For black or white mice, the serum creatinine concentration was 8.7 +/- 0.4 microM by HPLC but 44.9 +/- 1.9 microM by the lowest alkaline picrate method. Mouse urine creatinine concentrations were 3.24 +/- 0.19 mM by HPLC and 4.59 +/- 0.39 mM by the nearest alkaline picrate method. Rat serum creatinine concentrations analyzed by HPLC were about half the values obtained by AutoAnalyzer. Mouse and rat samples seemed to have substances which gave nonspecific color and thus interfered with the analysis of creatinine by the alkaline picrate methods. While the alkaline picrate analysis of creatinine was adequate for human samples, it was necessary to use HPLC to accurately measure rodent creatinine. The fractional excretion of creatinine was determined by measuring creatinine in mouse urine and plasma by both the kinetic and HPLC methods and comparing these values to urine and plasma inulin. Using the kinetic method, creatinine was cleared at 43 +/- 3% of the rate of inulin. Using the HPLC method, creatinine was cleared at 170 +/- 11% of the rate of inulin.     

An interferon-induced increase in cyclic AMP levels precedes the establishment of the antiviral state.

Mouse interferon induces an increase in cyclic AMP levels in interferon-sensitive mouse Ly cells but not in interferon-insensitive human KB-3 cells. The interferon-induced elevation in cyclic AMP precedes the induction of antiviral activity. Although interferon stimulation of the adenylate cyclase activity of Ly cell plasma membranes has not been detected, interferon is effective in stimulating this activity in plasma membranes from rat thyroid cells.     

Deoxyadenosine metabolism and cytotoxicity in cultured mouse T lymphoma cells: a model for immunodeficiency disease.

The inherited absence of either adenosine deaminase (ADA) or purine nucleoside phosphorylase is associated with severe immunological impairment. We have developed a cell culture model using a mouse T cell lymphoma to simulate ADA deficiency and to study the relationship between purine salvage enzymes and immune function. 2′-deoxyadenosine triphosphate (deoxyATP) levels have been shown to be greatly elevated in erythrocytes of immunodeficient, ADA-deficient patients, suggesting that deoxyadenosine is the potentially toxic substrate in ADA deficiency. Using a potent ADA inhibitor, we have demonstrated that deoxyadenosine is growth-inhibitory and cytotoxic to S49 cells, and that deoxyATP accumulates in these cells. Cell variants, unable to transport or phosphorylate deoxyadenosine, are much less sensitive to deoxyadenosine, indicating that intracellular phosphorylation of deoxyadenosine is required for the lethal effects.We have partially reversed the cytotoxic effects of deoxyadenosine with deoxycytidine in wild-type cells, but we cannot show any reversal in cell lines lacking deoxycytidine kinase. Adenosine (ado) kinase-deficient cells are extremely resistant to deoxyadenosine in the presence of deoxycytidine. This deoxycytidine reversal of deoxyadenosine toxicity is consistent with an inhibition of ribonucleotide reductase by deoxyATP, and we have shown that incubation of S49 cells with deoxyadenosine markedly reduces intracellular levels of deoxyCTP, deoxyGTP and TTP.Kinetics data in wild-type cells and in cell variants are consistent with the presence of two deoxyadenosine-phosphorylating activities — one associated with ado kinase and another associated with deoxycytidine kinase.The S49 cells appear to be a valid model for the simulation of ADA deficiency in cell culture, and from our results, we can suggest administration of deoxycytidine as a pharmacological regimen to circumvent the clinicopathologic symptoms in ADA deficiency.     

Adenosine kinase deficiency in tritiated deoxyadenosine-resistant mouse S49 lymphoma cell lines

Mutant sublines were derived of S49 mouse T-lymphoma cells that were resistant to tritiated deoxyadenosine. Twenty-five isolates that were selected in 1 microCi/ml of the nucleoside were cross-resistant to 6-thioguanine, were sensitive to HAT (hypoxanthine, aminopterin, and thymidine), and contained less than 1% of hypoxanthine phosphoribosyltransferase activity in wild-type cells. One of the mutant clones, S49-dA2, was further subjected to selection in a medium containing 2 microCi/ml tritiated deoxyadenosine and 1 microgram/ml deoxycoformycin, an inhibitor of adenosine deaminase. All resistant subclones were cross-resistant to tubercidin, 6-methylmercaptopurine riboside, and arabinosyladenine. One of the subclones, S49-12, was completely devoid of adenosine kinase and was partially deficient in deoxyadenosine kinase. This subclone, however, contained wild-type levels of deoxycytidine kinase. DEAE chromatography of the wild-type cell extracts revealed two deoxyadenosine phosphorylating activities, one of which coeluted with adenosine kinase and was the enzyme missing in S49-12. The other species phosphorylated both deoxyadenosine and deoxycytidine, of which deoxycytidine was the preferred substrate.     

Phorbol esters and cyclic AMP activate AMP deaminase in adult rat cardiac myocytes.

Using rapid deenergization as a probe for adenylate deaminase activity in intact adult rat cardiac myocytes, we have previously established that IMP formation is enhanced by alpha-adrenergic agonists. In the present study, the effect of adrenergic agents on adenylate deaminase was further characterized. Phenylephrine (PE)3 increased IMP production in a dose-dependent fashion with an EC50 of 8 x 10(-7) M. The response to PE was reversed within 10 min by the alpha 1-antagonist, prazosin. Likewise, adenylate deaminase was also activated in ventricular myocytes challenged with phorbol 12-myristate 13-acetate (PMA, EC50 = 5 nM); cardiac cells presented with 100 nM PMA increased IMP production from 4.4 +/- 0.5 (control) to 15.7 +/- 0.9 nmol/mg protein when subsequently deenergized. The effects of PMA and PE were attenuated 85 +/- 5% and 96 +/- 4%, respectively, by pretreatment of cells with 150 nM staurosporine, an inhibitor of protein kinase C. Furthermore, incubation of cardiac cells with 1 microM PMA for 24 h blunted the response to both PMA and phenylephrine 85-90%. Elevating cyclic AMP (cAMP) content to greater than 15 pmol/mg by treatment with forskolin or isoproterenol plus isobutylmethylxanthine also resulted in enhanced adenylate deaminase activity, but this stimulatory effect was not abolished by 24 h incubation with 5 microM PMA. Forskolin and PMA-induced increases in IMP production appeared to be additive. However, 0.5 microM isoproterenol inhibited the cellular response to phenylephrine by about 30% but did not affect PMA-stimulated adenylate deaminase activity. We conclude that both cAMP and protein kinase C stimulate adenylate deaminase, perhaps through selective activation of different isoforms. However, cAMP also exerts partial inhibition on alpha-adrenoreceptor-mediated increases in IMP production.     

Pyrimidine Salvage Pathways In Toxoplasma Gondii

ABSTRACT. Pyrimidine salvage enzyme activities in cell-free extracts of Toxoplasma gondii were assayed in order to determine which of these enzyme activities are present in these parasites. Enzyme activities that were detected included phosphoribosyltransferase activity towards uracil (but not cytosine or thymine), nucleoside phosphorylase activity towards uridine, deoxyuridine and thymidine (but not cytidine or deoxycytidine), deaminase activity towards cytidine and deoxycytidine (but not cytosine, cytidine 5'-monophosphate or deoxycytidine 5'-monophosphate), and nucleoside 5'-monophosphate phosphohydrolase activity towards all nucleotides tested. No nucleoside kinase or phosphotransferase activity was detected, indicating that T. gondii lack the ability to directly phosphorylate nucleosides. Toxoplasma gondii appear to have a single non-specific uridine phosphorylase enzyme which can catalyze the reversible phosphorolysis of uridine, deoxyuridine and thymidine, and a single cytidine deaminase activity which can deaminate both cytidine and deoxycytidine. These results indicate that pyrimidine salvage in T. gondii probably occurs via the following reactions: cytidine and deoxycytidine are deaminated by cytidine deaminase to uridine and deoxyuridine, respectively; uridine and deoxyuridine are cleaved to uracil by uridine phosphorylase; and uracil is metabolized to uridine 5'-monophosphate by uracil phosphoribosyltransferase. Thus, uridine 5'-monophosphate is the end-product of both de novo pyrimidine biosynthesis and pyrimidine salvage in T. gondii.     

Synthesis of thymidine nucleotides in Bacillus subtilis.

To investigate the synthesis of thymidine nucleotides in Bacillus subtilis, mutants that carried various combinations of thyA, thyB, and other mutations affecting pyrimidine metabolism were isolated. It was found that exogenously supplied deoxycytidine was converted to thymidine nucleotides. The present data suggest that deoxycytidine nucleotides are first deaminated to yield deoxyuridine nucleotides which can serve as substrates for both thyA- and thyB-coded synthetases. A deaminase activity for dCDP was found in crude extracts of B. subtilis. A mutant lacking the deaminase activity was unable to convert deoxycytidine nucleotides to thymidine nucleotides.     

The regulation of glucose transport by cAMP stimulators via three different mechanisms in rat and human adipocytes

The regulation of glucose transport by a beta-adrenergic agonist and other cAMP stimulators was assessed by kinetic analyses of 3-O-methylglucose (MG) transport in rat and human adipocytes and in isolated rat plasma membrane vesicles. Basal MG transport was biphasically affected by L-isoproterenol in rat adipocytes: lower concentrations (10-25 nM) of L-isoproterenol stimulated the basal rate by increasing the Vmax, but higher concentrations (0.5-2 microM) of L-isoproterenol inhibited the basal rate. On the other hand, the maximum insulin-stimulated MG transport rate was not affected by 25 nM L-isoproterenol, but was suppressed by 2 microM L-isoproterenol in rat adipocytes. In the presence of adenosine deaminase plus L-isoproterenol (25 nM and 2 microM), dibutyryl cyclic AMP (Bt2cAMP), 3-isobutyl-1-methylxanthine, or forskolin, both basal and the maximum rates of MG transport were suppressed in rat adipocytes. However, from kinetic experiments, both L-isoproterenol plus adenosine deaminase and Bt2cAMP decreased the Vmax. On the other hand, isobutymethylxanthine and forskolin decreased the Vmax as well as increased the K8. MG transport in plasma membrane vesicles was directly inhibited by either forskolin or isobutylmethylxanthine. In contrast, both 25 nM and 2 microM L-isoproterenol with or without adenosine deaminase, Bt2cAMP, or cAMP had no effect on MG transport in rat plasma membrane vesicles. In human adipocytes, L-isoproterenol always stimulated basal MG transport and did not suppress the maximum rate of MG transport, even though cAMP production was maximally stimulated by L-isoproterenol. Both adenosine deaminase plus L-isoproterenol and Bt2cAMP did not suppress the basal rate, but did show a modest suppression (40%) of the maximum insulin effect on MG transport in human adipocytes. However, both isobutylmethylxanthine and forskolin remarkably suppressed (85%) both the basal and the maximum rate of MG transport by both increasing the K8 and decreasing the Vmax. These results indicate MG transport in both rat and human adipocytes is regulated by 3 different mechanisms: (I) L-isoproterenol, a beta-adrenergic agonist, stimulates basal MG transport by increasing the Vmax, (II) cAMP mediates a decrease in MG transport by decreasing the Vmax, and (III) both forskolin and isobutylmethylxanthine also decrease MG transport by directly inhibiting the binding of MG molecules to transporters, resulting in a decrease in the Vmax and an increase in the K8.     

Modulation of guanine deaminase

1. Guanine deaminases purified from the 15000g supernatant fraction of iso-osmotic sucrose homogenates of rat and mouse liver and brain were tested for the influence of GTP and allantoin. 2. The suffixes A and B were assigned to the isoenzyme fractions eluted from DEAE-cellulose with the lower and the higher molarity of eluent respectively. Isoenzyme A from rat liver, the activity of which showed a sigmoid dependence on substrate saturation, was activated by GTP and inhibited by allantoin. Isoenzyme B, which had a hyperbolic substrate-saturation curve, was not influenced by GTP or allantoin. 3. Isoenzyme A from rat brain, the activity of which had a sigmoid dependence on substrate concentration, was stimulated by GTP. Isoenzyme B, which showed classical Michaelis-Menten kinetics, was inhibited by allantoin. 4. Mouse liver guanine deaminase was not influenced by either GTP or allantoin. 5. Isoenzyme A from mouse brain, which had a hyperbolic substrate-saturation curve, was not influenced by GTP or allantoin but isoenzyme B, with sigmoidal kinetics, was inhibited by allantoin. 6. Mg(2+) activated, or inhibited or did not have an effect on guanine deaminase, depending on the source of the enzyme. 7. The bearing of the above findings on the possible regulation of guanine deaminase activity in vivo is discussed.     

Do mammals make all their own inositol hexakisphosphate?

A highly specific and sensitive mass assay for inositol hexakisphosphate (InsP6) was characterized. This centres around phosphorylating InsP6 with [32P]ATP using a recombinant InsP6 kinase from Giardia lambia, followed by HPLC of the 32P-labelled products with an internal [3H]InsP7 standard. This assay was used to quantify InsP6 levels in a variety of biological samples.Concentrations of InsP6 in rat tissues varied from 10-20 microM (assuming 64% of wet weight of tissue is cytosol water), whereas using the same assumption axenic Dictyostelium discoideum cells contained 352 +/- 11 microM InsP6. HeLa cells were seeded at low density and grown to confluence, at which point they contained InsP6 levels per mg of protein similar to rat tissues. This amounted to 1.952 +/- 0.117 nmol InsP6 per culture dish, despite the cells being grown in serum shown to contain no detectable(less than 20 pmol per dish) InsP6. These results demonstrate that mammalian cells synthesize all their own InsP6. Human blood was analysed, and although the white cell fraction contained InsP6 at a concentration comparable with other tissues, in serum and platelet-free plasma no InsP6 was detected (<1 nM InsP6). Human urine was also examined, and also contained no detectable (<5 nM) InsP6. These results suggest that dietary studies purporting to measure InsP6 at micromolar concentrations in human plasma or urine may not have been quantifying this inositol phosphate. Therefore claims that administrating InsP6 in the diet or applying it topically can produce health benefits by increasing extracellular InsP6 levels may require reassessment.     

Anandamide stimulates phospholipase D activity in PC12 cells but not in NIH 3T3 fibroblasts

The endogenous cannabinoid arachidonoylethanolamide was previously reported to have no effects on the phospholipase D activity in Chinese hamster ovary cells expressing the human brain-specific cannabinoid receptor, while in mouse peritoneal cells, delta9-tetrahydrocannabinol stimulated this enzyme. In this work, arachidonoylethanolamide (0.1-1 microM) was found to stimulate the phospholipase D-mediated phospholipid hydrolysis in rat adrenal pheochromocytoma PC12 cells, but not in mouse NIH 3T3 fibroblasts. The phospholipase D-activating effects of arachidonoylethanolamide were comparable to those elicited by phorbol ester and nerve growth factor, while arachidonic acid (1 microM) had no effects. The results show that, depending on the cell type, arachidonoylethanolamide can be an activator of the phospholipase D system.