Role of S-adenosylhomocysteine in adenosine-mediated toxicity in cultured mouse T lymphoma cells

S-adenosylhomocysteine (SAH) is known to be a potent inhibitor of S-adenosylmethionine (SAM)-mediated reactions, of which SAH itself is a product. The immediate metabolic fate of SAH involves its hydrolysis to adenosine and L-homocysteine by the enzyme SAH hydrolase, but the reversibility of this reaction and its extremely low K_eq in the hydrolytic direction suggest that under certain conditions of adenosine excess, SAH might accumulate with significant cytotoxic effects. We have used a model system consisting of cultured S49 mouse lymphoma cells together with the adenosine deaminase (ADA) inhibitor, erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), to determine whether SAH is a mediator of adenosine cytotoxicity.Cells rendered resistant to adenosine-induced pyrimidine starvation by the addition of exogenous uridine or by the mutational loss of adenosine kinase are still sensitive to adenosine at concentrations >15 μM. We find that this effect is appreciably enhanced by the addition of L-homocysteine thiolactone to the culture medium. Cytotoxic concentrations of adenosine also cause significant elevations in intracellular levels of SAH, which are increased an additional several fold by 100μM exogenous L-homocysteine thiolactone. A fair correlation exists between a single time point determination of intracellular SAH and the degree of growth inhibition after 72 hr, but complicated time-dependent variations in SAH make it difficult to compare results obtained in the absence and presence of exogenous L-homocysteine thiolactone.In vivo DNA methylation in S49 cells is markedly inhibited by exposure of cells to concentrations of adenosine known to cause uridine-resistant cytotoxicity. This inhibition of methylation has been measured with short-term pulses of radiolabel, and correlates well with intracellular concentrations of SAH at all tested combinations of adenosine and L-homocysteine thiolactone. The results suggest that the uridine-resistant cytotoxic effects of adenosine on ADA-inhibited S49 cells are secondary to the inhibition of SAM-mediated methylation reactions by the adenosine metabolite SAH.     

Differential Effects of Nerve Growth Factor and Ciliary Neuronotrophic Factor on Catecholamine Storage and Catecholamine Synthesizing Enzymes of Cultured Rat Chromaffin Cells

The effects of nerve growth factor (NGF) and ciliary neuronotrophic factor (CNTF) on catecholamine content and in vitro activities of tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) were studied in adrenal chromaffin cells cultured from 8-day-old rats. Both NGF and CNTF enhanced chromaffin cell survival and partially prevented losses of adrenaline during the 4-day culture period in a dose-dependent manner. CNTF was more potent, although cellular levels of adrenaline and noradrenaline were not maintained. NGF did not add to the effect of CNTF. The effect of CNTF on catecholamine storage was not accompanied by changes in the activities of TH and PNMT. In contrast, NGF induced TH but not PNMT activity. These data indicate differences between the mechanisms by which NGF and CNTF affect adrenal chromaffin cells.     

NGF promotes copper accumulation required for optimum neurite outgrowth and protein methylation

The role of copper in biological phenomena that involve signal transduction is poorly understood. A well-defined cellular model of neuronal differentiation has been utilized to examine the requirement for copper during nerve growth factor (NGF) signal transduction that results in neurite outgrowth. Experiments demonstrate that NGF increases cellular copper content within 3 days of treatment. Copper chelators reduce the effects of NGF on neurite outgrowth and copper accumulation. The effects of tetraethylene pentamine (TEPA), a copper-specific chelator, are reversible by removal from the culture medium and/or by addition of equimolar copper chloride. Because previous work demonstrated that NGF increases protein methylation in PC12 cells, we examined whether TEPA also inhibits S-adenosylhomocysteine hydrolase (SAHH), an essential copper enzyme involved in all protein methylation reactions. In addition to direct in vitro inhibition of SAHH, we show that TEPA decreases protein arginine methyltransferase 1(PRMT1)-specific enzyme activity in PC12 cells and sympathetic neurons. These data comprise the first biochemical and cellular evidence to address the mechanism of copper involvement in neuronal differentiation.     

Protein Carboxyl Methylation in Synaptic Membrane of Rat Brain: The Possible Presence of Adenosine-Bound S-Adenosyl-L-Homocysteine Hydrolase in the Membrane

The effects of some neurotransmitters, adenosine (Ad), and homocysteine (Hcys) on protein carboxyl methylation in synaptic plasma membranes from rat cerebral cortex were examined. Neither any of the neurotransmitters nor Ad had a detectable effect. Incubation of membrane with DL-Hcys alone (5 X 10(-5) M), the combination of both Ad (5 X 10(-5)) and DL-Hcys (5 X 10(-5)), or S-adenosyl-L-homocysteine (SAH) (1 X 10(-6)) strongly decreased the methyl ester formation. The inhibitory effect of the combination of both compounds may be interpreted in terms of the increased SAH concentration due to the presence of SAH hydrolase in the membrane. The inhibitory effect of Hcys alone was blocked by preincubation with Ad deaminase or Neplanocin A, a potent inhibitor of SAH hydrolase, suggesting the presence of Ad-bound SAH hydrolase in the synaptic membrane. Ad-bound SAH hydrolase activity estimated by the inhibition of methylation in the presence of Hcys was located in the membrane fractions including synaptosomes, myelin, and microsomes (about 70%), but the SAH hydrolase activity estimated on the basis of the inhibitory effect of the combination of both Ad and Hcys was localized exclusively in the soluble fraction (about 90%). The distribution of the latter activity is coincident with that of SAH hydrolase reported to date. Incubation of the synaptic membrane with Hcys markedly increased the SAH concentration. The stimulatory effect of Hcys alone was blocked by Ad deaminase.     

Nerve growth factor-mediated induction of tyrosine hydroxylase and of neurite outgrowth in cultures of bovine adrenal chromaffin cells: dependence on developmental stage

Adrenal medullary cells were cultured in a serum-free medium from fetal, neonatal (calves), and adult bovine animals. Neurite outgrowth in response to nerve growth factor (NGF) was observed in cells obtained from fetuses up to a gestational age of 3 months but not in cultures from older animals. The tyrosine hydroxylase (TH) specific activity was found to depend on the cell density and corresponded, at a density of 2 × 10⁵ cells/cm², to the specific activity found in vivo. The TH specific activity increased about sevenfold from fetuses to adult animals. Administration of NGF in vitro caused an increase of the TH specific activity in fetal cells by up to 140% and in calf cells typically by 70–100%. Cultures from adult animals showed no significant TH increase in response to NGF. Scatchard analysis and kinetic studies of the NGF binding at 0°C to intact adrenal medullary cells cultured from calves or from adult bovine animals revealed the presence of only one class of receptors, having a dissociation constant (K_D) of 1 × 10 ⁹, M. There are 16,000 binding sites per cell. The affinity of the reeptors in vivo (determined in crude membrane preparations) did not alter during development, whereas the receptor density decreased with increasing fetal age, but was the same for calves and adults. Whereas the loss of NGF-mediated fiber outgrowth during development might be related to the reduction of receptor density, the disappearance of the NGF-mediated TH induction does not correlate with changes in the binding characteristics of NGF to the adrenal chromaffin cells.     

EFFECT OF GLUCOCORTICOIDS ON NERVE GROWTH FACTOR-MEDIATED ENZYME INDUCTION IN ORGAN CULTURES OF RAT SYMPATHETIC GANGLIA: ENHANCED RESPONSE AND REDUCED TIME REQUIREMENT TO INITIATE ENZYME INDUCTION

Abstract— After previous studies had shown that nerve growth factor produces a very similar change in the enzyme pattern of adrenergic neurons as does an increased activity of the preganglionic cholinergic nerves, the present experiments revealed that the nerve growth factor-mediated selective induction of TH and DBH is enhanced by glucocorticoids in a way similar to that mediated by acetylcholine via nicotinic receptors. Corticosterone (5 μM) produced not only an increase in the maximal response to NGF but shifted the concentration response curve of TH to NGF to the left. The potentiation effect was shown to be specific for glucocorticoids, since other steroid hormones like testosterone, β-estradiol and progesterone had no effect. Moreover, the glucocorticoid effect could be antagonized by cortexolone, suggesting an effect via glucocorticoid receptors. In addition to the potentiation of the nerve growth factor-mediated enzyme induction, glucocorticoids reduced the exposure time to NGF, necessary to initiate maximal TH induction, from 4 h to 10 min. The glucocorticoid potentiation of NGF-mediated specific enzyme induction is discussed in relation to the site and mechanism of action of NGF.     

Differential inhibition of nerve growth factor and epidermal growth factor effects on the PC12 pheochromocytoma line

Tests have been made of the action of the methyltransferase inhibitors 5'-S-methyl adenosine, 5'-S-(2-methyl-propyl)-adenosine, and 3-deaza- adenosine +/- L-homocysteine thiolactone, on nerve growth factor (NGF)- dependent events in the rat pheochromocytoma line PC12. Each of these agents inhibited NGF-dependent neurite outgrowth at concentrations of the order of millimolar. Slow initiation of neurite outgrowth over several days and more rapid regeneration of neurites (congruent to 1 d) were blocked, as was the priming mechanism necessary for genesis of neurites. The inhibitions were reversible in that PC12 cells maintained for several days in the presence of inhibitors grew neurites normally after washout of these agents. Other NGF-dependent responses of the PC12 line (i.e., induction of ornithine decarboxylase activity [over 4 h], enhancement of tyrosine hydroxylase phosphorylation [over 1 h], and rapid changes in cell surface morphology [30 s onward]) were inhibited by each of the agents. In contrast, corresponding epidermal growth factor-dependent responses in ornithine decarboxylase activity, phosphorylation, and cell surface morphology were not blocked, but instead either unaffected or enhanced, by the methylation inhibitors. These inhibitors did not act by blockade of binding of NGF to high- or low-affinity cell surface receptors, though they partially inhibited internalization of [125I]NGF. The inhibition of rapidly-induced NGF- dependent events and the differential inhibition of responses to NGF and epidermal growth factor imply that the methyltransferase inhibitors specifically block one of the first steps in the mechanistic pathway for NGF.     

Nerve Growth Factor and Dexamethasone Modulate Synthesis and Storage of Catecholamines in Cultured Rat Adrenal Medullary Cells: Dependence on Postnatal Age

Catecholamine content and in vitro activities of tyrosine hydroxylase (TH) and noradrenaline N-methyltransferase (NMT) were measured in cultures of isolated adrenal medullary cells from newborn and young postnatal rats to study the effects of the differentiation factors glucocorticoids and nerve growth factor (NGF). During the 4-day culture period the cellular catecholamine (CA) content and TH activity remained stable, whereas NMT activity dropped to about half of the initial level. In cells from 2- and 10-day-old rats 10 microM dexamethasone specifically prevented this loss in NMT activity. Furthermore, this glucocorticoid treatment increased, in a dose-dependent manner, the total CA content by 50-100% over control levels without changes in the adrenaline (A) proportion or TH activity. In contrast, NGF did not affect NMT activities at all. In cells from 10-day-old rats 100 ng/ml NFG elevated TH activity and total CA content to about 160% of controls and did not change the proportion of A. This increase in total CA content was linear with the NGF dose and required greater than 5 ng/ml NGF. In chromaffin cells from 2-day-old rats 100 ng/ml NGF affected neither TH activity nor the total content, whereas it significantly reduced the proportion of A by about 25%.     

Methyltransferase inhibitors block NGF-regulated survival and protein phosphorylation in sympathetic neurons.

Nerve growth factor (NGF) and elevated K+ concentrations (35 mM) support the survival of the same population of chick embryonic sympathetic neurons. We have used methyltransferase inhibitors, which block protein methylation in intact cells, to investigate the mechanism(s) by which NGF and high K+ exert their effects. Methyltransferase inhibitors selectively blocked NGF-but not high K+-mediated survival of neurons. The ability of neurons, plated on laminin, to respond rapidly to NGF with neurite outgrowth was used to demonstrate that the blockade of the effects of NGF by methyltransferase inhibitors was reversible. At the molecular level, we studied the rapid decrease in phosphorylation of p70, a 70-kd phosphoprotein of sympathetic neurons regulated by both NGF and high K+. Methyltransferase inhibitors blocked the decrease in p70 phosphorylation induced by NGF but not that by high K+. We conclude that the early molecular events of NGF-mediated neuronal survival differ from those of high K+-mediated neuronal survival in that they involve protein methylation, whereas at a later step, possibly at the level of protein phosphorylation, the two pathways leading to survival of sympathetic neurons converge.     

Increase in plasma homocysteine associated with parallel increases in plasma S-adenosylhomocysteine and lymphocyte DNA hypomethylation

S-Adenosylmethionine and S-adenosylhomocysteine (SAH), as the substrate and product of essential cellular methyltransferase reactions, are important metabolic indicators of cellular methylation status. Chronic elevation of SAH, secondary to the homocysteine-mediated reversal of the SAH hydrolase reaction, reduces methylation of DNA, RNA, proteins, and phospholipids. High affinity binding of SAH to the active site of cellular methyltransferases results in product inhibition of the enzyme. Using a sensitive new high pressure liquid chromatography method with coulometric electrochemical detection, plasma SAH levels in healthy young women were found to increase linearly with mild elevation in homocysteine levels (r = 0.73; p < 0.001); however, S-adenosylmethionine levels were not affected. Plasma SAH levels were positively correlated with intracellular lymphocyte SAH levels (r = 0.81; p < 0.001) and also with lymphocyte DNA hypomethylation (r = 0.74, p < 0.001). These results suggest that chronic elevation in plasma homocysteine levels, such as those associated with nutritional deficiencies or genetic polymorphisms in the folate pathway, may have an indirect and negative effect on cellular methylation reactions through a concomitant increase in intracellular SAH levels.     

Nerve growth factor-induced expression of the GTP cyclohydrolase I gene via Ras/MEK pathway in PC12D cells

Neurotrophins are essential for the development and survival of the catecholaminergic neurons. GTP cyclohydrolase I (GCH) is the first and rate-limiting enzyme in the biosynthesis of 5,6,7,8-tertahydrobiopterin (BH4), the required cofactor for tyrosine hydroxylase. Previously, we reported that TH requires the Ras/mitogen-activated protein kinase kinase (MEK) pathway for its induction by nerve growth factor (NGF). Here, we examined intracellular signals required for NGF-induced expression of the GCH gene in PC12D cells. The activity of GCH was increased up to 5-fold after the NGF treatment, and the increase was repressed by pretreatment with U0126, an MEK1/2 inhibitor, but not with protein kinase A (PKA), phosphoinositide 3-kinase (PI3K), p38 mitogen-activated protein kinase (MAPK), and c-Jun NH2-terminal kinase (JNK) inhibitors. Induction of GCH mRNA by NGF was also abolished by pretreatment with U0126. The human GCH promoter activity was significantly enhanced by NGF treatment. Deletion analysis showed that the 465-bp 5'-flanking region is responsible for NGF-enhanced promoter activity. These data suggest that the Ras-MEK pathway is required for coordinate expression of the GCH and TH genes induced by neurotrophins.     

The Ras-ERK pathway is required for the induction of neuronal nitric oxide synthase in differentiating PC12 cells

We have studied the role of MAP kinase pathways in neuronal nitric oxide synthase (nNOS) induction during the differentiation of PC12 cells. In nerve growth factor (NGF)-treated PC12 cells, we find nNOS induced at RNA and protein levels, resulting in increased NOS activity. We note that neither nNOS mRNA, nNOS protein nor NOS activity is induced by NGF treatment in cells that have been infected with a dominant negative Ras adenovirus. We have also used drugs that block MAP kinase pathways and assessed their ability to inhibit nNOS induction. Even though U0126 and PD98059 are both MEK inhibitors, we find that U0126, but not PD98059, blocks induction of nNOS protein and NOS activity in NGF-treated PC12 cells. Also, the p38 kinase inhibitor, SB203580, does not block nNOS induction in our clone of PC12 cells. Since the JNK pathway is not activated in NGF-treated PC12 cells, we conclude that the Ras-ERK pathway and not the p38 or JNK pathway is required for nNOS induction in NGF-treated PC12 cells. We find that U0126 is much more effective than PD98059 in blocking the Ras-ERK pathway, thereby explaining the discrepancy in nNOS inhibition. We conclude that the Ras-ERK pathway is required for nNOS induction.     

Partial purification and characterization of S-adenosylhomocysteine hydrolase isolated from Saccharomyces cerevisiae

S-Adenosylhomocysteine (SAH) hydrolase was purified 25-fold from bakers' yeast by chemical methods and column chromatography. The purified enzyme could readily synthesize SAH from adenosine and homocysteine, but could hydrolyze only negligible amounts of SAH. The purified enzyme showed no activity towards S-adenosylmethionine, methylthioadenosine, or adenosine. Several nucleotides, sulfhydryl compounds, and ribose could not replace adenosine or homocysteine in the reaction mixture. SAH could be hydrolyzed by SAH hydrolase if commercial adenosine deaminase was included in the reaction mixture. Under these conditions l-homocysteine could act as a product inhibitor. A number of compounds structurally similar to adenosine and homocysteine were found to inhibit synthesis of SAH from adenosine and homocysteine. The strongest inhibitors were adenine, adenosine-3'-monophosphate, adenosine-2'-monophosphate, adenosine diphosphate, adenosine triphosphate, and adenosine-5'-monophosphate. The biosynthetic and hydrolytic activity of SAH hydrolase in yeast cell ghosts was similar to the activity of the enzyme in vitro.     

Glucocorticoids and Nerve Growth Factor Differentially Modulate Cell Adhesion Molecule L1 Expression in PC12 Cells

Abstract: The differential expression of the cell adhesion molecule L1 by chromaffin cells has recently been suggested to be responsible for the segregation of chromaffin cells into homotypic catecholaminergic groups in the adrenal gland. The present study was undertaken to test the hypothesis that glucocorticoids, which increase in the adrenal gland during development, could be responsible for the repression of L1 in adrenergic chromaffin cells. PC12 cells were used as the experimental model, and relative L1 protein and mRNA levels were examined after treating the cells with glucocorticoids or NGF. Analysis of western blots indicated that glucocorticoids decreased the L1 protein levels by one-half, whereas NGF increased L1 protein levels ∼2.3-fold. In addition, the glucocorticoids inhibited both the NGF induction of the neurite outgrowth and the increase in L1 expression. Analysis of the mRNA levels by PCR and northern blots indicated that glucocorticoids reduced the L1 mRNA, whereas NGF increased the level of L1 mRNA. Maximal inhibition of L1 expression was observed at concentrations of 10⁻⁷ M dexamethasone, and the decrease occurred during the second day of treatment. The effects of dibutyryl cyclic AMP and phorbol ester on the glucocorticoid and NGF regulation of L1 protein were also examined. This is the first report indicating that L1 expression can be down-regulated by glucocorticoids. The results support the hypothesis that during development the repression of L1 in adrenergic chromaffin cells may be, in part, linked to the increase in glucocorticoid levels in the adrenal gland.     

Expression of tyrosine hydroxylase is epigenetically regulated in neural stem cells

Tyrosine hydroxylase (TH) is the first and rate-limiting enzyme in the biosynthesis of catecholamines, and its expression is regulated in a developmental stage- and cell type-specific manner. Our previous work suggested that the genetic elements responsible for cell type-specific expression of TH were in the repressor region of the TH promoter between −2187 and −1232 bp. To investigate the molecular mechanisms underlying the specificity of TH expression, the DNA methylation patterns of the CpG islands in the repressor region of the TH promoter were examined in human neural stem cells (NSCs) and dopaminergic neuron-like cells. Using a bisulfite sequencing method, we found that the cytosine residues of CpG islands within the NRSE-R site were specifically methylated in NSCs, but not in SH-SY5Y neuroblastoma cells. In NSCs, CpG methylation correlated with reduced TH gene expression, and inhibition of DNA methylation with 5-azacytidine restored TH expression. Furthermore, methyl-CpG binding domain proteins (MBDs) bound to the highly methylated X-1 and X-2 regions of the TH gene in NSCs. Taken together, these results suggest that region-specific methylation and MBDs play important roles in TH gene regulation in NSCs.     

Modulation of anxiety through blockade of anandamide hydrolysis

The psychoactive constituent of cannabis, Delta(9)-tetrahydrocannabinol, produces in humans subjective responses mediated by CB1 cannabinoid receptors, indicating that endogenous cannabinoids may contribute to the control of emotion. But the variable effects of Delta(9)-tetrahydrocannabinol obscure the interpretation of these results and limit the therapeutic potential of direct cannabinoid agonists. An alternative approach may be to develop drugs that amplify the effects of endogenous cannabinoids by preventing their inactivation. Here we describe a class of potent, selective and systemically active inhibitors of fatty acid amide hydrolase, the enzyme responsible for the degradation of the endogenous cannabinoid anandamide. Like clinically used anti-anxiety drugs, in rats the inhibitors exhibit benzodiazepine-like properties in the elevated zero-maze test and suppress isolation-induced vocalizations. These effects are accompanied by augmented brain levels of anandamide and are prevented by CB1 receptor blockade. Our results indicate that anandamide participates in the modulation of emotional states and point to fatty acid amide hydrolase inhibition as an innovative approach to anti-anxiety therapy.