Search PubMed⌕ Search

Biomedical subjects

R J Wurtman

Publications and source records attributed to R J Wurtman.

At least 19 recordsLinked to original sources
PubMed2005

Uridine enhances neurite outgrowth in nerve growth factor-differentiated PC12 [corrected].

During rapid cell growth the availability of phospholipid precursors like cytidine triphosphate and diacylglycerol can become limiting in the formation of key membrane constituents like phosphatidylcholine. Uridine, a normal plasma constituent, can be converted to cytidine triphosphate in PC12 [corrected] cells and intact brain, and has been shown to produce a resulting increase in phosphatidylcholine synthesis. To determine whether treatments that elevate uridine availability also thereby augment membrane production, we exposed PC12 [corrected] cells which had been differentiated by nerve growth factor to various concentrations of uridine, and measured the numbers of neurites the cells produced. After 4 but not 2 days uridine significantly and dose-dependently increased the number of neurites per cell. This increase was accompanied by increases in neurite branching and in levels of the neurite proteins neurofilament M [corrected] and neurofilament 70. Uridine treatment also increased intracellular levels of cytidine triphosphate, which suggests that uridine may affect neurite outgrowth by enhancing phosphatidylcholine synthesis. Uridine may also stimulate neuritogenesis by a second mechanism, since the increase in neurite outgrowth was mimicked by exposing the cells to uridine triphosphate, and could be blocked by various drugs known to antagonize P2Y receptors (suramin; Reactive Blue 2; pyridoxal-phosphate-6-azophenyl-2',4' disulfonic acid). Treatment of the cells with uridine or uridine triphosphate stimulated their accumulation of inositol phosphates, and this effect was also blocked by pyridoxal-phosphate-6-azophenyl-2',4' disulfonic acid. Moreover, degradation of nucleotides by apyrase blocked the stimulatory effect of uridine on neuritogenesis. Taken together these data indicate that uridine can regulate the output of neurites from differentiating PC12 [corrected] cells, and suggest that it does so in two ways, i.e. both by acting through cytidine triphosphate as a precursor for phosphatidylcholine biosynthesis and through uridine triphosphate as an agonist for P2Y receptors.

Analysis of Variance↗
PubMed2001

Melatonin treatment for age-related insomnia.

Older people typically exhibit poor sleep efficiency and reduced nocturnal plasma melatonin levels. The daytime administration of oral melatonin to younger people, in doses that raise their plasma melatonin levels to the nocturnal range, can accelerate sleep onset. We examined the ability of similar, physiological doses to restore nighttime melatonin levels and sleep efficiency in insomniac subjects over 50 yr old. In a double-blind, placebo-controlled study, subjects who slept normally (n = 15) or exhibited actigraphically confirmed decreases in sleep efficiency (n = 15) received, in randomized order, a placebo and three melatonin doses (0.1, 0.3, and 3.0 mg) orally 30 min before bedtime for a week. Treatments were separated by 1-wk washout periods. Sleep data were obtained by polysomnography on the last three nights of each treatment period. The physiologic melatonin dose (0.3 mg) restored sleep efficiency (P < 0.0001), acting principally in the midthird of the night; it also elevated plasma melatonin levels (P < 0.0008) to normal. The pharmacologic dose (3.0 mg), like the lowest dose (0.1 mg), also improved sleep; however, it induced hypothermia and caused plasma melatonin to remain elevated into the daylight hours. Although control subjects, like insomniacs, had low melatonin levels, their sleep was unaffected by any melatonin dose.

Age Factors↗
PubMed2000

Effect of oral CDP-choline on plasma choline and uridine levels in humans.

Twelve mildly hypertensive but otherwise normal fasting subjects received each of four treatments in random order: CDP-choline (citicoline; 500, 2000, and 4000 mg) or a placebo orally at 8:00 a.m. on four different treatment days. Eleven plasma samples from each subject, obtained just prior to treatment (8:00 a.m.) and 1-12 hr thereafter, were assayed for choline, cytidine, and uridine. Fasting terminated at noon with consumption of a light lunch that contained about 100 mg choline. Plasma choline exhibited dose-related increases in peak values and areas under the curves (AUCs), remaining significantly elevated, after each of the three doses, for 5, 8, and 10 hr, respectively. Plasma uridine was elevated significantly for 5-6 hr after all three doses, increasing by as much as 70-90% after the 500 mg dose, and by 100-120% after the 2000 mg dose. No further increase was noted when the dose was raised from 2000 to 4000 mg. Plasma cytidine was not reliably detectable, since it was less than twice blank, or less than 100 nM, at all of the doses. Uridine is known to enter the brain and to be converted to UTP; moreover, we found that uridine was converted directly to CTP in neuron-derived PC-12 cells. Hence, it seems likely that the circulating substrates through which oral citicoline increases membrane phosphatide synthesis in the brains of humans involve uridine and choline, and not cytidine and choline as in rats.

Administration, Oral↗
PubMed2000

Characterization of phentermine and related compounds as monoamine oxidase (MAO) inhibitors.

Phentermine was shown in the 1970s to inhibit the metabolism of serotonin by monoamine oxidase (MAO), but never was labeled as an MAO inhibitor; hence, it was widely used in combination with fenfluramine, and continues to be used, in violation of their labels, with other serotonin uptake blockers. We examined the effects of phentermine and several other unlabeled MAO inhibitors on MAO activities in rat lung, brain, and liver, and also the interactions of such drugs when administered together. Rat tissues were assayed for MAO-A and -B, using serotonin and beta-phenylethylamine as substrates. Phentermine inhibited serotonin-metabolizing (MAO-A) activity in all three tissues with K(i) values of 85-88 microM. These potencies were similar to those of the antidepressant MAO inhibitors iproniazid and moclobemide. When phentermine was mixed with other unlabeled reversible MAO inhibitors (e.g. pseudoephedrine, ephedrine, norephedrine; estradiol benzoate), the degree of MAO inhibition was additive. The cardiac valvular lesions and primary pulmonary hypertension that have been reported to be associated with fenfluramine-phentermine use may have resulted from the intermittent concurrent blockage of both serotonin uptake and metabolism.

Animals↗
PubMed2000

Amyloid precursor protein and membrane phospholipids in primary cortical neurons increase with development, or after exposure to nerve growth factor or Abeta(1-40).

We examined the relationships between membrane phospholipid levels, the secretion and expression of the amyloid precursor protein (APP), and the responses of both to nerve growth factor (NGF), Abeta(1-40) or Abeta(40-1) in developing cortical neurons cultured from rat embryos. Neuronal membrane phospholipid levels per cell, and phosphatidylcholine, phosphatidylserine, phosphatidylinositol and phosphatidylethanolamine increased individually between the first and seventh days of culturing. The amounts of APP holoprotein and APP mRNAs in the cells, as well as the amounts of soluble APP (APPs) secreted by them, also increased during neuronal development in vitro. The increases in APPs exceeded the increases in APP which, in turn, exceed those in phospholipid levels. The levels of APP holoprotein, but not of phospholipids, increased when neurons were grown in a choline-free medium, suggesting that increases in APP are not sufficient to stimulate changes in membrane phospholipids. Treatment of neuron cultures for four days with NGF or Abeta(1-40), but not with Abeta(40-1), dose-dependently increased membrane phospholipids, tau and GAP-43, as well as APP holoprotein and secreted APPs. These results indicate that agents, like NGF or Abeta(1-40), which enhance membrane phospholipid levels may promote neurite formation, APP expression and APPs secretion in primary neuronal cultures.

Amyloid beta-Peptides↗
PubMed2000

Sibutramine, a serotonin uptake inhibitor, increases dopamine concentrations in rat striatal and hypothalamic extracellular fluid.

We measured, using microdialysis, the effects of sibutramine, given intraperitoneally, on brain dopamine and serotonin flux into striatal and hypothalamic dialysates of freely moving rats, and on the uptake of [(3)H]-DA into striatal synaptosomes. For microdialysis experiments, samples collected every 30 min were assayed by high-pressure liquid chromatography, in a single run. Administration of a low dose of sibutramine (2.0 mg/kg, i.p) had no effect on dopamine or serotonin concentrations in striatal dialysates but higher doses increased both: 5 mg/kg increased these concentrations to 196+/-24% (p<0.01) and 221+/-28% (p<0.01) of baseline, respectively; 10 mg/kg increased dopamine to 260+/-66% (p<0.01) and serotonin to 160+/-20% (p<0.05) of baseline. In hypothalamus, the 5 mg/kg sibutramine dose increased the dopamine concentration to 186+/-40% (p<0.05) and that of serotonin to 312+/-86% (p<0.01) of baseline, while the 10 mg/kg (i.p.) dose increased dopamine to 392+/-115% (p<0.01), and serotonin to 329+/-104% (p<0.01) of baseline. In vitro, sibutramine blocked [(3)H]-dopamine uptake into striatal synaptosomes, with an IC(50) value of 3.8 microM. These findings indicate that sibutramine has at least as great an effect on brain extracellular dopamine levels as on brain serotonin, and suggest that the drug's antiobesity action may result from the changes it produces in brain dopamine as well as serotonin metabolism.

Amphetamine↗
PubMed2000

Regulation of APP synthesis and secretion by neuroimmunophilin ligands and cyclooxygenase inhibitors.

We and others previously showed that both the synthesis of the amyloid precursor protein (APP) and its processing (i.e., to amyloidogenic A beta peptides; soluble nonamyloidogenic APPs; and other APP fragments) are regulated by neurotransmitters. Transmitters that elevate cellular cAMP levels (like norepinephrine and prostaglandins, which act on beta-adrenergic receptors and prostaglandin E2 receptors respectively) enhance APP synthesis and the formation of amyloidogenic APP holoprotein. Transmitters that stimulate phosphatidylinositol hydrolysis (by activating muscarinic m1 or m3 receptors, serotoninergic 5HT2a or 5HT2c receptors, or metabotropic glutamate receptors of subtypes 1 or 5) increase the conversion of APP to soluble APPs, and decrease the formation of A beta. These findings suggest that drugs that regulate the activity of neurotransmitter receptors might be useful in preventing the excessive formation of A beta or other amyloid precursors in Alzheimer's disease. We now show that neuroimmunophilin ligands (like cyclosporin A or FK-506) and nonsteroidal antiinflammatory agents (NSAIDs), including cyclooxygenase (COX)-2 inhibitors, can also prevent APP overexpression and the overproduction of amyloidogenic peptides. We observe that the enhancement of APP overexpression by prostaglandin E2 is inhibited by neuroimmunophilin ligands like cyclosporin A or FK-506 (tacrolimus). We also find that the NSAIDs, which reduce prostaglandin synthesis by inhibiting COX-1 and -2 enzymes, might also be expected to lower APP levels. Our present data confirm that these drugs, as well as drugs that selectively inhibit COX-2, reduce the levels of amyloidogenic APP holoprotein in cultured neurons or in cultured astrocytes. We previously showed that elevations in cAMP, perhaps generated in response to prostaglandins, can suppress APPs secretion. The NSAIDs and COX inhibitors also increased levels of soluble APPs in the media of cultured astrocytes and neurons, perhaps acting by inhibition of prostaglandin production. Since APP holoprotein can be amyloidogenic, while APPs may be neurotrophic, our findings suggest that some neuroimmunophilin ligands, NSAIDs and COX-2 inhibitors might suppress amyloid formation and enhance neuronal regeneration in Alzheimer's disease.

Alzheimer Disease↗
PubMed2000

Cytidine (5')diphosphocholine modulates dopamine K(+)-evoked release in striatum measured by microdialysis.

Experiments were performed to determine whether exogenous cytidine (5')diphosphocholine (CDP-choline) could modify the release of dopamine (DA) in the striatum. Rats were divided into two groups, receiving either a standard diet (UAR 004) or the same diet supplemented with CDP-choline (250 mg/kg day) for 28 days. On the last day the dialysis probes were inserted in the striatum, and DA, homovanillic acid (HVA), and 3,4-dihydroxyphenylacetic acid (DOPAC) efflux were measured in the dialysis stream basally and during K+ depolarization (80 mM K+). Basal DA, HVA, and DOPAC did not show any difference between treated and untreated groups. Depolarization with K+ increased DA levels by up to 3,000% in the control group and by up to 4,770% in the CDP-choline-treated group (p < 0.05), and reduced extracellular HVA and DOPAC concentration by up to 45 and 35%, respectively, both in the untreated and CDP-choline-treated groups. These results show that long-term treatment with CDP-choline increases the K+ induced release DN and suggest, in accordance with previous research, that by providing exogenous choline and cytidine, CDP-choline modulates dopaminergic transmission.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1999

Intracellular and cell-surface distribution of amyloid precursor protein in cortical astrocytes.

Amyloid peptides that aggregate to form plaques in Alzheimer's disease are derived from secretory processing of the amyloid precursor protein (APP). Transport of APP to the cell surface may be prerequisite for non-amyloidogenic APP processing and the secretion of soluble APP (APPs), while missorting or reinternalization of APP to intracellular compartments can promote amyloid formation. In cultured astrocytes, APP mRNA and holoprotein are increased by elevations in cAMP levels, and 8-Bromo-cAMP promotes process formation on these cells. We now report that treatment of cultured astrocytes with 8-Bromo-cAMP increased intracellular and cell surface APP in the soma and perinuclear region as detected by immunolabeling with monoclonal antibody 22C11 and polyclonal antibody Kunitz-type protease inhibitor (KPI) (against the N-terminus and KPI domain of APP, respectively) and led to intense but discontinuous labelling of APP on the surface of astrocytic processes. Northern and Western blot analyses confirmed that 8-Bromo-cAMP treatment of cultured astrocytes also increased APP mRNA and KPI-containing APP holoprotein, implying that the intense APP immunolabeling observed in 8-Bromo-cAMP treated astrocytes was not derived from truncated forms of APP (e.g., APPs), but reflected high levels of APP holoprotein containing intact amyloid peptides. Discontinuous cell surface staining in process-bearing astrocytes may be caused by miscompartmentalization of APP related to rearrangement of the cytoskeleton. Inasmuch as intracellular APP is not accessible for non-amyloidogenic processing, we suggest that the increased immunoreactivity of intracellular APP in process-bearing astrocytes may predispose the cells to increased amyloid production.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1999

Enhancement of free fatty acid incorporation into phospholipids by choline plus cytidine.

Cytidine and choline, present in cytidine 5'-diphosphate choline (CDP-choline), are major precursors of the phosphatidylcholine found in cell membranes and important regulatory elements in phosphatide biosynthesis. Administration of CDP-choline to rats increases blood and brain cytidine and choline levels; this enhances the production of endogenous CDP-choline which then combines with fatty acids (as diacylglycerol), to yield phosphatidylcholine. We examined the effect of providing cytidine and choline on incorporation of free fatty acids into phosphatidylcholine and other major phospholipids in PC12 cells. Addition of equimolar cytidine and choline (100-500 microM) to [3H]-arachidonic acid (50 microM, 0.2 microCi, bound to bovine serum albumin) dose-dependently increased the accumulations of [3H]-phosphatidylcholine (PtdCho), [3H]-phosphatidylinositol (PtdIno) and [3H]-phosphatidylethanolamine (PtdEtn) (by up to 27+/-3%, 16+/-3% and 11+/-3%, respectively, means+/-S.E.M.). This effect was seen with 8-18 h of incubation. The incorporation of [3H]-oleic acid into [3H]-PtdCho was even more enhanced (by up to 42+/-3%) as were the incorporations of [14C]-choline and [3H]-glycerol. The effects of choline and cytidine were enhanced by 12-O-tetradecanoylphorbol-13-acetate (TPA, 1 microM), which activates CTP:phosphocholine cytidylyltransferase (CT) and facilitates choline uptake. Replacing choline by ethanolamine also enhanced the incorporation of [3H]-arachidonic acid into [3H]-PtdEtn, [3H]-PtdIno and [3H]-PtdCho. Arachidonic acid (10-200 microM) alone failed to affect the incorporation of [14C]-choline into phosphatidylcholine. We suggest that the increases in phospholipid synthesis caused by concurrent cytidine and choline supplementation enhance the incorporation of arachidonic acid and certain other fatty acids into the major glycerophospholipids. Removing these fatty acids as source of potentially toxic oxidation products could contribute to the beneficial effects of CDP-choline in treating stroke or other brain damage.

Animals↗
PubMed1999

Mechanisms whereby nerve growth factor increases diacylglycerol levels in differentiating PC12 cells.

We previously showed indirectly that the increase in diacylglycerol (DAG) levels caused by exposing differentiating PC12 cells to nerve growth factor (NGF) must derive mainly from de novo synthesis and, to a lesser and transient extent, from the hydrolysis of [3H]phosphatidylinositol (PI). To explore further the biochemical mechanisms of this increase, we measured, in PC12 cells, DAG synthesis from glycerol or various fatty acids; its liberation from phosphatidylcholine (PC); and the activities of various enzymes involved in DAG production and metabolism. Among cells exposed to NGF (0-116 h), the labeling of DAG from [3H]glycerol peaked earlier than that of [3H]PC, and the specific radioactivity of [3H]glycerol-labeled DAG was much higher than those of the [3H]phospholipids, indicating that [3H]DAG synthesis precedes [3H]phospholipid synthesis. NGF treatment also increased (by 50-330%) the incorporation of monounsaturated ([3H]oleic acid) and polyunsaturated ([14C]linoleic acid or [3H]arachidonic acid) fatty acids into DAG, and, by 15-70%, into PC. NGF treatment increased the activities of long chain acyl-CoA synthetases (LCASs), including oleoyl-CoA synthetase and arachidonoyl-CoA synthetase, by 150-580% over control, but cholinephosphotransferase activity rose by only 60%, suggesting that the synthesis of DAG in the cells was increased to a greater extent than its utilization. NGF did not promote the breakdown of newly formed [3H]PC to [3H]DAG, nor did it consistently affect the activities of phospholipase C or D. NGF did increase phospholipase A2 activity, however the hydrolysis catalyzed by this enzyme does not liberate DAG. Hence the major source of the increased DAG levels in PC12 cells exposed to NGF appears to be enhanced de novo DAG synthesis, probably initiated by the activation of LCASs, rather than the breakdown of PC or PI.

Animals↗
PubMed1999

Prostaglandin E2 stimulates amyloid precursor protein gene expression: inhibition by immunosuppressants.

Amyloid plaques that accumulate in the brains of patients with Alzheimer's disease (AD) are primarily composed of aggregates of amyloid peptides that are derived from the amyloid precursor protein (APP). Overexpression of APP in cell cultures increases the formation of amyloidogenic peptides and causes neurodegeneration and cognitive dysfunction in transgenic mice. We now report that activation of prostaglandin E2 (PGE2) receptors increases cAMP formation and stimulates overexpression of APP mRNA and holoprotein in primary cultures of cortical astrocytes. Levels of glial fibrillary acidic protein were also increased by PGE2 treatment, suggesting that these cultured astrocytes resemble reactive astrocytes found in vivo. The stimulation by PGE2 of APP synthesis was mimicked or blocked by activators or inhibitors, respectively, of protein kinase A. Actinomycin D or cycloheximide also inhibited the increase in APP holoprotein stimulated by PGE2. Treatment of astrocytes with 8-Bromo-cAMP or forskolin for 24 hr also stimulated APP overexpression in cultured astrocytes. The immunosuppressants cyclosporin A and FK-506 inhibited the increase in APP mRNA and holoprotein levels caused by PGE2 or by other treatments that elevated cellular cAMP levels; the inhibitory effect of FK-506 but not of cyclosporin A was attenuated by rapamycin. These results suggest that prostaglandins produced by brain injury or inflammation can activate APP transcription in astrocytes and that immunosuppressants may be used to prevent APP overexpression and possibly the pathophysiological processes underlying AD.

Amyloid beta-Protein Precursor↗
PubMed1999

Heterogeneous long chain acyl-CoA synthetases control distribution of individual fatty acids in newly-formed glycerolipids of neuronal cells undergoing neurite outgrowth.

Using PC12 cells undergoing neurite outgrowth, we studied the activation of various fatty acids, of different chain lengths and degrees of saturation, by long chain acyl-CoA synthetases (LCASs). Cells treated with nerve growth factor (NGF) were labeled with [3H]glycerol, [3H]oleic acid (OA) or [3H]arachidonic acid (AA) in the presence of other unlabeled fatty acids of endogenous or exogenous origin. Triacsin C (4.8 microM), an inhibitor of acyl-CoA synthetase, decreased the incorporation of exogenous [3H]OA into glycerolipids by 30-90%, and increased by about 60% the accumulation of free [3H]OA in the cells. However it did not affect the incorporation of endogenous fatty acids nor of exogenous [3H]AA into phospholipids, suggesting that LCASs which activate exogenous AA and at least some endogenous fatty acids are relatively insensitive to this drug. Activities of the LCAS that is specific for AA (ACS), or of the non-specific LCAS which activates OA and other fatty acids (OCS), were much higher in microsomal and cytoplasmic fractions than in mitochondria or nuclei. The Vmax and Km values of ACS and OCS in microsomes were 12 and 0.7 nmol/min/mg protein and 70 and 37 microM, respectively; and in cytoplasm, 6 and 0.6 nmol/min/mg protein and 38 and 60 microM, respectively. Triacsin C (2-33 microM) did not affect ACS activity in microsomal or cytoplasmal fractions, but inhibited OCS activities dose-dependently and competitively: IC50 and apparent Ki values were 13.5 microM and 14 microM in microsomes, and 3.8 microM and 4 microM in cytoplasm. NGF stimulated the activities of the LCASs, and, consistently, the incorporation of the various fatty acids into glycerolipids. These data indicate that LCASs are heterogeneous with respect to their intracellular locations, substrate specificities, kinetic characteristics and sensitivities to triacsin C; and that this heterogeneity affects the extents to which individual fatty acids are utilized to form glycerolipids.

Animals↗
PubMed1998

Effects of fenfluramine and phentermine (fen-phen) on dopamine and serotonin release in rat striatum: in vivo microdialysis study in conscious animals.

We measured the effects of acute or chronic administration of fenfluramine and phentermine, alone or in combination, on brain dopamine and serotonin release into striatal dialysates of freely moving rats. Samples collected every 30 min were assayed in a single run by high-pressure liquid chromatography. Acute or chronic administration of fenfluramine (1 mg/kg, i.p.) did not significantly change dopamine concentrations in rat striatal dialysates, but increased those of serotonin by 182% (acute) and 124% (chronic). Phentermine (2 mg/kg, i.p.), on the other hand, significantly increased dopamine concentrations by 52% (acute) and 80% (chronic) without affecting those of serotonin. Administration of the drugs in combination (fenfluramine 1 mg/kg and phentermine 2 mg/kg) amplified the effects of each, increasing striatal dopamine concentrations by 209% (acute) and serotonin concentrations by 330% (acute) and 299% (chronic).

Analysis of Variance↗
PubMed1998

Nerve growth factor stimulates diacylglycerol de novo synthesis and phosphatidylinositol hydrolysis in pheochromocytoma cells.

Induction of neurite outgrowth by treating pheochromocytoma cells (PC12 cells) with nerve growth factor (NGF) is associated with major increases in cellular levels of diacylglycerol (DAG), an essential and probably limiting precursor in phosphatidylcholine (PC) and phosphatidylethanolamine (PE) syntheses. To identify the sources of this DAG we examined the effects of NGF treatment on the conversion of [3H]oleic acid (OA) or [3H]glycerol to [3H]glycerolipids, and the turnover of these products in PC12 cells. In kinetic studies on [3H]OA incorporation, most of the radioactivity in the cells initially was free [3H]OA; then it appeared predominantly as [3H]DAG and, eventually, as large amounts of [3H]phospholipids (PLs). In NGF pre-treated cells, the increases in the levels of [3H]DAG (which were most prominent) and PLs were similar to those in unlabeled DAG and PLs. These effects of NGF could be partially blocked by an inhibitor (triacsin C) of long chain acyl-CoA synthetase. NGF pre-treatment also significantly enhanced the incorporation of [3H]glycerol into lipids, a pathway for de novo synthesis of glycerolipids. In studies on the degradation of [3H]OA-labeled lipids, the disappearance of [3H]OA-labeled neutral lipids exhibited an initial rapid phase and a subsequent stable phase. NGF treatment transiently promoted the hydrolysis of [3H]PI to [3H]DAG. These data suggest that the increases in DAG levels observed in PC12 cells exposed to NGF derive mainly from de novo synthesis and, to a lesser and transient extent, from the hydrolysis of [3H]PI.

Animals↗