Measurement of muscle protein synthesis by positron emission tomography with L-[methyl-11C]methionine.

Positron emission tomography (PET) with L-[methyl-11C]methionine was explored as an in vivo, noninvasive, quantitative method for measuring the protein synthesis rate (PSR) in paraspinal and hind limb muscles of anesthetized dogs. Approximately 25 mCi (1 Ci = 37 GBq) of L-[methyl-11C]methionine was injected intravenously, and serial images and arterial blood samples were acquired over 90 min. Data analysis was performed by fitting tissue- and metabolite-corrected arterial blood time-activity curves to a three-compartment model and assuming insignificant transamination and transmethylation in this tissue. PSR was calculated from fitted parameter values and plasma methionine concentrations. PSRs measured by PET were compared with arterio-venous (A-V) difference measurements across the hind limb during primed constant infusion (5-6 h) of L-[1-13C, methyl-2H3]methionine. Results of PET measurements demonstrated similar PSRs for paraspinal and hind limb muscles: 0.172 +/- 0.062 vs. 0.208 +/- 0.048 nmol-1.min-1.(g of muscle)-1 (P = not significant). PSR determined by the stable isotope technique was 0.27 +/- 0.050 nmol-1.min-1.(g of leg tissue)-1 (P < 0.07 from PET) and indicated that the contribution of transmethylation to total hind limb methionine utilization was approximately 10%. High levels of L-[methyl-11C]methionine utilization by bone marrow were observed. We conclude that muscle PSR can be measured in vivo by PET and that this approach offers promise for application in human metabolic studies.

Ca(2+)-independent reduction of N-methyl-D-aspartate channel activity by protein tyrosine phosphatase.

Regulation of ion channel function by intracellular processes is fundamental for controlling synaptic signaling and integration in the nervous system. Currents mediated by N-methyl-D-aspartate (NMDA) receptors decline during whole-cell recordings and this may be prevented by ATP. We show here that phosphorylation is necessary to maintain NMDA currents and that the decline is not dependent upon Ca2+. A protein tyrosine phosphatase or a peptide inhibitor of protein tyrosine kinase applied intracellularly caused a decrease in NMDA currents even when ATP was included. On the other hand, pretreating the neurons with a membrane-permeant tyrosine kinase inhibitor occluded the decline in NMDA currents when ATP was omitted. In inside-out patches, applying a protein tyrosine phosphatase to the cytoplasmic face of the patch caused a decrease in probability of opening of NMDA channels. Conversely, open probability was increased by a protein tyrosine phosphatase inhibitor. These results indicate that NMDA channel activity is reduced by a protein tyrosine phosphatase associated with the channel complex.

Regulation of N-methyl-D-aspartate-induced toxicity in the neostriatum: a role for metabotropic glutamate receptors?

Glutamate release activates multiple receptors that interact with each other and thus determine the response of the cell. Exploring these interactions is critical to developing an understanding of the functional consequences of synaptic transmission. Activation of metabotropic glutamate receptors (mGluRs) inhibits N-methyl-D-aspartate (NMDA)-evoked responses measured electrophysiologically in neostriatal slices. The present study examines the functional consequences of this regulation using infrared differential interference contrast videomicroscopy to measure and characterize glutamate receptor-induced cell swelling in a neostriatal brain slice preparation. This swelling is, in many cases, a prelude to necrotic cell death and the dye trypan blue was used to confirm that swelling can result in the death of neostriatal cells. Activation of mGluRs by the agonist 1-aminocyclopentane-1,3-dicarboxylic acid (tACPD) inhibited NMDA but not amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate-induced swelling. This regulation was cell-type specific as tACPD did not alter NMDA-induced swelling in pyramidal cells of the hippocampus. Importantly, these findings could be extended to in vivo preparations. Pretreatment with tACPD limited the size of lesions and associated behavioral deficits induced by intrastriatal administration of the NMDA receptor agonist quinolinic acid.

Extending the chemistry that supports genetic information transfer in vivo: phosphorothioate DNA, phosphorothioate RNA, 2'-O-methyl RNA, and methylphosphonate DNA.

DNA and RNA are the polynucleotides known to carry genetic information in life. Chemical variants of DNA and RNA backbones have been used in structure-function and biosynthesis studies in vitro, and in antisense pharmacology, where their properties of nuclease resistance and enhanced cellular uptake are important. This study addressed the question of whether the base(s) attached to artificial backbones encodes genetic information that can be transferred in vivo. Oligonucleotides containing chemical variants of DNA or RNA were used as primers for site-specific mutagenesis of bacteriophage f1. Progeny phage were scored both genetically and physically for the inheritance of information originally encoded by bases attached to the nonstandard backbones. Four artificial backbone chemistries were tested: phosphorothioate DNA, phosphorothioate RNA, 2'-O-methyl RNA and methylphosphonate DNA. All four were found capable of faithful information transfer from their attached bases when one or three artificial positions were flanked by normal DNA. Among oligonucleotides composed entirely of nonstandard backbones, only phosphorothioate DNA supported genetic information transfer in vivo.

Cloning and characterization of ERG25, the Saccharomyces cerevisiae gene encoding C-4 sterol methyl oxidase.

We have cloned the Saccharomyces cerevisiae C-4 sterol methyl oxidase ERG25 gene. The sterol methyl oxidase performs the first of three enzymic steps required to remove the two C-4 methyl groups leading to cholesterol (animal), ergosterol (fungal), and stigmasterol (plant) biosynthesis. An ergosterol auxotroph, erg25, which fails to demethylate and concomitantly accumulates 4,4-dimethylzy-mosterol, was isolated after mutagenesis. A complementing clone consisting of a 1.35-kb Dra I fragment encoded a 309-amino acid polypeptide (calculated molecular mass, 36.48 kDa). The amino acid sequence shows a C-terminal endoplasmic reticulum retrieval signal KKXX and three histidine-rich clusters found in eukaryotic membrane desaturases and in a bacterial alkane hydroxylase and xylene monooxygenase. The sterol profile of an ERG25 disruptant was consistent with the erg25 allele obtained by mutagenesis.

Latent N-methyl-D-aspartate receptors in the recurrent excitatory pathway between hippocampal CA1 pyramidal neurons: Ca(2+)-dependent activation by blocking A1 adenosine receptors.

When performed at increased external [Ca2+]/[Mg2+] ratio (2.5 mM/0.5 mM), temporary block of A1 adenosine receptors in hippocampus [by 8-cyclopentyltheophylline (CPT)] leads to a dramatic and irreversible change in the excitatory postsynaptic current (EPSC) evoked by Schaffer collateral/commissural (SCC) stimulation and recorded by in situ patch clamp in CA1 pyramidal neurons. The duration of the EPSC becomes stimulus dependent, increasing with increase in stimulus strength. The later occurring component of the EPSC is carried through N-methyl-D-aspartate (NMDA) receptor-operated channels but disappears under either the NMDA antagonist 2-amino-5-phosphonovaleric acid (APV) or the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). These findings indicate that the late component of the SCC-evoked EPSC is polysynaptic: predominantly non-NMDA receptor-mediated SCC inputs excite CA1 neurons that recurrently excite each other by predominantly NDMA receptor-mediated synapses. These recurrent connections are normally silent but become active after CPT treatment, leading to enhancement of the late component of the EPSC. The activity of these connections is maintained for at least 2 hr after CPT removal. When all functional NMDA receptors are blocked by dizocilpine maleate (MK-801), subsequent application of CPT leads to a partial reappearance of NMDA receptor-mediated EPSCs evoked by SCC stimulation, indicating that latent NMDA receptors are recruited. Altogether, these findings indicate the existence of a powerful system of NMDA receptor-mediated synaptic contacts in SCC input to hippocampal CA1 pyramidal neurons and probably also in reciprocal connections between these neurons, which in the usual preparation are kept latent by activity of A1 receptors.

Coupling the phosphotransferase system and the methyl-accepting chemotaxis protein-dependent chemotaxis signaling pathways of Escherichia coli.

Chemotactic responses in Escherichia coli are typically mediated by transmembrane receptors that monitor chemoeffector levels with periplasmic binding domains and communicate with the flagellar motors through two cytoplasmic proteins, CheA and CheY. CheA autophosphorylates and then donates its phosphate to CheY, which in turn controls flagellar rotation. E. coli also exhibits chemotactic responses to substrates that are transported by the phosphoenolpyruvate (PEP)-dependent carbohydrate phosphotransferase system (PTS). Unlike conventional chemoreception, PTS substrates are sensed during their uptake and concomitant phosphorylation by the cell. The phosphoryl groups are transferred from PEP to the carbohydrates through two common intermediates, enzyme I (EI) and phosphohistidine carrier protein (HPr), and then to sugar-specific enzymes II. We found that in mutant strains HPr-like proteins could substitute for HPr in transport but did not mediate chemotactic signaling. In in vitro assays, these proteins exhibited reduced phosphotransfer rates from EI, indicating that the phosphorylation state of EI might link the PTS phospho-relay to the flagellar signaling pathway. Tests with purified proteins revealed that unphosphorylated EI inhibited CheA autophosphorylation, whereas phosphorylated EI did not. These findings suggest the following model for signal transduction in PTS-dependent chemotaxis. During uptake of a PTS carbohydrate, EI is dephosphorylated more rapidly by HPr than it is phosphorylated at the expense of PEP. Consequently, unphosphorylated EI builds up and inhibits CheA autophosphorylation. This slows the flow of phosphates to CheY, eliciting an up-gradient swimming response by the cell.

Desensitization of N-methyl-D-aspartate receptors: a problem of interpretation.

The phenomenon of desensitization is universal, but its mechanism is still ill-understood and controversial. A recently published study [Lin, F. & Stevens, C. F. (1994) J. Neurosci, 14, 2153-2160] attempted to cast light on the mechanism of desensitization of N-methyl-D-aspartate (NMDA) receptors, in particular the vexed question of whether the channel must open before it can desensitize. During the desensitizing preexposure to agonist in those experiments, more desensitization was produced when channel openings were observed than when no openings were observed. The conclusion that "desensitization occurs more rapidly from the open state" unfortunately was based on a stochastic fallacy, and we present here a theoretical treatment and illustration showing that the observed behavior is predicted by a simple mechanism in which desensitization can occur only from a shut state.

Intracellular polyamines mediate inward rectification of Ca(2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors.

alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors that lack the glutamate receptor GluR2 subunit are Ca(2+)-permeable and exhibit inwardly rectifying current responses to kainate and AMPA. A proportion of cultured rat hippocampal neurons show similar Ca(2+)-permeable inwardly rectifying AMPA receptor currents. Inward rectification in these neurons was lost with intracellular dialysis and was not present in excised outside-out patches but was maintained in perforated-patch whole-cell recordings, suggesting that a diffusible cytoplasmic factor may be responsible for rectification. Inclusion of the naturally occurring polyamines spermine and spermidine in the recording pipette prevented loss of rectification in both whole-cell and excised-patch recordings; Mg2+ and putrescine were without effect. Inward rectification of Ca(2+)-permeable AMPA receptors may reflect voltage-dependent channel block by intracellular polyamines.

7-Chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine S,S-dioxide (IDRA 21), a congener of aniracetam, potently abates pharmacologically induced cognitive impairments in patas monkeys.

We report here on the ability of IDRA 21 and aniracetam, two negative allosteric modulators of glutamate-induced DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor desensitization, to attenuate alprazolam-induced learning deficit in patas monkeys working in a complex behavioral task. In one component of a multiple schedule (repeated acquisition or "learning"), patas monkeys acquired a different four-response chain each session by responding sequentially on three keys in the presence of four discriminative stimuli (geometric forms or numerals). In the other component (performance) the four-response chain was the same each session. The response chain in each component was maintained by food presentation under a fixed-ratio schedule. When alprazolam (0.1 or 0.32 mg/kg p.o.) was administered alone, this full allosteric modulator of gamma-aminobutyric acid type A (GABAA) receptors produced large decreases in the response rate and accuracy in the learning component of the task. IDRA 21 (3 or 5.6 mg/kg p.o.) and aniracetam (30 mg/kg p.o.) administered 60 min before alprazolam, having no effect when given alone, antagonized the large disruptive effects of alprazolam on learning. From dose-response studies, it can be estimated that IDRA 21 is approximately 10-fold more potent than aniracetam in antagonizing alprazolam-induced learning deficit. We conclude that IDRA 21, a chemically unrelated pharmacological congener of aniracetam, improves learning deficit induced in patas monkeys by the increase of GABAergic tone elicited by alprazolam. Very likely IDRA 21 exerts its behavioral effects by antagonizing AMPA receptor desensitization.

Molecular design of the N-methyl-D-aspartate receptor binding site for phencyclidine and dizolcipine.

The N-methyl-D-aspartate receptor (NMDAR), a pivotal entity for synaptic plasticity and excitotoxicity in the brain, is a target of psychotomimetic drugs such as phencyclidine (PCP) and dizolcipine (MK-801). In contrast, a related glutamate receptor, the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate/kainate receptor GluR1, is weakly sensitive to these drugs. Three point mutations on GluR1, mimicking homologous residues on the NMDAR, confer the PCP and MK-801 blockade properties that are characteristic of the NMDAR--namely, high potency, voltage dependence, and use dependence. The molecular determinants that specify the PCP block appear confined to the putative M2 transmembrane segment, whereas the sensitivity to MK-801 requires an interplay between residues from M2 and M3. Given the plausible involvement of the NMDAR in the etiology of several neurodegenerative diseases and in excitotoxic neuronal cell death, tailored glutamate receptors with specific properties may be models for designing and screening new drugs targeted to prevent glutamate-mediated neural damage.

Rapid acquisition of dendritic spines by visual thalamic neurons after blockade of N-methyl-D-aspartate receptors.

N-Methyl-D-aspartate (NMDA) receptors play an important role in the development of retinal axon arbors in the mammalian lateral geniculate nucleus (LGN). We investigated whether blockade of NMDA receptors in vivo or in vitro affects the dendritic development of LGN neurons during the period that retinogeniculate axons segregate into on-center and off-center sublaminae. Osmotic minipumps containing either the NMDA receptor antagonist D-2-amino-5-phosphonovaleric acid (D-APV) or saline were implanted in ferret kits at postnatal day 14. After 1 week, LGN neurons were intracellularly injected with Lucifer yellow. Infusion of D-APV in vivo led to an increase in the number of branch points and in the density of dendritic spines compared with age-matched normal or saline-treated animals. To examine the time course of spine formation, crystals of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate were placed in the LGN in brain slices from 14- to 18-day-old ferrets. Labeled LGN cell dendrites were imaged on-line in living slices by confocal microscopy, with slices maintained either in normal perfusion medium or with the addition of D-APV or NMDA to the medium. Addition of D-APV in vitro at doses specific for blocking NMDA receptors led to a > 6-fold net increase in spine density compared with control or NMDA-treated slices. Spines appeared within a few hours of NMDA receptor blockade, indicating a rapid local response by LGN cells in the absence of NMDA receptor activation. Thus, activity-dependent structural changes in postsynaptic cells act together with changes in presynaptic arbors to shape projection patterns and specific retinogeniculate connections.

Apoptosis and necrosis: two distinct events induced, respectively, by mild and intense insults with N-methyl-D-aspartate or nitric oxide/superoxide in cortical cell cultures.

N-Methyl-D-aspartate (NMDA) receptor-mediated neurotoxicity may depend, in part, on the generation of nitric oxide (NO.) and superoxide anion (O2.-), which react to form peroxynitrite (OONO-). This form of neurotoxicity is thought to contribute to a final common pathway of injury in a wide variety of acute and chronic neurologic disorders, including focal ischemia, trauma, epilepsy, Huntington disease, Alzheimer disease, amyotrophic lateral scelerosis, AIDS dementia, and other neurodegenerative diseases. Here, we report that exposure of cortical neurons to relatively short durations or low concentrations of NMDA, S-nitrosocysteine, or 3-morpholinosydnonimine, which generate low levels of peroxynitrite, induces a delayed form of neurotoxicity predominated by apoptotic features. Pretreatment with superoxide dismutase and catalase to scavenge O2.- partially prevents the apoptotic process triggered by S-nitrosocysteine or 3-morpholinosydnonimine. In contrast, intense exposure to high concentrations of NMDA or peroxynitrite induces necrotic cell damage characterized by acute swelling and lysis, which cannot be ameliorated by superoxide dismutase and catalase. Thus, depending on the intensity of the initial insult, NMDA or nitric oxide/superoxide can result in either apoptotic or necrotic neuronal cell damage.

5-Methyl-2'-deoxycytidine in single-stranded DNA can act in cis to signal de novo DNA methylation.

Methylation of cytosine residues in DNA plays an important role in regulating gene expression during vertebrate embryonic development. Conversely, disruption of normal patterns of methylation is common in tumors and occurs early in progression of some human cancers. In vertebrates, it appears that the same DNA methyltransferase maintains preexisting patterns of methylation during DNA replication and carries out de novo methylation to create new methylation patterns. There are several indications that inherent signals in DNA structure can act in vivo to initiate or block de novo methylation in adjacent DNA regions. To identify sequences capable of enhancing de novo methylation of DNA in vitro, we designed a series of oligodeoxyribonucleotide substrates with substrate cytosine residues in different sequence contexts. We obtained evidence that some 5-methylcytosine residues in these single-stranded DNAs can stimulate de novo methylation of adjacent sites by murine DNA 5-cytosine methyltransferase as effectively as 5-methylcytosine residues in double-stranded DNA stimulate maintenance methylation. This suggests that double-stranded DNA may not be the primary natural substrate for de novo methylation and that looped single-stranded structures formed during the normal course of DNA replication or repair serve as "nucleation" sites for de novo methylation of adjacent DNA regions.

Neurosteroids, via sigma receptors, modulate the [3H]norepinephrine release evoked by N-methyl-D-aspartate in the rat hippocampus.

N-Methyl-D-aspartate (NMDA, 200 microM) evokes the release of [3H]norepinephrine ([3H]NE) from preloaded hippocampal slices. This effect is potentiated by dehydroepiandrosterone sulfate (DHEA S), whereas it is inhibited by pregnenolone sulfate (PREG S) and the high-affinity sigma inverse agonist 1,3-di(2-tolyl)guanidine, at concentrations of > or = 100 nM. Neither 3 alpha-hydroxy-5 alpha-pregnan-20-one nor its sulfate ester modified NMDA-evoked [3H]NE overflow. The sigma antagonists haloperidol and 1-[2-(3,4-dichlorophenyl)-ethyl]-4-methylpiperazine, although inactive by themselves, completely prevented the effects of DHEA S, PREG S, and 1,3-di(2-tolyl)guanidine on NMDA-evoked [3H]NE release. Progesterone (100 nM) mimicked the antagonistic effect of haloperidol and 1-[2-(3,4-dichlorophenyl)ethyl]-4-methyl-piperazine. These results indicate that the tested steroid sulfate esters differentially affected the NMDA response in vitro and suggest that DHEA S acts as a sigma agonist, that PREG S acts as a sigma inverse agonist, and that progesterone may act as a sigma antagonist. Pertussis toxin, which inactivates the Gi/o types of guanine nucleotide-binding protein (Gi/o protein) function, suppresses both effects of DHEA S and PREG S. Since sigma 1 but not sigma 2 receptors are coupled to Gi/o proteins, the present results suggest that DHEA S and PREG S control the NMDA response via sigma 1 receptors.