Characterization of electrically evoked [3H]-D-aspartate release from hippocampal slices.

Electrical stimulation has certain advantages over chemical stimulation methods for the study of neurotransmitter release in brain slices. However, measuring detectable quantities of electrically evoked release of endogenous or radiolabeled markers of excitatory amino acid neurotransmitters has required current intensities or frequencies much higher than those usually required to study other transmitter systems. We demonstrate here that [3H]-D-aspartate (D-ASP) release can be detected from hippocampal slices at lower stimulation intensities in the presence of a glutamate reuptake inhibitor. Subsequently, we optimized the electrical stimulus parameters for characterizing electrically evoked D-ASP release. Under the experimental conditions described, greater than 90% of electrically evoked D-ASP release is calcium-dependent. Evoked D-ASP release is markedly reduced by pre-treating slices with the synaptic vesicle toxin bafilomycin A1 (BAF A1) or in the presence of 10-mM magnesium. Evoked D-ASP release is also reduced to variable degrees by N- and P/Q type voltage-sensitive calcium channel antagonists. Neither spontaneous efflux nor evoked D-ASP release were affected by NMDA, AMPA or group I metabotropic glutamate receptor (mGluR) antagonists. Evoked D-ASP release was reduced in the presence of an adenosine A1 receptor agonist and potentiated by treatment with a group I mGluR5 agonist. Evoked [3H]-D-ASP release was similar in magnitude to evoked [3H]-L-glutamate (L-GLU) release. Finally, in separate experiments using the same electrical stimulus parameters, more than 90% of electrically evoked endogenous L-GLU release was calcium dependent, a pattern similar to that observed for evoked [3H]-D-ASP release. Taken together, these results indicate that electrically evoked [3H]-D-ASP release mimics evoked glutamate release in brain slices under the experimental conditions employed in these studies.

d-Amphetamine attenuates decreased cerebral glucose utilization after unilateral sensorimotor cortex contusion in rats.

Unilateral contusion injury to the sensorimotor cortex causes, among other symptoms, a transient contralateral hindlimb hemiparesis in rats. A single i.p. 2 mg/kg dose of d-amphetamine (d-AMPH) 24 h after injury accelerates spontaneous recovery from this particular deficit. The mechanism(s) of spontaneous and d-AMPH enhanced recovery are unknown but alleviation of a neuronal depression has been proposed. This quantitative CMRglu study was designed to determine effects of cortical contusion injury and d-AMPH on CMRglu in cortical and subcortical structures. At 2 days after injury, CMRglu was significantly reduced compared to sham-operated controls only in structures ipsilateral to contusion. Affected structures included the caudate putamen, medial geniculate nucleus, lateral geniculate nucleus and the parietal cortex immediately posterior to injury. By 6 days post-contusion, the hypometabolism partially reversed in all structures. A single low dose of d-AMPH significantly alleviated the post-traumatic CMRglu reduction at 2 days after injury. Importantly, while this alleviation was not significant for any single structure, the main effect of treatment was highly significant. d-AMPH increased CMRglu at 2 days post-injury by 18-33% compared to contused/saline-treated rats. These results suggest that alleviation of neuronal metabolic depression may contribute to spontaneous and d-AMPH enhanced recovery.

Temporally changing patterns of hippocampal cerebral glucose utilization following sensorimotor cortical contusion in rats.

Unilateral sensorimotor cortical contusion significantly decreased ipsilateral hippocampal cerebral metabolic rates of glucose utilization (CMRglu) compared to sham controls at 2 and 16 days post injury. In contrast, hippocampal CMRglu was transiently increased at 6 days post injury. Both the increased and decreased CMRglu were predominantly localized to the hippocampal CA3 subfield ipsilateral to injury and were significantly different from sham controls in the dorsal but not ventral hippocampal formation.

Association of tissue-specific changes in translation elongation after cyclosporin with changes in elongation factor 2 phosphorylation.

In studies of cyclosporin (CsA) toxicity in Sprague-Dawley rats, CsA administered in vivo produced tissue-specific, dose-dependent changes in microsomal translation throughout the bodies of the animals. The most pronounced translation inhibition was in microsomes from the kidney, the organ in which dose-limiting CsA toxicity occurs. In contrast, translation was stimulated in microsomes from the liver. CsA produced changes at the level of translation elongation, which is regulated by the reversible phosphorylation of elongation factor 2 (EF2). Changes in translation elongation after CsA were found to be associated with, and most likely caused by, changes in EF2 phosphorylation. Reduced renal translation elongation was associated with increased EF2 phosphorylation, and increased hepatic elongation with decreased EF2 phosphorylation. EF2 is phosphorylated by Ca2+ calmodulin-dependent protein kinase III (PKIII). Phosphorylated EF2 is a substrate for protein phosphatase 2A (PP2A), but not calcineurin (protein phosphatase 2B or PP2B), the enzyme inhibited by CsA-cyclophilin complexes in T-cells. When CsA or inhibitors of PKIII (EGTA, trifluoperazine) were added in vitro to assays of EF2 phosphorylation in renal or hepatic cytoplasm, or to assays of renal or hepatic microsomal translation elongation, they were without significant effects. Addition in vitro of the PP2A inhibitor okadaic acid increased EF2 phosphorylation in renal and hepatic cytoplasms, but inconsistently produced an inhibition of microsomal translation. However, in less complex rabbit reticulocyte lysates, addition of okadaic acid inhibited PP2A, increased EF2 phosphorylation, and inhibited translation elongation. Furthermore, addition of EGTA and trifluoperazine to rabbit reticulocyte lysates inhibited Ca2+ calmodulin-dependent PKIII activity, decreased EF2 phosphorylation, and stimulated translation elongation. CsA acting alone or as a complex with cyclophilin could alter EF2 phosphorylation by affecting transcriptional regulation or the enzymatic activity of PKIII, PP2A or EF2. Changes in EF2 phosphorylation and translation in body tissues suggest that CsA causes widespread disturbances in phosphorylation and dephosphorylation pathways regulating cellular processes including transcription and translation factor activity. These disturbances may underlie the broad spectrum of toxicities observed during CsA therapy.

Dose- and age-dependent effects of prenatal ethanol exposure on hippocampal metabotropic-glutamate receptor-stimulated phosphoinositide hydrolysis.

Prenatal ethanol exposure reduces the density of the N-methyl-D-aspartate (NMDA) receptor agonist binding sites and decreases the capacity to elicit long-term potentiation (LTP) in hippocampal formation of 45-day-old rat offspring. We hypothesized that prenatal ethanol exposure would reduce metabotropic-glutamate receptor (mGluR)-activated phosphoinositide hydrolysis also. Sprague-Dawley rat dams were fed a liquid diet containing either 3.35% (v/v) ethanol or 5.0% ethanol throughout gestation. Control groups were pair-fed either isocalorically matched 0% ethanol liquid diets or lab chow ad libitum. (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD) stimulated inositol-1-phosphate (IP1) accumulation via activation of the mGluR in offspring whose mothers consumed the 3.35% ethanol liquid diet was not different compared with the control groups. Furthermore, trans-ACPD stimulated IP1 accumulation in 10- to 13-day-old offspring of the 5.0% ethanol diet group was not different compared with the control groups. However, trans-ACPD stimulated IP1 accumulation was reduced significantly in 56- to 82-day-old offspring of dams fed the 5.0% ethanol liquid diet compared with the control groups. In contrast, bethanechol stimulated IP1 accumulation, mediated via activation of muscarinic cholinergic receptors, was not affected by maternal consumption of either ethanol liquid diet. These results suggest both dose- and age-dependent effects of prenatal ethanol exposure on hippocampal responsiveness to trans-ACPD-activated phosphoinositide hydrolysis. Furthermore, the ability of the 3.35% ethanol diet to alter hippocampal NMDA receptors without altering the mGluR response suggests a differential sensitivity to the effects of ethanol exposure in utero among hippocampal glutamate receptor subtypes.(ABSTRACT TRUNCATED AT 250 WORDS)

Prenatal ethanol exposure during the last third of gestation in rat reduces hippocampal NMDA agonist binding site density in 45-day-old offspring.

The effect of ethanol exposure during different periods of prenatal or postnatal development on hippocampal N-methyl-D-aspartate (NMDA) receptor binding was studied in rat. Fetal rat pups were exposed to ethanol for different periods of time during gestation via maternal consumption of a 3.35% ethanol liquid diet. In a separate experiment, neonatal pups were fed 2.51 g ethanol/kg body weight/day from Postnatal Day (PD) 4 to PD 10 via intragastric feeding tube. These two ethanol administration paradigms produced average peak maternal and pup blood ethanol concentrations of 39 mg/dl and 57 mg/dl, respectively. At 45 days of age, offspring from each treatment group were sacrificed for measurements of hippocampal NMDA-sensitive [3H]-glutamate binding site density using in vitro radiohistochemical techniques. As observed previously, prenatal ethanol exposure throughout gestation resulted in NMDA-sensitive [3H]-glutamate binding site reductions in the apical dendritic field regions of dentate gyrus, hippocampal CA1 and subiculum of dorsal hippocampal formation compared to the ad lib or pair-fed control groups. NMDA-sensitive [3H]-glutamate binding was not different than control in rats exposed to ethanol during the first half of gestation only. Prenatal ethanol exposure during the last half or the last third of gestation resulted in NMDA-sensitive [3H]-glutamate binding site reductions comparable to the binding site reductions observed in rats exposed to ethanol throughout gestation. Hippocampal NMDA-sensitive [3H]-glutamate binding site density in postnatal ethanol-exposed rats was not different than the suckling or gastrostomy control groups.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro susceptibilities of Plasmodium falciparum to compounds which inhibit nucleotide metabolism.

A unique metabolic feature of malaria parasites is their restricted ability to synthesize nucleotides. These parasites are unable to synthesize the purine ring and must therefore obtain preformed purine bases and nucleosides from the host cell, the erythrocyte. On the other hand, pyrimidines must be synthesized de novo because of the inability of the parasites to salvage preformed pyrimidines. Thus, one would anticipate that the blockage of purine salvage or pyrimidine de novo synthesis should adversely affect parasite growth. This premise was tested in vitro with a total of 64 compounds, mostly purine and pyrimidine analogs, known to inhibit one or more steps of nucleotide synthesis. Of the 64 compounds, 22 produced a 50% inhibition of the growth of the human malaria parasite Plasmodium falciparum at a concentration of 50 microM or less. Inhibition of the growth of chloroquine-resistant clones of P. falciparum did not differ significantly from that of the growth of chloroquine-susceptible clones. Two of the compounds which effectively inhibited parasite growth, 6-mercaptopurine and 6-thioguanine, were found to be potent competitive inhibitors of a key purine-salvaging enzyme (hypoxanthine-guanine-xanthine phosphoribosyltransferase) of the parasite.

Characterization of adenine phosphoribosyltransferase from the human malaria parasite, Plasmodium falciparum.

Because of their inability to synthesize purines de novo, malaria parasites rely on purine phosphoribosyltransferases (PRTases) to convert purine bases salvaged from the host cell (the erythrocyte) into the corresponding purine nucleoside monophosphates. Our studies with late trophozoites of the human malaria parasite, Plasmodium falciparum, showed that virtually all of the purine PRTase activity is accounted for by two distinct enzymes. One enzyme utilizes hypoxanthine, guanine and xanthine (Queen, S.A., Vander Jagt, D. and Reyes, P. (1988) Mol. Biochem. Parasitol. 30, 123-134). The second enzyme utilizes only adenine and is the subject of this paper. This latter enzyme exhibits a biphasic pH-activity profile and is moderately to weakly inhibited by several divalent metal ions. Several of the properties of the P. falciparum enzyme were found to differ significantly from those of human erythrocyte adenine PRTase. (1) The molecular weight (18,000) of the parasite enzyme is smaller than that of the host cell enzyme. (2) The parasite enzyme, unlike the erythrocyte enzyme, is not significantly inhibited by sulfhydryl reagents. (3) 6-Mercaptopurine and 2,6-diaminopurine proved to be competitive inhibitors of the parasite enzyme (Ki 0.70 and 1.0 mM, respectively); on the other hand, the human enzyme is not inhibited by these agents. (4) The Km for adenine (0.80 microM) and 5-phosphoribosyl-1-pyrophosphate (0.70 microM) displayed by the parasite enzyme are significantly smaller than the corresponding Km values shown by the erythrocyte enzyme. These distinctions between the parasite and host enzymes point to the possibility that adenine PRTase of P. falciparum may represent a potential target for chemotherapeutic attack.

Properties and substrate specificity of a purine phosphoribosyltransferase from the human malaria parasite, Plasmodium falciparum.

The properties of a purine phosphoribosyltransferase from late trophozoites of the human malaria parasite, Plasmodium falciparum, are described. Enzyme activity with hypoxanthine, guanine and xanthine as substrates eluted in parallel during hydroxylapatite, size exclusion and DEAE-Sephadex chromatography as well as during chromatofocusing experiments. Furthermore, enzyme activity with all three purine substrates changed in parallel during heat inactivation of enzyme preparations and upon cold storage (4 degrees C) of the enzyme. When considered together, these results support the view that the phosphoribosyltransferase is capable of utilizing all three purine bases as substrates. Additional characterization revealed that the apparent molecular weight and isoelectric point of this enzyme are 55,500 and 6.2, respectively, and that the apparent Km for 5-phosphoribosyl-1-pyrophosphate ranges from 13.3 to 21.4 microM, depending on the purine base serving as substrate. The apparent Km values for hypoxanthine, guanine and xanthine were found to be 0.46, 0.30 and 29 microM, respectively. Other experiments showed that several divalent cations and sulfhydryl reagents produce a marked reduction of enzyme activity whereas dithiothreitol activates the enzyme. It should be noted that the ability to utilize xanthine as a substrate serves to distinguish the P. falciparum enzyme from its counterpart in the parasite's host cell, the human erythrocyte. The human enzyme shows only barely detectable activity with xanthine while the parasite enzyme displays similarly high levels of activity with all three purine substrates. Thus, the parasite enzyme might prove to be selectively susceptible to inhibition by xanthine analogs and related compounds.