Whole-brain glutamate metabolism evaluated by steady-state kinetics using a double-isotope procedure: effects of gabapentin

Cerebral rates of anaplerosis are known to be significant, yet the rates measured in vivo have been debated. In order to track glutamate metabolism in brain glutamatergic neurons and brain glia, for the first time unrestrained awake rats were continuously infused with a combination of H14CO3- and [1 - 13C]glucose in over 50 infusions ranging from 5 to 60 min. In whole-brain extracts from these animals, the appearance of 14C in brain glutamate and glutamine and appearance of 13C in the C-4 position of glutamate and glutamine were measured as a function of time. The rate of total neuronal glutamate turnover, the anaplerotic rate of synthesis of glutamine and glutamate from H14CO3-, flux through the glutamate/glutamine cycle, and a minimum estimate of whole-brain anaplerosis was obtained. The rate of synthesis of 14C-glutamate from H14CO3- was 1.29 +/- 0.11 nmoles/min/mg protein, whereas the rate of synthesis of 14C-glutamine was 1.48 +/- 0.10 nmoles/min/mg protein compared to total glutamate turnover of 9.39 +/- 0.73 nmoles/min/mg protein. From the turnover rate of glutamine, an upper limit for flux through the glutamate/glutamine cycle was estimated at 4.6 nmoles/min/mg protein. Synthesis of glutamine from H14CO3- was substantial, amounting to 32% of the glutamate/glutamine cycle. These rates were not significantly affected by a single injection of 100 mg/kg of the antiepileptic drug gabapentin. In contrast, acute administration of gabapentin significantly lowered incorporation of H14CO3- into glutamate and glutamine in excised rat retinas, suggesting metabolic effects of gabapentin may require chronic treatment and/or are restricted to brain areas enriched in target enzymes such as the cytosolic branched chain aminotransferase. We conclude that the brain has a high anaplerotic activity and that the combination of two tracers with different precursors affords unique insights into the compartmentation of cerebral metabolism.     

Differences in growth and water relations among Phaseolus vulgaris cultivars in response to induced drought stress

Relatively little ecophysiological research has been conducted to determine the responses to drought of Phaseolus vulgaris. Four bean cultivars (cvs.) from Brazil, A320, Carioca, Ouro Negro and Xodó were submitted to an imposed water deficit in order to evaluate the importance of some adaptive mechanisms of drought resistance through the analysis of growth parameters, water status, gas exchange and indicators of tolerance mechanisms at the cellular level. During the drought treatment, relative growth rates were more reduced for A320 and Xodó than Carioca and Ouro Negro. A320 closed its stomata very rapidly and complete stomatal closure was obtained at Psi(w)=-0.6 MPa, in contrast to the other cvs. where stomata were fully closed only at Psi(w)=-0.9 MPa. Net assimilation rates were closely related to stomatal conductances. Mechanisms at the cellular level appeared to be mostly important for higher tolerance. Carioca and Ouro Negro, when compared to A320 and Xodó, were characterized by having better drought tolerance mechanisms and higher tissue water retention capacity leading to a better growth under water deficits. The leaf dehydration rates of those cvs. were slow whereas those of the drought sensitive cvs. were rapid. The results were confirmed by the electrolyte leakage test and leaf osmotic potential measurements, which indicated higher membrane resistance and osmotic adjustment in the two tolerant cvs. Carioca and Ouro Negro. It appears from this study that despite being cultivated in the same geographical region, the four cvs. of P. vulgaris displayed somewhat different drought adaptive capacities for prolonged drought during the vegetative phase.     

Release of non-mast cell histamine from rat aorta

We previously reported that A23187 induces release of histamine from bovine intrapulmonary vein and provided pharmacological evidence against an involvement of mast cells as the source of histamine. This study was conducted to test more definitively the hypothesis that histamine is released from non-mast cell sources in blood vessels. The effects of A23187 on release of histamine were determined using rat aorta which does not contain mast cells. Aortic rings were mounted for recording of isometric tension, and following exposure to A23187 or vehicle, histamine in the bathing media was measured using enzyme immunoassay. A23187 (100 nmol/l - 10 micromol/l) induced concentration-related release of histamine from rings with endothelium. The accumulation of histamine in the bathing media induced by 10 microM A23187 reached plateau at 60 min (6.2 +/- 1.1 pmol/mg) and was markedly and significantly higher than vehicle control (0.4 +/- 0.1 pmol/mg, p < 0.05). Destruction of endothelium significantly inhibited A23187-induced histamine release (5.5 +/- 1.5 pmol/mg with endothelium, 1.1 +/- 0.3 pmol/mg without endothelium, p < 0.05). The results demonstrate that A23187 induces release of histamine from rat aorta which does not contain mast cells and that the release of histamine is largely dependent on the presence of endothelium.     

The human cDNA for a homologue of the plant enzyme 1-aminocyclopropane-1-carboxylate synthase encodes a protein lacking that activity

The sequences of genes encoding homologues of 1-aminocyclopropane-1-carboxylate (ACC) synthase, the first enzyme in the two-step biosynthetic pathway of the important plant hormone ethylene, have recently been found in Fugu rubripes and Homo sapiens (Peixoto et al., Gene 246 (2000) 275). ACC synthase (ACS) catalyzes the formation of ACC from S-adenosyl-L-methionine. ACC is oxidized to ethylene in the second and final step of ethylene biosynthesis. Profound physiological questions would be raised if it could be demonstrated that ACC is formed in animals, because there is no known function for ethylene in these organisms. We describe the cloning of the putative human ACS (PHACS) cDNA that encodes a 501 amino acid protein that exhibits 58% sequence identity to the putative Fugu ACS and approximately 30% sequence identity to plant ACSs. Purified recombinant PHACS, expressed in Pichia pastoris, contains bound pyridoxal-5'-phosphate (PLP), but does not catalyze the synthesis of ACC. PHACS does, however, catalyze the deamination of L-vinylglycine, a known side-reaction of apple ACS. Bioinformatic analysis indicates that PHACS is a member of the alpha-family of PLP-dependent enzymes. Molecular modeling data illustrate that the conservation of residues between PHACS and the plant ACSs is dispersed throughout its structure and that two active site residues that are important for ACS activity in plants are not conserved in PHACS.     

Intra-articular CD1c-expressing myeloid dendritic cells from rheumatoid arthritis patients express a unique set of T cell-attracting chemokines and spontaneously induce Th1, Th17 and Th2 cell activity

Introduction

Myeloid dendritic cells (mDCs) are potent T cell-activating antigen-presenting cells that have been suggested to play a crucial role in the regulation of immune responses in many disease states, including rheumatoid arthritis (RA). Despite this, studies that have reported on the capacity of naturally occurring circulating mDCs to regulate T cell activation in RA are still lacking. This study aimed to evaluate the phenotypic and functional properties of naturally occurring CD1c (BDCA-1)⁺ mDCs from synovial fluid (SF) compared to those from peripheral blood (PB) of RA patients.

Methods

CD1c⁺ mDC numbers and expression of costimulatory molecules were assessed by fluorescence-activated cell sorting (FACS) analysis in SF and PB from RA patients. Ex vivo secretion of 45 inflammatory mediators by mDCs from SF and PB of RA patients was determined by multiplex immunoassay. The capacity of mDCs from SF to activate autologous CD4⁺ T cells was measured.

Results

CD1c⁺ mDC numbers were significantly increased in SF versus PB of RA patients (mean 4.7% vs. 0.6%). mDCs from SF showed increased expression of antigen-presenting (human leukocyte antigen (HLA) class II, CD1c) and costimulatory molecules (CD80, CD86 and CD40). Numerous cytokines were equally abundantly produced by mDCs from both PB and SF (including IL-12, IL-23, IL-13, IL-21). SF mDCs secreted higher levels of interferon γ-inducible protein-10 (IP-10), monokine induced by interferon γ (MIG) and, thymus and activation-regulated chemokine (TARC), but lower macrophage-derived chemokine (MDC) levels compared to mDCs from PB. mDCs from SF displayed a strongly increased capacity to induce proliferation of CD4⁺ T cells associated with a strongly augmented IFNγ, IL-17, and IL-4 production.

Conclusions

This study suggests that increased numbers of CD1c⁺ mDCs in SF are involved in the inflammatory cascade intra-articularly by the secretion of specific T cell-attracting chemokines and the activation of self-reactive T cells.     

Effects of melatonin on acetylsalicylic acid induced gastroduodenal and jejunal mucosal injury

We evaluated the effects of melatonin on acetylsalicylic acid (ASA) induced gastroduodenal and jejunal mucosal injury. We used 40 postpubertal rats divided randomly into five groups of eight animals. The control group consisted of untreated animals. The Mel group was injected intraperitoneally (i.p.) with 5 mg/kg melatonin. The ASA group was injected i.p. with 200 mg/kg ASA. The ASA + Mel group was injected i.p. with 5 mg/kg melatonin 45 min after administering 200 mg/kg ASA i.p. The Mel + ASA group was injected i.p. with 5 mg/kg melatonin 45 min before administering 200 mg/kg ASA i.p. We found no statistically significant differences in mean histopathological scores in the ASA + Mel group compared to the ASA group. ASA caused shortened villi and loss of the apical villus in the duodenum. The histopathological score was increased and villus height was decreased in the ASA group compared to untreated controls. Treatment with melatonin attenuated the histological damage. In the ASA group, occasional areas showed erosion of villi in the jejunum; however, differences in mean histopathological score in ASA group compared to the other groups were not statistically significant. Malondialdehyde (MDA), glutathione (GSH) and superoxide dismutase (SOD) activities were measured in stomach, duodenal and jejunum tissue. We found increased MDA activity in both stomach and duodenal tissues in the ASA group compared to the control group (p < 0.05). We found no statistically significant changes in MDA levels in jejunal tissue in the ASA group compared to the control group. We found no change in SOD activity in either stomach or duodenal tissues in the ASA group compared to the control group. We observed decreased SOD activity in jejunal tissue in the ASA group compared to the control group (p < 0.05). We detected no change in GSH activity in stomach, duodenal or jejunal tissues in the ASA group compared to the control group. The stomach damage was less in melatonin treated groups, but the lesions were not completely eliminated. The jejunum in the ASA group retained a nearly normal appearance. We found that melatonin exhibited some healing effects on ASA induced duodenal mucosal injury.     

In vivo enzymatic activity of acetylCoA synthetase in skeletal muscle revealed by C turnover from hyperpolarized [1-C]acetate to [1-C]acetylcarnitine

Background

Acetate metabolism in skeletal muscle is regulated by acetylCoA synthetase (ACS). The main function of ACS is to provide cells with acetylCoA, a key molecule for numerous metabolic pathways including fatty acid and cholesterol synthesis and the Krebs cycle.

Methods

Hyperpolarized [1-¹³C]acetate prepared via dissolution dynamic nuclear polarization was injected intravenously at different concentrations into rats. The ¹³C magnetic resonance signals of [1-¹³C]acetate and [1-¹³C]acetylcarnitine were recorded in vivo for 1 min. The kinetic rate constants related to the transformation of acetate into acetylcarnitine were deduced from the 3 s time resolution measurements using two approaches, either mathematical modeling or relative metabolite ratios.

Results

Although separated by two biochemical transformations, a kinetic analysis of the ¹³C label flow from [1-¹³C]acetate to [1-¹³C]acetylcarnitine led to a unique determination of the activity of ACS. The in vivo Michaelis constants for ACS were K_M = 0.35 ± 0.13 mM and V_max = 0.199 ± 0.031 μmol/g/min.

Conclusions

The conversion rates from hyperpolarized acetate into acetylcarnitine were quantified in vivo and, although separated by two enzymatic reactions, these rates uniquely defined the activity of ACS. The conversion rates associated with ACS were obtained using two analytical approaches, both methods yielding similar results.

General significance

This study demonstrates the feasibility of directly measuring ACS activity in vivo and, since the activity of ACS can be affected by various pathological states such as cancer or diabetes, the proposed method could be used to non-invasively probe metabolic signatures of ACS in diseased tissue.     

The C57BL/6J Mouse Exhibits Sporadic Congenital Portosystemic Shunts

C57BL/6 mice are the most widely used strain of laboratory mice. Using in vivo proton Magnetic Resonance Spectroscopy (¹H MRS), we have repeatedly observed an abnormal neurochemical profile in the brains of both wild-type and genetically modified mice derived from the C57BL/6J strain, consisting of a several fold increase in cerebral glutamine and two fold decrease in myo-inositol. This strikingly abnormal neurochemical “phenotype” resembles that observed in chronic liver disease or portosystemic shunting and appeared to be independent of transgene, origin or chow and was not associated with liver failure. As many as 25% of animals displayed the abnormal neurochemical profile, questioning the reliability of this model for neurobiology. We conducted an independent study to determine if this neurochemical profile was associated with portosystemic shunting. Our results showed that 100% of the mice with high brain glutamine displayed portosystemic shunting by concomitant portal angiography while all mice with normal brain glutamine did not. Since portosystemic shunting is known to cause alterations in gene expression in many organs including the brain, we conclude that portosystemic shunting may be the most significant problem associated with C57BL/6J inbreeding both for its effect on the central nervous system and for its systemic repercussions.