Steady-state in Vivo Glutamate Dehydrogenase Activity in Rat Brain Measured by 15N NMR

Abstract

The in vivo activity of glutamate dehydrogenase (GDH) in the direction of reductive amination was measured in rat brain at steady-state concentrations of brain ammonia and glutamate after intravenous infusion of the substrate 15 This mitochondrial enzyme is present at a high level in rat brain, with an in vitro activity of 900 mol/h/g (1). GDH is believed to contribute to the synthesis of the metabolic and neurotransmitter pools of glutamate. While glutamine is also an important precursor of the neurotransmitter glutamate (2-4), there is evidence to suggest that the glutamate-glutamine cycle is not operating in a stoichiometric manner (5), and some de novo synthesis of glutamate from glucose is required to maintain the neurotransmitter pool (5, 6). GDH is a likely candidate for this role, since the equilibrium of the reaction favors the formation of glutamate.While GDH activities measured in cultured astrocytes and synaptosomal preparations have provided useful information (7-9), it is also important to measure the activity in intact brain, because the distribution of this enzyme according to cell type is controversial. Biochemical and histochemical studies show higher GDH levels in neurons (10 -12), while immunocytochemical studies show highest GDH immunoreactivity in astrocytes (13) N]glutamine were quantified in vitro for better spectral resolution. The results are discussed in relation to the role of GDH in glutamate replenishment. An explanation is offered for the apparent discrepancy between results obtained with labeled glucose and ammonia on the role of GDH in glutamate synthesis. EXPERIMENTAL PROCEDURESAnimal Preparation and Ammonia Infusion-Male Wistar rats (250 -300 g) were anesthetized by the intraperitoneal injection of sodium pentobarbital (Nembutal; 40 mg/kg body weight), and prepared for ammonia infusion through the femoral vein (21). Two infusion protocols were used to achieve different steady-state brain ammonia concentrations for measurement of in vivo GDH activities. One group of rats (Group I) was given infusion of 15 NH 4 Cl (Cambridge Isotopes: Ͼ97% enriched in 15 N in 1 M aqueous solution at pH 7.4) at a rate of 2.3 Ϯ 0.04 mmol/h/kg body weight. The infusion was continued (a) for the time indicated up to 6 h, or (b) for a fixed period of 3.1 Ϯ 0.18 h, followed by 14 NH 4 Cl infusion at the same rate for Յ3.2 h (chase period). In Group II, 15 NH 4 Cl (2 M) was infused at a rate of 3.3 Ϯ 0.07 mmol/h/kg for Յ6 h. In this group, ammonia concentration was increased to 2 M to keep the infusate volume below 1 ml/h. NMR-In vivo and in vitro 15N NMR spectra were obtained on a General Electric CSI-II spectrometer operating at 20.25 MHz for 15 N, as described previously (19,20). 15N chemical shifts are reported in ppm from nitromethane, with the negative sign indicating an upfield shift.

Late memory-related genes in the hippocampus revealed by RNA fingerprinting

“…Although our study did not directly assess the role of GDH and RYR2 in spatial memory, some hypotheses can be suggested based on previously identified functions. GDH is an enzyme, central to glutamate metabolism, which catalyzes the reversible conversion of ␣-ketoglutarate to glutamate (33,34). Increased steady-state levels of GDH mRNA, therefore, may reflect an increased turnover of the excitatory neurotransmitter glutamate, which has been implicated in learning and memory (35,36).…”

Non-Invasive Methods for Studying Brain Energy Metabolism: What They Show and What It Means

“…Thus, glutamine synthase flux reflects largely the glutamate/glutamine shuttle [46][47][48], but is also a means of ammonia detoxification, coupled to anaplerosis [48,54,55]. 'Mitochondrial exchange' between (mitochondrial) 2-oxoglutarate and (cytosolic) glutamate is due to aminotransferases [46,56] and also glutamate dehydrogenase [57,58]. Pyruvate recycling occurs in vivo [43], mainly in astrocytes [59].…”

“…of aspartate labelling) [46]. 15 N-MRS with infused 15 N-H 4 can be used to estimate flux through glutamate dehydrogenase [57,58], glutamine synthase [54,55,58] and also (after inhibition of glutamine synthase) Pi-activated glutaminase [67]; branchedchain aminotransferase is measurable using 15 N-leucine [68]. GABA shunt flux can be measured ex vivo from the labelling kinetics of GABA and glutamate [69].…”

In vivo N-15 MRS study of glutamate metabolism in the rat brain

“…The in vivo activity of glutamate dehydrogenase (GDH) in the direction of reductive amination was measured in the rat brain at steady-state concentrations of brain ammonia and glutamate after the intravenous infusion of the substrate 15 NH 4 + , using 15 N NMR [54]. The in vivo rate was determined from the steady-state fractional 15 N enrichment of brain ammonia, and the rate of the increase of brain [2- 15 N]glutamate/glutamine observed by 15 N NMR at 4.7 T. The in vivo GDH activity was 0.76–1.17 μmol/g/h, (corresponding to 0.013 – 0.02 μmol/g/min) at an 15 NH 4 Cl infusion rate of 2.3 μmol/g/h and was 1.1-1.2 μmol/g/h (0.018 – 0.02 μmol/g/min) at an 15 NH 4 Cl infusion rate of 3.3 μmol/g/h.…”

“…The results from the two laboratories are in good agreement when the difference in the ammonia infusion rates is considered. The low in vivo activity compared to the reported in vitro activity of GDH measured at enzyme-saturating concentrations of the substrates, 900 μmol/g/h [55], can most reasonably be attributed to the low in situ concentrations of ammonia and of 2-oxoglutarate (0.23 ± 0.05 mM) [40, 52, 54] relative to the K m values of the enzyme, 10–18 mM for NH 4 + and 0.2–1.5 mM for 2-oxoglutarate [56, 57]. The two MRS studies strongly suggest that there is net GDH-catalyzed glutamate synthesis from ammonia in hyperammonemic brain, although the rate is slow.…”