Voltammetric studies of basal and electrically evoked cathecolamine release from long‐term cultures of chromaffin cells

Abstract

Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a membrane peptidase expressed in a number of tissues such as kidney, prostate and brain. The brain form of GCPII (also known as N-acetylated-a-linked-acidic dipeptidase, NAALADase) cleaves N-acetyl-aspartyl glutamate to yield free glutamate. Animal model experiments show that inhibition of GCPII prevents neuronal cell death during experimental ischaemia. GCPII thus represents an important target for the treatment of neuronal damage caused by excess glutamate. We report the mapping of the entire coding region of GCPII and identification of the region sufficient and necessary for the production of active recombinant protein. Extracellular portion of human glutamate carboxypeptidase II (amino acids 44-750) was expressed in Drosophila Schneider's cells and purified to homogeneity. A novel assay for hydrolytic activity of GCPII, based on fluorimetric detection of released alpha-amino groups was established, and used for enzymological characterization of GCPII. The potential of this assay for high-throughput inhibitor testing was evaluated and pH dependence for the enzymatic activity have been analysed. Using a complete set of protected dipeptides, substrate specificity of recombinant GCPII was elucidated. Ac-Glu-Met, Ac-Asp-Met and surprisingly Ac-Ala-Met were identified as novel substrates for GCPII. The glycosylation has been found indispensable for the activity of the enzyme. A series of point mutants of the enzyme has been expressed and purified and the glycosylation sites critical for the proteolytic activity have been identified.Cerebral carbohydrate metabolism is intricately linked to glutamatergic action manifested by a large neuronal glutamate pool and a small glial glutamate pool. In the present study, the effect of deep barbiturate anesthesia on brain glucose transport, TCA cycle flux and glutamate/glutamine metabolism was assessed in the rat brain using 13 C MRS in conjunction with [1-13 C] glucose infusions at 9.4 Tesla. Brain glucose concentrations were elevated compared to a-chloralose anesthetized rats (Choi et al. 2001), consistent with a twofold reduced rate of glucose consumption, reaffirming that energy metabolism is substantial even under near-coma conditions. Several metabolic rates were extracted from the rate of label incorporation into amino acid and neurotransmitter using a mathematical model (Gruetter et al. 2001). The neuronal TCA cycle was similar to that in the glial compartment, 0.35 ± 0.03 and 0.26 ± 0.06 lmol/g/min, respectively, suggesting that the major action of pentobarbital is on neuronal energy metabolism. The apparent rate of glutamatergic neurotransmission, V NT , was measured for the first time and very low, 0.04 ± 0.01 lmol/g/min, even though glucose metabolism was still substantial, suggesting that the stoichiometry between glucose metabolism and glutamatergic neurotransmission is far from 1 : 1. The rate of pyruvate carboxylation was 0.03 ± 0.01 lmol/g/min. The exchange rate between cytosolic glutamate and mitochondri...