Jacob S. Valsborg

Astroglia contain a specific transport mechanism for N-acetyl-L-aspartate

Abstract : N‐Acetylaspartate (NAA) is the second most abundant amino acid in the adult brain. It is located and synthesized in neurons and probably degraded in the glia compartment, but the transport mechanisms are unknown. Rat primary neuron and astrocyte cell cultures were exposed to the L isomer of [3H]NAA and demonstrated concentration‐dependent uptake of [3H]NAA with a Km≈80 μM. However, Vmax was 23 ± 6.4 pmol/mg of protein/min in astrocytes but only 1.13 ± 0.4 pmol/mg of protein/min in neurons. The fact that neuron cultures contain 3‐5% astrocytes suggests that the uptake mechanism is expressed only in glial cells. The astrocyte uptake was temperature and sodium chloride dependent and specific for l‐NAA. The affinity for structural analogues was (IC50 in mM) as follows : l‐NAA (0.12) > N‐acetylaspartyglutamate (0.4) > N‐acetylglutamate (0.42) > l‐aspartate (>1) > l‐glutamate (>1) ≥dl‐threo‐β‐hydroxyaspartate > N‐acetyl‐l‐histidine. The naturally occurring amino acids showed no inhibitory effect at 1 mM. The glutamate transport blocker trans‐pyrrolidine‐2,4‐dicarboxylate exhibited an IC50 of 0.57 mM, whereas another specific glutamate transport inhibitor, dl‐threo‐β‐hydroxyaspartate, had an IC50 of >1 mM. The experiments suggest that NAA transport in brain parenchyma occurs by a novel type of sodium‐dependent carrier that is present only in glial cells.

N-Acetylaspartate Distribution in Rat Brain Striatum During Acute Brain Ischemia

Brain N-acetylaspartate (NAA) can be quantified by in vivo proton magnetic resonance spectroscopy (1H-MRS) and is used in clinical settings as a marker of neuronal density. It is, however, uncertain whether the change in brain NAA content in acute stroke is reliably measured by 1H-MRS and how NAA is distributed within the ischemic area. Rats were exposed to middle cerebral artery occlusion. Preischemic values of [NAA] in striatum were 11 mmol/L by 1H-MRS and 8 mmol/kg by HPLC. The methods showed a comparable reduction during the 8 hours of ischemia. The interstitial level of [NAA] ([NAA]e) was determined by microdialysis using [3H]NAA to assess in vivo recovery. After induction of ischemia, [NAA]e increased linearly from 70 µmol/L to a peak level of 2 mmol/L after 2 to 3 hours before declining to 0.7 mmol/L at 7 hours. For comparison, [NAA]e was measured in striatum during global ischemia, revealing that [NAA]e increased linearly to 4 mmol/L after 3 hours and this level was maintained for the next 4 h. From the change in in vivo recovery of the interstitial space volume marker [14C]mannitol, the relative amount of NAA distributed in the interstitial space was calculated to be 0.2% of the total brain NAA during normal conditions and only 2 to 6% during ischemia. It was concluded that the majority of brain NAA is intracellularly located during ischemia despite large increases of interstitial [NAA]. Thus, MR quantification of NAA during acute ischemia reflects primarily changes in intracellular levels of NAA.

Preparation of 14C-labelled 1,4-dideoxy-1,4-imino-D-arabinitol : Cyanosilylation of cyclic imines using KCN in a one-pot synthesis

A new method for C-C bond formation was developed based on in situ cyanosilylation of cyclic Schiff bases using KCN, TMSCl, KI and ZnI 2 . This method was used to prepare the potent α-glucosidase inhibitor 1,4-dideoxy-1,4-imino-D-arabinitol 14 C labelled at C-5.

Synthesis of (R) and (S) 14C-labelled ethyl nipecotate, for preparation of GABA uptake ligands

14C-labelled (R)- and (S)-ethyl 3-[carboxyl-14C]piperidinecarboxylate were synthesised in 3 steps, including a chiral HPLC separation, starting from 3-[carboxyl-14C]pyridine-carboxylate. The overall radiochemical yield of the labelled (R)- and (S)-enantiomers were 67% and 61%. The radiochemical purities were higher than 98% with specific radioactivities of 48 mCi/mmol.

Characterization of [3H]‐N‐[R‐(2‐ Benzothiazolyl)thio‐2‐propyl]‐2‐chloroadenosine ([3H]‐NNC 21‐0136) binding to rat brain: Profile of a novel selective agonist for adenosine A1 receptors

N‐[R‐(2‐Benzothiazolyl)thio‐2‐ propyl]‐2‐chloroadenosine (NNC 21‐0136) is a novel adenosine agonist with neuroprotectant properties in rodent models of focal and global ischemia that exhibits diminished cardiovascular side effects when compared to reference A1 agonists [Sheardown et al., 1995]. NNC 21‐0136 is shown to be a potent agonist of adenosine A1 receptors showing around 60‐ and 30‐fold selectivity over adenosine A2A and A3 receptors, respectively. In order to further characterize the central nervous system molecular target for NNC 21‐0136, the compound has been radiolabeled and utilized in receptor binding experiments. In vitro receptor autoradiography with [3H]‐NNC 21‐0136 revealed high levels of receptors in the CA1 region of the hippocampus, the granule cell layer of the cerebellar cortex, and moderate uniform levels throughout the cerebral cortex. [3H]‐NNC 21‐0136 binding to rat cerebral cortical membranes was reversible and saturable (Bmax = 0.98 ± 0.04 pmol/mg protein) to a high affinity site (Kd = 1.16 ± 0.06 nM) as determined by saturation binding experiments. [3H]‐NNC 21‐0136 binding was sensitive to guanosine‐5′‐O‐(3‐thio)triphosphate‐γ‐S and enhanced by the presence of divalent cations (Ca2+ and Mg2+). A highly significant correlation between the affinities of several compounds to displace [3H]‐NNC 21‐0136 binding as compared to [3H]‐N‐R‐(2‐phenylisopropyl)adenosine ([3H]‐R‐PIA) binding to adenosine A1 receptors in rat cortical membranes was observed. We conclude that [3H]‐NNC 21‐0136 is a new, selective radioligand for adenosine A1 receptors. Drug Dev. Res. 42:86–97, 1997.

14C-labelling of a novel antiischaemic adenosine A1 agonist at purine C-8

A 14 C-labelled form of 2-chloro-N-[(R)-(2-benzoxazolyl)thio-2-propyl]adenosine (1), a novel antiischaemic adenosine A 1 agonist, has been prepared in three steps from [8- 14 C]-9-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-2,6-dichloropurine (2). The overall radiochemical yield was 56%. The radiochemical purity was higher than 98% with a specific radioactivity of 36 mCi/mmol.