[3H]ac-RYYRWK-NH2, a novel specific radioligand for the nociceptin/orphanin FQ receptor.

The hexapeptide ac-RYYRWK-NH2 has been described as a potent partial agonist at the nociceptin (NC)/orphanin FQ receptor which has no affinity for mu-, kappa- or delta-opioid receptors. However, it is not clear whether ac-RYYRWK-NH2 is truly selective for the NC receptor, and ac-RYYRWK-NH2 has therefore been radiolabelled and characterised in receptor-binding experiments. Saturation experiments with [3H]ac-RYYRWK-NH2 binding to rat cortical membranes revealed a single high affinity site for [3H]ac-RYYRWK-NH2 (Kd=0.071 +/- 0.018 nM; Bmax=22+/-2 fmol/mg protein). Uncoupling of the G-proteins resulted in a significant 45% increase in Kd and no change in Bmax. [3H]ac-RYYRWK-NH2 binding to rat cortical membranes or to membranes from baby hamster kidney cells expressing human orphan opioid receptor-like (ORL1) was displaced by NC and ac-RYYRWK-NH2 to the same extent. The following rank order of potency was observed: ac-RYYRWK-NH2 > [Tyr14]NC-OH = NC-OH = NC-NH2 > NC, H-(1-13)-NH2 > NC(1-12)-NH2 >> NC(1-11)-NH2 and, thus, displayed a typical NC receptor pharmacology. Novel cyclic analogues of ac-RYYRWK-NH2 were prepared but these structures were much less active when compared to ac-RYYRWK-NH2. In vitro receptor autoradiography with [3H]ac-RYYRWK-NH2 to rat brain sections revealed high levels of binding in the cerebral cortex, amygdala, hypothalamus and superior colliculus, but low levels in the cerebellum and striatum. Overall, the regional distribution was very similar to that of [3H]NC. Ac-RYYRWK-NH2 seems indeed to be selective for the NC receptor and [3H]ac-RYYRWK-NH2 is a novel radioligand which may be useful for further exploring the pharmacology and receptor-ligand interaction of the NC receptor.

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

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 approximately 80 microM. 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-acetylaspartylglutamate (0.4) > N-acetylglutamate (0.42) > L-aspartate (>1) > L-glutamate (>1) > or = DL-threo-beta-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-beta-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 micromol/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.