Low dose of S+-ketamine prevents long-term potentiation in pain pathways under strong opioid analgesia in the rat spinal cord in vivo.

BACKGROUND: micro-Opioid receptor (MOR) agonists are strong antinociceptive drugs. Low, but not high doses of the MOR agonist fentanyl prevent synaptic long-term potentiation (LTP) in pain pathways. Block of spinal N-methyl-D-aspartate (NMDA) receptors prevent central sensitization. Here we tested whether the NMDA receptor antagonist S(+)-ketamine reduces C-fibre-evoked potentials and prevents induction of LTP despite high doses of fentanyl. METHODS: C-fibre-evoked field potentials were recorded in the superficial laminae I/II of the rat lumbar spinal cord. High-frequency stimulation (HFS) was applied to the sciatic nerve at C-fibre strength to induce LTP. S(+)-ketamine 5 mg kg(-1) was given 1 h before or after HFS. S(+)-ketamine 5 mg kg(-1) and fentanyl as a bolus (40 microg kg(-1)) followed by an infusion (96 microg kg(-1) h(-1)) were given before HFS to test the action of the combination of these drugs. RESULTS: HFS potentiated C-fibre-evoked field potentials to mean 173 (sem 15)% of control (n=7) for at least 1 h. Low-dose S(+)-ketamine given before HFS blocked the induction of LTP. S(+)-ketamine given after HFS had no effect on the maintenance of LTP. Low-dose S(+)-ketamine prevented induction of LTP under fentanyl-infusion. CONCLUSIONS: Low-dose S(+)-ketamine does not affect C-fibre-evoked potentials alone but blocks LTP induction in pain pathways. In contrast, high doses of opioids strongly reduce C-fibre-evoked potentials, but do not fully prevent LTP induction. In this animal study the combination of S(+)-ketamine with fentanyl reveals both a reduction of C-fibre-evoked potentials and prevention of LTP and seem therefore a better choice for perioperative pain management compared with the sole administration.

An enzyme-linked immunosorbent assay to quantify 14-3-3 proteins in the cerebrospinal fluid of suspected Creutzfeldt-Jakob disease patients.

The detection of 14-3-3 protein by Western immunoblot is a sensitive and specific cerebrospinal fluid marker of Creutzfeldt-Jakob disease (CJD). We developed a quantitative enzyme-linked immunosorbent assay (ELISA) that reliably detects 14-3-3 in cerebrospinal fluid. In a prospective study of 147 cerebrospinal fluid samples, the mean 14-3-3 concentration among pathologically confirmed CJD patients (28.0+/-20.6 ng/ml, n = 41) is significantly higher than the mean in the cerebrospinal fluid of those with other neurological disorders (3.1+/-2.9 ng/ ml, n = 84). At a cutoff value of 8.3 ng/ml, the ELISA has a sensitivity of 92.7% and a specificity of 97.6%. The 14-3-3 ELISA supports a diagnosis of CJD in patients who fulfill clinical criteria for possible CJD.

4-Aminobutyrate (GABA) transporters from the amine-polyamine-choline superfamily: substrate specificity and ligand recognition profile of the 4-aminobutyrate permease from Bacillus subtilis.

A previous study [Ferson, Wray and Fisher (1996) Mol. Microbiol. 22, 693-701] has shown that transposon-mediated disruption of a protein 47% identical to the Escherichia coli GABA (4-aminobutyrate) transporter abolishes the ability of nitrogen-limited culture conditions to induce expression of a GABA transport activity in Bacillus subtilis. Here it is demonstrated directly that the B. subtilis GABA permease (gabP) gene can complement the transport defect in the gabP-negative E. coli strain. Unexpectedly, the ligand-recognition profile of the B. subtilis GabP was found to differ substantially from that of the highly homologous E. coli GabP. Unlike the E. coli GabP, the B. subtilis GabP: (i) exhibits approx. equal preference for the 3-carbon (beta-alanine, Km=9.6 microM) and the 4-carbon (GABA, Km=37 microM) amino acids, and (ii) resists inhibition by bulky, conformationally constrained compounds (e.g. nipecotic acid, guvacine), which are active against GABA transporters from brain. The present study shows additionally that the B. subtilis GabP can translocate several open-chain GABA analogues (3-aminobutyrate, 3-aminopropanoate, cis-4-aminobutenoate) across the membrane via counterflow against [3H]GABA. Thus, consistent with the idea that the ligand-recognition domain of the B. subtilis GabP is less spacious than that of the close homologue from E. coli, the former exhibits more stringent requirements than the latter for substrate recognition and translocation. These distinct functional characteristics of the E. coli and B. subtilis GABA transporters provide a basis by which to identify ligand-recognition domains within the amine-polyamine-choline transporter superfamily.

Substrate specificity of the Escherichia coli 4-aminobutyrate carrier encoded by gabP. Uptake and counterflow of structurally diverse molecules.

Transport of 4-aminobutyrate into Escherichia coli is catalyzed by gab permease (GabP). Although published studies show that GabP is relatively specific, recognizing the common alpha-amino acids with low affinity, recent work from this laboratory indicates that a number of synthetic compounds are high affinity transport inhibitors (50% inhibition at 5-100 microM). Here we present evidence that many of these structurally heterogeneous compounds not only inhibit transport but also function as alternative GabP substrates (i.e. a set of observations inconsistent with the idea that the core of the GabP transport channel exhibits rigid structural specificity for the native substrate, 4-aminobutyrate.

Pyridine carboxylic acids as inhibitors and substrates of the Escherichia coli gab permease encoded by gabP.

Although considered selective for its natural substrate, 4-aminobutyrate, gab permease was inhibited by 1,2,3,6-tetrahydro-3-pyridinecarboxylate and 1,2,3,6-tetrahydro-4-pyridinecarboxylate. The former is a transported substrate, since its preloading into metabolically poisoned cells stimulated transient accumulation of 4-aminobutyrate via counterflow.

Ligand recognition properties of the Escherichia coli 4-aminobutyrate transporter encoded by gabP. Specificity of Gab permease for heterocyclic inhibitors.

4-aminobutyrate metabolism in Escherichia coli begins with transport across the cytoplasmic membrane via the GabP, which is encoded by gabP. Although GabP is specific and exhibits poor affinity for many cellular constituents such as the alpha-amino acids, the range of compounds recognized with high affinity has yet to be investigated. In order to address this gap in knowledge, we developed a gabP-negative host strain, which permits evaluation of test compounds for inhibitory effects on cloned GabP (expression inducible by isopropyl-1-thio-beta-D-galactopyranoside). Using this inducible expression system, three structurally distinct categories of high affinity transport inhibitor were identified. The structural dissimilarity of these inhibitors significantly alters our view of ligand recognition by GabP. Any complete model must now account for the observation that inhibition of 4-aminobutyrate transport can be mediated either (i) by open chain analogs of 4-aminobutyrate, (ii) by cyclic amino acid analogs, or (iii) by planar heterocyclic compounds lacking a carboxyl group. Such results do not support a previously sustainable view of GabP that features a restrictive ligand recognition domain, unable to accommodate structures that differ very much from the native substrate, 4-aminobutyrate.