Eric Clottes

Spinal 5-HT1A receptors differentially influence nociceptive processing according to the nature of the noxious stimulus in rats: effect of WAY-100635 on the antinociceptive activities of paracetamol, venlafaxine and 5-HT

&NA; The regulation of nociceptive processing by 5‐HT at the spinal level is intricate since the neurotransmitter has been implicated in both pro and antinociception. The aim of our study was to investigate, according to the nature of the noxious stimulus, how the blockade of spinal 5‐HT1A receptors could influence the antinociceptive actions of exogenous 5‐HT as well as two analgesics involving endogenous 5‐HT, paracetamol and venlafaxine. Rats were submitted either to the formalin test (tonic pain) or the paw pressure test (acute pain). WAY‐100635 (40 μg/rat, i.t.), a selective 5‐HT1A receptor antagonist, had no intrinsic action in either test. However, in the formalin test, it blocked the antinociceptive action of 5‐HT (50 μg/rat, i.t.) and paracetamol (300 mg/kg, i.v.) in both phases of biting/licking behaviour and that of venlafaxine (2.5 mg/kg, s.c.) in the late phase only. In the paw pressure test, the combination of sub‐effective doses of 5‐HT (0.01 μg/rat, i.t.), paracetamol (50 mg/kg, i.v.) or venlafaxine (20 mg/kg, s.c.) with WAY‐100635 led to a significant antinociceptive effect, which seems to depend on the reinforcement of the activity of inhibitory GABAergic interneurones. In conclusion, both direct stimulation of the spinal 5‐HT1A receptors by 5‐HT, and indirect stimulation using paracetamol or venlafaxine can differently influence pain transmission. We propose that the nature of the applied nociceptive stimulus would be responsible for the dual effect of the 5‐HT1A receptors rather than the hyperalgesic state or the supraspinal integration of the pain message.

Orally Administered Paracetamol Does Not Act Locally in the Rat Formalin Test Evidence for a Supraspinal, Serotonin-dependent Antinociceptive Mechanism

Background The mechanism of action of paracetamol (acetaminophen) remains elusive because it is still under discussion as to whether it acts locally and/or centrally. The primary aim of this study was to clarify its site(s) of action (central and/or local) using the rat formalin test. Methods Spontaneous biting and licking of the injected paw following intraplantar injection of formalin 2.5% was monitored during the two phases of nociceptive behavior (0–5 and 20–40 min after injection), and the authors examined the antinociceptive activity of paracetamol following oral, intravenous, intraplantar, and intrathecal administrations as well as the reversion of this effect by an intrathecal injection of WAY 100,635, a selective 5-HT1A receptor antagonist. Results The oral administration of paracetamol (300, 400 mg/kg) reduced nociceptive behavior in both phases (400 mg/kg: 36.9 ± 4.6% and 61.5 ± 5.2% of inhibition in phases I and II, respectively, P <0.05), whereas lower doses reduced primarily the score of the second phase of the test. Only high doses of 10 to 20 mg/kg intraplantarly administered paracetamol, which were ineffective when administered subcutaneously, produced a significant but limited reduction in the early phase of the test and had no effect on the second phase or any antiinflammatory activity. Thus, this local effect did not seem to participate in the antinociceptive action of 400 mg/kg orally given paracetamol, which was totally blocked in both phases by an intrathecal injection of 40 &mgr;g WAY 100,635 per rat. Such an inhibition was not observed when paracetamol (200 &mgr;g per rat) was intrathecally coinjected with WAY 100,635, whereas the antinociceptive action of 5-HT (50 &mgr;g per rat, intrathecally) during both phases of pain was inhibited by WAY 100,635 (intrathecally). Conclusions Orally administered paracetamol does not seem to exert any relevant local action in the formalin model of tonic pain in rats, but it might activate the serotonergic bulbospinal pathways via a supraspinal site of action that remains to be elucidated.

Identification and characterisation of a new human glucose-6-phosphatase isoform.

The liver endoplasmic reticulum glucose‐6‐phosphatase catalytic subunit (G6PC1) catalyses glucose 6‐phosphate hydrolysis during gluconeogenesis and glycogenolysis. The highest glucose‐6‐phosphatase activities are found in the liver and the kidney; there have been many reports of glucose 6‐phosphate hydrolysis in other tissues. We cloned a new G6Pase isoform (G6PC3) from human brain encoded by a six‐exon gene (chromosome 17q21). G6PC3 protein was able to hydrolyse glucose 6‐phosphate in transfected Chinese hamster ovary cells. The optimal pH for glucose 6‐phosphate hydrolysis was lower and the K m higher relative to G6PC1. G6PC3 preferentially hydrolyzed other substrates including pNPP and 2‐deoxy‐glucose‐6‐phosphate compared to the liver enzyme.

Acetaminophen recruits spinal p42/p44 MAPKs and GH/IGF-1 receptors to produce analgesia via the serotonergic system

The mechanism of action of acetaminophen is currently widely discussed. Direct inhibition of cyclooxygenase isoforms remains the commonly advanced hypothesis. We combined behavioral studies with molecular techniques to investigate the mechanism of action of acetaminophen in a model of tonic pain in rats. We show that acetaminophen indirectly stimulates spinal 5-hydroxytryptamine (5-HT)1A receptors in the formalin test, thereby increasing transcript and protein levels of low-affinity neurotrophin receptor, insulin-like growth factor-1 (IGF-1) receptor α subunit, and growth hormone receptor and reducing the amount of somatostatin 3 receptor (sst3R) mRNA. Those cellular events seem to be important for the antinociceptive activity of acetaminophen. Indeed, down-regulation of sst3R mRNA depends on acetaminophen-elicited, 5-HT1A receptordependent increase in neuronal extracellular signal-regulated kinase 1/2 (ERK1/2) activities that mediate antinociception. In addition, spinal growth hormone (GH) and IGF-1 receptors would also be involved in the antinociceptive activity of the analgesic at different degrees. Our results show the involvement of specific 5-HT1A receptor-dependent cellular events in acetaminophen-produced antinociception and consequently indicate that inhibition of cyclooxygenase activities is not the exclusive mechanism involved. Furthermore, we propose that the mechanisms of 5-HT1A receptor-elicited antinociception and the role of the spinal ERK1/2 pathway in nociception are more intricate than suspected so far and that the GH/IGF-1 axis is an interesting new player in the regulation of spinal nociception.

Denaturation by guanidinium chloride of dimeric MM-creatine kinase and its proteinase K-nicked form: evidence for a multiple-step process.

Cytosolic MM-creatine kinase is a homodimeric protein. Each monomer can be cleaved by proteinase K at an exposed surface loop, into two fragments K1 and K2, which remain associated. The nicked protein is thus a heterotetrameric protein, named (K1K2)2, made up of two heterodimers K1K2 linked together by their K1 subunit. In non-denaturing conditions, the cleaved protein does not present any measurable difference compared with uncleaved MM-creatine kinase, except for the loss of enzymatic activity. Comparative equilibrium denaturation of the two oligomeric proteins by guanidinium chloride indicates a multistep process with formation of either compact monomer or compact K1K2 dimer, a molten globule and a pre-molten globule state. In the case of the nicked-enzyme, the molten globule is composed of the two peptides K1 and K2, whereas in the pre-molten globule the interactions between K1 and K2 are too weak to maintain their cohesion. At low guanidinium chloride concentration, the proteinase K-nicked protein exhibits a higher accessibility of one of its tryptophan accompanied by a small decrease in its molar ellipticity suggesting a secondary structure loosening of the K1 peptide. Our results suggest that K1 and K2 are not strictly autonomous unfolding units and thus cannot be considered as independent domains.

High salt concentrations induce dissociation of dimeric rabbit muscle creatine kinase: Physico-chemical characterization of the monomeric species

Incubation of dimeric MM-creatine kinase (MM-CK) with high NaCl or LiCl concentrations results in dissociation of the subunits and complete enzyme inactivation. In NaCl, this process, which depends on protein concentration, may be described according to a two-state model where the dimer can be reversibly converted into compact folded monomers (D <--> 2M). At LiCl concentrations higher than 2-2.5 M, MM-CK is recovered in two monomeric states: an inactive compact species (M) and a more expanded form (EF), which represents 15-20% of the population. Thus, in LiCl, a three-state model (D <--> 2M --> 2EF) more adequately accounts for our experimental results. The monomeric species (M) obtained in NaCl and LiCl exhibits some properties of the molten globule state described in guanidine hydrochloride. Indeed, this form is compact and devoid of any enzymatic activity; it maintains a high degree of secondary structure and binds 8-anilino-1-naphthalenesulfonate. The formation of this intermediate induces the exposure of a second tryptophan (among the four present) which is located at the monomer-monomer interface in the native structure. In LiCl, the monomeric species (M) is irreversibly converted into a less compact form (EF) which seems to have lost a large part of its secondary structure.

Creatine kinase compactness and thiol accessibility during sodium dodecyl sulfate denaturation estimated by resonance energy transfer and 2-nitro-5-thiocyanobenzoic acid cleavage

We have investigated the effect of increasing sodium dodecyl sulfate (SDS) concentrations on rabbit muscle cytosolic creatine kinase structure by two methods. We have first determined the variation of accessibility of the thiol groups of the enzyme during SDS denaturation by a technique which involves an irreversible chemical modification of CK accessible thiol groups, followed by NTCB cleavage before the unmodified cysteines in 8 M urea (pH 9) and analysis of the peptides obtained by resolutive gel electrophoresis, without sequencing. We have determined that the order of accessibility of CK MM cysteine residues during SDS denaturation is Cys-282, Cys-145 and then Cys-253. The fourth cysteine residue, Cys-73, is never titrated even at high SDS/CK molar ratio. In contrast, the three last residues are simultaneously titrated when CK is denatured in guanidinium chloride. Thus, SDS-denatured CK seems to retain some residual organized structure. In order to confirm this hypothesis, compactness of the molecule was estimated by fluorescence energy transfer between CK tryptophans and AEDANS, an extrinsic fluorophore. The location of this fluorophore on the accessible thiol of Cys-282 was verified by the previous technique. The results of these experiments do indicate that SDS-denatured CK is more compact than CK completely unfolded in guanidinium chloride.

Rat liver glucose-6-phosphatase system: light scattering and chemical characterization.

Glucose-6-phosphatase is a multicomponent system located in the endoplasmic reticulum, involving both a catalytic subunit (G6PC) and several substrate and product carriers. The glucose-6-phosphate carrier is called G6PT1. Using light scattering, we determined K(D) values for phosphate and glucose transport in rat liver microsomes (45 and 33mM, respectively), G6PT1 K(D) being too low to be estimated by this technique. We provide evidence that phosphate transport may be carried out by an allosteric multisubunit translocase or by two distinct proteins. Using chemical modifications by sulfhydryl reagents with different solubility properties, we conclude that in G6PT1, one thiol group important for activity is facing the cytosol and could be Cys(121) or Cys(362). Moreover, a different glucose-6-phosphate translocase, representing 20% of total glucose-6-phosphate transport and insensitive to N-ethylmaleimide modification, could coexist with liver G6PT1. In the G6PC protein, an accessible thiol group is facing the cytosol and, according to structural predictions, could be Cys(284).

Involvement of a tyrosine residue in the ADP binding site of creatine kinase. A second‐derivative UV‐spectroscopy study

Second‐derivative spectroscopy was used to determine the percentage of tyrosine residues that are exposed to solvent in rabbit MM‐creatine kinase. Six residues, among the ten present per monomer, are solvent‐exposed. The presence of creatine in the incubation medium does not modify this value. However, this number is decreased by one when the enzyme is incubated with saturating concentrations of MgADP. A dissociation constant for MgADP can be estimated and the obtained value (0.085 mM) is comparable to the Km for this substrate. Thus, a tyrosine residue is located near the MgADP binding site or is masked during protein conformational change induced by adenyl nucleotide binding.