Involvement of nicotinergic mechanisms in thyrotropin-releasing hormone-induced neurologic recovery after concussive head injury in the mouse.

A behavioral study was performed in an attempt to understand the neuronal mechanisms involved in the thyrotropin-releasing hormone (TRH)-induced improvement of consciousness after concussive head injury in the mouse. Intravenous administration of TRH dose dependently shortened the duration of unconsciousness after concussion in the mouse (ED50 = 3.2 mg/kg). The improvement of recovery evoked by TRH (3 mg/kg i.v.) after concussion was not affected by i.p. pretreatment with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine, alpha-methyl-para-tyrosine, p-chlorophenylalanine, scopolamine or methylscopolamine. However, mecamylamine or hexamethonium i.p. pretreatment completely inhibited the TRH-induced improvement of outcome in traumatic brain injury. The results imply that TRH-induced improvement of recovery after concussion is not associated with increased activity of monoaminergic neurons in the brain. These results suggest that the inhibitory effect of TRH upon unconsciousness after concussion in mice is mainly produced by activation of central cholinergic systems via nicotinic receptors whereas muscarinic receptors seem to be not implicated.

4H-1,2,4-Pyridothiadiazine 1,1-dioxides and 2,3-dihydro-4H-1,2, 4-pyridothiadiazine 1,1-dioxides chemically related to diazoxide and cyclothiazide as powerful positive allosteric modulators of (R/S)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid receptors: design, synthesis, pharmacology, and structure-activity relationships.

A series of 4H-1,2,4-pyridothiadiazine 1,1-dioxides and 2, 3-dihydro-4H-1,2,4-pyridothiadiazine 1,1-dioxides bearing various alkyl and aryl substituents on the 2-, 3-, and 4-positions was synthesized and tested as possible positive allosteric modulators of the (R/S)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptors. Many compounds were found to be more potent than the reference compounds diazoxide and aniracetam as potentiators of the AMPA current in rat cortex mRNA-injected Xenopus oocytes. The most active compound, 4-ethyl-2,3-dihydro-4H-pyrido[3,2-e]-1,2, 4-thiadiazine 1,1-dioxide (31b), revealed an in vitro activity on Xenopus oocytes not far from that of cyclothiazide, the most potent allosteric modulator of AMPA receptors reported to date. Moreover, 31b, but not cyclothiazide, was found to potentiate the duration and the amplitude of the excitatory postsynaptic field potentials induced by electric stimulation in rat hippocampal slices. Such an effect could indicate, for 31b, but not for cyclothiazide, a possible interaction with postsynaptic AMPA receptor binding sites located on hippocampal CA1 neurons. Structure-activity relationships indicated that the structural requirements responsible for a biological activity on AMPA receptors are different from those responsible for an inhibitory activity on the insulin releasing process (putative ATP-sensitive K+-channel openers). For instance, 31b and other related dihydropyridothiadiazines were found to be ineffective as inhibitors of insulin release from rat pancreatic B-cells, in contrast to diazoxide and known pyridothiadiazines reported as ATP-sensitive K+-channel openers. Conversely, the pyridothiadiazines active on B-cells were found to be ineffective as potentiators of the AMPA currents in Xenopus oocytes. Thus, 31b appeared to be more specific than diazoxide as an AMPA receptor modulator. This compound may be considered as a new pharmacological tool, different from diazoxide and cyclothiazide, for studying AMPA receptors. Moreover, 31b can also constitute a new therapeutic agent for the treatment of cognitive disorders.

Suppression of nitric oxide formation by tyrosine kinase inhibitors in murine N9 microglia.

1. Microglial cells represent the first line of defence in the brain against infection and damage. However, under conditions of chronic inflammation and neurodegeneration, excessive activation of microglia can contribute to the neurodegenerative process by releasing a cornucopia of potentially cytotoxic substances including the cytotoxic free radical nitric oxide (NO). Although the cell signalling events implicated in NO formation in peripheral macrophages are well defined, events occurring in the phenotypically homologous cerebral microglial cell are not yet fully characterized. 2. In the present study, a cloned murine microglial cell line (N9), stimulated with combined lipopolysaccharide/interferon-gamma (LPS/IFN) incubation, was shown to produce a significant increase in NO formation, as measured by medium nitrite levels, during 8-72 h exposure. 3. LPS/IFN-stimulated NO production was partially inhibited with the nitric oxide synthase (NOS) competitive antagonists; N(omega)-nitro-L-arginine methyl ester and N(omega)-nitro-L-arginine. The ability of the selective inducible (iNOS) inhibitor, aminoguanidine, but not the selective 'neuronal-type' constitutive (cNOS) inhibitor 7-nitroindazole, to inhibit NO production suggested a primary role of iNOS in this response and was confirmed by immunolabelling of activated cells with a specific iNOS antibody. 4. A series of tyrosine kinase inhibitors, herbimycin A, genestein, tyrphostins, AG-126, AG-556 and the tyrosine phosphatase inhibitors, sodium orthovanadate and phenylarsine oxide, significantly attenuated LPS/IFN-mediated NO production. The serine/threonine kinase inhibitors, staursporine (protein kinase C), H-9 (cyclic GMP/cyclic AMP-dependent kinase) or serine/threonine phosphatase inhibitors, cyclosporin A (phosphatase 2B) and okadaic acid (phosphatase 1/2A), reduced NO formation by an apparent cytostatic mechanism, as determined by cellular reduction of 3-(4,5-dimethylthiazol-2-yi)-2,5-diphenyl-tetrazolium bromide (MTT). 5. The present results suggest that the co-ordinated activation of protein tyrosine kinases/phosphatases, and proximal signalling events implicating the interplay between serine-threonine kinases/phosphatases, is intricately linked with inflammatory mediated mechanisms of iNOS activation in microglial cells by regulating the activation of the transcription factor NFkappaB.

New prolyl endopeptidase inhibitors: in vitro and in vivo activities of azabicyclo[2.2.2]octane, azabicyclo[2.2.1]heptane, and perhydroindole derivatives.

A series of potent and selective prolylendopeptidase (PEP) inhibitors of the alpha-keto heterocyclic type has been obtained by replacing the classical central proline of 1-[1-(4-phenylbutanoyl)-L-prolyl]pyrrolidine (SUAM 1221,3) by non-natural amino acids PHI, ABO, and ABH. These 4-phenylbutanoyl side-chain-containing inhibitors exhibited potent in vitro inhibitory potencies with IC50 around 30 nM (compounds 24 and 25). Modulation of the side chain by replacement of the terminal phenyl ring by the dicyclopropyl moiety afforded derivatives 30 and 32 with improved potencies (IC50 between 10 and 20 nM). Furthermore, replacing the linear 4-phenylbutanoyl side chain by the (2-phenylcyclopropyl)carbonyl entity provided potent inhibitors with IC50 culminating at 0.9 nM on a rat cortex enzymatic preparation (compound 70). The configuration of the cyclopropyl ring had to be R,R in order to obtain not only a strong PEP inhibition in vitro but also a good activity in vivo, exemplified by inhibitor 68, which gave ID50 ip and po of 0.3 and 1 mg/kg, respectively. Finally, demonstration of the cognition-enhancing properties of compound 54 was given in the passive avoidance test using scopolamine-induced amnesia in the rat, where it dose dependently inhibited the scopolamine-induced decrease in avoidance response.

Dephosphorylation studies of SKNSH-SY 5Y cell Tau proteins by endogenous phosphatase activity.

Recent data have shown that the microtubule-associated Tau proteins are phosphorylated but to a lesser extent than PHF-Tau proteins which are the major components of Alzheimer's disease paired helical filaments. These normal Tau proteins are highly sensitive to the endogenous phosphatase activity during post-mortem delay. In order to understand the basic equilibrium between phosphatase and kinase activities, phosphorylation and dephosphorylation mechanisms of Tau proteins were studied in neuroblastoma cells. The present results demonstrate that an endogenous phosphatase activity is present and directed on Tau proteins in the SKNSH-SY 5Y cell extracts. Interestingly, the okadaic acid-induced hyperphosphorylated Tau proteins are more resistant to the phosphatase activity than the control Tau proteins. Our data emphasize the value of this in vitro cellular model for the study of biological conditions that control Tau protein phosphorylation levels.

Structure-activity relationships in a series of 2(1H)-quinolones bearing different acidic function in the 3-position: 6,7-dichloro-2(1H)-oxoquinoline-3-phosphonic acid, a new potent and selective AMPA/kainate antagonist with neuroprotective properties.

Recently, we reported the synthesis of 3-(sulfonylamino)-2(1H)-quinolones, a new series of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate and N-methyl-D-aspartic acid (NMDA)/glycine antagonists. By exploring the structure-activity relationships (SAR) in this series, we were able to identify the 6,7-dinitro derivative 6 as a potent and balanced antagonist at both receptors. Unfortunately, compound 6 was devoid of in vivo activity in mice anticonvulsant testing. To overcome this critical limitation, new compounds bearing various acidic moieties at the 3-position of the quinolone skeleton were synthesized and evaluated. The SAR of these new analogues indicated that not all acidic groups are acceptable at the 3-position: A rank order of potency going from carboxylic approximately phosphonic > tetrazole > mercaptoacetic > hydroxamic >> other heterocyclic acids was defined. In addition, the selectivity between the AMPA/kainate and NMDA/glycine sites is dependent on the nature of the substitution (nitro > chloro for AMPA selectivity), its position (5,7- > 6,7-pattern for glycine selectivity), and the distance between the quinolone moiety and the heteroatom bearing the acidic hydrogen (the longer the distance the more AMPA selective the compound). Among these new AMPA antagonists, we have identified 6,7-dichloro-2(1H)-oxoquinoline-3-phosphonic acid (24c) as a water soluble and selective compound endowed with an appealing pharmacological profile. Compared with the reference AMPA antagonist NBQX, the phosphonic acid 24c is much less potent in vitro but almost equipotent in vivo in the audiogenic seizures model after intraperitoneal administration. Moreover, unlike NBQX, compound 24c is also active after oral administration. In the gerbil global ischemia model, compound 24c shows a neuroprotective effect at 10 mg/kg/ip, equivalent to the reference NBQX.

Structure-activity relationships in a series of 3-sulfonylamino-2-(1H)-quinolones, as new AMPA/kainate and glycine antagonists.

This paper describes the design and synthesis of a new class of molecules, the 3-sulfonylamino-2-(1H)-quinolones, which are potent and selective antagonists at both the AMPA/kainate site as well as at the NMDA-associated glycine site. The molecules were characterized by their binding affinities to rat cortical membranes and by electrophysiology on Xenopus oocytes injected with mRNA isolated from rat cerebral cortex. The most potent compound 61 has an IC50 of 0.09 microM for binding at the AMPA/kainate site, and 0.16 microM in oocyte electrophysiology.

Allosteric potentiation by diazoxide of AMPA receptor currents and synaptic potentials.

Diazoxide (100-560 microM) reversibly increased the amplitude and duration of excitatory post-synaptic field potentials recorded in the dentate gyrus of hippocampal slices following stimulation of the perforant pathway. In rat cortex mRNA-injected Xenopus oocytes diazoxide (1-1000 microM) alone had little effect on membrane current, but rapidly and reversibly increased (up to 5-fold) current responses to (R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA, 30 microM), L-glutamate (100 microM), quisqualate (3 microM), kainate (100 microM) and domoate (3 microM), an effect that was neither mimicked by other activators of ATP-sensitive potassium channels nor blocked by glibenclamide. Diazoxide increased current amplitudes for all concentrations of the 'inactivating' ligands, AMPA, L-glutamate and quisqualate but had little effect on their EC50 values. In contrast, diazoxide increased the apparent potency of the 'non-inactivating' ligands, kainate and domoate, but increased the efficacy of saturating concentrations by only 10-20%. Diazoxide did not modify the competitive inhibition of AMPA and kainate currents by 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione (NBQX) and thus does not compete for the agonist site as do AMPA and kainate. Similarly, diazoxide neither inhibited the binding of [3H]AMPA or [3H]kainate to rat cortical membranes in competition experiments nor consistently modified the apparent [3H]AMPA affinity (Kd) or receptor density (Bmax) in saturation experiments. These data suggest that diazoxide acts at an allosteric site on the AMPA receptor/channel to potentiate activation in a manner dependent upon the properties of the excitatory agonist.

Comparative effects of aging process on phosphatidylcholine biosynthesis pathway: a key role for CTP-phosphocholine cytidylyltransferase?

The effects of aging on phosphatidylcholine (PtdCho) biosynthesis were investigated in liver and brain subcellular fractions of the rat, by studying the activity and regulation of CTP phosphocholine cytidylyltransferase (CT), the rate limiting enzyme in PtdCho biosyntheses. With both tissues, CT activity was present in cytosolic and microsomal fractions, but in brain, CT activity seemed to escape to an inhibiting feed back mechanism. In brain, CT activity was greater in the microsomal fraction, whilst in liver, a higher CT activity was seen in the cytosolic fraction. In liver fractions of aged animals, there was no significant change in CT activity or its sensitivity to negative feedback regulation, as compared to young animals. In contrast, a progressive age related decline in CT activity was observed in the brain microsomal fraction. Furthermore, the incorporation of newly formed CDPCho into PtdCho was also reduced in aged animals, and paralleled the decreased incorporation of choline in PtdCho. The age-related decrease in CT activity cannot be explained by product feed back inhibition or decreased diacylglycerol levels. Since PtdCho is a major membrane lipid, the reduction in CT activity may lead to a decreased membrane integrity and fluidity during the aging process, and these effects may be greater in neuronal cells.

Competitive inhibition by NBQX of kainate/AMPA receptor currents and excitatory synaptic potentials: importance of 6-nitro substitution.

We evaluated the inhibitory potencies at excitatory amino acid receptors of 2,3-dihydroxy-7-sulfamoyl-benzo[f]quinoxaline (BQX) and its 6-nitro derivative, NBQX. Currents activated by kainate or (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) in two-electrode voltage-clamp recordings of Xenopus oocytes injected with rat cortex mRNA were inhibited by BQX and NBQX: the apparent Ki values versus kainate were 14 microM and 78 nM, respectively, and versus AMPA were 23 microM and 63 nM, respectively. Thus, to a degree even more marked than with other quinoxalinedione derivatives, 6-nitro substitution of BQX to yield NBQX increases potency (200-fold) at the non-NMDA ionotropic receptor, but does not confer selectivity for kainate or AMPA. Schild analysis of the NBQX inhibition of the kainate and AMPA currents yielded pA2 values of 7.17 +/- 0.05 and 7.05 +/- 0.10, respectively, and slopes near unity confirming the competitive nature of the inhibition. Neither BQX nor NBQX significantly inhibited the current activated by glycine plus NMDA. The selectivity ratio of NBQX (greater than 5000-fold) is by far the greatest of any quinoxalinedione derivative antagonist of the kainate/AMPA receptor. BQX and NBQX also inhibited the excitatory postsynaptic field potentials mediated by kainate/AMPA receptors in the CA1 region of hippocampal slices after stimulation of the Schaffer collateral-commissural pathways with IC50 values of 130 and 0.90 microM, respectively. The 10-fold differences between the IC50 values in hippocampal slices and the Ki values in Xenopus oocytes correlate closely with data for other quinoxalinedione derivative antagonists.

Quinoxaline derivatives: structure-activity relationships and physiological implications of inhibition of N-methyl-D-aspartate and non-N-methyl-D-aspartate receptor-mediated currents and synaptic potentials.

The inhibitory potencies at excitatory amino acid (EAA) receptors of 11 quinoxaline derivatives were evaluated in two-electrode voltage-clamp recordings of Xenopus oocytes injected with rat cortex mRNA. Currents activated by kainate or (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) in Xenopus oocytes were inhibited competitively by all the quinoxaline derivatives, with apparent Ki values ranging from 0.27 to 300 microM against kainate and from 0.25 to 137 microM against AMPA. An excellent correlation was observed between inhibitory potencies of the quinoxaline derivatives against kainate and AMPA currents, in support of the contention that in this preparation these two agonists act at a single site. All 11 quinoxaline derivatives also inhibited current activated by the combination of glycine and N-methyl-D-aspartate (NMDA), apparently acting at the glycine site, and did so over a narrower range of apparent Ki values (0.37-8.1 microM). The correlation between the quinoxalines' kainate/AMPA potencies and their glycine/NMDA potencies was relatively weak. Thus, the quinoxaline derivatives were all good antagonists of glycine/NMDA currents and displayed a greater range of potencies against kainate and AMPA. The inhibitory effects of the six quinoxaline derivatives most potent in the Xenopus oocyte experiments were also tested against the excitatory postsynaptic field potential (EPSFP) recorded in the pyramidal cell dendritic field of the CA1 region of hippocampal slices after stimulation of the Schaffer collateral-commissural pathways. In slices superfused with "normal" medium (containing 1 mM Mg2+), in which the EPSFP is mediated primarily by non-NMDA receptors, IC50 values correlated closely with the Ki values against kainate/AMPA obtained in oocyte experiments but were approximately 8-fold higher. Similarly, in slices superfused with nominally Mg(2+)-free medium, in which the EPSFP is amplified due to a relief of the Mg2+ block of NMDA receptors, IC50 values correlated closely with the Ki values against glycine/NMDA obtained in oocyte experiments but were 60-fold higher. This comparison of results from the two experimental systems lends further support to the argument that hippocampal synaptic transmission is mediated postsynaptically by kainate/AMPA-type and NMDA/glycine-type EAA receptors that are pharmacologically indistinguishable from those expressed in mRNA-injected Xenopus oocytes. Furthermore, it suggests that EAA receptors in situ may be nearly saturated by high local concentrations of the endogenous ligands, a condition that would contribute substantially to the apparent non-NMDA receptor selectivity of certain quinoxaline derivatives.

[Is calcium a primum movens in the pathogenesis of cerebrovascular accidents?]. / Le calcium est-il un "Primum movens" dans la pathogénèse de l'accident vasculaire cérébral?

Concerning the physiological cascade associated with cerebral vascular accidents, numerous studies have been devoted to a search for a key element (outside the initial fall in blood perfusion) responsible for the noxious jumble that leads to tissue necrosis. As a fundamental element of cell physiology, calcium intervenes in a number of vital functions, but when present in excessive amounts it may engender disorders by accelerating reactions which it normally regulates. Thus, in cerebral tissue, and notably in acute ischaemia, calcium is held responsible for additional phenomena of coagulation, exacerbated neurotransmitter discharge or even cell poisoning by paralysis of mitochondrial respiration. Based on these data, attempts have been made to treat cerebral vascular accidents by the so-called calcium antagonists. Experimentally or clinically, the results obtained, although divergent, show that acting on calcium movements contributes to total or partial correction of the complex physiopathology of cerebral ischaemia. A new generation of calcium antagonists is needed to oppose the fundamental disorders engendered, notably those of mitochondrial function.