A M3 muscarinic receptor coupled to inositol phosphate formation in the rat cochlea?

Various neuroactive substances, including excitatory and inhibitory amino acids, biogenic amines and neuropeptides, were tested for their ability to stimulate the inositol phosphate (IPs) cascade in the presence of lithium in the rat cochlea. Among them, only the muscarinic agonists (carbachol and oxotremorine M) were able to stimulate the IPs formation in 12-day-old rat cochleas. The carbachol-elicited IPs formation was inhibited by muscarinic antagonists with the following relative order of potency: atropine greater than 4-DAMP much greater than pirenzepine greater than methoctramine = AF-DX 116. This pharmacological profile suggests that the activation of the M3 muscarinic receptor subtype is responsible for the increase in IPs synthesis in the rat cochlea. However, an interaction with a m5 receptor subtype could not be completely excluded. The unusual link of only one receptor subtype with the phosphoinositide breakdown in the cochlea, as opposed to the usual existence of several receptors coupled to this transduction system in other organs such as the brain, suggest a unique role for muscarinic agonists in the cochlea.

Common pharmacological and physico-chemical properties of 5-HT3 binding sites in the rat cerebral cortex and NG 108-15 clonal cells.

On account of the postulated existence of 5-HT3 receptor subtypes, the respective physico-chemical and pharmacological properties of specific binding sites for the potent 5-HT3 antagonist [3H]zacopride were compared using membranes from the rat posterior cortex or neuroblastoma-glioma NG 108-15 clonal cells. In both membrane preparations, [3H]zacopride bound to a single class of specific sites with a Kd close to 0.5 nM. However, the Bmax value in NG 108-15 cell membranes (970 +/- 194 fmol/mg protein) was approximately 50 times larger than that in cortical membranes (19 +/- 2 fmol/mg protein). The specific binding of [3H]zacopride was equally affected by temperature, pH and molarity of the assay medium, and equally insensitive to thiol- and disulfide-reagents (N-ethylmaleimide, p-chloromercuribenzene sulfonic acid, dithiothreitol) and GTP in cortical as well as NG 108-15 cell membranes. Determination of the molecular size of [3H]zacopride specific binding sites by radiation inactivation yielded values close to 35 kDa for both membrane preparations. Finally, a highly significant positive correlation (r = 0.979) was found between the respective pKi values of 34 different drugs for their inhibition of [3H]zacopride specific binding to cortical or NG 108-15 cell membranes. Among them, the most potent was S(-)zacopride (pKi = 9.55), followed by BRL 43964, ICS 205-930, quipazine, R(+)zacopride, GR 38032F and MDL 72222. Atypical antidepressants (mianserin, amoxapine) and neuroleptics (clotiapine, loxapine and clozapine) were active in rather low concentrations (pKi less than 6.5), suggesting that recognition of 5-HT3 sites might be relevant to part of the in vivo effects of these drugs. Such identical physico-chemical and pharmacological properties of [3H]zacopride specific binding in cortical and NG 108-15 cell membranes strongly suggest that the same 5-HT3 receptor (subtype?) exists in these two preparations.

High affinity binding site for (+) amphetamine in rat hypothalamus: Fact or artefact?

We studied the binding of (+)(3)H-amphetamine to membrane sites from rat hypothalamus. Our experiments demonstrate the possible existence of artefactual results in previous studies due to the inadequate use of a filtration technique. In addition, it seems that monoamine oxidase A might be a component of the complex mixture of binding sites which are recognized by (+)(3)H-amphetamine since harmaline and monoamine oxidase A selective inhibitors are good displacers of this ligand.

Immunocytochemical localization in rat brain of the enzyme that synthesizes ?-hydroxybutyric acid.

?-Hydroxybutyric acid, a reductive catabolite of GABA, has numerous neuropharmacological and neurophysiological properties when injected systematically to animals. Recently, a specific succinic semialdehyde reductase (SSR2) has been isolated from rat brain. This enzyme specifically produces [(3)H]?-hydroxybutyrate from [(3)H]GABA when incubated in vitro with rat brain tissue slices. A specific antibody against this enzyme has been raised in the rabbit and employed to localize by immunocytochemical procedures the sites of ?-hydroxybutyrate synthesis in two regions of rat brain, the nucleus Raphe dorsalis and the median hypothalamus. Light microscopy reveals the presence of numerous SSR2-positive reactions in the cytoplasm of fusiform or ovoid cells. High magnification shows that only neurons of varous sizes are stained; the cytoplasm is uniformly labelled with a few punctate deposits. At the electron microscopic level, some staining appears in the somata of neurons and in fibres or axonal terminals.

Multiple Cl(-)-independent binding sites for the excitatory amino acids: glutamate, aspartate and cysteine sulfinate in rat brain membranes.

As we have recently reported that Cl(-)-dependent glutamate (GLU) binding reflects GLU accumulation into membrane vesicles, the characteristics, kinetics and pharmacological specificities of L-[3H]glutamate (L-[3H]GLU) binding to crude rat brain synaptic membranes, were investigated in Cl(-)-free medium. L-[3H]GLU binding was systematically compared to that of L-[3H]cysteine sulfinate (L-[3H]CSA) and L-[3H]ASP), two other putative excitatory amino acids. A high affinity site was determined for each of these radioactive ligands (L-[3H]GLU: Kd = 0.14 microM, Bm = 3.4 pmol/mg protein; L-[3H]CSA: Kd = 0.07 microM, Bm = 2.2 pmol/mg protein; L-[3H]ASP: Kd = 5.8 microM, Bm = 31.2 pmol/mg protein). The pharmacological specificity of these Cl(-)-independent binding sites indicate the existence of at least 3 distinct high affinity sites, all different from the Cl(-)-dependent GLU binding 'site': one having a similar affinity for GLU and CSA, a second one preferring CSA, and a third one preferring ASP. Among the large quantity of structural analogs of the neuroexcitatory amino acids tested, only endogenous compounds (GLU, ASP and CSA) (except hydroxylamine-o-sulfate) were able to interact efficiently. No inhibition by classical agonists and antagonists (such as N-methyl-D-aspartate, quisqualate, kainate, 2-amino-4-phosphonobutyrate, or 2-amino-5-phosphonovalerate) was found. In addition to their high specificity, these Cl(-)-independent sites possess most other biochemical characteristics of receptor proteins.

Molecular pharmacology of niaprazine.

1. The pharmacological profile of niaprazine was investigated using in vitro ligand binding techniques. 2. Niaprazine exhibits a low affinity for the vesicular monoamine transporter and for D2, alpha 2, beta, H1 and muscarinic cholinergic receptors. Niaprazine, particularly the (+)stereoisomer, has a higher affinity for alpha 1 (Ki = 77 nM) and 5-HT2 (Ki = 25 nM) binding sites, but is poorly recognized by 5-HT1A and 5-HT1B binding sites (Ki sigma mciroM). In contrast, p-fluoro-phenylpiperazine, a major metabolite of niaprazine, exhibits a higher affinity for the 5-HT1 subclasses than for the 5HT2 class. 3. These results suggest that the pharmacological properties of niaprazine reflect both its non-reserpinic catecholamine depletor effect and its action on alpha 1 and 5-HT2 receptors. A role of p-fluoro-phenylpiperazine via 5-HT1 sites cannot be excluded.

High affinity binding sites for gamma-hydroxybutyric acid in rat brain.

The existence of a specific synthesizing enzyme for gamma-hydroxybutyric acid in rat brain has recently been reported. Here, for the first time, we demonstrate the presence of a high affinity, apparently specific binding site for this compound in the same tissue. This binding does not require Na+ and takes place optimally at pH 5.5. The bound gamma-hydroxybutyric acid is not displacable by GABA or baclofen. We report here on some structurally related compounds of GHB with a similar or better binding capacity than GHB itself. The number of binding sites increases with age up to adulthood and differs depending on the brain region. In primary tissue cultures of pure chicken neurones and glia, gamma-hydroxybutyric acid binding occurs exclusively in the neuronal preparations.

Investigation on central dopaminergic receptors (D-2) using the antagonistic properties of new benzamides.

The topography of the central dopaminergic receptor (D-2) has been studied using some analogues of tropapride, a new benzamide derivative, and sulpiride and clebopride as reference drugs. The compounds were compared by testing their ability to compete with [3H]spiperone in an in-vitro binding test and by measuring their potency as antagonists of apomorphine-induced climbing in mice. Tropapride was the most active compound, both in-vitro and in-vivo. With the amide group substituted in the 2-position of the tropane ring, the antidopaminergic activity of the compounds was much less than that of the 3-substituted derivatives. The interaction of the tropane derivatives with the D-2 receptor site is stereoselective as the equatorial stereoisomer was much more active than the axial isomer. The ethylene bridge present in the tropane derivatives but not in the corresponding piperidinyl analogues increases the affinity of the tropane derivatives for the D-2 receptor. Interaction with the D-2 receptor was confirmed as being Na+-dependent. The presence of a benzyl substituent on the basic nitrogen atom seems to be essential in the tropane series emphasizing the important role played in this series by the lipophilic auxiliary binding site postulated in Olson's model. In conclusion, the tropane skeleton may be considered a useful pharmacophoric group in the design of new dopaminergic drugs.

Isoflavones protect against diesel engine exhaust injury in organotypic culture of lung tissue.

The wide range of potential health beneficial effects of isoflavones, including a chemoprentive action, have prompted us to study the potential benefits of genistein and daidzein in an experimental model of environmental pollution impact on lung tissue. A diesel engine placed was used to generate reproducible emissions including both gaseous and particulate matters that are commonly found in urban atmospheres. Isoflavones were added to culture medium of rat lung slices 2 h prior to their exposure to pollutants for 3 h. Intracellular ATP and GSH levels, TNFα production, nucleosome assay and TUNEL labeling were monitored. Isoflavones showed almost total in vitro protection against inflammatory and pro-apoptotic responses in lung slices. Isoflavones 0.3 and 1 µmol/l protected against exhaust induced GSH depletion. Isoflavones 0.3 µmol/l appeared to exert the most beneficial effects. In conclusion, this study points out the potential interest of soy isoflavones consumption in polluted areas. Further studies should be undertaken to verify that similar effects could be obtained after in vivo administration of isoflavones.

[Mechanisms of action and biochemical toxicology of valproic acid]. / Mécanismes d'action et toxicologie biochimique de l'acide valproïque.

The first part of this article presents the hypotheses of the mechanism of action of the anti-epileptic drug, valproic acid (VPA). In the case of the GABAergic hypothesis, two major types of mechanism of action have been proposed, one at the pre-synaptic level, the other at the post-synaptic level. The action at the pre-synaptic level brings into play one or more enzymes of the GABA shunt. The action at the postsynaptic level consists of the potentiation of the inhibitory effect of GABA by VPA. This has justified the examination of the possible action of VPA at the level of the postsynaptic GABAergic receptor complex. The non-GABAergic hypotheses have been also considered to explain the anti-epileptic action of VPA, one hypothesis depends on the effects of VPA directly on the membrane, another hypothesis brings into play aspartate, and finally a hypothesis depending on the inhibition of aldehyde reductases. The second part of this article concerns the possible mechanism for the undesirable effects of VPA such as hyperammonaemia, hepatotoxicity and hypoglycaemia. The role played by beta- and omega-oxidation of VPA in the explanation of the undesirable effects of this molecule is particularly discussed.

Purification and characterization of G proteins from human brain: modification of GTPase activity upon phosphorylation.

Three G proteins from human brain membranes were purified to near homogeneity by conventional techniques including preparative electrophoresis. These G proteins were characterized by their ability to bind GTP, GDP and GTP analogs. Two of these proteins have molecular weights of 50,000 (G50) and 36,000 (G36), as determined on SDS-gels. G36 was ADP-ribosylated by pertussis toxin. Thus, G50 could represent a Gs alpha subunit, whereas G36 could be Gi alpha or Go alpha. G50 was phosphorylated by cAMP dependent protein kinase and protein kinase C. G36 was phosphorylated by a protein kinase independent of calcium and phospholipid, a proteolytic product of protein kinase C, analogous to protein kinase M. Phosphorylation of G36 by this protein kinase induced a dramatic decrease in its GTPase activity. The third G protein, of molecular weight 22,000 probably belongs to the group of monomeric G proteins possessing functional similarities with ras gene products. The regulation of G proteins involving calcium-dependent and independent pathways is delineated.

Subcellular distribution of gamma-hydroxybutyrate binding sites in rat brain principal localization in the synaptosomal fraction.

gamma-Hydroxybutyrate binding sites, first described on crude membranes from rat brain, have been further studied on subcellular fractions. The nerve ending fraction (fraction C) exhibits the maximal capacity for GHB binding. The two classes of binding sites (high and low affinities) described for the crude membrane preparation are enriched in this synaptosomal fraction. This result is further evidence in favor of a role for GHB as a neurotransmitter or neuromodulator in rat brain.

Positive cooperativity in high affinity binding sites for gamma-hydroxybutyric acid in rat brain.

High affinity binding sites for gamma-hydroxybutyrate have recently been shown to exist on crude membranes of rat brain. These sites exhibit a dissociation constant of 95 nM and a capacity of 557 fentomoles per mg protein. However, after more extensive washing of the crude membrane fraction and performing binding experiments at a lower concentration of radioactive GHB (below 20 nM), the existence of another binding site for GHB with a higher affinity than previously described was discovered. The data concerning this binding site are in favour of positive cooperative binding characteristics. This binding site may play a role in the mediation of the multiple physiological and pharmacological effects of GHB in the rat CNS and its presence provides additional evidence in favour of a neuromodulator or neurotransmitter role of GHB.

Ontogeny and distribution of specific succinic semialdehyde reductase apoenzyme in the rat brain.

The ontogeny and distribution in rat brain of specific succinic semialdehyde reductase is described. This enzyme is probably responsible for the synthesis of gamma-hydroxybutyrate in brain. The highest activities and levels of apoenzyme are found in cerebellum, olfactory bulb, septum and median hypothalamus. During neonatal development, the enzyme activity remains stable at least until 63 days of age. As the levels of other enzymes of the GABA shunt pathway increase during this same period, this result indicates that there is a relative decrease in the reductive pathway of succinic semialdehyde catabolism during development leading to gamma-hydroxybutyrate synthesis, compared to the oxidative pathway leading to succinate.

Regional and subcellular localization in rat brain of the enzymes that can synthesize gamma-hydroxybutyric acid.

Rat brain contains two major NADPH-linked aldehyde reductases that can reduce succinate semialdehyde to 4-hydroxybutyrate. One of these enzymes appears to be fairly specific for succinate semialdehyde and is not significantly inhibited by classic aldehyde reductase inhibitors such as barbiturates. The other enzyme can reduce several aromatic aldehydes and is strongly inhibited by barbiturates and branched-chain fatty acids. Using one such inhibitor, it was possible to distinguish between and measure the two enzyme activities separately in various rat brain regions and in subcellular fractions. Both enzymes are mainly cytoplasmic but there is some activity in the synaptosomal fraction. The activity of the specific succinic semialdehyde reductase is highest in the cerebellum, where it represents 21% of the total activity, and lowest in the cortex, where it represents about 11% of the total activity.