Yves Maurin

High-affinity 3H-substance P binding to longitudinal muscle membranes of the guinea pig small intestine

The binding of 3H-substance P (3H-SP) to longitudinal muscle membranes of the guinea pig small intestine has been characterized. The binding of 3H-SP exhibited a high affinity (Kd = 0.5nM). It was saturable (Bmax = 2 fmoles/mg tissue), reversible, and temperature-dependent. Kinetic studies and competition of 3H-SP binding by unlabeled SP yielded Kd and Ki values, respectively, which were in good agreement with the Kd calculated from saturation studies. The binding of 3H-SP appeared to be dependent on the presence of divalent cations in the incubation buffer. It was displaced by SP and various analogs and fragments in the rank order of SP greater than SP-(2-11) = SP-(3-11) greater than Nle11- SP = physalaemin greater than SP-(4-11) greater than SP-(5-11) greater than eledoisin much greater than SP-(7-11). Our results indicate that 3H-SP binds in longitudinal muscle of the guinea pig small intestine to a biologically relevant receptor which in many respects resembles the SP receptor characterized in the brain and the salivary gland of the rat.

Inhibition by yohimbine of the calcium-dependent evoked release of [3H]GABA in rat and mouse brain slices in vitro.

Calcium-dependent release of [3H]GABA was elicited by electrical stimulation in slices of rat and mouse cerebral cortex or by potassium stimulation in the mouse brain-stem. The stimulation-evoked release of [3H]GABA was inhibited by yohimbine in a concentration-dependent manner. High concentrations of other alpha-adrenoceptor antagonists such as phentolamine, RS 21361, idazoxan, rauwolscine and corynanthine also inhibited [3H]GABA release. This effect was not observed with pseudoyohimbine or prazosin. [3H]GABA release was not affected by exposure to the alpha-adrenoceptor agonists clonidine, M7, noradrenaline or methoxamine. In addition, clonidine did not antagonize the yohimbine-induced inhibition of [3H]GABA release. The inhibitory effect of yohimbine did not result from an interaction with endogenously released noradrenaline since the inhibition was still observed in reserpine-pretreated animals. It is concluded that yohimbine and other alpha 2-adrenoceptor antagonists inhibit the stimulation-evoked release of [3H]GABA through a mechanism which appears to be independent of the blockade of alpha 2-adrenoceptors and which does not involve an interaction with endogenous noradrenaline. The present results indicate that yohimbine exerts non-specific actions on the release of [3H]GABA and that similar effects can be observed with other alpha-adrenoceptor blocking agents in high concentrations. Consequently, when studying the effects of yohimbine and other alpha 2-adrenoceptor antagonists on noradrenergic neurotransmission, the possibility of non-specific effects should be taken into consideration, particularly in the high concentrations range.

Altered postnatal ontogeny of α1- and α2-Adrenoceptor binding sites in the brain of a convulsive mutant mouse (quaking)

Abstract Binding assays of [ 3 H]dihydroalprenolol ([ 3 H]DHA), [ 3 H]prazosin and [ 3 H]clonidine have been performed on whole brain (minus cerebellum) homogenates of the convulsive mutant mice quaking (qk) and the controls of the same strain (C57BL/6J:B6). In 70-day-old mutants (which fully exhibit the qk convulsive phenotype), the binding of [ 3 H]DHA to β-adrenoceptor binding sites was not different from the controls, whereas the binding capacities of [ 3 H]prazosin and [ 3 H]clonidine to α 1 - and α 2 -adrenoceptor sites, respectively, were greatly enhanced. The biphasic ontogenic pattern of α 2 -adrenoceptors had a greater amplitude in the brain of 30- to 90-day-old mutants than in the corresponding B6 controls. In mutants younger than 30 days or older than 90 days, the number of α 2 -adrenoceptor sites was not modified. The number of α 1 -adrenoceptor binding sites was increased in the brain of the mutants, only in animals older than 70 days. In younger mice, the postnatal modulation of α 1 -adrenoceptor sites was identical to the controls. Regional studies were performed in 70-day-old mice. [ 3 H]clonidine binding was increased in the brainstem of the mutants, and to a lesser extent in the cerebral cortex, while it was slightly diminished in the hypothalamic area. [ 3 H]prazosin binding was also increased in the brainstem of the mutants, and decreased in the olfactory bulbs. Our results suggest that the convulsions of the qk mutants are selectively associated with modifications of α- and not β-adrenoceptor binding sites. They suggest that although modifications of either subtype of α-adrenoceptor can be associated with the convulsive phenotype, the roles played respectively by α 1 - and α 2 -adrenoceptors might be different. The comparison of the ontogenic patterns of α 1 - and α 2 -adrenoceptors with the temporal development of the qk convulsive phenotype suggests that modifications of α 2 -adrenoceptors might be associated with the appearance of the convulsions, whereas modifications of α 1 -adrenoceptors might be linked to their maintenance. Similar modifications of both subtypes of α-adrenoceptors in the brainstem of the mutants emphasize the importance of this brain region in the convulsions of qk mice, as previously suggested on the basis of electrophysiological and neurochemical data.

Modulation of α1- and α2-adrenoceptor binding sites in the brain of audiogenic seizure susceptible mice (DBA/2J)

The binding of [3H]dihydroalprenolol ([3H]DHA), [3H]prazonin and [3H]clonidine was assayed in whole brain and various brain regions of audiogenic seizure (AS) susceptible DBA/2J (D2) mice aged 10, 24 and 50 days (i.e. before, during and after their period of AS susceptibility, respectively) and in age-matched C57BL/6J (B6) controls. In whole brain, at 24 days, [3H]DHA binding was similar in the two strains, while the binding of [3H]prazosin and [3H]clonidine was significantly lowered in D2 mice. No difference could be detected in 10 and 50 day old mice with any of the ligands. Regional studies indicated an involvement of the cerebral cortex, the olfactory bulbs and the brain-stem. α- (but not β-)adrenoceptor changes were concomitant with the AS susceptibility period. These changes were unevenly distributed in the brain of D2 mice; they suggest that α1- and α2-adrenoceptor subtypes might play different roles in the AS of the D2 mouse strain.

Supernumerary locus coeruleus neurons as a determinant of inherited epilepsy in the convulsive mutant mouse quaking

In quaking mice (a genetic model of epilepsy with an increased number of noradrenergic neurons) bilateral electrolytic coagulation of locus coeruleus (LC) in adult mice inhibited the convulsions elicited by somatic stimulations while neonatal 6-hydroxydopamine (6-OHDA) treatment remained ineffective upon the convulsions. Biochemical effects of the two treatments differed only in the brainstem where electrolytic lesion decreased while 6-OHDA treatment increased noradrenaline (NA) and 3-methoxy 4-hydroxyphenylethyleneglycol (MHPG) levels. Our results suggest that supernumerary LC neurons mediate the convulsions of the mutants through an action presumably restricted to the brainstem.

Paradoxical antagonism by bicuculline of the inhibition by baclofen of the electrically evoked release of [3H]GABA from rat cerebral cortex slices

The presynaptic regulation of the electrically evoked release of [3H]GABA was studied in the rat cerebral cortex. Among the GABA receptor agonists tested (GABA, SL 75102, muscimol, THIP, isoguvacine, (+/-)-baclofen), only (+/-)-baclofen inhibited the stimulation-evoked release of [3H]GABA. This effect of baclofen was stereoselective in favor of the (-) enantiomer. The inhibition by (+/-)-baclofen of the electrically evoked release of [3H]GABA was antagonized by bicuculline and picrotoxin. Our results suggest that the release of [3H]GABA in vitro can be modulated by a receptor-mediated mechanism which is sensitive to baclofen, bicuculline and picrotoxin but not to GABA, muscimol or THIP.

EFFECT OF TRICYCLIC ANTIDEPRESSANTS ON LYSOSOMAL SPHINGOMYELINASE ACTIVITY

Many cationic amphiphilic drugs give rise in vivo to drug-induced lipidosesl. Chronic treatments are required on experimental animals to obtain the typical lysosomal inclusions and accumulation of all major classes of lipids : cholesterol, phospholipids, triglycerides and gangliosides. In tissue cultures, the same lipid accumulation can be observed when incubating cultured cells for several days with the drugs in the culture medium. We have observed this for two types of cationic amphiphilic drugs, perhexiline maleate2 and tricyclic antidepressants3. Several mechanisms have been suggested : drug-binding to lipids impeding their degradation by lysosomal hydrolases, altered lysosomal pH by lysosomotropic drugs interfering with optimum conditions for enzyme activities. Thus the druginduced lipidoses could be due to the toxic effects of these drugs. Interestingly, as has been found by others studying AY 99444, among all the lysosomal enzymes tested, there was only a decrease in lysosomal sphingomyelinase activity2,3,5,7. In Niemann-Pick disease, it is known that several lipids other than sphingomyelin, including glycosphingolipids and cholesterol accumulate as a secondary consequence of the inborn error of metabolism involving sphingomyelinase. Thus, we wanted to determine whether sphingomyelinase activity could be decreased before any lipid accumulation, and whether it could be related to the mechanism of action of tricyclic antidepressants such as desipramine.

Effect of chronic lithium treatment on isolation-induced behavioral and biochemical effects in mice.

The following effects were induced in mice by a prolonged of isolation (6-7 weeks from weaning): (1) reduction in motor activity; (2) reduction in the effect of oxotremorine on rectal temperature; (3) increase in the response to salbutamol, a beta-adrenergic stimulant. Chronic lithium treatment (2 mg/ml in the drinking water during the last 3-4 weeks of isolation) prevented these phenomena. The number and affinity of beta-adrenergic receptors in the whole mouse brain (excluding the cerebellum) was unmodified by either isolation or by lithium. It is suggested that lithium blocks isolation-induced hypersensitivity, especially of the beta-adrenergic system; this mechanism may be involved in the therapeutic action of lithium.

Brain Gangliosides and Neurological Mutants

Modifications of glycolipids occur during development. Some glycosphingolipids are known to be stage-specific antigens during embryogenesis.Numerous surface antigens developmentally regulated and detected by monoclonal antibodies are glycosphingolipids,some of which being minor components of a cell. Striking modifications of the ganglioside profile occur during development of the nervous system; some of them appear to be linked to cellular proliferation (1), others to neuronal differentiation (1,2), and to myelinogenesis(3,4). Thus it is likely that,in the nervous system,these glycolipids play a role in mechanisms of cell-cell recognition and communication, hormone-receptor or growth factor-receptor interactions.