Roland Salesse

Study of orexins signal transduction pathways in rat olfactory mucosa and in olfactory sensory neurons-derived cell line Odora: Multiple orexin signalling pathways

Orexins A and B (OxA and OxB) are multifunctional neuropeptides implicated in the regulation of energy metabolism, wakefulness but also in a broad range of motivated behaviours. They signal through two G-protein-coupled receptors: orexin receptor 1 and 2 (Ox1R and Ox2R). The orexins and their receptors are present at all levels of the rat olfactory system: epithelium, bulb, piriform cortex but their signalling mechanisms remain unknown. We have studied orexins signal transduction pathways in the rat olfactory mucosa (OM) and in the Odora cell line derived from olfactory sensory neurons and heterologously expressing Ox1R or Ox2R. We have demonstrated by western blot and RT-PCR that multiple components of adenylyl cyclase (AC) and phospholipase C (PLC) signalling pathways were identical in OM and Odora cells. OxA and OxB induced a weak increase in IP3 in OM; they induced a significant rise in cAMP and IP3 in Odora transfected cells, suggesting the activation of AC and PLC pathways. Both OxA and OxB induced intracellular calcium elevation and transient activation of MAP kinases (ERK42/44) in Odora/Ox1R and Odora/Ox2R cells. These results suggest the existence of multiple orexins signalling pathways in Odora cells and probably in OM, corresponding to different possible roles of these peptides.

Adenylate cyclase and membrane fluidity

SummaryThe relationships between membrane fluidity as induced by drug addition and the stimulation of adenylate cyclase by hormones (mainly catecholamines), GTP, Gpp(NH)p and NaF are reviewed. In particular, the data corresponding to pigeon erythrocyte membranes are reviewed and compared with other data published in the literature. A brief summary of the theories involved in fluidity measurements and their significance at the molecular level is also given for anisotropy of fluorescence and electron spin resonance.One of the conclusions is that the cationic drugs and neutral alcohols by perturbing preferentially the inner half-layer of the bilayer induced in pigeon erythrocyte membrane correlated multiphasic changes on fluidity and adenylate cyclase activity.This and other experimental data concerning the regulation of the adenylate cyclase are discussed in regard to a new interpretation of cyclase stimulation: the repressor hypothesis. In cell membrane the catalytic unit C is repressed by its association with a repressor complex made of the hormone receptor R and the regulatory protein N. The activation of cyclase activity is the dissociation of the catalytic unit C from the repressor complex R.N according to the equilibrium: R.N.C (inactive) ⇌ R.N + C (active). Hormones, metal ions (magnesium), and nucleotides (GTP) are the allosteric ligands which shift this equilibrium towards the dissociation. state with the liberation of the active form, membrane-bound, C unit. Gpp(NH)p, fluoride and forskolin will also shift the equilibrium toward the right. GDP and free receptors favour the associated repressed state of the system.

In vitro activation of glycoprotein hormones Hybridization of subunits from thyrotropin, lutropin and human choriogonadotropin

In vitro assembly of thyrotropin alpha and beta subunits led to an increase in content of alpha helix and beta sheet very similar to that found for gonadotropins. This association-dependent active folding involved the burying of three tyrosine residues tentatively assigned to Tyr alpha 41, Tyr beta 37 and Tyr beta 59 and common to all studied glycoprotein hormones. In vitro hybridizations between alpha and beta subunits of various hormones (thyrotropin, lutropin and choriogonadotropin) from different species (ovine, bovine and human) triggered the same molecular events as assembly of homologous subunits: the burying of three tyrosine residues and the increase of periodic structure of the folding. These changes are slow, time-dependent processes. Rates and yields of hybrid formation measured by sedimentation analysis and difference spectroscopy of tyrosines are identical, within experimental error, with the rates and yields measured by the recovery of the biological activity either the stimulation of chick thyroids for thyrotropin-beta hybrids or binding to porcine testis receptors for gonadotropin-beta hybrids. Whatever the origin of the alpha subunit, the thyrotropin-beta hybrids were not able to bind to testis receptors although active on chick thyroids. Rates and yields of hybrid formation essentially depended on the origin of the beta subunit. All the hybrids could be dissociated at acid pH with rates similar to those of native hormone. The extension to thyrotropin and various hybrids of the structural features of the in vitro assembly already recognized for gonadotropins strengthens the hypothesis that one deals with a basic activation process which also occurs in vivo after the synthesis of the subunits.

Association-dependent active folding of alpha and beta subunits of lutropin (luteinizing hormone)☆

Abstract The kinetics of the structural transition of ovine lutropin (luteinizing hormone) from a dissociated and partially unfolded state to a biologically active, folded and associated state were studied from pH 1.8 to 6.5 by difference spectroscopy, circular dichroism, light scattering and ultracentrifugation under various conditions of temperature and ionic strength. Between pH 2.8 and 5.3 there is a thermodynamically reversible equilibrium between the two states of the hormone with a half-transition pH of 4.3 ± 0.1 at 26 °C. The interconversion of native folding to dissociated state is strictly first-order, and most of the kinetic results can be described in terms of two exponential decays with lifetimes of 20 and 100 seconds at pH 2 and 26 °C with one intermediate. A second intermediate with a short lifetime (1.5 s) is detected with stopped-flow experiments; its spectroscopic contribution is small. Refolding from the dissociated state is always second-order in the concentration range studied (12 to 110 μ m lutropin), with rates at 26 °C, 1.4 m −1 s−1 at pH 4.3 and 2.2 m −1 s−1 at pH 5.3. The temperature dependence of the rate constant of active folding at pH 5.3 corresponds to activation parameters ΔH ∗ = +23.5 kcal mol −1 and ΔS ∗ = +21.6 cal deg −1 mol−1. From these data and computer-simulation studies, a simplest possible mechanism is proposed that involves the formation of a loose complex between the α and β subunits of the hormone, followed by a slow step of formation of the active, folded state. In the pituitary gland this would be a necessary pathway towards the active form. The storage of the hormone in the gland would offer enough time for this activation step to proceed.

Effects of drugs on pigeon erythrocyte membrane and asymmetric control of adenylate cyclase by the lipid bilayer

In pigeon erythrocyte membrane, the beta-adrenergic receptor and the enzyme adenylate cyclase can be uncoupled in two different ways depending on the type of drug used. Cationic drugs: chlorpromazine, methochlorpromazine, tetracaine, n-octylamine and a neutral alcohol, octanol, abolished alprenolol receptor binding ability and in the same range of concentration of the drug, sensitized adenylate cyclase to fluoride or Gpp(NH)p stimulation. Anionic drugs: di- and trinitro-phenols, indomethacin and octanoic acid did not affect the total number of beta-adrenergic receptor sites and, with the exception of trinitrophenol, did not change the association constant for alprenolol but they abolished the stimulation of adenylate cyclase by isoproterenol, fluoride or Gpp(NH)p. These modifications of the adenylate cyclase system occurred in a range of drug concentration where cell shape and protection against hemolysis were also affected. As chemical composition varies widely from one drug to another, it is suggested that these effects are largely nonspecific and mediated by the lipid bilayer. They are probably related to a preferential sidedness of action of the drugs in the lipid bilayer, displaying the role of an asymmetric control of the adenylate cyclase system in the membrane by the two halves of this bilayer.

Change in the physical state of platelet plasma membranes upon ionophore A23187 activation. A fluorescence polarization study

Human platelets were isolated and fluorescence-labelled by 1,6-diphenylhexatriene. Diphenylhexatriene was essentially localized in the plasma membrane, as indicated by trinitrobenzenesulfonate-quenching experiments. A decrease of the fluorescence polarization of diphenylhexatriene was observed upon ionophore A23187 addition in the absence of aggregation. 0.3 microM ionophore allowed to reach the maximum rate of the decrease of fluorescence polarization; it also maximally stimulated the light transmission change, the serotonin release and the thromboxane B2 synthesis. The amplitude of the fluorescence polarization decrease was maximum at platelet concentrations between 4 X 10(7) and 7 X 10(7)/ml. The presence of Ca2+ in the medium increased the rate constant of the polarization change. Chlorpromazine (60 microM) completely inhibited this transition, but at 30 microM its inhibitory effect was reversed by Ca2+. The membrane events implied in platelet activation very likely lead to fluidization of the plasma membrane, perhaps by its fusion with the membranes of internal granules which are relatively depleted of cholesterol. Ca2+ plays a central role in the triggering of the observed effects at the membrane level.

Mesure des déclins de l'intensité et de l'anisotropie de fluorescence du pérylène en présence de membranes d'érythrocyte de pigeon: Comparaison avec un milieu isotrope visqueux

Summary Using synchrotron radiation as the excitation light, we studied the fluorescence parameters of perylene incubated with pigeon erythrocyte membranes and with an isotropic viscous medium, the Primol 342 oil. From 4 to 37°C, we observed a single lifetime of 4.5 ns in the oil and two with the membrane (τ1 = 1−1.4 ns and τ2 = 5.4−6.1 ns). The dependence upon temperature of the rotation correlation time of perylene (ϑ) in the oil was characteristic of an isotropic medium, whereas the limiting value of anitropy (r ∞) was zero. With the membrane, γ ∞ decreased from 0.14 to 0.06 and ϑ from 2.9 to 0.5 ns, indicating a greater amplitude and frequency of molecular motions. The addition of chlorpromazine, indomethacine, tetracaine, n-octylamine, octanol or octanoic acid to the membrane decreased the τ1 and τ2 values. This would stem from the desorganization of the membrane induced by the drugs.