Met-enkephalin receptor-mediated increase of membrane fluidity modulates nitric oxide (NO) and cGMP production in rat brain synaptosomes

Abstract

The association of [3H]-Met-enkephalin with synaptosomes isolated from rat brain cortex, when incubated for 30 min at 25 degrees C follows a sigmoid path with a Hill coefficient h = 1.25 +/- 0.04. Binding of Met-enkephalin into synaptosomes was saturable, with an apparent binding constant of 8.33 +/- 0.48 nM. At saturation, Met-enkephalin specific receptors corresponded to 65.5 +/- 7.2 nmol/mg synaptosomal protein. The Hill plot in combination with the biphasic nature of the curve to obtain the equilibrium constant, showed a moderate degree of positive cooperativity in the binding of Met-enkephalin into synaptosomes of at least one class of high affinity specific receptors. Met-enkephalin increased the lipid fluidity of synaptosomal membranes labelled with 1,6-diphenyl-1,3,5-hexatriene (DPH), as indicated by the steady-state fluorescence anisotropy [(ro/r)-1]-1. Arrhenius-type plots of [(ro/r)-1]-1 indicated that the lipid separation of the synaptosomal membranes at 23.4 +/- 1.2 degrees C was perturbed by Met-enkephalin such that the temperature was reduced to 15.8 +/- 0.8 degrees C. Naloxone reversed the fluidizing effect of Met-enkephalin, consistent with the receptor-mediated modulation of membrane fluidity. Naloxone alone had no effect on membrane fluidity. NO release and cGMP production by NO-synthase (NOS) and soluble guanylate cyclase (sGC), both located in the soluble fraction of synaptosomes (synaptosol) were decreased by 82% and 80% respectively, after treatment of synaptosomes with Met-enkephalin (10(-10)-10(-4) M). These effects were reversed by naloxone (10(-4) M) which alone was ineffective in changing NO and cGMP production.(ABSTRACT TRUNCATED AT 250 WORDS)