Met-enkephalin receptor-mediated increase of membrane fluidity modulates nitric oxide (NO) and cGMP production in rat brain synaptosomes.
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)
Deliconstantinos
G
G
Department of Experimental Physiology, University of Athens Medical School, Greece.
Villiotou
V
V
Stavrides
J C
JC
D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives* / Q000494:pharmacology; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism*; D006152:Cyclic GMP / Q000378:metabolism*; D004744:Enkephalin, Methionine / Q000378:metabolism / Q000494:pharmacology*; D005454:Fluorescence Polarization; D007700:Kinetics; D008560:Membrane Fluidity / Q000187:drug effects; D009270:Naloxone / Q000494:pharmacology*; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase; D051381:Rats; D011957:Receptors, Opioid / Q000187:drug effects / Q000502:physiology*; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism*; D013816:Thermodynamics; D019323:omega-N-Methylarginine
D011957:Receptors, Opioid; C033593:methionine-enkephalin receptor; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D009270:Naloxone; D004744:Enkephalin, Methionine; D001120:Arginine; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D006152:Cyclic GMP