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Nitric oxide (NO) modulates processes of synaptic transmission at pre- and postsynaptic levels. In the present work we studied the mechanisms of action of NO on [gamma-14C]amino-n-butyric acid ([14C]GABA) release in rat cortical synaptosomes. NO donors--S-nitroso-L-cysteine and hydroxylamine (but not sodium nitroprusside)--inhibited the neurotransmitter efflux in a concentration range from 10 microM to 1 mM. Nitrosocysteine completely and selectively suppressed the Ca2+-dependent (vesicular) [14C]GABA release, while not affecting the Ca2+-independent component of the [14C]GABA transport. The influence of NO donors was not related to activation of guanylyl cyclase, since the membrane-permeable cGMP analog dibutyryl-cGMP did not mimic and the guanylyl cyclase inhibitor methylene blue did not change the NO effects. In contrast, the membrane-permeable SH-reagent N-ethylmaleimide (NEM) resembled the effects of NO donors on the Ca2+-dependent [14C]GABA release. The degree of inhibition of the release by nitrosocysteine, hydroxylamine, and NEM correlated with their ability to oxidize intra-synaptosomal SH-groups. These data suggest that synaptosomal sulfhydryl groups are the target for NO action at the presynaptic level. The NO-induced oxidation of thiols may be involved in physiological and, especially, pathological effects of nitric oxide in the central nervous system.

The swelling of nerve terminals of rat brain in a hypotonic medium (230 mOsm) induced the potential-independent entrance of 45Ca2+ into synaptosomes and intrasynaptosomal mitochondria that changed the energy status of synaptosomes, the rate of O2 consumption and the content of ATP being decreased. The ratio ATP/ADP decreased from 6.5 +/- 0.26 (310 mOsm medium) to 3.1 +/- 0.18 (the medium 230 mOsm). Studies on the equilibrium distribution of K+ (86Rb+) and [3H]TPP+ showed that contents of these cations in the nerve terminals were virtually the same on incubation in both iso- and hypotonic media. This indicated that the swelling did not damage intrasynaptosomal mitochondria and plasma membranes of the synaptosomes. The inhibition of oxidative phosphorylation increased twofold the rate of glycolysis. The incubation of synaptosomes in calcium-free medium (230 mOsm) in the presence of EGTA (1 mM) prevented the inhibition of oxidative phosphorylation and synthesis of ATP by the osmotic swelling. Ruthenium Red (10 microM) in the medium 230 mOsm inhibited the entrance of 45Ca2+ into the intrasynaptosomal mitochondria and normalized the oxidative phosphorylation to the control level (310 mOsm medium). The decrease in the energy potential of synaptosomes induced by the hypoosmotic shock is suggested to be associated with the increase in Ca2+ content in the cytoplasm, its transport into the mitochondria, and the inhibitory effect on oxidative phosphorylation.

Incubation of rat brain synaptosomes at pH 6.0 in Ca2+-containing medium is associated with a decrease in the ATP content and the rate of oxygen consumption. ATP/ADP ratio decreased from 6.6 +/- 0.24 at pH 7.4 to 3.2 +/- 0.17 at pH 6.0. The content of 86Rb+ and [3H]tetraphenylphosphonium measured at pH 7.4 did not change after preincubation at pH 6.0, indicating the absence of lesion of synaptosomal plasma membranes and intrasynaptosomal mitochondria. Incubation with 1 mM EGTA in Ca2+-free medium as well as addition of 1 mM ouabain or 10 microM ruthenium red prevents the effect of acidosis. Similar results were obtained when 5 mM pyruvate was used as a mitochondrial substrate instead of glucose. It is suggested that acidosis-induced decrease in the ATP level is associated with the increase in Ca2+ concentration in the cytoplasm and its transport into mitochondria. Ouabain reverses this process due to activation of Na+/Ca2+ exchange.