ABSTRACT
Nitric oxide (NO), synthesized by neuronal NO synthase (NOS-I), plays essential physiological roles in the brain. The major molecular target for NO is soluble guanylyl cyclase (sGC), a heterodimeric hemoprotein composed of a larger alpha and a smaller beta subunit. Both subunits of sGC are needed to generate the second messenger cyclic GMP (cGMP). Here we show using subunit-specific antibodies and Western blot analysis that sGCalpha1 and sGCbeta1 protein subunits are present in all examined human brain regions. The relative distribution of the two subunits was similar and also correlated well with the known distribution of NOS-I. The highest expression levels of sGC were found in cortex, basal ganglia and the limbic system. These regions display the most prominent biochemical and histological changes during ageing. In cortex, a negative correlation between the amounts of sGC and age was found, while sex and post-mortem delay time did not affect sGC levels significantly. Our data suggest that sGCalpha1 and sGCbeta1 subunits are widely distributed in human brain, consistent with a major role in NO signaling. Moreover, the NO/cGMP pathway appears to be affected by ageing in the human brain.
Subject(s)
Brain/enzymology , Nerve Tissue Proteins/biosynthesis , Nitric Oxide Synthase/metabolism , Receptors, Cytoplasmic and Nuclear/biosynthesis , Aged , Aged, 80 and over , Aging/metabolism , Antibody Specificity , Blotting, Western , Brain/anatomy & histology , Brain/growth & development , Cyclic GMP/physiology , Enzyme Induction , Female , Guanylate Cyclase , Humans , Male , Middle Aged , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/immunology , Nitric Oxide/metabolism , Nitric Oxide Synthase Type II , Organ Specificity , Protein Subunits , Receptors, Cytoplasmic and Nuclear/chemistry , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/immunology , Second Messenger Systems/physiology , Soluble Guanylyl CyclaseABSTRACT
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.
Subject(s)
Brain/metabolism , Calcium/metabolism , Carbon Isotopes/metabolism , Cysteine/analogs & derivatives , Nitric Oxide Donors/pharmacology , S-Nitrosothiols , Synaptosomes/metabolism , gamma-Aminobutyric Acid/metabolism , src Homology Domains/physiology , Animals , Cyclic GMP/metabolism , Cysteine/pharmacology , Dibutyryl Cyclic GMP/metabolism , Dose-Response Relationship, Drug , Enzyme Activation , Enzyme Inhibitors/pharmacology , Ethylmaleimide/pharmacology , Guanylate Cyclase/metabolism , Hydroxylamine/pharmacology , Kinetics , Male , Methylene Blue/metabolism , Nitroprusside/pharmacology , Nitroso Compounds/pharmacology , Potassium/metabolism , Rats , Rats, Wistar , Sulfhydryl Reagents/pharmacology , Time FactorsABSTRACT
Nitric oxide (NO) is known to potentiate neurotransmitter release in several types of neuronal cells. In the present study, the influence of NO on the membrane potential of isolated nerve endings (synaptosomes) from rat brain was studied. NO donors--sodium nitroprusside (SNP), S-nitroso-L-cysteine (CysNO), and hydroxylamine (HA)--induced synaptosome depolarization monitored by decreasing accumulation of 86Rb+ and the lipophilic potential-sensitive probe [3H]tetraphenylphosphonium. SNP reduced plasma membrane potential by 3-5 mV with half-maximal effect at approximately 10 microM. More potent NO donors, CysNO and HA, led to significant depolarization of the plasma membrane at 10-100 microM concentrations and also induced depolarization of mitochondria at concentrations above 1 mM. At 10 microM-10 mM concentrations, NO donors inhibited potassium channels; CysNO and HA also suppressed the activity of the sodium pump. NO-induced depolarization was not blocked by guanylate cyclase inhibitor methylene blue and the permeable cGMP analog dibutyryl-cGMP did not affect the membrane potential. The effects of NO donors were mimicked by SH-modifying reagents including 5, 5'-dithio-bis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide (NEM). Non-permeable SH-reagent DTNB caused small depolarization resembling SNP action in its magnitude and kinetics. Significant decrease of potential in the presence of NEM, which permeates through the plasma membrane, was similar to that of CysNO and HA. The data suggest that in the presynaptic nerve endings, NO-induced depolarization of the plasma and mitochondrial membranes involves modification of protein SH-groups. The plasma membrane depolarization is due to the decreased potassium permeability and inhibition of the sodium pump.
