Marek Samochocki

ACTIVATION OF SEROTONERGIC 5-HT1A RECEPTOR REDUCES Ca2+- AND GLUTAMATERGIC RECEPTOR-EVOKED ARACHIDONIC ACID AND NO/cGMP RELEASE IN ADULT HIPPOCAMPUS

Stimulation of glutamatergic NMDA receptor in adult rat hippocampal synaptoneurosomes induces statistically significant Ca(2+)-dependent liberation of arachidonic acid (AA) and nitric oxide (NO)-activated cGMP synthesis. NMDA acting for 5 min at 100 microM markedly increases, by approx. 25%, Ca(2+)-mediated AA release from phospholipids of hippocampal synaptoneurosomes. Prolonged stimulation of NMDA receptor up to 10 min has smaller stimulatory effect and enhances AA release by about 6%. Moreover, NMDA activates NO-dependent cGMP production by approx. 5 times more than the Ca2+ itself. Release of both these second messengers is completely blocked by the competitive NMDA antagonist, APV (100 microM). The NMDA-mediated cGMP elevation completely depends on NO action, and is abolished by the specific inhibitor of NO synthase, NG-nitro-L-arginine. Moreover, serotonin at 10 microM in the presence of 10 microM pargyline, potently decreases both Ca(2+)- and NMDA receptor-mediated AA and cGMP release in hippocampal synaptoneurosomes. The agonist of 5-HT1A receptor, buspirone, in a way similar to serotonin itself, counteracts the Ca(2+)- and also NMDA receptor-evoked AA release and cGMP accumulation. An antagonist of 5-HT1A receptor, NAN-190, eliminates the effect of serotonin and buspirone on AA and NO/cGMP liberation. An antagonist of serotonergic 5-HT2 receptor, ketanserin, has no effect on the Ca2+ and serotonin action. These results indicate that serotonin, through 5-HT1A receptor, potently antagonizes the action of excitatory amino acid for AA release and NO/cGMP synthesis in the adult rat hippocampus. In conclusion, the interaction of serotonin with the glutamatergic system in the hippocampus may play an important role in the modulation of a signal transduction pathway, and by this molecular mechanism serotonin may exert a neuroprotective effect on hippocampal neurons.

Ca2+-Independent, Ca2+-Dependent, and Carbachol- Mediated Arachidonic Acid Release from Rat Brain Cortex Membrane

Synaptoneurosomes obtained from the cortex of rat brain prelabeled with [14C] arachidonic acid ([14C]AA) were used as a source of substrate and enzyme in studies on the regulation of AA release. A significant amount of AA is liberated in the presence of 2 mM EGTA, independently of Ca2+, primarily from phosphatidic acid and polyphosphoinositides (poly‐PI). Quinacrine, an inhibitor of phospholipase A2 (PLA2), suppressed AA release by about 60% and neomycin, a putative inhibitor of phospholipase C(PLC), reduced AA release by about 30%. An additive effect was exhibited when both inhibitors were given together. Ca2+ activated AA release. The level of Ca2+ present in the synaptoneurosomal preparation (endogenous level) and 5 μM CaC12 enhance AA liberation by approximately 25%, whereas 2 mM CaC12 resulted in a 50% increase in AA release relative to EGTA. The source for Ca2+‐dependent AA release is predominantly phosphatidylinositol (PI); however, a small pool may also be liberated from neutral lipids. Carbachol, an agonist of the cholinergic receptor, stimulated Ca2+‐dependent AA release by about 17%. Bradykinin enhanced the effect of carbachol by about 10–15%. This agonist‐mediated AA release occurs specifically from phosphoinositides (PI + poly‐PI). Quinacrine almost completely suppresses calcium‐ and carbachol‐mediated AA release. Neomycin inhibits this process by about 30% and totally suppresses the effect of bradykinin. Our results indicate that both phospholipases PLA2 and PLC with subsequent action of DAG lipase are responsible for Ca2+‐ independent AA release. Ca2+‐dependent and carbachol‐mediated AA liberation occurs mainly as the result of PLA2action. A small pool of AA is probably also released by PLC, which seems to be exclusively responsible for the effect of bradykinin.

Nitric Oxide: A Potent Mediator of Glutamatergic Neurotoxicity in Brain Ischemia

Nitric oxide (NO) is an important messenger molecule produced during activation of NMDA receptor. Within the last few years increasing attention has been focused on the N O function related to neuronal plasticity,lJ degeneration and neuronal death due to brain ischemia.3-5 The contrary data on the significance of N O in glutamate neurotoxicity in brain ischemia forced us to study the action of competitive inhibitor of N O synthase, NG-nitro-L-argnine (NNLA), in gerbil model of ischemia.

Modulatory action of arachidonic acid on GABAA/chloride channel receptor function in adult and aged brain cortex membranes

Effect of arachidonic acid on binding parameters of two binding sites on the GABAA receptor and on GABA activated Cl- uptake was investigated in synaptic plasma membrane and in synaptoneurosomes from brain cortex of adult (4-months old) and aged (27-months old) rats. The ligands used were [3H]muscimol, a GABA agonist and [35S]-t-butylbicyclophosphorothionate ([35S]TBPS), a convulsant that binds to the site near the chloride channel. Arachidonic acid increases significantly GABAA agonist binding and concomitantly decreases [35S]TBPS binding in a concentration dependent manner. The analysis of binding parameters in adult brain showed a significant decrease by AA of KD value for low but not for high affinity of [3H]muscimol binding. Concomitantly, AA enhances Bmax values for high affinity binding and has no effect on Bmax value for low affinity binding in synaptic plasma membrane (SPM) from adult brain. In synaptic plasma membrane from aged brain AA increases low and high affinity binding of agonist to GABAA receptor, modified significantly KD value by about 30 and 66%, respectively. On the other hand, AA significantly decreases of [35S]TBPS binding to chloride channel recognition site. Scatchards analysis indicates that this inhibition results from a decrease of total number of binding sites. Moreover, the affinity of [35S]TBPS binding was increased (KD = 37.0 nM for AA-treated membrane, as compared to 69.3 nM KD in control membrane). GABA-dependent chloride uptake into synaptoneurosomes is also inhibited by AA in a concentration dependent manner in adult brain. In aged brain synaptoneurosomes AA has similar inhibitory effect on GABA-activated chloride uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Serotonin, a potent modulator of arachidonic acid turnover, interaction with glutamatergic receptor in brain cortex

Brain cortex synaptoneurosomes actively incorporated [14C]arachidonic acid (AA) into lipids. Serotonin (5-HT), at a concentration range of 10 microM-1 mM, significantly stimulates the incorporation of AA mainly into phosphatidylinositol (PI) of brain cortex synaptoneurosomes. The stimulation rate of AA incorporation by 5-HT was the same in the presence and absence of lysophosphatidylinositol (LPI). However, in the absence of LPI some stimulation of AA uptake was also observed into phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid. Buspirone, an agonist of 5-HT1A receptor, has a similar effect on AA incorporation into membrane lipids as serotonin itself. Moreover, ketanserin, an antagonist of 5-HT2 receptor, also induces activation of AA incorporation into membrane lipids. On the other hand, glutamate, in a concentration dependent manner, significantly inhibits AA uptake into PI and also has some inhibitory action on AA uptake into the other lipids. Serotonin itself and the agonist of 5-HT1A receptor through the activation of AA turnover counteract glutamate-induced inhibition of AA uptake into lipids of brain cortex. Our results indicated that serotonin directly, through the specific receptors, or indirectly, through the interaction with glutamatergic receptors, modulates turnover and the level of arachidonic acid in the brain.

Aging Diminishes Serotonin-Stimulated Arachdonic Acid Uptake and Cholinergic Receptor-Activated Arachidonic Acid Release in Rat Brain Cortex Membrane

Abstract: Synaptoneurosomal and synaptosomal fractions from the brain cortex of adult (4‐month‐old) and aged (27‐month‐old) rats were used for studies on the uptake and subsequent release of [14C]arachidonic acid ([14C]AA) from brain lipids. The incorporation of AA and the pattern of its uptake into lipids of the aged brain cortex synapto‐neurosomes and synaptosomes were not significantly different when compared with those in the adult brain cortex fractions. Serotonin (5‐HT), at 10 μM to 1 μM in the presence of pargyline and the agonist of the 5‐HT1A receptor, buspirone, stimulated AA uptake into membrane lipids, mainly into phosphatidylinositol, by about 40% exclusively in adult brain synaptoneurosomes. Aging significantly diminished the effect of 5‐HT on AA uptake. Synaptoneurosomal and synaptosomal fractions prelabeled with [14C]AA were used subsequently for investigation of voltage‐dependent, muscarinic and 5‐HT receptor‐mediated AA release. Aging diminished markedly carbachol‐stimulated Ca2+‐dependent AA liberation from membrane lipids of synaptoneurosomes and synaptosomes. Moreover, aging decreased voltage‐dependent and 5‐HT2 receptor‐mediated AA release. These results show that aging affects receptor‐dependent AA uptake and pre‐and postsynaptic receptor‐mediated AA release. These modulations of AA incorporation and release in aged brain may be of patho‐physiological significance, in view of the importance of these processes for signal transmission in the brain. The changes of receptor‐dependent processes of deacylation and reacylation may be responsible for alteration in the function of neuronal cells and may affect learning and memory ability and brain plasticity during aging.

Carbachol-Stimulated Release of Arachidonic Acid and Eicosanoids from Brain Cortex Synaptoneurosome Lipids of Adult and Aged Rats

Synaptoneurosomes from the brain cortex of adult rats (4 months old) and aged rats (27 months old), prelabeled with [14C]arachidonic acid (AA), were used as the source of enzyme(s) and substrates to study the effect of a cholinergic agonist on the release of AA and eicosanoids. In synaptoneurosomes from adult brains, carbachol, the nonhydrolyzable analog of acetylcholine, increased AA release by 16% in the presence of 2 mM calcium. This agonist-mediated AA release occurred specifically from phosphatidylinositol (PI). Concomitantly, carbachol in the presence of 2 mM Ca2+ significantly activated the formation of 15-HETE and PGF2 alpha. This effect of carbachol on the level of eicosanoids was also observed in the presence of endogenous calcium. In synaptoneurosomes from aged brains, carbachol had no effect on the release of AA and eicosanoids. The results of studies involving inhibitors of phospholipase A2 (PLA2) and phospholipase C (PLC) suggested that PLA2 is almost completely responsible for the Ca(2+)-dependent, carbachol-mediated AA liberation. The distribution of labeled AA in the lipids after incubation of synaptoneurosomes in the presence of 2 Mm Ca2+ and carbachol indicated that in aged synaptoneurosomes, the muscarinic receptor-mediated degradation of phosphoinositides through phospholipase C is preserved, but the turnover of the phosphoinositide cycle is probably suppressed. These results indicate that aging significantly affects the population of cholinergic-muscarinic receptors coupled to PLA2.

Age‐Related Changes of GABA‐Activated Chloride Channel Properties in Brain Cortex Synaptic Plasma Membrane: Evidence for Phospholipase Involvement

Abstract: Brain aging decreases binding of tert‐butylbicyclophosphorothionate (TBPS), a specific ligand for Cl− channels, but has no effect on Cl− influx. Detailed studies on the kinetics of TBPS dissociation allowed the characterization of Cl− channel properties. Aging lowers, exclusively in the presence of GABA agonist, muscimol, the half‐life of the fast phase of TBPS dissociation, indicating an opening time of receptor‐dependent Cl− channels shorter than that in adult brain. The half‐life of the slow phase of TBPS dissociation is significantly lower in aged brain in the presence and absence of muscimol. These results suggest a sustained Cl− current, including also the other channel(s) not connected with GABAA receptor activation. The analysis of biphasic TBPS dissociation demonstrates a lowered number of binding sites resulted in the reduction of the number of Cl− channels in the “open” state. This may explain an observed decrease of TBPS binding in aged brain. One of the possible factors involved in modification of GABAA receptor behavior during aging may be arachidonic acid or diacylglycerol, known to be accumulated in aged brain. The action of these compounds on the Cl− channel, observed in this study, correlates well with the effect of aging.

The negative coupling between serotonin and muscarinic receptor(s) for arachidonic acid and inositol phosphates release in brain cortex synaptoneurosomes. Effect of aging

In the present study we have investigated the relationship between serotonergic receptor(s) (5-HTR) and muscarinic cholinergic receptor (mAChR) activation, leading to the arachidonic acid (AA) release and inositol phosphates (IPs) formation in adult and aged brain cortex synaptoneurosomes. It was observed that serotonin (5-HT) almost completely inhibits carbachol-stimulated AA release in adult brain. This negative coupling between 5-HTR and mAChR probably depends on the direct stimulation of the acylation reaction coupled with 5-HT1AR. In the aged brain this type of interaction does not occur because aging eliminates carbachol and 5-HT effect. On the other hand, in aged brain, both 5-HT and carbachol more actively stimulate IPs accumulation than in adult brain. Serotonin inhibits carbachol-stimulated IPs release to the level observed during 5-HT2R activation. Our study indicates for the first time, the negative coupling between 5-HTR and mAChR for AA liberation in adult brain and a lack of this kind of receptors interaction in senescent brain. In adult brain, 5-HT1AR-stimulated AA incorporation is responsible for this coupling. Moreover, serotonin through the stimulation of 5-HT2R, suppresses mAChR-dependent IPs liberation in adult and aged brain similarly. The interactions between these two neurotransmitter receptors lead to the modification of lipid mediators formation and may have important implications in alteration of signal transduction in adult and in aged brain.

Nitric oxide responsible for NMDA receptor-evoked inhibition of arachidonic acid incorporation into lipids of brain membrane.

The activation of the glutamatergic NMDA receptor has no effect on arachidonic acid release from cortical synaptoneurosomal lipids prelabeled with [1-14C]arachidonic acid ([14C]AA). However, activation of NMDA receptor leads to the reduction of AA incorporation into rat brain cortex synaptoneurosomal membrane phosphatidylinositol (PI). The competitive NMDA receptor antagonist, 2-amino-5-phosphovaleric acid (APV), completely eliminates the effect of NMDA on this process. More precise analysis of the sequence of events leading to NMDA-induced decrease of AA incorporation indicates that this process is significantly blocked by voltage-gated sodium and calcium channels inhibitors, such as tetrodotoxin (TTX) and omega-conotoxin (CTX), respectively. Then the antagonist of inositol trisphosphate receptor, TMB-8, totally abolishes the effect of NMDA on AA incorporation into PI. The lowering of AA incorporation evoked by NMDA is significantly diminished by nitric oxide (NO) synthase inhibitor, NG-nitro-L- arginine (NNLA). Further studies were carried out with NO donor(s) to explain the mechanism of NO action in the inhibition of AA incorporation into PI. Our results suggest the following sequence of events: opening of voltage-dependent sodium and calcium channels, subsequent activation of PI-4,5-bisphosphate-specific phospholipase C (PLC), elevation of inositol trisphosphate (IP3)-sensitive calcium ions, stimulation of NO production and NO-mediated S-nitrosylation, or free radical effect on enzymes involved in AA incorporation. Our data suggest that NO-mediated events may be responsible for NMDA-evoked inhibition of AA incorporation into PI of synaptoneurosomal membrane.