Local inputs to aldosterone-sensitive neurons of the nucleus tractus solitarius.

Aldosterone-sensitive neurons in the nucleus tractus solitarius (NTS) become activated during sodium depletion and could be key neural elements regulating sodium intake. The afferent inputs to these neurons have not yet been defined, but one source may be neurons in the area postrema, a neighboring circumventricular organ that innervates the NTS and exerts a powerful inhibitory influence on sodium appetite [Contreras RJ, Stetson PW (1981) Changes in salt intake after lesions of the area postrema and the nucleus of the solitary tract in rats. Brain Res 211:355-366]. After an anterograde axonal tracer was injected into the area postrema in rats, sections through the NTS were immunolabeled for the enzyme 11-beta-hydroxysteroid dehydrogenase type 2 (HSD2), a marker for aldosterone-sensitive neurons, and examined by confocal microscopy. We found that some of the aldosterone-sensitive neurons received close appositions from processes originating in the area postrema, suggesting that input to the HSD2 neurons could be involved in the inhibition of sodium appetite by this site. Axonal varicosities originating from the area postrema also made close appositions with other neurons in the medial NTS, including the neurotensin-immunoreactive neurons in the dorsomedial NTS. Besides these projections, a dense field of neurotensinergic axon terminals overlapped the distribution of the HSD2 neurons. Neurotensin-immunoreactive axon terminals were identified in close apposition to the dendrites and cell bodies of some HSD2 neurons, as well as unlabeled neurons lying in the same zone within the medial NTS. A local microcircuit involving the area postrema, HSD2 neurons, and neurotensinergic neurons may play a major role in the regulation of sodium appetite.

Presynaptic N-methyl-D-aspartate receptor activation inhibits neurotransmitter release through nitric oxide formation in rat hippocampal nerve terminals.

In brain synapses, nitric oxide synthase activation is coupled to N-methyl-D-aspartate-mediated calcium entry at postsynaptic densities through regulatory protein complexes, however a presynaptic equivalent to this signaling mechanism has not yet been identified. Novel evidence indicates that N-methyl-D-aspartate glutamate receptors may play a presynaptic role in synaptic plasticity. Thus, we investigated whether ionotropic glutamate receptor activation in isolated nerve terminals regulates neurotransmitter release, through nitric oxide formation. N-Methyl-D-aspartate dose-dependently inhibited the release of glutamate evoked by 4-aminopyridine (IC(50)=155 microM), and this effect was reversed by the N-methyl-D-aspartate receptor antagonist D-(-)-2-amino-5-phosphopentanoic acid and by the nitric oxide synthase inhibitor, L-nitroarginine, in synaptosomes isolated from whole hippocampus, CA3 and CA1 areas, but not from the dentate gyrus. In contrast, the 4-aminopyridine-evoked release of glutamate was reduced by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid or kainate by a nitric oxide-independent mechanism, since it was not blocked by L-nitroarginine, and N-methyl-D-aspartate, but not alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid or kainate, significantly increased cGMP formation. Presynaptic N-methyl-D-aspartate receptors are probably involved since removing extracellular nitric oxide with the scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide did not block the depression of glutamate release by N-methyl-D-aspartate. The mechanism underlying this depression involves the inhibition of synaptic vesicle exocytosis since N-methyl-D-aspartate/nitric oxide inhibited the release of [3H]glutamate and [14C]GABA evoked by hypertonic sucrose. The results also suggest that presynaptic N-methyl-D-aspartate receptors may function as auto- and heteroreceptors.

Both protein kinase G dependent and independent mechanisms are involved in the modulation of glutamate release by nitric oxide in rat hippocampal nerve terminals.

We compared the effects of sodium nitroprusside (SNP), and of 8-bromo guanosine 3',5'-cyclic monophosphate (8-BrcGMP), on the 4-aminopyridine (4-AP)-evoked Ca2+-dependent release of glutamate from hippocampal nerve terminals and further investigated the role of protein kinase G (PKG) in this mechanism. SNP and 8-BrcGMP dose-dependently inhibited glutamate release, however SNP concentrations ([SNP]) > 500 microM abolished the 4-AP evoked release, whereas 8-BrcGMP maximally inhibited the release by about 30%. The inhibition of glutamate release at low concentrations of SNP (< or = 5 microM) was of about 20%, and was reversed by Rp-8(4-chlorophenylthio)guanosine-3',5'-cyclic-monophosphorotioate ) (RpCPTcGMP, 50 nM), but the inhibition at higher concentrations (5 < SNP < or = 50 microM) was insensitive to the PKG inhibitor, but sensitive to [1 H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one] (ODQ), which partially prevented the inhibition. [SNP] > 50 microM strongly inhibited glutamate release, and this was not reversed by either inhibitor. Furthermore, [SNP] < or = 50 microM enhanced cGMP formation, and the observed effects were not related to either decreased Ca2+ entry or ATP/ADP levels. Our results indicate that NO/PKG is the signaling pathway underlying the inhibition of glutamate release at low concentrations of NO, and imply that other NO-dependent, but PKG-independent, mechanisms are activated and have complementary roles at higher NO concentrations.

Nitric oxide differentially affects the exocytotic and the carrier-mediated release of [3H] gamma-aminobutyric acid in rat hippocampal synaptosomes.

We studied the effects of nitric oxide (NO) on the Ca2+-dependent KCl-evoked release of gamma-aminobutyric acid (GABA) by rat hippocampal synaptosomes, measured in the presence of 1-(2-(((diphenyl-methylene)amino)oxy)ethyl)-1,2,5, 6-tetrahydro-3-pyridine-carboxylic acid (NNC-711), which blocks the GABA carrier. Under these conditions, the NO donor, hydroxylamine, up to 1 mM, inhibited the Ca2+-dependent exocytotic GABA release, but did not affect the basal release. However, in the absence of NNC-711, hydroxylamine concentrations higher than 30 microM caused a two-fold increase in the basal release of GABA, and the KCl-evoked release of GABA was higher than in the presence of NNC-711 because both exocytotic and carrier-mediated release occur. Thus, it is expected that when both release mechanisms are operative, NO inhibits the exocytotic release and stimulates the carrier-mediated release, and the overall effect is an increased liberation of the neurotransmitter from the nerve terminals.

Modulation of glutamate release from rat hippocampal synaptosomes by nitric oxide.

We used hippocampal synaptosomes to study the effect of NO originating from NO donors and from the activation of the NO synthase on the Ca2+-dependent release of glutamate due to 4-aminopyridine (4-AP) depolarization. We distinguished between the effects of NO on the exocytotic and on the carrier-mediated release of glutamate, which we found to be related to an increase in cGMP content and to a reduction of the ATP/ADP ratio, respectively. The NO donor hydroxylamine, at concentrations < or = 0.3 mM, inhibited the Ca2+-dependent glutamate release evoked by 4-AP, and addition of the NO donor, NOC-7, had a similar effect, which was reversed by the NO scavenger, carboxy-PTIO. Increasing the activity of NO synthase by addition of L-arginine also led to a decrease in the Ca2+-dependent release of glutamate induced by 4-AP, and this effect was reversed by inhibiting NO synthase with NG-nitro-L-arginine. This depression of the exocytotic release of glutamate was accompanied by an increase in cGMP levels due to the stimulation of soluble guanylyl cyclase by NO, produced either by the NO donors (hydroxylamine <0.3 mM) or by the endogenous NO synthase, but no significant decrease in ATP/ADP ratio was observed. However, at concentrations > or = 0.3 mM, hydroxylamine drastically increased the basal release and completely inhibited the Ca2+-dependent release of glutamate (IC50 = 168 microM). At these higher levels of NO, cGMP levels dropped to about 40% of the maximal values obtained at lower concentrations, and the ATP/ADP ratio decreased to about 50% (at 0.3 mM hydroxylamine). The large increase in the basal release could be partially inhibited by L-trans-2,4-PDC, previously loaded into the synaptosomes, suggesting that the nonexocytotic basal release occurred by reversal of the glutamate carrier. Therefore, the increase in cGMP induced by NO stimulation of the guanylyl cyclase decreases the exocytotic release of glutamate, but higher NO levels reduce the ATP/ADP ratio by inhibiting mitochondrial function, which therefore causes the massive release of cytosolic glutamate through the glutamate carrier.

Dispersal of proteolipid macroaggregates with trifluoroacetic acid and analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis.

The propensity of highly purified proteolipids to form macroaggregates in aqueous solutions, especially when heated with sodium dodecyl sulfate (SDS), with or without thiol reagents, has made qualitative and quantitative analyses of individual species by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) difficult and unreliable. Comparisons of proteolipid profiles from liver, brain, and cultured human keratinocytes demonstrate that 40-72% of the total proteolipid in SDS-PAGE sample buffer is in the form of macroaggregates. Treatment of proteolipids with neat trifluoroacetic acid (TFA) followed by removal of the TFA and incubation in cold SDS-PAGE sample buffer causes complete dispersal of the macroaggregates and allows recovery of virtually all of the proteolipid applied to gels (increasing yields by as much as 3.6 times, depending on tissue type). Gels of TFA-treated samples display differences not only in the relative amounts of individual species but also in novel species not found in untreated samples. Eluted macroaggregates treated with TFA display the same SDS-PAGE banding profiles as TFA-treated whole proteolipids. Hence, routine TFA treatment of proteolipids prior to SDS-PAGE increases total proteolipid yields, allows reliable quantitation of individual apoprotein species, and reveals species previously obscured by the formation of macroaggregates.