Immunolocalization of nitric oxide synthase isoforms in human archival and rat tissues, and cultured cells.

Nitric oxide (NO) exerts important physiological and pathological roles in humans. The study of NO requires the immunolocalization of its synthesizing enzymes, neuronal, endothelial and inducible NO synthases (NOS). NOS are labile to formalin-fixation and paraffin-embedding, which are used to prepare human archival tissues. This lability has made NOS immunohistochemical studies difficult, and a detailed protocol is not yet available. We describe here a protocol for the immunolocalization of NOS isoforms in human archival cerebellum and non-nervous tissues, and in rat tissues and cultured cells. Neuronal NOS antigenicity in human archival and rat nervous tissue sections was microwave-retrieved in 50 mM Tris-HCl buffer, pH 9.5, for 20 min at 900 W. Neuronal NOS was expressed in stellate, basket, Purkinje and granule cells in human and rat cerebellum. Archival and frozen human cerebellar sections showed the same neuronal NOS staining pattern. Archival cerebellar sections not subjected to antigen retrieval stained weakly. Antigenicity of inducible NOS in human lung was best retrieved in 10 mM sodium citrate buffer, pH 6.0, for 15 min at 900 W. Inflammatory cells in a human lung tuberculoma were strongly stained by anti-inducible NOS antibody. Anti-endothelial NOS strongly stained kidney glomeruli. Cultured PC12 cells were strongly stained by anti-neuronal NOS without antigen retrieving. The present immunohistochemistry protocol is easy to perform, timeless, and suitable for the localization of NOS isoforms in nervous and non-nervous tissues, in human archival and rat tissues. It has been extensively used in our laboratory, and is also appropriate for other antigens.

Immunolocalization of nitric oxide synthase isoforms in human archivaland rat tissues, and cultured cells

Nitric oxide (NO) exerts important physiological and pathological roles in humans. The study of NO requires the immunolocalization of its synthesizing enzymes, neuronal, endothelial and inducible NO synthases (NOS). NOS are labile to formalin-fixation and paraffin-embedding, which are used to prepare human archival tissues. This lability has made NOS immunohistochemical studies difficult, and a detailed protocol is not yet available. We describe here a protocol for the immunolocalization of NOS isoforms in human archival cerebellum and non-nervous tissues, and in rat tissues and cultured cells. Neuronal NOS antigenicity in human archival and rat nervous tissue sections was microwave-retrieved in 50mM Tris–HCl buffer, pH 9.5, for 20 min at 900 W. Neuronal NOS was expressed in stellate, basket, Purkinje and granule cells in human and rat cerebellum. Archival and frozen human cerebellar sections showed the same neuronal NOS staining pattern. Archival cerebellar sections not subjected to antigen retrieval stained weakly. Antigenicity of inducible NOS in human lung was best retrieved in 10mMsodium citrate buffer,pH6.0, for 15 min at 900 W.Inflammatory cells in ahumanlung tuberculoma were strongly stained by anti-inducible NOS antibody. Anti-endothelial NOS strongly stained kidney glomeruli.Cultured PC12 cells were strongly stained by anti-neuronal NOS without antigen retrieving. The present immunohistochemistry protocol is easy to perform, timeless, and suitable for the localization of NOS isoforms in nervous and non-nervous tissues, in human archival and rat tissues. It has been extensively used in our laboratory, and is also appropriate for other antigens.

Evidence that nitric oxide regulates the acute effects of ethanol on rat N-methyl-D-aspartate receptors in vitro.

Acute exposure to ethanol has been shown to inhibit the function of N-methyl-D-aspartate (NMDA) receptors (NMDAR). However, the mechanism by which ethanol produces inhibition of NMDAR and the factors that modulate this effect are not completely understood. Nitric oxide (NO) is an important modulator of NMDAR function in the hippocampus. Therefore, we examined the effects of NO donors on the ethanol-induced inhibition of NMDAR. Primary cultures of hippocampal neurons were prepared from postnatal day 3 rats. After 7 days in culture, NMDAR currents were recorded by using whole-cell patch-clamp electrophysiological techniques. Effects of acute exposure to ethanol on these currents were assessed in the absence and presence of NO donors. We found that the NO donors 1-hydroxy-2-oxo-3-(N-ethyl-2-aminoethyl)-e-ethyl-1-triazene (NOC-12, 100 microM) and S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 500 microM) inhibit currents gated by 100 microM NMDA plus 0.5 microM glycine. The inhibitory effect of NOC-12 on NMDAR currents could not be observed when 100 microM of the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) was present. Importantly, it was found that ethanol inhibits NMDAR responses to a significantly lesser extent in the presence of these donors. Ethanol (65 mM) inhibited NMDAR responses by 42+/-2%. In the presence of NOC-12 or SNAP, ethanol inhibited NMDAR responses by 21+/-4% and 11+/-7%, respectively. The effect of NOC-12 on ethanol's actions on NMDAR currents was blocked by PTIO. Our results suggest that NO is a novel modulator of the acute effects of ethanol on NMDAR function.

Genetic ablation of the t-SNARE SNAP-25 distinguishes mechanisms of neuroexocytosis.

Axon outgrowth during development and neurotransmitter release depends on exocytotic mechanisms, although what protein machinery is common to or differentiates these processes remains unclear. Here we show that the neural t-SNARE (target-membrane-associated-soluble N-ethylmaleimide fusion protein attachment protein (SNAP) receptor) SNAP-25 is not required for nerve growth or stimulus-independent neurotransmitter release, but is essential for evoked synaptic transmission at neuromuscular junctions and central synapses. These results demonstrate that the development of neurotransmission requires the recruitment of a specialized SNARE core complex to meet the demands of regulated exocytosis.