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This study tested whether NADPH-oxidase activity, expression, and functional effects on vascular tone are influenced by gender in the rat cerebral circulation and whether such differences are estrogen-dependent.

Parvalbumin‐containing fast‐spiking interneurons in the cerebral cortex exhibit widespread electrical coupling, as do somatostatin‐containing low‐threshold spiking interneurons. Besides the classical neurotransmitter γ‐aminobutyric acid, these cortical interneurons may also release various neuropeptides including substance P (SP), as well as the freely diffusible messenger nitric oxide (NO). To investigate whether these two networks of interneurons might interact via these nonclassical messengers, we performed immunocytochemistry for SP and NO signaling pathways in rat somatic sensory cortex. SP was found in a subset of parvalbumin‐positive cells concentrated in layers IV and V, whereas its receptor, NK1, was found in a subset of somatostatin‐containing neurons (and also, at much lower levels, in a disjoint subset of parvalbumin‐containing neurons). Only 4% of SP‐containing axon terminals were apposed to NK1‐positive dendrites, suggesting that in the cerebral cortex, SP may act predominantly as a paracrine neuromediator. Nitric oxide synthase‐I (NOS‐I), the synthetic enzyme for NO, was found almost exclusively in NK1‐positive neurons; 95% of intensely somatostatin/NK1‐positive neurons were also positive for NOS‐I, and 94% of NOS‐positive neurons were also positive for NK1. Immunoreactivity for soluble guanylyl cyclase (the NO receptor) was at high levels in the apical dendrites of layer V pyramidal neurons and in parvalbumin/SP‐positive neurons. These data point to a novel reciprocal chemical interaction between two inhibitory networks in the rat neocortex. J. Comp. Neurol. 441:288–301, 2001. © 2001 Wiley‐Liss, Inc.

The diffusible messenger nitric oxide (NO) is implicated in auditory processing. It acts in the brain largely through activation of soluble guanylyl cyclase (sGC), a heterodimer comprised of α and β subunits. The authors used immunohistochemistry to study the NO/guanosine 3′,5′‐cyclic monophosphate (cGMP) pathway in the cochlear nucleus of Sprague‐Dawley rats. Central fibers of the cochlear nerve were stained for neuronal nitric oxide synthase (NOS‐I) but not for sGCβ. Within the ventral cochlear nucleus, a large fraction of principal cells were immunopositive for both NOS‐I and sGCβ; these cells could be seen at times receiving contacts from NOS‐I‐positive fibers. sGC staining of somatic cytoplasm extended into the distal dendritic tree. At variance with this pattern, NOS‐I was concentrated mainly in somata. Double‐labeling experiments showed that most of the principal neurons expressed both antigens. By contrast, in the granule cell domain, small cells that were immunopositive for NOS‐I rarely corresponded to those that were immunopositive for sGC. To assess whether NOS‐I and sGC immunoreactivities colocalize with their respective catalytic activities, the authors performed multiple labeling with L‐citrulline (a by‐product of the formation of NO from L‐arginine) and cGMP, respectively. L‐citrulline was restricted to NOS‐I‐positive elements, and the large majority of NOS‐expressing neurons were positive for citrulline. Multiple labeling revealed that almost all sGC‐positive neurons also accumulated cGMP both in the ventral cochlear nucleus and in the granule cell domain. These data suggest that NO is a signaling molecule in the cochlear nucleus, perhaps functioning in both a paracrine manner and an autocrine manner. J. Comp. Neurol. 431:1–10, 2001. © 2001 Wiley‐Liss, Inc.

Endothelial nitric-oxide synthase (NOS-III) is defined as being strictly dependent on Ca2+/calmodulin (CaM) for activity, although NO release from endothelial cells has been reported to also occur at intracellular free Ca2+levels that are substimulatory for the purified enzyme. We demonstrate here that NOS-III, but neither NOS-I nor -II, is rapidly and strongly activated and phosphorylated on both Ser and Thr in the presence of cGMP-dependent protein kinase II (cGK II) and the catalytic subunit of cAMP-dependent protein kinase (cAK) in vitro. Phosphopeptide analysis by mass spectrometry identified Ser1177, as well as Ser633which is situated in a recently defined CaM autoinhibitory domain within the flavin-binding region of human NOS-III. Phosphoamino acid analysis identified a putative phosphorylation site at Thr495in the CaM-binding domain. Importantly, both cAK and cGK phosphorylation of NOS-IIIin vitrocaused a highly reproducible partial (10–20%) NOS-III activation which was independent of Ca2+/CaM, and as much as a 4-fold increase in Vmaxin the presence of Ca2+/CaM. cAK stimulation in intact endothelial cells also increased both Ca2+/CaM-independent and -dependent activation of NOS-III. These data collectively provide new evidence for cAK and cGK stimulation of both Ca2+/CaM-independent and -dependent NOS-III activity, and suggest possible cross-talk between the NO and prostaglandin I2pathways and a positive feedback mechanism for NO/cGMP signaling.

The biosynthesis of nitric oxide (NO) is catalyzed by homodimeric NO synthases (NOS). For unknown reasons, all NOS co-purify with substoichiometric amounts of (6 R)-5,6,7,8-tetrahydrobiopterin (H4Bip) and require additional H4Bip for maximal activity. We examined the effects of H4Bip and pterin-derived inhibitors (anti-pterins) on purified neuronal NOS-I quaternary structure and H4Bip content. During l-arginine turnover, NOS-I dimers time dependently dissociated into inactive monomers, paralleled by a loss of enzyme-associated pterin. Dimer dissociation was inhibited when saturating levels of H4Bip were added during catalysis. Similar results were obtained with pterin-free NOS-I expressed in Escherichia coli. This stabilizing effect of H4Bip was mimicked by the anti-pterin 2-amino-4,6-dioxo-3,4,5,6,8,8a,9,10-octahydro-oxazolo[1,2f]-pteridine (PHS-32), which also displaced NOS-associated H4Bip in a competitive manner. Surprisingly, H4Bip not only dissociated from NOS during catalysis, but was only partially recovered in the solute (50.0 ± 16.5% of control at 20 min). NOS-associated H4Bip appeared to react with a NOS catalysis product to a derivative distinct from dihydrobiopterin or biopterin. Under identical conditions, reagent H4Bip was chemically stable and fully recovered (95.5 ± 3.4% of control). A similar loss of both reagent and enzyme-bound H4Bip and dimer content was observed by NO generated from spermine NONOate. In conclusion, we propose a role for H4Bip as a dimer-stabilizing factor of neuronal NOS during catalysis, possibly by interfering with enzyme destabilizing products.

OBJECTIVES

Vascular endothelial cells and neutrophils synthesize and release potent vasodilatatory factors, i.e. endothelium‐derived relaxing factors (EDRF) and neutrophil‐derived relaxing factors (NDRF). One EDRF has been identified as nitric oxide (NO) derived from arginine. We studied the synthesis and release of NO from human neutrophils stimulated with the chemotactic peptide N‐formyl‐L‐methionyl‐L‐leucyl‐L‐phenylalanine, platelet activating factor or leukotriene B4. The formation and release of NO was enhanced several‐fold in the presence of superoxide dismutase, probably by inhibiting superoxide‐induced breakdown of NO. The formation and release of NO but not the formation of superoxide anions was decreased in neutrophils pretreated with L‐canavanine, an inhibitor of arginine‐utilizing enzymes. Our data suggest that at least one NDRF is identical with NO or another labile NO containing compound derived from arginine.

Several low molecular weight compounds are capable of activating soluble guanylyl cyclase. Recent evidence suggests that some of these are formed under physiological conditions: the nitric oxide radical, carbon monoxide and the hydroxyl radical. Thus, multiple signal transduction pathways appear to exist that form a family of guanylyl cyclase activating factors and thereby regulate the intracellular cyclic guanosine 3′, 5′-monophosphate level.

Complementary DNA clones corresponding to human brain nitric oxide (NO) synthase have been isolated. The deduced amino acid sequence revealed an overall identity with rat brain NO synthase of about 93% and contained all suggested consensus sites for binding of the co-factors. The cDNA transfected COS-1 cells showed significant NO synthase activity with the typical co-factor requirements. Unexpectedly, messenger RNA levels of this isoform of NO synthase was more abundant in human skeletal muscle than human brain. Moreover, we detected high NO synthase activity and the expressed protein in human skeletal muscle by Western blot analysis, indicating a possible novel function of NO in skeletal muscle.

Nitric oxide (NO) acts as a neuronal messenger which activates soluble guanylyl cyclase (SGC) in neighboring cells and produces a wide range of physiological effects in the central nervous system (CNS). Using immunocytochemical and histochemical stains, we have characterized the NO/SGC system in the rabbit retina and to a lesser extent, in monkey retina. Based on staining patterns observed with an antibody to nitric oxide synthase (NOS) type I and a histochemical marker for NADPH diaphorase, a metabolic intermediate required for NOS activity, three major classes of neurons appear to generate NO in the rabbit retina. These include two subclasses of sparsely distributed wide field amacrine cells, rod and cone photoreceptors, and a subpopulation of ganglion cells. Equivalent cell populations were labled in monkey retina. An antibody to SGC (tested only in rabbit retina), labeled large arrays of cone photoreceptors in the outer nuclear layer, both amacrine and bipolar cells in the inner nuclear layer (INL), as well as populations of neurons in the ganglion cell layer. These data suggest that the ability to generate NO is restricted to relatively few neurons in the inner retina and to photoreceptor cells in the outer retina; while presumptive target cells, containing pools of SGC, are widespread and form contiguous fields across the inner and outer nuclear layers (ONL) as well as the ganglion cell layer.