Inter-isoformal regulation of nitric oxide synthase through heteromeric dimerization

A 130-kD protein that coimmunoprecipitates with the tight junction protein ZO-1 was bulk purified from Madin-Darby canine kidney (MDCK) cells and subjected to partial endopeptidase digestion and amino acid sequencing. A resulting 19-amino acid sequence provided the basis for screening canine cDNA libraries. Five overlapping clones contained a single open reading frame of 2,694 bp coding for a protein of 898 amino acids with a predicted molecular mass of 98,414 daltons. Sequence analysis showed that this protein contains three PSD-95/SAP90, discs-large, ZO-1 (PDZ) domains, a src homology (SH3) domain, and a region similar to guanylate kinase, making it homologous to ZO-1, ZO-2, the discs large tumor suppressor gene product of Drosophila, and other members of the MAGUK family of proteins. Like ZO-1 and ZO-2, the novel protein contains a COOH-terminal acidic domain and a basic region between the first and second PDZ domains. Unlike ZO-1 and ZO-2, this protein displays a proline-rich region between PDZ2 and PDZ3 and apparently contains no alternatively spliced domain. MDCK cells stably transfected with an epitope-tagged construct expressed the exogenous polypeptide at an apparent molecular mass of approximately 130 kD. Moreover, this protein colocalized with ZO-1 at tight junctions by immunofluorescence and immunoelectron microscopy. In vitro affinity analyses demonstrated that recombinant 130-kD protein directly interacts with ZO-1 and the cytoplasmic domain of occludin, but not with ZO-2. We propose that this protein be named ZO-3.     

Down-regulation of neuronal nitric oxide synthase by nitric oxide after oxygen-glucose deprivation in rat forebrain slices

The precise role that nitric oxide (NO) plays in the mechanisms of ischemic brain damage remains to be established. The expression of the inducible isoform (iNOS) of NO synthase (NOS) has been demonstrated not only in blood and glial cells using in vivo models of brain ischemia-reperfusion but also in neurons in rat forebrain slices exposed to oxygen-glucose deprivation (OGD). We have used this experimental model to study the effect of OGD on the neuronal isoform of NOS (nNOS) and iNOS. In OGD-exposed rat forebrain slices, a decrease in the calcium-dependent NOS activity was found 180 min after the OGD period, which was parallel to the increase during this period in calcium-independent NOS activity. Both dexamethasone and cycloheximide, which completely inhibited the induction of the calcium-independent NOS activity, caused a 40-70% recovery in calcium-dependent NOS activity when compared with slices collected immediately after OGD. The NO scavenger oxyhemoglobin produced complete recovery of calcium-dependent NOS activity, suggesting that NO formed after OGD is responsible for this down-regulation. Consistently, exposure to the NO donor (Z)-1-[(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-iu m-1,2-diolate (DETA-NONOate) for 180 min caused a decrease in the calcium-dependent NOS activity present in control rat forebrain slices. Furthermore, OGD and DETA-NONOate caused a decrease in level of both nNOS mRNA and protein. In summary, our results indicate that iNOS expression down-regulates nNOS activity in rat brain slices exposed to OGD. These studies suggest important and complex interactions between NOS isoforms, the elucidation of which may provide further insights into the physiological and pathophysiological events that occur during and after cerebral ischemia.     

Demonstration of action-potential-producing cells in the rat pineal gland in vitro and their regulation by norepinephrine and nitric oxide

PURPOSE: The purpose of this study was to analyse donor and time dependent variations in the frequencies of radiation-induced aberrations in chromosomes 1 and 2. MATERIALS AND METHODS: Human lymphocytes from two donors were irradiated with 1 and 2 Gy of X-rays. Chromosomal aberrations were scored in chromosomes 1 and 2 painted with different fluorochromes and in Giemsa stained cells. Two time displaced experiments were performed with lymphocytes of each donor. RESULTS: In cells of both donors chromosome 1 was generally more frequently involved in translocations than chromosome 2. This result was not always reproducible. Chromosome 2 showed a higher frequency of acentric fragments, especially following a dose of 1 Gy. Again interexperimental variations were observed. No differences between the two chromosomes were seen with regard to other aberration types. Both chromosomes showed less dicentrics and more acentric fragments than proportional to their DNA content. CONCLUSIONS: Chromosomes 1 and 2 show a different sensitivity to ionizing radiation. The difference is dependent on the aberration type and is not always reproducible. This variability could contribute to the difficulty in reaching a consensus regarding the radiosensitivity of individual chromosomes.     

Role of nitric oxide in rat nephrotoxic nephritis: comparison between inducible and constitutive nitric oxide synthase

Nitric oxide (NO), generated by inducible NO synthase (iNOS) in migrating macrophages, is increased in glomerulonephritis. This study investigates the effect of NO inhibition on rat nephrotoxic nephritis (NTN) to clarify the role of NO production in glomerular damage. NTN was induced in Sprague Dawley rats by an injection of an anti-glomerular basement membrane (GBM) antibody. Urinary nitrite excretion and nitrite release from kidney slices (5.47 +/- 1.19 versus 2.15 +/- 0.73 nmol/mg protein, NTN versus Control, P < 0.05) were increased in NTN on day 2. Glomerular macrophage infiltration and intercellular adhesion molecule (ICAM)-1 expression increased from day 2. iNOS expression was increased in interstitial macrophages. Glomerular endothelial cell NOS (ecNOS) expression evaluated by counting immunogold particles along GBM was suppressed (0.06 +/- 0.02 versus 0.35 +/- 0.04 gold/micron GBM, P < 0.0001). Glomerular damage developed progressively. NG-nitro-L-arginine methyl ester (L-NAME), which inhibits both iNOS and ecNOS and aminoguanidine (AG), a relatively selective inhibitor for iNOS, equally suppressed nitrite in urine and renal tissue. Glomerular ICAM-1 expression and macrophage infiltration were reduced by L-NAME, but not by AG. Expression of ecNOS was significantly increased by L-NAME (0.91 +/- 0.08, P < 0.0001 versus NTN), but slightly by AG (0.18 +/- 0.04). AG significantly and L-NAME slightly attenuated the glomerular damage at day 4. In conclusion, suppression of iNOS prevents glomerular damage in the early stage of NTN. Treatment by L-NAME reduces macrophage infiltration by suppression of ICAM-1 expression, which may be explained by an increase in ecNOS expression.     

Post-transcriptional regulation of endothelial nitric oxide synthase mRNA stability by Rho GTPase

The mechanism by which 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors increase endothelial nitric oxide synthase (eNOS) expression is unknown. To determine whether changes in isoprenoid synthesis affects eNOS expression, human endothelial cells were treated with the HMG-CoA reductase inhibitor, mevastatin (1-10 microM), in the presence of L-mevalonate (200 microM), geranylgeranylpyrophosphate (GGPP, 1-10 microM), farnesylpyrophosphate (FPP, 5-10 microM), or low density lipoprotein (LDL, 1 mg/ml). Mevastatin increased eNOS mRNA and protein levels by 305 +/- 15% and 180 +/- 11%, respectively. Co-treatment with L-mevalonate or GGPP, but not FPP or LDL, reversed mevastatin's effects. Because Rho GTPases undergo geranylgeranyl modification, we investigated whether Rho regulates eNOS expression. Immunoblot analyses and [35S]GTPgammaS-binding assays revealed that mevastatin inhibited Rho membrane translocation and GTP binding activity by 60 +/- 5% and 78 +/- 6%, both of which were reversed by co-treatment with GGPP but not FPP. Furthermore, inhibition of Rho by Clostridium botulinum C3 transferase (50 microg/ml) or by overexpression of a dominant-negative N19RhoA mutant increased eNOS expression. In contrast, activation of Rho by Escherichia coli cytotoxic necrotizing factor-1 (200 ng/ml) decreased eNOS expression. These findings indicate that Rho negatively regulates eNOS expression and that HMG-CoA reductase inhibitors up-regulate eNOS expression by blocking Rho geranylgeranylation, which is necessary for its membrane-associated activity.     

Characteristics of nitric oxide synthase type I of rat cerebellar astrocytes

We have previously reported that stimulation of astrocyte cultures by particular agonists and calcium ionophores induces cyclic GMP formation through activation of a constitutive nitric oxide synthase (NOS) and that astrocytes from cerebellum show the largest response. In the present work we have used rat cerebellar astrocyteenriched primary cultures to identify and characterise the isoform of NOS expressed in these cells. The specific NOS activity in astrocyte homogenates, determined by conversion of [3H]arginine to [3H]citrulline, was ten times lower than in homogenates from cerebellar granule neurons. Upon centrifugation at 100,000 g, the astroglial activity was recovered in the supernatant, whereas in neurons around 30% of the activity remained particulate. The cytosolic NOS activities of both astrocytes and granule neurons displayed the same Km for L-arginine, dependency of calcium, and sensitivity to NOS inhibitors. Expression of NOS-I in astrocyte cytosolic fractions was revealed by Western blot with a specific polyclonal antiserum against recombinant NOS-I. Double immunofluorescence labelling using anti-glial fibrillary acidic protein (GFAP) and anti-NOS-I antibodies revealed that a minor population of the GFAP-positive cells, usually in clusters, presented a strong NOS-I immunostaining that was predominantly located around the nuclei and had a granular appearance, indicating association with the endoplasmic reticulum-Golgi system. Astrocytes of stellate morphology also showed immunoreactivity in the processes. Similar staining was observed with the avidin-biotin-peroxidase complex using different anti-NOS-I antisera. With this method the majority of cells showed a weak NOS-I immunoreactivity around the nuclei and cytosol. A similar pattern was observed with the NADPH-diaphorase reaction. These results demonstrate that the NOS-I expressed in astrocytes presents the same biochemical characteristics as the predominant neuronal isoform but may differ in intracellular location.     

Inhibition of nitric oxide synthase attenuates osmotic thirst in the rat

Changes in water intake after intraperitoneal injection of a nitric oxide synthase (NOS) inhibitor was studied in the rat. Administration of NW-nitro-L arginine methyl ester (L-NAME) at a dose of 50 mg/kg attenuated osmotic thirst induced by intraperitoneal injection of hypertonic saline, but did not affect spontaneous intake of water and thirst induced by subcutaneous injection of angiotension II. Pretreatment with L-arginine significantly attenuated the inhibition of osmotic thirst evoked with subsequent L-NAME. Administration of NW-nitro-D-arginine methyl ester (D-NAME) altered neither the spontaneous nor the osmotic drinking behavior. These findings suggest that NO may affect the osmotically induced drinking.     

Growth factor induction of nitric oxide synthase in rat pheochromocytoma cells

Members of the beta isozyme subfamily of phosphatidylinositol-specific phospholipase C (PLC) are stimulated by alpha subunits and betagamma dimers of heterotrimeric guanine-nucleotide-binding proteins (G proteins). Myeloid differentiated human HL-60 granulocytes and bovine neutrophils contain a soluble phospholipase C, which is stimulated by the metabolically stable GTP analogue guanosine (5'-->O)-3-thiotriphosphate (GTP[S]). To identify the component(s) involved in mediating this stimulation, the relevant polypeptide(s) was resolved from endogenous phospholipase C and purified from bovine neutrophil cytosol by measuring its ability to confer GTP[S] stimulation to exogenous recombinant PLCbeta2. The resolved factor, which behaved as 48-kDa protein upon gel filtration, stimulated PLCbeta2 but not PLCbeta1 or PLCdelta1. Activation of phosphatidylinositol 4-phosphate 5-kinase was not involved in this stimulation. The purified stimulatory factor consisted of two polypeptides of molecular masses of approximately 23 kDa and 26 kDa. The protein stimulated a deletion mutant of PLCbeta2 that lacked a carboxyl-terminal region necessary for stimulation by members of the alpha(q) subfamily of the G-protein alpha subunits. The results of this study suggest that a GTP-binding protein distinct from alpha(q) subunits, probably a low-molecular-mass GTP-binding protein associated with a regulatory protein, is involved in isozyme-specific activation of PLCbeta2.     

Up-regulation of inducible nitric oxide synthase and production of nitric oxide by the Swarm rat and human chondrosarcoma

Phospholipids are the major constituents of cell membranes, and have numerous structural and functional roles in the nervous system. Although the metabolic pathways responsible for the syntheses of the phosphatides phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), and phosphatidylserine (PtdSer) are well understood, the mechanisms controlling these pathways in neural tissue have not been fully characterized. Recent studies have suggested that the main factors controlling PtdCho and PtdEtn synthesis by the Kennedy cycle tend to be the intracellular levels of key substrates for the biosynthetic enzymes, or changes in the activities of the rate-limiting enzymes. Moreover, different control mechanisms may operate, depending upon the functional state of the tissue.     

Expression and regulation of protein inhibitor of neuronal nitric oxide synthase in ventilatory muscles

Rice tungro bacilliform virus (RTBV) is a plant pararetrovirus and a member of the Caulimoviridae family and closely related to viruses in the Badnavirus genus. The coat protein of RTBV is part of the large polyprotein encoded by open reading frame 3 (ORF3). ORF3 of an RTBV isolate from Malaysia was sequenced (accession no. AF076470) and compared with published sequences for the region that encodes the coat protein or proteins. Molecular mass of virion proteins was determined by mass spectrometry (matrix-assisted laser desorption/ionization-TOF) performed on purified virus particles from three RTBV isolates from Malaysia. The N- and C-terminal amino acid sequences of the coat protein were deduced from the mass spectral analysis, leading to the conclusion that purified virions contain a single coat protein of 37 kDa. The location of the coat protein domain in ORF3 was reinforced as a result of immunodetection reactions using antibodies raised against six different segments of ORF3 using Western immunoblots after SDS-PAGE and isoelectrofocusing of proteins purified from RTBV particles. These studies demonstrate that RTBV coat protein is released from the polyprotein as a single coat protein of 37 kDa.     

Distribution of nitric oxide synthase and nitric oxide-receptive, cyclic GMP-producing structures in the rat brain

The structures capable of synthesizing cyclic GMP in response to nitric oxide in the rat brain were compared relative to the anatomical localization of neuronal nitric oxide synthase. In order to do this, we used brain slices incubated in vitro, where cyclic GMP-synthesis was stimulated using sodium nitroprusside as a nitric oxide-donor compound, in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. Nitric oxide-stimulated cyclic GMP synthesis was found in cells and fibers, but was especially prominent in varicose fibers throughout the rat brain. Fibers containing the nitric oxide-stimulated cyclic GMP production were present in virtually every area of the rat brain although there were large regional variations in the density of the fiber networks. When compared with the localization of nitric oxide synthase, it was observed that although nitric oxide-responsive and the nitric oxide-producing structures were found in similar locations in general this distribution was complementary. Only occasionally was nitric oxide-mediated cyclic GMP synthesis observed in structures which also contained nitric oxide synthase. We conclude that the nitric oxide-responsive soluble guanylyl cyclase and nitric oxide synthase are usually juxtaposed at very short distances in the rat brain. These findings very strongly support the proposed role of nitric oxide as an endogenous activator of the soluble guanylyl cyclase in the central nervous system and convincingly demonstrate the presence of the nitric oxide-cyclic GMP signal transduction pathway in virtually every area of the rat brain.     

Dynamic changes in NADPH-diaphorase staining reflect activity of nitric oxide synthase: evidence for a dopaminergic regulation of striatal nitric oxide release

In fixed tissue, neuronal NADPH-diaphorase staining results from nitric oxide synthase (NOS) activity. Neuronal NOS only synthesizes nitric oxide once activated by the binding of Ca2+/calmodulin. We show here that neuronal NADPH-diaphorase staining is also dependent on Ca2+/calmodulin, implying that only activated NOS is detected. In addition, in bovine pulmonary endothelial cells, carbachol and bradykinin dramatically and rapidly increase the intensity of NADPH-diaphorase staining. Furthermore, administration of MK801, an NMDA antagonist, decreases neuronal NADPH-diaphorase staining. This suggests that the intensity of the NADPH-diaphorase staining is related to the level of enzyme activation at the moment of tissue fixation. The potential of exploiting this observation to detect cellular activation of NOS is illustrated by the observations that the intensity of NADPH-diaphorase staining in rat striatal neurones is decreased following systemic treatment with the D1-like dopamine receptor antagonist SCH23390, and increased by the D2-like antagonist eticlopride. These results therefore provide strong evidence that the NADPH-diaphorase reaction can be used to monitor NOS activity at a cellular level of resolution, and reveal a dopaminergic regulation of NOS activity in the striatum mediated by D1-like and D2-like dopamine receptors.     

Adrenal control of erectile function and nitric oxide synthase in the rat penis

Penile erection is a nitric oxide (NO)-mediated process that has been shown to be androgen dependent in rats. Castration reduces the activity of the penile enzyme involved in NO synthesis, nitric oxide synthase (NOS). To determine whether adrenal androgens and/or corticosteroids contribute to this control, the following groups of Fischer 344 adult male rats (n = 5-7) were studied: 1) intact, 2) castrated, 3) adrenalectomized alone, 4) castrated/adrenalectomized, 5) castrated/adrenalectomized with aldosterone (1.25 mg/kg, s.c.) and hydrocortisone (12 mg/kg, s.c.), 6) castrated/adrenalectomized with dihydrotestosterone (1.2-cm SILASTIC-brand tubing pellet; Dow Corning, Midland, MI), 7) castrated/adrenalectomized with dehydroepiandrosterone (2-cm tubing), 8) castrated/adrenalectomized with aldosterone (1.25 mg/kg, s.c.), and 9) castrated/adrenalectomized with hydrocortisone (12 mg/kg, s.c.). After 1 week, EFS was applied, and the maximal intracavernosal pressure (MIP) and mean arterial pressure (MAP) were recorded. The MIP/MAP ratio in the adrenalectomized group (0.37) was reduced to values found in the castrated group (0.40). The values in both groups were significantly less than those in intact controls (0.75). The most significant reduction in MIP/MAP was seen in the adrenalectomized/castrated group (0.16). Erectile response in animals submitted to adrenalectomy and castration was restored close to intact values with the administration of hydrocortisone and aldosterone (0.63). Similar results were obtained by the administration of either of the substances alone (0.56 and 0.67, respectively). Penile NOS activity assayed by the L-arginine/citrulline conversion was decreased by 55% in the castrated group compared with that in the intact group, but was not further reduced in the adrenalectomized/castrated or adrenalectomized groups. Penile neuronal NOS protein content, estimated by Western blot, was decreased only in the adrenalectomized/castrated animals (35%), and endothelial NOS content was not affected. These data suggest that the rat adrenal gland contributes to the maintenance of the erectile mechanism and may affect neuronal NOS content in the penis in the rat model. The possibility that hypotension may play a role in the erectile dysfunction observed in adrenalectomized rats cannot be discarded.     

Memory-related changes of nitric oxide synthase activity and nitrite level in rat brain

The changes of nitric oxide synthase (NOS) activity and nitrite level in rat brain regions after spatial learning were investigated. NOS activity was assayed by conversion of [3H]L-arginine to [3H]L-citrulline, and a sensitive fluorometric assay for quantification of nitrite was used. Compared with sham-trained rats, NOS activity and nitrite level in hippocampus and cortex, and also the nitrite level in cerebellum, was elevated significantly one day after rats had learnt a water-rewarded spatial alteration task. These results suggest a spatial memory-related changes of endogenous NO in rat brain, and support the idea that NO participates in learning and memory processes.     

Fluorescence-based measurement of nitric oxide synthase activity in activated rat macrophages using dichlorofluorescin

This study examined the sleep and mood differences between premenopausal and perimenopausal women matched for age and sociodemographic variables. Wrist actigraphy, Profile of Mood State (POMS), State-Trait Anxiety Inventory (STAI), a sleep questionnaire, and responses to a sleep diary were recorded for a period of 1 week. It was found that the sleep disruption of perimenopausal subjects was significantly greater than that of the premenopausal group (p < 0.05). Overall, the perimenopausal group demonstrated a significant increase in sleep disruption and mood alterations when compared with the premenopausal group. Actigraphic data showed that perimenopausal subjects experienced longer and more numerous arousals resulting in significantly less sleep (p < 0.05). In addition, perimenopausal subjects scored significantly higher (p < 0.05) on the STAI and significantly lower on the Vigor subscale of the POMS (p < 0.01) than premenopausal subjects. Correlational analyses indicated that sleep and mood changes were significantly related in the perimenopausal group, but not in the premenopausal group. Taken together, these results suggest that the mood changes experienced by the perimenopausal group may be mediated by sleep disruption.