Regulation of Multimeric Structure of NO-synthase as a New Factor of Organogenesis

The effect of NO on organogenesis in Drosophila is discussed. A new model of regulation of the activity of NO-producing enzyme, NO synthase is described, which takes into account endogenous synthesis of its reduced isoform. The reduced isoform of NO synthase is capable of suppressing the enzymatic activity of full-sized NO synthase during formation of a heterodimer in vivo and in vitro. The reduced form of this enzyme inhibits the antiproliferative effect of the full-sized NO synthase isoform during formation of eye structure in Drosophila by affecting the pathways of cell cycle regulation. The reduced form of NO synthase is an endogenous dominant-negative factor of regulation of the NO synthase activity in development of Drosophila.     

Suppressive effect of endogenous regucalcin on nitric oxide synthase activity in cloned rat hepatoma H4-II-E cells overexpressing regucalcin

The role of endogenous regucalcin, which is a regulatory protein in calcium signaling, in the regulation of nitric oxide (NO) synthase activity in the cloned rat hepatoma H4-II-E cells was investigated. Hepatoma cells were cultured for 24-72 h in the presence of fetal bovine serum (FBS; 10%). NO synthase activity in the 5,500 g supernatant of cell homogenate was significantly increased by the addition of calcium chloride (10 microM) and calmodulin (2.5 microg/ml) in the enzyme reaction mixture. The presence of trifluoperazine (TFP; 50 microM), an antagonist of calmodulin, inhibited the effect of calcium (10 microM) addition in increasing NO synthase activity, indicating the existence of Ca(2+)/calmodulin-dependent NO synthase in hepatoma cells. NO synthase activity was significantly decreased by the addition of regucalcin (10(-8) or 10(-7) M) in the reaction mixture without or with Ca(2+)/calmodulin addition. The effect of regucalcin (10(-7) M) in decreasing NO synthase activity was also seen in the presence of TFP (50 microM) or EGTA (1 mM). The presence of anti-regucalcin monoclonal antibody (10-50 ng/ml) in the reaction mixture caused a significant elevation of NO synthase activity. NO synthase activity was significantly suppressed in the hepatoma cells (transfectants) overexpressing regucalcin. This decrease was completely abolished in the presence of anti-regucalcin monoclonal antibody (50 ng/ml) in the reaction mixture. Moreover, the effect of Ca(2+)/calmodulin addition in increasing NO synthase activity in the hepatoma cells (wild-type) was completely prevented in transfectants. The present study demonstrates that endogenous regucalcin has a suppressive effect on NO synthase activity in the cloned rat hepatoma H4-II-E cells.     

Effect of nitric oxide synthesis inhibition on mouse liver and brain metallothionein expression

The role of nitric oxide (NO) production on metallothionein (MT) regulation in the liver and the brain has been studied in mice by means of the administration of nitric oxide synthase (NOS) inhibitors. Mice injected with either the arginine analog N^G-monomethyl-L-arginine (L-NMMA) or the heme binding compound 7-nitro indazole (7-NI) showed consistently increased liver MT-I mRNA and MT-I+II total protein levels, suggesting that NO is involved in the hepatic MT regulation. In agreement with the liver results, in situ hybridization analysis demonstrated a significant upregulation of the brain MT-I isoform in areas such as the cerebrum cortex, neuronal CA1-CA3 layers and dentate gyrus of the hippocampus, and Purkinje cell layer of the cerebellum, in 7-NI treated mice. The same trend was observed for the brain specific isoform, MT-III, but to a much lower extent. The effect of NOS inhibition was also evaluated in a MT-inducing condition, namely during immobilization stress. In both the liver and the brain, stress upregulated the MT-I isoform, and 7-NI significantly reduced or even blunted the MT-I response to stress, suggesting a mediating role of NO on MT-I regulation during stress. Stress also increased the MT-III mRNA levels in some brain areas, an effect blunted by the concomitant administration of 7-NI, which in some areas even decreased MT-III mRNA levels below the saline injected mice. Results in primary culture of neurons and astrocytes demonstrate significant effects of the NOS inhibitors in some experimental conditions. The present results suggest that NO may have some role on MT regulation in both the liver and the brain.     

Effects of VIP and NO on the motor activity of vascularly perfused rat proximal colon

The effects of vasoactive intestinal polypeptide (VIP) and nitric oxide (NO) on the motor activity of the rat proximal colon were examined in an ex vivo model of vascularly perfused rat proximal colon. VIP reduced motor activity and this inhibitory effect was not altered by either atropine, hexamethonium, tetrodotoxin (TTX) nor TTX plus acetylcholine (ACh), but was completely antagonized by NO synthase inhibitor N(G)-nitro-L-arginine (L-NA) and by VIP receptor antagonist, VIP(10-28). These results suggest that VIP may exert a direct inhibitory effect on the motor activity of the rat proximal colon via a VIP receptor located on the smooth muscle and this effect is mediated by NO but not by cholinergic pathways. Atropine and hexamethonium reduced but ACh stimulated motor activity and the effect of ACh was not changed by TTX, suggesting that the cholinergic pathway may exert a direct stimulatory effect on motor activity. Single injection of TTX, VIP(10-28) or L-NA induced a marked increase in motor activity, suggesting that the motor activity of rat proximal colon is tonically suppressed by VIP and NO generating pathways, and elimination of inhibitory neurotransmission by TTX may induce an abnormal increase of the motor activity. The interaction between VIP and NO in regulation of motor activity was further examined by a measurement of NO release from vascularly perfused rat proximal colon. Results showed that NO release was significantly increased during infusion of VIP and this response was reversed by L-NA. These results suggest that VIP generating neurons may inhibit colonic motility by stimulating endogenous NO production in either smooth muscle cells or nerve terminals.     

Suppressive role of endogenous regucalcin in the enhancement of nitric oxide synthase activity in liver cytosol of normal and regucalcin transgenic rats

The suppressive role of endogenous regucalcin, which is a regulatory protein of calcium signaling, in the enhancement of nitric oxide (NO) synthase activity in the liver cytosol of rats was investigated. The enzyme activity was measured in a reaction mixture containing either vehicle or calcium chloride (1-20 microM) in the absence or presence of regucalcin (0.1, 0.25, or 0.5 microM). NO synthase activity was significantly increased by the addition of calcium (5-20 microM). This increase was completely abolished in the presence of trifluoperazine (TFP; 10-50 microM), an antagonist of Ca(2+)/calmodulin. The addition of regucalcin (0.1-0.5 microM) caused a significant fall in the calcium-increased enzyme activity. The effect of regucalcin (0.25 microM) in decreasing NO synthase activity was seen in the presence of ethylene glycol bis-(2-aminoethylether) N,N,N',N'-tetraacetic acid (EGTA, 1 mM) or TFP (20 microM), indicating that regucalcin acts independent on Ca(2+)/calmodulin. NO synthase activity was significantly raised in the presence of anti-regucalcin monoclonal antibody (10-50 ng/ml) in the reaction mixture. The effect of the antibody (50 ng/ml) or calcium (10 microM) in elevating NO synthase activity in the liver cytosol of normal rats was not seen in the liver cytosol obtained from regucalcin transgenic rats. Moreover, the increase in NO synthase activity in the liver cytosol of normal rats induced by a single intraperitoneal administration of calcium (5.0 mg/100 g body weight) was significantly enhanced in the presence of anti-regucalcin monoclonal antibody (50 ng/ml) in the reaction mixture. The administration of calcium caused a significant increase in regucalcin level in the liver cytosol of normal rats. The present study demonstrated that endogenous regucalcin plays a suppressive role in the enhancement of NO synthase activity in the liver cytosol of rats.     

Effect of Nitroprusside (Nitric Oxide) on Endogenous Dopamine Release from Rat Striatal Slices

It is becoming apparent that the synthesis of nitric oxide (NO) from L-arginine not only explains endothelium-dependent vascular relaxation, but is a widespread mechanism for the regulation of cell function and communication. We examined the role of NO on the endogenous dopamine (DA) release from rat striatum. Nitroprusside, in the concentration range of 3-100 microM, induced a dose-dependent increase in the endogenous DA release from rat striatal slices. The maximal response was 330% over the baseline release. A higher concentration of nitroprusside (300 microM) produced an inhibitory effect on the spontaneous release of DA. L-Arginine (10 and 100 microM), a substrate in the NO-forming enzyme system, also produced an elevation of DA release. L-Arginine-induced DA release was attenuated by NG-monomethyl-L-arginine, an inhibitor of NO synthase. NADPH (1 microM), a cofactor of NO synthase, enhanced L-arginine-induced DA release. These results suggest a possible involvement of NO in the DA release process in rat striatum.     

Aspirin reduces endothelial cell senescence

We report here the effect of aspirin on the onset of replicative senescence. Endothelial cells that were cultured until cumulative population doublings 40 showed clear signs of aging. Incubation with aspirin inhibited senescence-associated beta-galactosidase activity and increased telomerase activity. Along with the delayed onset of senescence, aspirin decreased reactive oxygen species and increased nitric oxide (NO) and cGMP levels. Furthermore, aspirin reduced the elaboration of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO synthase, and up-regulated the activity of dimethylarginine dimethylaminohydrolase, the enzyme that degrades ADMA. These effects were specific in that other nonsteroidal anti-inflammatory drugs, such as ibuprofen or acetaminophen, did not prevent the onset of endothelial senescence. The NO synthase inhibitor l-NAME, but not its inactive d-enantiomer, led to complete inhibition of aspirin-delayed senescence. These findings demonstrate that aspirin delays the onset of endothelial senescence by preventing a decrease in NO formation/generation. This might provide a therapeutic strategy aimed at blocking aging-induced NO inhibition.     

Regulation of neuronal nitric oxide synthase by histone,protamine, and myelin basic protein

We examined the effects of endogenous basic proteins rich in the amino acidL-arginine on neuronal NO synthase activity by monitoring cyclic GMP formation in intact neuron-like neuroblastoma N1E-115 cells. Histone, protamine and myelin basic protein significantly stimulated cyclic GMP formation, both in a time- and concentration-dependent manner. These effects were blocked by hemoglobin and NO synthase inhibitors. Removal of the extracellular/intracellular Ca²⁺ gradient by a Ca²⁺ chelator completely abolished the cyclic GMP responses elicited by histone and protamine, suggesting that influex of extracellular Ca²⁺ might be involved in their activation of NO synthase. The effects of myelin basic protein on cyclic GMP formation, however, appeared to be due to Ca²⁺ release from intracellular stores. In cytosolic preparations of rat cerebellum, these basic proteins inhibited the metabolism ofL-arginine intoL-citrulline by NO synthase. We conclude from our findings that endogenous basic proteins might be involved in the regulation of neuronal NO synthase activity. Their effects on the enzyme could be either stimulatory or inhibitory, depending on whether the basic proteins exert their effects extracellularly or intracellularly, respectively.     

Nitric oxide and carotid body chemoreception.

Nitric oxide (NO) has been proposed as an inhibitory modulator of carotid body chemosensory responses to hypoxia. It is believed that NO modulates carotid chemoreception by several mechanisms, which include the control of carotid body vascular tone and oxygen delivery and reduction of the excitability of chemoreceptor cells and petrosal sensory neurons. In addition to the well-known inhibitory effect, we found that NO has a dual (dose-dependent) effect on carotid chemoreception depending on the oxygen pressure level. During hypoxia, NO is primarily an inhibitory modulator of carotid chemoreception, while in normoxia NO increased the chemosensory activity. This excitatory effect produced by NO is likely mediated by an impairment of mitochondrial electron transport and oxidative phosphorylation, which increases the chemosensory activity. The recent findings that mitochondria contain an isoform of NO synthase, which produces significant amounts of NO for regulating their own respiration, suggest that NO may be important for the regulation of mitochondrial energy metabolism and oxygen sensing in the CB.     

Mitochondrial nitric oxide synthase is constitutively active and is functionally upregulated in hypoxia

Nitric oxide is a potent modulator of mitochondrial respiration, ATP synthesis, and K_ATP channel activity. Recent studies show the presence of a potentionally new isoform of the nitric oxide synthase (NOS) enzyme in mitochondria, although doubts have emerged regarding the physiological relevance of mitochondrial NOS (mtNOS). The aim of the present study were to: (i) examine the existence and distribution of mtNOS in mouse tissues using three independent methods, (ii) characterize the cross-reaction of mtNOS with antibodies against the known isoforms of NOS, and (iii) investigate the effect of hypoxia on mtNOS activity. Nitric oxide synthase activity was measured in isolated brain and liver mitochondria using the arginine to citrulline conversion assay. Mitochondrial NOS activity in the brain was significantly higher than in the liver. The calmodulin inhibitor calmidazolium completely inhibited mtNOS activity. In animals previously subjected to hypoxia, mtNOS activity was significantly higher than in the normoxic controls. Antibodies against the endothelial (eNOS), but not the neuronal or inducible isoform of NOS, showed positive immunoblotting. Immunogold labeling of eNOS located the enzyme in the matrix and the inner membrane using electron microscopy. We conclude that mtNOS is a constitutively active eNOS-like isoform and is involved in altered mitochondrial regulation during hypoxia.     

Constitutive nitric oxide synthase from cerebellum is reversibly inhibited by nitric oxide formed from L-arginine.

The objective of this study was to determine whether constitutive nitric oxide (NO) synthase from rat cerebellum could be regulated by the two products of the reaction, NO and L-citrulline, utilizing L-arginine as substrate. NO synthase activity was determined by monitoring the formation of 3H-citrulline from 3H-L-arginine in the presence of added cofactors. The rate of citrulline formation in enzyme reaction mixtures was non-linear. Addition of superoxide dismutase (SOD; 100 units) inhibited NO synthase activity and made the rate of product formation more non-linear, whereas addition of oxyhemoglobin (HbO2; 30 microM) increased NO synthase activity, made the rate of product formation linear and also abolished the effect of SOD. Added NO (10 microM) inhibited NO synthase activity and this inhibition was potentiated by SOD and abolished by HbO2. Added L-citrulline (1 mM) did not alter NO synthase activity. The two NO donors, S-nitroso-N-acetylpenicillamine (200 microM) and N-methyl-N'-nitro-N-nitrosoguanidine (200 microM) mimicked the inhibitory effect of NO and inhibition of NO synthase activity by NO was reversible. These observations indicate clearly that NO formed during the NO synthase reaction or added to the enzyme reaction mixture causes a reversible inhibition of NO synthase activity. Thus, NO may function as a negative feedback modulator of its own synthesis.     

Cadmium decreases crown root number by decreasing endogenous nitric oxide,which is indispensable for crown root primordia initiation in rice seedlings

Cadmium (Cd) is toxic to crown roots (CR), which are essential for maintaining normal growth and development in rice seedlings. Nitric oxide (NO) is an important signaling molecule that plays a pivotal role in plant root organogenesis. Here, the effects of Cd on endogenous NO content and root growth conditions were studied in rice seedlings. Results showed that similar to the NO scavenger, cPTIO, Cd significantly decreased endogenous NO content and CR number in rice seedlings, and these decreases were recoverable with the application of sodium nitroprusside (SNP, a NO donor). Microscopic analysis of root collars revealed that treatment with Cd and cPTIO inhibited CR primordia initiation. In contrast, although SNP partially recovered Cd-caused inhibition of CR elongation, treatment with cPTIO had no effect on CR elongation. l-NMMA, a widely used nitric oxide synthase (NOS) inhibitor, decreased endogenous NO content and CR number significantly, while tungstate, a nitrate reductase (NR) inhibitor, had no effect on endogenous NO content and CR number. Moreover, enzyme activity assays indicated that treatment with SNP inhibited NOS activity significantly, but had no effect on NR activity. All these results support the conclusions that a critical endogenous NO concentration is indispensable for rice CR primordia initiation rather than elongation, NOS is the main source for endogenous NO generation, and Cd decreases CR number by inhibiting NOS activity and thus decreasing endogenous NO content in rice seedlings.     

Dimethylarginine dimethylaminohydrolase and endothelial dysfunction in failing hearts

Congestive heart failure (CHF) is associated with impaired endothelium-dependent nitric oxide (NO)-mediated vasodilation (endothelial dysfunction). We hypothesized that coronary endothelial dysfunction in CHF may be due in part to decreased dimethylarginine dimethylaminohydrolase (DDAH), the enzyme that degrades endogenous inhibitors of NO synthase (NOS), including asymmetric dimethylarginine. Coronary blood flow and the endothelium-dependent vasodilator response to acetylcholine were studied in dogs in which CHF was produced by rapid ventricular pacing for 4 wk. Coronary flow and myocardial O2 consumption at rest and during treadmill exercise were decreased after development of CHF, and the vasodilator response to intracoronary acetylcholine (75 microg/min) was decreased by 39 +/- 5%. DDAH activity and DDAH isoform 2 (DDAH-2) protein content were decreased by 53 +/- 13% and 58 +/- 14%, respectively, in hearts with CHF, whereas endothelial NOS and DDAH isoform 1 (DDAH-1) were increased. Caveolin-1 and protein arginine N-methyltransferase 1, the enzyme that produces asymmetric dimethylarginine, were unchanged. Immunohistochemical staining showed DDAH-1 strongly expressed in coronary endothelium and smooth muscle and in the sarcolemma of cardiac myocytes. In cultured human endothelial cells, DDAH-1 was uniformly distributed in the cytosol and nucleus, whereas DDAH-2 was found only in the cytosol. Decreased DDAH activity and DDAH-2 protein expression may cause accumulation of endogenous inhibitors of endothelial NOS, thereby contributing to endothelial dysfunction in the failing heart.     

Effects of nitric oxide on growth of maize seedling leaves in the presence or absence of ultraviolet-B radiation

The leaves of maize seedlings were used to measure leaf biomass including leaf length, width and weight, and to examine the relationship between nitric oxide (NO) synthase activity in microsomes and cytosol to the exo- and endo-beta-glucanase activity during growth. It was found that ultraviolet-B radiation (UV-B radiation) strongly induced nitric oxide synthase (NOS) activity but caused both a decrease of leaf biomass and exo- or endo-beta-glucanase activity. In contrast, the NOS inhibitor and NO donor largely decreased the activity of NOS in non-irradiated seedlings. The inhibitor also reduced exo- and endo-beta-glucanase activity and leaf biomass while the donor increased the enzyme activity and leaf biomass under normal conditions. Alternatively, under ultraviolet-B, the additional inhibitor of NOS and NO donor appeared to compromise the effects of ultraviolet-B on glucanase activity and leaf biomass, making the relationship between NOS activity and glucanase activity negatively correlated. This suggests that the changes of NOS activity showed a positive correlation to glucanase activity and leaf biomass in the absence of ultraviolet-B, but a negative correlation to ultraviolet-B irradiation and NO donor treatment alone. It is assumed that exo- and endogenous NO is responsible for the up-regulation of regular growth and development without ultraviolet-B. Under UV-B radiation, however, it might function as a signaling molecule of ultraviolet-B inhibiting leaf growth of maize seedlings to carry out stress-signaling transduction.     

Receptor-regulated dynamic S-nitrosylation of endothelial nitric-oxide synthase in vascular endothelial cells

The endothelial isoform of nitric-oxide synthase (eNOS) is regulated by a complex pattern of post-translational modifications. In these studies, we show that eNOS is dynamically regulated by S-nitrosylation, the covalent adduction of nitric oxide (NO)-derived nitrosyl groups to the cysteine thiols of proteins. We report that eNOS is tonically S-nitrosylated in resting bovine aortic endothelial cells and that the enzyme undergoes rapid transient denitrosylation after addition of the eNOS agonist, vascular endothelial growth factor. eNOS is thereafter progressively renitrosylated to basal levels. The receptor-mediated decrease in eNOS S-nitrosylation is inversely related to enzyme phosphorylation at Ser(1179), a site associated with eNOS activation. We also document that targeting of eNOS to the cell membrane is required for eNOS S-nitrosylation. Acylation-deficient mutant eNOS, which is targeted to the cytosol, does not undergo S-nitrosylation. Using purified eNOS, we show that eNOS S-nitrosylation by exogenous NO donors inhibits enzyme activity and that eNOS inhibition is reversed by denitrosylation. We determine that the cysteines of the zinc-tetrathiolate that comprise the eNOS dimer interface are the targets of S-nitrosylation. Mutation of the zinc-tetrathiolate cysteines eliminates eNOS S-nitrosylation but does not eliminate NO synthase activity, arguing strongly that disruption of the zinc-tetrathiolate does not necessarily lead to eNOS monomerization in vivo. Taken together, these studies suggest that eNOS S-nitrosylation may represent an important mechanism for regulation of NO signaling pathways in the vascular wall.     

Excitatory Amino Acid Receptors Coupled to the Nitric Oxide/Cyclic GMP Pathway in Rat Cerebellum During Development

The coupling of excitatory amino acid receptors to the formation of nitric oxide (NO) from arginine during the postnatal development of rat cerebellum was assayed in slice preparations by measuring cyclic GMP accumulation. In the immature tissue, N-methyl-D-aspartate (NMDA) and glutamate were highly efficacious agonists, whereas alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and quisqualate evoked only small responses. The effect of glutamate at all concentrations tested (up to 10 mM) was abolished by the NMDA antagonist, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801). In adult slices, AMPA and quisqualate were much more effective and their effects were inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione, an antagonist for ionotropic non-NMDA receptors, whereas the apparent efficacy of NMDA was greatly reduced. The major changes took place between 8 and 14 days postnatum and, in the case of NMDA, part of the loss of sensitivity appeared to reflect a decline in the ambient levels of glycine with age. Moreover, a component of the response to glutamate in the adult was resistant to MK-801. Cyclic GMP accumulations induced by NMDA and non-NMDA agonists alike were Ca(2+)-dependent and could be antagonized by competitive NO synthase inhibitors in an arginine-sensitive manner, indicating that they are all mediated by NO formation. With one of the inhibitors, L-NG-nitroarginine, a highly potent component (IC50 = 6 nM) evident in slices from rats of up to 8 days old was lost during maturation, indicating that there may be a NO synthase isoform which is prominent only in the immature tissue. Cyclic GMP levels in adult slices under "basal" conditions were reduced markedly by blocking NMDA receptors, by inhibiting action potentials with tetrodotoxin, or by NO synthase inhibition, suggesting that the endogenous transmitter released during spontaneous synaptic activity acts mainly through NMDA receptors to trigger NO formation.     

Heme insertion, assembly, and activation of apo-neuronal nitric-oxide synthase in vitro

It has been established that in the case of inducible NO synthase (NOS), a functionally active homodimer is assembled from the heme-deficient monomeric apo-NOS in vitro by the addition of heme, whereas the heme-deficient neuronal isoform (apo-nNOS) is at best only partially activated. In the current study we have discovered that reactive oxygen species, which can be removed by the addition of superoxide dismutase and catalase, destroy the heme and limit the activation of apo-nNOS in vitro. With the use of these improved conditions, we show for the first time that heme insertion is a rapid process that results in formation of a heme-bound monomeric nNOS that is able to form the ferrous-CO P450 complex but is unable to synthesize NO. A slow process requiring more than 90 min is required for dimerization and activation of this P450 intermediate to give an enzyme with a specific activity of approximately 1100 nmol of NO formed/min/mg of protein, similar to that of the native enzyme. Interestingly, the dimer is not SDS-resistant and is not the same dimer that forms in vivo. These studies indicate at least two intermediates in the assembly of nNOS and advance our understanding of the regulation of nNOS.     

Involvement of sphingosine kinase in TNF-alpha-stimulated tetrahydrobiopterin biosynthesis in C6 glioma cells

In C6 glioma cells, the sphingolipid second messenger ceramide potentiates expression of inducible nitric-oxide synthase (iNOS) induced by tumor necrosis factor alpha (TNF-alpha) without affecting GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme in the biosynthesis of 6(R)-5,6,7,8-tetrahydrobiopterin (BH(4)), a cofactor required for iNOS activity. TNF-alpha also stimulates sphingosine kinase, the enzyme that phosphorylates sphingosine to form sphingosine-1-phosphate (SPP), a further metabolite of ceramide. Several clones of C6 cells, expressing widely varying levels of sphingosine kinase, were used to examine the role of SPP in regulation of GTPCH and BH(4) biosynthesis. Overexpression of sphingosine kinase, with concomitant increased endogenous SPP levels, potentiated the effect of TNF-alpha on GTPCH expression and activity and BH(4) biosynthesis. In contrast, enforced expression of sphingosine kinase had no effect on iNOS expression or NO formation. Furthermore, N,N-dimethylsphingosine, a potent sphingosine kinase inhibitor, completely eliminated the increased GTPCH activity and expression induced by TNF-alpha. Surprisingly, we found that, although C6 cells can secrete SPP, which is enhanced by TNF-alpha, treatment of C6 cells with exogenous SPP or dihydro-SPP had no affect on BH(4) biosynthesis. However, both SPP and dihydro-SPP markedly stimulated ERK 1/2 in C6 cells, which express cell surface SPP receptors. Interestingly, although this ERK activation was blocked by PD98059, which also reduced cellular proliferation induced by enforced expression of sphingosine kinase, PD98059 had no effect on GTPCH activity. Collectively, these results suggest that only intracellularly generated SPP plays a role in regulation of GTPCH and BH(4) levels.     

Circadian organization in male mice lacking the gene for endothelial nitric oxide synthase (eNOS-/-)

Circadian (approximately 24 h) rhythms in physiology and behavior are generated by the bilateral suprachiasmatic nucleus (SCN) of the anterior hypothalamus. For these endogenous rhythms to be synchronized with the external environment, light information must be transmitted to pacemaker cells within the SCN. This transmission of light information is accomplished via a direct retino-hypothalamic tract (RHT). Nitric oxide (NO), an endogenous gas that functions as a neurotransmitter, has been implicated as a messenger necessary for photic entrainment. Three isoforms of the enzyme that form NO, NO synthase, have been identified (a) in neurons (nNOS), (b) in the endothelial lining of blood vessels (eNOS), and (c) as an inducible form in macrophages (iNOS). The present study was undertaken to determine the specific role of eNOS in circadian organization and photic entrainment. Wild-type (WT) and eNOS-/- mice were initially entrained to a 14:10 light:dark (LD) cycle. After 3 weeks, the LD cycle was phase advanced. After an additional 3 weeks, animals were held in constant darkness (DD). eNOS-/- animals did not exhibit a deficit in the ability to entrain to the LD cycle, phase-shift locomotor activity, or free-run in constant conditions. Animals held in DD were killed after light exposure during either the subjective day or the subjective night to assess c-fos induction in the SCN. Light exposure during the subjective night increased c-fos protein expression in the SCN of both WT and eNOS-/- mice relative to animals killed after light exposure during the subjective day. Taken together, these findings suggest that endothelial isoform of NOS may not be necessary for photic entrainment in mice.     

Functional coupling of a Ca2+/calmodulin-dependent nitric oxide synthase and a soluble guanylyl cyclase in vertebrate photoreceptor cells.

Electrophysiological recordings on retinal rod cells, horizontal cells and on-bipolar cells indicate that exogenous nitric oxide (NO) has neuromodulatory effects in the vertebrate retina. We report here endogenous NO formation in mammalian photoreceptor cells. Photoreceptor NO synthase resembled the neuronal NOS type I from mammalian brain. NOS activity utilized the substrate L-arginine (Km = 4 microM) and the cofactors NADPH, FAD, FMN and tetrahydrobiopterin. The activity showed a complete dependence on the free calcium concentration ([Ca2+]) and was mediated by calmodulin. NO synthase activity was sufficient to activate an endogenous soluble guanylyl cyclase that copurified in photoreceptor preparations. This functional coupling was strictly controlled by the free [Ca2+] (EC50 = 0.84 microM). Activation of the soluble guanylyl cyclase by endogenous NO was up to 100% of the maximal activation of this enzyme observed with the exogenous NO donor compound sodium nitroprusside. This NO/cGMP pathway was predominantly localized in inner and not in outer segments of photoreceptors. Immunocytochemically, we localized NO synthase type I mainly in the ellipsoid region of the inner segments and a soluble guanylyl cyclase in cell bodies of cone photoreceptor cells. We conclude that in photoreceptors endogenous NO is functionally coupled to a soluble guanylyl cyclase and suggest that it has a neuromodulatory role in visual transduction and in synaptic transmission in the outer retina.