Regulation of NHE3 by nitric oxide in Caco-2 cells

The effect of nitric oxide (NO) on Na+/H+ exchange (NHE) activity was investigated utilizing Caco-2 cells as an experimental model. Incubation of Caco-2 cells with 10(-3) M S-nitroso-N-acetylpenicillamine (SNAP), a conventional donor of NO, for 20 min resulted in a approximately 45% dose-dependent decrease in NHE activity, as determined by assay of ethylisopropylamiloride-sensitive 22Na uptake. A similar decrease in NHE activity was observed utilizing another NO-specific donor, sodium nitroprusside. SNAP-mediated inhibition of NHE activity was not secondary to a loss of cell viability. NHE3 activity was significantly reduced by SNAP (P < 0.05), whereas NHE2 activity was essentially unaltered. The effects of SNAP were mediated by the cGMP-dependent signal transduction pathway as follows: 1) LY-83583 and 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), specific inhibitors of soluble guanylate cyclase, blocked the inhibitory effect of SNAP on NHE; 2) 8-bromo-cGMP mimicked the effects of SNAP on NHE activity; 3) the SNAP-induced decrease in NHE activity was counteracted by a specific protein kinase G inhibitor, KT-5823 (1 microM); 4) chelerythrine chloride (2 microM) or calphostin C (200 nM), specific protein kinase C inhibitors, did not affect inhibition of NHE activity by SNAP; 5) there was no cross activation by the protein kinase A-dependent pathway, as the inhibitory effects of SNAP were not blocked by Rp-cAMPS (25 microM), a specific protein kinase A inhibitor. These data provide novel evidence that NO inhibits NHE3 activity via activation of soluble guanylate cyclase, resulting in an increase in intracellular cGMP levels and activation of protein kinase G.     

Insulin increases NO-stimulated guanylate cyclase activity in cultured VSMC while raising redox potential

Insulin acutely stimulates cyclic guanosine monophosphate (cGMP) production in primary confluent cultured vascular smooth muscle cells (VSMC) from canine femoral artery, but the mechanism is not known. These cells contain the inducible isoform of nitric oxide (NO) synthase (iNOS), and insulin-stimulated cGMP production in confluent cultured cells is blocked by the NOS inhibitor, N(G)-monomethyl-L-arginine (L-NMMA). In the present study, it is shown that iNOS is also present in freshly dispersed VSMC from this artery, indicating that iNOS expression in cultured VSMC is not an artifact of the culture process. Insulin did not stimulate NOS activity in primary confluent cultured cells because it did not affect citrulline or combined NO(-)(3)/NO(-)(2) production. To see whether insulin required the permissive presence of NO to stimulate cGMP production, iNOS and basal cGMP production were inhibited with L-NMMA, and the cells were incubated with or without 1 nM insulin and/or the NO donor, S-nitroso-N-acetyl-D,L-penicillamine (SNAP) at a concentration (0.1 microM) that restored cGMP production to the basal value. In the presence of L-NMMA, insulin no longer affected cGMP production but when insulin was added to L-NMMA plus SNAP, cGMP production was increased by 69% (P < 0.05 vs. L-NMMA plus SNAP). Insulin, which increases glucose uptake by these cells, increased the cell lactate content and the lactate-to-pyruvate ratio (LPR) by 81 and 97%, respectively (both P < 0.05), indicating that the hormone increased aerobic glycolysis and the redox potential. The effects of insulin on LPR and cGMP production were blocked by removing glucose or by adding 2-deoxyglucose to the incubation media and were duplicated by the reducing substrate, beta-hydroxybutyrate. We conclude that insulin does not acutely affect iNOS activity in these VSMC but it does augment cGMP production induced by the NO already present in the cell while increasing aerobic glycolysis and the cell redox potential.     

Natriuretic peptides and nitric oxide stimulate cGMP synthesis in different cellular compartments

Cyclic nucleotide-gated (CNG) channels are a family of ion channels activated by the binding of cyclic nucleotides. Endogenous channels have been used to measure cyclic nucleotide signals in photoreceptor outer segments and olfactory cilia for decades. Here we have investigated the subcellular localization of cGMP signals by monitoring CNG channel activity in response to agonists that activate either particulate or soluble guanylyl cyclase. CNG channels were heterologously expressed in either human embryonic kidney (HEK)-293 cells that stably overexpress a particulate guanylyl cyclase (HEK-NPRA cells), or cultured vascular smooth muscle cells (VSMCs). Atrial natriuretic peptide (ANP) was used to activate the particulate guanylyl cyclase and the nitric oxide donor S-nitroso-n-acetylpenicillamine (SNAP) was used to activate the soluble guanylyl cyclase. CNG channel activity was monitored by measuring Ca2+ or Mn2+ influx through the channels using the fluorescent dye, fura-2. We found that in HEK-NPRA cells, ANP-induced increases in cGMP levels activated CNG channels in a dose-dependent manner (0.05-10 nM), whereas SNAP (0.01-100 microM) induced increases in cGMP levels triggered little or no activation of CNG channels (P < 0.01). After pretreatment with 100 microM 3-isobutyl-1-methylxanthine (IBMX), a nonspecific phosphodiesterase inhibitor, ANP-induced Mn2+ influx through CNG channels was significantly enhanced, while SNAP-induced Mn2+ influx remained small. In contrast, we found that in the presence of IBMX, both 1 nM ANP and 100 microM SNAP triggered similar increases in total cGMP levels. We next sought to determine if cGMP signals are compartmentalized in VSMCs, which endogenously express particulate and soluble guanylyl cyclase. We found that 10 nM ANP induced activation of CNG channels more readily than 100 muM SNAP; whereas 100 microM SNAP triggered higher levels of total cellular cGMP accumulation. These results suggest that cGMP signals are spatially segregated within cells, and that the functional compartmentalization of cGMP signals may underlie the unique actions of ANP and nitric oxide.     

Laser ablation reveals regulation of ciliary activity by serotonergic neurons in molluscan embryos

Early in embryonic development, the pond snail Helisoma trivolvis exhibits a rotational behavior that is generated by beating of cilia in the dorsolateral and pedal bands. Although previous anatomical and pharmacological studies provided indirect evidence that a pair of serotonergic neurons, Embryonic Neurons C1 (ENC1s), is involved in regulating embryonic rotation, direct evidence linking ENC1 to ciliary function is still lacking. In the present study, we used laser microbeams to perturb ENC1 in vivo while monitoring ciliary activity in identified ciliary bands. A laser treatment protocol to specifically ablate ENC1 without damaging the surrounding cells was established. Unilateral laser treatment of ENC1 caused transient increases in the activity of the pedal and ipsidorsolateral cilia, lasting 30-50 min. In contrast, activity of cilia that were not anatomically associated with ENC1 was unaffected by laser treatment. Mianserin, an effective serotonin antagonist in Helisoma ciliated cells, decreased the overall CBF of pedal and dorsolateral cilia by reducing the occurrence of spontaneous CBF surges in these cilia. Finally, the cilioexcitatory action of ENC1 laser treatment was mimicked by serotonin and reduced in the presence of mianserin. These results suggest that laser treatment provokes a release of serotonin from ENC1, resulting in a prolonged elevation of activity in the target ciliary cells. We conclude that, in addition to their previously established role in regulating neurodevelopment, ENC1s also function as serotonergic motor neurons to regulate ciliary activity, and therefore the rotational behavior of early embryos.     

Nitric oxide induces apoptotic death of cardiomyocytes via a cyclic-GMP-dependent pathway

Recently, we have reported that excess amounts of nitric oxide (NO) produced by inducible NO synthase are involved in the development of myocardial damage in rats with induced myocarditis. However, there remain many problems to be solved concerning its mechanism of action. In this study, we examined whether NO induces apoptotic cell death in cardiomyocytes. Cultured neonatal rat cardiomyocytes were exposed to S-nitroso-N-acetylpenicillamine (SNAP) and (+/-)-E-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamine (NOR 3), as NO donors, or 8-bromo-cyclic GMP (cGMP), an analog of cGMP which functions as a second messenger in cells stimulated by NO. DNA fragmentation was confirmed by electron microscopy, by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method, and by agarose gel electrophoresis. Exogenously supplied SNAP or NOR 3 induced cardiomyocyte apoptosis in a dose- and time-dependent manner. Cardiomyocytes exposed to SNAP displayed typical features of apoptosis as demonstrated by electron microscopy. Treatment of the cells with 8-bromo-cGMP also induced apoptosis. In cardiomyocytes, SNAP-induced apoptosis was completely blocked by a PKG inhibitor (KT5823) and by a soluble guanylate cyclase inhibitor (ODQ) and was suppressed by hemoglobin and was completely blocked by ZVAD-FMK, a caspase inhibitor. These results show that NO-mediated apoptosis of cardiomyocytes is cGMP dependent and that caspases are involved in this process.     

NO and cGMP mediate angiotensin AT2 receptor-induced renal renin inhibition in young rats

We hypothesized that angiotensin subtype-2 receptor (AT(2)R) inhibits renal renin biosynthesis in young rats via nitric oxide (NO). We monitored changes in renal NO, cGMP, renal renin content (RRC), and ANG II in 4-wk-old rats in response to low sodium (LNa(+)) intake alone and combined with 8-h direct renal cortical administration of AT(1) receptor blocker valsartan (VAL), AT(2)R blocker PD123319 (PD), NO synthase inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME), NO donor S-nitroso-N-acetyl penicillamine (SNAP), or guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazolo[4,2-alpha] quinoxaline-1-one (ODQ). In addition, we monitored renal endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) in response to VAL or PD. LNa(+), VAL, PD, l-NAME, and ODQ increased RRC, ANG II, and renin mRNA. PD and l-NAME decreased NO and cGMP, while SNAP reduced RRC, ANG II, renin mRNA, and reversed the effects of PD. PD also reduced eNOS and nNOS protein and mRNA. Combined treatment with PD, l-NAME, or ODQ and VAL reversed the effects of VAL and caused further increase in RRC, ANG II, renin mRNA, and protein. ODQ reversed the effects of SNAP. These data demonstrate that the renal AT(2) receptor decreases renal renin biosynthesis and ANG II production in young rats. Reversal of the PD effects by SNAP and SNAP effects by ODQ confirms that NO and cGMP mediate the AT(2) receptor inhibition of renal renin production.     

Nitric oxide-evoked glutamate release and cGMP production in cerebellar slices: control by presynaptic 5-HT1D receptors

We previously reported that pre- and postsynaptic 5-hydroxytryptamine (5-HT) receptors effectively control glutamatergic transmission in adult rat cerebellum. To investigate where 5-HT acts in the glutamate ionotropic receptors/nitric oxide/guanosine 3',5'-cyclic monophosphate (cGMP) pathway, in the present study 5-HT modulation of the cGMP response to the nitric oxide donor S-nitroso-penicillamine (SNAP) was studied in adult rat cerebellar slices. While cGMP elevation produced by high-micromolar SNAP was insensitive to 5-HT, 1 microM SNAP, expected to release nitric oxide in the low-nanomolar concentration range, elicited cGMP production and endogenous glutamate release both of which could be prevented by activating presynaptic 5-HT1D receptors. Released nitric oxide appeared responsible for cGMP production and glutamate release evoked by 1 microM SNAP, as both the effects were mimicked by the structurally unrelated nitric oxide donor 2-(N,N-diethylamino)-diazenolate-2-oxide (0.1 microM). Dependency of the 1 microM SNAP-evoked release of glutamate on external Ca2+, sensitivity to presynaptic release-regulating receptors and dependency on ionotropic glutamate receptor functioning, suggest that nitric oxide stimulates exocytotic-like, activity-dependent glutamate release. Activation of ionotropic glutamate receptors/nitric oxide synthase/guanylyl cyclase pathway by endogenously released glutamate was involved in the cGMP response to 1 microM SNAP, as blockade of NMDA/non-NMDA receptors, nitric oxide synthase or guanylyl cyclase, abolished the cGMP response. To conclude, in adult rat cerebellar slices low-nanomolar exogenous nitric oxide could facilitate glutamate exocytotic-like release possibly from parallel fibers that subsequently activated the glutamate ionotropic receptors/nitric oxide/cGMP pathway. Presynaptic 5-HT1D receptors could regulate the nitric oxide-evoked release of glutamate and subsequent cGMP production.     

Exogenous NO triggers preconditioning via a cGMP- and mitoKATP-dependent mechanism

Exogenous nitric oxide (NO) triggers a preconditioning-like effect in heart via a pathway that is dependent on reactive oxygen species. This study examined the signaling pathway by which the NO donor S-nitroso-N-acetylpenicillamine (SNAP, 2 microM) triggers its anti-infarct effect. Isolated rabbit hearts experienced 30 min of regional ischemia and 120 min of subsequent reperfusion. Infarct size was determined by triphenyltetrazolium chloride staining. Infarct size was reduced from 30.5 +/- 3.0% of the risk zone in control hearts to 10.2 +/- 2.0% in SNAP-treated hearts. Bracketing the SNAP infusion with either the guanylyl cyclase blocker 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (2 microM) or the mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channel blocker 5-hydroxydecanoate (200 microM) completely blocked the infarct-sparing effect of SNAP (34.3 +/- 3.8 and 32.2 +/- 1.6% infarction, respectively). Pretreatment of hearts with 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (10 microM), which is a cell-permeable cGMP analog that activates protein kinase G, mimicked the preconditioning effect of SNAP by reducing infarct size to 7.5 +/- 1.1% of the risk zone. This salutary effect was abolished by either the free radical scavenger N-(2-mercaptopropionyl)glycine (1 mM) or 5-hydroxydecanoate (100 microM; 28.9 +/- 2.7 and 33.6 +/- 5.0% infarction of the risk zone, respectively). To confirm these functional data and the effect of SNAP on the guanylyl cyclase-protein kinase G signaling pathway, cGMP levels were measured. SNAP increased the level from 0.18 +/- 0.04 to 0.61 +/- 0.14 pmol/mg of protein (P < 0.05). These data suggest that exogenous NO triggers the preconditioning effect by initiating a cascade of events including stimulation of guanylyl cyclase to make cGMP, activation of protein kinase G, opening of mitoK(ATP) channels, and, finally, production of reactive oxygen species.     

Dissociation of cGMP accumulation and relaxation in myometrial smooth muscle: effects of S-nitroso-N-acetylpenicillamine and 3-morpholinosyndonimine

In guinea pig, primate and man, nitric oxide (NO)-induced regulation of myometrial smooth muscle contraction is distinct from other smooth muscles because cyclic guanosine 3',5'-cyclic monophosphate (cGMP) accumulation is neither necessary nor sufficient to relax the tissue. To further our understanding of the mechanism of action of NO in myometrium, we employed the NO donors, S-nitroso-N-acetylpenicillamine (SNAP), and 3-morpholinosyndonimine (SIN-1) proposed to relax airway smooth muscle by disparate mechanisms involving elevation in intracellular calcium ([Ca(2+)](i)) or cGMP accumulation, respectively. Treatment of guinea pig myometrial smooth muscle with either NO donor at concentrations thought to produce maximal relaxation of smooth muscles resulted in significant elevations in cGMP that were accompanied by phosphorylation of the cGMP-dependent protein kinase substrate vasodilator-stimulated phosphoprotein (VASP), shown here for the first time to be present and phosphorylated in myometrium. Stimulation of myometrial strips with oxytocin (OT, 1 microM) produced an immediate increase in contractile force that persisted in the continued presence of the agonist. Addition of SNAP (100 microM) in the presence of OT relaxed the tissue completely as might be expected of an NO donor. SIN-1 failed to relax the myometrium at any concentration tested up to 300 microM. In Fura-2 loaded myometrial cells prepared from guinea pig, addition of SNAP (100 microM) in the absence of other agonists caused a significant, reproducible elevation of intracellular calcium while SIN-1 employed under the same conditions did not. Our data further support the notion that NO action in myometrium is distinct from that in other smooth muscles and underscores the possibility that discrete regional changes in [Ca(2+)](i), rather than cGMP, signal NO-induced relaxation of the muscle.     

Modulation of Cl-/OH- exchange activity in Caco-2 cells by nitric oxide

The present studies were undertaken to determine the direct effects of nitric oxide (NO) released from an exogenous donor, S-nitroso-N-acetyl pencillamine (SNAP) on Cl-/OH- exchange activity in human Caco-2 cells. Our results demonstrate that NO inhibits Cl-/OH- exchange activity in Caco-2 cells via cGMP-dependent protein kinases G (PKG) and C (PKC) signal-transduction pathways. Our data in support of this conclusion can be outlined as follows: 1) incubation of Caco-2 cells with SNAP (500 microM) for 30 min resulted in approximately 50% inhibition of DIDS-sensitive 36Cl uptake; 2) soluble guanylate cyclase inhibitors Ly-83583 and (1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one significantly blocked the inhibition of Cl-/OH- exchange activity by SNAP; 3) addition of 8-bromo-cGMP (8-BrcGMP) mimicked the effects of SNAP; 4) specific PKG inhibitor KT-5823 significantly inhibited the decrease in Cl-/OH- exchange activity in response to either SNAP or 8-BrcGMP; 5) Cl-/OH-exchange activity in Caco-2 cells in response to SNAP was not altered in the presence of protein kinase A (PKA) inhibitor (Rp-cAMPS), demonstrating that the PKA pathway was not involved; 6) the effect of NO on Cl-/OH- exchange activity was mediated by PKC, because each of the two PKC inhibitors chelerythrine chloride and calphostin C blocked the SNAP-mediated inhibition of Cl-/OH- exchange activity; 7) SO/OH- exchange in Caco-2 cells was unaffected by SNAP. Our results suggest that NO-induced inhibition of Cl-/OH- exchange may play an important role in the pathophysiology of diarrhea associated with inflammatory bowel diseases.     

Bradykinin and ATP stimulate L-arginine uptake and nitric oxide release in vascular endothelial cells.

The effects of bradykinin and ATP on L-arginine transport and nitric oxide (NO) production were studied in porcine aortic endothelial cells cultured and perfused on microcarriers and deprived of L-arginine for 24 h. Stimulation of cells with bradykinin (100 nM) or ATP (100 microM) resulted in a rapid increase in L-arginine uptake and NO release. In the presence of nitro-L-arginine (100 microM), an inhibitor of NO synthase, the stimulatory effect of bradykinin on L-arginine uptake was partially inhibited while NO release was completely abolished. Nitro-L-arginine alone was not an inhibitor of basal L-arginine transport, suggesting that its inhibitory action was not directly on the L-arginine transporter but a result of the inhibition of NO generation. These data indicate that during agonist-stimulated NO production there is a concomitant increase in the transport of L-arginine into endothelial cells providing a mechanism for the continual generation of NO.     

Ginsenoside Re promotes human sperm capacitation through nitric oxide-dependent pathway

The regulation of sperm capacitation is important for successful fertilization. Ginsenosides, the biologically effective components of ginseng, have been found to enhance intracellular nitric oxide (NO) production and the latter has recently been indicated to play a significant role in modulation of sperm functions. We investigated the effect of Ginsenoside Re on human sperm capacitation in vitro and the mechanism by which the Ginsenosides play their roles. Spermatozoa were separated by Percoll and incubated with 0, 1, 10, or 100 microM of Ginsenoside Re. The percentages of spontaneous and lysophosphatidylcholine (LPC)-induced acrosome reaction (AR), as a measure of sperm capacitation, were assayed with fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA). The intracellular cGMP level was measured by [(3)H] cGMP radioimmunoassay system. The results showed that the percentages of both spontaneous and LPC-induced AR and intracellular cGMP level were significantly enhanced by Ginsenoside Re with a concentration-dependent manner. Sodium nitroprusside (SNP, 100 nM), a NO donor, mimicked the effects of Ginsenoside Re. And pretreatment with a NOS inhibitor N(omega)-nitro-l-arginine methyl ester (L-NAME, 100 microM) or a NO scavenger N-acetyl-l-cysteine (LNAC, 1 mM) completely blocked the effects of Ginsenoside Re. Furthermore, the AR-inducing effect of Ginsenoside Re was significantly reduced in the presence of the soluble guanylate cyclase inhibitor LY83583 or cGMP-dependent protein kinase (PCK) inhibitor KT5823, whereas addition of the cGMP analogue 8-Br-cGMP significantly increased the AR of human spermatozoa. Data suggested that Ginsenoside Re is beneficial to sperm capacitation and AR, and that the effect is accomplished through NO/cGMP/PKG pathway.     

Endothelin-3 stimulates production of endothelium-derived nitric oxide via phosphoinositide breakdown.

Cultured bovine endothelial cells (EC) have specific receptors for endothelin (ET)-3 functionally coupled to phosphoinositide breakdown. We studied whether ET-3 stimulates synthesis of nitric oxide (NO), an endothelium-derived relaxing factor that activates soluble guanylate cyclase in EC, and whether the ET-3-induced NO formation involves G-proteins. ET-3 dose-dependently stimulated production of intracellular cGMP in EC, of which effects were abolished by pretreatment with NG-monomethyl L-arginine, an inhibitor of NO synthesis, and methylene blue, an inhibitor of soluble guanylate cyclase. The stimulatory effects of ET-3 on cGMP production, inositol trisphosphate formation and increase in cytosolic free Ca2+ concentration were similarly blocked by pretreatment with pertussis toxin (PTX). These data suggest that ET-3 induces synthesis of NO mediated by phosphoinositide breakdown via PTX-sensitive G-protein in EC.     

Expression of endothelial nitric oxide synthase in ciliated epithelia of rats.

Endothelial nitric oxide synthase (eNOS), originally found in the endothelium of vascular tissue, also exists in other cell types, including ciliated epithelia of airways. The eNOS is ultrastructurally localized to the basal body of the microtubules of the cilia, and nitric oxide (NO) stimulates ciliary beat frequency (CBF). We examined whether the expression of eNOS is present in ciliated cells of other organs. Western blotting analysis revealed that eNOS was expressed in the rat cerebrum, lung, trachea, testis, and oviduct. Immunohistochemical staining showed that eNOS was localized in the ciliated epithelia of airways, oviduct, testis, and ependymal cells of brain in addition to the endothelium and smooth muscle of the vasculature. To confirm the activation of eNOS in the ciliated epithelia, we examined the effect of L-arginine (L-Arg), the substrate of NOS, on the production of nitrite and nitrate (NOx) in the cultured explants of rat trachea. L-Arg (100 microM) increased NOx levels significantly (p<0.05). In explants exposed to inhibitors of NOS, the effect of l-Arg on the production of NOx was blocked. These findings suggest that epithelial NO plays an important role in signal transduction associated with ciliary functions.     

Regulation of ciliary beat frequency by the nitric oxide signaling pathway in mouse nasal and tracheal epithelial cells

Objectives

Our purpose was to investigate the role of the nitric oxide (NO) signaling pathway in the regulation of ciliary beat frequency (CBF) in mouse nasal and tracheal epithelial cells.

Methods

We studied the effects of the NO donor l-arginine (L-Arg) and specific inhibitors of the NO signaling pathway on CBF of both nasal and tracheal epithelial cells by using high-speed digital microscopy. We also examined eNOS, sGC β, PKG I and acetylated α tubulin expression in native mouse nasal and tracheal epithelium using immunohistochemical methods.

Results

L-Arg significantly increased CBF of cultured nasal and tracheal epithelial cells, and the effects were blocked by pretreatment with N^G-nitro-l-arginine methyl ester (L-NAME), a NOS inhibitor, with LY-83583, a sGC inhibitor, or with KT-5823, a PKG inhibitor. Positive immunostaining for NO signaling molecules including eNOS, sGC β and PKG I was observed in either nasal or tracheal ciliated epithelium.

Conclusion

NO plays a role in regulating CBF of mouse respiratory epithelial cells via a eNOS–NO–sGC β–cGMP–PKG I pathway.     

Nitric oxide contributes to the development of a post-injury Th2 T-cell phenotype and immune dysfunction

Severe injury induces immune dysfunction resulting in increased susceptibility to opportunistic infections. Previous studies from our laboratory have demonstrated that post-burn immunosuppression is mediated by nitric oxide (NO) due to the increased expression of macrophage inducible nitric oxide synthase (iNOS). In contrast, others suggest that injury causes a phenotypic imbalance in the regulation of Th1- and Th2 immune responses. It is unclear whether or not these apparently divergent mediators of immunosuppression are interrelated. To study this, C57BL/6 mice were subjected to major burn injury and splenocytes were isolated 7 days later and stimulated with antiCD3. Burn injury induced NO-mediated suppression of proliferative responses that was reversed in the presence of the NOS inhibitor L-monomethyl-L-arginine and subsequently mimicked by the addition of the NO donor, S-nitroso-N-acetyl-penicillamine (SNAP). SNAP also dose-dependently suppressed IFN-gamma and IL-2 (Th1), but not IL-4 and IL-10 (Th2) production. Delaying the addition of SNAP to the cultures by 24 h prevented the suppression of IFN-gamma production. The Th2 shift in immune phenotype was independent of cGMP and apoptosis. The addition of SNAP to cell cultures also induced apoptosis, attenuated mitochondrial oxidative metabolism and induced mitochondrial membrane depolarization. However, these detrimental cellular effects of NO were observed only at supra-physiologic concentrations (>250 microM). In conclusion, these findings support the concept that NO induces suppression of cell-mediated immune responses by selective action on Th1 T cells, thereby promoting a Th2 response.     

Multiple potassium channels mediate nitric oxide-induced inhibition of rat vascular smooth muscle cell proliferation.

Several nitric oxide (NO) effects in the cardiovascular system are mediated by soluble guanylate cyclase (sGC) activation but potassium channels (KC) are also emerging as important effectors of NO actions. We investigated the relationship among vascular smooth muscle cell proliferation, NO, cyclic GMP, and KC using the A7r5 smooth muscle cell line derived from rat aorta. NO donors (two nitrosothiols, S-nitroso-acetyl-d,l-penicillamine, SNAP, and S-nitroso-glutathione, GSNO, and an organic nitrate, glyceryl trinitrate, GTN; 1-1000 microM) dose-dependently inhibited cell proliferation. ODQ (a selective inhibitor of sGC; 0.1 and 1 microM) and KT5823 (a selective inhibitor of cGMP-dependent protein kinase, 1 microM) prevented NO effects, confirming that sGC is a key target. In this report, we show that tetraethylammonium (TEA, a non-selective blocker of KC, 300 microM), and 4-aminopyridine (a selective blocker of voltage-dependent KC, 100 microM) prevented SNAP inhibitory effects on cell proliferation, whereas glibenclamide (a selective blocker of ATP-dependent KC, 1 microM) was ineffective. Iberiotoxin (a selective blocker of high conductance calcium-activated KC, 100 nM), as well charybdotoxin (a blocker of high and intermediate conductance calcium-activated KC, 100 nM) and apamine (a selective blocker of small conductance calcium-activated KC, 100 nM), blocked the antiproliferative effect induced by SNAP. NS1619 (an opener of high conductance calcium-activated KC, 1-100 microM), inhibited cell proliferation. In addition, sub-effective concentrations of ODQ (100 nM) and TEA (10 microM) synergized in blocking SNAP antiproliferative effects. Thus, voltage-dependent and calcium-activated but not ATP-dependent KC appear to have a prominent role, besides sGC activation, in NO-induced inhibition of vascular smooth muscle cell proliferation.     

Inhibitory effects of NO on carotid body: contribution of neural and endothelial nitric oxide synthase isoforms

We tested the hypothesis that nitric oxide (NO) produced within the carotid body is a tonic inhibitor of chemoreception and determined the contribution of neuronal and endothelial nitric oxide synthase (eNOS) isoforms to the inhibitory NO effect. Accordingly, we studied the effect of NO generated from S-nitroso-N-acetylpenicillamide (SNAP) and compared the effects of the nonselective inhibitor N(omega)-nitro-l-arginine methyl ester (l-NAME) and the selective nNOS inhibitor 1-(2-trifluoromethylphenyl)-imidazole (TRIM) on chemosensory dose-response curves induced by nicotine and NaCN and responses to hypoxia (Po(2) approximately 30 Torr). CBs excised from pentobarbitone-anesthetized cats were perfused in vitro with Tyrode at 38 degrees C and pH 7.40, and chemosensory discharges were recorded from the carotid sinus nerve. SNAP (100 microM) reduced the responses to nicotine and NaCN. l-NAME (1 mM) enhanced the responses to nicotine and NaCN by increasing their duration, but TRIM (100 microM) only enhanced the responses to high doses of NaCN. The amplitude of the response to hypoxia was enhanced by l-NAME but not by TRIM. Our results suggest that both isoforms contribute to the NO action, but eNOS being the main source for NO in the cat CB and exerting a tonic effect upon chemoreceptor activity.     

Methylene blue blocks cGMP production and disrupts directed migration of microglia to nerve lesions in the leech CNS

Migration and accumulation of microglial cells at sites of injury are important for nerve repair. Recent studies on the leech central nervous system (CNS), in which synapse regeneration is successful, have shown that nitric oxide (NO) generated immediately after injury by endothelial nitric oxide synthase (eNOS) stops migrating microglia at the lesion. The present study obtained results indicating that NO may act earlier, on microglia migration, and aimed to determine mechanisms underlying NO's effects. Injury induced cGMP immunoreactivity at the lesion in a pattern similar to that of eNOS activity, immunoreactivity, and microglial cell accumulation, which were all focused there. The soluble guanylate cyclase (sGC) inhibitor methylene blue (MB) at 60 microM abolished cGMP immunoreactivity at lesions and blocked microglial cell migration and accumulation without interfering with axon conduction. Time-lapse video microscopy of microglia in living nerve cords showed MB did not reduce cell movement but reduced directed movement, with significantly more cells moving away from the lesion or reversing direction and fewer cells moving toward the lesion. The results indicate a new role for NO, directing the microglial cell migration as well as stopping it, and show that NO's action may be mediated by cGMP.     

eNOS, nNOS, cGMP and protein kinase G mediate the inhibitory effect of pancreastatin, a chromogranin A-derived peptide, on growth and proliferation of hepatoma cells

Pancreastatin (PST), a chromogranin A-derived peptide, has an anti-insulin metabolic effect and inhibits growth and proliferation by producing nitric oxide (NO) in HTC rat hepatoma cells. When NO production is blocked, a proliferative effect prevails due to the activation a Galphaq/11-phospholipase C-beta (PLC-beta) pathway, which leads to an increase in [Ca2+]i, protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) activation. The aim of the present study was to investigate the NO synthase (NOS) isoform that mediates these effects of PST on HTC hepatoma cells and the possible roles of cyclic GMP (cGMP) and cGMP-dependent protein kinase. DNA and protein synthesis in response to PST were measured as [3H]-thymidine and [3H]-leucine incorporation in the presence of various pharmacological inhibitors: N-monomethyl-L-arginine (NMLA, nonspecific NOS inhibitor), L-NIO (endothelial nitric oxide synthase (eNOS) inhibitor), espermidine (neuronal nitric oxide synthase (nNOS) inhibitor), LY83583 (guanylyl cyclase inhibitor), and KT5823 (protein kinase G inhibitor, (PKG)). L-NIO, similarly to NMLA, reverted the inhibitory effect of PST on hepatoma cell into a stimulatory effect on growth and proliferation. Nevertheless, espermidine also prevented the inhibitory effect of PST, but there was no stimulation of growth and proliferation. When guanylyl cyclase activity was blocked, there was again a reversion of the inhibitory effect into a stimulatory action, suggesting that the effect of NO was mediated by the production of cGMP. PKG inhibition prevented the inhibitory effect of PST, but there was no stimulatory effect. Therefore, the inhibitory effect of PST on growth and proliferation of hepatoma cells may be mainly mediated by eNOS activation. In turn, the effect of NO may be mediated by cGMP, whereas other pathways in addition to PKG activation seem to mediate the inhibition of DNA and protein synthesis by PST in HTC hepatoma cells.