Silencing of Kv4.1 potassium channels inhibits cell proliferation of tumorigenic human mammary epithelial cells

Potassium channel activity has been shown to facilitate cell proliferation in cancer cells. In the present study, the role of Kv4.1 channels in immortal and tumorigenic human mammary epithelial cells was investigated. Kv4.1 protein expression was positively correlated with tumorigenicity. Moreover, transfection with siRNAs targeting Kv4.1 mRNA suppressed proliferation of tumorigenic mammary epithelial cells. Experiments using mRNA isolated from human breast cancer tissues revealed that the level of Kv4.1 mRNA expression varied depending on the stage of the tumor. Kv4.1 protein expression increased during stages T2 and T3 compared to normal tissue. These results demonstrated that Kv4.1 plays a role in proliferation of tumorigenic human mammary epithelial cells. In addition, elevated Kv4.1 expression may be useful as a diagnostic marker for staging mammary tumors and selective blockers of Kv4.1 may serve to suppress tumor cell proliferation.     

Nitric oxide irreversibly inhibits cytochrome oxidase at low oxygen concentrations: evidence for inverse oxygen concentration-dependent peroxynitrite formation

The present study shows that nitric oxide (NO) irreversibly inhibits purified cytochrome oxidase in a reverse oxygen concentration-dependent manner. The inhibition is dramatically protected by a peroxynitrite scavenger, suggesting that peroxynitrite is formed from the reaction of NO with cytochrome oxidase at low oxygen concentration, and that peroxynitrite is involved in irreversible cytochrome oxidase inactivation. Production of nitroxyl anion or superoxide was tested as potential mechanisms underlying the conversion of NO to peroxynitrite. A nitroxyl anion scavenger potently protected the irreversible inhibition, whereas a superoxide dismutase did not provide protective effect, suggesting that the peroxynitrite was formed from nitroxyl anion rather than the reaction of NO with superoxide.     

Nitric oxide inhibits cell growth in cultured human thyrocytes

Effect of NO induced by interleukin-1 (IL-1) or IL-1/interferon-_γ (IL-1/IFN-_γ) was investigated on cell growth using primary cultures of human thyrocytes. Cytokine-induced NO production was associated not only with an increase in cyclic GMP (cGMP) formation but also with an inhibition of cell growth determined by bromo-deoxyuridine (Br-dU) incorporation into DNA. When NO synthesis was blocked by N^G-monomethyl-L-arginine (L-MMA), cGMP formation was prevented in parallel with NO production and inversely a restoration of cell growth was evident. S-nitroso-N-acetyl-penicillamine, a NO donor, but not a cell permeable cGMP analog, 8-bromo-cGMP, inhibited cell growth in a dose-dependent manner. The present findings strongly indicate that endogenous NO produced by the cytokine treatment as well as exogenous NO, has a cGMP-independent inhibitory action on human thyrocyte growth.     

Nitric oxide mediates low magnesium inhibition of osteoblast-like cell proliferation

An adequate intake of magnesium (Mg) is important for bone cell activity and contributes to the prevention of osteoporosis. Because (a) Mg is mitogenic for osteoblasts and (b) reduction of osteoblast proliferation is detected in osteoporosis, we investigated the influence of different concentrations of extracellular Mg on osteoblast-like SaOS-2 cell behavior. We found that low Mg inhibited SaOS-2 cell proliferation by increasing the release of nitric oxide through the up-regulation of inducible nitric oxide synthase (iNOS). Indeed, both pharmacological inhibition with the iNOS inhibitor l-N(6)-(iminoethyl)-lysine-HCl and genetic silencing of iNOS by small interfering RNA restored the normal proliferation rate of the cells. Because a moderate induction of nitric oxide is sufficient to potentiate bone resorption and a relative deficiency in osteoblast proliferation can result in their inadequate activity, we conclude that maintaining Mg homeostasis is relevant to ensure osteoblast function and, therefore, to prevent osteoporosis.     

GABAB receptor activation inhibits Ca2+-activated potassium channels in synaptosomes: involvement of G-proteins

86Rb-efflux assay from preloaded synaptosomes of rat cerebral cortex was developed to study the effect of GABAB receptor agonist baclofen on Ca2+-activated K+-channels. Depolarization (100 mM K) of 86Rb-loaded synaptosomes in physiological buffer increased Ca2+-activated 86Rb-efflux by 400%. The 86Rb-efflux was blocked by quinine sulphate, tetraethylammonium and La3+ indicating the involvement of Ca2+-activated K+-channels. (-)Baclofen inhibited Ca2+-activated 86Rb-efflux in a stereospecific manner. The inhibitory effect of (-)baclofen was mediated by GABAB receptor activation, since it was blocked by GABAB antagonist phaclofen, but not by bicuculline. Further, pertussis toxin also blocked the ability of baclofen or depolarizing action to affect Ca2+-activated K+-channels. These results suggest that baclofen inhibits Ca2+-activated K+-channels in synaptosomes and these channels are regulated by G-proteins. This assay may provide an ideal in vitro model to study GABAB receptor pharmacology.     

Nitric oxide inhibits proliferation but increases life-span of T lymphocytes in tumour-bearing rats

 Nitric oxide (NO) has been shown to inhibit the proliferation of lymphocytes. However, in tumour-bearing rats treated with the immunomodulator OM 163, the regressing nodules were heavily infiltrated by T lymphocytes, although they contained high levels of NO. We show here that NO, while inhibiting the proliferation of lymphocytes, increased their life-span, pointing to the ambivalence of this molecule in the course of tumour growth and regression. Received: 16 October 1997 / Accepted: 8 January 1998     

Nitric oxide involvement in pancreatic beta cell apoptosis by glibenclamide.

Glibenclamide as a second-generation compound of sulfonylurea has widely been used in the treatment of type 2 diabetes patients. It has been shown that it induces apoptosis in beta cells, which is partially mediated by Ca(2+) influx. Here, we investigated the role of nitric oxide (NO) and nitric oxide synthase (NOS) isoforms on glibenclamide-induced apoptosis in rat insulinoma cells. Our results showed that glibenclamide induces NO generation (measured as nitrite) that is accompanied with decrease of cell viability in a defined concentration of glibenclamide. The effects of glibenclamide on cell viability were partially inhibited after treatment with N(G)-nitro-L-arginine methyl ester (L-NAME), inhibitor more selective for constitutive nitric oxide synthase, and in the presence of D600--a blocker of voltage-gated L-type Ca(2+) channels inhibited Ca(2+) influx into beta cells, whereas aminoguanidine (AG), a preferential inhibitor of inducible NOS, was significantly less effective. Analysis of DNA fragmentation by electrophoresis and staining with Hoechest 33342 and propidium iodide showed that L-NAME, but not AG, prevented DNA fragmentation and decreased the number of cells with condensed and fragmented nuclei. It revealed that the effects of glibenclamide on apoptosis were partially inhibited by treatment with L-NAME. In conclusion, we have shown that NO production in glibenclamide treated cells may be involved in the induction of apoptotic cell death in pure beta cell line and it may be due to Ca(2+) dependent activation of constitutive NOS isoforms.     

Nitric oxide inhibits mammalian methylmalonyl-CoA mutase

Methylmalonyl-CoA mutase is a key enzyme in intermediary metabolism, and children deficient in enzyme activity have severe metabolic acidosis. We found that nitric oxide (NO) inhibits methylmalonyl-CoA mutase activity in rodent cell extracts. The inhibition of enzyme activity occurred within minutes and was not prevented by thiols, suggesting that enzyme inhibition was not occurring via NO reaction with cysteine residues to form nitrosothiol groups. Enzyme inhibition was dependent on the presence of substrate, implying that NO was reacting with cobalamin(II) (Cbl(II)) and/or the deoxyadenosyl radical (.CH(2)-Ado), both of which are generated from the co-factor of the enzyme, 5'-deoxyadenosyl-cobalamin (AdoCbl), on substrate binding. Consistent with this hypothesis was the finding that high micromolar concentrations (> or =600 microm) of oxygen also inhibited enzyme activity. To study the mechanism of NO reaction with AdoCbl, we simulated the enzymatic reaction by photolyzing AdoCbl, and found that even at low NO concentrations, NO reacted with both the generated Cbl(II) and .CH(2)-Ado indicating that NO could effectively compete with the back formation of AdoCbl. Thus, NO inhibition of methylmalonyl-CoA mutase appeared to be from the reaction of NO with both AdoCbl intermediates (Cbl(II) and .CH(2)-Ado) generated during the enzymatic reaction. The inhibition of methylmalonyl-CoA mutase by NO was likely of physiological relevance because a NO donor inhibited enzyme activity in intact cells, and scavenging NO from cells or inhibiting cellular NO synthesis increased methylmalonyl-CoA mutase activity when measured subsequently in cell extracts.     

Nitric oxide inhibits wound collagen synthesis

Nitric oxide (NO) is a messenger molecule which regulates many physiological functions like immunity, vascular tone and serves as a neurotransmitter. Although it is known to participate in healing process, its role in collagen synthesis is not clear. Therefore, the present investigation was done to study the role of NO in wound collagen synthesis. Rats received full thickness, circular (8 mm), transdermal wounds which were treated with NO releaser, sodium nitroprusside (SNP, 0.001 100 M) topically for 5 days. Wound collagen content estimated in terms of hydroxyproline (HP) and confirmed histochemically was decreased significantly by all SNP doses. L-Arginine, a substrate for nitric oxide synthase (NOS) when applied topically decreased collagen content of the wounded tissues. N-Nitro-L-arginine methyl ester (L-NAME), a competitive inhibitor of NOS, increased wound collagen content significantly as compared to untreated and SNP treated animal wounds when administered intraperitoneally at the doses 3, 10 and 30 mg/kg. Furthermore, histological findings also demonstrated laying down of thick collagen bundles and proliferation of fibroblasts together with prominent angiogenesis in L-NAME treated wound tissues as compared to untreated and SNP treated tissues. N-nitro-D-arginine methyl ester, an inactive isomer, was found to have no effect on wound collagen levels. When L-arginine was administered in L-NAME pretreated rats, it significantly elevated wound HP content. The results indicate that NO plays an important role in regulating the collagen biosynthesis in skin model of a healing wound.     

Nitric oxide inhibits oocyte meiotic maturation

Recently, we have found that the nitrate/nitrite concentrations in preovulatory follicles significantly decrease after hCG injection and that inducible nitric oxide synthase (iNOS) plays a main role in the decrease of the intrafollicular nitric oxide (NO) concentration. The purpose of the present study was to investigate the role of NO on oocyte meiotic maturation and to consider the physiological means of the decrease in intrafollicular NO concentration. Immature rats received 15 IU of eCG, and ovaries were removed under ether anesthesia 48 h later. Each ovary was bluntly divided into five or six pieces containing from four to seven preovulatory follicles under the microscope and then incubated with hCG, aminoguanidine (AG; an iNOS inhibitor), or S-nitroso-L-acetyl penicillamine (SNAP; an NO donor) for 5 h. After incubation, preovulatory follicles were punctured, and germinal vesicle breakdown (GVBD) was observed. Also, cGMP concentrations in these follicles were measured. Next, denuded oocytes were recovered from preovulatory follicles at 48 h after injection of 15 IU of eCG and incubated with SNAP with or without ferrous hemoglobin. Every 30 min up to 12 h, GVBD was observed. Both AG and hCG promoted GVBD, and SNAP prevented this effect. In addition, AG decreased intrafollicular cGMP levels, and the concomitant addition of SNAP prevented this decrease. Finally, SNAP dose-dependently inhibited GVBD in denuded oocyte, and this effect of SNAP was reversed by the addition of hemoglobin. We conclude that the iNOS-NO-(cGMP) axis may play an important role in oocyte meiotic maturation.     

Nitric oxide stimulates PC12 cell proliferation via cGMP and inhibits at higher concentrations mainly via energy depletion.

We investigated the mechanisms by which nitric oxide (NO) from an NO donor (DETA/NO) regulates proliferation of pheochromocytoma PC12 cells. The NO donor stimulated proliferation at low concentrations, but reversibly and completely inhibited proliferation at higher concentrations. The stimulation (but not the inhibition) of proliferation was apparently due to NO stimulation of soluble guanylate cyclase to produce cGMP, as it was prevented by a specific cyclase inhibitor (ODQ), and replicated by a cell-permeable form of cGMP. The NO-induced cytostasis was not reversed by inhibitors of MEK kinase or poly(ADP-ribose)polymerase, or by treatments that bypass inhibition of ribonucleotide reductase or ornithine decarboxylase. Cytostatic concentrations of DETA/NO strongly inhibited respiration of PC12 cells, and specific respiratory inhibitors (rotenone, myxothiazol, or azide) caused complete cytostasis. Uridine and pyruvate reversed the cytostasis induced by the specific respiratory inhibitors, but not that induced by DETA/NO. However, the combination of uridine, pyruvate, and N-acetyl-cysteine did reverse DETA/NO-induced cytostasis. DETA/NO strongly and progressively inhibited glycolysis measured by glucose consumption, lactate production, and ATP level, and a specific glycolytic inhibitor (5 mM 2-deoxy-d-glucose) caused complete cytostasis. Our results indicate that NO at low concentrations increases cell proliferation via cGMP, while high concentrations of NO block proliferation via inhibition of both glycolysis and respiration, causing energy depletion.     

Nitric oxide and ATP-sensitive potassium channels mediate lipopolysaccharide-induced depression of central respiratory-like activity in brain slices

Infection may result in early abnormalities in respiratory movement, and the mechanism may involve central and peripheral factors. Peripheral mechanisms include lung injury and alterations in electrolytes and body temperature, but the central mechanisms remain unclear. In the present study, brainstem slices harvested from rats were stimulated with lipopolysaccharide at different doses. Central respiratory activities as demonstrated by electrophysiological activity of the hypoglossal rootlets were examined and the mechanisms were investigated by inhibiting nitric oxide synthase and ATP-sensitive potassium channels. As a result, 0.5 μg/ml lipopolysaccharide mainly caused inhibitory responses in both the frequency and the output intensity, while 5 μg/ml lipopolysaccharide caused an early frequency increase followed by delayed decreases in both the frequency and the output intensity. At both concentrations the inhibitory responses were fully reversed by inhibition of nitric oxide synthase with Nω-nitro-L-arginine methyl ester hydrochloride (20 μM), and by inhibition of ATP- sensitive potassium channels with glybenclamide (100 μM). These results show that direct lipopolysaccharide challenge altered central respiratory activity in dose- and time- related manners. Nitric oxide synthase and ATP-sensitive potassium channels may be involved in the respiratory changes.     

Nitric oxide involvement in Drosophila immunity.

The augmented production of nitric oxide (NO) was observed during the hemocyte-mediated melanotic encapsulation responses of Drosophila melanogaster and D. teissieri. When introduced into the hemocoel of D. melanogaster larvae, NO activated the gene encoding the antimicrobial peptide Diptericin. These observations, together with previous studies documenting the production of superoxide anion (O(*-)(2)) and H(2)O(2) in immune-challenged Drosophila, provide evidence that reactive intermediates of both oxygen (ROI) and nitrogen (RNI) constitute a part of the cytotoxic arsenal employed by Drosophila in defense against both microbial pathogens and eukaryotic parasites. These ROI and RNI appear to represent an evolutionarily conserved innate immune response that is mediated by regulatory proteins that are homologous to those of mammalian species.     

Dexmedetomidine directly inhibits vascular ATP-sensitive potassium channels

AimsDexmedetomidine is reported to have an effect on peripheral vasoconstriction; however, the exact mechanisms underlying this process are unclear. In this study, we hypothesized that dexmedetomidine-induced inhibition of vascular ATP-sensitive K⁺ (K_ATP) channels may be associated with this vasoconstriction. To test this hypothesis, we investigated the effects of dexmedetomidine on vascular K_ATP-channel activity at the single-channel level.Main methodsWe used cell-attached and inside-out patch-clamp configurations to examine the effects of dexmedetomidine on the activities of native rat vascular K_ATP channels, recombinant K_ATP channels with different combinations of various inwardly rectifying potassium channels (Kir6.0 family: Kir6.1, 6.2) and sulfonylurea receptor subunits (SUR1, 2A, 2B), and SUR-deficient channels derived from a truncated isoform of Kir6.2 subunit, namely, Kir6.2ΔC36 channels.Key findingsDexmedetomidine was observed to inhibit the native rat vascular K_ATP channels in both cell-attached and inside-out configurations. This drug also inhibited the activity of all types of recombinant SUR/Kir6.0 K_ATP channels as well as Kir6.2ΔC36 channels with equivalent potency.SignificanceThese results indicate that dexmedetomidine directly inhibits K_ATP channels through the Kir6.0 subunit.     

Nitric oxide down-regulates MKP-3 mRNA levels: involvement in endothelial cell protection from apoptosis

MAP kinase-dependent phosphorylation processes have been shown to interfere with the degradation of the antiapoptotic protein Bcl-2. The cytosolic MAP kinase phosphatase MAP kinase phosphatase-3 (MKP-3) induces apoptosis of endothelial cells in response to tumor necrosis factor alpha (TNFalpha) via dephosphorylation of the MAP kinase ERK1/2, leading to Bcl-2 proteolysis. Here we report that the endothelial cell survival factor nitric oxide (NO) down-regulated MKP-3 by destabilization of MKP-3 mRNA. This effect of NO was paralleled by a decrease in MKP-3 protein levels. Moreover, ERK1/2 was found to be protected against TNFalpha-induced dephosphorylation by coincubation of endothelial cells with the NO donor. Subsequently, both the decrease in Bcl-2 protein levels and the mitochondrial release of cytochrome c in response to TNFalpha were largely prevented by exogenous NO. In cells overexpressing MKP-3, no differences in phosphatase activity in the presence or absence of NO were found, excluding potential posttranslational modifications of MKP-3 protein by NO. These data demonstrate that upstream of the S-nitrosylation of caspase-3, NO exerts additional antiapoptotic effects in endothelial cells, which rely on the down-regulation of MKP-3 mRNA.     

Nitric oxide inhibits the proliferation of T-helper 1 and 2 lymphocytes without reduction in cytokine secretion

To study the effect of nitric oxide (NO) on the activity of Th subsets, cloned Th1 and Th2 lymphocytes were stimulated in the presence of an NO donor. NO, when present from the start of incubation, inhibited the proliferation of both Th subsets dose-dependently, achieving complete inhibition at a relatively low level. The addition of NO 24 h after the onset of T cell stimulation also resulted in reduced proliferation of both Th subsets, suggesting that NO affects a late process during T cell activation. Stimulation of T cells in the presence of NO did not induce apoptosis at the concentrations that completely inhibited proliferation, although apoptosis became evident at higher NO concentrations. The secretion of several cytokines (i.e., IFN-gamma, IL-4, and IL-5) was slightly upregulated, while IL-2 production was modestly inhibited in the presence of NO. However, exogenous IL-2 did not reverse the NO-induced inhibition of T cell proliferation, nor did additional stimulation with phorbol esters. Finally, expression of IL-2R was modestly decreased in the presence of NO, although TCR expression was not affected. These studies demonstrate that relatively low concentrations of NO induce a strong and specific inhibition of T cell proliferation in both Th subsets, suggesting that local NO production may regulate Th-mediated tissue inflammation.