Costanzo Costamagna

Artemisinin inhibits inducible nitric oxide synthase and nuclear factor NF-kB activation

Artemisinin is a natural product used as an alternative drug in the treatment of severe and multidrug‐resistant malaria. In the present work we show that artemisinin shares with other sesquiterpene lactones the ability to inhibit the activation of the nuclear factor NF‐kB: by this mechanism, artemisinin, as well as parthenolide, inhibits nitric oxide synthesis in cytokine‐stimulated human astrocytoma T67 cells. These results suggest that artemisinin, in addition to its antiparasitic properties, could also exert a therapeutic effect on neurological complications of malaria.

Insulin Stimulates Nitric Oxide Synthesis in Human Platelets and, Through Nitric Oxide, Increases Platelet Concentrations of Both Guanosine-3′, 5′-Cyclic Monophosphate and Adenosine-3′, 5′-Cyclic Monophosphate

The insulin-induced platelet anti-aggregating effect is attributed to a nitric oxide (NO)-mediated increase of cyclic guanosine monophosphate (cGMP). The aim of this work, carried out in human platelets, is to show whether insulin increases NO synthesis in platelets and whether it enhances not only cGMP but also cyclic adenosine monophosphate (cAMP) in these cells. We observed that 1) insulin dose-dependently increases NO production, evaluated as citrulline synthesis from Larginine (n = 4, P = 0.015); 2) insulin dose-dependently increases not only cGMP but also cAMP: for instance, after 8 min of insulin incubation at 1,920 pmol/l, cAMP increased from 39.8 ± 1.4 to 121.3 ± 12.6 pmol/l/109platelets (in = 16, P = 0.0001);3) when insulin is incubated for 120 min, the increase of cGMP and cAMP shows a plateau between 2 and 20 min, and while the effect on cGMP is significant until 120 min, the effect on cAMP is no more significant at 60 and 120 min; 4) insulin increases the effects on cAMP of the adenylate cyclase agonists Iloprost and forskolin (n = 5, P = 0.0001) and enhances their platelet anti-aggregating effects (n = 6 and 8, respectively; P = 0.0001); and 5) the inhibition of NO synthase by NG-monomethyl-L-arginine blunts both the insulin effects on basal cGMP and cAMP (n = 4) and those on the Iloprost- and forskolin-induced cAMP increase (n = 5). Thus, insulin increases NO synthesis in human platelets, and, through NO, enhances both cGMP and cAMP. The platelet antiaggregating effect exerted by insulin is, therefore, a NO-mediated phenomenon involving both cGMP and cAMP.

Follicular fluid proteins stimulate nitric oxide (NO) synthesis in human sperm: A possible role for NO in acrosomal reaction

Nitric oxide (NO) is a free radical involved in the regulation of several functions of the male genitourinary system. It is produced by neurons and the endothelium and epithelia of reproductive system; it mediates penile erection and regulates sperm motility, viability, and metabolism. Here we show that human spermatozoa exhibit a detectable NO synthase (NOS) activity, measured both as ability of the intact sperm and cell lysate to convert L‐[3H]arginine into L‐[3H]citrulline and as 24 h accumulation of extracellular nitrite in intact sperm suspensions. NOS activity (identified as an endothelial isoform) was inhibited by L‐canavanine and NG‐monomethyl‐L‐arginine, and nitrite accumulation was inhibited by the NO scavenger hemoglobin; both enzyme activity and nitrite production were increased by a 24 h incubation of spermatozoa with protein‐enriched extracts of human follicular fluid (PFF); a significant increase of citrulline synthesis was observed only after a 4 h incubation with 40% PFF, a time period during which acrosomal reactivity was significantly increased. PFF‐induced acrosomal reaction was inhibited by L‐canavanine and hemoglobin, and the NO donors sodium nitroprusside (SNP), S‐nitroso‐N‐acetyl‐penicillamine (SNAP), and DETA NONOate were able to increase the percentage of reacted spermatozoa. Our results suggest that NO synthesized by human sperm may play a role in follicular fluid–induced acrosomal reaction. J Cell Physiol 178:85–92, 1999.

Signaling Pathway of Nitric Oxide-Induced Acrosome Reaction in Human Spermatozoa

Abstract Nitric oxide (NO) has been recently shown to modulate in vitro motility, viability, the acrosome reaction (AR), and metabolism of spermatozoa in various mammalian species, but the mechanism or mechanisms through which it influences sperm functions has not been clarified. In human capacitated spermatozoa, both the intracellular cGMP level and the percentage of AR-positive cells were significantly increased after 4 h of incubation with the NO donor, sodium nitroprusside (SNP). SNP-induced AR was significantly reduced in the presence of the soluble guanylate cyclase (sGC) inhibitors, LY83583 and ODQ; this block was bypassed by adding 8-bromo-cGMP, a cell-permeating cGMP analogue, to the incubation medium. Finally, Rp-8-Br-cGMPS and Rp-8-pCPT-cGMPS, two inhibitors of the cGMP-dependent protein kinases (PKGs), inhibited the SNP-induced AR. Furthermore, SNP-induced AR did not occur in Ca2+-free medium or in the presence of the protein kinase C (PKC) inhibitor, calphostin C. This study suggests that the AR-inducing effect of exogenous NO on capacitated human spermatozoa is accomplished via stimulation of an NO-sensitive sGC, cGMP synthesis, and PKG activation. In this effect the activation of PKC is also involved, and the presence of extracellular Ca2+ is required.

Modulation of guinea‐pig cardiac L‐type calcium current by nitric oxide synthase inhibitors

1 Electrophysiological (whole‐cell clamp) techniques were used to study the effect of NO synthase (NOS) inhibitors on guinea‐pig ventricular calcium current (ICa), and biochemical measurements (Western blot and citrulline synthesis) were made to investigate the possible mechanisms of action. 2 The two NOS inhibitors, NG‐monomethyl‐L‐arginine (L‐NMMA, 1 mM) and NG‐nitro‐L‐arginine (L‐NNA, 1 mM), induced a rapid increase in ICa when applied to the external solution. D‐NMMA (1 mM), the stereoisomer of L‐NMMA, which has no effect on NOS, did not enhance ICa. 3 Western blot experiments gave no indication of the presence of inducible NOS protein (iNOS) in our cell preparation, neither immediately after dissociation nor after more than 24 h. Statistically, there was no significant difference between electrophysiological experiments performed on freshly dissociated cells and experiments performed the next day. Moreover cells prepared and kept in the presence of dexamethasone (3 μM), to inhibit the expression of iNOS, gave the same response to L‐NMMA as control cells. 4 The stimulatory effect of L‐NMMA (1 mM) on basal ICa was reversed by competition with higher doses (5 mM) of externally applied L‐arginine, the natural substrate of NOS. The effect of L‐NMMA was also eliminated by L‐arginine in the patch pipette solution. 5 Intracellular perfusion with GDPβS (0.5 mM), which stabilizes the G‐proteins in the inactive state, did not affect the L‐NMMA‐induced stimulation of ICa. 6 Carbachol (1 μM) reduced the ICa previously stimulated by L‐NMMA, and intracellular cGMP (10 μM) prevented L‐NMMA enhancement. 7 Simultaneous treatment with L‐NMMA and isoprenaline (1 μM) induced a non‐cumulative enhancement of ICa that could not be reversed by carbachol (1 μM). 8 NO synthesis, measured by the formation of [3H]citrulline from L‐[3H]arginine during a 15 min incubation, showed a relatively high basal NO production, which was inhibited by L‐NMMA but not affected by carbachol. 9 These results suggest that inhibitors of NOS are able to modulate the basal ventricular ICa in the absence of a receptor‐mediated pathway, and that NO might be required for the muscarinic reduction of ICa under isoprenaline stimulation, even if NO production is not directly controlled by the muscarinic pathway.

Crocidolite asbestos inhibits pentose phosphate oxidative pathway and glucose 6-phosphate dehydrogenase activity in human lung epithelial cells

The cytotoxicity of asbestos has been related to its ability to increase the production of reactive oxygen species (ROS), via the iron-catalyzed reduction of oxygen and/or the activation of NADPH oxidase. The pentose phosphate pathway (PPP) is generally activated by the cell exposure to oxidant molecules. Contrary to our expectations, asbestos (crocidolite) fibers caused a dose- and time-dependent inhibition of PPP and decreased its activation by an oxidative stress in human lung epithelial cells A549. In parallel, the intracellular activity of the PPP rate-limiting enzyme, glucose 6-phosphate dehydrogenase (G6PD), was significantly diminished by crocidolite exposure. This inhibition was selective, as the activity of other PPP and glycolysis enzymes was not modified, and was not attributable to a decreased expression of G6PD. On the opposite, the incubation with glass fibers MMVF10 did not modify PPP and G6PD activity. PPP and G6PD inhibition did not correlate with the increased nitric oxide (NO) production elicited by crocidolite in A549 cells. Experiments with the purified enzyme suggest that crocidolite inhibits G6PD by directly interacting with the protein. We propose here a new mechanism of asbestos-evoked oxidative stress, wherein fibers increase the intracellular ROS levels also by inhibiting the main antioxidant pathway of the cell.

Human vascular smooth muscle cells express a constitutive nitric oxide synthase that insulin rapidly activates, thus increasing guanosine 3′ : 5′-cyclic monophosphate and adenosine 3′ : 5′-cyclic monophosphate concentrations

Aims/hypothesis. Insulin incubation of human vascular smooth muscle cells (hVSMC) for 120 min increases both guanosine 3′ : 5′-cyclic monophosphate (cGMP) and adenosine 3′ : 5′-cyclic monophosphate (cAMP) and these effects are blocked by inhibiting nitric oxide synthase (NOS). These data suggest that insulin activates a constitutive Ca2+-dependent NOS (cNOS), not described at yet in hVSMC. To test this hypothesis, we evaluated in hVSMC: i) the kinetics of the insulin-induced enhancement of the two cyclic nucleotides; ii) the ability of nitric oxide (NO) to increase both cyclic nucleotides; iii) NO involvement in the short-term influence of insulin on both cyclic nucleotides; iv) the ability of insulin to increase NO production in a few minutes; v) the presence of a cNOS activity; vi) the expression of mRNA for cNOS. Methods. In hVSMC incubated with insulin, NO donors and the Ca2+ ionophore ionomycin, we measured cAMP and cGMP (RIA); in hVSMC incubated with insulin and ionomycin we measured NO, evaluated as l-(3H)-citrulline production from l-(3H)-arginine; by northern blot hybridization, we measured the expression of cNOS mRNA. Results. i) By incubating hVSMC with 2 nmol/l insulin for 0–240 min, we observed an increase of both cGMP and cAMP (ANOVA: p = 0.0001). Cyclic GMP rose from 0.74 ± 0.01 to 2.62 ± 0.10 pmol/106 cells at 30 min (p = 0.0001); cAMP rose from 0.9 ± 0.09 to 11.65 ± 0.74 pmol/106 cells at 15 min (p = 0.0001). ii) Sodium nitroprusside (100 μmol/l) and glyceryltrinitrate (100 μmol/l) increased both cGMP and cAMP (p = 0.0001). iii) The effects of insulin on cyclic nucleotides were blocked by NOS inhibition. iv) An increase of NO was observed by incubating hVSMC for 5 min with 2 nmol/l insulin (p = 0.0001). v) Ionomycin (1 μmol/l) enhanced NO production (p = 0.0001) and increased both cyclic nucleotides (p = 0.0001). vi) hVSMC expressed mRNA of cNOS. Conclusion/interpretation. Human VSMC express cNOS, which is rapidly activated by insulin with a consequent increase of both cGMP and cAMP, suggesting that insulin-induced vasodilation in vivo is not entirely endothelium-mediated. [Diabetologia (1999) 42: 831–839]

Na+/H+ exchanger activity is increased in doxorubicin-resistant human colon cancer cells and its modulation modifies the sensitivity of the cells to doxorubicin.

Multidrug resistant (MDR) tumor cells exhibit an altered pH gradient across different cell compartments, which favors a reduced intracellular accumulation of antineoplastic drugs and a decreased therapeutic effect. In our study, we have observed that the activity and expression of Na+/H+ exchanger (NHE), which is involved in the homeostasis of intracellular pH (pHi), are increased in doxorubicin‐resistant (HT29‐dx) human colon carcinoma cells in comparison with doxorubicin‐sensitive HT29 cells. The pHi was significantly higher in HT29‐dx cells, which accumulated less doxorubicin than HT29 cells. The NHE inhibitor 5‐(N‐ethyl‐N‐isopropyl)amiloride (EIPA) significantly reduced the pHi value and increased the intracellular accumulation of doxorubicin in both cell populations: in the presence of EIPA HT29‐dx cells accumulated as much drug as control HT29 cells. On the other hand, monensin, a Na+/H+ ionophore mimicking NHE activation, and phorbol 12‐myristate 13‐acetate (PMA), which stimulates NHE, significantly increased the pHi and decreased the drug accumulation in HT29 cells to values similar to those observed in control HT29‐dx cells. EIPA potentiated the cytotoxic effect of doxorubicin in HT29 cells, and made HT29‐dx cells as sensitive to the cytotoxic effect of the drug as control HT29 cells. Instead, PMA and monensin made HT29 cells as insensitive to doxorubicin as HT29‐dx cells. These results suggest that in MDR cells the higher cytosolic pH is likely to decrease drug accumulation, and that such resistance can be reverted by inhibiting the NHE activity. This result opens the possibility to revert MDR with the clinical use of NHE inhibitors.

Activation of Nuclear Factor-κB Pathway by Simvastatin and RhoA Silencing Increases Doxorubicin Cytotoxicity in Human Colon Cancer HT29 Cells

Doxorubicin efficacy in cancer therapy is hampered by the dose-dependent side effects, which may be overcome by reducing the drugs dose and increasing its efficacy. In the present work, we suggest that the activation of the nuclear factor-κB (NF-κB) pathway and of nitric-oxide (NO) synthase increases the doxorubicin efficacy in human colon cancer HT29 cells. To induce NF-κB, we took into account the effect of doxorubicin itself and of the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor simvastatin; as NF-κB inhibitors, we chose the sesquiterpene lactones parthenolide and artemisinin. Simvastatin increased the NF-κB activity and NO synthesis, elicited the tyrosine nitration of the multidrug resistance-related protein 3, and enhanced the doxorubicin intracellular accumulation and cytotoxicity. Simvastatin potentiated the effect of doxorubicin on the NF-κB pathway and the inducible NO synthase expression. The effects of simvastatin were due to the inhibition of the small G-protein RhoA and of its effector Rho kinase. Parthenolide and artemisinin prevented all of the statin effects by inducing RhoA/Rho kinase activation. On the other hand, they did not reduce the NF-κB translocation and doxorubicin intracellular content when RhoA was silenced by small interfering RNA (siRNA). It is interesting that RhoA siRNA was sufficient to increase NF-κB translocation, NO synthase activity, doxorubicin accumulation, and cytotoxicity also in non-stimulated cells. Our results suggest that artemisinin, a widely used antimalarial drug, may impair the response to doxorubicin in colon cancer cells; on the contrary, simvastatin and RhoA siRNA may represent future therapeutic approaches to improve doxorubicin efficacy, reducing the risk of doxorubicin-dependent adverse effects.

The NADPH oxidase inhibitor apocynin induces nitric oxide synthesis via oxidative stress.

We have recently shown that apocynin elicits an oxidative stress in N11 mouse glial cells and other cell types. Here we report that apocynin increased the accumulation of nitrite, the stable derivative of nitric oxide (NO), in the extracellular medium of N11 cell cultures, and the NO synthase (NOS) activity in cell lysates. The increased synthesis of NO was associated with increased expression of inducible NOS (iNOS) mRNA, increased nuclear translocation of the redox-sensitive transcription factor NF-kappa B and decreased intracellular level of its inhibitor IkB alpha. These effects, accompanied by increased production of H2O2, were very similar to those observed after incubation with bacterial lipopolysaccharide (LPS) and were inhibited by catalase. These results suggest that apocynin, similarly to LPS, induces increased NO synthesis by eliciting a generation of reactive oxygen species (ROS), which in turn causes NF-kappa B activation and increased expression of iNOS. Therefore, the increased bioavailability of NO reported in the literature after in vivo or in vitro treatments with apocynin might depend, at least partly, on the drug-elicited induction of iNOS, and not only on the inhibition of NADPH oxidase and the subsequent decreased scavenging of NO by oxidase-derived ROS, as it is often supposed.