Chloroquine stimulates nitric oxide synthesis in murine, porcine, and human endothelial cells.

Nitric oxide (NO) is a free radical involved in the regulation of many cell functions and in the expression of several diseases. We have found that the antimalarial and antiinflammatory drug, chloroquine, is able to stimulate NO synthase (NOS) activity in murine, porcine, and human endothelial cells in vitro: the increase of enzyme activity is dependent on a de novo synthesis of some regulatory protein, as it is inhibited by cycloheximide but is not accompanied by an increased expression of inducible or constitutive NOS isoforms. Increased NO synthesis is, at least partly, responsible for chloroquine-induced inhibition of cell proliferation: indeed, NOS inhibitors revert the drug-evoked blockage of mitogenesis and ornithine decarboxylase activity in murine and porcine endothelial cells. The NOS-activating effect of chloroquine is dependent on its weak base properties, as it is exerted also by ammonium chloride, another lysosomotropic agent. Both compounds activate NOS by limiting the availability of iron: their stimulating effects on NO synthesis and inhibiting action on cell proliferation are reverted by iron supplementation with ferric nitrilotriacetate, and are mimicked by incubation with desferrioxamine. Our results suggest that NO synthesis can be stimulated in endothelial cells by chloroquine via an impairment of iron metabolism.

Retinoic acid-induced differentiation in a human neuroblastoma cell line is associated with an increase in nitric oxide synthesis.

The human neuroblastoma cell line SK-N-BE, after incubation with 10 microM retinoic acid (RA) or 20 nM phorbol 12-myristate 13-acetate (PMA), underwent biochemical and morphological signs of differentiation within 10-14 days. In parallel, SK-N-BE cells produced significantly higher amounts of nitric oxide (NO) in comparison with controls, as assessed by the measurement of nitrite and nitrate in the culture supernatant and of NO synthase (NOS) activity in the cell lysates (measured as ability to convert [3H]arginine into [3H]citrulline and as NADPH diaphorase activity). Nitrite/nitrate production was abolished by adding the NO scavenger hemoglobin in the culture medium and was inhibited by aminoguanidine (AG, a selective inhibitor of the inducible NOS isoform) but not by the less selective inhibitor NG-nitro-L-arginine methylester (NAME). Western blotting experiments with monoclonal antibodies against the ncNOS and iNOS isoforms suggest that RA-elicited NOS activation is not attributable to an increased expression of the protein. NAME and AG were not able to revert inhibition of proliferation induced by RA, and the NO donor sodium nitroprusside did not mimic the effect of RA and PMA. These data indicate that increased NO synthesis does not mediate RA- or PMA-induced differentiation but may be an additional marker of differentiation into sympathetic-like neuronal cells.

Reversal of doxorubicin resistance by the amiloride analogue EIPA in multidrug resistant human colon carcinoma cells.

Although multidrug resistance (mdr) may arise through a variety of mechanisms, the most widely studied and accepted form is associated with an increased concentration of P-glycoprotein (P-gp), a 170 kd protein found in the membrane fraction of a number of mammalian cells. Since mdr seems to be related to the ability of resistant cells to extrude drugs and the circumvention of mdr is supposed to be due to the restored ability to accumulate drugs, membrane has been regarded as the crucial site for such a regulation and an important role for membrane ion exchangers has been suggested. The aim of this work was to elucidate whether the Na+/H+ antiporter is involved in the mechanism of regulation and circumvention of mdr and if 5-(N-ethyl-N-isopropyl) amiloride (EIPA), a selective inhibitor of the Na+/H+ exchanger, can modulate the functional expression of the mdr phenotype. The effect of EIPA on doxorubicin (DX) resistant cells (LoVo/DX) obtained from a human colon adenocarcinoma cell line (LoVo) was studied. EIPA at concentrations ranging from 10 to 50 mu M was able to increase the antibiotic cytotoxicity in the resistant Lovo/DX cells. The reversal of DX resistance paralleled an increase of the ability of the cells to accumulate the drug. Both drug loading and sensitivity to the inhibitory effect of DX on cell proliferation were restored by EIPA in a dose-dependent way. These results suggest a new mechanism of mdr reversal and indicate that amiloride and its derivatives may be useful in reversing DX resistance and in enhancing the clinical effectiveness of chemotherapeutics.

Correlation between nitric oxide synthase activity and reduced glutathione level in human and murine endothelial cells.

The synthesis of nitric oxide (NO), detected as citrulline production, in human (HUVEC) and murine (tEnd.1) endothelial cells correlated with intracellular GSH. tEnd.1, which exhibited an intracellular GSH level 2.5-fold higher than HUVEC, showed a citrulline production (basally and after ionomycin stimulation) 5-8 times higher than human cells. Ionomycinelicited citrulline synthesis in tEnd.1 cells increased 2.4-fold after loading with GSH, and decreased dose-dependently after GSH depletion. Cell loading with N-(2-mercaptopropionyl)-glycine neither significantly increased citrulline production nor relieved the effect of GSH depletion.

Erythrocyte stages of Plasmodium falciparum exhibit a high nitric oxide synthase (NOS) activity and release an NOS-inducing soluble factor.

Nitric oxide (NO), a highly diffusible cellular mediator involved in a wide range of biological effects, has been indicated as one of the cytotoxic agents released by leukocytes to counteract malaria infection. On the other hand, NO has been implicated as a mediator of the neuropathological symptoms of cerebral malaria. In such circumstances NO production has been thought to be induced in host tissues by host-derived cytokines. Here we provide evidence for the first time that human red blood cells infected by Plasmodium falciparum (IRBC) synthesize NO. The synthesis of NO (measured as citrulline and nitrate production) appeared to be very high in comparison with human endothelial cells; no citrulline and nitrate production was detectable in noninfected red blood cells. The NO synthase (NOS) activity was very high in the lysate of IRBC (while not measurable in that of normal red blood cells) and was inhibited in a dose-dependent way by three different NOS inhibitors (L-canavanine, NG-amino-L-arginine, and NG-nitro-L-arginine). NOS activity in P. falciparum IRBC is Ca++ independent, and the enzyme shows an apparent molecular mass < 100 kD, suggesting that the parasite expresses an isoform different from those found in mammalian cells. IRBC release a soluble factor able to induce NOS in human endothelial cells. Such NOS-inducing activity is not tissue specific, is time and dose dependent, requires de novo protein synthesis, and is probably associated with a thermolabile protein having a molecular mass > 100 kD. Our data suggest that an increased NO synthesis in P. falciparum malaria can be directly elicited by soluble factor(s) by the blood stages of the parasite, without necessarily requiring the intervention of host cytokines.

Middle T antigen-transformed endothelial cells exhibit an increased activity of nitric oxide synthase.

Endothelioma cell lines transformed by polyoma virus middle T antigen (mTa) cause cavernous hemangiomas in syngeneic mice by recruitment of host cells. The production of nitric oxide (NO), as measured by nitrite and citrulline production, was significantly higher in mTa-transformed endothelial cells in comparison with nontransformed control cells. The maximal activity of NO synthase (NOS) was about 200-fold higher in cell lysates from the tEnd.1 endothelioma cell line than in lysates from nontransformed controls, whereas the affinity for arginine did not differ. The biochemical characterization of NOS and the study of mRNA transcripts indicate that tEnd.1 cells express both the inducible and the constitutive isoforms. NOS hyperactivity is not a simple consequence of cell transformation but needs a tissue-specific mTa expression. Since tEnd.1-conditioned medium induces NOS activity in normal endothelial cells, most likely NOS hyperactivity in endothelioma cells is attributable to the release of a soluble factor. This NOS-activating factor, which seems to be an anionic protein, could stimulate tEnd.1 cells to express NOS by an autocrine way. By the same mechanism, tEnd.1 cells could induce NOS in the neighboring endothelial cells, and NO release could play a role in the hemangioma development. Such hypothesis is confirmed by our in vivo experiments, showing that the administration of the NOS inhibitor L-canavanine to endothelioma-bearing mice significantly reduced both the volume and the relapse time of the tumor.

S35b, a new phenylsulfonylfuroxan compound, inhibits thrombin-induced synthesis of platelet-activating factor and prostacyclin in human endothelial cells.

Endothelial cells (EC) produce platelet activating factor (PAF) and prostacyclin (PGI2) in response to inflammatory agents such as thrombin. Upon cell stimulation a calcium-dependent phospholipase A2 (PLA2) is activated which hydrolyzes a membrane phospholipid to yield 1-0-alkyl-2-lyso-sn-glycero-3-phospho-choline (lyso-PAF) and free arachidonic acid. Lyso-PAF is in turn converted into PAF by a specific acetyltransferase and arachidonic acid is metabolized via cyclic endoperoxides to PGI2. In the present study we report that S35b (4-methyl-3-phenylsulfonylfuroxan), a new phenyl-sulfonylfuroxan compound with potent antiaggregatory effect, inhibits thrombin-induced PAF synthesis and acetyltransferase activation as well as PGI2 production in human umbilical vein endothelial cells (HUVEC) in a concentration-dependent way. Additionally, we show that S35b stimulates the production of cyclic GMP (cGMP) in HUVEC in a concentration- and time-dependent manner. At high concentration, S35b potentiates the cAMP increase induced by iloprost or forskolin without having a significant influence on cAMP level itself. Potentiation of cAMP increase during agonist-induced EC stimulation seems not to be important for the effect of S35b on cellular function as the compound is active in inhibiting PAF production when endothelial cells are pretreated with indomethacin to block PGI2 synthesis. The increase of cGMP evoked by S35b may account for the effect on endothelial cell function.