Divergent effects of nitric oxide on airway epithelial cell activation

Nitric oxide (NO*) is a gaseous mediator synthesized by nitric oxide synthases. NO* is involved in the modulation of inflammation, but its role in airway inflammation remains controversial. We investigated the role of NO* in the synthesis of the chemokines interleukin-8 and monocyte chemotactic protein-1, and of intercellular adhesion molecule-1 by human airway epithelial cells. normal human bronchial epithelial cells and the bronchial epithelial cell line BEAS-2B were used. interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) secretion and intercellular adhesion molecule-1 (ICAM-1) expression were measured by ELISA. mRNA was assessed by semiquantitative RTI-PCR. Interleukin-8 secretion was significantly reduced after 24h incubation with the NO* donor, sodium nitroprusside. The effect was dose-dependent. Similar results were obtained with S-nitroso-N-D,L-penicillamine and S-nitroso-L-glutathione. Inhibition of endogenous NO* with the nitric oxide synthase inhibitor N-nitro-L-arginine-methyl-ester caused an increase in IL-8 secretion by lipopolysaccharide- and cytokine-stimulated BEAS-2B cells. Sodium nitroprusside also caused a reduction in monocyte chemotactic protein-1 secretion by both cell types. In contrast, intercellular adhesion molecule-1 expression was upregulated by sodium nitroprusside. RTI-PCR results indicate that the modulation of protein levels was paralleled by modification in mRNA levels. NO* has divergent effects on the synthesis of different inflammatory mediators in human bronchial epithelial cells.     

Possible involvement of NO in the stimulating effect of pifithrins on survival of hemopoietic clonogenic cells

Pifithrin α (PFTα), one of the first known low molecular weight modulators of activity of tumor suppressor p53, increases survival of hemopoietic clonogenic cells (evaluated by the criterion of formation of endogenous spleen CFU-C8 colonies in irradiated animals). This effect appeared when PFTα was administered either before or after irradiation. Increase in CFU-C8 was also observed after administration of two PFTα analogs, derivatives of 2-amino-4,5,6,7-tetrahy-drobenzothiazole. These included a parent compound, 2-ATBT (2-amino-4,5,6,7-tetrahydrobenzothiazole), which is used for synthesis of PFTα, and a product of its intramolecular cyclization under physiological conditions, cyclo-PFT (2-(4-methylphenyl)imidazo[2,1-b]-5,6,7,8-tetrahydrobenzothiazole). Earlier we found that many low molecular weight compounds increasing number of CFU-C8 (e.g. isothiourea derivatives) demonstrate NO inhibitory activity. Such activity was also found in 2-ATBT and cyclo-PFT by means of EPR spectroscopy of NO. These compounds caused more than twofold inhibition of NO production in vivo. Thus, it has been demonstrated that PFTα and its structural analogs increase survival of hemopoietic clonogenic cells in vivo, and NO may play a role in the mechanism of this effect.     

Effect of cortisone-resistant thymocytes on endogenous colony formation in inbred mice]

Endogenous colonies in the spleen of sublethally irradiated (CBA X C57BL/6) E1 hybrid mice were recorded after the injection of thymocytes and the lymph node cells from the hydrocortisone-treated and intact CBA mice. Cortisone-resistant thymocytes failed to suppress the endogenous colony formation, whereas the lymph node cells produced a distinct suppressive effect on the endogenous colonies. After the injection of cortisone-resistant thymocytes the number of colonies in the spleen of individual recipients was double that in control irradiated hybrids.     

Effect of the U-2 fraction of splenic extract from Horsfield's terrapin on the hematopoiesis of mammals

Intraperitoneal administration of a spleen extract from Testudo horsfieldi and its U-2 fraction increases the number of endogenous splenic haemopoietic colonies. The U-2 fraction administered to irradiated (4 Gy) mice increases the number of bone marrow CFUs. Bone marrow cells of exposed (4 Gy) mice preincubated in vitro with the U-2 fraction also increase the number of exogenous colonies in the recipient's spleen.     

Clonogenic hemopoietic cells (CFU-S) in mouse pre- and postnatal ontogeny

Content of three classes of clonogenic haemopoietic cells (CFU-S-7, CFU-S-11 and CFU-S-ep) was determined in haemopoietic organs of mouse during embryogenesis (10, 14 and 18 day) and postnatal ontogenesis (2, 3 and 7 day, 1, 2, 3 and 18 month). CFU-S-7 and CFU-S-11 that from big splenic colonies on 7th and 11th days of transplantation are present in liver, spleen and bone marrow at all developmental stages. However their concentration and CFU-S-7 CFU-S-11 ratio change in haemopoietic organs. CFU-S-ep that form small colonies on 11th day are observed before birth in liver and spleen and 1 week after birth there and also in bone marrow but are practically absent from haemopoietic organs of older animals. Thus, CFU-S compartment structure is characterized by definite ratio of its subpopulations. It seems to reflect functional state of haemopoietic system during development.     

Effect of estradiol on splenic repopulation by endogenous and exogenous hemopoietic cells in irradiated mice

Estradiol treatment of irradiated mice during repopulation of their spleens by endogenous hemopoietic cells reduced the number of myelocytic colonies and increased the numbers of erythropoietic and undifferentiated colonies. The inhibitory effects of the hormone on myelopoiesis were not dependent on stimulation of erythropoiesis, since they occurred in the absence of erythropoiesis in mice made polycythemic by hypertransfusion. Treatment of bone marrow donors with estradiol reduced the ability of their marrow cells to form spleen colonies, particularly reducing the proportion of myelopoietic colonies relative to the total number of colonies of all types. Conversely erythropoietic colonies, though reduced in absolute number, were increased in relative number. Such treatment also decreased the volume and cell content of the marrow cavity through stimulation of endosteal bone formation. Estradiol treatment of lethally irradiated recipient mice did not detectably alter the total numbers or types of hemopoietic spleen colonies formed in these animals from transplanted marrow cells; however, without estradiol treatment, myelopoietic colonies were so few and erythropoietic colonies so numerous that the effects of the hormones may have been undetectable by the methods employed. The sex of the donor or recipient mouse did not affect the numbers or types of colonies formed, suggesting that endogenous levels of estradiol were too low to exert effects dectectable by the methods used. However, since our mice were only 8 weeks old, the data do not exclude the possibility that older female mice, with higher levels of estradiol, would have differed from males in relative numbers of myelopoietic as compared with erythropoietic colonies.     

Cells with marrow and spleen repopulating ability and forming spleen colonies on day 16, 12, and 8 are sequentially ordered on the basis of increasing rhodamine 123 retention

Mouse bone marrow cells (BMC) were subjected to countercurrent centrifugal elutriation and subsequently separated on the basis of light scatter and fluorescence intensity after being labeled with the supravital dye Rhodamine 123 (Rh-123). The sorted cells were then assayed for their in vivo spleen colony-forming ability (day -8, -12, and -16 CFU-S) and their ability to repopulate the bone marrow or spleen over a 13-day period with CFU-S-12, CFU-GM, or nucleated cells. Cells with marrow repopulating ability (MRA), as measured by the ability of the sorted cells to repopulate the marrow with secondary CFU-S-12 or CFU-GM, had low affinity for Rh-123. These cells showed minimal spleen colony-forming ability, and the ratio of MRA to CFU-S-12 in this preparation was 309. Cells with spleen repopulating ability (SRA), CFU-S-16, CFU-S-12, and CFU-S-8 retained increasing amounts of Rh-123, respectively, and CFU-S-8 were almost exclusively found among cells with high Rh-123 affinity. These cells also included about half of all day-12 CFU-S, and the ratio of MRA to day-12 CFU-S was 0. The results show that MRA cells, SRA cells, CFU-S-16, CFU-S-12, and CFU-S-8 can be sequentially ordered on the basis of increasing mitochondrial activity. The data also demonstrate for the first time, and without the application of negative selection by the use of cytostatic agents, that MRA cells are a separate class of primitive hemopoietic stem cells that fully meet the criteria of pre-CFU-S.     

Factors influencing hematopoietic spleen colony formation in irradiated mice. IV. The effect of erythropoietic stimuli

A variety of erythropoietic stimuli influenced the number of endogenous spleen colonies in irradiated mice and the number of transplantable colony forming cells in the spleen and marrow of unirradiated mice. Bleeding was the most effective stimulus. Bleeding before irradiation resulted in a 30-fold increase in endogenous spleen colonies and in increases in spleen weight, spleen iron and iododeoxyuridine uptake and volume of packed red cells ten days after irradiation. Bleeding unirradiated mice produced a 10-fold increase in the number of transplantable colony forming cells in the spleen and a slight decrease in the total number in the humerus. Bleeding before irradiation resulted in a significant reduction in 30-day post irradiation deaths, an effect abolished by splenectomy. Plasma from bled mice induced an increase in endogenous colonies when injected before irradiation into normal mice. Injection of erythropoietin, testosterone or testosterone plus cobalt induced effects which were, in general, qualitatively similar to those of bleeding, although they were less effective quantitatively. Except for a slight effect induced by ten injections of erythropoietin, post-irradiation stimulation in normal mice proved ineffective. Erythropoietin increased colony numbers and spleen iron uptake when given after irradiation to hypertransfused mice. The results of these studies do not support the concept that the colony forming cell and the erythropoietin sensitive cell are separate entities.     

Defective transient endogenous spleen colony formation in S1/S1d mice.

WCB6F1 mice of the genotype S1/S1d did not form transient 5-day endogenous spleen colonies following midlethal irradiation, either spontaneously or in response to postirradiation bleeding. Their hematologically normal (+/+) littermates produced colonies equivalent in number and morphologic type to a normal strain (D2B6F1), as evaluated by both macroscopic and microscopic criteria. Bone marrow cells from S1/S1d mice, when transplanted into lethally irradiated +/+ mice, were able to generate equivalent numbers of transient endogenous spleen colonies (TE-CFUs), as compared to that obtained when syngeneic +/+ marrow cells were injected into lethally irradiated +/+ recipients. A defective growth of an early class of hematopoietic progenitor cells, resulting in the clinical course of the S1/S1d anemia is suggested and confirms previous reports on the microenvironmental nature of this abnormality.     

Effect of a neutrophilia-inducing tumor on hemopoietic stem cells in mice

The influence of neutrophilic stimulation on hemopoietic stem cells was studied in mice with tumor-induced neutrophilia. Transfusions of marrow cells from normal and neutrophilic tumor-bearing mice into lethally irradiated normal and tumor-bearing mice were performed. The number and the erythroid:granuloid (E:G) ratio of day 7 colonies in the recipient spleens and bones as well as the size of spleen colonies of recipient animals were determined. The E:G ratio of spleen and bone marrow colonies between normal and tumor-bearing mouse recipients and the number of spleen colonies did not differ significantly in either experiment. However, spleen colonies which developed in tumor-bearing irradiated mice were significantly larger than those which developed in normal recipients in both experiments. These studies indicated that while the line of differentiation taken by hemopoietic stem cells was not affected by the neutrophilic influence of the tumor, the tumor-bearing host environment appeared to enhance proliferation of transfused stem cells and/or their descendants. The stimulators of granulocytopoiesis in this model of neutrophilia appear to act on a population of progenitor cells more mature than the stem cells capable of forming 7-day colonies in the spleen and bone marrow of irradiated recipient mice.     

Radioprotective effect of E. coli endotoxin on hematopoietic stem cells is partially suppressed by inhibiting production of nitric oxide by administering N-omega-nitro-L-arginine

Lipopolysaccharide of E. coli (LPS) injected to mice one day before the total gamma-irradiation caused a substantial increase in the level of endogeneous colonies formed in the spleen. It is known that this type of endotoxin may result in considerable production of nitric oxide from macrophages in different tissues. Therefore it is possible that the activation of hemopoietic stem cells after LPS-treatment was completely or partially stimulated by nitric oxide, which is the most important physiological mediator, or by action some other mediators (cytokines and growth factors) produced by hemopoietic microenvironment elements in response to the action of nitric oxide. This assumption was checked in experience with combined treatment of mice by LPS and a nonspecific inhibitor of nitric oxide production--N omega-nitro-1-arginine (NA). NA used in high dose (250 mg/kg) reduced partially (approximately by 30%) the LPS-increased level of spleen endogeneous colonies. When LPS was injected to mice 15 minutes after gamma-irradiation, this led to a slight increase in level of spleen colonies. In case when LPS was used together with NA after gamma-irradiation, this increase was still found.     

The effect of erythropoietin on the growth and development of spleen colony-forming cells

The progressive growth and development of spleen colonies was studied in heavily irradiated host mice in which erythropoiesis was modified by various procedures. Erythropoietic activity in non-polycythemic hosts bearing spleen colonies was not increased by injections of exogenous erythropoietin. Detectable levels of erythropoietin were found in the heavily irradiated host mice suggesting that the failure of exogenous erythropoietin to modify erythropoiesis was because the host mice were already maximally stimulated by the high endogenous erythropoietin levels. Spleen colonies do not become erythroid in polycythemic mice. The injection of exogenous erythropoietin into heavily irradiated polycythemic hosts did not decrease the total number of spleen colonies produced by a given bone marrow transplant, as would be expected if erythropoietin acted directly on the colony-forming cells. Comparison of growth curves for colony-forming cells in the spleens of polycythemic hosts either receiving or not receiving erythropoietin indicated that the overall doubling time of colony-forming cells during the first ten days after transplantation was not changed by the daily injection of erythropoietin. These experiments are consistent with the concept that erythropoietin is necessary for the development of erythroid colonies. Erythropoietin acts upon some progeny of the colony-forming cell rather than the colony-forming cell itself.     

Effect of the cell components of Staphylococcus aureus on the functional activity of hemopoietic cells in mice

The data on the stimulating action of S. aureus cells, strains B-243, 2287, Wood-46, Cowan I, as well as cell-wall peptidoglycan, on the formation of endogenous colonies in the spleen of sublethally irradiated mice are presented. Teichoic acid, S. aureus ribosomal and cytoplasmic antigens produced no such effect. Whole S. aureus cells and their components were incapable of activating transitory colonies in the spleen of sublethally irradiated mice. After immunization with cell walls, peptidoglycan and protein A the mice showed a rise in the absolute and relative content of blood-forming stem cells in the marrow and the spleen. Killed S. aureus cells increased the relative content of blood-forming stem cells in the marrow, while in the spleen a rise in both absolute and relative content of such cells occurred, which was detected in the exocolonization test.     

Possible role of nitric oxide in radiation-induced salivary gland dysfunction

In this study, we developed a murine model of xerostomia to elucidate the mechanism of radiation-induced salivary gland dysfunction and determined the levels of nitric oxide (NO) in the salivary glands to assess its involvement in the salivary dysfunction induced by radiation. In addition, an inhibitor of NO synthesis was administered to the model in vivo, and its effect on saliva secretion was investigated. Salivary gland irradiation at a dose of 15 Gy caused a significant decrease in secretion compared to unirradiated salivary glands. There were no marked differences between the irradiated mice and unirradiated mice in water or food consumption or in body weight changes. The NO levels in the cultured salivary gland epithelial cells were increased by treatment with a combination of interferon gamma (Ifng), interleukin 1-beta (Il1b), and tumor necrosis factor alpha (Tnfa). Irradiation increased the NO level in the salivary gland tissue. The presence of N(G)-monomethyl-l-arginine acetate (l-NMMA), an inhibitor of NO synthesis, caused a decrease in the NO level in cultured salivary gland tissues after irradiation. Administration of l-NMMA to irradiated mice improved saliva secretion. These results suggest that excessive production of NO induced by radiation is involved in the formation of radiation-induced xerostomia. The finding that administration of an inhibitor of NO synthesis ameliorated the dysfunction of irradiated salivary glands indicates that NO plays a role as a mediator of the dry mouth symptoms that occur after irradiation.     

Cloning stem cells in the bone marrow of irradiated mice]

Different amount of intact or irradiated bone marrow from syngenous donors was administered to mice irradiated with a lethal dose. There was revealed a linear dependence of the number of the 8-9-day colonies grown in the bone marrow of the femur on the amount of the administered cells, and an exponential dependence on the irradiation dose. Regularity of the stem cell cloning in the bone marrow was analogous to such in the spleen. Radiosensitivity of the colony-forming units (CFU) differed depending on the site (the spleen, the bone marrow) of their colony formation. The CFU settling in the marrow proved to be more radioresistant (D(0) equalled 160-200 P) in comparison with the CFU settling in the spleen (D(0) constituted 80-100 P). It is supposed that a different radiosensitivity of the CFU was caused by the presence of heterogenic population of the stem cells and also by specific peculiarities of the organ (the spleen, the bone marrow) in which the colonies formed.     

Nitric oxide donor increases the efficiency of cytostatic therapy and retards the development of drug resistance.

The potentiality to increase the chemotherapeutic effectiveness of some cytostatics in low, subtherapeutic doses in combination with nitric oxide (NO) donor has been shown. This type of combined therapy results in significant increase in life span and number of survivors among mice bearing leukemias P388 and L-1210. A similar effect was observed for intracerebral leukemia P388 transplantation. In this case the life span of mice treated with cyclophosphamide and NO donor increased by three times in comparison to therapy with cyclophosphamide alone. The coinjection of nitric oxide donor and cytostatics improved the antimetastatic activity of the cytostatics: the index of melanoma B16 metastasis inhibition at the cyclophosphamide monotherapy is 50%; on addition of NO donor the index is over 80%. Comparative studies of NO donor (organic nitrate) and a similar compound in which ONO(2) moieties were replaced by OH groups demonstrated that the presence of NO(2) is required for adjuvant activity of compounds and confirmed that nitric oxide modifies the antitumor effects of cytostatics. It is shown also that nitric oxide donor retards the development of drug resistance to cyclophosphamide.     

Enzymatic synthesis of tea theaflavin derivatives and their anti-inflammatory and cytotoxic activities

Derivatives based on a benzotropolone skeleton (9-26) have been prepared by the enzymatic coupling (horseradish peroxidase/H2O2) of selected pairs of compounds (1-8), one with a vic-trihydroxyphenyl moiety, and the other with an ortho-dihydroxyphenyl structure. Some of these compounds have been found to inhibit TPA-induced mice ear edema, nitric oxide (NO) synthesis, and arachidonic acid release by LPS-stimulated RAW 264.7 cells. Their cytotoxic activities against KYSE 150 and 510 human esophageal squamous cell carcinoma and HT 29 human colon cancer cells were also evaluated.     

Nitric oxide is involved in integration of UV‐B absorbing compounds among parts of clonal plants under a heterogeneous UV‐B environment

In nature, ultraviolet‐B (UV‐B) radiation is highly heterogeneous, both spatially and temporally. Plants exposed to UV‐B radiation produce UV‐B absorbing compounds that function as a protective filter. For clonal plants under heterogeneous UV‐B radiation conditions, integration among ramets can allow irradiated ramets to benefit un‐irradiated ramets by causing them to increase their UV‐B absorbing compounds content. In this study, we evaluated integration between pairs of clonal ramets of Glechoma longituba under heterogeneous or homogeneous UV‐B conditions. We determined the levels of UV‐B absorbing compounds, nitric oxide (NO) and hydrogen peroxide (H₂O₂) and measured the activity of phenylalanine ammonia‐lyase (PAL) in connected ramet pairs under homogeneous or heterogeneous UV‐B conditions. Under heterogeneous UV‐B conditions, the UV‐B absorbing compounds content increased in leaves of irradiated and un‐irradiated ramets, but not in the connecting stolons. The NO content increased in irradiated and un‐irradiated leaves and stolons, but the H₂O₂ content did not. Application of NO synthesis inhibitors and an NO blocker to irradiated ramets blocked the increase in UV‐B absorbing compounds and PAL activity in un‐irradiated ramets. These results suggested that NO is involved in the integration process for UV‐B absorbing compounds among ramets. Our findings suggested that a UV‐B‐induced increase in NO transmits a signal to un‐irradiated ramets via the stolon, leading to an increase in PAL activity and UV‐B absorbing compounds content. The internal translocation of signal enables members of clonal networks to function as a whole unit and to mount an efficient defensive response to localized UV‐B radiation.     

Competitive antagonism of nitric oxide synthetase causes weight loss in mice.

These studies demonstrate that the competitive antagonist of nitric oxide synthesis, L-NG-nitro-arginine methyl ester (NO Arg ME), produces an L-arginine reversible decrease in food intake in mice. NO Arg ME also blocked the feeding effect of the potent orexigenic peptide, neuropeptide Y. NO Arg ME produced weight loss when administered over 5 days. The studies suggest that nitric oxide is a physiological modulator of food intake and that nitric oxide synthetase inhibitors may be useful in the management of obesity.     

Nitric oxide and Fenton/Haber-Weiss chemistry: nitric oxide is a potent antioxidant at physiological concentrations

We have examined the action of nitric oxide (NO) on the ability of Fenton's reagent (ferrous iron and hydrogen peroxide), to oxidize a number of organic optical probes. We found that NO is able to arrest the oxidation of organic compounds at concentrations of NO found in brain, in vivo. We present evidence that Fenton's reagent proceeds via a ferryl intermediate ([Fe[double bond]O]2+), before the generation of hydroxyl radical *OH. NO reacts rapidly with this ferryl, blocking the production of *OH. We propose that NO has an important role in protecting biological tissues, and the brain in particular, from Fenton chemistry.