Generation of Nitric Oxide by Peripheral Blood Leukocytes and Platelets in Healthy Humans and Patients with Thermal Trauma

The generation of nitric oxide (NO) by human peripheral blood leukocytes and platelets has been studied in healthy subjects and patients with burns (with the affected area varying from 10 to 45% of the body surface). Differential centrifugation was used to isolate leukocytes and platelets from the blood. The leukocyte suspension was diluted with a complete medium to a concentration of 1 × 10⁷ cells/ml, and the platelet suspension, to 1 × 10⁸ cells/ml; the suspensions were then cultured for 15 h (37°C). The concentration of nitrite, an NO metabolite, was determined using the Griss reaction. The relative production of NO by leukocytes of healthy subjects and patients was 0.75 ± 0.06 and 2.93 ± 0.16 mol/l, respectively (p < 0.001), and its relative production by platelets of healthy subjects and patients was 2.15 ± 0.14 and 3.62 ± 0.13 mol/l, respectively (p < 0.01). The absolute generation of NO by leukocytes of healthy subjects and patients is 0.47 ± 0.05 and 3.02 ± 0.28 mol/l, respectively (p < 0.001), and its absolute generation by platelets of healthy subjects and patients was 7.70 ± 0.55 and 14.68 ± 0.84 mol/l, respectively (p < 0.001). Thus, the absolute production of NO by platelets is 16 times higher than the absolute production of NO by leukocytes of healthy subjects. Stress increases the generation of NO by both leukocytes and platelets. The absolute generation of NO by platelets in thermal trauma is positively correlated with the plasma content of fibrinogen in the patients.     

Elicitor-induced nitric oxide burst is essential for triggering catharanthine synthesis in Catharanthus roseus suspension cells

Elicitor prepared from the cell walls of Penicillium citrinum induced multiple responses in Catharanthus roseus suspension cells, including rapid generation of nitric oxide (NO), sequentially followed by enhancement of catharanthine production by C. roseus cells. Elicitor-induced catharanthine biosynthesis was blocked by NO-specific scavenger 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and nitric oxide synthase (NOS) inhibitor S,S-1,3-phenylene-bis(1,2-ethanediyl)-bis-isothiourea (PBITU). PBITU also strongly inhibited elicitor-induced NO generation by C. roseus suspension cells. The inhibiting effect of PBITU on elicitor-induced catharanthine production was reversed by external application of NO via the NO-donor sodium nitroprusside. The results strongly suggested that NO, generated by NOS or NOS-like enzymes in C. roseus suspension cells when treated with the fungal elicitor, was essential for triggering catharanthine synthesis.     

Nitric oxide functions in both methyl jasmonate signaling and abscisic acid signaling in Arabidopsis guard cells

Intracellular components in methyl jasmonate (MeJA) signaling remain largely unknown, to compare those in well-understood abscisic acid (ABA) signaling. We have reported that nitric oxide (NO) is a signaling component in MeJA-induced stomatal closure, as well as ABA-induced stomatal closure in the previous study. To gain further information about the role of NO in the guard cell signaling, NO production was examined in an ABA- and MeJA-insensitive Arabidopsis mutant, rcn1. Neither MeJA nor ABA induced NO production in rcn1 guard cells. Our data suggest that NO functions downstream of the branch point of MeJA and ABA signaling in Arabidopsis guard cells.Key words: abscisic acid, Arabidopsis thaliana, guard cells, methyl jasmonate, nitric oxideStomatal pores that are formed by pairs of guard cells respond to various environmental stimuli including plant hormones. Some signal components commonly function in MeJA- and ABA-induced stomatal closing signals,¹ such as cytosolic alkalization, ROS generation and cytosolic free calcium ion elevation. Recently, we demonstrated that NO functions in MeJA signaling, as well as ABA signaling in guard cells.²NO production by nitric oxide synthase (NOS) and nitrate reductase (NR) plays important roles in physiological processes in plants.^3,4 It has been shown that NO functions downstream of ROS production in ABA signaling in guard cells.⁵ NO mediates elevation of cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt), inactivation of inward-rectifying K⁺ channels and activation of S-type anion channels,⁶ which are known to be key factors in MeJA- and ABA-induced stomatal closure.^2,7–9It has been reported that ROS was not induced by MeJA and ABA in the MeJA- and ABA-insensitive mutant, rcn1 in which the regulatory subunit A of protein phosphatase 2A, RCN1, is impaired.^7,10 We examined NO production induced by MeJA and ABA in rcn1 guard cells (Fig. 1). NO production by MeJA and ABA was impaired in rcn1 mutant (p = 0.87 and 0.25 for MeJA and ABA, respectively) in contrast to wild type. On the other hand, the NO donor, SNP induced stomatal closure both in wild type and rcn1 mutant (data not shown). These results are consistent with our previous results, i.e., NO is involved in both MeJA- and ABA-induced stomatal closure and functions downstream of the branching point of MeJA and ABA signaling in Arabidopsis guard cells.⁷ Our finding implies that protein phosphatase 2A might positively regulate NO levels in guard cells (Fig. 2).Open in a separate windowFigure 1Impairment of MeJA- and ABA-induced NO production in rcn1 guard cells. (A) Effects of MeJA (n = 10) and ABA (n = 9) on NO production in wild-type guard cells. (B) Effects of MeJA (n = 7) and ABA (n = 7) on NO production in rcn1 guard cells. The vertical scale represents the percentage of diaminofluorescein-2 diacetate (DAF-2 DA) fluorescent levels when fluorescent intensities of MeJA- or ABA-treated cells are normalized to control value taken as 100% for each experiment. Each datum was obtained from at least 30 guard cells. Error bars represent standard errors. Significance of differences between data sets was assessed by Student''s t-test analysis in this paper. We regarded differences at the level of p < 0.05 as significant.Open in a separate windowFigure 2A model of signal interaction in MeJA-induced and ABA-induced stomatal closure. Neither MeJA nor ABA induces ROS production, NO production, I_Kin and stomatal closure in rcn1 mutant. These results suggest that NO functions downstream of the branch point of MeJA signaling and ABA signaling in Arabidopsis guard cells.     

Electrochemical study of the intracellular transduction of vascular endothelial growth factor induced nitric oxide synthase activity using a multi-channel biocompatible microelectrode array

Background

Nitric oxide (NO) plays a major role in physiology as a biological mediator. NO has been identified in nervous, immune and vascular systems and is a critical parameter in numerous pathologies, such as cancer. This article describes the electrochemical biomeasurements of NO synthase (NOS) activity from cultured endothelial cells using a multiple microelectrode array.

Methods

Firstly, the effect of biocompatible fibronectin coating on electrochemical measurements was investigated. Secondly, endothelial cells were deposited on the fibronectin coated sensor and NO release was triggered with vascular endothelial growth factor (VEGF). N^G-nitro-l-arginine methyl ester (L-NAME) was used as an inhibitor of NO production, and different kinase blockers were investigated. Change in NOS activity was quantified using differential pulse voltammetry before and after addition of VEGF.

Results

Our results show that carefully applied layers of fibronectin have a very limited effect on electrochemistry and that VEGF induces an increase in NOS activity that is mainly mediated through the phosphatidylinositol 3 kinase (PI-3), and not by the extracellular signal-regulated kinases 1/2. Results obtained using electrochemical sensors were supported by wound healing assay demonstrating the critical role of phosphatidylinositol 3 kinase and extracellular signal-regulated kinases 1/2 for angiogenesis.

Conclusion

Electrochemical study of the intracellular transduction of the VEGF signal leading to NO synthesis was achieved, showing the critical role of PI-3 kinase.

General significance

This study presents an electrochemical sensor allowing measurements of NOS activity in cell cultures and tissue samples.     

l-Ascorbic-acid biosynthesis in the euryhaline diatom Cyclotella cryptica

l-Ascorbic acid (AA) production in cells of Cyclotella cryptica Reimann, Lewin, Guillard (Bacillariophyceae) is enhanced when darkadapted cells are exposed to light.Heterotrophically grown cells incubated with d-[6-³H,6-¹⁴C]glucose and d-[1-³H,6-¹⁴C]glucose (2 h in dark followed by 15 h light) produced labeled AA with significantly different ratios of ³H and ¹⁴C. Comparisons of labeling patterns in AA and chitin-derived d-glucosamine support a path of conversion in Cyclotella from d-glucose to AA that inverts the carbon chain of the sugar. This process resembles similar conversions found in AA-synthesizing animals and species from two other algal classes.Abbreviations AA l-Ascorbic acid - glc d-glucose - glcN d-glucosamine     

Suppression of cytokine production by supernatants from CD8+ lymphocytes activated by mycobacterial fractions: The role of interleukins 4 and 6

Supernatants derived from CD8⁺ lymphocytes treated with mycobacterial components, or the partially purified carbohydrates from these supernatants, increased the production of IL-4 and IL-6 by mononuclear cells. The addition of anti-IL4 or anti-IL6 antibodies to LPS stimulated MN cells incubated with supernatants from CD8⁺ lymphocytes or carbohydrates resulted in the restoration of other cytokine production by these MN cells. Recombinant IL-4 and IL-6 on their own suppressed the production of IL-1, TNF-, IL-2 and IFN- by mononuclear cells. Such suppression could be reversed with antibodies to IL-4 and IL-6. The addition of rIL-4 and rIL-6 did not increase the suppression of cytokine production induced by suppressor supernatants or carbohydrates. Interleukin 4 decreased the production of IL-6 by MN cells; whilst IL-6 suppressed IL-4 production in a dose dependent manner. Both effects could be reversed with the appropriate antisera. Our results suggest that mycobacteria could evade host immunity by inducing the production of IL-4 and IL-6 by host mononuclear cells. These cytokines, in turn, would suppress the production of other cytokines necessary for effective cellular immunity.Abbreviations IL-1 interleukin 1 - IL-2 interleukin 2 - IL-4 interleukin 4 - IL-6 interleukin 6 - IFN- gamma interferon - TNF- tumour necrosis factor alpha - MN cells mononuclear cells - NAL's non-adherent lymphocytes - LPS lipopolysaccharide - PMA phorbol myristate acetate - RIA radioimmunoassay - ELISA enzyme-linked immunosorbent assay - rIL-4 recombinant interlukin-4 - rIL-6 recombinant interleukin-6 - U/ml units per millilitre - g/ml micrograms per millilitre - ng/ml nanograms per millilitre     

NO detection in biological samples: Differentiation of NO and NO using infrared laser spectroscopy

Accurate characterization of the biochemical pathways of nitric oxide (NO) is essential for investigations in the field of NO research. To analyze the different reaction pathways of enzymatic and non-enzymatic NO formation, determination of the source of NO is crucial. Measuring NO-related products in biological samples distinguishing between ¹⁴NO and ¹⁵NO offers the opportunity to specifically analyze NO signaling in blood and tissue. The aim of this study was to establish a highly sensitive technique for the specific measurement of NO in an isotopologue-selective manner in biological samples.With the cavity leak-out spectroscopy setup (CALOS) a differentiation between ¹⁴NO and ¹⁵NO is feasible. We describe here the employment of this method for measurements in biological samples. Certified gas mixtures of ¹⁴NO/N₂ and ¹⁵NO/N₂ were used to calibrate the system. and of aqueous and biological samples were reduced in a triiodide solution, and the NO released was detected via CALOS. Gas-phase chemiluminescence detection (CLD) was used for evaluation.The correlation received for both methods for the detection of NO in the gas phase was r = 0.999, p < 0.0001. Results obtained using aqueous and biological samples verified that CALOS enables NO measurements with high accuracy (detection limit for 0.3 pmol and 0.5 pmol; correlation ¹⁴NO: p < 0.0001, r = 0.975, ¹⁵NO: p < 0.0001, r = 0.969).The CALOS assay represents an extension of NO measurements in biological samples, allowing specific investigations of enzymatic and non-enzymatic NO formation and metabolism in a variety of samples.     

Active transport of proline into washed cells of Rhodopseudomonas sphaeroides f. sp. denitrificans grown with nitrate

Washed cells of Rhodopseudomonas sphaeroides f. sp. denitrificans, prepared from cultures grown anaerobically in light with NO ₃ ^- as the terminal acceptor, readily incorporated [¹⁴C]-proline both in light and in the dark. The proline uptake was coupled to the reduction of either NO ₃ ^- , NO ₂ ^- , N₂O or O₂. Light stimulated the accumulation of proline in these cells. The addition of NO ₃ ^- to washed cells in light decreased the K _m for proline from 40 M to 5.7 M. Proline transport was inhibited by antimycin A, 2-n-heptyl-4-hydroxyquinoline-N-oxide both in light and in the dark with nitrate indicating that electron transfer from both denitrification and photosynthesis are involved in this uptake. Inhibition by carbonyl cyanide-m-chlorophenyl hydrazone and 2.4-dinitrophenol indicate that proline transport is energy dependent. The H⁺/proline stoichiometry increased from 1 to 2.5 when the external pH was increased from 6.0 to 8.0. Under these conditions pro increased but p decreased markedly above pH 7.0.Abbreviations TPP⁺ Tetraphenylphosphonium bromide - EDTA ethylenediamine-tetra-acetic acid - CCCP carbonyl cyanide-m-chlorophenyl hydrazone - DNP 2,4-dinitrophenol - HOQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - DBMIB dibromo-methyl-isopropyl-p-benzoquinone - DCCD N,N-dicyclohexylcarbodiimide     

Nitric oxide is a positive regulator of the Warburg effect in ovarian cancer cells

Ovarian cancer (OVCA) is among the most lethal gynecological cancers leading to high mortality rates among women. Increasing evidence indicate that cancer cells undergo metabolic transformation during tumorigenesis and growth through nutrients and growth factors available in tumor microenvironment. This altered metabolic rewiring further enhances tumor progression. Recent studies have begun to unravel the role of amino acids in the tumor microenvironment on the proliferation of cancer cells. One critically important, yet often overlooked, component to tumor growth is the metabolic reprogramming of nitric oxide (NO) pathways in cancer cells. Multiple lines of evidence support the link between NO and tumor growth in some cancers, including pancreas, breast and ovarian. However, the multifaceted role of NO in the metabolism of OVCA is unclear and direct demonstration of NO''s role in modulating OVCA cells'' metabolism is lacking. This study aims at indentifying the mechanistic links between NO and OVCA metabolism. We uncover a role of NO in modulating OVCA metabolism: NO positively regulates the Warburg effect, which postulates increased glycolysis along with reduced mitochondrial activity under aerobic conditions in cancer cells. Through both NO synthesis inhibition (using L-arginine deprivation, arginine is a substrate for NO synthase (NOS), which catalyzes NO synthesis; using L-Name, a NOS inhibitor) and NO donor (using DETA-NONOate) analysis, we show that NO not only positively regulates tumor growth but also inhibits mitochondrial respiration in OVCA cells, shifting these cells towards glycolysis to maintain their ATP production. Additionally, NO led to an increase in TCA cycle flux and glutaminolysis, suggesting that NO decreases ROS levels by increasing NADPH and glutathione levels. Our results place NO as a central player in the metabolism of OVCA cells. Understanding the effects of NO on cancer cell metabolism can lead to the development of NO targeting drugs for OVCAs.Despite recent medical and pharmaceutical advances in cancer research, ovarian cancer (OVCA) remains one of the most deadly gynecological malignancies, with most of the cancer first detected in late stages when metastasis has already occurred.¹ Only 20% of OVCA patients are diagnosed when cancer has not spread past the ovaries; in the other 80% of cases, the cancer has metastasized, most frequently to the peritoneum.² Platinum-based preoperative chemotherapy is the standard of care of early stage disease, and surgical resection along with platinum-based postoperative chemotherapy is the standard of care for late stage disease.¹ However, many platinum-based chemotherapy drugs come with unwanted side effects. Therefore, an alternative therapy for OVCA is needed.Nitric oxide (NO) shows promise either as a cancer therapeutic agent by itself or as a target of cancer therapies.³ This may be because NO can act as a signaling molecule or as a source of oxidative and nitrosative stress.⁴ NO can stimulate mitochondrial biogenesis through PGC-1-related coactivator⁵ and increase mitochondrial function.^{6, 7} In follicular thyroid carcinoma cells, S-nitroso-N-acetyl-D,L-penicillamine (SNAP), a NO donor, was shown to increase the expression of genes involved in mitochondrial biogenesis.^{8, 9} A 14-day treatment of lung carcinoma cells with dipropylenetriamine NONOate (DETA-NONOate), another NO donor, increased cell migration compared with the absence of treatment.¹⁰ In breast cancer cells, exogenous NO increased cell proliferation, as well as cyclin-D1 and ornithine decarboxylase expression.¹¹ In prostate cancer cells, NO was shown to inhibit androgen receptor-dependent promoter activity and proliferation of androgen-dependent cells, indicating that NO would select for the development of prostate cancer cells that are androgen-independent.¹² NO has even been shown to inhibit mitochondrial ATP production, and therefore inhibit apoptosis, as ATP is necessary for the apoptotic process.¹³ Moreover, inducible nitric oxide synthase (iNOS) knockout mice had less tumor formation than wild-type mice, indicating that NO promotes lung tumorigenesis.¹⁴ On the other hand, NO production, as induced by proinflammatory cytokines, induced apoptosis in OVCA cells.³ NOS overexpression by transfection of a plasmid containing NOS-3 DNA resulted in increased cell death in HepG2 cells.¹⁵ In another study, NO was implicated in N-(4-hydroxyphenyl) retinamide-mediated apoptosis.¹⁶ Finally, iNOS expression in p53-depleted mice increased apoptosis of lymphoma cells compared with p53-deficient mice without iNOS expression.¹⁷ Therefore, NO has been seen to have both an anti-tumorigenic as well as a pro-tumorigenic effect.Arginine, a conditionally essential amino acid used to produce NO, is also a potential target for cancer therapy. L-arginine is normally produced by the body; however, in some diseased states, more arginine than what the body normally produces is required.¹⁸ Arginine sources include protein breakdown or directly from the diet, in addition to de novo synthesis.¹⁹ In the de novo production of L-arginine, citrulline and aspartate are first converted to argininosuccinate by arginase, which is then split into arginine and fumarate by argininosuccinate lyase.²⁰ L-arginine can also be converted to citrulline and NO through NO synthase (NOS).¹⁹ Some cancer cells, including melanoma and hepatocellular carcinoma, do not express argininosuccinate synthase (ASS), an enzyme involved in arginine production and thus rely on exogenous arginine.¹⁹ For these cancers, arginine-deprivation therapy is being heavily explored as a treatment.^{21, 22} OVCA cells have been shown to express ASS.²³ In fact, OVCA cells were shown to have increased expression of ASS compared with normal ovarian surface epithelium.²⁴ As OVCA can synthesize arginine de novo, strategies which target arginine''s conversion into citrulline are needed for regulating OVCA tumor growth.Recent studies suggest that cancer cells undergo metabolic reprogramming, which drives cancer cells'' growth and progression.^{25, 26, 27, 28, 29, 30, 31, 32, 33} One critically important, yet often overlooked, component to tumor growth is the metabolic rewiring of NO pathways in OVCA cells. Despite considerable investigation on NO''s regulation of cancer cell proliferation and growth, mechanistic details regarding the effect of NO on cancer cell metabolism is still lacking: specifically, how NO affects glycolysis, TCA cycle flux, and ROS production. Studies on the effects of NO on cancer cell metabolism have mainly focused on the effect of NO on mitochondrial respiration.^{34, 35, 36, 37} NO has been shown to inhibit cytochrome c oxidase (COX) in the mitochondria of breast cancer cells, as well as decrease oxygen consumption rate.^{37, 38, 39} Moncada and colleagues studied the effect of NO on the metabolism of rat cortical astrocytes and neurons, two cells with different glycolytic capacities. They showed that NO decreased ATP concentration, which led to an increase in glycolysis in astrocytes, but not in neurons, indicating that glycolytic capacity affects the metabolic response of these cells to NO.⁴⁰ NO was shown to reduce ATP production via OXPHOS in rat reticulocytes, cells that produce 90% of their ATP from OXPHOS.⁴¹ Endothelial NOS (eNOS) was shown to have a role in the upregulation of GLUT4 transporters by AMPK and AICAR in the heart muscle.⁴² Additionally, NO can serve to stabilize HIF-1α in hypoxic conditions through S-nitrosylation of PHD2,⁴ and as HIF-1α upregulates GLUT transporters and glycolysis,⁴³ NO may affect the metabolism of cancer cells.Although NO is found to affect glycolysis of normal cells, how NO modulates glycolysis of OVCA cells is less understood. The multifaceted role of NO in the metabolism of OVCA is unclear, and direct demonstration of NO''s role in modulating the metabolism of OVCA cells is lacking. This study aims at understanding the mechanistic links between NO and the overall cancer metabolism – specifically, its effects on glycolysis, TCA cycle, OXPHOS, and ROS production – of OVCA cells. Our results show that NO decreases mitochondrial respiration, forcing OVCA cells to undergo higher glycolytic rates to maintain ATP production levels. Our work is the first to illustrate the central role of NO on OVCA metabolism – specifically, showing how NO (i) positively regulates the Warburg effect in OVCA cell, (ii) maintains low ROS levels by upregulating NADPH generation, and (ii) negatively alters mitochondrial respiration, thus promoting cancer growth and proliferation. Our work is also unique in that it is the first to explore the effects of NO on TCA cycle flux and glutaminolysis, potentially also affecting ROS levels by affecting antioxidant levels. In conclusion, by elucidating the effects of NO on cancer metabolism and ROS levels, we have a better understanding of the different mechanisms by which NO affects cancer cell growth. This understanding may lead to potentially useful therapies to halt cancer progression.     

Nitric oxide and nitrite are likely mediators of pollen interactions

The ability of plants to produce nitric oxide (NO) is now well recognised. In plants, NO is involved in the control of organ development and in regulating some of their physiological functions. We have recently shown that pollen generates NO in a constitutive manner and have measured both intra- and extracellular production of this radical. Furthermore, we have shown that nitrite accumulates in the media surrounding the pollen and have suggested that the generation of these signaling molecules may be important for the normal interaction between the pollen grain and the stigma on which it alights. However, pollen grains inevitably come into contact with other tissues, including those of animals and it is likely that the NO produced will influence the behavior of the cells associated with these tissues. Such non-animal-derived, NO-mediated effects on mammalian cells may not be restricted to pollen and plant debris and fungal spore-derived NO may elicit similar effects.Key words: allergy, fungal spores, nitric oxide, nitrite, pollenNitric oxide (NO) has been recognised as a signaling molecule for 20+ years, but its roles in controlling cellular activity are far from fully understood. In plants, NO is involved in numerous biological processes¹ including seed germination,² floral development,³ the control of stomatal closure⁴ and root gravitropism⁵ and is also known to affect gene expression.⁶ Recently, we showed that pollen of Arabidopsis, Senecio and Tradescantia produces NO,⁷ and speculated that its role in this specific context is to help orchestrate early signaling events of the pollen-stigma interaction.^7,8 We subsequently showed that NO generation by pollen is more widespread among angiosperms and not just restricted to the species that were first investigated.⁹ Obviously, this intracellular generation of NO could influence the internal biochemistry of the pollen grain and pollen tube. However, for it to impact on other tissues, such as the stigma, on which the pollen grains alight during pollination, the NO generated would have to be released into the extracellular matrix.To demonstrate that pollen grains do indeed release NO to their surroundings we employed a water soluble derivative of the fluorescent NO probe, diaminofluorescein (DAF), to show that the 525 nm emission of the surrounding solution increased with time and that this fluorescence could be removed by scavenging the NO released from the pollen with compounds such as 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl 3-oxide (PTIO). Thus, it is quite conceivable that, in vivo, NO produced by pollen moves into the extracellular matrix where it exerts an influence on the activity of cells in the adjacent tissues. Interestingly, in vitro rehydration of the pollen (analagous to the regulated hydration of pollen on the stigma) was needed before NO evolution could be measured. Normally, some form of specific stimulation, such as that which occurs either during pathogenesis¹⁰ or which results from the increased hormone levels observed during stomatal closure,¹¹ is required to initiate NO production by plant tissues. Thus, it is interesting here, that water appears to be the signaling cue to initiate constitutive NO release by the pollen.As a result of its free radical nature, NO is notoriously difficult to measure. As the chemistry involved in their reactivity has become better understood, doubts have been raised concerning the specificity of many of the fluorescent probes that have been used for its detection.¹² Commonly the fluorescent NO probe, DAF, is used, but similar alternative probes such as diamino-rhodamine (DAR) have recently also been described.¹³ Here, Figure 1 shows the NO-dependent fluorescence of DAR4M-AM-infused Brassica napus pollen and the associated temporal increase in the fluorescence of the extracellular medium containing a cell impermeable form of the dye. Despite the use of these different dye-based probes, it has still proved important to use other approaches to detect pollen NO production to refute the possibility that similarly reactive free radicals other than NO are responsible for the increased fluorescence observed. We have, therefore, confirmed our fluorescence measurements using electron paramagnetic resonance (EPR) techniques⁹ which have also indicated the presence of NO. Thus, the use of both fluorescent probe and EPR approaches point to the generation and release of NO from the pollen of all the plant species studied.Open in a separate windowFigure 1The diamino-rhodamine dyes, DAR4M-AM (cell permeable) and DAR4M (cell impermeable), can be used to detect intra- and extracellular pollen-derived nitric oxide (NO) respectively. Aqueous suspensions of Brassica napus sp. pollen were incubated for 15 min at room temp in 10 µM DAR4M-AM either without (A) or with (B) 200 µM of the NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). In each case, after removal of the excess dye and resuspension of the pollen in 10% (v/v) glycerol, the accumulated DAR4M-AM fluorescence signals within the pollen grains were detected by spinning disc, laser scanning, confocal microscopy with excitation at 560 nm and emission detection at 575 nm. The extracellular accumulation with time of the NO-associated fluorescence signal of the dye, DAR4M, in the media was also followed spectrophotometrically (C). Using the same excitation and emission detection wavelengths, the fluorescence of aqueous suspensions of the pollen in 10 µM DAR4M either without (Ci) or with (Cii) 200 “M cPTIO was monitored over a 10 min. period at room temp. The output fluorescence signal with time is presented in relative units.An additional NO detection technique based on ozone chemiluminescence was also used to confirm the data obtained.⁹ Unlike the fluorescence and EPR approaches which measure the accumulated production of NO, this method detects the steady-state levels of NO at any given time. However, as these levels proved to be very low and not readily detectable by this approach, we altered the assay conditions so as to measure the nitrite that accumulated as a result of NO oxidation in the extracellular media. While the nitrite that accumulated in the media could have done so as a result of being directly excreted by the pollen, the results obtained were in accordance with the earlier observations that pollen evolves NO.⁹ Neither should nitrite be dismissed as a mere downstream by-product. Not only is it the substrate for the production of NO by enzymes such as nitrate reductase,¹⁴ it can also act as a cell signaling molecule in its own right¹⁵ effecting increased cGMP production, increases in different cytochrome P450 activities and the induction of specific gene expression.Having established that pollen produces NO and nitrite, the mechanisms underlying their generation and subsequent signaling require determination. In mammalian cells the production of NO by a family of nitric oxide synthase enzymes is well understood.¹⁶ However, attempts to find plant homologues have so far proved unsuccessful, with the sole proposed candidate¹⁷ having now been shown to be a G protein.^18–20 Nitrate reductase is clearly one source of NO in plants,^11,14 but whether other enzymes exist which are similarly involved remains a matter for debate and discovery. Obviously, as plant NO synthesising enzymes are identified their function in the generation of NO and nitrite in pollen will need to be established.Originally,⁷ we suggested that pollen-derived NO is integral to the pollen-stigma interaction and this now needs to be determined. Nevertheless, the NO and nitrite released externally by pollen may also affect the cells of any moist tissues on which pollen grains land. Such cells may include, for example, those lining mammalian nasal passages. It is well established that NO helps orchestrate the activity of cells involved in human immune responses¹⁶ and this begs the question as to whether or not pollen-produced NO alters these responses during, for example, the onset of the symptoms of hayfever? Many plant cells produce NO, particular during stress and after wounding²¹ and damaged plant tissues that come into contact with human cells in environments that create such debris also have the potential to elicit similar responses. The reaction of mammalian cells to fungi, which are known to possess NOS enzymes²² and whose spores are a main contributor to asthma,²³ may also be similarly mediated.To conclude, pollen grains appear to generate both NO and nitrite constitutively. Determining the functional significance and ramifications of this production in terms of both endogenous and exogenous cell signaling is an important focus for future research.     

Serine racemase deficiency attenuates choroidal neovascularization and reduces nitric oxide and VEGF levels by retinal pigment epithelial cells

Choroidal neovascularization (CNV) is a leading cause of blindness in age‐related macular degeneration. Production of vascular endothelial growth factor (VEGF) and macrophage recruitment by retinal pigment epithelial cells (RPE) significantly contributes to the process of CNV in an experimental CNV model. Serine racemase (SR) is expressed in retinal neurons and glial cells, and its product, d ‐serine, is an endogenous co‐agonist of N‐methyl‐d ‐aspartate receptor. Activation of the receptor results in production of nitric oxide (^.NO), a molecule that promotes retinal and choroidal neovascularization. These observations suggest possible roles of SR in CNV. With laser‐injured CNV mice, we found that inactivation of SR‐coding gene (Srr_null) significantly reduced CNV volume, neovascular density, and invading macrophages. We exploited the underlying mechanism in vivo and ex vivo. RPE from wild‐type (WT) mice expressed SR. To explore the possible downstream target of SR inactivation, we showed that choroid/RPE homogenates extracted from laser‐injured Srr_null mice contained less inducible nitric oxide synthase and decreased phospho‐VEGFR2 compared to amounts in WT mice. In vitro, inflammation‐primed WT RPEs expressed more inducible NOS, produced more^.NO and VEGF than did inflammation‐primed Srr_null RPEs. When co‐cultured with inflammation‐primed Srr_null RPE, significantly fewer RF/6A‐a cell line of choroidal endothelial cell, migrated to the opposite side of the insert membrane than did cells co‐cultured with pre‐treated WT RPE. Altogether, SR deficiency reduces RPE response to laser‐induced inflammatory stimuli, resulting in decreased production of a cascade of pro‐angiogenic cytokines, including^.NO and VEGF, and reduced macrophage recruitment, which contribute synergistically to attenuated angiogenesis.

     

Endothelin-1 Inhibits Thick Ascending Limb Transport via Akt-stimulated Nitric Oxide Production

Endothelin-1 inhibits sodium reabsorption in the thick ascending limb (THAL) via stimulation of nitric oxide (NO) production. The mechanism whereby endothelin-1 stimulates THAL NO is unknown. We hypothesized that endothelin-1 stimulates THAL NO production by activating phosphatidylinositol 3-kinase (PI3K), stimulating Akt activity, and phosphorylating NOS3 at Ser¹¹⁷⁷. This enhances NO production and inhibits sodium transport. We measured 1) NO production by fluorescence microscopy using DAF2-DA, 2) Akt activity using a fluorescence resonance energy transfer-based Akt reporter, 3) phosphorylated NOS3 and Akt by Western blotting, and 4) NKCC2 activity by fluorescence microscopy. In isolated THAL, endothelin-1 (1 nmol/liter) increased NO production from 0.23 ± 0.24 to 2.81 ± 0.32 fluorescence units/min (p < 0.001; n = 5) but failed to stimulate NO production in THALs isolated from NOS3^–/– mice. Wortmannin (150 nmol/liter), a PI3K inhibitor, reduced endothelin-1-stimulated NO by 83% (0.49 ± 0.13 versus 3.31 ± 0.49 fluorescence units/min for endothelin-1 alone; p < 0.006; n = 5). Endothelin-1 stimulated Akt activity by 0.16 ± 0.02 arbitrary units as measured by fluorescence resonance energy transfer (p < 0.001; n = 5) and increased phosphorylation of Akt at Ser⁴⁷³ by 56 ± 11% (p < 0.002; n = 7). Dominant-negative Akt blocked endothelin-1-induced NO by 60 ± 8% (p < 0.001 versus control; n = 6), and an Akt inhibitor had a similar effect. Endothelin-1 increased phosphorylation of NOS3 at Ser¹¹⁷⁷ by 89 ± 24% (p < 0.01; n = 7) but had no effect on Ser⁶³³. Endothelin-1 inhibited NKCC2 activity, an effect that was blocked by dominant-negative Akt and NOS inhibition. We conclude that endothelin-1 stimulates THAL NO production by activating PI3K, stimulating Akt activity, and phosphorylating NOS3 at Ser¹¹⁷⁷. This enhances NO production and inhibits sodium transport.Nitric oxide (NO) augments salt and water excretion by the kidney (1–6). NO produced by both NOS1 and NOS3 (neuronal and endothelial NOS²) contributes to this effect (7–9). Endothelin-1 appears to be one factor that stimulates NO production by both enzymes in the kidney (7–10). Inhibition of endothelin-induced NOS activation can cause salt-sensitive hypertension (6). The thick ascending limb reabsorbs ∼30% of the filtered NaCl, and improper regulation of sodium reabsorption by this segment has been implicated in salt-sensitive hypertension (11, 12). Thus, studying the effects of endothelin-1 on the thick ascending limb is physiologically significant.Endothelin-1 inhibits thick ascending limb NaCl reabsorption via stimulation of NO (9). NO has been shown to inhibit apical Na⁺-K⁺-2Cl^– co-transport (NKCC2) (13), the main route for sodium entry in this segment and the first step in NaCl absorption (14, 15). The thick ascending limb expresses all three NOS isoforms. The actions of endothelin-1 are likely due to NOS3 activation because 1) this isoform is responsible for regulating thick ascending limb NaCl reabsorption (8), and 2) endothelin-1 stimulates NOS3 expression in the thick ascending limb (16). However, whether endothelin-1 acutely stimulates NO production via NOS3 activation in the thick ascending limb is uncertain.NOS3 can be activated by several signaling pathways, including those that involve Ca²⁺/calmodulin and phosphatidylinositol 3-kinase (PI3K). In endothelial cells, both pathways are important. However, in the thick ascending limb, only the latter has been shown to activate NOS3 (17, 18). Thus, the signaling cascades that activate NOS3 in the thick ascending limb and endothelial cells likely differ (19). The mechanisms by which endothelin-1 stimulates NOS3 and inhibits sodium transport in this segment are unknown. We hypothesized that endothelin-1 stimulates thick ascending limb NO production by activating PI3K, stimulating Akt activity, and phosphorylating NOS3 at Ser¹¹⁷⁷. This enhances NO production and inhibits sodium transport.     

Nitric oxide production is not required for dihydrosphingosine-induced cell death in tobacco BY-2 cells

Sphinganine or dihydrosphingosine (d18:0, DHS), one of the most abundant free sphingoid Long Chain Base (LCB) in plants, is known to induce a calcium-dependent programmed cell death (PCD) in tobacco BY-2 cells. We have recently shown that DHS triggers a production of H₂O₂, via the activation of NADPH oxidase(s). However, this production of H₂O₂ is not correlated with the DHS-induced cell death but would rather be associated with basal cell defense mechanisms. In the present study, we extend our current knowledge of the DHS signaling pathway, by demonstrating that DHS also promotes a production of nitric oxide (NO) in tobacco BY-2 cells. As for H₂O₂, this NO production is not necessary for cell death induction.Key words: tobacco BY-2 cells, sphingolipids, LCBs, dihydrosphingosine, sphinganine, apoptosis, programmed cell death (PCD), nitric oxide (NO)These last few years, it has been demonstrated in plants that long chain bases (LCBs), the sphingolipid precursors, are important regulators of different cellular processes including programmed cell death (PCD).^1–3 Indeed, plant treatment with fumonisin B1 or AAL toxin, two mycotoxins that disrupt sphingolipid metabolism, leads to an accumulation of the dihydrosphingosine (d18:0, DHS), one of the most abundant free LCB in plants and correlatively to the induction of cell death symptoms.^4,5 A more recent study shows a rapid and sustained increase of phytosphingosine (t18:0), due to a de novo synthesis from DHS, when Arabidopsis thaliana leaves are inoculated with the avirulent strain Pseudomonas syringae pv. tomato (avrRpm1), known to induce a localized PCD called hypersensitive response (HR).⁶ More direct evidences were obtained from experiments on Arabidopsis cells where external application of 100 µM C2-ceramide, a non-natural acylated LCB, induced PCD in a calcium (Ca²⁺)-dependent manner.⁷ Recently, we have shown that DHS elicited rapid Ca²⁺ increases both in the cytosol and the nucleus of tobacco BY-2 cells and correlatively induced apoptotic-like response. Interestingly, blocking nuclear Ca²⁺ changes without affecting the cytosolic Ca²⁺ increases prevented DHS-induced PCD.⁸Besides calcium ions, reactive oxygen species (ROS) have also been suggested to play an important role in the control of PCD induced by sphingolipids in plants.⁹ Thus, the C2-ceramide-induced PCD in Arabidopsis is preceded by an increase in H₂O₂.⁷ However, inhibition of ROS production by catalase, a ROS-scavenging enzyme, did not prevent C2-ceramide-induced cell death, suggesting that this PCD is independent of ROS generation. Moreover, we recently showed in tobacco BY-2 cells that DHS triggers a dose-dependent production of H₂O₂ via activation of a NADPH oxidase.¹⁰ The DHS-induced cytosolic Ca²⁺ transient is required for this H₂O₂ production while the nuclear calcium variation is not necessary. In agreement with the results of Townley et al. blocking the ROS production using diphenyleniodonium (DPI), a known inhibitor of NADPH oxidases, does not prevent DHS-induced cell death. Gene expression analysis of defense-related genes, using real-time quantitative PCR (RT-qPCR) experiments, rather indicates that H₂O₂ generation is likely associated with basal defense mechanisms.¹⁰In the present study, we further investigated the DHS signaling cascade leading to cell death in tobacco BY-2 cells, by evaluating the involvement of another key signaling molecule i.e., nitric oxide (NO). In plants, NO is known to play important roles in numerous physiological processes including germination, root growth, stomatal closing and adapative response to biotic and abiotic stresses (reviewed in ref. ^11–14). NO has also been shown to be implicated in the induction of PCD in animal cells,¹⁵ in yeast,¹⁶ as well as in plant cells, in which it is required for tracheid differentiation¹⁷ or HR activation.^18,19 Interestingly in the latter case, the balance between NO and H₂O₂ production appears to be crucial to induce cell death.²⁰ Here we show in tobacco BY-2 cells that although DHS elicits a production of NO, this production is not necessary for the induction of PCD.     

Effect of Nitric Oxide on Poliovirus Infection of Two Human Cell Lines

The role of nitric oxide after poliovirus infection of the human HeLa (carcinoma) and U937 (promonocytic) cell lines has been analyzed. Both types of cells produced detectable levels of nitric oxide after poliovirus infection. However, this production was not sufficient to limit viral productivity. On the other hand, pretreatment with the nitric oxide donor glycerine trinitrate lengthened the course of poliovirus infection.It has been demonstrated that nitric oxide (NO) plays an important role in defense against a wide spectrum of microbial pathogens (22). Nevertheless, the antiviral activity of NO has not been observed until recently (6, 10). In those first reports, murine macrophages produced NO after activation with gamma interferon and resisted infection with herpes simplex virus type 1 (HSV-1) (6), vaccinia virus, or ectromelia virus (10). Further reports pointed to NO as a first line of defense against infections in murine systems with RNA viruses (e.g., vesicular stomatitis virus [4, 12], Friend leukemia virus [3], encephalomyocarditis virus [8]; Sindbis virus [SV] [25], or Japanese encephalitis virus [15]) and DNA viruses, such as HSV-1 (6) or vaccinia virus (9, 24). Nevertheless, in some cases the effect of the production of NO in cultured cells is difficult to extrapolate to animals systems (14, 23).As regards human cells, the role of NO after viral infection remains to be unveiled. NO produced by human B cells seemed to inhibit Epstein-Barr virus reactivation (20). Moreover, NO donors can inhibit human immunodeficiency virus type 1 (HIV-1) replication in human peripheral blood mononuclear cells (5). Nevertheless, Koka et al. (11) suggest that some pathologic effects that appeared in the central nervous system after HIV-1 infection could be due to the toxic effect of NO. NO constitutively produced by activated human promonocytic U937 cells plays a role in resistance to H-1 autonomous parvovirus infection (17). Infection with HSV-1 of U937 cells differentiated with the phorbol ester 12-myristate 13-acetate induced the production of significant levels of NO; however, this NO production did not change viral production (16).Despite the protective effect of NO against certain viral infections, a number of recent studies indicate a harmful role of NO in many systems. Thus, NO seems to play an important role in the development of pneumonia triggered by influenza virus in mice (2) and in pathogenesis in mice infected with the tick-borne encephalitis flavivirus (13). Furthermore, it has been reported that infection of mice with coxsackievirus B3 (CVB3) induced NO in the heart, aggravating the course of the viral myocarditis (21). These results are in conflict with those of Lowenstein et al. (19), who observed that NO ameliorated the effect of CVB3 infection in mice. In a recent work, Adler et al. (1) showed that HSV-1-induced pneumonia in mice could be suppressed by the inhibitor of inducible nitric oxide synthase (iNOS), N^ω-monomethyl-l-arginine (l-NMMA). Considering all these controversial results, the question of whether NO acts as an inhibitor of viral replication or as a harmful agent remains unanswered.We have studied the effect of NO on poliovirus infection. To this end, human promonocytic U937 cells were cultured in RPMI 1640 (Life Technologies, Paisley, United Kingdom) and supplemented with 10% heat-inactivated fetal calf serum. HeLa cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% newborn calf serum. Poliovirus type 1 (Mahoney strain) was propagated in HeLa cells. Initially, the accumulation of NO in both human cell cultures after addition of the NO donor glycerin trinitrate (GTN) (Merck, Darmstadt, Germany) was studied. Fig. ​Fig.1A1A shows dose-dependent levels of NO, which increased during the course of incubation, detected in both HeLa and U937 cells. For further assays, cells were preincubated with 4 mg of GTN/ml for 12 h, since higher concentrations produced cytotoxicity, as observed by trypan blue staining (data not shown). In order to study the poliovirus-induced NO, HeLa and U937 cells were infected at multiplicities of infection (MOI) of 0.5 and 5 PFU/cell, respectively. The formation of NO was measured as described by Green et al. (7). In each individual experiment, aliquots of U937 or HeLa supernatants (0.1 ml), uninfected or infected with poliovirus, were incubated, in triplicate, in flat-bottom 96-well culture plates and mixed with the same amount of Greiss reagent (0.1% naphthyl-ethylenediamine dihydrochloride [Sigma] in distilled water and 1% sulfanilamide [Sigma] in 5% phosphoric acid [vol/vol]). Subsequently, this mixture was incubated for 10 min at room temperature and the optical density at 550 nm was measured in an MR 5000 microplate reader (Dynatech, Billingshurst, West Sussex, United Kingdom). As illustrated in Fig. ​Fig.1B,1B, the infection induced a slight but significant production of NO. Incubation with 2 mM l-NMMA (Calbiochem-Novabiochem Corporation, San Diego, Calif.) decreased the production of NO induced by the viral infection (Fig. ​(Fig.1B).1B). Addition of monomethyl-d-arginine (Calbiochem) as a control of specificity did not exert any effect on the accumulation of NO (data not shown). Furthermore, infection of GTN-pretreated cells did not modify the levels of NO produced with GTN alone, suggesting that maximal levels of NO had been reached or that exogenous NO addition could inhibit cellular iNOS. Open in a separate windowFIG. 1Poliovirus induces NO production in human cells. (A) Treatment of HeLa and U937 cell cultures with GTN produces NO accumulation. Cells (10⁵ per ml) were incubated at 37°C in the presence or absence of the NO donor. At the indicated times, NO production was assayed as detailed in the text. (B) HeLa or U937 cells (10⁵) were infected with poliovirus at MOIs of 0.5 and 5 PFU/cell, respectively. Subsequently, cultures were incubated at 37°C in the presence or absence of 4 mg of GTN/ml. In parallel, cultures were preincubated for 4 h with 2 mM l-NMMA (striped bars). At 20 h p.i. the accumulation of NO was assayed. Values are means ± standard deviations of three experiments, performed in triplicate.The implication of this endogenous NO production in HeLa and U937 cells after poliovirus infection is shown in Table ​Table1.1. Treatment of the cultures with 2 mM l-NMMA altered neither the production of infectious poliovirus particles nor the cellular death observed by plaque assay and cell counting, respectively. On the other hand, and in agreement with previous findings (12), exogenous NO supplied by 12 h of pretreatment with 4 mg of GTN/ml produced an increase of cell viability and a 3.9- or 15-fold decrease in the PFU produced in HeLa and U937 cells, respectively, analyzed by means of a plaque assay performed on HeLa cell monolayers. This reduction of infectious particles was not due to a direct inhibitory effect of GTN on poliovirus input, since pretreatment of 5 × 10⁶ poliovirus particles with 16 mg of GTN/ml for 5 h did not alter the subsequent infectivity of the virus (data not shown). Altogether, these results indicate that the addition of NO decreases poliovirus infection in both the HeLa and U937 human cell lines. However, the low level of endogenous NO production induced after the infection does not seem to be sufficient to alter the course of poliovirus infection. Morphological studies confirmed these results (data not shown).

TABLE 1

Pretreatment of HeLa and U937 cells with GTN protects from poliovirus infection^a

Influence of hydrological seasonality on bacterioplankton in two neotropical floodplain lakes

Seasonal fluctuation in river stage strongly affects the ecological functioning of tropical floodplain lakes. This study was conducted to assess the influence of hydrological seasonality on bacterial production and abundance in two floodplain lakes of the Autana River, a blackwater river in the Middle Orinoco basin, Venezuela. Water samples for nutrient chemistry, chlorophyll a, and microbiological determinations were collected in two floodplain lakes and in the mainstem of the river during 1997–98. DOC and chlorophyll a concentrations were similar between mainstem and lake sites during high water when river and lakes were well connected but became different during the period of low water when the interaction was minimal. Higher values of bacterial production were observed in the floodplain lakes (0.62–1.03 g C l^–1 h^–1) compared to the mainstem sites (0.17–0.19 g C l^–1 h^–1) during the period of low water, while during the period of high water river and lake sites showed similar levels (0.04 g C l^–1 h^–1). Bacterial numbers followed bacterial production in the floodplain lakes, reaching higher numbers during the period of low water (1.41–2.40 × 10⁶ cells ml^–1). Availability of substrate and inorganic nutrients, pH, and inputs and losses of bacterial cells could be determining the observed seasonal patterns in bacterial production and abundance. The Autana lakes exhibited a strong seasonal pattern in the chemical and biological conditions, showing higher productivity during the lentic phase that lasted between 5 and 6 months.     

Lipopolysaccharide enhances asymmetrical production of cytokines and nitric oxide by left and right cerebral cortical microglial cells in BALB/C mice

Lipopolysaccharide (LPS)‐induced inflammatory factors production by the cerebral cortical glial cells in two sides of the murine brain are different. To determine if microglial cells, a subset of glial cells, are involved in asymmetric production, interleukin‐6 (IL‐6), interleukin‐1β (IL‐1β) and nitric oxide (NO) responses to LPS by microglial cells in the right and left cerebral cortices were examined. Primary microglial cells were isolated from BALB/C neonatal mice, treated with LPS (10 µg ml^?1) for 24 h and examined for IL‐6, IL‐1β and NO production. At untreated state, the levels of IL‐6, IL‐1β and NO showed no statistical difference between left and right. However, after LPS treatment, the levels of IL‐6, IL‐1β and NO for the right microglial cells was statistically significant higher than the left (P < 0·05). Our results denote that enhanced production of IL‐6, IL‐1β and NO after LPS treatment in microglia is directly proportional to their basal‐state levels, and right cortical microglia produce higher levels of IL‐6, IL‐1β and NO than left cortical microglia. Copyright © 2011 John Wiley & Sons, Ltd.     

Parallel regulation of arginine transport and nitric oxide synthesis by angiotensin II in vascular smooth muscle cells role of protein kinase C

Summary Experiments were performed to characterize arginine transport in vascular smooth muscle cells (SMCs) and the effect of angiotensin II (Ang II) on this process. In addition, the role of arginine transport in the cytokineinduced nitric oxide (NO) production was assessed. Arginine transport takes place through Na⁺-independent (60%) and Na⁺-dependent pathways (40%). The Na⁺-independent arginine uptake appears to be mediated by system y⁺ because of its sensitivity to cationic amino acids such as lysine, ornithine and homoarginine. The transport system was relatively insensitive to acidification of the extracellular medium. By contrast, the Na⁺-dependent pathway is consistent with system B^0,+ since it was inhibited by both cationic and neutral amino acids (i.e., glutamine, phenylalanine, and asparagine), and did not accept Li⁺ as a Na⁺ replacement. Treatment of SMCs with 100nM Ang II significantly inhibited the Na⁺-dependent arginine transport without affecting systems y⁺, A, and L. This effect occurred in a dose-dependent manner (IC₅₀ of 8.9 ± 0.9nM) and is mediated by the AT-1 receptor subtype because it was blocked by DUP 753, a non-peptide antagonist of this receptor. The inhibition of system B^0,+ by Ang II is mediated by protein kinase C (PKC) because it was mimicked by phorbol esters (phorbol 12-myristate 13-acetate) and was inhibited by staurosporine. Ang II also inhibited the IL-1 induced nitrite accumulation by SMCs. This action was also inhibited by staurosporine and reproduced with phorbol esters, suggesting a coupling between arginine uptake and NO synthesis through a PKC-dependent mechanism. However, arginine supplementation in the medium (10mM) failed to prevent the inhibitory action of Ang II on NO synthesis. These findings suggest that although Ang II inhibits concomitantly arginine transport and NO synthesis in SMCs, the reduction of NO synthesis is not associated with alterations in the cellular transport of arginine.Abbreviations Arg arginine - Orn ornithine - HmR homoarginine - Lys lysine - Gln glutamine - Asn asparagine - His histidine - Phe phenylalanine - Leu leucine - Cys Cysteine - Ala alanine - Ser serine - Thr threonine - Glu glutamate - mAIB -methyl-aminoisobutyric acid - BCH bicycloaminoheptane     

Proton translocation during denitrification in Rhodopseudomonas sphaeroides f. denitrificans

Proton translocation during the reduction of NO ₃ ^- , NO ₂ ^- , N₂O and O₂, with endogenous substrates, in washed cells of Rhodopseudomonas sphaeroides f. denitrificans was investigated by an oxidant pulse method. On adding NO ₂ ^- to washed cells, anaerobically in the dark, an alkalinization occurred in the reaction mixture followed by acidification. When NO ₃ ^- , N₂O or O₂ was added to cells in the dark or with these compounds and NO ₂ ^- in light an acidification only was observed. Proton translocation was inhibited by carbonyl cyanide-m-chlorophenyl hydrazone.Valinomycin treated cells produced acid in response to the addition of either NO ₃ ^- , NO ₂ ^- , N₂O or O₂. The proton extrusion stoichiometry ( ratios) in illuminated cells were as follows: NO ₃ ^- 0.5N₂, 4.82; NO ₂ ^- 0.5N₂, 5.43; N₂ON₂, 6.20; and O₂H₂O, 6.43. In the dark the comparable values were 3.99, 4.10, 4.17 and 3.95. Thus, illuminated cells produced higher values than those in the dark, indicating a close link between photosynthesis and denitrification in the generation of proton gradients across the bacterial cell membranes.When reduced benzyl viologen was the electron donor in the presence of 1 mM N-ethylmaleimide and 0.5 mM 2-n-heptyl-4-hydroxyquinoline-N-oxide in the dark, the addition of either NO ₃ ^- , NO ₂ ^- or N₂O to washed cells resulted in a rapid alkalinization of the reaction mixture. The stoichiometries for proton consumption, ratios without a permeant ion were NO ₃ ^- NO ₂ ^- ,-1.95; NO ₂ ^- 0.5 N₂O,-3.03 and N₂ON₂,-2.02. The data indicate that these reductions occur on the periplasmic side of the cytoplasmic membrane.Abbreviations BVH reduced benzyl viologen - CCCP carbonyl cyanide m-chlorophenyl hydrazone - DIECA N, N-diethyl-dithiocarbamate - HOQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - NEM N-ethylmaleimide     

Effect of irradiance, temperature and salinity on growth and toxin production by Nodularia spumigena

The object of this work was to determine, using a full-factorial experiment, the influence of temperature, irradiance and salinity on growth and hepatotoxin production by Nodularia spumigena, isolated from Lake Alexandrina in the south-east of South Australia. Higher levels of biomass (determined as particulate organic carbon, POC), toxin production and intracellular toxin concentration per mg POC were produced under light limited conditions (30 mol m^–2 s^–1) and at salinities equal to or greater than those experienced in Lake Alexandrina. Both highest biomass and total toxin production rates were recorded at temperatures equal to or greater than those of the lake (20 and 30°C). The temperature at which maximum biomass and toxin production was recorded decreased from 30°C for cultures grown at 30 mol m^–2 s^–1 to 20°C when grown at 80 mol m^–2 s^–1. In contrast, intracellular toxin per mg POC was highest at the lowest growth temperature, 10°C, at both 30 and 80 mol m^–2 s^–1. It appears that the optimum temperature for biosynthetic pathways used in the production of toxin is lower than the optimum temperature for those pathways associated with growth. Intracellular toxin levels were higher in cells cultured at 10°C/30 mol m^–2 s^–1 whereas the majority of the toxin was extracellular in cells grown at 30°C/30 mol m^–2 s^–1. This implies that the highest concentration of toxin in lake water would occur under high temperature and high irradiance conditions. Individual environmental parameters of salinity, irradiance and temperature were all shown to influence growth and toxin production. Notwithstanding, the overall influence of these three parameters on toxin production was mediated through their effect upon growth rate.