An electrochemical sensor array system for the direct, simultaneous in vitro monitoring of nitric oxide and superoxide production by cultured cells

Herein, we evaluated in vitro the anti-leishmanial activity of betulin derivatives in Venezuelan isolates of Leishmania amazonensis, isolated from patients with therapeutic failure. Methods: We analyzed promastigote in vitro susceptibility as well as the cytotoxicity and selectivity of the evaluated compounds. Additionally, the activity of selected compounds was determined in intracellular amastigotes. Finally, to gain hints on their potential mechanism of action, the effect of the most promising compounds on plasma and mitochondrial membrane potential, and nitric oxide and superoxide production by infected macrophages was determined. Results: From the tested 28 compounds, those numbered 18 and 22 were chosen for additional studies. Both 18 and 22 were active (GI50 ≤ 2 µM, cytotoxic CC50 > 45 µM, SI > 20) for the reference strain LTB0016 and for patient isolates. The results suggest that 18 significantly depolarized the plasma membrane potential (p < 0.05) and the mitochondrial membrane potential (p < 0.05) when compared to untreated cells. Although neither 18 nor 22 induced nitric oxide production in infected macrophages, 18 induced superoxide production in infected macrophages. Conclusion: Our results suggest that due to their efficacy and selectivity against intracellular parasites and the potential mechanisms underlying their leishmanicidal effect, the compounds 18 and 22 could be used as tools for designing new chemotherapies against leishmaniasis.

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Bioelectrochemical signal monitoring of in-vitro cultured cells by means of an automated microsystem based on solid state sensor-array

Biosensors and Bioelectronics ◽

10.1016/s0956-5663(03)00040-x ◽

2003 ◽

Vol 18 (5-6) ◽

pp. 621-626 ◽

Cited By ~ 43

Author(s):

Leandro Lorenzelli ◽

Benno Margesin ◽

Sergio Martinoia ◽

M.T Tedesco ◽

Maurizio Valle

Keyword(s):

Solid State ◽

Sensor Array ◽

Cultured Cells ◽

Solid State Sensor ◽

Signal Monitoring

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Evidence of nitric oxide, a flow-dependent factor, being a trigger of liver regeneration in rats

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y98-128 ◽

1998 ◽

Vol 76 (12) ◽

pp. 1072-1079 ◽

Cited By ~ 32

Author(s):

H Helen Wang ◽

W Wayne Lautt

Keyword(s):

Nitric Oxide ◽

Shear Stress ◽

Liver Regeneration ◽

Partial Hepatectomy ◽

Cultured Cells ◽

Portal Blood Flow ◽

Liver Weight ◽

Proliferative Effect

The hypothesis tested was that the hemodynamic consequence of partial hepatectomy (PHX) triggers the cascade of events that leads to liver regeneration. After PHX, all the portal flow must go through the remaining vascular bed, thus producing increased shear stress and release of nitric oxide (NO), which then initiates the next stages of the regeneration process. As an index of triggering of the regeneration cascade, we used an in vitro bioassay detecting the appearance of proliferating factors (PFs; various growth factors, cytokines, and hormones) in plasma 4 h after two-thirds PHX in rats. PF levels, assessed using proliferation of cultured hepatocytes, were elevated in two-thirds PHX rats, fully blocked by the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME), and restored by L-arginine. L-NAME inhibited liver weight restoration at 48 h but resulted in high mortality. L-NAME lacked toxic effects in non-PHX rats. NO was directly antiproliferative on cultured cells, suggesting that the proliferative effect of NO in vivo was secondary to the activation of other proliferative stimuli. The data support the hypothesis that vascular shear stress induced release of NO following PHX serves as a primary trigger to initiate the regeneration process.Key words: shear stress, portal blood flow, hyperplasia, hepatic partial hepatectomy.

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On-chip multi-electrochemical sensor array platform for simultaneous screening of nitric oxide and peroxynitrite

Lab on a Chip ◽

10.1039/c0lc00585a ◽

2011 ◽

Vol 11 (7) ◽

pp. 1342 ◽

Cited By ~ 39

Author(s):

Damien Quinton ◽

Aurélie Girard ◽

Loan To Thi Kim ◽

Vincent Raimbault ◽

Laurent Griscom ◽

...

Keyword(s):

Nitric Oxide ◽

Electrochemical Sensor ◽

Sensor Array ◽

Array Platform ◽

On Chip

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Lamprey GnRH-III Acts on Its Putative Receptor via Nitric Oxide to Release Follicle-Stimulating Hormone Specifically

Experimental Biology and Medicine ◽

10.1177/153537020222700910 ◽

2002 ◽

Vol 227 (9) ◽

pp. 786-793 ◽

Cited By ~ 12

Author(s):

W.H. Yu ◽

S. Karanth ◽

C.A. Mastronardi ◽

S. Sealfon ◽

C. Dean ◽

...

Keyword(s):

Nitric Oxide ◽

Cultured Cells ◽

Follicle Stimulating Hormone ◽

The Other ◽

Male Rats ◽

Other Hand ◽

Lh Rh ◽

Stimulating Hormone

Lamprey gonadotropin-releasing hormone-III (I-GnRH-III), the putative follicle-stimulating hormone (FSH)-releasing factor (FSHRF), exerts a preferential FSH-releasing activity in rats both in vitro and in vivo. To test the hypothesis that I-GnRH-III acts on its own receptors to stimulate gonadotropin release, the functional activity of this peptide at mammalian (m) leutinizing hormone (LH)RH receptors transfected to COS cells was tested. I-GnRH-III activated m-LHRH receptors only at a minimal effective concentration (MEC) of 10–6 M, whereas m-LHRH was active at a MEC of 10–9 M, at least 1,000 times less than that required for I-GnRH-III. In 4-day monolayer cultured cells, I-GnRH-III was similarly extremely weak in releasing either LH or FSH, and, in fact, it released LH at a lower concentration (10–7 M) than that required for FSH release (10–6 M). In this assay, m-LHRH released both FSH and LH significantly at the lowest concentration tested (10–10 M). On the other hand, I-GnRH-III had a high potency to selectively release FSH and not LH from hemipituitaries of male rats. The results suggest that the cultured cells were devoid of FSHRF receptors, thereby resulting in a pattern of FSH and LH release caused by the LHRH receptor. On the other hand, the putative FSH-releasing factor receptor accounts for the selective FSH release by I-GnRH-III when tested on hemipituitaries. Removal of calcium from the medium plus the addition of EGTA, a calcium chelator, suppressed the release of gonadotropins induced by either I-GnRH-III or LHRH, indicating that calcium is required for the action of either peptide. Previous results showed that sodium nitroprusside, a releaser of nitric oxide (NO), causes the release of both FSH and LH from hemipituitaries incubated in vitro. In the present experiments, a competitive inhibitor of NO synthase, L-NG-monomethyl-l-arginine (300 μM) blocked the action of I-GnRH-III or partially purified FSHRF. The results indicate that I-GnRH-III and FSHRF act on putative FSHRF receptors by a calcium-dependent NO pathway.

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20-Hydroxyeicosatetraenoic acid causes endothelial dysfunction via eNOS uncoupling

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.01172.2007 ◽

2008 ◽

Vol 294 (2) ◽

pp. H1018-H1026 ◽

Cited By ~ 104

Author(s):

Jennifer Cheng ◽

Jing-Song Ou ◽

Harpreet Singh ◽

John R. Falck ◽

Dubasi Narsimhaswamy ◽

...

Keyword(s):

Nitric Oxide ◽

Endothelial Dysfunction ◽

Calcium Ionophore ◽

Normal Function ◽

Superoxide Production ◽

Hydroxyeicosatetraenoic Acid ◽

Cellular Mechanisms ◽

Enos Uncoupling

Nitric oxide (NO), generated from l-arginine by endothelial nitric oxide synthase (eNOS), is a key endothelial-derived factor whose bioavailability is essential to the normal function of the endothelium. Endothelium dysfunction is characterized by loss of NO bioavailability because of either reduced formation or accelerated degradation of NO. We have recently reported that overexpression of vascular cytochrome P-450 (CYP) 4A in rats caused hypertension and endothelial dysfunction driven by increased production of 20-hydroxyeicosatetraenoic acid (20-HETE), a major vasoconstrictor eicosanoid in the microcirculation. To further explore cellular mechanisms underlying CYP4A-20-HETE-driven endothelial dysfunction, the interactions between 20-HETE and the eNOS-NO system were examined in vitro. Addition of 20-HETE to endothelial cells at concentrations as low as 1 nM reduced calcium ionophore-stimulated NO release by 50%. This reduction was associated with a significant increase in superoxide production. The increase in superoxide in response to 20-HETE was prevented by NG-nitro-l-arginine methyl ester, suggesting that uncoupled eNOS is a source of this superoxide. The response to 20-HETE was specific in that 19-HETE did not affect NO or superoxide production, and, in fact, the response to 20-HETE could be competitively antagonized by 19(R)-HETE. 20-HETE had no effect on phosphorylation of eNOS protein at serine-1179 or threonine-497 following addition of calcium ionophore; however, 20-HETE inhibited association of eNOS with 90-kDa heat shock protein (HSP90). In vivo, impaired acetylcholine-induced relaxation in arteries overexpressing CYP4A was associated with a marked reduction in the levels of phosphorylated vasodilator-stimulated phosphoprotein, an indicator of bioactive NO, that was reversed by inhibition of 20-HETE synthesis or action. Because association of HSP90 with eNOS is critical for eNOS activation and coupled enzyme activity, inhibition of this association by 20-HETE may underlie the mechanism, at least in part, by which increased CYP4A expression and activity cause endothelial dysfunction.

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Interaction of Yersinia enterocolitica biovar 1A with cultured cells in vitro does not reflect the two previously identified clonal groups

Journal of Medical Microbiology ◽

10.1099/jmm.0.056077-0 ◽

2013 ◽

Vol 62 (12) ◽

pp. 1807-1814 ◽

Cited By ~ 3

Author(s):

Mahesh S. Dhar ◽

Jugsharan S. Virdi

Keyword(s):

Nitric Oxide ◽

Yersinia Enterocolitica ◽

Cultured Cells ◽

Clonal Group ◽

Group A ◽

Repetitive Extragenic Palindrome ◽

Group B ◽

The Individual ◽

Oxide Synthase

Yersinia enterocolitica biovar 1A strains have been delineated into two clonal groups (A and B) based on repetitive extragenic palindrome- and enterobacterial repetitive intergenic consensus-PCR genotyping. The present study investigated the interaction of Y. enterocolitica biovar 1A strains with cultured cells in vitro by their ability to adhere, invade and survive within these cells. The response of macrophages to these strains was also studied by quantifying the expression of inducible nitric oxide synthase, production of nitric oxide and cytokines, and activation of NFκB. The survival rate of clonal group B strains inside macrophages was significantly higher than that of clonal group A strains. In addition, strains harbouring the fepA gene showed better survival inside macrophages. However, the production of nitric oxide and cytokines and activation of NFκB did not show any significant differences between the two clonal groups. In this study, interaction of Y. enterocolitica biovar 1A with cultured cells in vitro did not reflect the previously identified clonal groups, but was more dependent on the characteristics of the individual strains. Therefore, a combination of genotype and phenotype data must be used to characterize this extremely heterogeneous organism.

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Induction of Haemeoxygenase-1 Improves FFA-Induced Endothelial Dysfunction in Rat Aorta

Cellular Physiology and Biochemistry ◽

10.1159/000373946 ◽

2015 ◽

Vol 35 (3) ◽

pp. 1230-1240 ◽

Cited By ~ 9

Author(s):

Fang Han ◽

Zongguang Hui ◽

Shuxian Zhang ◽

Ningning Hou ◽

Yali Wang ◽

...

Keyword(s):

Nitric Oxide ◽

Endothelial Dysfunction ◽

High Sensitivity ◽

Adenosine Monophosphate ◽

Rat Aorta ◽

Superoxide Production ◽

Protective Mechanism ◽

No Production ◽

Beneficial Effects

Background: The induction of haemeoxygenase-1 (HO-1) exerts beneficial effects in the setting of endothelial dysfunction in obesity. High free fatty acid (FFA) levels are a common feature of obesity and are the primary cause of endothelial dysfunction. The objective of our study was to explore the effects of HO-1 induction on FFA-induced endothelial dysfunction in rats. Methods: Rats received FFA treatment with either cobalt protoporphyrin (CoPP) to induce HO-1 or stannous protoporphyrin (SnPP) to inhibit HO-1. Endothelial function was determined by measuring endothelium-dependent vasodilatation (EDV). Nitric oxide (NO) production, superoxide production and nuclear factor (NF)-κB expression in the aorta were each determined. The levels of adenosine monophosphate (AMP)-activated kinase (AMPK) and endothelial nitric oxide synthase (eNOS) expression in endothelial cells were determined via Western blotting. Results: Induction of HO-1 by CoPP decreased circulating FFA, high-sensitivity C-reactive protein and malondialdehyde levels and increased serum adiponectin and glutathione levels compared with the FFA group (P<0.05). High FFA levels resulted in EDV impairment, which was improved by HO-1 induction (P<0.05). Induction of HO-1 increased NO levels and reduced aortic superoxide production and NF-κB expression compared with the FFA group. The FFA group exhibited decreased AMPK expression and eNOS phosphorylation, both of which were enhanced via HO-1 induction (P<0.05). The beneficial effects of CoPP on EDV were partially attenuated in vitro in the presence of inhibitors of AMPK, phosphatidylinositol 3-kinase (PI3K), and eNOS. Conclusions: HO-1 induction with CoPP improves FFA-induced endothelial dysfunction in the rat aorta. The protective mechanism appears to be related to the activation of the AMPK-PI3K-eNOS pathway as a result of increased adiponectin levels as well as decreased inflammation and oxidative stress.

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Evaluation of in vitro Activation of Bovine Endometrial and Vaginal Epithelial and Blood Mononuclear Cells to Produce Nitric Oxide in Response to Mycoplasma bovis, Mycoplasma bovigenitalium and Ureaplasma diversum

Acta veterinaria ◽

10.2478/acve-2021-0012 ◽

2021 ◽

Vol 71 (2) ◽

pp. 137-146

Author(s):

Regiani Nascimento Gagno Pôrto ◽

Ana Paula Junqueira-Kipnis ◽

Marco Antonio de Oliveira Viu ◽

Rafaela Cavalcanti Teixeira ◽

Maria Lucia Gambarini

Keyword(s):

Nitric Oxide ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Cultured Cells ◽

Culture Broth ◽

Mycoplasma Bovis ◽

No Production ◽

Endometrial Cells ◽

Ureaplasma Diversum

Abstract Genital mycoplasmosis is a condition present in bovine production systems, and the most important agents involved are Mycoplasma bovis, Mycoplasma bovigenitalium and Ureaplasma diversum. Some aspects of their pathogenesis remain unclear. This study was designed in order to evaluate their ability to stimulate mononuclear cells from the endometrium, vagina and peripheral blood of cycling and healthy cows to produce nitric oxide (NO). Cellular cultures of endometrial, vaginal and peripheral blood cells from 33 healthy cows were cultivated with Mycoplasma bovis, Mycoplasma bovigenitalium and Ureaplasma diversum originated from the 4th passage in culture broth and the NO production was measured by the Greiss reaction. Confirmation of the presence of mononuclear cells and of the agents during and after the NO assay was done by Giemsa stained smears and further cultivation and detection by PCR reaction. Mononuclear cells from all samples produced NO. Mycoplasma bovigenitalium stimulated higher NO production than the others (p<0.05). Endometrial cells produced less NO than vaginal or blood cultured cells. In conclusion, it seems that Mycoplasma bovis, Mycoplasma bovigenitalium, and Ureaplasma diversum are able to activate mononuclear cells and induce the production of NO, thus suggesting that this pathway is elicited in response to the primary infection by these agents. More studies are necessary to verify why these agents remain in the bovine reproductive tract for long periods and how they reassume deleterious effects.

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