1-3-dihydroxy-4-4-5-5-tetramethyl-2-(4-carboxyphenyl)tetrahydroimidazole and 4-5-diaminofluorescein-diacetate

Nitric oxide (NO) has been demonstrated to be a principal effector molecule responsible for mediating intracellular killing of Leishmania parasites, the causative organism of leishmaniasis. As measurement of intracellular NO remains a challenge to biologists, we have developed a flow cytometric approach to perform real time biological detection of NO within Leishmania parasites and parasitized macrophages using a membrane permeable derivative of diaminofluorescein [4,5-diaminofluorescein diacetate (DAF-2DA)]. Initially, assay optimization was performed in Leishmania donovani promastigotes, assay specificity being confirmed using both a NO donor [S-nitroso-N-acetyl-penicillamine (SNAP)] and a NO scavenger [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, C-PTIO]. Using 40 μM DAF-2DA, basal levels of intracellular NO were measured which varied in different Leishmania species; addition of conventional anti-leishmanial drugs, antimony and miltefosine translated into a dramatic increase in DAF-2T fluorescence. Furthermore, the assay also measured levels of NO in macrophages, but needed a 20 fold lower concentration of DAF-2DA, being 2 μM. Following parasitization, levels of NO decreased which was normalized following treatment with anti-leishmanial drugs. Similarly monocytes of patients with visceral leishmaniasis at disease presentation showed decreased levels of NO which too reverted on completion of treatment. Taken together, this study opens new perspectives of research regarding monocyte function and provides a real time approach for monitoring the effect of anti-leishmanial compounds.

Topics: Adolescent; Adult; Aged; Animals; Antimony Potassium Tartrate; Antiprotozoal Agents; Benzoates; Female; Flow Cytometry; Fluorescein; Humans; Imidazoles; In Vitro Techniques; Leishmania; Leishmaniasis, Visceral; Macrophages, Peritoneal; Male; Mice; Mice, Inbred BALB C; Middle Aged; Monocytes; Nitric Oxide; Phosphorylcholine; Species Specificity; Young Adult

Nitric oxide (NO) controls diverse functions in many cells and organs of animals. It is also produced in plants and has a variety of effects, but little is known about their underlying mechanisms. In the present study, we have discovered a role for NO in the regulation of pollen tube growth, a fast tip-growing cellular system. Pollen tubes must be precisely oriented inside the anatomically complex female ovary in order to deliver sperm. We hypothesized that NO could play a role in this guidance and tested this hypothesis by challenging the growth of pollen tubes with an external NO point source. When a critical concentration was sensed, the growth rate was reduced and the growth axis underwent a subsequent sharp reorientation, after which normal growth was attained. This response was abrogated in the presence of the NO scavenger CPTIO and affected by drugs interfering in the cGMP signaling pathway. The sensitivity threshold of the response was significantly augmented by sildenafil citrate (SC), an inhibitor of cGMP-specific phosphodiesterases in animals. NO distribution inside pollen tubes was investigated using DAF2-DA and was shown to occur mostly in peroxisomes. Peroxisomes are normally excluded from the tip of pollen tubes and little if any NO is found in the cytosol of that region. Our data indicate that the rate and orientation of pollen tube growth is regulated by NO levels at the pollen tube tip and suggest that this NO function is mediated by cGMP.

Topics: 1-Methyl-3-isobutylxanthine; Benzoates; Cyclic GMP; Dose-Response Relationship, Drug; Flowers; Fluorescein; Imidazoles; Lilium; Nitric Oxide; Peroxisomes; Phosphodiesterase Inhibitors; Piperazines; Purines; Sildenafil Citrate; Sulfones

Nitric oxide (NO) generated by either endothelial nitric oxide synthase (eNOS) or inducible nitric oxide synthase (iNOS) may be involved in prostate tumorigenesis through the inhibition of reactive oxygen species (ROS)-induced apoptosis. Multicellular DU-145 prostate tumor spheroids endogenously generated NO that paralleled the production of ROS. With increasing spheroid size, eNOS expression was downregulated, whereas an upregulation of iNOS expression was observed. In parallel, NO generation declined, as evaluated by the NO indicator diaminofluorescein-2 diacetate (DAF-2DA), suggesting that NO generation in DU-145 tumor spheroids is mainly mediated by eNOS. Elevation of ROS by treatment of tumor spheroids with either buthionine sulfoximine (BSO) or hydrogen peroxide resulted in upregulation of eNOS, whereas iNOS was downregulated. Furthermore, eNOS expression was increased by epidermal growth factor (EGF) in a redox-sensitive manner. Upregulation of eNOS after treatment with hydrogen peroxide was apparently transduced through receptor tyrosine kinase signaling pathways since it was abolished by the protein kinase C (PKC) inhibitor bisindolylmaleimide-1 (BIM-1), the p21(ras) inhibitor S-trans-trans-farnesylthiosalicylic acid (FTS), the c-Raf inhibitor ZM 336372 and PD98059, which inhibits ERK1/2 activation. Endogenous NO may serve to escape from oxidative stress-induced apoptosis since treatment of tumor spheroids with the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl imidazoline-1-oxyl 3-oxide (carboxy-PTIO) as well as the NO synthase inhibitor N-omega-amino-L-arginine (L-NAA) increased cleaved caspase-3. Consequently, lowering intracellular NO levels with either L-NAA or PTIO significantly raised ROS levels, indicating that endogenously generated NO may play a role as a ROS scavenger, thereby protecting exponentially growing tumor spheroids from ROS-induced apoptosis.

Topics: Antioxidants; Apoptosis; Arginine; Benzoates; Buthionine Sulfoximine; Caspase 3; Caspases; Enzyme Inhibitors; Epidermal Growth Factor; Fluorescein; Free Radical Scavengers; Humans; Hydrogen Peroxide; Imidazoles; Immunoenzyme Techniques; Indicators and Reagents; Male; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Prostatic Neoplasms; Reactive Oxygen Species; Signal Transduction; Spheroids, Cellular; Tumor Cells, Cultured; Vitamin E

Nitric oxide (NO) is an important signal transmitter with multiple haemodynamic functions in the kidney. Study of these is complicated by the difficulty in measuring NO directly or visualizing its production. Recently the synthesis of a group of new NO-sensitive fluorescent dyes, diaminofluoresceins (DAF), suitable for imaging applications has been reported. We attempted to use one DAF (DAF-2 DA) to investigate the relationship between endothelial calcium, NO production and afferent arteriolar reactivity.. We used the isolated, perfused juxtamedullary nephron preparation (JMN) and loaded the afferent arteriolar endothelium with Fura-2 AM and DAF-2 DA (4,5-diaminofluorescein-2-diacetyl). After in vitro calibration of the imaging system, we measured Fura-2 and DAF-2 fluorescence in single endothelial cells of afferent arterioles (AA) perfused at a pressure of 100 mmHg.. Carboxy-2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (carboxy-PTIO) (10-3 m), a specific NO scavenger, decreased DAF-2 fluorescence in the endothelium by 16.1% and the mid-afferent arteriolar diameter by 10.2%, and increased endothelial calcium by 17.8%. Nomega-nitro-l-arginine methyl ester (l-NAME) (10-4 m) decreased fluorescence intensity of DAF-2 by 18.6%, increased cellular calcium level by 19.7% and constricted the vessels by 11.6%. Addition of carbachol (10-4 m) increased average DAF-2 fluorescence by 22.8% and endothelial calcium concentration by 28.9%, whereas the arteriolar diameter remained essentially unchanged. Carbachol failed to increase DAF-2 fluorescence when administered after l-NAME pre-treatment.. We conclude that endothelial NO homeostasis is an important determinant of AA reactivity and suggest that DAF are suitable for real-time imaging of afferent arteriolar NO production in the isolated, perfused JMN and may be used in combination with calcium-sensitive fluorophores. We have found that NO reduction by carboxy-PTIO or l-NAME increases endothelial calcium, suggesting involvement of calcium signalling in an autocrine NO production feedback in the endothelium. This method should help to further clarify the role of endothelial NO in renal haemodynamics.

Topics: Animals; Arterioles; Benzoates; Calcium; Carbachol; Endothelium; Enzyme Inhibitors; Fluorescein; Fluorescence; Free Radical Scavengers; Imidazoles; Indicators and Reagents; Kidney Medulla; Male; Nephrons; NG-Nitroarginine Methyl Ester; Nitric Oxide; Rats; Rats, Sprague-Dawley

Topics: Abscisic Acid; Benzoates; Drug Synergism; Fabaceae; Fluorescein; Imidazoles; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Plant Epidermis; Plant Growth Regulators; Signal Transduction