Evidence that nitric oxide synthase is involved in progesterone-induced acrosomal exocytosis in mouse spermatozoa

In a recent work, we detected nitric oxide synthase (NO synthase) in the acrosome and tail of mouse and human spermatozoa by an immunofluorescence technique. Also, NO-synthase inhibitors added during sperm capacitationin vitro reduced the percentage of oocytes fertilized in vitro, suggesting a role for NO synthase in sperm function. Therefore, in the present study the effect of three NO-synthase inhibitors, NG-nitro-L-arginine methyl ester (L-NAME), NG-nitro-D-arginine methyl ester (D-NAME) and L-NG-nitro-arginine (NO2-arg), and of a nitric oxide donor, spermine-NONOate, on the progesterone-induced acrosome reaction of mouse sperm was examined. NO-synthase inhibitors were added at 0, 60 or 90 min during capacitation; at 120 min, mouse epididymal spermatozoa were exposed to 15 µM progesterone for another 15 min. In another set of experiments, different concentrations of spermine-NONOate were added to capacitated spermatozoa for 15 min; in these experiments, progesterone was not included. NO2-arg and L-NAME blocked progesterone-induced exocytosis regardless of the time at which these inhibitors were added. Moreover, D-NAME did not inhibit exocytosis. In contrast, spermine-NONOate stimulated the acrosomal exocytosis in vitro directly. These results provide evidence that mouse sperm NO synthase participates in the progesterone-induced acrosome reactionin vitro and that nitric oxide induces this event.

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Endothelium-dependent vasodilation of cerebral arteries is altered with simulated microgravity through nitric oxide synthase and EDHF mechanisms

Journal of Applied Physiology ◽

10.1152/japplphysiol.00941.2005 ◽

2006 ◽

Vol 101 (1) ◽

pp. 348-353 ◽

Cited By ~ 21

Author(s):

Rhonda D. Prisby ◽

M. Keith Wilkerson ◽

Elke M. Sokoya ◽

Robert M. Bryan ◽

Emily Wilson ◽

...

Keyword(s):

Nitric Oxide ◽

Methyl Ester ◽

Nitric Oxide Synthase ◽

Cerebral Perfusion ◽

Simulated Microgravity ◽

Cerebral Arteries ◽

Caveolin 1 ◽

Fluid Shifts ◽

Oxide Synthase

Cephalic elevations in arterial pressure associated with microgravity and prolonged bed rest alter cerebrovascular autoregulation in humans. Using the head-down tail-suspended (HDT) rat to chronically induce headward fluid shifts and elevate cerebral artery pressure, previous work has likewise shown cerebral perfusion to be diminished. The purpose of this study was to test the hypothesis that 2 wk of HDT reduces cerebral artery vasodilation. To test this hypothesis, dose-response relations for endothelium-dependent (2-methylthioadenosine triphosphate and bradykinin) and endothelium-independent (nitroprusside) vasodilation were determined in vitro in middle cerebral arteries (MCAs) from HDT and control rats. All in vitro measurements were done in the presence and absence of the nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester (10−5 M) and cyclooxygenase inhibitor indomethacin (10−5 M). MCA caveolin-1 protein content was measured by immunoblot analysis. Endothelium-dependent vasodilation to 2-methylthioadenosine triphosphate and bradykinin were both lower in MCAs from HDT rats. These lower vasodilator responses were abolished with NG-nitro-l-arginine methyl ester but were unaffected by indomethacin. In addition, HDT was associated with lower levels of MCA caveolin-1 protein. Endothelium-independent vasodilation was not altered by HDT. These results indicate that chronic cephalic fluid shifts diminish endothelium-dependent vasodilation through alterations in the endothelial nitric oxide synthase signaling mechanism. Such decrements in endothelium-dependent vasodilation of cerebral arteries could contribute to the elevations in cerebral vascular resistance and reductions in cerebral perfusion that occur after conditions of simulated microgravity in HDT rats.

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Role of nitric oxide on in vitro human eosinophil migration11Abbreviations: AMT, 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine; fMLP, N-formyl-methionyl-leucyl-phenylalanine; cGMP, db-cGMP, dibutyryl cyclic GMP; d-NAME, Nω-nitro-d-arginine methyl ester; l-NAME, Nω-nitro-l-arginine methyl ester; l-NIL, l-N6-(1-iminoethyl) lysine; NO, nitric oxide; NOS, nitric oxide synthase; ODQ, 1H-[1,2,4]-oxidiazolo[4,3-a] quinoxalin-1-one; and TRIM, 1-(2-trifluoromethylphenyl) imidazole.

Biochemical Pharmacology ◽

10.1016/s0006-2952(01)00782-1 ◽

2001 ◽

Vol 62 (10) ◽

pp. 1417-1421 ◽

Cited By ~ 26

Author(s):

Sara M. Thomazzi ◽

Heloisa H.A. Ferreira ◽

Nicola Conran ◽

Gilberto De Nucci ◽

Edson Antunes

Keyword(s):

Nitric Oxide ◽

Methyl Ester ◽

Nitric Oxide Synthase ◽

Cyclic Gmp ◽

Human Eosinophil ◽

Oxide Synthase

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Dilation of rat diaphragmatic arterioles by flow and hypoxia: roles of nitric oxide and prostaglandins

Journal of Applied Physiology ◽

10.1152/jappl.1999.86.5.1644 ◽

1999 ◽

Vol 86 (5) ◽

pp. 1644-1650 ◽

Cited By ~ 18

Author(s):

Michael E. Ward

Keyword(s):

Nitric Oxide ◽

Methyl Ester ◽

Nitric Oxide Synthase ◽

Concurrent Treatment ◽

Nitric Oxide Release ◽

Luminal Flow ◽

Hypoxic Vasodilation ◽

Oxide Synthase ◽

Endothelial Nitric Oxide

The in vitro responses to ACh, flow, and hypoxia were studied in arterioles isolated from the diaphragms of rats. The endothelium was removed in some vessels by low-pressure air perfusion. In endothelium-intact arterioles, pressurized to 70 mmHg in the absence of luminal flow, ACh (10−5 M) elicited dilation (from 103 ± 10 to 156 ± 13 μm). The response to ACh was eliminated by endothelial ablation and by the nitric oxide synthase antagonists N G-nitro-l-arginine (l-NNA; 10−5 M) and N G-nitro-l-arginine methyl ester (l-NAME, 10−5 M) but not by indomethacin (10−5 M). Increases in luminal flow (5–35 μl/min in 5 μl/min steps) at constant distending pressure (70 mmHg) elicited dilation (from 98 ± 8 to 159 ± 12 μm) in endothelium-intact arterioles. The response to flow was partially inhibited byl-NNA,l-NAME, and indomethacin and eliminated by endothelial ablation and by concurrent treatment withl-NAME and indomethacin. The response to hypoxia was determined by reducing the periarteriolar[Formula: see text] from 100 to 25–30 Torr by changing the composition of the gas used to bubble the superfusing solution. Hypoxia elicited dilation (from 110 ± 9 to 165 ± 12 μm) in endothelium-intact arterioles but not in arterioles from which the endothelium had been removed. Hypoxic vasodilation was eliminated by treatment with indomethacin and was not affected byl-NAME orl-NNA. In rat diaphragmatic arterioles, the response to ACh is dependent on endothelial nitric oxide release, whereas the response to hypoxia is mediated by endothelium-derived prostaglandins. Flow-dilation requires that both nitric oxide and cyclooxygenase pathways be intact.

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Expression of the inducible nitric oxide synthase gene in diaphragm and skeletal muscle

Journal of Applied Physiology ◽

10.1152/jappl.1996.81.6.2415 ◽

1996 ◽

Vol 81 (6) ◽

pp. 2415-2420 ◽

Cited By ~ 42

Author(s):

Marita Thompson ◽

Lisa Becker ◽

Debbie Bryant ◽

Gary Williams ◽

Daniel Levin ◽

...

Keyword(s):

Nitric Oxide ◽

Skeletal Muscle ◽

Nitric Oxide Synthase ◽

Inducible Nitric Oxide Synthase ◽

No Synthase ◽

Inos Mrna ◽

Pump Failure ◽

Oxide Synthase ◽

Inducible Nitric Oxide

Thompson, Marita, Lisa Becker, Debbie Bryant, Gary Williams, Daniel Levin, Linda Margraf, and Brett P. Giroir. Expression of the inducible nitric oxide synthase gene in diaphragm and skeletal muscle. J. Appl. Physiol. 81(6): 2415–2420, 1996.—Nitric oxide (NO) is a pluripotent molecule that can be secreted by skeletal muscle through the activity of the neuronal constitutive isoform of NO synthase. To determine whether skeletal muscle and diaphragm might also express the macrophage-inducible form of NO synthase (iNOS) during provocative states, we examined tissue from mice at serial times after intravenous administration of Escherichia coli endotoxin. In these studies, iNOS mRNA was strongly expressed in the diaphragm and skeletal muscle of mice 4 h after intravenous endotoxin and was significantly diminished by 8 h after challenge. Induction of iNOS mRNA was followed by expression of iNOS immunoreactive protein on Western immunoblots. Increased iNOS activity was demonstrated by conversion of arginine to citrulline. Immunochemical analysis of diaphragmatic explants exposed to endotoxin in vitro revealed specific iNOS staining in myocytes, in addition to macrophages and endothelium. These results may be important in understanding the pathogenesis of respiratory pump failure during septic shock, as well as skeletal muscle injury during inflammation or metabolic stress.

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Estrogen reduces mouse cerebral artery tone through endothelial NOS- and cyclooxygenase-dependent mechanisms

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2000.279.2.h511 ◽

2000 ◽

Vol 279 (2) ◽

pp. H511-H519 ◽

Cited By ~ 104

Author(s):

Greg G. Geary ◽

Diana N. Krause ◽

Sue P. Duckles

Keyword(s):

Nitric Oxide ◽

Methyl Ester ◽

Nitric Oxide Synthase ◽

Cerebral Artery ◽

Cerebral Arteries ◽

Estrogen Replacement ◽

Luminal Diameter ◽

Small Resistance ◽

Oxide Synthase

Gender and estrogen status are known to influence the incidence and severity of cerebrovascular disease. The vasoprotective effects of estrogen are thought to include both nitric oxide-dependent and independent mechanisms. Therefore, using small, resistance-sized arteries pressurized in vitro, the present study determined the effect of gender and estrogen status on myogenic reactivity of mouse cerebral arteries. Luminal diameter was measured in middle cerebral artery segments from males and from females that were either untreated, ovariectomized (OVX), or OVX with estrogen replacement (OVX + EST). The maximal passive diameters of arteries from all four groups were similar. In response to increases in transmural pressure, diameters of arteries from males and OVX females were smaller compared with diameters of arteries from either untreated or OVX + EST females. In the presence of N G-nitro-l-arginine methyl ester, artery diameters decreased in all groups, but diameters remained significantly smaller in arteries from males and OVX females compared with untreated and OVX + EST females. After endothelium removal or when inhibition of nitric oxide synthase and cyclooxygenase were combined, differences in diameters of arteries from OVX and OVX + EST were abolished. These data suggest that chronic estrogen treatment modulates myogenic reactivity of mouse cerebral arteries through both endothelium-derived cyclooxygenase- and nitric oxide synthase-dependent mechanisms.

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Erhöhte NF-κB- und iNOS-Expression in Keratozyten von Keratokonuspatienten – Hinweise auf eine entzündliche Komponente?

Klinische Monatsblätter für Augenheilkunde ◽

10.1055/a-1002-0100 ◽

2019 ◽

Cited By ~ 2

Author(s):

Tanja Stachon ◽

Lorenz Latta ◽

Krasimir Kolev ◽

Berthold Seitz ◽

Achim Langenbucher ◽

...

Keyword(s):

Nitric Oxide ◽

Nitric Oxide Synthase ◽

Nuclear Factor Kappa B ◽

No Synthase ◽

Inos Expression ◽

Nuclear Factor ◽

Nos Inhibitors ◽

Inos Inhibitor ◽

Oxide Synthase

Zusammenfassung Hintergrund In den letzten Jahren mehren sich Hinweise auf eine entzündliche Komponente beim Keratokonus (KC). Ein Schlüsselgen bei entzündlichen Prozessen ist der Nuclear Factor Kappa B (NF-κB). NF-κB ist ein Transkriptionsfaktor, der unter anderem das Enzym Nitric Oxide Synthase (NOS), das mit dem konkurrierenden Enzym Arginase (Arg) bei entzündlichen Prozessen involviert ist, aktiviert. Ziel dieser Studie war es, die Isotypen von NOS und Arginase zu analysieren, die Expression NF-κB, NOS und Arginase sowie den regulativen Mechanismus von NOS und Arginase in Keratozyten von Keratokonuspatienten mithilfe des Inhibitors 1400W in vitro zu untersuchen. Methoden Primäre humane Keratozyten wurden durch enzymatische Behandlung mit Kollagenase A aus humanen Korneoskleralscheiben (n = 8) und von Explantaten von geplanten perforierenden Keratoplastiken (KC-Patienten) isoliert (n = 8) und in DMEM/F12-Kulturmedium, versetzt mit 5% fetalem Kälberserum, kultiviert. Die Expression von NF-κB, NOS und Arginase wurden mit quantitativer PCR (qPCR) und Westernblot-Analyse (WB) untersucht. Nitrit- und Ureakonzentrationen im Zellkulturüberstand wurden nach Zugabe des NOS-Inhibitors 1400W (0 – 40 µM) analysiert. Ergebnisse In den Keratozyten wurden ausschließlich die Isotypen iNOS (induzierbare NO-Synthase) und Arg-II nachgewiesen. Die mRNA-Expression von NF-κB und iNOS waren in KC-Keratozyten höher als in normalen Zellen (p = 0,0135 und p = 0,0001), während in der Arg-II-Expression keine Unterschiede messbar waren. Im WB war bei NF-κB eine höhere Bandenintensität messbar (p = 0,0012), bei iNOS konnten keine Unterschiede in der Bandenintensität nachgewiesen werden. Im Überstand der KC-Keratozyten wurden geringere Konzentrationen von Nitrit und Urea nach Zugabe des Inhibitors 1400W gemessen (p = ≤ 0,014), nicht jedoch bei normalen Zellen (p ≥ 0,178). Schlussfolgerung Aufgrund der erhöhten Expression von NF-κB und iNOS muss von einer inflammatorischen Komponente beim Keratokonus ausgegangen werden. Die unterschiedliche Regulation der KC-Keratozyten durch den iNOS-Inhibitor 1400W legt eine veränderte metabolische Aktivität nahe, die durch entzündliche Prozesse hervorgerufen werden kann.

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