scholarly journals Expression and localization of nitric oxide synthase isoforms during porcine oocyte growth and acquisition of meiotic competence

2009 ◽  
Vol 54 (No. 4) ◽  
pp. 137-149 ◽  
Author(s):  
E. Chmelíková ◽  
M. Sedmíková ◽  
J. Petr ◽  
T. Kott ◽  
V. Lánská ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Intracellular Localization ◽  
Growth Period ◽  
Fluorescence Signal ◽  
Oocyte Growth ◽  
Porcine Oocyte ◽  
Oxide Synthase ◽  
Nos Isoforms ◽  
Meiotic Competence

Reproduction biotechnologies depend on the use of fully meiotically competent oocytes. Growing oocytes without full meiotic competence are an interesting potential source due to their quantity, but the mechanisms regulating the processes of acquisition of meiotic competence have not been clarified to date. Nitric oxide synthase (NOS) and its product, nitric oxide (NO), may possibly play a role. Understanding the precise NO regulatory mechanism is therefore important for the development of <I>in vitro</I> growth methods. The objective of this work was to detect changes in the expression of NOS isoforms and their mRNA expression and changes in the intracellular localization of separate NOS isoforms during the growth period of the porcine oocyte, and also to determine whether these changes are related to the process of meiotic competence acquisition. mRNA for all NOS isoforms was already detected in oocytes at the beginning of their growth and was present in them until they completed their growth period. mRNA for iNOS and eNOS was also observed in granulosa and cumulus cells from these oocytes. But nNOS mRNA was not demonstrated in these types of cells. Pig oocytes and their surrounding cells contained all NOS proteins. Their amounts increased and localization changed with the acquisition of meiotic competence. nNOS was localized mainly in the cortex in meiotically incompetent oocytes, while meiotically competent oocytes contained nNOS in the nucleus as well. iNOS protein was distributed in the cytoplasm and nucleus in all oocytes, and meiotically incompetent oocytes contained iNOS in the nucleolus as well. eNOS protein was distributed in oocytes in the form of fine granules with a strong fluorescence signal. Protein was concentrated in the nuclear area in meiotically incompetent oocytes and also in the periphery in oocytes with partially and fully-developed meiotic competence. All these findings indicate that NOS isoforms may significantly influence the acquisition of meiotic competence in porcine oocytes.

Nitric oxide synthase isoforms and the effect of their inhibition on meiotic maturation of porcine oocytes

Zygote ◽  
2010 ◽  
Vol 18 (3) ◽  
pp. 235-244 ◽  
Author(s):  
Eva Chmelíková ◽  
Michal Ješeta ◽  
Markéta Sedmíková ◽  
Jaroslav Petr ◽  
Lenka Tůmová ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Intracellular Localization ◽  
Cumulus Cells ◽  
Meiotic Maturation ◽  
Control Group ◽  
Oocyte Cytoplasm ◽  
Oxide Synthase ◽  
Nos Isoforms ◽  
Experimental Group

SummaryIn this paper we assessed: (i) the change in nitric oxide synthase (NOS) isoforms' expression and intracellular localization and in NOS mRNA in porcine oocytes during meiotic maturation; (ii) the effect of NOS inhibition by Nω-nitro-l-arginine methyl ester (l-NAME) and aminoguanidine (AG) on meiotic maturation of cumulus–oocyte complexes (COC) as well as denuded oocytes (DO); and (iii) nitric oxide (NO) formation in COC. All three NOS isoforms (eNOS, iNOS and nNOS) and NOS mRNA (eNOS mRNA, iNOS mRNA and nNOS mRNA) were found in both porcine oocytes and their cumulus cells except for nNOS mRNA, which was not detected in the cumulus cells. NOS isoforms differed in their intracellular localization in the oocyte: while iNOS protein was dispersed in the oocyte cytoplasm, nNOS was localized in the oocyte cytoplasm and in germinal vesicles (GV) and eNOS was present in dots in the cytoplasm, GV and was associated with meiotic spindles. l-NAME inhibitor significantly suppressed metaphase (M)I to MII transition (5.0 mM experimental group: 34.9% MI, control group: 9.5% MI) and at the highest concentration (10.0 mM) also affected GV breakdown (GVBD); in contrast also AG inhibited primarily GVBD. The majority of the oocytes (10.0 mM experimental group: 60.8%, control group: 1.2%) was not able to resume meiosis. AG significantly inhibited GVBD in DO, but l-NAME had no significant effect on the GVBD of these cells. During meiotic maturation, NO is formed in COC and the NO formed by cumulus cells is necessary for the process of GVBD.

Selected Contribution: Differential role of nitric oxide synthase isoforms in fever of different etiologies: studies using Nosgene-deficient mice

2003 ◽  
Vol 94 (6) ◽  
pp. 2534-2544 ◽  
Author(s):  
Wieslaw Kozak ◽  
Anna Kozak
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Corn Oil ◽  
Normal Male ◽  
Wild Type ◽  
Oxide Synthase ◽  
And Control ◽  
Nos Isoforms ◽  
Deficient Mice

Male C57BL/6J mice deficient in nitric oxide synthase (NOS) genes (knockout) and control (wild-type) mice were implanted intra-abdominally with battery-operated miniature biotelemeters (model VMFH MiniMitter, Sunriver, OR) to monitor changes in body temperature. Intravenous injection of lipopolysaccharide (LPS; 50 μg/kg) was used to trigger fever in response to systemic inflammation in mice. To induce a febrile response to localized inflammation, the mice were injected subcutaneously with pure turpentine oil (30 μl/animal) into the left hindlimb. Oral administration (gavage) of N G-monomethyl-l-arginine (l-NMMA) for 3 days (80 mg · kg−1 · day−1in corn oil) before injection of pyrogens was used to inhibit all three NOSs ( N G-monomethyl-d-arginine acetate salt and corn oil were used as control). In normal male C57BL/6J mice, l-NMMA inhibited the LPS-induced fever by ∼60%, whereas it augmented fever by ∼65% in mice injected with turpentine. Challenging the respective NOS knockout mice with LPS and with l-NMMA revealed that inducible NOS and neuronal NOS isoforms are responsible for the induction of fever to LPS, whereas endothelial NOS (eNOS) is not involved. In contrast, none of the NOS isoforms appeared to trigger fever to turpentine. Inhibition of eNOS, however, exacerbates fever in mice treated with l-NMMA and turpentine, indicating that eNOS participates in the antipyretic mechanism. These data support the hypothesis that nitric oxide is a regulator of fever. Its action differs, however, depending on the pyrogen used and the NOS isoform.

Expression of nitric oxide synthase isoforms in normal ventilatory and limb muscles

1997 ◽  
Vol 83 (2) ◽  
pp. 348-353 ◽  
Author(s):  
Sabah N. A. Hussain ◽  
Qasim El-Dwairi ◽  
Mohammed N. Abdul-Hussain ◽  
Dalia Sakkal
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Fiber Type ◽  
Nos Inhibitors ◽  
Limb Muscles ◽  
Fiber Type Distribution ◽  
Neuronal Nos ◽  
Weak Expression ◽  
Oxide Synthase ◽  
Nos Isoforms

Hussain, Sabah N. A., Qasim El-Dwairi, Mohammed N. Abdul-Hussain, and Dalia Sakkal. Expression of nitric oxide synthase isoforms in normal ventilatory and limb muscles. J. Appl. Physiol. 83(2): 348–353, 1997.—Nitric oxide (NO), an important messenger molecule with widespread actions, is synthesized by NO synthases (NOS). In this study, we investigated the correlation between fiber type and NOS activity among ventilatory and limb muscles of various species. We also assessed the presence of the three NOS isoforms in normal skeletal muscles and how various NOS inhibitors influence muscle NOS activity. NOS activity was detected in various muscles; however, NOS activity in rabbits and rats varied significantly among different muscles. Immunoblotting of muscle samples indicated the presence of both the neuronal NOS and the endothelial NOS isoforms but not the cytokine-inducible NOS isoform. However, these isoforms were expressed to different degrees in various muscles. Although the neuronal NOS isoform was detectable in the canine diaphragm, very weak expression was detected in rabbit, rat, and mouse diaphragms. The endothelial NOS isoform was detected in the rat and mouse diaphragms but not in the canine and rabbit diaphragms. We also found that N G-nitro-l-arginine methyl ester, 7-nitroindazole, and S-methylisothiourea were stronger inhibitors of muscle NOS activity than was aminoguanidine. These results indicate the presence of different degrees of constitutive NOS expression in normal ventilatory and limb muscles of various species. Our data also indicate that muscle NOS activity is not determined by fiber type distribution but by other not yet identified factors. The functional significance of this expression remains to be assessed.

Fire Ant Venom Alkaloid, Isosolenopsin A, a Potent and Selective Inhibitor of Neuronal Nitric Oxide Synthase

2003 ◽  
Vol 22 (2) ◽  
pp. 81-86 ◽  
Author(s):  
G. B. Yi ◽  
D. Mc Clendon ◽  
D. Desaiah ◽  
J. Goddard ◽  
A. Lister ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Inhibitory Activity ◽  
Systemic Toxicity ◽  
Fire Ant ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Nos Inhibitors ◽  
Oxide Synthase ◽  
Nos Isoforms

Massive, multiple fire ant, Solenopsis invicta, stings are often treated aggressively, particularly in the elderly, despite limited evidence of systemic toxicity due to the venom. Over 95% of the S. invicta venom is composed of piperidine alkaloid components, whose toxicity, if any, is unknown. To assess a possible pharmacological basis for systemic toxicity, an alkaloid-rich, protein-free methanol extract of the venom from whole ants was assayed for inhibitory activity on the following nitric oxide synthase (NOS) isoforms, rat cerebellar neuronal (n NOS), bovine recombinant endothelial (e NOS), and murine recombinant immunologic (i NOS). Cytosolic NOS activity was determined by measuring the conversion of [3H]arginine to [3H]citrulline in vitro. Rat n NOS activity was inhibited significantly and in a concentration-dependent manner by the alkaloid-rich venom extract. For n NOS, enzyme activity was inhibited by approximately 50% with 0.33 ± 0.06 μgg of this venom extract, and over 95% inhibition of the three isoforms, n NOS, e NOS, and i NOS, was found with doses of 60 μg in 60-μl reaction mixture. These results indicate that the alkaloid components of S. invicta venom can produce potent inhibition of all three major NOS isoforms. Isosolenopsin A ( cis-2-methyl-6-undecylpiperidine), a naturally occurring fire ant piperidine alkaloid, was synthesized and tested for inhibitory activity against the three NOS isoforms. Enzyme activities for n NOS and e NOS were over 95% inhibited with 1000 μM of isosolenopsin A, whereas the activity of i NOS was inhibited by only about 20% at the same concentration. The IC50 for each of three NOS isoforms was approximately 18 ± 3.9 μM for n NOS, 156 ± 10 μM for e NOS, and >1000 μM for i NOS, respectively. Kinetic studies showed isosolenopsin A inhibition to be noncompetitive with L-arginine ( Ki = 19 ± 2 μM). The potency of isosolenopsin A as an inhibitor of n NOS compares favorably with the inhibitory potency of widely used n NOS inhibitors. Inhibition of NOS isoforms by isosolenopsin A and structurally similar compounds may have toxicological significance with respect to adverse reactions to fire ant stings.

Expression of nitric oxide synthase isoforms and detection of nitric oxide in rat placenta

2002 ◽  
Vol 282 (4) ◽  
pp. C762-C767 ◽  
Author(s):  
Tatsuya Takizawa ◽  
Hiroshi Yoshikawa ◽  
Miho Yamada ◽  
Hidetoshi Morita
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Pcr Analysis ◽  
No Production ◽  
Epr Spectrum ◽  
Pregnant Rats ◽  
Paramagnetic Resonance ◽  
Triplet Signal ◽  
Oxide Synthase ◽  
Nos Isoforms

Nitric oxide (NO) production in the rat placenta was monitored and quantified by electron paramagnetic resonance (EPR) spectroscopy with hemoglobin and an Fe- N-(dithiocarboxy)sarcosine (DTCS) complex as NO-trapping reagents. Expression of nitric oxide synthase (NOS) isoforms was also examined by quantitative RT-PCR analysis. The EPR spectrum of the placenta with hemoglobin trapping showed a three-line hyperfine structure ( g = 2.008 and a = 1.66-mT). The EPR signal was diminished after the placenta was homogenized or the NOS inhibitor l-NAME was administered to pregnant rats. Therefore, the specific signal was definitely identified as being derived from endogenous NO spin-trapped by hemoglobin, and the EPR spectrum showed that the NO adduct existed as a pentacoordinate α-NO heme species. The EPR spectrum of the placenta with Fe-DTCS trapping showed a triplet signal ( g = 2.038) derived from an NO-Fe-DTCS complex. The height of the triplet signal did not vary significantly with gestational stage during the last few days of gestation. At the gestational stages examined, the level of NOS II mRNA expression was significantly higher than that of NOS III mRNA. NOS II expression in term ( day 21.5) placenta was significantly increased compared with that in preterm ( day 19.5) placenta ( P < 0.01, n = 4 or 5). These results suggest that NOS II is the predominant producer of NO in the placenta and that NOS II-generated NO plays significant roles in the maintenance of placental functions immediately before birth.

Attenuation of nitric oxide synthase isoform expression by mild hypothermia after focal cerebral ischemia: variations depending on timing of cooling

2003 ◽  
Vol 98 (6) ◽  
pp. 1271-1276 ◽  
Author(s):  
Murat Karabiyikoglu ◽  
Hyung Soo Han ◽  
Midori A. Yenari ◽  
Gary K. Steinberg
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Mild Hypothermia ◽  
Inos Expression ◽  
Western Blots ◽  
Neuronal Marker ◽  
Content Type ◽  
Oxide Synthase ◽  
Nos Isoforms ◽  
Fine Print

Object. In this study the authors examined the influence of mild hypothermia on early expression of nitric oxide synthase (NOS) isoforms and peroxynitrite generation after experimental stroke. Methods. In 82 male Sprague—Dawley rats, middle cerebral artery occlusion was performed for 2 hours by using the intraluminal suture model. The rats were maintained at their normal body temperature or exposed to 2 hours of intraischemic or postischemic (2-hour delay) mild hypothermia. Brains were collected 2, 6, and 24 hours after onset of ischemia for immunohistochemical and Western blot analysis of neuronal (n)NOS and inducible (i)NOS expression and peroxynitrite generation. Conclusions. Western blots showed significantly increased nNOS and iNOS expression in the ischemic cortex at 2, 6, and 24 hours compared with sham-operated animals. The NOS expression was highest at 24 hours. Postischemic hypothermia attenuated nNOS expression at 6 and 24 hours to a greater extent than intraischemic hypothermia. Intraischemic hypothermia reduced iNOS expression at both 2 and 24 hours, whereas postischemic hypothermia decreased iNOS expression at 24 hours. Results of immunohistochemical studies showed that nNOS colocalized with the neuronal marker MAP-2 at all time points, whereas iNOS was initially localized to vessels, and then localized to activated microglia by 24 hours. Intraischemic but not postischemic hypothermia decreased the number of nitrotyrosine-positive cells in the ischemic cortex at 24 hours. Mild hypothermia significantly but differentially attenuates increases in NOS isoforms, with more robust nNOS suppression when cooling is delayed. This may have important implications for understanding the mechanism of hypothermic neuroprotection and for stroke therapy.

scholarly journals Expression of Constitutive Nitric Oxide Synthase Isoforms in Varicose Vein Wall; Preliminary Results

2011 ◽  
Vol 2011 ◽  
pp. 1-6 ◽  
Author(s):  
Zora Haviarová ◽  
Andrea Janegová ◽  
Pavel Janega ◽  
Štefan Durdík ◽  
Peter Kováč ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Varicose Vein ◽  
Structural Changes ◽  
Varicose Veins ◽  
Control Group ◽  
Color Analysis ◽  
Vein Wall ◽  
Oxide Synthase ◽  
Nos Isoforms

There are conflicting findings in literature about the structural changes of the primary varicose veins. NO (a potent vasodilatator) is synthesized by nitric oxide synthase (NOS). From 3 known NOS isoforms the two are constitutional: eNOS (endothelial NOS) and nNOS (neuronal NOS). 10 varicose and 10 control vein samples were processed by standard light microscopy and immuno-histochemica techniques using rabbit polyclonal antibodies against eNOS and nNOS. Antibodies expression was evaluated semiquantitatively and proved morphometrically by 2D-image analysis. total area of NOS isoforms expressions was determined by color analysis and color digital subtraction. The results showed discontinuous and significantly lower expression of both NOS isoforms the in the tunica media of varicose veins compared with the control group. For the statistical analysis the unpaired -test was used. Our results suppose lower NO levels in varicose vein wall, deducing that varicose dilatation is due to other mechanism, and they contradict the results of previously published similar works.

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