• 대한수의학회지
• /
• 제43권4호
• /
• pp.551-554
• /
• 2003

Nitric oxide synthase (NOS) has been reported in uterus. We examined the expression of the NOS isoforms, constitutive endothelial (eNOS) and inducible NOS (iNOS), in bovine uterus by immunohistochemistry. eNOS immunoreactivity was localized predominantly to the endothelial cells that line uterine microvessels and to endometrial glandular epithelial cells, but was barely detectable in endometrial stromal cells. iNOS immunostaining was detected in glandular epithelial and stromal cells in the endometrium and in the endothelial cells of myometrial blood vessels. These findings suggest that both eNOS and iNOS may play important roles in the physiology of the uterus, possibly by generating NO.

• Toxicological Research
• /
• 제24권3호
• /
• pp.169-174
• /
• 2008

This study examined the mechanisms underlying the effects of the vasorelaxation active substance(VAS), dimethyladenosine-5'-L-arabinose, and its partial purification fraction on nitric oxide synthase in improving erectile dysfunction with particular focus on the nitric oxide (NO)-cGMP pathways. Two rat models, 9-month-old SD rats and 11-month-old SD rats, were given VAS(40 mg/kg per day) for 4 days, The aqueous fraction of silworm male pupae extract; semi-purified VAS(100 mg/kg per day) for 10 days, respectively. The NOS activities of the following three enzymes were examined: neuronal NO synthase(nNOS), inducible NOS(iNOS), endothelial NOS(eNOS), vascular endothelial growth factor on endothelial cells(VEGF) and anti-inflammation effect of Tumor necrosis factor-$\alpha$. The results showed increases in the nitric oxide synthase activities. Western blotting of the tissue homogenate showed an increase in the nNOS level in the brain and tongue, and an increase in the endothelial NO synthase(eNOS) level in penis. However, there was little association with VEGF production in HUVEC endothelial cells and no relationship with TNF-$\alpha$ which showed low levels.

• Journal of Applied Biological Chemistry
• /
• 제48권3호
• /
• pp.120-126
• /
• 2005

Endothelial nitric oxide synthase (eNOS) plays a critical role in vascular biology and pathophysiology. Its activity is regulated by multiple mechanisms such as calcium/calmodulin, protein-protein interactions, sub-cellular locations and phosphorylation at various sites. Phosphorylation of eNOS-Ser1177 (based on mouse sequence) has been identified as an important mechanism of eNOS activation. However, signaling pathway leading to it phosphorylation remains controversial. The regulation of eNOS-Ser1177 phosphorylation by Src and protein kinase A (PKA) was investigated in the present study using cultured mouse aorta endothelial cells. Expression of a constitutively active Src mutant in the cells enhanced phosphorylation of eNOS and protein kinase B (Akt). The Src-stimulated phosphorylation was not attenuated by the expression of a dominant negative PKA regulatory subunit. Neither activation nor inhibition of PKA activity had any significant effect on tyrosine phosphorylation of activation or inactivation site in Src. Based on the results of this study, it is suggested that Src/Akt pathway and PKA signaling may regulate eNOS phosphorylation independently. The existence of multiple mechanisms for eNOS phosphorylation may guarantee endothelial nitric oxide production in various cellular contexts which is essential for maintenance of vascular health.

• 대한의생명과학회지
• /
• 제19권3호
• /
• pp.180-187
• /
• 2013

Hypoxia is an integral part of the environment during luteolysis. In this study we examined whether hypoxia could directly stimulate endothelial cells to produce nitric oxide (NO). Endothelial cells were cultured in hypoxic (5% $O_2$) or normoxic (20% $O_2$) conditions and the levels of total NO, inducible NO and endothelial NO was measured. We found that hypoxia but not normoxia upregulated NO production. The increased NO levels correlated with increased inducible NO synthase (iNOS) expression whereas expression of endothelial NOS (eNOS) expression remained constant. Addition of the iNOS specific inhibitor 1400W to hypoxic cultures prevented NO production suggesting that hypoxia-induced NO production in endothelial cells was due mainly to upregulation of iNOS. We also found that prostaglandin $F_{2{\alpha}}$ (PGF) production was unaffected by hypoxia suggesting that upregulation of NO was not due to increased synthesis of PGF. In summary, we report that endothelial cells cultured under hypoxic conditions produce NO via the iNOS pathway. This study provides the importance of the relation between the hypoxic environment and the induction of NO by endothelial cells during regression of the corpus luteum in the ovary.

• Journal of Ginseng Research
• /
• 제37권1호
• /
• pp.64-73
• /
• 2013

Korean red ginseng water extract (KG-WE) has known beneficial effects on the cardiovascular system via inducting nitric oxide (NO) production in endothelium. Endothelial arginase inhibits the activity of endothelial nitric oxide synthase (eNOS) by substrate depletion, thereby reducing NO bioavailability and contributing to vascular diseases including hypertension, aging, and atherosclerosis. In the present study, we demonstrate that KG-WE inhibits arginase activity and negatively regulates NO production and reactive oxygen species generation in endothelium. This is associated with increased dimerization of eNOS without affecting the protein expression levels of either arginase or eNOS. In a vascular tension assay, when aortas isolated from wild type mice were incubated with KG-WE, NO-dependent enhanced vasorelaxation was observed. Furthermore, KG-WE administered via by drinking water to atherogenic model mice being fed high cholesterol diet improved impaired vascular function. Taken together, these results suggest that KG-WE may exert vasoprotective effects through augmentation of NO signaling by inhibiting arginase. Therefore, KG-WE may be useful in the treatment of vascular diseases derived from endothelial dysfunction, such as atherosclerosis.

• Environmental Analysis Health and Toxicology
• /
• 제29권
• /
• pp.10.1-10.6
• /
• 2014

Objectives Cigarette smoking had been recorded as the main cause of impaired endothelium-dependent vasodilation in smokers by reducing nitric oxide (NO), a production of endothelial nitric oxide synthase (eNOS). However, the mechanism of NO impairment via eNOS activity is unclear until now. In this study, cell passage is suggested to be a relevant factor to eNOS expression under cigarette smoking stress. Methods Bovine aortic endothelial cells (BAECs) were chosen as the research subject with passages ranking from 6 to 9 (6P to 9P). After exposure of cigarette smoking extract (CSE) solution, MTT assay and Western blot method were performed to check the cell viability as well as eNOS protein concentration. In these experiments, four concentrations of CSE at 0.5, 1, 2, and 4% were selected for treatment. Results Our results showed that cells almost died at 4% of CSE. Besides, eNOS protein mass had a linear decrease under the increase of CSE concentration. In addition, the effect of CSE on eNOS expression was dissimilar between different passages. Conclusions This study indicated that CSE had effect on both cell viability and eNOS expression. Besides, a reduction in protein mass was matched with the decrease of cell viability due to CSE tress. Last but not least, the response of eNOS protein to different concentration of CSE at different passages was disparate, making the hypothesis about cell passage related inhibition of eNOS caused by CSE solution.

• Journal of Applied Biological Chemistry
• /
• 제48권4호
• /
• pp.178-182
• /
• 2005

Nitric oxide (NO) produced from endothelial cells plays a critical role in vascular physiology. The regulation of endothelial NO synthase (eNOS) involves various mechanisms including multiple Ser/Thr phosphorylations. Recently, eNOS-Ser617 was newly recognized to be phosphorylated in response to humoral factors including vascular endothelial growth factor. However, it remains unknown whether and how eNOS-Ser617 phosphorylation is stimulated by shear stress, the primary stimulus of endothelial NO production. This issue was explored in the present study using cultured bovine aortic endothelial cells (BAECs). Over-expression of a constitutively active protein kinase B(Akt) mutant in BAECs increased Ser617 phosphorylation while constitutively active protein kinase A mutant had no effect. When BAECs were subjected to an arterial level of laminar shear stress, eNOS-Ser617 phosphorylation was clearly increased in a time-dependent manner. Shear stress also stimulated Akt phosphorylation at Thr308, one of the key regulatory sites. The time courses of eNOS-Ser617 and Akt-Thr308 phosphorylations appeared to be very similar. These results suggested that eNOS-Ser617 phosphorylation, mediated by Akt, is a physiological response to the mechanical shear stress, involved in the regulation of NO production in endothelial cells.

• The Korean Journal of Physiology and Pharmacology
• /
• 제10권3호
• /
• pp.155-159
• /
• 2006

Among the Shc proteins, p66shc is known to be related to oxidative stress responses and regulation of the production of reactive oxygen species (ROS). The present study was undertaken to investigate the role of p66shc on endothelial nitric oxide synthase (eNOS) activity in the mouse embryonic fibroblasts (MEFs). When wild type (WT) or p66shc (-/-) MEFs were transfected with full length of eNOS cDNA, the expression and activity of eNOS protein were higher in the p66shc (-/-) MEFs. These phenomena were reversed by reconstitution of p66shc cDNA transfection in the p66shc (-/-) MEFs. The basal superoxide production in the p66shc (-/-) MEFs was not significantly different from that of WT of MEFs. However, superoxide production induced by NADPH in the p66shc (-/-) MEF was lesser than that in WT MEFs. When compared with WT MEFs, cell lysate of p66shc (-/-) MEFs showed significantly increased H-ras activity without change of endogenous H-ras expression. Our findings suggest the pivotal role of p66shc adaptor protein played in inhibition of endothelial nitric oxide production via modulation of the expression and/or activity of eNOS protein.

• 동의생리병리학회지
• /
• 제20권3호
• /
• pp.680-684
• /
• 2006

This study was designed to investigate effects of KH-304 in improving erectile dysfunction (ED), particularly in terms of nitric oxide (NO)-cGMP pathways. After oral administration of the KH-304 water extract, 1OOmg, 300mg, 500mg or 700mg per 1 kg of Dody weigh for 10days, We examined the expression and activity of two enzyme: neuronal NO synthase (nNOS), endothelial NO synthase (eNOS) and that act upon the major NO-cGMP signaling pathway in penile tissue. Effect of KH-304 on COMP degradation was also examined using bovine vascular smooth muscle cells pretreated with an NO donor, S-nitroso-N-Acetylpenicillamine (SNAP), Also, it examined the endothelial NO synthase (eNOS) for seaching effecting period (100mg, 300mg/kg for 10 and 30days) and peak intracavernous pressures (ICPS) in penile tissues rabbit copus cavernosum contracted by 10-6 M phenylephrine. The severely reduced peak intracavernous pressures (ICPS) in penile tissues were restored completely after KH-304 treatment, and KH-304 treatment significantly made the latency period earlier. Furthermore, the penile expression levels of nNOS, eNOS dependent NOS activities and COMP concentrations were increased significantly in the KH-304 100, 300mg treated rats. These results suggest that KH-304 with high expression of NOS may be useful in erectile dysfunction.

• Applied Microscopy
• /
• 제38권3호
• /
• pp.221-233
• /
• 2008

산화질소는 생물체내에서 생리적이나 병리학적으로 중요한 역할을 한다고 알려져 있으며, 특히 침샘조직에서 침분비작용과 샘혈류 조절에 중요한 인자의 하나로 관여함이 알려져 있다. 산화질소합성효소 (nitric oxide synthase, NOS)는 동위효소로서 내피산화질소합성효소 (endothelial NOS, eNOS), 신경산화질소합성효소 (neuronal NOS, nNOS)와 유도산화질소합성효소 (inducible NOS, iNOS)가 있으며, 세포내에서 내인성산화질소를 합성한다고 알려져 있다. 그러나 산화질소합성효소의 세포내 분포에 관한 전자현미경적 연구는 매우 드물며, 흰쥐 침샘에서의 산화질소생산효소(NOS)에 대한 전자현미경적 연구는 없었다. 흰쥐 침샘에서 NOS의 세포내 분포를 규명하기 위하여 면역전자현미경방법을 이용한 금입자표지법을 시행하여 아래와 같은 결과를 얻었다. eNOS에 양성 면역반응을 보이는 구조는 침샘의 분비세포 중 장액세포에 있는 전자밀도가 높은 분비과립이었으며, 점액분비세포의 점액분비과립에서는 비교적 약한 면역반응성이 관찰되었다. 즉 턱밑샘과 혀밑샘을 구성하고 있는 두 종류의 분비세포 중 장액세포의 분비과립에 금입자가 비교적 많이 표지되었으며, 점액세포의 분비과립에서는 적은 수의 금입자가 관찰되었고, 침샘의 소엽속관 (intralobular duct)의 전자밀도가 높은 분비과립에서도 금입자가 표지된 것이 관찰되었다. 귀밑샘에서도 장액세포의 분비과립과 소엽속관의 분비과립에 금입자가 표지되었다. nNOS의 양성 면역반응은 턱밑샘에서 점액세포의 분비과립에서만 약간의 금입자가 관찰되었으며, 턱밑샘, 혀밑샘 및 귀밑샘의 분비세포와 분비관세포에서는 iNOS에 대한 양성 면역반응이 관찰되지 않았다. 흰쥐 침샘에서 산화질소합성효소 중 eNOS는 침샘분비세포의 분비과립에 존재하며, 특히 전자밀도가 높은 장액성분비과립에 주로 분포하고 있으며, 분비관 중에서 소엽속관에도 분포하고 있는 것이 관찰되었으나, 다른 동위효소인 nNOS와 iNOS는 거의 관찰되지 않았다. 산화질소합성효소가 흰쥐 침샘분비세포의 분비과립과 소엽속관의 분비과립에 분포하고 있는 것으로 보아 침샘에서 산화질소가 침의 생산과 분비에 중요한 역할을 담당하는 것으로 생각된다.