Regulation of endothelial nitric oxide synthase by PGD2 in the developing choroid

2000 ◽  
Vol 278 (1) ◽  
pp. H60-H66 ◽  
Author(s):  
Isabelle Dumont ◽  
Pierre Hardy ◽  
Krishna G. Peri ◽  
Xin Hou ◽  
Stéphane Molotchnikoff ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Blood Flow ◽  
Nitric Oxide Synthase ◽  
No Production ◽  
Enos Expression ◽  
Choroidal Blood Flow ◽  
Nitrite Production ◽  
Enos Mrna ◽  
Oxide Synthase

We investigated if prostaglandins might regulate the increased choroidal endothelial (e) nitric oxide synthase (NOS) expression in the perinate. Prostaglandins, eNOS mRNA, immunoreactive protein and activity, and nitrite [stable metabolite of nitric oxide (NO)] production were markedly higher in newborn (1 day old) than juvenile (6–8 wk old) pig choroid. Treatment of isolated newborn choroids with the prostaglandin synthase inhibitor ibuprofen for 24 h reduced eNOS mRNA and nitrite production to values in juveniles. This effect was equally observed with the PGD2 receptor (DP) blocker BW A868C and was prevented by cotreatment with PGD2 but not other prostaglandins; similar observations were made on NOS activity in vivo. PGD2 also increased eNOS expression on choroids of juveniles, and this effect was blocked by BW A868C. The manifestation of this upregulation of eNOS by PGD2 on the control of choroidal vasomotor response was tested by using NO-dependent vasorelaxants, ACh, bradykinin (Bk), and substance P (SP). ACh-, Bk-, and SP-elicited choroidal vasorelaxation was greater in saline-treated newborn than juvenile pigs. Ibuprofen (24 h) decreased ACh-, Bk-, and SP-evoked vasorelaxation in newborns, whereas PGD2 increased that in juveniles and prevented the ibuprofen-induced attenuated relaxation in newborns; infusion of N ω-monomethyl-l-arginine in choroids of those animals treated with PGD2 reversed the augmented vasorelaxation to ACh, Bk, and SP. Finally, PGD2-induced upregulation of NOS in the perinate was also reflected by curtailed choroidal blood flow autoregulatory response to increased perfusion pressure. In conclusion, PGD2 exhibits a major role in upregulating eNOS expression and activity in the choroid, which in turn results in greater NO-mediated vasorelaxation; a new mechanism for eNOS regulation via DP is hereby disclosed. The relationship between PGD2 and eNOS in the developing subject provides an explanation for the interactive role of these two factors in the absent choroidal blood flow autoregulation in the perinate.

scholarly journals Protein kinase Cδ regulates endothelial nitric oxide synthase expression via Akt activation and nitric oxide generation

2008 ◽  
Vol 294 (3) ◽  
pp. L582-L591 ◽  
Author(s):  
Neetu Sud ◽  
Stephen Wedgwood ◽  
Stephen M. Black
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Endothelial Nitric Oxide Synthase ◽  
Promoter Activity ◽  
Dominant Negative ◽  
No Production ◽  
Enos Expression ◽  
Akt Activation ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

In this study, we explore the roles of the delta isoform of PKC (PKCδ) in the regulation of endothelial nitric oxide synthase (eNOS) activity in pulmonary arterial endothelial cells isolated from fetal lambs (FPAECs). Pharmacological inhibition of PKCδ with either rottlerin or with the peptide, δV1-1, acutely attenuated NO production, and this was associated with a decrease in phosphorylation of eNOS at Ser1177 (S1177). The chronic effects of PKCδ inhibition using either rottlerin or the overexpression of a dominant negative PKCδ mutant included the downregulation of eNOS gene expression that was manifested by a decrease in both eNOS promoter activity and protein expression after 24 h of treatment. We also found that PKCδ inhibition blunted Akt activation as observed by a reduction in phosphorylated Akt at position Ser473. Thus, we conclude that PKCδ is actively involved in the activation of Akt. To determine the effect of Akt on eNOS signaling, we overexpressed a dominant negative mutant of Akt and determined its effect of NO generation, eNOS expression, and phosphorylation of eNOS at S1177. Our results demonstrated that Akt inhibition was associated with decreased NO production that correlated with reduced phosphorylation of eNOS at S1177, and decreased eNOS promoter activity. We next evaluated the effect of endogenously produced NO on eNOS expression by incubating FPAECs with the eNOS inhibitor 2-ethyl-2-thiopseudourea (ETU). ETU significantly inhibited NO production, eNOS promoter activity, and eNOS protein levels. Together, our data indicate involvement of PKCδ-mediated Akt activation and NO generation in maintaining eNOS expression.

Endothelin stimulates endothelial nitric oxide synthase expression in the thick ascending limb

2004 ◽  
Vol 287 (2) ◽  
pp. F231-F235 ◽  
Author(s):  
Marcela Herrera ◽  
Jeffrey L. Garvin
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Receptor Antagonist ◽  
Endothelial Nitric Oxide Synthase ◽  
No Production ◽  
Thick Ascending Limb ◽  
Enos Expression ◽  
No Release ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Endothelin-1 (ET-1) acutely inhibits NaCl reabsorption by the thick ascending limb (THAL) by activating the ETB receptor, stimulating endothelial nitric oxide synthase (eNOS), and releasing nitric oxide (NO). In nonrenal tissue, chronic exposure to ET-1 stimulates eNOS expression via the ETB receptor and activation of phosphatidylinositol 3-kinase (PI3K). We hypothesized that ET-1 increases eNOS expression in the THAL by binding to ETB receptors and stimulating PI3K. In primary cultures of medullary THALs treated for 24 h, eNOS expression increased by 36 ± 18% with 0.01 nM ET-1, 123 ± 30% with 0.1 nM ( P < 0.05; n = 5), and 71 ± 30% with 1 nM, whereas 10 nM had no effect. BQ-788, a selective ETB receptor antagonist, completely blocked stimulation of eNOS expression caused by 0.1 nM ET-1 (12 ± 25 vs. 120 ± 40% for ET-1 alone; P < 0.05; n = 5). BQ-123, a selective ETA receptor antagonist, did not affect the increase in eNOS caused by 0.1 nM ET-1. Sarafotoxin c (S6c; 0.1 μM), a selective ETB receptor agonist, increased eNOS expression by 77 ± 30% ( P < 0.05; n = 6). Wortmannin (0.01 μM), a PI3K inhibitor, completely blocked the stimulatory effect of 0.1 μM S6c (77 ± 30 vs. −28 ± 9%; P < 0.05; n = 6). To test whether the increase in eNOS expression heightens activity, we measured NO release in response to simultaneous treatment with l-arginine, ionomycin, and clonidine using a NO-sensitive electrode. NO release by control cells was 337 ± 61 and 690 ± 126 pA in ET-1-treated cells ( P < 0.05; n = 5). Taken together, these data suggest that ET-1 stimulates THAL eNOS, activating ETB receptors and PI3K and thereby increasing NO production.

Inducible nitric oxide synthase in the epithelial epididymal cells of the rat

1997 ◽  
Vol 9 (8) ◽  
pp. 789 ◽  
Author(s):  
Barbara Wiszniewska ◽  
Rafal Kurzawa ◽  
Andrzej Ciechanowicz ◽  
Boguslaw Machalinski
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Epithelial Cells ◽  
Inducible Nitric Oxide Synthase ◽  
Cultured Cells ◽  
No Production ◽  
Nitrite Production ◽  
Oxide Synthase ◽  
Inducible Nos ◽  
Inducible Nitric Oxide

The expression of mRNA for inducible nitric oxide synthase (iNOS) in rat epithelial cells of epididymis was investigated with reverse transcription followed by polymerase chain reaction. Immunocytochemical reaction for iNOS was performed to confirm the enzyme’ s localization in the epididymal epithelium. Additionally, an indirect spectrophotometric method for nitric oxide (NO) determination was applied for measurement of nitrite production by cultured epididymal epithelial cells. Inducible NOS mRNA was detected in freshly isolated epithelial cells, in cultured cells without stimulation as well as in cultured cells after stimulation by lipopolysaccharide and interferon-gamma. Inducible NOS immunoreactivity was observed in the apical part of epithelial cells of epididymal sections and in the cytoplasm of cells in culture. Release of nitrite was observedin vitro in both the unstimulated and stimulated cells of caput (1·44 ± 0·94 v. 4·37 ± 2·42 µM) and cauda (0·69 ± 1·21 v. 5·21 ± 2·76 µM) epididymis (P < 0·001). To the best of our knowledge, this is the first study to demonstrate iNOS in the epididymal epithelial cells of the rat. Nitric oxide released by epididymal epithelial cells may act on cells and tissues located nearby. The results may help explain epididymal function: sperm storage, passage and maturation. Excessive epididymal NO production may also play a role in the inflammatory infertility of the male. Extra keyword: iNOS

Expression of endothelial nitric oxide synthase in the postnatal developing porcine kidney

2001 ◽  
Vol 280 (5) ◽  
pp. R1269-R1275 ◽  
Author(s):  
Michael J. Solhaug ◽  
Usa Kullaprawithaya ◽  
Xui Q. Dong ◽  
Ke-Wen Dong
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Protein Content ◽  
Endothelial Nitric Oxide Synthase ◽  
Enos Expression ◽  
Developmentally Regulated ◽  
Enos Mrna ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide ◽  
Enos Protein

The postnatal pattern of renal endothelial nitric oxide synthase (eNOS) is unknown. The purpose of this study was to characterize eNOS expression during maturation and compare this to neuronal NOS (nNOS). The experiments measured whole kidney eNOS mRNA expression by RT-PCR and protein content by Western blot, as well as cortical and medullary protein content in piglets at selected postnatal ages and in adult pigs. Whole kidney eNOS mRNA was compared with nNOS. Whole kidney eNOS expression decreased from the newborn to its lowest at 7 days, returning by 14 days to adult levels. This eNOS mRNA pattern contrasted with nNOS, which was highest at birth, and progressively decreased to its lowest level in the adult. At birth, cortical eNOS protein was greater than medullary, contrasting with the adult pattern of equivalent levels. In conclusion eNOS is developmentally regulated during early renal maturation and may critically participate in renal function during this period. The eNOS developmental pattern differs from nNOS, suggesting that these isoforms may have different regulatory factors and functional contributions in the postnatal kidney.

Short-term exposure to homocysteine depresses rat aortic contractility by an endothelium-dependent mechanism

2000 ◽  
Vol 78 (6) ◽  
pp. 500-506 ◽  
Author(s):  
S Wang ◽  
G Wright ◽  
J Harrah ◽  
R Touchon ◽  
W McCumbee ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Smooth Muscle ◽  
Nitric Oxide Synthase ◽  
Contractile Response ◽  
Fold Increase ◽  
No Production ◽  
Short Term ◽  
Short Term Exposure ◽  
Oxide Synthase

The effect of short-term exposure to homocysteine (Hcy) on the contractile characteristics of rat aortic tissue was assessed both in vitro and in vivo. The contractile response of Hcy-treated aortic rings in culture for 1 or 4 days was unchanged from control responses. By comparison, aortic rings from animals injected with Hcy showed marked attenuation of response compared with controls injected with saline, cysteine or methionine. The contractile response to K+ was decreased within 24 hours of Hcy injection, whereas the response to both K+ (-27%) and noradrenaline (-56%) was significantly decreased by 4 days. In contrast, the contractile response to phorbol-12,13-dibutyrate was not different between Hcy and control groups. Intimal rubbing completely restored the responsiveness of Hcy-treated tissue to K+ and noradrenaline. By comparison, L-NAME only partially restored contractile responsiveness, while the cyclooxygenase inhibitor indomethacin had no effect on contractile attenuation induced by Hcy. Western blot analysis showed a 2-fold increase of endothelial nitric oxide synthase (eNOS) and a 3-fold increase in inducible nitric oxide synthase (iNOS) protein expression in the aortic endothelial cells from Hcy-injected rats. The results indicate an early detectable effect of Hcy on the in vivo contractile properties of vascular smooth muscle. The effect is endothelium-mediated and may vary depending on the agonist studied. The mechanism is uncertain but appears to involve increased nitric oxide (NO) production. Finally, the data suggest that attenuation of contraction may not be due to a direct effect of Hcy but that the compound is modified or acts indirectly in vivo.Key words: nitric oxide, nitric oxide synthase, in vivo, smooth muscle.

scholarly journals Nitric oxide regulates nitric oxide synthase-2 gene expression by inhibiting NF-κB binding to DNA

1997 ◽  
Vol 322 (2) ◽  
pp. 609-613 ◽  
Author(s):  
Song Kyu PARK ◽  
Hsin Lee LIN ◽  
Sean MURPHY
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Interferon Γ ◽  
No Production ◽  
No Donor ◽  
Nitric Oxide Synthase 2 ◽  
Oxide Synthase ◽  
Spermine Nonoate ◽  
Dna Complex

Treatment of astroglial cells with interleukin 1β and interferon γ transcriptionally activates the nitric oxide synthase (NOS)-2 gene. The duration of mRNA expression is brief because of transcript instability. In addition, NO donors reduce the expression of NOS-2 mRNA dramatically by reducing the rate of transcription. In this study we observed that the NO donor, spermine NONOate did not inhibit the activation and translocation of NF-κB, a key transcription factor in the induction of NOS-2, but inhibited formation of the NF-κB–DNA complex. This effect was reversed by methaemoglobin (acting as an NO trap) and by the reducing agent dithiothreitol. Formation of the interferon-regulatory factor–DNA complex was unaffected by NO. These results suggest that NO can modulate its own production by interfering with NF-κB interaction with the promoter region of the NOS gene, a negative feedback effect that may be important for limiting NO production in vivo.

Abstract 1333: Modulation Of Cardiac Beta-adrenergic Responsiveness By The Neuronal Nitric Oxide Synthase/Sarcolemmal Calcium Pump Complex

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Tamer M Mohamed ◽  
Delvac Oceandy ◽  
Nasser Alatwi ◽  
Florence Baudoin ◽  
Elizabeth J Cartwright ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Neuronal Nitric Oxide Synthase ◽  
Neonatal Rat ◽  
No Production ◽  
Adrenergic Stimulation ◽  
Rat Cardiomyocytes ◽  
Adrenergic Response ◽  
Oxide Synthase

The pivotal role of neuronal nitric oxide synthase (nNOS) in regulating cardiac function has only recently been unveiled. Notably, others have shown that responsiveness to β-adrenergic stimulation is dependent on nNOS activity. In a cellular model, we showed that the Ca 2+ /calmodulin-dependent nNOS activity is reduced by overexpression of isoform 4b of the plasma membrane Ca 2+ /Calmodulin-dependent Ca 2+ -pump (PMCA4b), which binds to nNOS. We demonstrated that PMCA4b overexpression in the heart reduced β-adrenergic responsiveness in vivo via an nNOS dependent mechanism (Oceandy et al, Circulation 2007). Here we investigated the cellular mechanisms of the regulation of the β-adrenergic response by PMCA4b. We used an adenoviral system to overexpress PMCA4b (PMCA4b cells) or LacZ (control, C) in neonatal rat cardiomyocytes. PMCA4b cells showed an 18±5% and 24±5% reduction in nitric oxide (DAF-FM fluorescence) and cGMP levels, respectively (n=6, p<0.05 each) compared to C demonstrating the regulation of NO production by the PMCA4b in this system. Since nNOS has been shown to regulate phospholamban (PLB) phosphorylation, we examined phosphorylation of PLB at Ser16. PMCA4b cells showed a significant increase in Ser16-PLB at baseline (66±17%, p<0.05) compared to C. As a result of increased baseline Ser16-PLB in PMCA4b cells, β-adrenergic stimulation of PMCA4b cells using 2μM isoproter-enol (IP) showed reduced relative induction in Ser16-PLB (23±10% vs. 78±19% in C; n=5, p<0.05). Further analysis in adult cardiomyocytes isolated from our PMCA4b transgenic mice (PMCA4b TG) demonstrated that PMCA4b TG showed 3-fold higher Ser16-PLB phosphorylation at baseline compared to wild type (WT) myocytes and the relative response following β-adrenergic stimulation was significantly reduced (1.2±0.2 fold induction after IP treatment in PMCA4b TG, vs. 3.1±0.7 in WT, n=5, p<0.05). Thus, PMCA4b regulates NO production from nNOS, which in turn modulates cGMP levels and PLB phosphorylation. These findings provide mechanistic insight into the regulation of the β-adrenergic response in the heart by PMCA4b and place this Ca 2+ -pump upstream of the recently described pathway linking nNOS and Ser16-PLB phosphorylation and downstream of the β-adrenergic receptor(s).

Evaluation of islet heme oxygenase-CO and nitric oxide synthase-NO pathways during acute endotoxemia

2001 ◽  
Vol 280 (5) ◽  
pp. C1242-C1254 ◽  
Author(s):  
Ragnar Henningsson ◽  
Per Alm ◽  
Ingmar Lundquist
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Heme Oxygenase ◽  
Insulin Response ◽  
Glucagon Secretion ◽  
No Production ◽  
Compensatory Mechanisms ◽  
Oxide Synthase

We investigated, by a combined in vivo and in vitro approach, the temporal changes of islet nitric oxide synthase (NOS)-derived nitric oxide (NO) and heme oxygenase (HO)-derived carbon monoxide (CO) production in relation to insulin and glucagon secretion during acute endotoxemia induced by lipopolysaccharide (LPS) in mice. Basal plasma glucagon, islet cAMP and cGMP content after in vitro incubation, the insulin response to glucose in vivo and in vitro, and the insulin and glucagon responses to the adenylate cyclase activator forskolin were greatly increased after LPS. Immunoblots demonstrated expression of inducible NOS (iNOS), inducible HO (HO-1), and an increased expression of constitutive HO (HO-2) in islet tissue. Immunocytochemistry revealed a marked expression of iNOS in many β-cells, but only in single α-cells after LPS. Moreover, biochemical analysis showed a time dependent and markedly increased production of NO and CO in these islets. Addition of a NOS inhibitor to such islets evoked a marked potentiation of glucose-stimulated insulin release. Finally, after incubation in vitro, a marked suppression of NO production by both exogenous CO and glucagon was observed in control islets. This effect occurred independently of a concomitant inhibition of guanylyl cyclase. We suggest that the impairing effect of increased production of islet NO on insulin secretion during acute endotoxemia is antagonized by increased activities of the islet cAMP and HO-CO systems, constituting important compensatory mechanisms against the noxious and diabetogenic actions of NO in endocrine pancreas.

In vivo pharmacological evaluation off two novel type II (inducible) nitric oxide synthase inhibitors

1995 ◽  
Vol 73 (5) ◽  
pp. 665-669 ◽  
Author(s):  
W. Ross Tracey ◽  
Masaki Nakane ◽  
Fatima Basha ◽  
George Carter
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Inducible Nitric Oxide Synthase ◽  
No Production ◽  
Type Ii ◽  
Nos Inhibitors ◽  
Oxide Synthase ◽  
Dose Dependent ◽  
Inducible Nitric Oxide

Selective type II (inducible) nitric oxide synthase (NOS) inhibitors have several potential therapeutic applications, including treatment of sepsis, diabetes, and autoimmune diseases. The ability of two novel, selective inhibitors of type II NOS, S-ethylisothiourea (EIT) and 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT), to inhibit type II NOS function in vivo was studied in lipopolysaccharide (LPS) treated rats. Type II NOS activity was assessed by measuring changes in plasma nitrite and nitrate concentrations ([NOx]). Both EIT and AMT elicited a dose-dependent and >95% inhibition of the LPS-induced increase in plasma [NOx]. The ED50 values for EIT and AMT were 0.4 and 0.2 mg/kg, respectively. In addition, the administration of LPS and either NOS inhibitor resulted in a dose-dependent increase in animal mortality; neither compound was lethal when administered alone. Pretreatment with L-arginine (but not D-arginine) prevented the mortality, while not affecting the type II NOS-dependent NO production, suggesting the toxicity may be due to inhibition of one of the other NOS isoforms (endothelial or neuronal). Thus, although EIT and AMT are potent inhibitors of type II NOS function in vivo, type II NOS inhibitors of even greater selectivity may need to be developed for therapeutic applications.Key words: nitric oxide, nitrite, nitrate, sepsis, lipopolysaccharide.

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