Presence Of eNOS Research Articles

Influence of endothelial nitric oxide synthase gene intron 4 VNTR polymorphism on serum nitric oxide level among healthy south-Indian population

Background: Nitric oxide (endothelium derived relaxing factor) is synthesized from L-arginine by nitric oxide synthases. Endothelial NOS is responsible for most of the nitric oxide produced under physiological conditions. Acting via cGMP NO cause smooth muscle relaxation, prevents platelet aggregation and acts as an anti-inflammatory agent thereby playing a vital role in regulating vascular tone. Various studies have postulated the role of Nitricoxide in vascular disorders like hypertension and atherosclerosis. This study is aimed to find out the influence of endothelial nitric oxide synthase gene intron 4 VNTR polymorphism on Serum nitric oxide level among healthy South-Indian Population. Genotype analysis was done on 282 randomly selected healthy individuals by polymerase chain reaction. Phenotype analysis of eNOS activity was done by measuring serum NOx level. Significantly lower eNOS activity (10.5 versus 18.3, P value is < 0.00001) among ‘a’ alleles when compared to ‘b’ alleles. We have found the presence of eNOS ‘a’ allele is found to decrease serum NOx level. As the distribution of ‘a’allele is lower among the population, a larger study is needed to find the role of eNOS intron4 gene in maintaining serum Nitric oxide level.

Melatonin Increases Fetal Weight in Wild-Type Mice but Not in Mouse Models of Fetal Growth Restriction.

Fetal growth restriction (FGR) presents with an increased risk of stillbirth and childhood and adulthood morbidity. Melatonin, a neurohormone and antioxidant, has been suggested as having therapeutic benefit in FGR. We tested the hypothesis that melatonin would increase fetal growth in two mouse models of FGR which together represent a spectrum of the placental phenotypes in this complication: namely the endothelial nitric oxide synthase knockout mouse (eNOS-/-) which presents with abnormal uteroplacental blood flow, and the placental specific Igf2 knockout mouse (P0+/-) which demonstrates aberrant placental morphology akin to human FGR. Melatonin (5 μg/ml) was administered via drinking water from embryonic day (E)12.5 in C57Bl/6J wild-type (WT), eNOS-/-, and P0+/- mice. Melatonin supplementation significantly increased fetal weight in WT, but not eNOS-/- or P0+/- mice at E18.5. Melatonin did, however, significantly increase abdominal circumference in P0+/- mice. Melatonin had no effect on placental weight in any group. Uterine arteries from eNOS-/- mice demonstrated aberrant function compared with WT but melatonin treatment did not affect uterine artery vascular reactivity in either of these genotypes. Umbilical arteries from melatonin treated P0+/- mice demonstrated increased relaxation in response to the nitric oxide donor SNP compared with control. The increased fetal weight in WT mice and abdominal circumference in P0+/-, together with the lack of any effect in eNOS-/-, suggest that the presence of eNOS is required for the growth promoting effects of melatonin. This study supports further work on the possibility of melatonin as a treatment for FGR.

Association of eNOS and ACE gene polymorphisms as a genetic risk factor in gestational diabetes in Iranian women.

Gestational diabetes mellitus (GDM) is the most popular metabolic disease during pregnancy. The aim of the present study was to investigate any possible association between eNOS Glu298Asp and ACE I/D gene polymorphisms and the risk of GDM in a group of Iranian pregnant women. In this case-control study 204 pregnant women were recruited (94 cases and 110 controls). Genomic DNA was isolated from whole blood and genotyping was performed by the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR- RFLP) and only PCR for eNOS and ACE polymorphisms respectively. Frequencies of GT and TT genotype of eNOS polymorphism among women with and without GDM were 67.90% vs. 74.47 and 7.41% vs. 8.51% respectively (P = 0.4). Corresponding figures for DD genotype of ACE polymorphism among GDM patients was more than that in healthy women (51.65% vs. 63.81% respectively). Conversely, ACE heterozygote genotype was more common in diabetic women (35.16% vs. 26.67% respectively). Although these differences were not statistically significant (P = 0.2). Our study showed that there is no association between the presence of eNOS and ACE gene polymorphisms and developing gestational diabetes mellitus among pregnant women in our population. Further longitudinal and multicenter studies should be carried out to assess the exact metabolic effects of these polymorphisms.

Concomitant presence of endothelial nitric oxide 894T and angiotensin II‐converting enzyme D alleles are associated with diabetic nephropathy in a Kurdish population from Western Iran

The present study investigated the influence of insertion (I)/deletion (D) polymorphism of the angiotensin II-converting enzyme (ACE) gene in combination with endothelial nitric oxide (eNOS) G894T polymorphism on the predisposition to diabetic nephropathy (DN). Using polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (PCR-RFLP) method, the ACE and eNOS polymorphisms were genotyped in 72 microalbuminuric, 68 macroalbuminuric and 72 normoalbuinuric type 2 diabetes mellitus (T2DM) patients from Western Iran. The presence of eNOS T or ACE D allele was not associated with increased risk of macroalbuminuria (odds ratio (OR) = 1.36, P = 0.27 and OR = 1.6, P = 0.062, respectively). However, in the presence of both alleles there was a trend towards increased risk of macroalbuminuria (fivefold, P = 0.05). Our study indicates that the concomitant presence of both ACE D and eNOS T alleles tends to be associated with an elevation risk of macroalbuminuria compared with the presence of each polymorphism alone. This risk could be attributed to the increasing activity of ACE and angiotensin II level in the presence of D allele and decreasing NO production in the presence of T allele accelerating diabetic nephropathy.

Ischemic preconditioning activates caveolae-mediated eNOS signalling and elicits cardioprotection through S-nitrosylation of the subsarcolemmal mitochondria

BackgroundRecent studies suggest that caveolae transduce endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) signalling in ischemic preconditioning (IPC) hearts. And the NO-mediated protein S-nitrosylation (SNO) has been shown...

Functional Significance of Cytosolic Endothelial Nitric-oxide Synthase (eNOS): REGULATION OF HYPERPERMEABILITY

Endothelial NOS (eNOS)-derived NO is a key factor in regulating microvascular permeability. We demonstrated previously that eNOS translocation from the plasma membrane to the cytosol is required for hyperpermeability. Herein, we tested the hypothesis that eNOS activation in the cytosol is necessary for agonist-induced hyperpermeability. To study the fundamental properties of endothelial cell monolayer permeability, we generated ECV-304 cells that stably express cDNA constructs targeting eNOS to the cytosol or plasma membrane. eNOS-transfected ECV-304 cells recapitulate the eNOS translocation and permeability properties of postcapillary venular endothelial cells (Sánchez, F. A., Rana, R., Kim, D. D., Iwahashi, T., Zheng, R., Lal, B. K., Gordon, D. M., Meininger, C. J., and Durán, W. N. (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 6849-6853). We used platelet-activating factor (PAF) as a proinflammatory agonist. PAF activated eNOS by increasing phosphorylation of Ser-1177 and inducing dephosphorylation of Thr-495, increasing NO production, and elevating permeability to FITC-dextran 70 in monolayers of cells expressing wild-type and cytosolic eNOS. PAF failed to increase permeability to FITC-dextran 70 in monolayers of cells transfected with eNOS targeted to the plasma membrane. Interestingly, this occurred despite eNOS Ser-1177 phosphorylation and production of comparable amounts of NO. Our results demonstrate that the presence of eNOS in the cytosol is necessary for PAF-induced hyperpermeability. Our data provide new insights into the dynamics of eNOS and eNOS-derived NO in the process of inflammation.

The concomitant presence of eNOS 894 T and ACE D alleles is associated with diabetic nephropathy and its progression

The concomitant presence of eNOS 894 T and ACE D alleles is associated with diabetic nephropathy and its progression

Localization of eNOS in the Olfactory Epithelium of the Rat

Nitric oxide (NO) is a free radical and produced from L-arginine by nitric oxide synthase (NOS). Since NO is recently suggested to be involved in olfactory perception, the expression of eNOS, an isoform of NOS, was examined in the rat olfactory epithelium. The activity of NADPH-diaphorase was also examined as a marker of NOS. In the dorsomedial region of the nasal cavity, intensely positive reactions for NADPH-diaphorase were observed in the entire cytoplasm of sensory cells (olfactory cells). By immunohistochemistry, intensely positive reactions for eNOS were also found in the dorsomedial region of the nasal cavity. These reactions were observed on the free border of the olfactory epithelium. By immunoelectron microscopy, positive reactions for eNOS were found in the cilia of olfactory cells. In addition, in situ hybridization analysis of the olfactory epithelium revealed the expression of eNOS mRNA in the olfactory cells. These results indicate the presence of eNOS in the olfactory cells of the rat, and differential expression of eNOS in the olfactory epithelium depending on the regions of the nasal cavity. In addition, NO produced by eNOS may be involved in olfactory perception in the cilia of olfactory cells.

Endothelial nitric oxide synthase in the amphibian, Xenopus tropicalis

Nitric oxide (NO) is generated by NO synthase (NOS) of which there are three isoforms: neuronal NOS, inducible NOS, and endothelial NOS (eNOS or NOS III). The presence of eNOS and endothelial NO signalling in fish and amphibian blood vessels has been controversial. This study utilised the genome of Xenopus (Silurana tropicalis) to clone eNOS and determine its tissue expression, and then establish if eNOS is involved in vascular regulation. The open reading frame of Xenopus eNOS (XteNOS) cDNA encoded an 1177 amino acid protein that showed closest structural identity to mammalian eNOS. XteNOS mRNA expression was highest in lung and skeletal muscle, with lower expression in the liver, gut, kidney, heart and brain. No discernable XteNOS mRNA expression was observed in the lateral and dorsal aortae. Western analysis of lung protein using an affinity‐purified anti‐XteNOS produced a single band at approximately 140 kDa, which is similar to mammalian eNOS. Immunohistochemistry showed XteNOS immunoreactivity in the collecting duct of the kidney and the lung parenchyma, but not in the endothelium of blood vessels of the kidney or mesentery. Myography using endothelium‐denuded lateral aorta of X. tropicalis found an acetylcholine‐induced NO‐mediated vasodilation that is most likely attributed to perivascular nitrergic nerves. Thus, Xenopus has an eNOS gene that is not expressed in the vascular endothelium.

Cyclosporine A-induced nitration of tyrosine 34 MnSOD in endothelial cells: role of mitochondrial superoxide

Cyclosporine A (CsA) has represented a fundamental therapeutic weapon in immunosuppression for the past three decades. However, its clinical use is not devoid of side effects, among which hypertension and vascular injury represent a major drawback. Endothelial cells are able to generate reactive oxygen and nitrogen species upon exposure to CsA, including formation of peroxynitrite. This may result in endothelial cell toxicity and increased tyrosine nitration. We have now studied the subcellular origin of superoxide formation in endothelial cells treated with CsA and the biochemical consequences for the function of mitochondrial enzymes. By using electron spin resonance and endothelial cells lacking functional mitochondria, we showed that superoxide anion is generated in mitochondria. This was associated with an effect of CsA on bioenergetic parameters: increased mitochondrial membrane potential and inhibition of cellular respiration. In addition, CsA inhibited the activity of the mitochondrial enzymes aconitase and manganese superoxide dismutase (MnSOD). The use of murine lung endothelial cells deficient in endothelial nitric oxide synthase (eNOS) and NOS/peroxynitrite inhibitors allowed us to establish that the presence of eNOS and concomitant NO synthesis and peroxynitrite formation were essential for CsA induced nitration and inhibition of MnSOD activity. As the latter has been shown to become inactivated by nitration, we sought to identify this modification by mass spectrometry analysis. We found that CsA induced specific MnSOD tyrosine 34 nitration both in the recombinant protein and in endothelial cells overexpressing MnSOD. We propose that CsA induced endothelial damage may be related to increased mitochondrial superoxide formation and subsequent peroxynitrite-dependent nitroxidative damage, specifically targeting MnSOD. The inactivation of this key antioxidant enzyme by tyrosine nitration represents a pathophysiological cellular mechanism contributing to self-perpetuation and amplification of CsA-related vascular toxicity.

Endothelial NOS is required for SDF-1α/CXCR4-mediated peripheral endothelial adhesion of c-kit+ bone marrow stem cells

In the era of intravascular approaches for regenerative cell therapy, the underlying mechanisms of stem cell migration to non-marrow tissue have not been clarified. We hypothesized that next to a local inflammatory response implying adhesion molecule expression, endothelial nitric oxide synthase (eNOS)-dependent signaling is required for stromal- cell-derived factor-1 alpha (SDF-1α)-induced adhesion of c-kit+ cells to the vascular endothelium. SDF-1α/tumor necrosis factor-alpha (TNF-α)-induced c-kit+-cell shape change and migration capacity was studied in vitro using immunohistochemistry and Boyden chamber assays. In vivo interaction of c-kit+ cells from bone marrow with the endothelium in response to SDF-1α/TNF-α stimulation was visualized in the cremaster muscle microcirculation of wild-type (WT) and eNOS (−/−) mice using intravital fluorescence microscopy. In addition, NOS activity was inhibited with N-nitro-L-arginine-methylester-hydrochloride in WT mice. To reveal c-kit+-specific adhesion behavior, endogenous leukocytes (EL) and c-kit+ cells from peripheral blood served as control. Moreover, intercellular adhesion molecule-1 (ICAM-1) and CXCR4 were blocked systemically to determine their role in inflammation-related c-kit+-cell adhesion. In vitro, SDF-1α enhanced c-kit+-cell migration. In vivo, SDF-1α alone triggered endothelial rolling—not firm adherence—of c-kit+ cells in WT mice. While TNF-α alone had little effect on adhesion of c-kit+ cells, it induced maximum endothelial EL adherence. However, after combined treatment with SDF-1α+TNF-α, endothelial adhesion of c-kit+ cells increased independent of their origin, while EL adhesion was not further incremented. Systemic treatment with anti-ICAM-1 and anti-CXCR4-monoclonal antibody completely abolished endothelial c-kit+-cell adhesion. In N-nitro-L-arginine-methylester-hydrochloride-treated WT mice as well as in eNOS (−/−) mice, firm endothelial adhesion of c-kit+ cells was entirely abrogated, while EL adhesion was significantly increased. The chemokine SDF-1α mediates firm adhesion c-kit+ cells only in the presence of TNF-α stimulation via an ICAM-1- and CXCR4-dependent mechanism. The presence of eNOS appears to be a crucial and specific factor for firm c-kit+-cell adhesion to the vascular endothelium.

Abstract 974: Endothelial NOS (eNOS) Regulates Mitochondrial Oxidative Stress in Fetal Pulmonary Artery Endothelial Cells (PAEC)

Background : Endothelial NOS regulates pulmonary vasodilation at birth by synthesis and release of NO. Exposure of fetal lung to O 2 results in increased oxidative phoshporylation and ATP release at birth. ATP stimulates NO release from eNOS. Superoxide (O 2 − ) is generated by mitochondria as a byproduct of oxidative phosphorylation. The regulation of mitochondrial O 2 during this transition and its role in impaired vasodilation that occurs in persistent pulmonary hypertension of newborn (PPHN) remain unknown. Objective : We investigated the hypothesis that targeting of eNOS to mitochondrial outer membrane regulates O 2 − in fetal PAEC in response to ATP and O 2 . Methods : Studies were done in isolated PAEC and pulmonary arteries (PA) from fetal lambs. PPHN was induced by prenatal ductal constriction for 8 days. Association of eNOS with outer membrane protein, porin and chaperone, hsp90 was determined by immunoprecipitation and double label immuno-fluorescence. Mitochondrial NO and O 2 − levels were determined by DAF-2 and mito-HE reagents. Relaxation responses of PA rings to ATP and NO were determined in the presence/absence of decoy peptides that inhibit eNOS association with hsp90 and porin and mitochondrial targeted ubiquinones. Results : Mitochondria from PAEC showed presence of eNOS on outer membrane but no nNOS. Exposure of PAEC to ATP or O 2 increased the association of eNOS with porin and hsp90 in the mitochondria. ATP increased mitochondrial NO release. Inhibition of eNOS-hsp90 association or eNOS-porin association with decoy peptides that mimic specific eNOS domains required for these interactions resulted in increased mitochondrial O 2 − and attenuated the relaxation response of PA rings to ATP and NO. PAEC from PPHN lambs showed increased basal O 2 consumption, decreased NO-regulation of O 2 consumption and increased mitochondrial O 2 − . PA rings from PPHN lambs showed impaired relaxation responses to ATP and NO and improved relaxation when treated with mitochondrial targeted ubiquinone. Conclusion : Interaction of eNOS with mitochondria plays an important role in the regulation of O 2 − in PAEC during postnatal transition to the O 2 rich environment. Pulmonary hypertension is associated with impaired targeting of eNOS and increase in mitchondrial O 2 − .

Arginase: a modulator of myocardial function

when nitric oxide (NO) donors were first administered to isolated, normal cardiomyocytes, a negative, cGMP-dependent inotropic effect was observed, whereas inhibitors of NO synthases (NOS) had no effect. Further studies then demonstrated a biphasic contractile response to exogenous NO and cGMP,

Endothelial NOS is main mediator for shear stress-dependent angiogenesis in skeletal muscle after prazosin administration.

The increase of wall shear stress in capillaries by oral administration of the alpha1-adrenergic receptor antagonist prazosin induces angiogenesis in skeletal muscles. Because endothelial nitric oxide synthase (eNOS) is upregulated in response to elevated wall shear stress, we investigated the relevance of eNOS for prazosin-induced angiogenesis in skeletal muscles. Prazosin and/or the NOS inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME) were given to C57BL/6 wild-type mice and eNOS-knockout mice for 14 days. The capillary-to-fiber (C/F) ratio and capillary density (CD; no. of capillaries/mm2) were determined in frozen sections from extensor digitorum longus (EDL) muscles of these mice. Immunoblotting was performed to quantify eNOS expression in endothelial cells isolated from skeletal muscles, whereas VEGF (after precipitation with heparin-agarose) and neuronal NOS (nNOS) concentrations were determined in EDL solubilizates. In EDL muscles of C57BL/6 mice treated for 14 days, the C/F ratio was 28% higher after prazosin administration and 11% higher after prazosin and L-NAME feeding, whereas the CD increased by 21 and 13%, respectively. The C/F ratio was highest after day 4 of prazosin treatment and decreased gradually to almost constant values after day 8. Prazosin administration led to elevation of eNOS expression. VEGF levels were lowest at day 4, whereas nNOS values decreased after day 8. In EDL muscles of eNOS-knockout mice, no significant changes in C/F ratio, CD, or VEGF and nNOS expression were observed in response to prazosin administration. Our data suggest that the presence of eNOS is essential for prazosin-induced angiogenesis in skeletal muscle, albeit other signaling molecules might partially compensate for or contribute to this angiogenic activity. Furthermore, subsequent remodeling of the capillary system accompanied by sequential downregulation of VEGF and nNOS in skeletal muscle fibers characterizes shear stress-dependent angiogenesis.

Bradykinin limits infarction when administered as an adjunct to reperfusion in mouse heart: the role of PI3K, Akt and eNOS

Attenuation of reperfusion injury by growth factors has recently been linked to recruitment of phosphatidylinositol-3 kinase (PI3K) and protein kinase B (Akt), a pathway also linked to the phosphorylation of eNOS by bradykinin. We, therefore, hypothesised that bradykinin would limit infarct size when given as an adjunct to reperfusion. Using an isolated perfused mouse heart model of ischaemia/reperfusion injury, we show that 100 nmol/l bradykinin, administered upon reperfusion, attenuates infarct size (32 ± 2% to 22 ± 2%, P < 0.01). This protection was abrogated by concomitant administration of the PI3K inhibitor, wortmannin (100 nmol/l), whereas wortmannin alone had no impact upon infarct size (31 ± 3% and 30 ± 1%, respectively). In eNOS knockout hearts, bradykinin was not seen to be protective (31 ± 2% versus 32 ± 2%), yet knockout hearts could be rescued with the nitric oxide donor, S-nitroso- N-acetyl penicillamine (SNAP) (1 μmol/l) (17 ± 4%, P < 0.01). Using western blot analysis, we show that bradykinin administration results in rapid, robust phosphorylation of both Akt and eNOS, greater than that seen in control hearts upon reperfusion (Akt/eNOS phosphorylation: 68 ± 7/122 ± 29 AU versus 32 ± 5/47 ± 10 AU respectively, P < 0.01). This pattern of Akt phosphorylation was mimicked in the absence of eNOS, whereas Akt phosphorylation was inhibited by wortmannin. Exogenous nitric oxide administration had no impact upon Akt phosphorylation. Therefore, we demonstrate that exogenous bradykinin, administered at reperfusion, limits infarct size with concomitant rapid phosphorylation of Akt and eNOS, and that this protection is dependent upon the presence of eNOS. These results may open new avenues for research into clinical limitation of reperfusion injury following acute myocardial infarction.

Mechanism of eNOS gene transfer inhibition of vascular smooth muscle cell proliferation.

Endothelial nitric oxide synthase (eNOS) is responsible for the production of nitric oxide (NO) in blood vessels. NO has been shown to be involved in the inhibition of vascular smooth muscle cell (VSMC) proliferation. In the present study, the eNOS gene was transferred into rat aortic smooth muscle cells by using an adenoviral vector, and the effect of endogenously produced NO on VSMC proliferation was investigated. The presence of eNOS in eNOS-transfected cells was confirmed by immunocytochemistry and Western blot analysis. eNOS transfection resulted in inhibition of VSMC proliferation. This effect was accompanied by increased levels of p53 and p21. This effect was abrogated in the presence of the protein kinase A (PKA) inhibitor Rp-8-bromoadenosine 3',5'-cyclic monophosphothioate. The increased levels of p53 and p21 observed in eNOS-transfected cells were reduced in the presence of the PKA inhibitor. These data suggest that p21 and p53 play a role in the inhibition of proliferation in eNOS-transfected cells and that levels of these two proteins are regulated by PKA.

Localization of nitric oxide synthase in the periodontal tissues of orthodontically moved and stationary teeth

There is ample evidence that the inflammatory response and vascular supply of the periodontal tissues is crucial to mediating the effects of applied force. The connection between mechanical forces and vascular activity, however, is not well defined. Nitric oxide (NO), a molecule only recently recognized as a chemical messenger within biologic systems, has been shown to be central to these processes in many parts of the body, but not during tooth movement. Therefore, the aim of this investigation was to identify the sites of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) activity in the periodontal tissues of moved and stationary teeth immunohistochemically. Our data show that the periodontal tissues of stationary rat molar teeth exhibit the presence of less eNOS and iNOS. There is an increased amount of eNOS produced after rat molar tooth movement in blood vessel walls. Similarly, increased amounts of iNOS produced after rat molar tooth movement in the periodontal ligament and connective tissue between roots of moved teeth as well as around blood vessels could be seen. The presence of eNOS and iNOS was consistently seen in the odontoblast layer of pulp chambers of moved and stationary teeth. Our results indicate a possible role of NO in periodontal remodeling during tooth movement.

Endothelial nitric oxide synthase and caveolin-1 are co-localized in sinusoidal endothelial fenestrae.

Nitric oxide is synthesized in diverse mammalian tissues by a family of calmodulin-dependent nitric oxide synthases (NOS). Caveolin, the principal structural protein in caveolae, interacts with endothelial NOS leading to enzyme inhibition in a reversible process modulated by Ca++-calmodulin. The aim of the present study was to clarify the ultrastructural localization of eNOS and caveolin-1 in hepatic sinusoidal endothelium by an electron immunogold method. Male Wistar rats were used. Liver tissues and hepatic sinusoidal endothelial cells isolated from rat livers by collagenase infusion were studied. For immunohistochemistry, liver specimens were reacted with anti-eNOS or anti-caveolin-1 antibody. The ultrastructural localization of eNOS or caveolin-1 was identified by electron microscopy using an immunogold post-embedding method. Immunohistochemical studies using liver tissues localized endothelial NOS in hepatic sinusoidal lining cells, portal veins and hepatic arteries; and caveolin-1 in sinusoidal lining cells, bile canaliculi, portal vein and hepatic arteries. Immunogold particles indicating the presence of eNOS and caveolin-1 were demonstrated on the plasma membrane of sinusoidal endothelial fenestrae in liver tissue and also in isolated sinusoidal endothelial cells. Endothelial NOS and caveolin are co-localized on sinusoidal endothelial fenestrae, suggesting that interaction of the two may modulate cellular regulation of NO synthesis.

Localization of the e-NOS enzyme in endothelial cells and odontoblasts of healthy human dental pulp

Nitric oxide synthases (NOS) are important enzymes present in different cells such as endothelial cells, macrophages, etc. Recently, it has been found that nitric oxide (NO) is responsible for vasodilation, blood pressure regulation, platelet aggregation, cardiac contractility, and the mediation of immunity during bacterial infections and inflammation. However, the production and role of NO in various structures of the oral cavity have not been investigated extensively. The aim of this study was to evaluate the presence of e-NOS in healthy human odontoblasts and endothelial cells of the dental pulp. Twenty healthy human dental pulps were collected and frozen and pulp slices were obtained using a cryostat. The e-NOS enzyme was revealed by immunohistochemical analysis and the enzyme level was detected by Western blotting and mRNA expression by RT-PCR. The immunohistochemical results demonstrated, for the first time, the presence of e-NOS in odontoblasts and in endothelial cells. The presence of e-NOS m-RNA was confirmed by RT-PCR and the expression of the protein by Western blotting. These results clearly show that the e-NOS enzyme is present in both odontoblasts and endothelial cells of healthy human pulp. The presence of e-NOS in the odontoblast and endothelial cells of the dental pulp may mediate local vasodilation and cell proliferation.

Endothelial nitric oxide synthetase (eNOS) in astrocytes: Another source of nitric oxide in neocortex

The distribution of the endothelial form of nitric oxide synthetase (eNOS) was examined in the visual cortex of three species of primate and in the rat using immunocytochemistry. Labeled cells were found in both the gray and white matter. These cells were stellate in appearance and labeled cell processes were seen contacting blood vessels or the pia, suggesting that, by morphological criteria, the cells were astrocytes. All eNOS positive cells were double labeled with an antibody against S100beta. Although all cells were double labeled in the white matter, in the gray matter, some S100beta positive cells did not contain detectable levels of eNOS. eNOS positive astrocytic processes appeared to form prominent and distinctive structures next to neurons, especially in cortical layer IIIC. We postulate that these eNOS-positive structures form astrocytic perisynaptic sheaths on neuronal somas in the cortex. If this is true, then nitric oxide can influence neuronal transmission directly at axosomatic synapses in the cortex. In addition, the presence of eNOS in astrocytes and in their processes that contact blood vessels suggests that the link between local cortical activity and changes in cerebral blood flow could be mediated by astrocytic release of nitric oxide.