Presence Of Nitric Oxide Synthase Inhibitor Research Articles

NO-mediated activation of KATP channels contributes to cutaneous thermal hyperemia in young adults

Local skin heating to 42°C causes cutaneous thermal hyperemia largely via nitric oxide (NO) synthase (NOS)-related mechanisms. We assessed the hypothesis that ATP-sensitive K+ (KATP) channels interact with NOS to mediate cutaneous thermal hyperemia. In 13 young adults (6 women, 7 men), cutaneous vascular conductance (CVC) was measured at four intradermal microdialysis sites that were continuously perfused with 1) lactated Ringer solution (control), 2) 5 mM glibenclamide (KATP channel blocker), 3) 20 mM NG-nitro-l-arginine methyl ester (NOS inhibitor), or 4) a combination of KATP channel blocker and NOS inhibitor. Local skin heating to 42°C was administered at all four treatment sites to elicit cutaneous thermal hyperemia. Thirty minutes after the local heating, 1.25 mM pinacidil (KATP channel opener) and subsequently 25 mM sodium nitroprusside (NO donor) were administered to three of the four sites (each 25-30 min). The local heating-induced prolonged elevation in CVC was attenuated by glibenclamide (19%), but the transient initial peak was not. However, glibenclamide had no effect on the prolonged elevation in CVC in the presence of NOS inhibition. Pinacidil caused an elevation in CVC, but this response was abolished at the glibenclamide-treated skin site, demonstrating its effectiveness as a KATP channel blocker. The pinacidil-induced increase in CVC was unaffected by NOS inhibition, whereas the increase in CVC elicited by sodium nitroprusside was partly (15%) inhibited by glibenclamide. In summary, we showed an interactive effect of KATP channels and NOS for the plateau of cutaneous thermal hyperemia. This interplay may reflect a vascular smooth muscle cell KATP channel activation by NO.

Severe Hypouricemia Impairs Endothelium-Dependent Vasodilatation and Reduces Blood Pressure in Healthy Young Men: A Randomized, Placebo-Controlled, and Crossover Study.

BackgroundUric acid (UA) is a plasmatic antioxidant that has possible effects on blood pressure. The effects of UA on endothelial function are unclear. We hypothesize that endothelial function is not impaired unless significant UA depletion is achieved through selective xanthine oxidase inhibition with febuxostat and recombinant uricase (rasburicase).Methods and ResultsMicrovascular hyperemia, induced by iontophoresis of acetylcholine and sodium nitroprusside, and heating‐induced local hyperemia after iontophoresis of saline and a specific nitric oxide synthase inhibitor were assessed by laser Doppler imaging. Blood pressure and renin‐angiotensin system markers were measured, and arterial stiffness was assessed. CRP (C‐reactive protein), allantoin, chlorotyrosine/tyrosine ratio, homocitrulline/lysine ratio, myeloperoxidase activity, malondialdehyde, and interleukin‐8 were used to characterize inflammation and oxidative stress. Seventeen young healthy men were enrolled in a randomized, double‐blind, placebo‐controlled, 3‐way crossover study. The 3 compared conditions were placebo, febuxostat alone, and febuxostat together with rasburicase. The allantoin (μmol/L)/UA (μmol/L) ratio differed between sessions (P<0.0001). During the febuxostat‐rasburicase session, heating‐induced hyperemia became altered in the presence of nitric oxide synthase inhibition; and systolic blood pressure, angiotensin II, and myeloperoxidase activity decreased (P≤0.03 versus febuxostat). The aldosterone concentration decreased in the febuxostat‐rasburicase group (P=0.01). Malondialdehyde increased when UA concentration decreased (both P<0.01 for febuxostat and febuxostat‐rasburicase versus placebo). Other parameters remained unchanged.ConclusionsA large and short‐term decrease in UA in humans alters heat‐induced endothelium‐dependent microvascular vasodilation, slightly reduces systolic blood pressure through renin‐angiotensin system activity reduction, and markedly reduces myeloperoxidase activity when compared with moderate UA reduction. A moderate or severe hypouricemia leads to an increase in lipid peroxidation through loss of antioxidant capacity of plasma.Clinical Trial Registration URL: http://www.clinicaltrials.gov. Unique identifier: NCT03395977.

Activation of voltage-gated sodium channel triggers vasorelaxation of mice mesenteric arteries

The resistance arteries, essential for the microcirculation, are able to rapidly change their diameters to control blood flow. These changes or vasomotricity depend on two opposite processes which are flow-mediated dilatation and myogenic tone. At cellular level, numerous ion channels, such as Ca2+ channels and K+ channels are involved in the control of vascular tone (VT). Voltage-gated Na+ channels (Nav) have also been reported in blood vessel, but their implication in VT remains puzzling. Figueroa and al. (2007) have demonstrated that Nav expressed in mouse cremaster arterioles are involved in endothelial function. The aim of our study is to identify the Nav subtypes expressed in resistance artery and to define their implication in VT by combining physiological and pharmacological approaches. We used mesenteric arteries (MA) from 5-month-old C57BL/6 J male and female mice to characterize Nav expression at transcriptional level by RT-qPCR and at protein level by histoimmunostaining. To investigate their place in VF, wire myography and arteriography were employed. Three transcripts encoding Nav1.2 (scn2a), Nav1.3 (scn3a) and Nav1.5 (scn5a) were identified in MA. The immunolabelling of the cardiac subtype, Nav1.5 confirmed its expression in MA. Interestingly, we showed that Nav activation with veratridine (VTD) induced the vasorelaxation of pre-stimulated MA with U46619. Since the VTD-evoked relaxation response is totally obviated in presence of NO synthase inhibitor, L-NNA, we suggest that the opening of Nav expressed in endothelial cells and subsequent Na+ entry induce the NO pathway activation. Our data highlight the contribution of Nav in VT. Further experiments will be necessary to define the role of each Nav subtype in VTD-induced vasorelaxant response and to characterize the cellular pathway by which the NO synthase is activated by Na+ entry.

In Endothelial Cells, the Activation or Stimulation of Soluble Guanylyl Cyclase Induces the Nitric Oxide Production by a Mechanism Dependent of Nitric Oxide Synthase Activation.

In endothelial cells, investigate if the soluble guanylate cyclase (sGC) activation or stimulation is able to potentiate the relaxation in vessels. Aortic and coronary rings with and without endothelium were placed in a myograph and cumulative concentration-effect curves for DETA-NO or ataciguat were performed. Nitric oxide (NO) were measured by fluorescence or by selective electrode in human umbilical endothelial cells (HUVECs) in response to some treatments, including ataciguat, 8-Br-cGMP and A23187. The presence of the endothelium potentiated the relaxation induced by DETA-NO in aortic and coronary rings. In addition, in aortic rings the endothelium potentiated the relaxation induced by ataciguat. In the presence of nitric oxide synthase (NOS) inhibitor, the endothelium effect was abolished to DETA-NO or ataciguat, in both vessels. Ataciguat, 8-Br-cGMP and A23187 were able to induce NO production in HUVECs cells. In the presence of NOS inhibitor, the NO production induced by ataciguat and 8-Br-cGMP was abolished. Our results suggest that in aortic and coronary rings the endothelium potentiates the relaxation induced by activation or stimulation of sGC through a mechanism dependent of NOS activation.This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

CORM-401 induces calcium signalling, NO increase and activation of pentose phosphate pathway in endothelial cells.

Carbon monoxide-releasing molecules (CO-RMs) induce nitric oxide (NO) release (which requires NADPH), and Ca2+ -dependent signalling; however, their contribution in mediating endothelial responses to CO-RMs is not clear. Here, we studied the effects of CO liberated from CORM-401 on NO production, calcium signalling and pentose phosphate pathway (PPP) activity in human endothelial cell line (EA.hy926). CORM-401 induced NO production and two types of calcium signalling: a peak-like calcium signal and a gradual increase in cytosolic calcium. CORM-401-induced peak-like calcium signal, originating from endoplasmic reticulum, was reduced by thapsigargin, a SERCA inhibitor, and by dantrolene, a ryanodine receptors (RyR) inhibitor. In contrast, the phospholipase C inhibitor U73122 did not significantly affect peak-like calcium signalling, but a slow and progressive CORM-401-induced increase in cytosolic calcium was dependent on store-operated calcium entrance. CORM-401 augmented coupling of endoplasmic reticulum and plasmalemmal store-operated calcium channels. Interestingly, in the presence of NO synthase inhibitor (l-NAME) CORM-401-induced increases in NO and cytosolic calcium were both abrogated. CORM-401-induced calcium signalling was also inhibited by superoxide dismutase (poly(ethylene glycol)-SOD). Furthermore, CORM-401 accelerated PPP, increased NADPH concentration and decreased the ratio of reduced to oxidized glutathione (GSH/GSSG). Importantly, CORM-401-induced NO increase was inhibited by the PPP inhibitor 6-aminonicotinamide (6-AN), but neither by dantrolene nor by an inhibitor of large-conductance calcium-regulated potassium ion channel (paxilline). The results identify the primary role of CO-induced NO increase in the regulation of endothelial calcium signalling, that may have important consequences in controlling endothelial function.

Residual NO modulates contractile responses and membrane potential in isolated rat mesenteric arteries

Shear stress or vasocontriction causes endothelial nitric oxide (NO) release resulting in the regulation of vascular smooth muscle tone in small resistance arteries. Generation of NO is inhibited by nitric oxide synthase (NOS) inhibitors. In this study, we investigated the effect of residual NO, released even in the presence of NOS inhibitors, on the membrane depolarization and phenylephrine-induced contractions of smooth muscle. For this purpose, we used hydroxocobalamin (HC), an NO scavenger, in the presence of NOS inhibitiors, Nω-nitro- L-arginine (L-NA) or Nω-nitro-L-arginine methyl ester (L-NAME) in mesenteric arteries isolated from rats. Phenylephrine (0,01–10 μM), an α1-adrenoceptor agonist, led to depolarisation and concentration-dependent contraction in mesenteric arteries. The depolarisation and contractile responses were augmented by L-NA or L-NAME. Hydroxocobalamine (HC) or carboxy-PTIO (c-PTIO) also caused to further increase the membrane depolarization and contractions induced by phenylephrine in the presence of NOS inhibitors. Chemical removal of endothelium by saponin, tyrosin kinase inhibitor erbstatin A, but not calmodulin inhibitor calmidazolium inhibited the additional membrane depolarisation and contractile responses induced by L-NA or L-NAME and L-NA or L-NAME plus HC. These findings show that residual NO modulates the contractile responses in isolated rat mesenteric arteries by exerting a tonic inhibitor effect on the depolarization and vasoconstriction induced by phenylephrine.

Combined oral contraceptive-induced hypertension is accompanied by endothelial dysfunction and upregulated intrarenal angiotensin II type 1 receptor gene expression.

Combined oral contraceptive (COC) use is associated with increased risk of developing hypertension. Activation of the intrarenal renin-angiotensin system (RAS) and endothelial dysfunction play an important role in the development of hypertension. We tested the hypothesis that COC causes hypertension that is associated with endothelial dysfunction and upregulation of intrarenal angiotensin-converting enzyme 1 (Ace1) and angiotensin II type 1 receptor (At1r). Female Sprague-Dawley rats aged 12weeks received (p.o.) olive oil (control) and a combination of 0.1μg ethinylestradiol and 1.0μg norgestrel (low COC) or 1.0μg ethinylestradiol and 10.0μg norgestrel (high COC) daily for 6weeks. Blood pressure was recorded by tail cuff plethysmography. Expression of genes in kidney cortex was determined by quantitative real-time polymerase chain reaction. COC treatment led to increased blood pressure, circulating uric acid, C-reactive protein and plasminogen activator inhibitor-1, renal uric acid, and expression of renal Ace1 and At1r. COC treatment resulted in increased contractile responses to phenylephrine in endothelium-denuded aortic rings. Endothelium-dependent relaxation responses to acetylcholine, but not endothelium-independent relaxation responses to nitric oxide (NO) donation by sodium nitroprusside, were attenuated in COC-exposed rings. Impaired relaxation responses to acetylcholine were masked by the presence of NO synthase inhibitor (L-NAME) in the COC-exposed rings, whereas the responses to acetylcholine in the presence of selective cyclooxygenase-2 inhibitor (NS-398) were enhanced. These findings indicate that COC induces hypertension that is accompanied by endothelial dysfunction, upregulated intrarenal Ace1 and At1r expression, and elevated proinflammatory biomarkers.

Resting tension is important for the contractile response but not to vasodilatation in renal hypertensive rat aorta

Nitric oxide (NO) produced by the NO‐Synthase (NOS) controls vascular tone and systemic arterial pressure. Shear stress‐mediated phosphorylation of NOS regulates NOS activity. Phosphorylation of Ser1177 (P‐Ser) activates NOS and Thr495 (P‐Thr) inactivates NOS. Protein kinase Akt/PI3K and c‐Src are enzymes that can phosphorylate these residues, but the signaling in renal hypertensive (2K‐1C) rat aorta submitted to increasing resting tension is not known. This study aimed to evaluate the effects of the resting tension of 1.5 g (control) and 3.0 g on the eNOS phosphorylation in phenylephrine (PE)‐induced contractile response in isolated 2K‐1C rat aorta. Concentration‐effect curves for PE were constructed in aortas under resting tension of 1.5 g and 3.0 g in the absence or presence of NOS inhibitor (L‐NAME, 100 μM). On the top of maintained contraction to PE, concentration‐effect curves for acetylcholine (ACh) were constructed. The expression of P‐Ser and P‐Thr were evaluated by Western Blot. The maximum effect induced by PE on resting tension of 1.5 g was lower in 2K‐1C (1.2 ± 0.2g, n=7; p&lt;0.001) than in normotensive 2K (2.2 ± 0.1g; n=5). This effect was normalized by NOS inhibition with L‐NAME. Both P‐Ser and P‐Thr expressions were higher in 2K‐1C than in 2K aortas. In 2K‐1C the increased P‐Ser was inhibited by PI3K/Akt inhibitor (wortmannin, 20 μM) and the increased P‐Thr was inhibited by c‐Src inhibitor (PP1, 10 μM). Under resting tension of 3.0 g, PE‐induced contractile response was similar in 2K‐1C and 2K aortas. Increased resting tension from 1.5 g to 3.0 decreased P‐Ser and increased P‐Thr in 2K aorta, but it was not modified in 2K‐1C aorta. ACh‐induced relaxation was lower in 2K‐1C (81.4 ± 4.7%; n=7; p&lt;0.001) than in 2K (100.5 ± 2.4%; n=5), that was not modified under 3.0 g resting tension in 2K and 2K‐1C. Taken together, our results demonstrate that the increasing resting tension from 1.5 g to 3.0 g decreases eNOS phosporylation in the activation site (P‐Ser) in 2K rat aorta. The anti‐contractile effect induced by PE in 2K‐1C rat aorta under 1.5 g tension is due to eNOS activation in a PI3K/Akt dependent way. The anti‐contractile effect is abolished by increasing the resting tension to 3.0 g due to P‐Thr in a c‐Src dependent way. These results indicate that resting tension is important for the contractile response, but not to vasodilatation in renal hypertensive (2K‐1C) rat aorta.Support or Funding InformationSupported by FAPESP, CNPq and CAPES.

Stress response to cadmium and manganese in Paracentrotus lividus developing embryos is mediated by nitric oxide

Increasing concentrations of contaminants, often resulting from anthropogenic activities, have been reported to occur in the marine environment and affect marine organisms. Among these, the metal ions cadmium and manganese have been shown to induce developmental delay and abnormalities, mainly reflecting skeleton elongation perturbation, in the sea urchin Paracentrotus lividus, an established model for toxicological studies. Here, we provide evidence that the physiological messenger nitric oxide (NO), formed by l-arginine oxidation by NO synthase (NOS), mediates the stress response induced by cadmium and manganese in sea urchins. When NO levels were lowered by inhibiting NOS, the proportion of abnormal plutei increased. Quantitative expression of a panel of 19 genes involved in stress response, skeletogenesis, detoxification and multidrug efflux processes was followed at different developmental stages and under different conditions: metals alone, metals in the presence of NOS inhibitor, NO donor and NOS inhibitor alone. These data allowed the identification of different classes of genes whose metal-induced transcriptional expression was directly or indirectly mediated by NO. These results open new perspectives on the role of NO as a sensor of different stress agents in sea urchin developing embryos.

The production of vasoconstriction-induced residual NO modulates perfusion pressure in rat mesenteric vascular bed

In the presence of nitric oxide synthase (NOS) inhibitors, the contribution of residual NO to endothelium-dependent relaxation induced by chemical agonists acetylcholine and bradykinin has been documented in resistance vessels. However, the contribution of residual NO to the vasodilatation in response to pressure and fluid shear stress is not well understood. In this study, to demonstrate the activity of residual NO, we applied a NO scavenger, hydroxocobalamin (HCX), on the phenylephrine-induced increase in perfusion pressure in the presence of NOS inhibitors, Nω-nitro-L-arginine (L-NA) or Nω-nitro-L-arginine methyl ester (L-NAME) in the rat perfused mesenteric bed. The perfusion pressure was increased by phenylephrine (1-2 µM), an α1-adrenoceptor agonist. This increase was augmented by the addition of L-NA or L-NAME. In the presence of any NOS inhibitors, the application of hydroxocobalamin (100 µM) further increased the perfusion pressure. The removal of endothelium by saponin (50 mg/L) and the use of a non-selective protein kinase inhibitor, staurosporine (5 nM), and a tyrosine kinase inhibitor, erbstatin A (30 µM), but not a calmodulin inhibitor, calmidazolium (0.5 µM), inhibited the additional pressor responses induced by L-NA or L-NAME and a combination of either of them with hydroxocobalamine. These findings show that there could be a NOS inhibitor-resistant residual NO production in response to pressure in the rat mesenteric vascular bed. This residual NO production may be associated with the activation of tyrosine kinase and protein kinases, but not calmodulin. Finally, this pressure-induced residual NO exerts a modulatory role against vasoconstriction induced by phenylephrine.

Arginine NO-dependent and NO-independent effects on hemodynamics

l-arginine administration decreases mean arterial blood pressure (MABP), presumably by excess nitric oxide (NO) synthesis. However, some reports indicate that d-arginine, not a substrate of NO synthase (NOS), also induces hypotension. To clarify this phenomenon, the hemodynamic effects of l- and d-arginine and their modification by NOS inhibition with l-nitroarginine methyl ester (l-NAME) were assessed. MABP, cardiac output, stroke volume, heart rate and systemic vascular resistance were recorded in Sprague-Dawley rats under urethane or ketamine/diazepam anesthesia, with or without blockade of NO synthesis by l-NAME. Both stereoisomers of arginine induced a dose-related drop in MABP of similar magnitude and time course, but recovery from hypotension was slower in l-arginine than in d-arginine. The hypotension induced by both stereoisomers was due to a decrease in systemic vascular resistance (SVR) with increase in cardiac output (CO) and stroke volume (SV). Administration of l-NAME induced a pronounced increase in MABP and SVR, with decreases in CO and heart rate (HR). Infusion of l-arginine after l-NAME significantly decreased MABP and SVR at the highest dose while d-arginine failed to do so. After l-NAME, MABP was significantly lower under l-arginine than under d-arginine at all doses. These experiments suggest a dual mechanism in the hypotensive effect of l-arginine: a NO independent action on vascular resistance shared with d-arginine, and a NO dependent mechanism that becomes evident in the presence of NOS inhibition with l-NAME. Cardiac effects of NO do not appear to play a role in l-arginine hypotension.

The (pro)renin receptor blocker handle region peptide upregulates endothelium-derived contractile factors in aliskiren-treated diabetic transgenic (mREN2)27 rats

Elevated prorenin levels associate with microvascular complications in patients with diabetes mellitus, possibly because prorenin affects vascular function in diabetes mellitus, for example by generating angiotensins following its binding to the (pro)renin receptor [(P)RR]. Here we evaluated whether the renin inhibitor aliskiren, with or without the putative (P)RR antagonist handle region peptide (HRP) improved the disturbed vascular function in diabetic TGR(mREN2)27 rats, a high-prorenin, high-(P)RR hypertensive model. Telemetry transmitters were implanted to monitor blood pressure. After 3 weeks of treatment, rats were sacrificed, and iliac and mesenteric arteries were removed to evaluate vascular reactivity. Diabetes mellitus enhanced the contractile response to nitric oxide synthase (NOS) blockade, potentiated the response to phenylephrine, diminished the effectiveness of endothelin type A (ETA) receptor blockade and allowed acetylcholine to display constrictor, cyclo-oxygenase-2 mediated, endothelium-dependent responses in the presence of NOS inhibition and blockers of endothelium-derived hyperpolarizing factors. Aliskiren normalized blood pressure, suppressed renin activity, and reversed the above vascular effects, with the exception of the altered effectiveness of ETA receptor blockade. Remarkably, when adding HRP on top of aliskiren, its beneficial vascular effects either disappeared or were greatly diminished, although HRP did not alter the effect of aliskiren on blood pressure and renin activity. Renin inhibition improves vascular dysfunction in diabetic hypertensive rats, and HRP counteracts this effect independently of blood pressure and angiotensin. (P)RR blockade therefore is unlikely to be a new tool to further suppress the renin-angiotensin system (RAS) on top of existing RAS blockers.

Apocynum venetum leaf extract, an antihypertensive herb, inhibits rat aortic contraction induced by angiotensin II: A nitric oxide and superoxide connection

Ethnopharmacological relevanceThe leaves extract of Apocynum venetum (AVLE), also known as “luobuma”, have long been used in traditional Chinese medicine to treat hypertension and depression in parts of China and it has been shown to possess anti-oxidant and anti-lipid peroxidation effects. AVLE (10μg/ml) has been reported to have a long-lasting endothelium-dependent relaxant effect and this effect has been proposed to be due to its nitric oxide(NO)-releasing and superoxide anion(SOA)-scavenging properties. Aim of the studyThe present study seeks to evaluate the differential actions of AVLE extract between Ang II- and PE-induced vasoconstriction and the involvement of superoxide anions. Materials and methodsSingle dose of Ang II (100nM and 1nM)- or PE (0.1μM)-induced contraction were assessed in both endothelium-intact and -denuded aortic rings after pre-incubation of AVLE (10μg/ml) for 15min. The experiment was repeated in either the presence of NO synthase inhibitor, l-NAME (300μM) or selective AT1 receptor inhibitor, losartan (0.1nM), or superoxide scavenger, tiron (1mM) or a combination of l-NAME and AVLE. Superoxide production was measured by using enhanced-chemiluminescence assay. ResultsWe have demonstrated that AVLE (10μg/ml) effectively suppressed the Ang II-induced contraction (100nM and 1nM) of both endothelium-intact and -denuded rat aortic rings. In endothelium-intact rings, l-NAME, reversed AVLE-induced inhibition of Ang II-contraction. PE-induced contraction was significantly inhibited by AVLE in endothelium-intact rings, but not in endothelium-denuded rings. The inhibition by AVLE of PE-induced contraction was totally abolished in the presence of l-NAME. Ang II-induced SOA production concentration dependently with the optimal effect seen at 100nM of Ang II, and AVLE (0.3, 1, 10μg/ml) reduced this effect. SOA production in Ang II-stimulated rings was significantly higher than unstimulated control rings, while PE did not stimulate SOA production at all. SOA formation in the presence of Ang II was also inhibited in the presence of SOD (superoxide scavenger), DPI (NADPH inhibitor) and losartan (specific AT1 receptor antagonist). ConclusionThese results collectively suggest that the ability of AVLE in inhibiting Ang II-induced contraction via its SOA scavenging properties and nitric oxide releasing effect may account for its usage as an antihypertensive treatment in traditional folk medicine.

Abstract 17410: Pre-Treatment with Liposomal 6R-BH4 but not 6S-BH4 Induces in vivo Cardioprotection Through Maintenance of Nitric Oxide Synthase Coupling

Tetrahydrobiopterin (BH4) is an essential co-factor for nitric oxide synthase (NOS) activity and BH4 depletion leads to endothelial NOS (eNOS) uncoupling with increased superoxide (O 2 -. ) generation. Myocardial BH4 levels are decreased with ischemia/reperfusion (I/R) and have been hypothesized to play a critical role in modulating eNOS activity and myocardial salvage. BH4 treatment exerts myocardial protection; however, questions remain regarding the related mechanism and role of NOS coupling versus nonspecific antioxidative effects of BH4. To address this issue, rats were subjected to 60-min in vivo coronary artery occlusion and varying periods (10-min, 20-min or 24-hr) of reperfusion with or without liposomal 6R-BH4 or 6S-BH4 supplementation (1 mg/kg, iv, 5-min before ischemia). Myocardial NO and O 2 -. production were measured in frozen heart sections using the fluorescence probes (CuFL and DHE, respectively) in the absence or presence of NOS inhibitor or SOD mimetic. Myocardial infarct size was determined 24-hr after reperfusion and compared with NOS inhibitor (L-NAME) or 6R-BH4 or 6S-BH4 (that lacks NOS co-factor activity). Compared to vehicle group, 6R-BH4 treatment caused an increase in NO with decrease in O 2 -. in I/R hearts. Postischemic NO or O 2 -. -derived fluorescence was largely abolished by added NOS inhibitor L-NAME (1 mM) or the SOD-mimetic MnTBAP (50 μM), respectively. 6R-BH4 reduced myocardial infarct size by ∼50% compared to vehicle group (31±2% vs. 59±2%, n=8). L-NAME pre-treatment (10 mg/kg, iv, 15-min before vehicle or 6R-BH4) had no effect on vehicle group, but it abolished the infarct-limiting effect of 6R-BH4 treatment. Compared to vehicle, 6S-BH4 had minimal effect on myocardial infarction (59±2% vs. 50±3%, n=6-8). Thus, these findings demonstrate that the protective effect of BH4 is primarily due to restoration of NOS coupling with suppression of O 2 -. and enhancement of NO production from the enzyme.

Molecular and biochemical characterization of nitric oxide synthase isoforms and their intracellular distribution in human peripheral blood mononuclear cells

Nitric oxide synthase (NOS) expression and catalytic status in human peripheral blood mononuclear cells (PBMCs) is debatable, while its sub-cellular distribution remains unascertained. The present study characterizes NOS transcripts by real time PCR, NOS protein by immunoprecipitation (IP)/Western blot (WB), nitric oxide (NO) generation by DAF-2DA and NOS sub-cellular distribution by immunogold electron microscopy in resting PBMCs, monocytes and lymphocytes obtained from healthy donors. We observed constitutive expression of full length NOS isoforms (nNOS, iNOS and eNOS) in PBMCs: with the highest expression of iNOS in comparison to nNOS and eNOS. Isolated monocytes expressed more eNOS transcript and protein as compared to nNOS and iNOS. Lymphocytes however had more iNOS transcripts and protein than nNOS and eNOS. NOS was catalytically active in PBMCs, monocytes as well as in lymphocytes as evident by NO generation in the presence of substrate and cofactors, which was significantly reduced in the presence of NOS inhibitor. Immunogold electron microscopy and morphometric analysis revealed the distinct pattern of NOS distribution in monocytes and lymphocytes and also exhibited differences in the nuclear–cytoplasmic ratio. nNOS localization was much more in the cytosol than in the nucleus among both monocytes and lymphocytes. Interestingly, iNOS distribution was comparable in both cytosol and nucleus among monocytes, but in lymphocytes iNOS was predominantly localized to the cytosol. The present study exhibits constitutive presence of all the NOS isoforms in PBMCs and reports the distinct pattern of NOS distribution among monocytes and lymphocytes.

A flow-cytometry assisted segregation of responding and non-responding population of endothelial cells for enhanced detection of intracellular nitric oxide production

Nitric oxide (NO) is an important paracrine substance released by the endothelium to regulate vasomotor tone. The constitutive levels of endothelium dependent NO production is low. However, it is induced significantly in response to certain environmental and biological stimuli. An accurate evaluation of such stimulus induced NO release is of pharmacological significance. We observed that the sensitivity of NO detection in endothelial cells is compromised by baseline fluorescence emanated from non-activated cells resulting in ambiguous detection. In order to measure NO levels in activated population independent of non-activated cells, we segregated DAF-FM loaded cells based on their fluorescence intensity using flow-cytometry. Specific agonists like bradykinin, VEGF and insulin enhanced the proportion of activated cells. This effect was partially blocked in presence of NO synthase inhibitor, N G-nitro- l-arginine-methyl ester ( l-NAME). We demonstrate that the fluorescence yield of activated population serves as a sensitive measure to evaluate agonist induced nitric oxide production in endothelial cells. Such increase in NO production in activated cells was also associated with increased eNOS phosphorylation at Ser-1177. While the endothelial cells showed heterogeneity with respect to NO production, immuno-phenotyping for endothelial cell-surface markers revealed a homogenous population.

The role of nitric oxide in the treatment of tumours with aminolaevulinic acid-induced photodynamic therapy

Photodynamic therapy (PDT) is a local cancer treatment which induces cell death by the interaction of light with a photosensitizing drug. Previous studies indicate that nitric oxide (NO) plays a role in Photofrin-PDT, but this has not been investigated in aminolaevulinic acid (ALA)-PDT. The current study determines whether inhibition of nitric oxide synthase (NOS) activity modulates treatment responses to ALA-PDT, in tumours displaying differential levels of NO. Murine tumours with low (EMT6) or high (RIF-1) NO levels were implanted into the cremaster muscle of BALB/c or C3H/HeN mice respectively. Animals were prepared for in vivo microscopy 7–14 days later. Mice received oral ALA (200 mg/kg) 4 h before PDT. l-NAME, l-NNA or 1400 W (10 mg/kg) were administered via the jugular vein 5 min before PDT. NOS inhibition ( l-NAME or l-NNA) combined with ALA-PDT in RIF-1 tumours demonstrated enhanced damage to both the tumour and normal microvasculature, with increased macromolecular leak and reduction in vessel diameter, whereas ALA-PDT alone had no effect. In contrast, EMT6 tumours responded to ALA-PDT alone but sensitivity was not enhanced in the presence of NOS inhibition. 1400 W combined with ALA-PDT induced similar microvascular effects to l-NAME in both tumours, but were less pronounced. The data demonstrates that NO has an important role in events likely to be critical for treatment response, sensitivity and therapeutic outcome of ALA-PDT.

Inhibition of Nitric Oxide Synthases Abrogates Pregnancy-Induced Uterine Vascular Expansive Remodeling

Background/Aims: It was the aim of this study to test the hypothesis that hypertension and/or inhibition of nitric oxide (NO) synthases alters uterine vascular remodeling during pregnancy. Methods: Using a model of hypertension (NO synthase inhibition with L-NAME) in nonpregnant and pregnant rats, comparisons were made with age-matched controls, as well as with animals receiving hydralazine along with L-NAME to maintain normotension in the presence of NO synthase inhibition. Circumferential and axial remodeling of large (main uterine, MUA) and small (premyometrial radial) arteries were quantified and compared. Results:L-NAME treatment prevented expansive circumferential remodeling of the MUA; cotreatment with hydralazine was without effect. Circumferential remodeling of smaller premyometrial radial arteries was also significantly attenuated in hypertensive pregnant animals, while premyometrial radial arteries from rats receiving hydralazine with L-NAME were of intermediate diameter. Neither hypertension nor NO synthase inhibition had any effect on the substantial (200–300%) axial growth of MUA or premyometrial radial arteries. Conclusion: NO plays a major role in facilitating pregnancy-induced expansive remodeling in the uterine circulation, particularly in larger arteries. Some beneficial effects of hydralazine on expansive circumferential remodeling were noted in smaller radial vessels, and these may be linked to its prevention of systemic hypertension and/or to local effects on the arterial wall. Neither NO synthase inhibition nor hypertension had any effect on arterial longitudinal growth.

Abstract 378: Gene Delivery of H11 Kinase/Hsp22 Provides Cardioprotection by A Nitric Oxide-dependent Mechanism

H11 kinase/Hsp22 (H11K) is a small heat shock protein (hsp) which is upregulated in ischemic heart disease, both in animal models and in patients. Cardiac-specific overexpression of H11K in a transgenic (TG) mouse provides protection against ischemic damage that is equivalent to ischemic preconditioning (IPC). Because studies in mice can not always be translated to patients, we examined the role of H11K in mediating cardiomyopathy in a swine model. An adenovirus harboring the H11K sequence, or a LacZ control, was injected in the potential area-at-risk (AAR) perfused by the left circumflex coronary artery (CA) in chronically instrumented swine (n=5/group). Three days after injection, CA occlusion (O) for 60 min, followed by 3 days of reperfusion (R), induced myocardial infarction. Although the AAR was similar between groups, the MI/AAR was reduced significantly (P&lt;0.05) in the H11K-injected group (32±5%) compared to the LacZ group (50±5%). Three days following reperfusion, regional wall thickening in the ischemic region was reduced by 58±2% of baseline in H11K group vs. 82±7% in LacZ (P&lt;0.05). Compared to LacZ, H11K-injected myocardium showed a significant (P&lt;0.05) 4-fold increase in H11K, and a 2- to 4-fold increase in the expression of the inducible form of nitric oxide synthase (NOS), a key mediator of IPC. To substantiate a role of NOS, the effects of CAO/CAR were examined in H11K-injected pigs (n=4) after pretreatment with 35 mg/kg of the NOS inhibitor L-NNA. In these pigs, the cardioprotection of H11K was abolished, i.e., MI/AAR was 67±6% (NS vs. LacZ control). These results were further substantiated in the TG mouse, pretreated or not with L-NNA for 2 days. After 45 min no-flow ischemia followed by 6h reperfusion, the MI/AAR was 5.0±0.4% in non-treated TG and 36±6% in L-NNA-treated TG mice (P&lt;0.01). Therefore, overexpression of H11K in a large mammalian model of ischemic heart disease induces significant cardioprotection. The mechanism involves upregulation of iNOS, since cardioprotection is no longer evident in the presence of NOS inhibition with L-NNA.

Globular adiponectin increases cGMP formation in blood platelets independently of nitric oxide.

Platelet-derived nitric oxide (NO) has been shown to play conflicting roles in platelet function, although it is accepted that NO mediates its actions through soluble guanylyl cyclase (sGC). This confusion concerning the roles of platelet NO may have arisen because of an uncharacterized mechanism for activation of sGC. To examine the ability of the novel platelet agonist globular adiponectin (gAd) to stimulate the NO-independent cGMP-protein kinase G (PKG) signaling cascade. We used three independent markers of NO signaling, [(3)H]l-citrulline production, cGMP accrual, and immunoblotting of vasodilator-stimulated phosphoprotein (VASP), to examine the NO signaling cascade in response to gAd. gAd increased platelet cGMP formation, resulting in a dose- and time-dependent increase in phospho-VASP(157/239). Phosphorylation of VASP in response to gAd was mediated by both protein kinase A and PKG. Importantly, cGMP formation occurred in the absence of NO synthase (NOS) activation and in the presence of NOS inhibitors. Indeed, inhibition of the NOS signaling cascade had no influence on gAd-mediated platelet aggregation. Exploration of the mechanism demonstrated that NO-independent cGMP formation, phosphorylation of VASP and association of sGCalpha(1) with heat shock protein-90 induced by gAd were blocked under conditions that inhibited Src kinases, implying a tyrosine kinase-dependent mechanism. Indeed, sGCalpha1 was reversibly tyrosine phosphorylated in response to gAd, collagen, and collagen-related peptide, an effect that required Src kinases and downstream Ca(2+) mobilization. These data demonstrate activation of the platelet cGMP signaling cascade by a novel tyrosine kinase-dependent mechanism in the absence of NO.