Nω-nitro L-arginine Research Articles

The rapid release of corticosterone from the adrenal induced by ACTH is mediated by nitric oxide acting by prostaglandin E2.

The adrenal cortex is a major stress organ in mammals that reacts rapidly to a multitude of external and internal stressors. Adrenocorticotropin (ACTH) is the main stimulator of the adrenal cortex, activating corticosteroid synthesis and secretion. We evaluated the mechanism of action of ACTH on adrenals of male rats, preserving the architecture of the gland in vitro. We demonstrated that both sodium nitroprusside (NP), a nitric oxide (NO) donor, and ACTH stimulate corticosterone release. NO mediated the acute response to ACTH because Nomega-nitro-l-arginine methyl ester, a NO synthase inhibitor, and hemoglobin, a NO scavenger, blocked the stimulation of corticosterone release induced by ACTH. NP stimulated prostaglandin E release, which in turn stimulated corticosterone release from the adrenal. Additionally, indomethacin, which inhibits cyclooxygenase, and thereby, prostaglandin release, prevented corticosterone release from the adrenal induced by both NP and ACTH, demonstrating that prostaglandins mediate acute corticosterone release. Corticosterone content in adrenals after incubation with ACTH or NP was lower than in control glands, indicating that any de novo synthesis of corticosterone during this period was not sufficient to keep up with the release of the stored hormone. The release induced by ACTH or NP depleted the corticosterone content in the adrenal by approximately 40% compared with the content of glands incubated in buffer. The mechanism of rapid release is as follows: NO produced by NO synthase activation by ACTH activates cyclooxygenase, which generates PGE(2), which in turn releases corticosterone stored in microvesicles and other organelles.

Soluble Guanylate Cyclase Activator Reverses Acute Pulmonary Hypertension and Augments the Pulmonary Vasodilator Response to Inhaled Nitric Oxide in Awake Lambs

Inhaled nitric oxide (NO) is a potent and selective pulmonary vasodilator, which induces cGMP synthesis by activating soluble guanylate cyclase (sGC) in ventilated lung regions. Carbon monoxide (CO) has also been proposed to influence smooth muscle tone via activation of sGC. We examined whether direct stimulation of sGC by BAY 41-2272 would produce pulmonary vasodilation and augment the pulmonary responses to inhaled NO or CO. In awake, instrumented lambs, the thromboxane analogue U-46619 was intravenously administered to increase mean pulmonary arterial pressure to 35 mm Hg. Intravenous infusion of BAY 41-2272 (0.03, 0.1, and 0.3 mg x kg(-1) x h(-1)) reduced mean pulmonary arterial pressure and pulmonary vascular resistance and increased transpulmonary cGMP release in a dose-dependent manner. Larger doses of BAY 41-2272 also produced systemic vasodilation and elevated the cardiac index. N(omega)-nitro-l-arginine methyl ester abolished the systemic but not the pulmonary vasodilator effects of BAY 41-2272. Furthermore, infusing BAY 41-2272 at 0.1 mg x kg(-1) x h(-1) potentiated and prolonged the pulmonary vasodilation induced by inhaled NO (2, 10, and 20 ppm). In contrast, inhaled CO (50, 250, and 500 ppm) had no effect on U-46619-induced pulmonary vasoconstriction before or during administration of BAY 41-2272. In lambs with acute pulmonary hypertension, BAY 41-2272 is a potent pulmonary vasodilator that augments and prolongs the pulmonary vasodilator response to inhaled NO. Direct pharmacological stimulation of sGC, either alone or in combination with inhaled NO, may provide a novel approach for the treatment of pulmonary hypertension.

The fusicoccin-induced accumulation of nitric oxide in sycamore cultured cells is not required for the toxin-stimulated stress-related responses

Fusicoccin (FC), a well-known phytotoxin acting as a 14-3-3 protein-mediated activator of the plasma membrane H +-ATPase, has been recently shown to induce a number of stress-related responses in sycamore ( Acer pseudoplatanus L.) cultured cells. In this paper, we show that FC induces nitric oxide (NO) production, resulting in an accumulation of NO in the culture medium and in the cells. The accumulation is completely abolished by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), unaffected by the nitric oxide synthase inhibitor Nω-nitro- l-arginine (NNA) and almost completely suppressed by the nitrate reductase inhibitor sodium tungstate. Scavenging of NO by cPTIO does not prevent the stimulating effect of FC on stress-related responses, namely the H 2O 2 production, the leakage of cytochrome c from the mitochondria, the accumulation of cytosolic 14-3-3s and of the endoplasmic reticulum-resident molecular chaperone binding protein (BiP). This suggests that NO does not act as a signal for the other FC-induced responses here considered, but rather its production can be regarded as another symptom of a stress condition brought about by the toxin.

Brain mitochondrial nitric oxide synthase: in vitro and in vivo inhibition by chlorpromazine

Mouse brain mitochondria have a nitric oxide synthase (mtNOS) of 147 kDa that reacts with anti-nNOS antibodies and that shows an enzymatic activity of 0.31–0.48 nmol NO/min mg protein. Addition of chlorpromazine to brain submitochondrial membranes inhibited mtNOS activity (IC 50 = 2.0 ± 0.1 μM). Brain mitochondria isolated from chlorpromazine-treated mice (10 mg/kg, i.p.) show a marked (48%) inhibition of mtNOS activity and a markedly increased state 3 respiration (40 and 29% with malate–glutamate and succinate as substrates, respectively). Respiration of mitochondria isolated from control mice was 16% decreased by arginine and 56% increased by NNA ( N ω-nitro- l-arginine) indicating a regulatory activity of mtNOS and NO on mitochondrial respiration. Similarly, mitochondrial H 2O 2 production was 55% decreased by NNA. The effect of NNA on mitochondrial respiration and H 2O 2 production was significantly lower in chlorpromazine-added mitochondria and absent in mitochondria isolated from chlorpromazine-treated mice. Results indicate that chlorpromazine inhibits brain mtNOS activity in vitro and can exert the same action in vivo.

Cerebral vascular dysfunction mediated by superoxide in hyperhomocysteinemic mice.

Hyperhomocysteinemia is an emerging risk factor for stroke, but little is known about effects of hyperhomocysteinemia on cerebral vascular function. We tested the hypothesis that chronic hyperhomocysteinemia in mice causes endothelial dysfunction in cerebral arterioles through a mechanism that involves superoxide. Mice heterozygous for a targeted disruption of the cystathionine beta-synthase gene (Cbs+/-) and their wild type littermates (Cbs+/+) were fed either a control diet or a high-methionine diet for 10 to 12 months. Plasma total homocysteine was elevated with the high-methionine diet compared with the control diet for both Cbs+/+ (7.9+/-1.0 versus 5.0+/-0.5 micromol/L; P<0.05) and Cbs+/- (20.5+/-3.1 versus 8.2+/-0.9 micromol/L; P<0.001) mice. Dilatation of cerebral arterioles ( approximately 30 microm baseline diameter) was measured in vivo in response to the endothelium-dependent dilator acetylcholine or the endothelium-independent dilator nitroprusside. Vasodilatation to acetylcholine was impaired with the high-methionine diet compared with the control diet for both Cbs+/+ and Cbs+/- mice (P<0.01). Dilatation of arterioles to acetylcholine was restored toward normal by the superoxide scavenger tiron (P<0.05). Vasodilatation to nitroprusside was not influenced by Cbs genotype or diet. Dihydroethidium (DHE) staining for vascular superoxide was elevated in Cbs+/- mice fed the high-methionine diet and was inhibited by apocynin or Nomega-nitro-l-arginine methyl ester, implicating NAD(P)H oxidase and nitric oxide synthase as potential sources of superoxide. Superoxide is a key mediator of endothelial dysfunction in the cerebral circulation during diet-induced hyperhomocysteinemia.

Vasorelaxation induced by common edible tropical plant extracts in isolated rat aorta and mesenteric vascular bed

In this study, the vasodilatory actions of nine edible tropical plant extracts were investigated. Ipomoea batatas (sweet potato leaf), Piper betle (betel leaf), Anacardium occidentale (cashew leaf), Gynandropsis gynandra (maman leaf), Carica papaya (papaya leaf), and Mentha arvensis (mint leaf) extracts exhibited more than 50% relaxing effect on aortic ring preparations, while Piper betle and Cymbopogon citratus (lemongrass stalk) showed comparable vasorelaxation on isolated perfused mesenteric artery preparation. The vascular effect on the aortic ring preparations were mainly endothelium-dependent, and mediated by nitric oxide (NO) as supported by the inhibition of action in the presence of N ω-nitro- l-arginine (NOLA), an nitric oxide synthase (NOS) inhibitor, or by the removal of endothelium. In contrast, vasodilatory actions in resistance vessels (perfused mesenteric vascular beds) appear to involve several biochemical mediators, including NO, prostanoids, and endothelium-dependent hyperpolarizing factors (EDHFs). Total phenolic contents and antioxidant capacities varied among different extracts and found to be independent of vascular relaxation effects. This study demonstrates that many edible plants common in Asian diets to possess potential health benefits, affording protection at the vascular endothelium level.

Structural diversity of NADPH diaphorase‐reactive enteral networks in Stylommatophora (Gastropoda, Pulmonata)

Abstract. In this work we investigated the involvement of putative nitric oxide (NO)‐forming neurons in enteric plexuses of stylommatophoran gastropods. The nitric oxide synthase (NOS)‐containing cells were detected by NADPH diaphorase (NADPHd) histochemistry in the entreral nervous systems of several stylommatophoran species (Achatinacea: Achatina fulica, Helicacea: Cepaea hortensis, Cepaea nemoralis, Discus rotundatus, Helicella obvia, Helix lucorum, Helix lutescens, Monachoides umbrosa, Trichia hispida, Zebrina detrita, Succineacea: Succinea putris, Vertiliginacea: Clausilia dubia, Zonitacea: Arion ater, Arion subfuscus, Limax maximus). We detected the NO synthesis of isolated midintestinal segments by Griess's quantification of nitrite, one end product of NO. Effects of the NOS substrate L‐arginine and the NOS inhibitor Nω‐nitro‐L‐arginine (NOARG) were also tested on nitrite production. We found NADPHd‐reactive neurons and extrinsic nerves with NADPHd‐stained fibers within the myenteric and submucosal networks of the midintestine of investigated members of Helicacea, Succineacea, and Vertiliginacea families. These networks innervated the midintestinal musculature and several nerve cells of the myenteric and submucosal plexi. In investigated members of Achatinacea and Zonitacea, NADPHd‐stained networks were not detectable within the digestive tract. Administration of 1 mM L‐arginine elevated, whereas 2 mM of NOARG diminished, the nitrite levels of the NADPHd‐stained networks containing midintestine in C. nemoralis and H. lucorum. Enteral NADPHd staining was not detected in A. ater and L. maximus, and the nitrite production was not affected by L‐arginine. Our results indicate a possible, but evolutionarily not conserved, NO‐mediated enteral transmission in stylommatophoran gastropods.

Reduced NOS3 phosphorylation mediates reduced NO/cGMP signaling in mesenteric arteries of deoxycorticosterone acetate-salt hypertensive rats.

Salt-sensitive hypertension is associated with impaired NO/cGMP signaling. We hypothesized that increased superoxide production by NADPH oxidase and altered endothelial NO synthase (NOS3) phosphorylation determine endothelial dysfunction in hypertension. Experiments tested if NO/cGMP signaling and NOS3 serine phosphorylation are decreased and NADPH oxidase activity is increased in mesenteric arteries from deoxycorticosterone acetate (DOCA)-salt rats compared with arteries from placebo rats. Concentration response curves to phenylephrine were performed in mesenteric arteries in the presence and absence of Nomega-nitro-L-arginine (LNA) and antioxidants to determine the influence of basal NO and superoxide production on vascular tone. LNA increased phenylephrine sensitivity in arteries from placebo, but not DOCA-salt rats, regardless of antioxidant treatment. To determine basal cGMP production, mesenteric arteries were incubated with 3-isobutyl-1-methylxanthine in the presence or absence of LNA, sodium nitroprusside (SNP), antioxidants, or tetrahydrobiopterin. NOS-dependent cGMP production was reduced in arteries from DOCA-salt rats compared with arteries from placebo rats and was not restored by acute treatment with antioxidants or tetrahydrobiopterin. SNP-induced cGMP production was similar between groups as was NADPH oxidase activity, measured by lucigenin chemiluminescence, in mesenteric arteries. Expression and phosphorylation of NOS3 were examined by Western blotting. Phosphorylation of NOS3 was decreased in arteries from DOCA-salt rats compared with placebo at serine residues 1179 and 635. These findings indicate that diminished NO/cGMP signaling in mesenteric arteries from DOCA-salt rats is caused by reduced phosphorylation of NOS3 at serine 1179 and serine 635, rather than NO scavenging by superoxide.

A Possible Stimulatory Effect of FMRFamide on Neural Nitric Oxide Production in the Central Nervous System of Helix lucorum L.

The anatomical and functional relationship between neurons expressing nitric oxide (NO) synthase and molluscan cardioexcitatory (FMRFamide)-like neuropeptides was studied in the central ganglia of Helix lucorum (Pulmonata, Gastropoda), applying NADPHdiaphorase (NADPHd) histochemistry to visualize NO synthase and immunocytochemistry to demonstrate FMRFamide (FMRFa) at the light microscopic level. The NO production of the ganglia was detected by the colorimetric Griess determination of nitrite, a breakdown product of NO. Effects of the NO synthase substrate amino acid L-arginine, the NO synthase inhibitor Nω-nitro-L-arginine (NOARG), synthetic FMRFa and the FMRFa sensitive ion channel blocker amiloride hydrochloride on nitrite production were also tested. NADPHd reaction labeled nerve cells and fibers in the procerebra, mesocerebra and metacerebra within the cerebral ganglia, and cell clusters in the postcerebral ganglia. FMRFa immunolabeling could be observed within subpopulations of NADPHd positive cells and in pericellular varicose fibers surrounding NADPHd stained neurons. Nitrite production of the ganglia was stimulated by L-arginine (10– 20 mM) but was decreased by NOARG (1–2 mM). Synthetic FMRFa (0.830–3.340 mM) increased the nitrite production in a dose dependent manner, but was ineffective in the presence of NOARG. Amiloride hydrochloride (7.890 mM) reduced the FMRFa evoked nitrite production in all ganglia. This is the first description of an anatomical relationship between putative NO producing and FMRFa containing cells, suggesting a possible regulatory role of FMRFa in the NO mediated signaling in an invertebrate nervous system.

Nitric oxide inhibits H2O2-induced transferrin receptor-dependent apoptosis in endothelial cells: Role of ubiquitin-proteasome pathway.

We investigated here the mechanism of cytoprotection of nitric oxide (*NO) in bovine aortic endothelial cells treated with H2O2. NONOates were used as *NO donors that released *NO slowly at a well defined rate in the extracellular and intracellular milieus. H2O2-mediated intracellular dichlorofluorescein fluorescence and apoptosis were enhanced by the transferrin receptor (TfR)-mediated iron uptake. *NO inhibited the TfR-mediated iron uptake, dichlorofluorescein fluorescence, and apoptosis in H2O2-treated cells. *NO increased the proteasomal activity and degradation of nitrated TfR via ubiquitination. Nomega-nitro-L-arginine methyl ester, a nonspecific inhibitor of endogenous *NO biosynthesis, decreased the trypsin-like activity of 26S proteasome. *NO, by activating proteolysis, mitigates TfR-dependent iron uptake, dichlorodihydrofluorescein oxidation, and apoptosis in H2O2-treated bovine aortic endothelial cells. The relevance of biological nitration on redox signaling is discussed.

Functional consequences of endothelial nitric oxide synthase uncoupling in congestive cardiac failure.

Impaired endothelium-mediated vasodilatation (EMVD) in congestive cardiac failure (CCF) has been linked to decreased nitric oxide (NO) bioavailability because of its interaction with vascular superoxide (O2*-), derived predominantly from NAD(P)H-dependent oxidases. When uncoupled from essential cofactors, endothelial nitric oxide synthase (eNOS) produces O2*-. We studied the functional consequences of eNOS uncoupling in relation to EMVD in patients with CCF. We employed the platelet as a compartmentalized ex-vivo model to examine O2*- and NO production. When eNOS is functioning normally, incorporation of Nomega-Nitro-L-Arginine methyl ester (L-NAME, 1 mmol/L), results in increased O2*- detection, as inhibition of NO production prevents NO scavenging of O2*-. This was observed in controls and 9 of the CCF patients, in whom O2*- detection increased by 63% and 101%, respectively. In the remaining 9 CCF patients, incorporation of L-NAME reduced O2*- production by 39%, indicating O2*- production by eNOS uncoupling. Detection of platelet-derived NO was significantly greater in eNOS-coupled platelets compared with the uncoupled group (2.8+/-1.4 versus 0.9+/-0.4 pmol/108 platelets, P=0.04). Endothelium-dependent and -independent vasodilator responses to acetylcholine and sodium nitroprusside recorded using venous occlusion plethysmography were significantly impaired in patients exhibiting eNOS uncoupling. This study provides first evidence that platelet eNOS can become uncoupled in human CCF. Impaired endothelium-dependent and -independent vasodilator responses and diminished platelet-derived NO production occurred in association with enzyme uncoupling.

Long-term treatment with N(omega)-nitro-L-arginine methyl ester causes arteriosclerotic coronary lesions in endothelial nitric oxide synthase-deficient mice.

N(omega)-nitro-L-arginine methyl ester (l-NAME) is widely used to inhibit endothelial synthesis of NO in vivo. However, it is controversial whether the long-term vascular effects of l-NAME are mediated primarily by inhibition of endothelial NO synthesis. We addressed this point in mice that are deficient in the endothelial NO synthase gene (eNOS-KO mice). Wild-type and eNOS-KO mice received l-NAME in drinking water for 8 weeks. In wild-type mice, long-term treatment with l-NAME caused significant medial thickening and perivascular fibrosis in coronary microvessels but not in large coronary arteries. Importantly, in eNOS-KO mice, treatment with l-NAME also caused an extent of medial thickening and perivascular fibrosis in coronary microvessels that was comparable to that in wild-type mice and that was not prevented by supplementation of L-arginine. Vascular NO and cGMP levels were not significantly reduced by l-NAME treatment, and no expression of inducible or neuronal NO synthase was noted in microvessels of eNOS-KO mice, suggesting an involvement of NO-independent mechanisms. Treatment with l-NAME caused an upregulation of vascular ACE and an increase in cardiac lucigenin chemiluminescence that were comparable in both strains and that were abolished by simultaneous treatment with temocapril (ACE inhibitor) or CS866 (angiotensin II type 1 receptor antagonist) along with the suppression of vascular lesion formation. These results provide the first direct evidence that the long-term vascular effects of l-NAME are not mediated by simple inhibition of endothelial NO synthesis. Direct upregulation of local ACE and increased oxidative stress appear to be involved in the long-term vascular effects of l-NAME in vivo.

Heart rate variability and baroreflex function in AT2 receptor-disrupted mice.

We adapted telemetry and sequence analysis employed in humans to mice and measured heart rate variability and the spontaneous baroreflex sensitivity in angiotensin II type 2 (AT2) receptor-deleted (AT2 -/-) and wild-type (AT2 +/+) mice with either deoxycorticosterone acetate (DOCA)-salt hypertension or N(omega)-nitro-L-arginine methylester hydrochloride (L-NAME) hypertension. Mean arterial pressure leveled during the day at 101+/-1 mm Hg and during the night at 109+/-1 mm Hg in AT2 receptor-deleted mice, compared with 98+/-2 mm Hg (day) and 104+/-2 mm Hg (night) in wild-type mice. Mean arterial pressure increased in AT2 receptor-deleted mice with L-NAME to 114+/-1 mm Hg (day) and 121+/-1 mm Hg (night), compared with 105+/-2 mm Hg (day) and 111+/-2 mm Hg (night), respectively. DOCA-salt also increased day and night blood pressures in AT2 receptor-deleted mice to a greater degree than in wild-type mice. Heart rate variability in the time and frequency domain was not different between AT2 receptor-deleted mice and AT2 receptor-deleted mice at baseline. Systolic blood pressure variability in the low frequency band was lower in AT2 receptor-deleted mice (0.6+/-0.1 ms2 versus 3.9+/-1.3 ms2) than in wild-type mice. Baroreceptor-heart rate reflex sensitivity was significantly increased in AT2 receptor-deleted mice compared with wild-type mice (3.4+/-0.6 versus 2.1+/-0.5 ms/mm Hg). These differences remained after DOCA-salt and L-NAME treatments. We conclude that activation of the AT2 receptor impairs arterial baroreceptor reflex function, probably by a central action. These data support the existence of an inhibitory central effect of the AT2 receptor on baroreflex function.

The cardiovascular and renal effects of acute and chronic inhibition of nitric oxide production in fetal sheep.

The acute and long-term effects of blockade of nitric oxide (NO) production were studied in six chronically catheterised fetal sheep aged from 116 and 118 days; six untreated fetal sheep received injections of saline. Injection of 10 mg (kg maternal body wt)(-1) of the nitric oxide synthase (NOS) inhibitor N(omega)-nitro-L-arginine (NOLA) to the fetus, caused an immediate rise in fetal mean arterial pressure (MAP, P < 0.005) and a reflex fall in fetal heart rate (FHR, P < 0.001). Plasma renin concentration (PRC) fell from 8.4 +/- 3.3 to 1.5 +/- 0.3 ng ml(-1) h(-1) (P < 0.001) and was dependent on MAP (P = 0.001). Glomerular filtration rate (GFR) tended to increase, but renal blood flow (RBF) velocity decreased (P < 0.001). Thus filtration fraction (FF) increased (P < 0.025). Urine flow and sodium excretion increased (P < 0.001 for both). Fractional sodium reabsorption decreased (P < 0.05). In fetuses treated with NOLA, arterial pressure was found to affect glomerular haemodynamics and renal tubular handling of sodium. No such relationships were observed in untreated fetuses. The vascular responses to acetylcholine tended to be less (P = 0.07) and the responses to noradrenaline were enhanced in NOLA-treated fetuses. There were no changes in untreated fetuses. Fetuses were then injected twice daily with either 5 mg kg(-1) NOLA or saline for the next 2 days. On the 4th day, injection of 10 mg kg(-1) NOLA did not have any effects on MAP, FHR or renal function. However, the pressor responses to angiotensin II (Ang II) were enhanced (P < 0.005), as was the response to noradrenaline but to a lesser extent. It is concluded that endothelial production of NO maintains normal fetal blood pressure, renal vascular resistance and fetal renal function. When NO production was blocked by repeated injections of NOLA, other vasodilator pathways took over the maintenance of cardiovascular and renal vascular tone. However, alterations in both cardiovascular and renal function were still present. That is, there was increased pressor sensitivity to exogenous Ang II and unmasking of effects of arterial pressure on glomerular and tubular function.

Altered nitric oxide synthase 3 distribution in mesenteric arteries of hypertensive rats.

A high-salt (HS) diet and angiotensin II (Ang II) are both associated with the development of hypertension and impaired endothelial function. We hypothesize that alterations in nitric oxide synthase (NOS) activity or subcellular localization of NOS 3 protein may contribute to endothelial dysfunction in salt-dependent hypertension. To test this hypothesis, two models of salt-dependent hypertension were studied: DOCA-salt and Ang II. For Ang II hypertension, rats were divided into 4 groups: control on normal or HS diet, Ang II-infused on normal or HS diet. The mesenteric arterial bed was isolated and frozen in liquid nitrogen. Frozen arteries were homogenized and separated into cytosolic and particulate fractions. NOS activity was assayed by determining the conversion of (3)H-arginine to (3)H-citrulline in the absence and presence of the NOS inhibitor, Nomega-nitro-L-arginine. NOS 3 protein expression was significantly increased in the cytosol of arteries from DOCA-salt compared with placebo rats and in Ang II-infused and Ang HS rats compared with control. NOS 3 expression in the particulate fraction was comparable among all groups. NOS activity (pmol/30 min/total protein) was significantly increased in the cytosolic fraction of arteries from DOCA-salt rats compared with placebo and in Ang HS rats compared with control. NOS activity was comparable in the particulate fraction in all rat groups. In conclusion, there is an altered subcellular distribution of NOS 3 in salt-dependent hypertension that may contribute to the development of hypertension and endothelial dysfunction.

Nitric oxide induces dilation of rat aorta via inhibition of rho-kinase signaling.

NO induces vasodilation through cGMP-dependent protein kinase--dependent and --independent mechanisms. A recent study demonstrated that recombinant cGMP-dependent protein kinase can phosphorylate the small G protein, RhoA, thus inhibiting its activity. Additionally, sodium nitroprusside was found to reverse the phenylephrine-induced translocation of RhoA, which is further indicative of the inhibition of RhoA activity. RhoA is known to be involved in the Ca(2+) sensitization of vascular smooth muscle through the actions of one of its downstream effectors, Rho-kinase. This study examined whether NO endogenously induces the relaxation of intact rat aorta via the inhibition of the Rho-kinase--mediated Ca(2+)-sensitizing pathway. Endogenous Rho-kinase inhibitor activity was inhibited by the selective compound Y-27632. Treatment of endothelium-intact rat aorta with Y-27632 (1 micromol/L) resulted in an attenuation of maximal force generated in response to phenylephrine. In endothelium-denuded rings, however, 1 micromol/L Y-27632 was ineffective at inhibiting the phenylephrine-induced contraction. Additionally, 1 micromol/L Y-27632 was significantly less effective at inhibiting the phenylephrine-induced contraction of endothelium-intact rings in the presence of inhibitors of NO synthase or guanylate cyclase (N(omega)-nitro-L-arginine and 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one, respectively). Interestingly, sodium nitroprusside restored the ability of 1 micromol/L Y-27632 to attenuate phenylephrine-induced contraction. Rho-kinase inhibition was also found to increase the sensitivity of the endothelium-denuded aorta to sodium nitroprusside. These data demonstrate that NO inhibits Rho-kinase activity in the intact rat aorta, supporting the hypothesis that endogenous NO-mediated vasodilation occurs through the inhibition of Rho-kinase constrictor activity in the intact rat aorta.

Nitric oxide produced by THAL nitric oxide synthase inhibits TGF.

Nitric oxide (NO) produced by neuronal NO synthase (nNOS) in the macula densa decreases tubuloglomerular feedback (TGF). NO produced by NOS in the thick ascending limb (THAL) inhibits NaCl transport. We hypothesized that NO produced by NOS in the THAL reaches the macula densa and inhibits TGF. Rabbit afferent arterioles and attached macula densa were simultaneously microperfused in vitro. TGF response was determined by measuring afferent arteriole diameter before and after increasing NaCl in the macula densa perfusate. When the nNOS inhibitor 7-nitroindazole (7-NI) (10 micromol/L) was added to the macula densa lumen, it increased TGF from 2.3 +/- 0.2 to 3.5 +/- 0.5 microm (P<0.02; n=6). In the presence of 7-NI, N(omega)-nitro-L-arginine methyl ester (L-NAME) (1 mmol/L) enhanced TGF from 2.6 +/- 0.3 to 4.0 +/- 0.5 microm (P<0.02; n=6) when the macula densa was perfused orthograde via the THAL, whereas it had no effect on TGF when the macula densa was perfused retrograde via the distal tubule (DT). Inhibition of macula densa soluble guanylate cyclase with LY83583 (1 micromol/L) blocked the effect of NO produced by THAL NOS when the macula densa was perfused via the THAL. We concluded that NO produced by THAL NOS acts as a paracrine factor, reaching the macula densa and inhibiting TGF.

Abrogated leptin-induced cardiac contractile response in ventricular myocytes under spontaneous hypertension: role of Jak/STAT pathway.

Leptin regulates cardiovascular function. Leptin levels are elevated in obesity and hypertension and may play a role in cardiovascular dysfunctions in these comorbidities. This study was designed to determine the influence of hypertension on the cardiac contractile response of leptin. Mechanical and intracellular Ca(2+) properties were evaluated using an IonOptix system in ventricular myocytes from spontaneously hypertensive (SHR) and age-matched Wistar Kyoto (WKY) rats. The contractile properties included peak shortening (PS), duration and maximal velocity of shortening/relengthening (TPS/TR(90), +/-dL/dt), and fura-fluorescence intensity change (DeltaFFI). NO and nitric oxide synthase (NOS) activity were assessed by the Griess and the (3)H-arginine/citrulline conversion assays, respectively. The leptin receptor (Ob-R) and the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway were evaluated by Western blot analysis. SHR animals displayed significantly elevated blood pressure and plasma leptin levels. Leptin elicited a concentration-dependent inhibition of PS and DeltaFFI in WKY, but not in SHR myocytes. Leptin did not affect TPS, TR(90), or +/- dL/dt. The difference in leptin-induced contractile response between the WKY and the SHR groups was abolished by the NOS inhibitor, Nomega-nitro-L-arginine methyl ester (L-NAME), but not by elevated extracellular Ca(2+). Either the JAK2 inhibitor AG-490 or the mitogen-activated protein (MAP) kinase inhibitor SB203580 abrogated the leptin-induced response in the WKY myocytes, whereas AG-490 unmasked a negative response in PS in the SHR myocytes. SHR myocytes displayed similar Ob-R protein abundance and basal NO levels, a blunted leptin-induced increase in NOS activity as well as enhanced basal STAT3 levels compared with the WKY group. These data indicate that the leptin-induced cardiac contractile response is abolished by spontaneous hypertension, possibly because of mechanisms involving altered JAK/STAT, MAP kinase signaling, and NO response.

Dietary polyunsaturated fatty acid and antioxidant modulation of vascular dysfunction in the spontaneously hypertensive rat

Two currently available edible oils–olive and canola–and two oil blends of plant origin having different n-3/n-6 polyunsaturated fatty acid (PUFA) ratios were evaluated for their ability to modify vascular dysfunction in the spontaneously hypertensive rat (SHR). Synthetic diets supplemented with test oils (5% w/w) were fed for 12 weeks, and segments of thoracic aorta used to assess vascular function. Vessels from the SHR displayed a spontaneous constrictor response after the inhibition of endothelial cell nitric oxide (NO) with Nω-nitro-L-arginine (NOLA). Dietary α -linoleate enrichment led to a reduction (P<0.05) in this abnormality with a dietary n-3/n-6 PUFA ratio of 1.0 (blend-1) yielding the best outcome. Relaxation to acetylcholine (ACh) was unaffected by dietary lipid supplementation. NOLA treated rings also displayed contractions to ACh that were abolished by indomethacin, thromboxane antagonists SQ29548, picotamide and flavonoids kaempferol and quercetin. In contrast, α -tocopherol, rutin and the lipoxygenase inhibitor esculetin resulted in only partial (30–55%) inhibition, and were ineffective against the NOLA-induced contraction suggesting the operation of different biochemical mechanisms in mediating the spontaneous and Ach-induced contractions. Results implicate plant-based oils and antioxidants as potential modulators of vascular function.

Flow-induced dilation of human coronary arterioles: important role of Ca(2+)-activated K(+) channels.

Flow-induced vasodilation (FID) is a physiological mechanism for regulating coronary flow and is mediated largely by nitric oxide (NO) in animals. Because hyperpolarizing mechanisms may play a greater role than NO in the microcirculation, we hypothesized that hyperpolarization contributes importantly to FID of human coronary arterioles. Arterioles from atria or ventricles were cannulated for videomicroscopy. Membrane potential of vascular smooth muscle cells (VSMCs) was measured simultaneously. After constriction with endothelin-1, increases in flow induced an endothelium-dependent vasodilation. Nomega-Nitro-L-arginine methyl ester 10(-4) mol/L modestly impaired FID of arterioles from patients without coronary artery disease (CAD), whereas no inhibition was seen in arterioles from patients with CAD. Indomethacin 10(-5) mol/L was without effect, but 40 mmol/L KCl attenuated maximal FID. Tetraethylammonium 10(-3) mol/L but not glibenclamide 10(-6) mol/L reduced FID. Charybdotoxin 10(-8) mol/L impaired both FID (15+/-3% versus 75+/-12%, P<0.05) and hyperpolarization (-32+/-2 mV [from -28+/-2 mV after endothelin-1] versus -42+/-2 mV [-27+/-2 mV], P<0.05). Miconazole 10(-6) mol/L or 17-octadecynoic acid 10(-5) mol/L reduced FID. By multivariate analysis, age was an independent predictor for the reduced FID. Conclusions-We conclude that shear stress induces endothelium-dependent vasodilation, hyperpolarizing VSMCs through opening Ca(2+)-activated K(+) channels in human coronary arterioles. In subjects without CAD, NO contributes to FID. NO and prostaglandins play no role in patients with CAD; rather, cytochrome P450 metabolites are involved. This is consistent with a role for endothelium-derived hyperpolarizing factor in FID of the human coronary microcirculation.