NO-mediated Relaxation Research Articles

Losartan Prevents Portal Hypertension-Induced, Redox-Mediated Endothelial Dysfunction in the Mesenteric Artery in Rats

Advanced stages of portal hypertension are characterized by generalized vasodilatation and a hyperdynamic syndrome that leads to complications such as hepatopulmonary syndrome. We assessed the endothelial function--particularly the formation of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF)--in rats following common bile duct ligation (CBDL) to determine the underlying mechanisms of these processes. Reactivity of mesenteric artery rings from male Wistar rats was determined in organ chambers. The expression levels of connexins (Cx) (Cx37, Cx40, Cx43), intermediate and small conductance Ca(2+)-activated K(+) channels (IK(Ca), SK(Ca)), endothelial NO synthase (eNOS), NADPH oxidase subunits, and nitrotyrosines were assessed by immunohistochemistry in mesenteric and pulmonary arteries. The vascular formation of reactive oxygen species (ROS) was evaluated using dihydroethidine. Control rats or those that had undergone CBDL were given either the NADPH oxidase inhibitor apocynin or the angiotensin II receptor type 1 antagonist losartan. Decreased EDHF-mediated relaxations to acetylcholine and red wine polyphenols were observed in CBDL rats, compared with controls, whereas the level of NO-mediated relaxation was similar. Impaired EDHF-mediated relaxations were associated with reduced vascular expression of Cx37, Cx40, Cx43, IK(Ca) and SK(Ca); increased expression of eNOS and NADPH oxidase subunits; and increased vascular formation of ROS and peroxynitrites. These effects were prevented by exposure to apocynin or losartan. CBDL is associated with reduced EDHF-mediated relaxations in the mesenteric artery, whereas NO-mediated relaxations persisted. These findings indicate that impaired EDHF-mediated relaxation involves an excessive vascular oxidative stress, most likely following activation of angiotensin II type 1 receptors.

Effects of 45-nm silver nanoparticles on coronary endothelial cells and isolated rat aortic rings

This study was undertaken to determine whether silver nanoparticles (Ag-45nm NPs) induce selective and specific biological effects, such as induction of proliferation and nitric oxide (NO) production, and cytotoxicity in coronary endothelial cells (CECs), and regulation of vascular tone in isolated rat aortic rings. Physical characterization of Ag-45nm NPs by transmission electron microscopy (TEM) demonstrated that nanoparticles ranging in size from 10 to 90nm had biological effects on CECs. Increasing concentrations of Ag-45nm NPs exerted a dual effect on cell proliferation whereby proliferation was inhibited at low concentrations of NPs and stimulated at high concentrations. The effects of high, but not low, concentrations of Ag-45nm NPs were dependent on NO because the effects were partially blocked by NG-nitro-l-arginine methyl ester (L-NAME). We have also shown that high, but not low, concentrations of Ag-45nm NPs induce NO-dependent proliferation through activation of endothelial nitric oxide synthase (eNOS) by phosphorylation of Serine 1177. Moreover, the antiproliferative and proliferative effects of Ag-45nm NPs were concentration-dependent and inversely correlated with cellular toxicity. In isolated rat aortic rings, a low concentration of NPs induced vasoconstriction and a high concentration stimulated vasodilation. The physiologic effects induced by a low concentration of Ag-45nm NPs inhibited acetylcholine- (ACh-) induced NO-mediated relaxation. Vasodilation induced by a high concentration of NPs was partially abolished by L-NAME pretreatment. When the endothelium was removed from the rings, all physiologic responses were blocked. These results clearly demonstrate that the NPs have selective and specific effects on the vascular endothelium in a concentration-dependent manner and suggest that opposite effects could be associated with NPs of different sizes.

Attenuated store-operated Ca2+ entry underpins the dual inhibition of nitric oxide and EDHF-type relaxations by iodinated contrast media

Our objective was to investigate whether alterations in endothelial Ca(2+) homeostasis contribute to the clinical toxicity of iodinated radiographic contrast media (IRCM) by modulating nitric oxide (NO) production and the endothelium-derived hyperpolarizing factor (EDHF) phenomenon. The triiodinated monomer iohexol caused concentration-dependent reductions in store-operated Ca(2+) entry (SOCE) in rabbit aortic valve endothelium incubated in Ca(2+)-free buffer with cyclopiazonic acid (CPA, 30 microM) to deplete endoplasmic reticulum Ca(2+) stores. This action was mimicked by Gd(3+) ions and 2-aminoethoxydiphenyl borate, two established inhibitors of SOCE, whereas Ca(2+) entry was unaffected by the osmotic agent mannitol. Immunohistochemistry demonstrated that iohexol did not prevent CPA-evoked membrane clustering of Orai1, the key pore element of the store-operated Ca(2+) channel (SOC) apparatus. In myograph studies with rabbit iliac artery rings, iohexol, and the hexaiodinated dimer iodixanol (both at 90 mg I/mL) attenuated NO-mediated and EDHF-type arterial relaxations evoked by CPA, but did not affect EDHF-type relaxations to acetylcholine, whose principal mode of action is to mobilize Ca(2+) via inositol 1,4,5-trisphosphate (InsP(3))-induced Ca(2+) release. Iohexol also exerted inhibitory effects on NO-mediated relaxation and smooth muscle contraction that were not evident with iodixanol. The data support the hypothesis that IRCM induce generalized endothelial dysfunction by inhibiting Ca(2+) influx via SOCs rather than their assembly. The presence of organically bound iodine, rather than osmolar effects, may underpin this previously unrecognized phenomenon. In contrast, direct effects of IRCM on smooth muscle function may correlate with osmolarity rather than iodine concentration.

Role of gender and estrogen receptors in the rat aorta endothelium-dependent relaxation to red wine polyphenols

Regular intake of moderate amounts of beverages rich in polyphenols such as red wine is associated with a protective effect on the vascular system, in part, by increasing the endothelial formation of nitric oxide (NO), a major vasoprotective factor. Since estrogens are potent inducers of NO formation and polyphenols have been shown to have phytoestrogen properties, we determined whether estrogen receptors mediate the stimulatory effect of red wine polyphenols (RWPs) on the endothelial formation of NO using isolated rat aortic rings and cultured endothelial cells. RWPs caused endothelium-dependent relaxations, which were more pronounced in the aorta of female than male rats. Increased relaxations were also observed to acetylcholine but not to sodium nitroprusside. Relaxations to RWPs were abolished by nitro l-arginine and MnTMPyP, markedly reduced by polyethyleneglycol-catalase and wortmannin, and not affected by the estrogen antagonist ICI 182,780 in aortic rings from males and females. eNOS expression was higher in aortic sections of female than male rats. RWPs caused the phosphorylation of Akt and eNOS in endothelial cells, which was unaffected by ICI 182,780. Thus, RWPs cause redox-sensitive PI3-kinase/Akt-dependent NO-mediated relaxations, which are more pronounced in the aorta of female than male rats; an effect most likely due to the increased expression level of eNOS rather than activation of estrogen receptors.

Tempol normalizes aortic relaxation but not elevated isoprostanes or PGE2 in copper‐deficient rats

Copper deficiency promotes accumulation of reactive oxygen species, which likely impair nitric oxide (NO)‐ mediated relaxation as well as trigger vascular inflammation. We hypothesize that an antioxidant supplementation would diminish oxidative stress, restore aortic relaxation, and limit pro‐inflammatory prostaglandin E2 (PGE2) synthesis during Cu deficiency. Weanling male Sprague Dawley rats were fed purified diets which were either copper‐adequate (Cu‐A; 6.3 μg Cu/g diet); copper‐deficient (Cu‐D; 0.3 μg Cu/g diet), or the Cu‐D diet combined with the SOD mimetic Tempol (Cu‐D/T; 1mM in drinking water) for 4 weeks. Relaxation of aortic rings was measured with force displacement transducers coupled to a Grass polygraph. Acetylcholine‐induced NO‐mediated relaxation was significantly less in the Cu‐D (pD2 = 6.77 ± 0.10) vs Cu‐A (pD2 = 8.18 ± 0.30); the decrease was normalized in Cu‐D/Tempol (pD2 = 7.75 ± 0.16) rats. Serum isoprostanes (index of oxidative stress) and PGE2 were higher in both the Cu‐D and Cu‐D/T groups vs Cu‐A controls. Thus, Cu‐deficency significantly impaired NO‐mediated relaxation and tempol supplementation restored aortic responsiveness. Our data also suggest a role for copper as a modulator of oxidative stress and inflammation independent of SOD activity or NO‐derived oxidants. Support: NIH DK055030.

Prolactin family regulate vascular tone through the modulation of nitric oxide production

Prolactin (PRL) family, include, placental lactogen (PL), growth hormone (GH) and prolactin, have been associated with reproduction, lactation, differentiation and angiogenesis. However, little is known about their effects upon the vasculature. The present study was designed to determine the regulatory effects that PL, GH and PRL exert on the vascular tone and, the association with endothelial nitric oxide (NO) production, a free radical that promotes vasodilation. In this study we used isolated rat aortic rings sub maximally precontracted with phenylephrine and, treated with known vasoactive substances, acetylcholine (ACh) to elicit NO‐mediated relaxation or an endothelium‐independent relaxation NO donor, as controls. PL and GH, promoted only vasodilation‐endothelium dependent, directly associated with an increase of NO production, these actions were comparable with ACh‐induce relaxation. PRL stimulated vasoconstriction and did not modify the basal levels of NO. We are first to report, opposite actions of PL and GH with respect to PRL, in this vascular bed. Further investigation are underway in our laboratory to fully know the role of PL, GH and PRL upon vascular endothelium, which may contribute to the understanding and control of cardiovascular pathologies, like coronary ischemia, that colud resolve through stimulation of vasodilaton. Supported by PROMEP /103.5/07/2574 and CONACyT 84576.

Crataegus Special Extract WS 1442 Causes Endothelium-dependent Relaxation via a Redox-sensitive Src- and Akt-dependent Activation of Endothelial NO Synthase but Not via Activation of Estrogen Receptors

This study determined whether the Crataegus (Hawthorn species) special extract WS 1442 stimulates the endothelial formation of nitric oxide (NO), a vasoprotective factor, and characterized the underlying mechanism. Vascular reactivity was assessed in porcine coronary artery rings, reactive oxygen species (ROS) formation in artery sections by microscopy, and phosphorylation of Akt and endothelial NO synthase (eNOS) in endothelial cells by Western blot analysis. WS 1442 caused endothelium-dependent relaxations in coronary artery rings, which were reduced by N-nitro-L-arginine (a competitive inhibitor of NO synthase) and by charybdotoxin plus apamin (two inhibitors of endothelium-derived hyperpolarizing factor-mediated responses). Relaxations to WS 1442 were inhibited by intracellular ROS scavengers and inhibitors of Src and PI3-kinase, but not by an estrogen receptor antagonist. WS 1442 stimulated the endothelial formation of ROS in artery sections, and a redox-sensitive phosphorylation of Akt and eNOS in endothelial cells. WS 1442 induced endothelium-dependent NO-mediated relaxations of coronary artery rings through the redox-sensitive Src/PI3-kinase/Akt-dependent phosphorylation of eNOS.

Role of Calcium-Activated Potassium Channels with Small Conductance in Bradykinin-Induced Vasodilation of Porcine Retinal Arterioles

Endothelial dysfunction and impaired vasodilation may be involved in the pathogenesis of retinal vascular diseases. In the present study, the mechanisms underlying bradykinin vasodilation were examined and whether calcium-activated potassium channels of small (SK(Ca)) and intermediate (IK(Ca)) conductance are involved in regulation of endothelium-dependent vasodilation in retinal arterioles was investigated. Porcine retinal arterioles (diameter approximately 112 microm, N = 119) were mounted in microvascular myographs for isometric tension recordings. The arterioles were contracted with the thromboxane analogue, U46619, and concentration-response curves were constructed for bradykinin and a novel opener of SK(Ca) and IK(Ca) channels, NS309. In U46619-contracted arterioles, bradykinin and NS309 induced concentration-dependent relaxations. In vessels without endothelium, bradykinin relaxation was abolished and NS309 relaxation was attenuated. Inhibition of NO synthase with asymmetric dimethylarginine and/or cyclooxygenase with indomethacin markedly reduced bradykinin and NS309 relaxation. NO synthase and cyclooxygenase inhibition together with oxyhemoglobin abolished bradykinin relaxation and attenuated NS309 relaxation. Blocking of SK(Ca) and IK(Ca) channels with apamin plus charybdotoxin or blocking of SK(Ca) channels alone in the absence and the presence of indomethacin markedly reduced bradykinin and NS309 relaxation, whereas blocking of IK(Ca) channels had no significant effect. In vessels without endothelium, blocking of SK(Ca) channels alone had no effect on sodium nitroprusside-induced relaxation. In porcine retinal arterioles, NO and prostaglandins mediate endothelium-dependent relaxation to bradykinin and NS309. Moreover, these findings suggest that SK(Ca) channels contribute to NO-mediated relaxation induced by bradykinin and NS309 and, hence, may play an important role in retinal arterial endothelial function.

Epigallocatechin-3-gallate relaxes the isolated bovine ophthalmic artery: Involvement of phosphoinositide 3-kinase-Akt-nitric oxide/cGMP signalling pathway

The present study investigates the direct action and the underlying mechanism(s) of epigallocatechin-3-gallate (EGCG) vasomotor effects on the bovine isolated ophthalmic artery. Adjacent rings were cut from each artery and mounted in a wire miograph system for isometric recording. Concentration–response curves for EGCG were constructed by adding cumulative concentrations of the drug to arterial rings pre-contracted with 5-HT (1 µM). Effects of mechanical endothelial cell removal and of selective blockers of the nitric oxide (NO)/cGMP pathways were investigated on the EGCG relaxant responses. EGCG relaxed ophthalmic arteries and maximum relaxation was 78.4 ± 2.64%. Mechanical removal of endothelium, blockade of soluble guanylyl cyclase by 1H-1,2,4-oxadiazolo [4,3-a]quinoxalin-1-one (ODQ, 1 and 5 µM) or inhibition of nitric oxide (NO) synthase by N G-nitro- L-arginine (L-NAME, 50 and 100 µM) reduced significantly the relaxant response to catechin; moreover, the NO donor S-nitroso- N-acetylpenicillamine (SNAP, 100 µM) significantly increased the vasorelaxant responses to EGCG. Relaxation to EGCG was inhibited by iberiotoxin (200 nM), a blocker of big-conductance Ca 2+-activated K + (BK Ca) channel, whereas the blockade of K ATP channel by glibenclamide (5 µM) and of small-conductance Ca 2+-activated K + (SK Ca) channel by apamin (100 nM) elicited no effect. Interestingly, also inhibition of phosphoinositide-3-kinase (PI3K) by wortmannin (100 nM) and of Akt by SH6 (1 µM) markedly decreased the EGCG-evoked vasorelaxation. These data suggest that EGCG induced vasorelaxation in ophthalmic arteries with endothelium-intact via the activation of the NO/cGMP signalling pathway and defined an intriguing role for PI3K and Akt as upstream mediators for activation of NO-mediated relaxant responses.

ENOS Activation Induced by a Polyphenol-Rich Grape Skin Extract in Porcine Coronary Arteries

Background/Aims: Drinking red wine is associated with a decreased mortality from coronary heart diseases. This study examined whether polyphenols contained in a grape skin extract (GSE) triggered the endothelial formation of nitric oxide (NO) and investigated the underlying mechanism. Methods: Vascular reactivity was assessed in organ chambers using porcine coronary artery rings in the presence of indomethacin (a cyclooxygenase inhibitor) and charybdotoxin plus apamin (inhibitors of endothelium-derived hyperpolarizing factor-mediated responses). The phosphorylation level of Src, Akt and endothelial NO synthase (eNOS) were assessed by Western blot analysis, and the formation of reactive oxygen species (ROS) was investigated using dihydroethidine and dichlorodihydrofluorescein. Results: GSE-induced endothelium-dependent relaxations were abolished by N^G-nitro-L-arginine (an eNOS inhibitor) and ODQ (a soluble guanylyl cyclase inhibitor), and they were reduced by MnTMPyP, polyethyleneglycol catalase, PP2 (an inhibitor of Src kinase) and wortmannin (an inhibitor of phosphoinositide 3-kinase). GSE caused phosphorylation of Src, which was prevented by MnTMPyP. It also caused phosphorylation of Akt and eNOS, which were prevented by MnTMPyP, polyethyleneglycol catalase, PP2, wortmannin and LY294002. GSE elicited the formation of ROS in native and cultured endothelial cells, which was prevented by MnTMPyP. Conclusions: GSE causes endothelium-dependent NO-mediated relaxations of coronary arteries. This effect involves the intracellular formation of ROS in endothelial cells leading to the Src kinase/phosphoinositide 3-kinase/Akt-dependent phosphorylation of eNOS.

Effects of diabetes and elevated glucose on nitrergic relaxations in the isolated duodenum of the rat

Nitrergic relaxations of the isolated duodenum, induced by streptozotocin, were investigated in the experimental 8-week diabetes rat model. The effects of elevated glucose were also examined in the incubated duodenal muscles (in Krebs-Henseleit solution containing 44 mM glucose for 6 h) taken from nondiabetic rats. The relaxations induced by electrical field stimulation (EFS) and nicotine were significantly reduced in diabetic rats compared with control rats. Incubating of duodenal tissues in medium containing elevated glucose revealed significantly impaired relaxations to EFS and nicotine compared to responses obtained after normal glucose incubation. However, the relaxant responses to sodium nitroprusside and papaverine were similar in all groups. Incubating in hyperosmolar solutions containing sucrose, the relaxant responses were not affected. In conclusion, impairment of NO-mediated relaxations in diabetes may be related to hyperglycemia. The alterations caused by elevated glucose are not due to a hyperosmotic effect because the same concentration of sucrose had no effect on the relaxations.

Abnormalities of endothelium-dependent responses in mesenteric arteries from Otsuka Long-Evans Tokushima Fatty (OLETF) rats are improved by chronic treatment with thromboxane A2 synthase inhibitor

Thromboxane A2 (TXA2) is thought to contribute to the development of diabetic complications. We tested the hypothesis that the impaired endothelial function seen in Otsuka Long-Evans Tokushima Fatty (OLETF) rats (a type 2 diabetic model) might be improved by chronic treatment with ozagrel, a TXA2 synthase inhibitor. In mesenteric arteries from OLETF rats (40–46 weeks old) [vs. those from age-matched Long-Evans Tokushima Otsuka (LETO) rats]: (1) ACh-induced endothelium-dependent relaxation, NO-mediated relaxation, and endothelium-derived hyperpolarizing factor (EDHF)-type relaxation were all reduced; (2) ACh-induced cyclooxygenase-dependent contraction was enhanced; (3) endothelium-derived contracting factor (EDCF)-mediated contraction was enhanced; (4) ACh-stimulated nitrite production was reduced but the nitrate/nitrite ratio was increased; and (5) ACh-stimulated production of TXA2 was increased. Chronic treatment with ozagrel (100mg/kg/day for 4 weeks, starting when they were 36–42 weeks of age) partly corrected the above abnormalities. These results suggest that ozagrel has normalizing effects on endothelial functions in OLETF mesenteric arteries, at least partly by increasing endothelium-derived relaxing factors (i.e., NO and EDHF) signaling and reducing EDCF signaling.

Up-Regulation of NO/cGMP Signaling Pathway in Corpus Cavernosum of Sickle Cell Disease Transgenic Mice

Priapism is defined as prolonged and persistent penile erection, unassociated with sexual interest or stimulation. This condition is common among males with sickle cell disease (SCD), however the molecular mechanisms involved in this syndrome are not well understood. Penile nitric oxide (NO), originating from both neurons and endothelium, modulates penile vascular and smooth muscle relaxation. NO, by binding sGC, converts cyclic guanosine triphosphate (cGTP) to cyclic guanosine monophosphate (cGMP), relaxing vascular smooth muscle and causing penile erection. The Berkeley mouse model is a well-accepted animal model of severe anemia that displays features of priapism similar to those seen in humans. These animals have a decreased activity of the enzyme responsible for cGMP hydrolysis, phosphodiesterase type 5A (PDE5) in the penis, suggesting that dysregulated NO signaling may lead to priapism in this mice model. Thus, the aim of this study was to evaluate the cavernosum smooth muscle relaxation mediated by the NO-cGMP signaling pathway in the SCD transgenic mouse. SS mice and normal C57BL/6 mice (control) penile tissues were removed at the level of attachment of the corporeal body; tunica albuginea was then carefully opened from its proximal extremity toward the penile shaft, and the erectile tissue within the corpus cavernosum (CC) was surgically dissected free. The strips were mounted in 10 ml organ baths containing Krebs solution at 37°C that was continuously bubbled with a mixture of 95% oxygen and 5% carbon dioxide (pH 7.4), and vertically suspended between two metal hooks. The tissues were stretched to a resting tension of 2.5 mN and allowed to equilibrate for 60 min. Changes in isometric force were recorded using a PowerLab 400 Data Acquisition System (Software Chart, version 5.2, AD Instruments, Colorado Springs, Colo). In a first set of experiments, cumulative concentration-response curves were constructed for a muscarinic agonist, acetylcholine (ACh; 0.001–1 μM), the NO donor compound, sodium nitroprusside (SNP; 0.001–10 μM) and the NO-independent stimulator of soluble guanylyl cyclase (sGC), BAY 41–2272 (0.001–10 μM), in corpus cavernosum (CC) precontracted with phenylephrine (PE; 10 μM). In a second set of experiments, to evaluate the nitrergic cavernosal relaxations, an electrical field stimulation (EFS; 1–32 Hz; 50 V; 10-s trains) was used. EFS were applied in strips placed between two platinum ring electrodes connected to a Grass S88 stimulator (Astro-Med Industrial Park, RI, U.S.A.). The endothelium-dependent NO-mediated relaxing responses to ACh induced concentration-dependent CC relaxations, and both potency (pEC50; 7.27 ± 0.09) and maximal responses (Emax; 78 ± 5%) were significantly higher in SS mice, compared with control mice (6.85 ± 0.07 and 60 ± 3%, respectively). EFS-induced relaxations in SS mice CC were significantly increased in all frequency studies compared to control mice. The endothelium-independent NOmediated relaxing responses to SNP produced concentration-dependent relaxation in isolated mice CC and both pEC50 (6.30 ± 0.09) and Emax (113 ± 6%) were significantly increased in the SS group when compared to the control group (5.80 ± 0.15 and 83 ± 4%, respectively). The NO-independent sGC stimulator, BAY 41–2272, induced significant increases in both pEC50 (7.07 ± 0.05) and Emax (124 ± 5 %) in the SS group when compared to the control group (6.78 ± 0.06 and 99 ± 2 %). In summary, these data demonstrate that SS mice exhibit an excessive relaxation response in CC, possibly by up-regulation of the NO-cGMP signaling pathway, reflecting in uncontrolled erection function. As such this dysregulated NO-pathway may constitute a potential therapeutic target to treat priapism in SCD individuals.

Poly (ADP-ribose) Polymerase Inhibition Improves Endothelial Dysfunction Induced by Hyperhomocysteinemia in Rats

We investigated the possible protective effect of poly (ADP-ribose) polymerase (PARP) inhibition in preventing endothelial dysfunction induced by hyperhomocysteinemia (Hhcy). Sprague-Dawley rats were divided into Hhcy group, Hhcy + 3-aminobenzamide(3-AB) group, control group and control + 3-AB group. A high-methionine diet was given to induce hyperhomocysteinemia. In Hhcy + 3-AB and control + 3-AB groups, rats were injected intraperitoneally with 3-AB (inhibitor of PARP). After 45 days, ultrastructural changes of aortas were observed by transmission electron microscope. Vascular reactivity of thoracic aortic rings was measured in organ chambers. PARP activity was detected. The levels of plasma total homocysteine, nitrite/nitrate, endothelin (ET)-1 and malondialdehyde were assayed. Rats in Hhcy group developed severe hyperhomocysteinemia and significant loss of endothelial function as measured by both vascular rings and levels of nitrite/nitrate and ET-1. Malondialdehyde levels increased significantly in Hhcy rats compared with control rats. 3-AB improved Ach-induced, NO-mediated vascular relaxation and stabilized the level of nitrite/nitrate and ET-1. Obvious improvement of ultrastructure can be observed in Hhcy + 3-AB group. These results suggest that pharmacological inhibition of PARP prevents the development of endothelial dysfunction in rats with hyperhomocysteinemia which may represent a novel approach to improve vascular dysfunction associated with hyperhomocysteinemia.

Cilostazol improves endothelial dysfunction by increasing endothelium-derived hyperpolarizing factor response in mesenteric arteries from Type 2 diabetic rats

Diabetes mellitus impairs endothelial function, an effect that can be considered a hallmark of the development of cardiovascular diseases in diabetics. Cilostazol, a selective phosphodiesterase 3 inhibitor, is currently used to treat patients with diabetic vascular complications. However, the effects of cilostazol on responses mediated by endothelium-derived relaxing [in particular, nitric oxide (NO) and hyperpolarizing factors (EDHF)] and contracting factors remain unclear. Here, we hypothesized that cilostazol could improve endothelial dysfunctions in mesenteric arteries isolated from type 2 diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Using cilostazol-treated (100 mg/kg/day for 4 weeks) or -untreated OLETF and control (Long Evans Tokushima Otsuka) rats, we examined the acetylcholine-induced endothelium-dependent responses and the cell-permeant cyclic adenosine monophosphate (cAMP) analog-induced relaxations in the superior mesenteric artery. We also determined blood parameters in these animals. In OLETF rats, chronic treatment with cilostazol reduced the blood levels of triglyceride, non-esterified fatty acids, and leptin, and increased antioxidant capacity, but did not alter the blood glucose or insulin levels. In studies on mesenteric arteries from cilostazol-treated OLETF animals, the cilostazol treatment improved: (a) the acetylcholine-induced EDHF-mediated relaxation and (b) the cAMP-mediated relaxation. However, cilostazol did not alter the NO-mediated relaxation or the endothelium-derived contracting factor-mediated contraction. These results suggest that cilostazol improves endothelial functions in OLETF mesenteric arteries by increasing EDHF signaling, and that it normalizes some metabolic abnormalities in OLETF rats. On that basis, cilostazol may prove to be a potent drug for the clinical treatment of diabetic vasculopathy.

Inhibition by lithium of the nitrergic relaxation of rat anococcygeus muscle

In the present study, we evaluated the effect of lithium on the nitric oxide (NO)-mediated nonadrenergic noncholinergic (NANC) relaxation of rat anococcygeus muscle. The isolated precontracted (phenylephrine, 7.5 μM) rat anococcygeus muscle were relaxed via electrical field stimulation (5 Hz) in the absence or presence of lithium (0.5, 1, and 5 mM) or in tissues excised from ex vivo lithium (600 mg/L in drinking water for 30 days)-treated animals. Effects of the NO synthase (NOS) inhibitor l-NAME (0.03 and 100 μM) or guanylyl cyclase inhibitor ODQ (1 μM) and NO precursor l-arginine (1 mM) on relaxations were investigated. Effect of either in vitro (1 and 5 mM) or ex vivo lithium treatment on relaxation to the NO donor sodium nitroprusside (SNP; 0.1–1000 μM) was also investigated on phenylephrine-contracted strips. The NANC relaxation was significantly reduced by in vitro (1 and 5 mM; up to P < 0.01) and ex vivo lithium treatment ( P < 0.001). l-NAME (100 μM and 1 mM) and ODQ (1 and 10 μM) significantly inhibited NANC relaxations in either control or lithium-treated strips. Combination of lithium (0.5 mM) with l-NAME (0.03 μM) significantly ( P < 0.001) reduced the NANC relaxation. Although 1 mM l-arginine had no effect on relaxations, it prevented their inhibition by both in vitro (1 and 5 mM) and ex vivo lithium of relaxations. SNP produced concentration-dependent relaxation in precontracted rat anococcygeus muscle which was not altered by lithium treatment. Reverse transcription polymerase chain reaction (RT-PCR) revealed a significant increase in the neuronal NOS expression in the anococcygeus muscle of ex vivo lithium-treated animals compared with controls. Our experiments suggested that both ex vivo and in vitro lithium administration attenuated the NO-mediated neurogenic relaxation of isolated rat anococcygeus muscle.

Impairment of NO-Dependent Relaxation in Intralobar Pulmonary Arteries: Comparison of Urban Particulate Matter and Manufactured Nanoparticles

Background and ObjectivesBecause pulmonary circulation is the primary vascular target of inhaled particulate matter (PM), and nitric oxide is a major vasculoprotective agent, in this study we investigated the effect of various particles on the NO–cyclic guanosine monophosphate (cGMP) pathway in pulmonary arteries.MethodsWe used intrapulmonary arteries and/or endothelial cells, either exposed in vitro to particles or removed from PM-instilled animals for assessment of vasomotricity, cGMP and reactive oxygen species (ROS) levels, and cytokine/chemokine release.ResultsEndothelial NO-dependent relaxation and cGMP accumulation induced by acetylcholine (ACh) were both decreased after 24 hr exposure of rat intrapulmonary arteries to standard reference material 1648 (SRM1648; urban PM). Relaxation due to NO donors was also decreased by SRM1648, whereas responsiveness to cGMP analogue remained unaffected. Unlike SRM1648, ultrafine carbon black and ultrafine and fine titanium dioxide (TiO2) manufactured particles did not impair NO-mediated relaxation. SRM1648-induced decrease in relaxation response to ACh was prevented by dexamethasone (an anti-inflammatory agent) but not by antioxidants. Accordingly, SRM1648 increased the release of proinflammatory mediators (tumor necrosis factor-α, interleukin-8) from intrapulmonary arteries or pulmonary artery endothelial cells, but did not elevate ROS levels within intrapulmonary arteries. Decreased relaxation in response to ACh was also evidenced in intrapulmonary arteries removed from rats intratracheally instilled with SRM1648, but not with fine TiO2.ConclusionIn contrast to manufactured particles (including nanoparticles), urban PM impairs NO but not cGMP responsiveness in intrapulmonary arteries. We attribute this effect to oxidative-stress–independent inflammatory response, resulting in decreased guanylyl cyclase activation by NO. Such impairment of the NO pathway may contribute to urban-PM–induced cardiovascular dysfunction.

Muscarinic receptor M1and phosphodiesterase 1 are key determinants in pulmonary vascular dysfunction following perinatal hypoxia in mice

Perinatal adverse events such as limitation of nutrients or oxygen supply are associated with the occurrence of diseases in adulthood, like cardiovascular diseases and diabetes. We investigated the long-term effects of perinatal hypoxia on the lung circulation, with particular attention to the nitric oxide (NO)/cGMP pathway. Mice were placed under hypoxia in utero 5 days before delivery and for 5 days after birth. Pups were then bred in normoxia until adulthood. Adults born in hypoxia displayed an altered regulation of pulmonary vascular tone with higher right ventricular pressure in normoxia and increased sensitivity to acute hypoxia compared with controls. Perinatal hypoxia dramatically decreased endothelium-dependent relaxation induced by ACh in adult pulmonary arteries (PAs) but did not influence NO-mediated endothelium-independent relaxation. The M(3) muscarinic receptor was implicated in the relaxing action of ACh and M(1) muscarinic receptor (M(1)AChR) in its vasoconstrictive effects. Pirenzepine or telenzepine, two preferential inhibitors of M(1)AChR, abolished the adverse effects of perinatal hypoxia on ACh-induced relaxation. M(1)AChR mRNA expression was increased in lungs and PAs of mice born in hypoxia. The phosphodiesterase 1 (PDE1) inhibitor vinpocetine also reversed the decrease in ACh-induced relaxation following perinatal hypoxia, suggesting that M(1)AChR-mediated alteration of ACh-induced relaxation is due to the activation of calcium-dependent PDE1. Therefore, perinatal hypoxia leads to an altered pulmonary circulation in adulthood with vascular dysfunction characterized by impaired endothelium-dependent relaxation and M(1)AChR plays a predominant role. This raises the possibility that muscarinic receptors could be key determinants in pulmonary vascular diseases in relation to "perinatal imprinting."

The effects of interleukin-6 on the contraction and relaxation responses of the cavernous smooth muscle from rats

The purpose of this study is to elucidate the effect of IL-6 on the vasomotor reactivity of the corpus cavernosum of the rats. The strips were either left untreated or treated with 1 ng/ml of IL-6 for 60 min. By increasing concentrations of phenylephrine, acetylcholine, or sodium nitroprusside, we assessed concentration–contraction or relaxation responses. The IL-6-treated strips were incubated for 30 min with or without l-NAME (NW-nitro-l-arginine methyl ester), l-arginine, indomethacin, BQ-123 (an endothelin receptor A inhibitor), or SQ 29,548 (a thromboxane A2 [TXA2] receptor blocker), and the effects on phenylephrine-induced contraction or acetylcholine-induced relaxation of phenylephrine-induced contraction were measured. The contractile responses to phenylephrine were significantly enhanced in the IL-6-treated strips, compared with the IL-6-nontreated strips, and the relaxation responses to acetylcholine were significantly inhibited in the IL-6-treated group compared with the IL-6-nontreated group. But after endothelial denudation, there was no difference between the IL-6-treated strips and the IL-6-nontreated strips on the contraction–relaxation responses to phenylephrine or acetylcholine. The relaxation responses to sodium nitroprusside were not inhibited in both groups. l-NAME completely inhibited the relaxation response to acetylcholine in the IL-6-treated strips, as well as the IL-6-nontreated strips. Indomethacin and SQ 29,548 significantly inhibited the increased contractile responses to phenylephrine in the IL-6-treated strips. But BQ 123 rarely affected the same responses. l-arginine reversed the inhibited relaxation responses to acetylcholine in the IL-6-treated strips. Therefore, IL-6 inhibits endothelium-dependent, NO-mediated relaxation and also enhances α1-adrenergic receptor-mediated contraction via an endothelium-dependent TXA2-mediated mechanism in the corpus cavernosum of the rat.

Inhibition of superoxide anion‐mediated impairment of endothelium by treatment with luteolin in rat mesenteric artery

This study was designed (i) to test whether endothelium‐derived hyperpolarizing factor (EDHF) and nitric oxide (NO) mediated vasorelaxation and hyperpolarization of vascular smooth muscle cell (VSMC) are impaired by superoxide anion, and (ii) to investigate the vasoprotection of luteolin against impaired endothelial function. Rat mesenteric arterial rings were isolated for isometric force recording and electrophysiological studies. Visualization of superoxide production was detected by fluorescent dye dihydroethidium. We found that 300μM pyrogallol decreased the relaxation and hyperpolarization of VSMC. Luteolin attenuated the loss of EDHF‐ and NO‐dependent vasorelaxation and the impairment of hyperpolarization to ACh induced by pyrogallol. Oxidative fluorescent microtopography showed that luteolin significantly reduced the superoxide levels induced by pyrogallol. The results suggest that exposure to superoxide anion impairs ACh‐induced relaxation and hyperpolarization of SMC in resistance arteries via impairing both EDHF‐ and NO‐mediated relaxation. Luteolin protects resistance arteries against superoxide anion caused injury, which implies that luteolin maybe effective in the therapy of vascular endothelium injury associated with oxidative stress.This work was supported by grant Y206179 from Zhejiang Provincial National Science Foundation of China.