Vascular smooth muscle contraction is an independent regulator of endothelial nitric oxide production

This investigation was conducted to determine whether endothelial nitric oxide (NO) production is regulated by vascular smooth muscle contraction. Unperfused ring segments of rat aorta and mesenteric artery were studied using isometric tension recording (n = 6-8 in all experiments). Following a reference contraction to K+ 80 mM (100%), arteries were left either unstimulated or stimulated by different concentrations of K+ or prostaglandin F2alpha (PGF2alpha) to induce different levels of vascular precontraction. N(G)-nitro-L-arginine methyl ester (L-NAME 0.1-300 microM) or NS 2028 (0.03-3 microM), which is a new specific inhibitor of the NO-sensitive guanylate cyclase, was then added at increasing concentrations to evaluate endothelial NO production. L-NAME and NS 2028 produced a concentration-dependent vasoconstrictor response which was progressively enhanced with increasing levels of precontraction. For L-NAME, this amounted in aorta to (% of reference contraction): 35+/-1% and 105 +/- 4% (precontraction by K(+) 20 and 30 mM) and 22+/-1%, 89+/-1%, 138+/-1% and 146+/-2% (precontraction by PGF2alpha 0.5, 1, 2 and 3 microM). A similar coupling was found in the mesenteric artery. A precontraction as little as 2% was enough to trigger a vasoconstrictor response to L-NAME. In contrast, L-NAME and NS 2028 had no effect in non-contracted arteries, not even when passive mechanical stretch was increased by 100%. The results suggest (i) that endothelial NO formation is progressively increased with increasing vascular tone, and (ii) that vascular isometric contraction per se stimulates endothelial NO formation. It is concluded, that active vascular smooth muscle contraction is an independent regulator of endothelial NO production.     

Halothane but not isoflurane attenuates interleukin 1beta-induced nitric oxide synthase in vascular smooth muscle

BACKGROUND: Inducible nitric oxide synthase (iNOS) is induced by endotoxin or cytokines, such as interleukin (IL)-1, through a protein synthesis pathway. Halothane reportedly inhibits protein synthesis in various tissues. The aim of the current study was to examine the effect of halothane on the IL-1beta-evoked induction of NOS in vascular smooth muscle. METHODS: After removal of the endothelium, arterial rings of rat aorta were mounted in an isometric force recording system. The effects of halothane (1.0-3.0%) or isoflurane (3.0%) on IL-1beta (20 ng/ml)-induced inhibition of the contractile responses to KCl (30 mM) and phenylephrine (10(-9)-10(-5) M) were studied. The cyclic guanosine monophosphate and cyclic adenosine monophosphate contents were determined by radioimmunoassay. Expression of iNOS and iNOS mRNA were measured by Western or Northern blot analysis, respectively. RESULTS: Halothane (1.0-3.0%) but not isoflurane (3%) significantly reduced the ML-1beta-induced inhibition of contraction in a concentration-dependent manner. The cyclic guanosine monophosphate content of the vascular smooth muscle increased significantly after a 5-h exposure to IL-1beta. Halothane at 3.0% significantly inhibited the increase in cyclic guanosine monophosphate content induced by IL-1beta. Halothane had no effect on cyclic adenosine monophosphate content. IL-1beta-induced expression of iNOS and iNOS mRNA in the rat aorta were inhibited significantly by halothane. CONCLUSION: The current study demonstrated that halothane but not isoflurane inhibits IL-1beta-stimulated hyporesponsiveness to vasoconstrictive agents in vascular smooth muscle and that this inhibitory effect of halothane involves the inhibition of iNOS mRNA expression. Thus, these findings suggest that halothane may have some sites to affect nitric oxide-signaling pathway.     

The anti-platelet agent,ticlopidine, upregulates interleukin-1-Beta-stimulated nitric oxide production in cultured rat vascular smooth muscle cells

BACKGROUND: Hemodialysis patients who had been treated with anti-platelet aggregation drugs, including ticlopidine, sometimes developed hypotension. The mechanism by which ticlopidine lowers the blood pressure in hemodialysis patients is unclear. To elucidate the mechanism of the action of this drug, we investigated cytokine-stimulated nitric oxide (NO) metabolism by ticlopidine in cultured rat vascular smooth muscle cells (VSMC). METHODS: Nitrite, a stable metabolite of NO, and intracellular cAMP and cGMP contents were assayed by the Griess method and enzyme immunoassay, respectively. iNOS mRNA and protein expressions were analyzed by Northern blotting and Western blotting. RESULTS: Ticlopidine enhanced interleukin-1beta (IL-1beta)-induced nitrite production in a dose- and time-dependent manner. The mRNA and protein expressions of inducible NO synthase were upregulated by ticlopidine in a dose- and time-dependent manner. IL-1beta alone stimulated both intracellular cAMP and cGMP contents, and the addition of ticlopidine further enhanced their contents. KT 5720, a selective inhibitor of protein kinase A, but not KT 5823, a selective inhibitor of protein kinase G, abolished the enhancement of IL-1beta-induced nitrite production by ticlopidine. In addition, a phosphodiesterase inhibitor, isobutylmethylxanthine, enhanced IL-1beta and ticlopidine induced nitrite production. CONCLUSION: We concluded that ticlopidine enhanced the IL-1beta-induced NO production via cAMP and subsequent activation of protein kinase A in cultured rat VSMC.     

Effects of estrogen on nitric oxide synthase expression in rat aorta allograft and smooth muscle cells.

BACKGROUND: We find that chronic estradiol treatment inhibits the development of transplant arteriosclerosis (TA). The mechanism of this inhibition remains unclear. The objective of this study is to investigate in a non-cyclosporin-requiring TA model whether estradiol-17beta treatment modulates the expression of both endothelial nitric oxide synthase (ecNOS) and inducible nitric oxide synthase (iNOS) in the early phase following transplantation. METHODS: Orthotopic abdominal aorta allograft transplantation was performed in male rats using Brown-Norway rats as donors and Lewis rats as recipients. The recipients (n = 50) were treated with estradiol 20 microg/kg/day or placebo by osmotic minipump from 2 days prior to surgery until sacrifice on post-operative days 1, 3, 7, 14, and 21. The allografts were harvested and cross-sections of the vascular tissues were used for immunohistochemical staining of ecNOS and iNOS. The effects of estradiol on cytokine-induced (tumor necrosis factor-alpha and interleukin-1 beta iNOS protein and messenger RNA (mRNA) expression were also evaluated on rat aorta smooth muscle cells by Western blotting and RT-PCR in vitro, respectively. RESULTS: The expression of ecNOS and iNOS was graded semiquantitatively from 0 to +3. Estrogen elevates ecNOS expression in the intima in the early phase following transplantation, 0.85 +/- 0.14 (day 7) and 1.08 +/- 0.11 (day 14) vs 1.53 +/- 0.25 (day 7) and 1.60 +/- 0.17 (day 14) for placebo and estradiol treated groups respectively, p < 0.01. Estrogen suppresses iNOS expression in neointima (0.67 +/- 0.17 vs 0.24 +/- 0.04, p < 0.01, day 14), media (1.03 +/- 0.15 vs 0.4 +/- 0.09, p < 0.01, day 7), and adventitia (1.55 +/- 0.12 vs 1.02 +/- 0.10, p < 0.05, day 14) in the same phase. Estradiol treatment inhibits cytokine-induced iNOS mRNA expression in cultured smooth muscle cells. CONCLUSIONS: Chronic estrogen treatment modulates both ecNOS and iNOS expression in the early phase following transplantation. This is associated with the estrogen-protective effects on TA.     

Halothane attenuates the endothelial Ca2+ increase and vasorelaxation of vascular smooth muscle in the rat aorta

Isolated spiral strips of rat thoracic aorta with endothelium were suspended for isometric tension recordings in a physiological salt solution. Endothelium-dependent vasorelaxation was elicited by carbachol 10(-6) and 10(-5) mol litre-1 during norepinephrine-induced contractions, and the effects of 1.5% and 3% halothane were evaluated with concomitant measurement of [Ca2+]i using fura-2-Ca2+ fluorescence. The effects of halothane on endothelium-dependent relaxation were compared with those of nitro G-L-arginine methyl ester 10(-4) mol litre- 1 (L-NAME: an inhibitor of nitric oxide synthase). Carbachol reduced norepinephrine-induced contractions in a concentration-dependent manner, but augmented the norepinephrine-induced increase in [Ca2+]i in endothelium intact strips. In contrast, carbachol did not influence muscle tension or [Ca2+]i when the endothelium was completely denuded. Although 3% halothane and L-NAME 10(-4) mol litre-1 inhibited carbachol- induced vasorelaxation in a similar manner, halothane inhibited carbachol-induced increases in [Ca2+]i. These results indicate that halothane inhibited a carbachol-induced increase in [Ca2+]i in the endothelium, which subsequently attenuated the decrease in muscle tension.      

Biphasic effect of nitric oxide on testosterone and cyclic GMP production by purified rat Leydig cells cultured in vitro

Nitric oxide (NO) biphasically modulates osteoclast function and sperm motility by exerting a positive effect at low concentrations and a negative effect at high concentrations. We therefore tested whether NO exerts a comparable effect on testosterone secretion by cultured rat Leydig cells. Three NO-donors, S-nitroso-N-acetylpenicillamine (SNAP), diethylamine/nitric oxide complex sodium salt (DEA/NO) and diethylenetriamine nitric oxide adduct (DETA/NO) were administered in a wide range of concentrations (10(-8)-10(-3) M for 3 h) to Percoll-purified Leydig cells from adult rats. These drugs raised testosterone and cGMP secretion when used at low concentrations (10(-8)-10(-5) M); however, they inhibited testosterone, but did not affect cGMP, secretion at concentrations higher than 10(-5) M. Administration of the NO scavenger haemoglobin (160 micrograms/mL) prevented both the stimulatory and the inhibitory effect of these drugs. Nitrite accumulation was measured as a marker of NO released by the drugs in our in vitro system; it fell within the range of control media in the presence of NO-donor concentrations lower than 10(-5) M, but was several-fold higher in the media of cells treated with concentrations of the NO-donors greater than 10(-5) M. These data show that (1) NO exerts a biphasic effect on testosterone secretion, which is stimulatory at low and inhibitory at high concentrations; (2) the stimulatory effect of NO is mediated by cGMP, the classic second messenger for NO action.     

Basal and acetylcholine-stimulated nitric oxide formation mediates relaxation of rabbit cavernous smooth muscle

Externally applied acetylcholine (ACh) in human corpus cavernosum has been shown to cause endothelium-dependent smooth muscle relaxation. Changes in isometric tension in rabbit cavernous smooth muscle strips mounted in organ bath chambers were monitored in the presence of blocking agents. Nitric oxide (NO) is known as an endothelium-derived relaxation factor (EDRF). Addition of specific inhibitors of nitric oxide synthesis, such as L-n-monomethyl arginine (L-NMMA) at 5 x 10(-4) mol/l.. or L-n-nitro arginine (L-NOARG) at 2 x 10(-4) mol/l. to strips precontracted with phenylephrine (PE) at 3.16 x 10(-6) mol/l. led to significant increases in tension. In the presence of L-NMMA or L-NOARG, relaxing effects of ACh at 10(-8)-3.16 x 10(-5) mol/l. mediated by muscarinic receptors were almost completely abolished. These data indicate that rabbit cavernous smooth muscle is under the control of basal NO release. They constitute strong evidence that cholinergically induced endothelial formation of NO plays a crucial role in relaxing cavernous smooth muscle.     

The effect of nitric oxide and peroxynitrite on rabbit cavernosal smooth muscle relaxation

 Nitric oxide (NO) mediates penile erection by inducing cavernosal smooth muscle relaxation. Superoxide anion (O₂ ⁻) can influence the activity of NO by reacting with it to produce peroxynitrite (PN). This is a highly reactive species that is known to attack a variety of biological targets. It is far more reactive and damaging than its precursors. We therefore, investigated the effect of PN on rabbit cavernosal smooth muscle relaxation and compared it to NO. Cavernosal strips from nine adult New Zealand White rabbits were excised (n=12 strips for each arm of the study) and mounted in organ baths. After pre-contraction with phenylephrine (PE) (100 μM) the strips were exposed to either NO or PN (1–100 μM) and subsequent smooth muscle relaxations monitored. Some tissues were incubated with oxadiazoloquinoxalin-1-one (ODQ; 10 μM), an inhibitor of guanylyl cyclase, before the addition of NO or PN. NO and PN induced concentration-dependent relaxations in all strips. However, PN (IC₅₀: 26 ± 3.6 μM) was significantly less potent than NO (IC₅₀: 11 ± 0.7 μM) [P < 0.01]. Relaxation induced by NO was immediate and short-lived, with the tension returning to its original level. In contrast, PN-initiated relaxations were of a slower onset and more prolonged, with the tissues unable to recover tension. However, after several washouts the tissues were fully responsive to PE. Both NO- and PN-mediated relaxations were inhibited by ODQ, suggesting the involvement of cGMP in this process. Although PN mediates cavernosal smooth muscle relaxation, it is much less potent than NO. As PN is thought to play a role in a variety of pathologies where erectile dysfunction is prominent, it may also contribute to the pathogenesis of erectile dysfunction.     

The relaxing effects of barbiturates in vascular smooth muscle of rat aorta

The effects of thiamylal and pentobarbital on contractions mediated through the influx of extracellular Ca⁺⁺ and the release of intracellularly stored Ca⁺⁺ were compared in rat aortic strips. Thiamylal (3 × 10^–5M to 10^–3M) and pentobarbital (10^–4 to 10^–3M) significantly attenuated the contraction induced by KCl (20mM), and shifted the dose-response curve for Ca⁺⁺ of KCl (20mM)-treated strips downwards and to the right. Caffeine (10^–2M)-induced contraction was significantly attenuated by thiamylal at concentrations greater than 10^–4M and by pentobarbital at 3 × 10^–4M. Only a high concentration (10^–3M) of these barbiturates significantly inhibited the contractions induced by norepinephrine (NE, 10^–6M) in Ca⁺⁺-free medium. Contraction of strips without endothelium by a Ca⁺⁺ ionophore, A23187 (5 × 10^–6M), in the presence of a Ca channel blocker, was relaxed by high concentrations of thiamylal (3 × 10^–4M to 10^–3M) and pentobarbital (10^–3M). It is concluded that thiamylal inhibits contraction through an intracellular action as well as a Ca channel-blocking action in vascular smooth muscle of rat aorta. However, the intracellular action of pentobarbital is less potent than that of thiamylal.(Nishiwada M, Nakamura K, Hatano Y, et al.: The relaxing effects of barbiturates in vascular smooth muscle of rat aorta. J Anesth 5: 380–387, 1991)     

Endothelial and neuronal-derived nitric oxide mediated relaxation of corpus cavernosal smooth muscle in a rat, in vitro, model of erectile function

We set out to establish a simple, reproducible, rat in vitro model of erectile function and to use this to demonstrate the functional importance of both neuronal- and endothelial-derived nitric oxide within this animal. Two corpora cavernosal smooth muscle strips were harvested from sexually mature male Wistar rats and mounted in an organ bath for measurement of isometric tension. Following contraction with noradrenaline the strips were relaxed by the addition of either acetylcholine or sodium nitroprusside. Electrical field stimulation was performed in the presence of atropine and guanethidine. Relaxation responses were repeated in the presence of methylene blue, L-arginine, L-NNA and haemoglobin +/- L-arginine. Methylene blue abolishes the relaxation to acetylcholine and EFS; L-NNA and haemoglobin cause a significant impairment in the relaxation response. L-arginine reverses the effect of haemoglobin. In conclusion, the inhibitory, relaxant stimulus of rat corpora cavernosa is due to both neuronal nitric oxide and endothelial-derived nitric oxide released in response to cholinergic stimulation.     

Acetate relaxation of isolated vascular smooth muscle

The vasorelaxant effects of acetate in arginine vasopressin (AVP)-contracted rat tail artery strips were examined in order to study mechanism of action. Dose-dependent relaxation by acetate was found in the clinically important range of 4 to 16 mM. Relaxation was not due to complexing of ionized calcium, persisted after mechanical removal of the endothelium, and was not altered by pretreatment with indomethacin. Although acetate also inhibited contraction by alpha-1 and alpha-2 agonists, the relaxant effect was not altered by destruction of sympathetic nerve terminals using 6-hydroxydopamine. The degree of relaxation in this model by various anions correlated with their lyotropic properties; however, the vasorelaxant effect of acetate exceeded that which would be expected on the basis of its position in the lyotropic series. The vasorelaxant effect of acetate was shared by other short-chain fatty acids that can be conjugated with coenzyme A (CoA), such as propionate and malonate. In contrast, a much lesser or absent relaxant effect was found with nonfatty-acid precursors of acetyl CoA, such as pyruvate, lactate, and alanine. The vasorelaxant effect of acetate was abolished by pretreatment with DIDS, an inhibitor of organic anion uptake, suggesting that cellular uptake of acetate is essential to its vasorelaxant action. The results suggest that the relaxant effect of acetate in vascular smooth muscle is non-specific, is not mediated by prostaglandins, does not depend upon the presence of either endothelium or the sympathetic nervous system, and may be due to metabolism of acetate to acetyl CoA with attendant conversion of ATP to AMP.     

Thiopental and propofol impair relaxation produced by ATP-sensitive potassium channel openers in the rat aorta

ATP-sensitive potassium channel openers are used as vasodilators in the treatment of cardiovascular disorders. The effects of i.v. anaesthetics on arterial relaxation induced by ATP-sensitive potassium channel openers have not been studied. Therefore, in this study, we have examined if thiopental (thiopentone) and propofol affect the vascular response to the ATP-sensitive potassium channel openers, cromakalim and pinacidil, in the isolated rat aorta. Rings of rat thoracic aortas without endothelium were suspended for isometric force recording. Concentration-response curves were obtained in a cumulative manner. During submaximal contractions with phenylephrine 0.3 microgramsmol litre-1, relaxation after cromakalim 0.1-30 microgramsmol litre-1, pinacidil 0.1-30 microgramsmol litre-1 and papaverine 0.1-300 microgramsmol litre-1 was demonstrated. Thiopental 30-300 microgramsmol litre-1, propofol 10-100 microgramsmol litre-1, 10% Intralipid 45 microliters or glibenclamide 5 microgramsmol litre-1 were applied 15 min before addition of phenylephrine. During contractions with phenylephrine, cromakalim and pinacidil induced concentration-dependent relaxation. A selective ATP-sensitive potassium channel antagonist, glibenclamide 5 microgramsmol litre-1, abolished this relaxation, whereas it did not affect relaxation produced by papaverine. Thiopental concentrations > 30 microgramsmol litre-1 significantly impaired relaxation produced by cromakalim or pinacidil. Propofol concentrations > 10 microgramsmol litre-1 also significantly reduced relaxation produced by cromakalim or pinacidil, whereas Intralipid was ineffective. Thiopental 300 microgramsmol litre-1 and propofol 100 microgramsmol litre-1 did not alter relaxation produced by papaverine. These results suggest that the i.v. anaesthetics, thiopental and propofol, impaired vasodilatation mediated by ATP-sensitive potassium channels in vascular smooth muscle cells.      

Complete loss of functional smooth muscle cells precedes vascular remodeling in rat aorta allografts

BACKGROUND: The functional consequences of vascular remodeling in rat aorta allografts were studied at different times after transplantation (Tx). METHODS: At days 1, 3, 7, 14, 28, and 56 after Tx, rat aorta allografts (Dark Agouti [DA]-to-Lewis) were mounted as isolated organs, and their contractile properties tested with phenylephrine, KCl, or endothelin-1. Controls were native DA-aortae and DA-syngeneic grafts. Changes in alpha smooth muscle actin and morphology were assessed by immunoblotting and histology. RESULTS: PostTx syngeneic grafts presented similar functional and morphological properties to native aortae. In allografts, no morphological changes was detected at day 7 after Tx, but phenylephrine-induced vasoconstriction was reduced by 60%. Signs of medial smooth muscle cell (SMC) loss and adventitial inflammation were observed at day 14 after Tx, without neointima formation. A complete loss of contractile property was observed at day 28 after Tx in association with a 75% decrease in alpha-SMC actin, severe adventitial inflammation, and reduced medial cellularity. At this time, neointima was restricted to both edges of allografts. At day 56 after Tx, allografts were also not functional and exhibited neointima on their entire length. All these changes were prevented by treating recipients with cyclosporine (7.5 mg/kg/day). CONCLUSION: These results indicate that, after Tx, the contractile property of rat aorta allografts is altered before manifest vascular remodeling. Because this can be prevented by cyclosporine, it most likely reflects an acute rejection of SMC. These results also show that vascular graft dysfunction can be used to monitor the development of rejection in the rat aorta allograft model.     

Involvement of cyclic nucleotides in renal artery smooth muscle relaxation

The elevation of vascular smooth muscle tone in the renal arteries during kidney transplantation and nephron-sparing surgery plays a major role in postsurgical organ dysfunction. Therefore, a better understanding of the intracellular mechanisms of contraction and relaxation is of fundamental interest to improve urological treatment. The present study was designed to investigate the complex intracellular system of cyclic nucleotides involved in the regulation of smooth muscle relaxation by using swine renal artery rings in the Schuler organ bath. Phenylephrine (PE) induced dose-dependent and fully reversible isometric contractions with a threshold concentration of 10 nM and an EC(50) of 804 nM. The receptor was identified as alpha(1A)-subtype by the selective antagonist WB4101. Increasing the intracellular concentration of cyclic 3':5'-adenosine monophosphate (cAMP) by dibutyryl-cAMP (5 mM) and forskolin (5 micro M) resulted in a decreased contractiltity of 48.0% and 76.3%, respectively. Elevation of the cytosolic content of cyclic 3':5'-guanosine monophosphate (cGMP) using dibutyryl-cGMP (1 mM), sodium nitroprusside (100 micro M) and SIN-1 (100 micro M) decreased the average PE-induced contraction by 16.4%, 41.9% and 62.4%, respectively. The unselective phosphodiesterase inhibitors theophylline (1 mM), papaverine (100 micro M) and IBMX (5 mM) reduced the PE-induced contraction by 37.3%, 93.1% and 95.5%, respectively. Furthermore, selective inhibition of phosphodiesterases by milrinone (PDE(3)-selective) resulted in a decreased contractility by 1.3% (50 micro M), 29.5% (100 micro M) and 93.5% (5 mM), and using rolipram (PDE(4) selective), the PE-induced contraction was inhibited by 57.9% (50 micro M) and 81.9% (100 micro M). The results suggest the involvement of cAMP and cGMP in the relaxation of renal artery smooth muscle cells. Moreover, phosphodiesterases, especially PDE(3) and PDE(4), seem to play a critical role in the regulation of renal artery smooth muscle tone.     

体外培养的人腹主动脉瘤血管平滑肌细胞中一氧化氮的生成和诱生型一氧化氮合酶的表达

目的探讨体外培养的人腹主动脉瘤 (AAA)血管平滑肌细胞 (SMC)及其表达诱生型一氧化氮合酶 (iNOS)和培养液中生成一氧化氮 (NO)的情况。方法 0 0 2 %Ⅰ型胶原酶消化法进行AAA SMC原代培养 ,平滑肌α 肌动蛋白 (α SMA)鉴定SMC并绘制AAA SMC的增殖曲线 ;免疫细胞化学方法检测AAA SMC中iNOS蛋白的表达 ,并测定原代及 2代细胞培养液中亚硝酸盐和硝酸盐的浓度之和 (NO2 - NO3 -,NOX)。结果酶消化法成功培养AAA SMC ,α SMA阳性率为 4 5 %± 5 8% ,传代培养发现AAA SMC增殖力有限 ;AAA SMC中iNOS蛋白阳性率 86 7%± 4 6 % ,细胞培养液中存在高浓度的NOX。结论AAA SMC存在异常增殖但增殖力有限 ,且细胞可能存在表型变化 ;AAA SMC中iNOS蛋白的高表达及NO的过量生成 ,提示由SMC生成的过量NO可能在腹主动脉瘤发病机制中具有重要作用。     

Human recombinant erythropoietin inhibits interleukin-1beta-stimulated nitric oxide and cyclic guanosine monophosphate production in cultured rat vascular smooth-muscle cells.

BACKGROUND: Recently rat vascular smooth-muscle cells (VSMC) have been shown to possess Epo receptor, and respond to various cytokines for producing nitric oxide (NO). In the present study we examined the effect of pharmacological dose of human recombinant erythropoietin (rHuEpo) on the IL-1beta-induced NO and cGMP production as well as inducible nitric oxide synthase (iNOS) in cultured rat VSMC. METHODS: Nitrite, a stable metabolite of NO, and intracellular cGMP contents were assayed by Griess method and enzyme immunoassay. iNOS mRNA expression was analysed by Northern blotting. RESULTS: RHuEpo inhibited IL-1beta-induced nitrite production in a dose- and time-dependent manner with concomitant changes of intracellular cGMP contents. On the other hand, rHuEpo did not inhibit atrial natriuretic peptide- (ANP) or sodium nitroprusside (SNP)-induced nitrite and cGMP production at all. While rHuEpo inhibited IL-1beta-induced iNOS mRNA expression, rHuEpo vehicle did not affect IL-1beta-induced iNOS mRNA expression. CONCLUSIONS: It is suggested that a pharmacological dose of rHuEpo inhibits IL-1beta-induced NO and cGMP production as well as iNOS mRNA expression, presumably via the Epo receptor.     

Involvement of endothelial nitric oxide synthase in the impaired endothelium-dependent relaxation of cavernous smooth muscle in hypercholesterolemic rabbit

The present study was designed to evaluate whether functional impairment and/or protein expression of constitutive nitric oxide synthase (cNOS; endothelial NOS [eNOS] and neuronal NOS[nNOS]) was involved in impairment of endothelium-dependent relaxation of cavernous smooth muscle in hypercholesterolemic rabbits. New Zealand White rabbits were randomly divided into control and experimental groups. The control group (n=20) received a regular diet, while the two experimental groups (n=20 for each) were fed a 2% cholesterol diet for 4 and 8 weeks, respectively. We conducted isometric tension studies with endothelium-dependent and endothelium-independent vasodilators with or without preincubation with L-arginine and nonadrenergic, noncholinergic (NANC)-selective electrical field stimulation on isolated strips of corpus cavernosum. Expression of cNOS (eNOS and nNOS) protein was assessed by Western blot analysis. cNOS activities in both cytosolic and particulate fractions were measured by determining the conversion of L-[U-14C] arginine to L-[U-14C] citrulline. Blood levels of cholesterol were significantly higher (P < 0.01) in the experimental groups than in the control group. The relaxation responses to endothelium-dependent agents (acetylcholine and adenosine 5'-diphosphate [ADP]) were significantly reduced (P < 0.05) in both experimental groups, regardless of their incubation with L-arginine, compared with the control group. However, no differences were found among the three groups in the relaxation response to endothelium-independent agents (papaverine and nitroprusside) and to NANC-selective electrical field stimulation. There was no difference in immunoreactive nNOS from cytosolic and particulate fractions between the cavernous tissues of the control and experimental groups. nNOS protein levels in the particulate fractions were markedly lower than in the cytosolic fractions. The particulate cNOS activity was significantly decreased (P < 0.05) in the experimental groups compared with the control group, while the cytosolic cNOS activity in the experimental groups was not different from that found in the control group. Therefore, it is concluded that functional impairment of eNOS, rather than of nNOS, may lead to impairment of cavernous smooth muscle relaxation in response to endothelium-mediated stimuli in hypercholesterolemic rabbits.