Endothelium-dependent vasorelaxation independent of nitric oxide and K(+) release in isolated renal arteries of rats

1. We investigated whether K(+) can act as an endothelium-derived hyperpolarizing factor (EDHF) in isolated small renal arteries of Wistar-Kyoto rats. 2. Acetylcholine (0.001 - 3 microM) caused relaxations that were abolished by removal of the endothelium. However, acetylcholine-induced relaxations were not affected by the nitric oxide (NO) synthase inhibitor N:(omega)-nitro-L-arginine methyl ester (L-NAME, 100 microM), by L-NAME plus the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, 1 microM) or by L-NAME plus the cyclo-oxygenase inhibitor indomethacin (10 microM). In rings precontracted with high-K(+)(60 mM) physiological salt solution in the presence of L-NAME, acetylcholine-induced relaxations were abolished. 3. L-NAME-resistant relaxations were abolished by the large-conductance Ca(2+)-activated K(+) channel inhibitor charybdotoxin plus the small-conductance Ca(2+)-activated K(+) channel inhibitor apamin, while the inward rectifier K(+) channel inhibitor Ba(2+) or the gap junction inhibitor 18alpha-glycyrrhetinic acid had no effect. Acetylcholine-induced relaxation was unchanged by ouabain (10 microM) but was partially inhibited by a higher concentration (100 microM). 4. In half of the tissues tested, K(+)(10 mM) itself produced L-NAME-resistant relaxations that were blocked by ouabain (10 microM) and partially reduced by charybdotoxin plus apamin, but not affected by 18alpha-glycyrrhetinic acid or Ba(2+). However, K(+) did not induce relaxations in endothelium-denuded tissues. 5. In conclusion, acetylcholine-induced relaxations in this tissue are largely dependent upon hyperpolarization mechanisms that are initiated in the endothelium but do not depend upon NO release. K(+) release cannot account for endothelium-dependent relaxation and cannot be an EDHF in this artery. However, K(+) itself can initiate endothelium-dependent relaxations via a different pathway from acetylcholine, but the mechanisms of K(+)-induced relaxations remain to be clarified.     

Endothelium-dependent relaxation by substance P in human isolated omental arteries and veins: relative contribution of prostanoids,nitric oxide and hyperpolarization

1. The objective of the present study was to investigate human omental arteries and veins with respect to: (i) the contractile effect of the thromboxane A₂ analogue U46619, (ii) endothelium-dependency and mediators of the relaxing effect of substance P (SP) and acetylcholine (ACh).
2. Changes in isometric tension in response to administration of U46619, SP and ACh were measured in human isolated omental arteries and veins with and without endothelium. To investigate the mechanism of action of SP, the SP-induced relaxation was measured in the presence of indomethacin (cyclo-oxygenase inhibitor), N^G-monomethyl-L-arginine (L-NMMA, nitric oxide-synthase inhibitor), KCl (inhibitor of endothelium-dependent hyperpolarization), tetraethylammonium (TEA; non-selective inhibitor of K⁺-channels, with some preference for the high conductance Ca²⁺-activated K⁺-channel, BK_Ca), glibenclamide (inhibitor of the ATP-sensitive K⁺-channel) and/or clotrimazole (inhibitor of the cytochrome P450-system and the intermediate conductance Ca²⁺-activated K⁺-channel, IK_Ca).
3. U46619 contracted both the artery and the vein segments. Endothelium removal did not alter the contraction.
4. ACh caused neither contraction nor relaxation in artery and vein segments precontracted with U46619.
5. In both artery and vein segments precontracted with U46619, SP produced endothelium-dependent relaxation. The relaxation was unaffected by indomethacin, but was incompletely reduced by L-NMMA and KCl respectively. The L-NMMA-resistent relaxation was abolished in the presence of KCl.
6. TEA inhibited the SP-induced relaxation in artery and vein segments both in the presence and absence of L-NMMA and indomethacin, while glibenclamide and clotrimazole had no effect.
7. In conclusion, the SP-induced relaxation in human omental arteries and veins seems to be mediated via NO and endothelium-dependent hyperpolarization. K_ATP and I_KCa are probably not involved in the hyperpolarization, but activation of BK_Ca may contribute to the hyperpolarization. Prostanoid synthesis and the cytochrome P450-system are probably not involved in the SP-induced relaxation in this area.

     

Endothelium-dependent contraction in intrapulmonary arteries: mediation by endothelial NK1 receptors and TXA2.

1. We have examined whether three natural tachykinins, substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) induce an endothelium-dependent contraction (EDC) in the rabbit isolated intrapulmonary artery. 2. Removal of the endothelium almost abolished the contraction induced by SP (10(-8) M) while it did not attenuate the contraction induced by SP (10(-7) M), NKA (10(-9) - 10(-7) M) or NKB (10(-8) and 10(-7) M). 3. The EDC induced by SP (10(-8) M) was abolished by NK1 antagonists (FK-888, CP-96345, CP-99994 and SR-140333) but not by an NK2 antagonist (SR-48968). 4. The EDC induced by SP was attenuated by cyclo-oxygenase inhibitors (aspirin and indomethacin), thromboxane A2 (TXA2) synthetase inhibitors (OKY-046, KY-234 and KY-063) and a TXA2 antagonist (S-1452). 5. The rank order of potency causing endothelium-independent contraction (EIC) was NKA > NKB > SP. The EIC induced by SP (10(-7) M) was attenuated by an NK2 antagonist but not by NK1 antagonists, cyclo-oxygenase inhibitors, TXA2 synthetase inhibitors or a TXA2 antagonist. 6. In conclusion, SP at 10(-8) M induces EDC via endothelial NK1 receptors and TXA2 production, and SP at 10(-7) M induces EIC via NK2 receptors in the rabbit intrapulmonary artery.     

Tonic inhibitory action by nitric oxide on spontaneous mechanical activity in rat proximal colon: involvement of cyclic GMP and apamin-sensitive K+ channels.

1. The cellular mechanisms by which endogenous nitric oxide (NO) modulates spontaneous motility were investigated in rat isolated proximal colon. The mechanical activity was detected as changes in intraluminal pressure. 2. Apamin (1-100 nM) produced a concentration-dependent increase in the amplitude of the spontaneous pressure waves. The maximal contractile effect was of the same degree as that produced by Nomega-nitro-L-arginine methyl ester (L-NAME) (100 microM) and the joint application of apamin plus L-NAME had no additive effects. Apamin (0.1 microM) reduced the inhibitory effects (i.e. reduction in the amplitude of the pressure waves) induced by sodium nitroprusside (SNP) (1 nM - 10 microM) or 8-Br-cyclic GMP (1-100 microM). 3. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (0.1-5 microM), inhibitor of NO-stimulated guanylate cyclase, produced a concentration-dependent increase of the spontaneous contractions. ODQ (1 microM) in the presence of apamin (0.1 microM) did not produce any further increase in the contraction amplitude, whereas after L-NAME (100 microM) it decreased the spontaneous contractions. ODQ (1 microM) reduced the SNP inhibitory effects. 4. Zaprinast (1-50 microM), inhibitor of cyclic GMP phosphodiesterase, produced a concentration-dependent decrease of the spontaneous contractions. The effects of zaprinast were significantly reduced in the presence of apamin (0.1 microM) or L-NAME (100 microM). 5. These results suggest that small conductance Ca2+-dependent K+ channels and cyclic GMP are involved in the modulation of the spontaneous contractile activity in rat proximal colon. Cyclic GMP production system and opening of apamin-sensitive K+ channels appear to work sequentially in transducing an endogenous NO signal.     

Mechanisms involved in relaxation induced by exogenous nitric oxide in pig coronary arteries.

The aim of the present study was to analyze the mechanisms involved in the vasodilator responses elicited by nitric oxide (NO) in segments of porcine posterior descending coronary artery. Exogenous NO (0.1-30 microM) induced concentration-dependent relaxations in segments precontracted with a concentration of the thromboxane A2 mimetic, U-46619 (30-300 nM) that produced a contraction 70% (submaximal contraction) of that elicited by 75 mM K+ (maximal contraction). The relaxations were almost abolished by 6-anilinoquinoline-5,8-quinone (LY-83583, 10 microM), an inhibitor of guanylate cyclase, and with precontraction with 40 or 60 mM K+. Relaxations were reduced by 5 mM tetraethylammonium (TEA), a blocker of Ca(2+)-activated K+ channels (Kca channels) and unaltered by ouabain (500 microM), 4-aminopyridine (1 mM), glibenclamide (10 microM), apamin (1 microM) and charybdotoxin (0.3 microM), inhibitors of sodium pump, voltage-sensitive, ATP-sensitive, small-conductance Kca and large-conductance KcaK+ channels, respectively. These results suggest that the relaxation caused by exogenous NO is mediated by guanylate cyclase activation, with only a slight participation of a hyperpolarizing mechanism mediated by activation of Kca channels.     

Endothelium-dependent relaxation and hyperpolarization evoked by bradykinin in canine coronary arteries: enhancement by exercise-training.

1. Kinins, which are produced locally in arterial walls, stimulate the release of endothelium-derived vasodilator substances. Therefore, they may participate in the metabolic adaptation to chronic exercise that occurs in the coronary circulation. Experiments were designed to compare the reactivity to bradykinin in coronary arteries isolated from sedentary and exercised-trained dogs (for 8-10 weeks). 2. The organ chambers used in this study were designed for measurement of isometric tension and cell membrane potential with glass microelectrodes. Rings of canine isolated coronary arteries with endothelium were suspended in the organ chambers filled with modified Krebs-Ringer bicarbonate solution (37 degrees C, gassed with 5% CO2 in 95 O2), and were all treated with indomethacin to prevent interference from prostaglandins. 3. Bradykinin evoked concentration-dependent relaxations of the coronary arteries. However, the kinin was significantly less potent in relaxing coronary arteries from the sedentary dogs than those from the trained ones. 4. In the presence of NG-nitro-L-arginine (an inhibitor of nitric oxide synthases), concentration-relaxation curves to bradykinin were shifted to the right in both types of preparations. Nonetheless, the peptide was still significantly more potent in arteries from exercise-trained animals. 5. In the electrophysiological experiments, concentration-hyperpolarization curves to bradykinin obtained in arteries from sedentary dogs were also significantly to the right of those in vessels from exercise-trained animals. Thus, in arteries from exercised animals, bradykinin more potently evoked the release of both nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF). 7. The angiotensin converting enzyme (ACE)-inhibitor, perindoprilat, shifted to the left the concentration-relaxation curves to bradykinin obtained under control conditions and in the presence of NG-nitro-L-arginine. The concentration-hyperpolarization curves to bradykinin were also shifted to the left by perindoprilat. The shift induced by the ACE-inhibitor in either type of preparation was not significantly different. 8. These findings demonstrate that exercise-training augments the sensitivity of the coronary artery of the dog to the endothelium-dependent effects of bradykinin. This sensitization to bradykinin may reflect an increased role of both NO and EDHF, and is not the consequence of differences in ACE activity in the receptor compartment.     

Vascular smooth muscle relaxation mediated by nitric oxide donors: a comparison with acetylcholine, nitric oxide and nitroxyl ion

1. Vasorelaxant properties of three nitric oxide (NO) donor drugs (glyceryl trinitrate, sodium nitroprusside and spermine NONOate) in mouse aorta (phenylephrine pre-contracted) were compared with those of endothelium-derived NO (generated with acetylcholine), NO free radical (NO*; NO gas solution) and nitroxyl ion (NO(-); from Angeli's salt). 2. The soluble guanylate cyclase inhibitor, ODQ (1H-(1,2,4-)oxadiazolo(4,3-a)-quinoxalin-1-one; 0.3, 1 and 10 microM), concentration-dependently inhibited responses to all agents. 10 microM ODQ abolished responses to acetylcholine and glyceryl trinitrate, almost abolished responses to sodium nitroprusside but produced parallel shifts (to a higher concentration range; no depression in maxima) in the concentration-response curves for NO gas solution, Angeli's salt and spermine NONOate. 3. The NO* scavengers, carboxy-PTIO, (2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide; 100 microM) and hydroxocobalamin (100 microM), both inhibited responses to NO gas solution and to the three NO donor drugs, but not Angeli's salt. Hydroxocobalamin, but not carboxy-PTIO, also inhibited responses to acetylcholine. 4. The NO(-) inhibitor, L-cysteine (3 mM), inhibited responses to Angeli's salt, acetylcholine and the three NO donor drugs, but not NO gas solution. 5. The data suggest that, in mouse aorta, responses to all three NO donors involve (i) activation of soluble guanylate cyclase, but to differing degrees and (ii) generation of both NO* and NO(-). Glyceryl trinitrate and sodium nitroprusside, which generate NO following tissue bioactivation, have profiles resembling the profile of endothelium-derived NO more than that of exogenous NO. Spermine NONOate, which generates NO spontaneously outside the tissue, was the drug that most closely resembled (but was not identical to) exogenous NO.     

Endothelium-dependent relaxation to acetylcholine in the rabbit basilar artery: importance of membrane hyperpolarization.

1. Muscarinic stimulation of isolated, preconstricted segments of the basilar artery, with either acetylcholine or carbachol, was followed by endothelium-dependent smooth muscle relaxation and membrane hyperpolarization. 2. Smooth muscle relaxation to acetylcholine was stimulated in the presence of lower concentrations than the associated hyperpolarization (EC50 values 3.2 microM and 31.6 microM, respectively), and was sustained during agonist application, while the hyperpolarization was relatively transient. 3. Repeated exposure to acetylcholine was associated with loss of membrane hyperpolarization, while smooth muscle relaxation was unaltered. Following a second exposure to 100 microM acetylcholine, mean hyperpolarization was markedly depressed from 8.5 to 2 mV, and subsequent exposures failed to induce any hyperpolarization. Relaxations with a similar amplitude and rate of development, were recorded with each subsequent addition of acetylcholine. 4. The competitive substrate inhibitors for nitric oxide synthase, L-NG-monomethyl arginine (100 microM L-NMMA) or L-NG-nitro arginine methyl ester (100 microM L-NAME), modified the form and amplitude of both the relaxation and the hyperpolarization to acetylcholine. In the majority of experiments, both the hyperpolarization and the relaxation were almost totally abolished. 5. Neither nitric oxide, applied directly in physiological salt solution, nor sodium nitroprusside, produced smooth muscle hyperpolarization except in high concentrations. Reproducible, small amplitude (around 2 mV) hyperpolarization followed the application of either NO gas (15 microM) or sodium nitroprusside (100 microM), both of which induced almost maximal smooth muscle relaxation. 6. These data show that muscarinic stimulation of endothelial cells in the rabbit basilar artery is followed by both smooth muscle hyperpolarization and relaxation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endothelium-dependent relaxation induced by angiotensin II and histamine in isolated arteries of dog.

In helical strips of dog renal and mesenteric arteries pre-contracted with prostaglandin F2 alpha (PGF2 alpha), endothelium-dependent relaxations were investigated. Removal of the endothelium was shown histologically by staining with silver nitrate and functionally by testing the inability of acetylcholine to induce arterial relaxations. When the endothelium was removed, relaxation of renal arteries to angiotensin (Ang) II was markedly suppressed, whereas relaxations induced by PGI2 or isoprenaline were attenuated only slightly. Removal of the endothelium attenuated the relaxant response of mesenteric arteries to histamine but did not significantly alter the response to PGI2. Treatment with indomethacin caused an additional attenuation of the relaxant response to histamine or a reversal of the Ang II-induced relaxation to a contraction in the arterial strips, from which the endothelium had been removed. Relaxation of renal arteries induced by Ang II and of mesenteric arteries induced by histamine is postulated to result from PGI2 released from the arterial wall. Therefore, it appears that the endothelium is a major site but not the only site responsible for drug-induced release of PGI2.     

Urocortin-induced endothelium-dependent relaxation of rat coronary artery: role of nitric oxide and K+ channels

1. The mechanisms underlying the vasodilator response to urocortin are incompletely understood. The present study was designed to examine the role of endothelial nitric oxide and Ba(2+)-sensitive K(+) channels in the endothelium-dependent component of urocortin-induced relaxation in the rat left anterior descending coronary artery. 2. Urocortin induced both endothelium-dependent and -independent relaxation with respective pD(2) of 8.64+/-0.03 and 7.90+/-0.10. Removal of endothelium reduced the relaxing potency of urocortin. In rings pretreated with 10(-4) M N(G)-nitro-L-arginine methyl ester, 10(-5) M methylene blue or 10(-5) M ODQ, the urocortin-induced relaxation was similar to that observed in endothelium-denuded rings. L-Arginine (5x10(-4) M) antagonized the effect of N(G)-nitro-L-arginine methyl ester. 3. The relaxant response to urocortin was reduced in endothelium-intact rings preconstricted by 3.5x10(-2) M K(+) and abolished when extracellular K(+) was raised to 5x10(-2) M. Pretreatment with 10(-4) M BaCl(2) significantly inhibited urocortin-induced relaxation. Combined treatment with 10(-4) M BaCl(2) plus 10(-4) M N(G)-nitro-L-arginine methyl ester did not cause further inhibition. In urocortin (10(-8) M)-relaxed rings, BaCl(2) induced concentration-dependent reversal in vessel tone. Tertiapin-Q (10(-6) M) also attenuated urocortin-induced relaxation. In contrast, BaCl(2) did not alter urocortin-induced relaxation in endothelium-denuded rings. 4. In endothelium-denuded rings, hydroxylamine- and nitroprusside-induced relaxation was inhibited by 10(-4) M BaCl(2), but not by 10(-6) M tertiapin-Q. 5. The endothelium of the coronary artery was moderately stained with the antiserum against urocortin. 6. Taken together, the present results indicate that the urocortin-induced endothelium-dependent relaxation of rat coronary arteries is likely attributable to endothelial nitric oxide and subsequent activation of Ba(2+)- or tertiapin-Q-sensitive K(+) channels. The urocortin-induced endothelium-dependent relaxation appears to be mediated by cyclic GMP-dependent mechanisms.     

Urocortin舒张自发性高血压大鼠胸主动脉与NO和K~+通道的关系

目的探讨Urocortin(Ucn)对自发性高血压大鼠(SHR)胸主动脉舒缩功能的作用及机制。方法采用体外血管灌流,观察Ucn对SHR胸主动脉的舒张作用,以及左旋硝基精氨酸甲酯(N(ω)n itro-L-argin ine methyl ester,L-NAME)、亚甲蓝(M ethylene B lue,MB)和格列本脲(G lybenc lam ide)对其舒张作用的影响。结果Ucn(1 nmol.L-1~1μmol.L-1)可明显舒张内皮完整和去内皮SHR胸主动脉(P<0.01),此作用具有剂量依赖性;一氧化氮(NO)合成酶抑制剂L-NAME(100μmol.L-1)和鸟苷酸环化酶(GC)抑制剂MB部分抑制Ucn舒张血管的作用,而且增强去甲肾上腺素(NE)产生的收缩反应。ATP敏感钾通道(KATP)阻断剂格列本脲(10μmol.L-1)可减弱Ucn的舒血管作用。结论Ucn对SHR血管具有内皮依赖性和非内皮依赖性舒张作用,此作用部分是Ucn增加血管内皮细胞NO水平实现的,并且与NO-cGMP通路和KATP通道有关。     

Endothelium-dependent calcium-induced relaxation in the presence of Ca2+-antagonists in canine depolarized coronary arteries.

1. We examined the mechanisms underlying Ca2+-induced relaxation in the presence of clentiazem, a new Ca2+-antagonist, in depolarized coronary arteries of the dog. 2. Ca2+ (3 x 10(-5)-3 x 10(-3) M) caused an unexpected relaxation in the presence of a high concentration of clentiazem (10(-6) M) in coronary, but not in mesenteric or renal arteries. 3. The Ca2+-induced relaxation was also observed in the presence of established Ca2+-antagonists such as diltiazem (3 x 10(-6) M), nifedipine (3 x 10(-8) M) and verapamil (3 x 10(-6) M). 4. The Ca2+-induced relaxation was inhibited by removal of the endothelium, treatment with oxyhaemoglobin (1.5 x 10(-6) M) or methylene blue (10(-5) M), but not by treatment with indomethacin (5 x 10(-6) M). 5. The Ca2+-induced relaxation was observed in an endothelium-denuded coronary artery segment when closely apposed to an endothelium-containing segment of coronary or mesenteric artery. 6. These results suggest that Ca2+-induced relaxation in the presence of high concentrations of Ca2+-antagonists is mediated through endothelium-derived relaxing factor (EDRF). In addition, Ca2+-antagonists do not affect the Ca2+-influx necessary for the release and/or synthesis of EDRF.     

Partial mediation by nitric oxide of the relaxation of human isolated detrusor strips in response to electrical field stimulation.

1. A method for reproducing relaxation of human isolated detrusor smooth muscle in vitro in response to electrical field stimulation is described. 2. The parameters of stimulation associated with relaxation were those which would be expected to give a largely nerve-mediated response: the relaxations were not reduced by tetrodotoxin (3 x 10(-7) M) and were therefore not dependent on voltage sensitive sodium channels. 3. The relaxations were decreased (mean 74.1%) by nitro L-arginine (NOARG, 10(-5) M). 4. Methylene blue (10(-5) M), an inhibitor of soluble guanylate cyclase, abolished the relaxations. 5. These results indicate that there may be a relaxation mechanism in the human bladder which is at least partly mediated via the production of nitric oxide.     

Endothelium-dependent relaxation in response to low concentrations of bradykinin is enhanced by phosphoramidon, bosentan and BQ-123 in bovine coronary arteries in vitro.

An endothelin (ET)-converting enzyme inhibitor phosphoramidon (10 microM), an ET(AB)-receptor antagonist bosentan (10 microM) and an ET(A)-receptor antagonist BQ-123 (1 microM) potentiated endothelium-dependent relaxation of bovine coronary arteries in response to bradykinin (BK) at femtomolar to picomolar concentrations, but not at nanomolar concentrations. BQ-788 (3 microM), an ET(B)-receptor antagonist, showed no significant effects on fM-nM BK-induced relaxation. These results suggest that the endothelium-dependent relaxation of isolated bovine coronary arteries induced by very low concentrations of BK is partly regulated by a complex mechanism involving the ET(A)-receptor antagonism.     

Role of nitric oxide and K+ channels in relaxation induced by polygodial in rabbit corpus cavernosum in vitro

This study examines the relaxation produced by the sesquiterpene polygodial and compares its action with those caused by acetylcholine (ACh) and sodium nitroprusside (SNP) in the rabbit corpus cavernosum (RbCC) in vitro. RbCC was set up in a 5-ml bath containing Krebs solution at 37 degrees C, at pH 7.2, and under 2 g of tension. Polygodial, ACh, and SNP elicited graded relaxation in RbCC with mean EC50 values of 46.70 microM, 0.38 microM, and 0.30 microM, respectively. The nitric oxide (NO) synthase inhibitor L-NOARG and the guanylate cyclase inhibitors LY 83583 and ODQ markedly inhibited the relaxation induced by polygodial (% of inhibition of 79, 48, and 51, respectively) and those caused by ACh (% of inhibition of 100, 49, and 32, respectively). Tetraethylammonium (TEA) and glibenclamide inhibited the relaxation induced by polygodial (52% and 43%, respectively), but only TEA caused shift to the right on ACh-mediated relaxation. In contrast, apamin, charybdotoxin, and 4-aminopyridine or the protein kinase A inhibitor KT 5720 all failed to affect either polygodial or ACh-mediated relaxation in these preparations. The authors concluded that polygodial produced graded relaxation in the RbCC in vitro via a mechanism that was partially dependent on the release of NO or a NO-derived substance through an activation of guanylate cyclase but was independent of adenylate cyclase mechanism. In addition, the opening of K+ channels sensitive to TEA and glibenclamide, but not those sensitive to apamin, 4-aminopyridine, or charybdotoxin, also contributed to the relaxant action produced by polygodial in the RbCC.     

Endothelium-dependent hyperpolarization and relaxation in mesenteric arteries of middle-aged rats: influence of oestrogen.

1. We determined whether gender and/or oestrogen deficiency affect endothelium-dependent hyperpolarization and relaxation in mesenteric arteries isolated from middle-aged (44 - 45 week old) rats. 2. The hyperpolarizing response to acetylcholine (ACh) was significantly greater in females than in males. Ovariectomy caused a marked reduction in ACh-induced hyperpolarization in female arteries, and this was improved by 17 beta-oestradiol replacement therapy. 3. ACh-induced relaxations in female arteries were not significantly different from those observed in male rats, and were unaffected by ovariectomy, regardless of whether indomethacin was present. However, when endothelial nitric oxide synthase (eNOS) was blocked with N(G)-nitro-L-arginine, the sensitivity and maximum relaxant response to ACh was significantly higher in intact females compared with males and ovariectomized females. Treatment with 17 beta-oestradiol prevented the reduced vasorelaxant response in ovariectomized females. 4. Immunohistochemical examination for eNOS showed no apparent difference in eNOS protein expression in the endothelium of arteries between intact and ovariectomized females. 5. Since circulating concentrations of oestrogen were essentially low in middle-aged female rats, the present results suggest that subtle changes from a critical concentration of oestrogen at this age may strongly affect the vascular actions of endothelium-derived hyperpolarizing factor without effect on eNOS expression and activity.     

Nitric oxide- and nitric oxide donors-induced relaxation and its modulation by oxidative stress in piglet pulmonary arteries.

Inhaled nitric oxide (iNO) is widely used in the treatment of pulmonary hypertension while inhaled NO donors have been suggested as an alternative therapy. The differential susceptibility to inactivation by oxidative stress and oxyhaemoglobin of NO and two NO donors, sodium nitroprusside (SNP) and S-nitroso-N-acetyl-penicillamine (SNAP) were analysed in isolated endothelium-denuded pulmonary arteries from 2-week-old piglets stimulated with U46619. NO, SNAP and SNP relaxed the arteries (pIC(30)=7.73+/-0.12, 7.26+/-0.17 and 6.43+/-0.13, respectively) but NO was not detected electrochemically in the bath after the addition of SNP and only at concentrations at which SNAP produced more than 50% relaxation. The sGC inhibitor ODQ (10(-6) M) or the sarcoplasmic Ca(2+)-ATPase thapsigargin (2x10(-6) M) markedly inhibited the relaxation induced by NO, SNAP and SNP. Addition of oxyhaemoglobin (3x10(-7) M) or diethyldithiocarbamate (1 mM) markedly inhibited NO- (pIC(30)=6.88+/-0.07 and 6.92+/-0.18, respectively), weakly inhibited SNAP- and had no effect on SNP-induced relaxation. Xanthine oxidase (5 mu ml(-1)) plus hypoxanthine (10(-4) M) markedly inhibited NO- (pIC(30)=6.96+/-0.12) but not SNAP- or SNP-induced relaxation. Superoxide dismutase (SOD), MnCl(2), diphenileneiodonium and exposing the luminal surface of the rings outwards (inversion) potentiated the relaxant responses of NO (pIC(30)=8.52+/-0.16, 8.23+/-0.11, 8.01+/-0.11 and 8.20+/-0.10, respectively). However, SOD did not modify the NO detected by the electrode and had no effect on SNAP- or SNP-induced relaxation. Therefore, the kinetics and local distribution of NO release of NO donors influence the susceptibility to the scavenging effects of oxyhaemoglobin and superoxide.     

Involvement of nitric oxide in endothelium-dependent, phasic relaxation caused by histamine in monkey cerebral arteries.

Monkey cerebral artery strips partially contracted with prostaglandin F2 alpha responded to histamine with biphasic patterns of relaxation. The delayed and sustained relaxation was suppressed by cimetidine, whereas the phasic response was abolished by treatment with chlorpheniramine and NG-nitro-L-arginine (L-NA), a nitric oxide (NO) synthase inhibitor. The inhibition by L-NA was reversed by L-arginine. D-NA was without effect. Endothelium denudation abolished the phasic relaxation. We hypothesized that endothelium-dependent, phasic relaxations caused by histamine are mediated by NO that is released by H1-receptor stimulation, whereas the sustained relaxation is associated with the activation of H2-receptors in the smooth muscle of monkey cerebral arteries.     

Endothelium-dependent relaxation of rabbit middle cerebral artery to a histamine H3-agonist is reduced by inhibitors of nitric oxide and prostacyclin synthesis.

1. The possible involvement of prostanoids and endothelium-derived relaxing factor (EDRF) in the vasodilatation induced by a histamine H3-agonist was examined in the rabbit perfused middle cerebral artery preconstricted with K+ (50 mM). 2. The endothelium-dependent relaxation to (R)-alpha-methylhistamine [(R)-alpha-MeHA] was competitively antagonized by thioperamide (an H3-antagonist) with a pA2 of 9.05, but unaffected by propranolol, atropine, L- and D-sulpiride. This effect was stereoselective since the (S)-isomer was 100 times less potent than the (R)-isomer. 3. Two inhibitors of nitric oxide synthesis, NG-nitro-L-arginine methyl ester (L-NAME) and NG-monomethyl-L-arginine (L-NMMA), inhibited the relaxation induced by (R)-alpha-methylhistamine. The inhibitory effects of 10(-5) M NG-nitro-L-arginine methyl ester and 10(-5) M NG-monomethyl-L-arginine were reversed by equimolar concentrations of L-arginine, but strongly enhanced by 10(-4) M tranylcypromine. Tranylcypromine alone (10(-5) M-10(-4) M) partially reduced the (R)-alpha-methylhistamine-induced relaxation. Both dexamethasone and indomethacin also inhibited this relaxation. 4. The results suggest that the H3-mediated relaxation of the rabbit middle cerebral artery may involve release of both a prostanoid, probably prostacyclin, and endothelium-derived relaxing factor. The relaxant effects of these two endogenous compounds appear to be synergistic.     

Endothelium-dependent hyperpolarization of smooth muscle cells in rabbit femoral arteries is not mediated by EDRF (nitric oxide)

Summary Acetylcholine elicits an endothelium-dependent hyperpolarization of vascular smooth muscle cells. The experiments reported here tested the hypothesis that this hyperpolarization is mediated by the endothelium-derived relaxant factor (EDRF) identified as nitric oxide. Membrane potential was recorded with standard glass microelectrodes in smooth muscle cells in segments of rabbit femoral arteries. In endothelium-intact vessels, smooth muscle cells (resting potential: –67.0 ± 1.3 mV) hyperpolarized significantly (P < 0.001) by 5.7 ± 0.9 mV in response to acetylcholine (1 M). Inhibition of EDRF, either in the presence of hemoglobin or by pretreatment with gossypol, attenuated the relaxation elicited by acetylcholine in endothelium-intact segments precontracted with 0.1 M noradrenaline but had no significant effect on either the control membrane potential (–62.2 ± 1.9 mV and –68.5 ± 2.1 mV, respectively) or the hyperpolarization in response to acetylcholine (5.0 ± 1.6 mV and 5.8 ± 1.6 mV, respectively). In contrast, in vessel segments with the endothelium removed, the hyperpolarization in response to acetylcholine was abolished although the control membrane potential (–68.0 ± 5.1 mV) was not significantly different from that in endotheliumintact vessels. Sodium nitroprusside, an endothelium-dependent vasodilator and exogenous analog of EDRF, also had no significant effect on membrane potential. The lack of response to acetylcholine was not merely the result of nonspecific damage to the smooth muscle cells: vessel segments without endothelium were still able to hyperpolarize in response to various other vasodilators. These results suggest that the endothelium-dependent hyperpolarization of vascular smooth muscle cells in response to acetylcholine is not mediated by EDRF.Send offprint requests to R. Busse at the above address