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.     

Intestinal relaxation by endothelin isopeptides: involvement of Ca(2+)-activated K+ channels.

Our previous studies have shown that endothelin-1 (ET-1) induces an initial relaxation followed by a contraction in the guinea-pig ileum. To test whether other ET isopeptides (ET-2, ET-3, vasoactive intestinal contractor (VIC) and sarafotoxin S6b) and big ET-1, the ET-1 precursor, also induce similar biphasic responses, we compared their effects in isolated guinea-pig ileum. In addition, the mechanism of initial relaxation was studied. At 1-100 nM, ET-1, ET-2 and VIC were equipotent in producing the biphasic responses. S6b also produced similar biphasic responses, except that only a relaxation was elicited at 1 nM. ET-3 was approximately 30- to 100-fold less active than ET-1 in producing the contraction, whereas it was as potent as ET-1 in producing relaxation. Big ET-1 induced a relaxation of slower onset and longer duration, followed by a weak contraction at concentrations higher than 30 nM. The initial relaxation produced by ET-1 was not affected by pretreatment with L-NAME (NW-nitro-L-arginine methyl ester), hemoglobin, 9-AC (anthracene-9-carboxylic acid), SITS (4-acetamido-4'-isothiocyanatostilbene-2-2'-disulfonic acid), glibenclamide, ouabain, phorbol 12,13-dibutyrate, sodium nitroprusside, human atrial natriuretic peptide (hANP) or forskolin, whereas it was abolished by pretreatment with apamin. Although phorbol 12,13-dibutyrate pretreatment had no significant effect on the biphasic response of ET-1, it rapidly reversed the sustained contraction produced by ET-1. These results indicate that the initial relaxation is caused by the activation of Ca(2+)-activated K+ channels.     

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。     

Predominant role of A1 adenosine receptors in mediating adenosine induced vasodilatation of rat diaphragmatic arterioles: involvement of nitric oxide and the ATP-dependent K+ channels

1. We investigated, by intravital microscopy in rats, the role of the subtypes of adenosine receptors A₁ (A₁/AR) and A₂ (A₂AR) in mediating adenosine-induced vasodilatation of second and third order arterioles of the diaphragm.
2. Adenosine, and the A₁AR selective agonists R(−)-N⁶-(2-phenylisopropyl)-adenosine (R-PIA) and N⁶-cyclo-pentyl-adenosine (CPA) induced a similar concentration-dependent dilatation of diaphragmatic arterioles. The non selective A₂AR subtype agonist N⁶-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl) ethyl]adenosine (DPMA) also dilated diaphragmatic arterioles but induced a significantly smaller dilatation than adenosine. By contrast the selective A_2aAR subtype agonist 2-[p-(2-carboxyethyl)phenyl amino]-5′-N-ethyl carboxamido adenosine (CGS 21680) did not modify diaphragmatic arteriolar diameter.
3. The non selective adenosine receptor antagonist 1,3-dipropyl-8-p-sulphophenylxanthine (SPX, 100 μM) and the selective A₁AR antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPX, 50 nM) significantly attenuated adenosine-induced dilatation of diaphragmatic arterioles. By contrast, adenosine significantly dilated diaphragmatic arterioles in the presence of A₂AR antagonist 3,7-dimethyl-1-propargylxanthine (DMPX, 10 μM).
4. The dilatation induced by adenosine was unchanged by the mast cell stabilizing agent sodium cromoglycate (cromolyn, 10 μM).
5. The nitric oxide (NO) synthase inhibitor N^ω-nitro-L-arginine (L-NOARG, 300 μM) attenuated the dilatation induced by adenosine, and by the A₁AR and A₂AR agonists.
6. The ATP-dependent K⁺ channel blocker glibenclamide (3 μM) significantly attenuated diaphragmatic arteriolar dilatation induced by adenosine and by the A₁AR agonists R-PIA and CPA. By contrast, glibenclamide did not significantly modify arteriolar dilatation induced by the A₂AR agonist DPMA.
7. These findings suggest that adenosine-induced dilatation of diaphragmatic arterioles in the rat is predominantly mediated by the A₁AR, via the release of NO and activation of the ATP-dependent K⁺ channels.

     

Cannabinoid agonists induce relaxation in the bovine ophthalmic artery: evidences for CB1 receptors, nitric oxide and potassium channels

Glaucoma pathophysiology appears to involve vascular deficits, which may contribute to initiation and progression of the disease. Anandamide, the endogenous cannabinoid ligand, and WIN55212-2, a synthetic cannabinoid agonist, are able to evoke concentration-dependent relaxations in bovine ophthalmic artery rings, precontracted with 5-hydroxytryptamine (5-HT) (1 microM). Endothelium removal reduces cannabinoid agonist potency and efficacy. The selective cannabinoid 1 (CB1) receptor antagonists SR141716A (100 nM) and AM251 (100 nM) cause a shift to the right in the concentration-response curves to anandamide and WIN55212-2 in arterial rings both in the presence and in the absence of endothelium. In endothelium-intact arteries, the nitric oxide synthase inhibitor, N(G)-monomethyl-L-arginine (L-NMMA, 300 microM), completely blocked the anandamide- and WIN55212-2-relaxant responses; by contrast, the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP, 100 microM) induced an increase in vasorelaxant responses to cannabinoid agonists. Relaxations to anandamide and WIN55212-2 were inhibited by iberiotoxin (IbTX, 200 nM), a blocker of large conductance, Ca2+-activated K+ channel (BK(Ca)), and by 4-aminopyridine (4-AP; 1 mM), a blocker of delayed rectifier K+ channel, whereas the blockade of K(ATP) channels by glibenclamide (5 microM) and of small conductance Ca2+-activated K+ channels (SK(Ca)) by apamin (100 nM) did not produce any effects. These data suggest that anandamide and WIN55212-2 relax the bovine ophthalmic artery by involving CB1 the cannabinoid receptor-sensitive pathway. In endothelium-intact arteries, relaxation occurs through activation of nitric oxide synthase cyclic GMP and Ca2+-activated K+ channels. They also cause endothelium-independent relaxation by involving potassium channel opening.     

Superoxide anion and K+ channels mediate electrical stimulation-induced relaxation in the rat basilar artery.

Electrical field stimulation (a single pulse, 0.2 ms) caused a rapid relaxation of rat basilar artery segments precontracted with different agents, but not with 30 mM KCl. This relaxation was not modified by endothelium removal, 10 microM tetrodotoxin, 1 microM propranolol, 1 microM atropine, 30 microM indomethacin, 10 microM methylene blue, 100 microM N(G)-nitro-L-arginine methyl ester or 1 microM cimetidine but it was significantly reduced by 50 and 100 U/ml superoxide dismutase. Charybdotoxin (0.1 and 0.2 microM), a blocker of large-conductance Ca2+-activated K+ channels (BK(Ca)), decreased the relaxation elicited by electrical stimulation, whereas it was unaltered by 10 microM glibenclamide or 1 microM apamin, blockers of ATP-sensitive (K(ATP)) or small-conductance K(Ca) channels, respectively. Thapsigargin (0.01 and 0.1 microM), an inhibitor of sarcoplasmic reticulum Ca2+-ATPase, increased the electrical stimulation-induced relaxation, which was nearly abolished by charybdotoxin. These results show that electrical stimulation induces endothelium-independent and non-neurogenic relaxations in the rat basilar artery. This response appears to involve generation of superoxide anion, increase of cytosolic free Ca2+ concentration and subsequent activation of BK(Ca) channels.     

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.     

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.     

Role of K+ channels in the vasodilator response to bradykinin in the rat heart.

1. The role of K+ channels in the nitric oxide (NO)-independent coronary vasodilator effect of bradykinin was examined in the Langendorff heart preparation in which nitroarginine was used to inhibit NO synthesis and elevate perfusion pressure; cyclo-oxygenase was inhibited with indomethacin. 2. The K+ channel inhibitors, tetraethylammonium, procaine and charybdotoxin, but not glibenclamide, further increased perfusion pressure suggesting a role for K+ channels, other than ATP-sensitive K+ channels, in the regulation of coronary vascular tone under the experimental conditions adopted here. 3. The non-specific K+ channel inhibitors, tetraethylammonium and procaine, reduced vasodilator responses to bradykinin and cromakalim but not those to nitroprusside in the perfused heart treated with nitroarginine and indomethacin. 4. Glibenclamide, an inhibitor of ATP-sensitive K+ channels, reduced vasodilator responses to cromakalim but did not affect those to bradykinin or nitroprusside. 5. Charybdotoxin, an antagonist of Ca(2+)-activated K+ channels, inhibited responses to bradykinin but did not affect those to cromakalim or nitroprusside. 6. Nifedipine inhibited vasodilator responses to bradykinin and cromakalim without affecting those to nitroprusside. 7. Inhibition of cytochrome P450 with clotrimazole reduced responses to bradykinin but did not modify those to cromakalim or nitroprusside. 8. These results suggest that bradykinin utilizes a Ca(2+)-activated K+ channel to produce vasodilatation in the rat heart.     

Cyclic GMP-independent relaxation of rat pulmonary artery by spermine NONOate, a diazeniumdiolate nitric oxide donor

In rat pulmonary artery pre-contracted with phenylephrine, the mechanisms of relaxation to the nitric oxide (NO) donor, spermine NONOate, were investigated. Responses to spermine NONOate were only partially blocked by the soluble guanylate cyclase inhibitor, ODQ (1H:-[1,2,4]Oxadiazolo-[4,3,-a]quinoxalin-1-one) at concentrations up to 30 microM. Ten microM ODQ gave maximal inhibition. Endothelium removal had no effect on the potency of spermine NONOate or its inhibition by ODQ. The protein kinase G inhibitor, Rp-8-Br-cGMPS (100 microM), caused minimal inhibition of spermine NONOate despite causing marked inhibition of glyceryl trinitrate and isosorbide dinitrate. Spermine NONOate (100 microM) caused a 35 fold increase in guanosine 3'5' cyclic monophosphate (cyclic GMP) above basal levels in pulmonary artery rings. ODQ (3 microM) abolished this cyclic GMP production but did not inhibit corresponding relaxant responses. Similar results were seen with another NONOate (MAHMA NONOate; 10 microM). ODQ-resistant relaxation to spermine NONOate (i. e. relaxation seen in the presence of 10 microM ODQ) was inhibited by potassium (80 mM), charybdotoxin (300 nM), iberiotoxin (300 nM), apamin (100 nM), ouabain (1 mM) or thapsigargin (100 nM) but not by 4-aminopyridine (3 mM), glybenclamide (10 microM) or diltiazem (10 microM). Potassium, charybdotoxin, ouabain and thapsigargin also inhibited ODQ-resistant relaxation to FK409 ((+/-)-E:-4-ethyl-2-[E:-hydroxyimino]-5-nitro-3-hexenamide). We conclude that, on rat pulmonary artery, spermine NONOate can produce cyclic GMP-independent relaxation that involves, at least in part, activation of Na(+)/K(+)-ATPase, sarco-endoplasmic reticulum Ca(2+)-ATPase and calcium-activated potassium channels.     

Nitric oxide, prostanoid and non-NO, non-prostanoid involvement in acetylcholine relaxation of isolated human small arteries

The main purpose of the study was to clarify to which extent nitric oxide (NO) contributes to acetylcholine (ACh) induced relaxation of human subcutaneous small arteries. Arterial segments were mounted in myographs for recording of isometric tension, NO concentration and smooth muscle membrane potential. In noradrenaline-contracted arteries, ACh induced endothelium-dependent relaxations. The NO synthase inhibitor, N(G)-nitro-L-arginine (L-NOARG) had a small significant effect on the concentration-response curves for ACh, and in the presence of L-NOARG, indomethacin only caused a small additional rightward shift in the ACh relaxation. The NO scavenger, oxyhaemoglobin attenuated relaxations for ACh and for the NO donor S-nitroso-N-acetylpenicillamine (SNAP). Inhibition of guanylyl cyclase with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ), and inhibition of protein kinase G with beta-phenyl-1, N2-etheno-8-bromoguanosine- 3', 5'- cyclic monophosphorothioate, Rp-isomer, slightly attenuated ACh relaxation, but abolished SNAP induced relaxation. ACh induced relaxation without increases in the free NO concentration. In contrast, for equivalent relaxation, SNAP increased the NO concentration 32+/-8 nM. ACh hyperpolarized the arterial smooth muscle cells with 11.4+/-1.3 mV and 10.5+/-1.3 mV in the absence and presence of L-NOARG, respectively. SNAP only elicited a hyperpolarization of 1.6+/-0.9 mV. In the presence of indomethacin and L-NOARG, ACh relaxation was almost unaffected by lipoxygenase inhibition with nordihydroguaiaretic acid, or cytochrome P450 inhibition with 17-octadecynoic acid or econazole. ACh relaxation was strongly reduced by the combination of charybdotoxin and apamin, but small increments in the extracellular potassium concentration induced no relaxations. The study demonstrates that the NO/L-arginine pathway is present in human subcutaneous small arteries and to a limited extent is involved in ACh induced relaxation. The study also suggests a small contribution of arachidonic acid metabolites. However, ACh relaxation is mainly dependent on a non-NO, non-prostanoid endothelium dependent hyperpolarization. British Journal of Pharmacology (2000) 129, 184 - 192     

Contribution of nitric oxide, cyclic GMP and K+ channels to acetylcholine-induced dilatation of rat conduit and resistance arteries

1. We compared the effects of inhibiting nitric oxide synthase (NOS), soluble guanylate cyclase (sGC) and K+ channel activation on dilator responses to acetylcholine (ACh) in rat resistance (hindquarters) and conduit arteries (thoracic aorta). 2. In rat perfused hindquarters, the NO synthase inhibitor N omega-nitro-L-arginine (L-NNA; 1 mmol/L) partially inhibited the ACh-induced dilatation and the combination of L-NNA + haemoglobin (Hb; 20 mumol/L), a NO scavenger, did not further affect the response. Exposure to high K+ (30 mmol/L) also inhibited the response to ACh and this response was further reduced by L-NNA + high K+. Surprisingly, when applied alone 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of sGC, did not affect responses to ACh, whereas treatment with ODQ + high K+ markedly impaired dilatation. 3. In aortic rings precontracted with phenylephrine (PE; 0.01-1 mumol/L), the maximum relaxation to ACh was significantly reduced by L-NNA (0.1 mmol/L) and further inhibited by L-NNA + Hb (20 mumol/L). At 10 mumol/L, ODQ alone inhibited the maximum relaxation to ACh, which was further reduced by ODQ + high K+ (30 mmol/L). High K+ caused a smaller but significant inhibition of ACh-induced relaxation. 4. These results suggest that NO and cGMP play a relatively greater role in ACh-induced dilatation of the aorta compared with the hindquarters resistance vasculature and are consistent with the hypothesis that a non-NO endothelium-derived hyperpolarizing factor (endothelium-derived hyperpolarizing factor; EDHF) makes a relatively greater contribution to dilatation of resistance vessels than in conduit arteries. The data suggest that when sGC is inhibited, a compensatory mechanism involving K+ channel opening by NO can largely maintain ACh-induced vasodilator responses of resistance vessels. Furthermore, when NO synthesis is blocked, a non-NO EDHF may play a role in ACh-induced dilatation of the resistance vasculature.     

Evidence that different mechanisms underlie smooth muscle relaxation to nitric oxide and nitric oxide donors in the rabbit isolated carotid artery

1. The endothelium-dependent relaxants acetylcholine (ACh; 0.03–10 μM) and {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 (0.03–10 μM), and nitric oxide (NO), applied either as authentic NO (0.01–10 μM) or as the NO donors 3-morpholino-sydnonimine (SIN-1; 0.1–10 μM) and S-nitroso-N-acetylpenicillamine (SNAP; 0.1–10 μM), each evoked concentration-dependent relaxation in phenylephrine stimulated (1–3 μM; mean contraction and depolarization, 45.8±5.3 mV and 31.5±3.3 mN; n=10) segments of rabbit isolated carotid artery. In each case, relaxation closely correlated with repolarization of the smooth muscle membrane potential and stimulated a maximal reversal of around 95% and 98% of the phenylephrine-induced depolarization and contraction, respectively.
2. In tissues stimulated with 30 mM KCl rather than phenylephrine, smooth muscle hyperpolarization and relaxation to ACh, {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187, authentic NO and the NO donors were dissociated. Whereas the hyperpolarization was reduced by 75–80% to around a total of 10 mV, relaxation was only inhibited by 35% (n=4–7 in each case; P<0.01). The responses which persisted to ACh and {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 in the presence of 30 mM KCl were abolished by either the NO synthase inhibitor L-N^G-nitroarginine methyl ester (L-NAME; 100 μM) or the inhibitor of soluble guanylyl cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 μM; 10 min; n=4 in each case; P<0.01).
3. Exposure to ODQ significantly attenuated both repolarization and relaxation to ACh, {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 and authentic NO, reducing the maximum changes in both membrane potential and tension to each relaxant to around 60% of control values (n=4 in each case; P<0.01). In contrast, ODQ almost completely inhibited repolarization and relaxation to SIN-1 and SNAP, reducing the maximum responses to around 8% in each case (n=3–5; P<0.01).
4. The potassium channel blockers glibenclamide (10 μM), iberiotoxin (100 nM) and apamin (50 nM), alone or in combination, had no significant effect on relaxation to ACh, {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187, authentic NO, or the NO donors SIN-1 and SNAP (n=4 in each case; P>0.05). Charybdotoxin (ChTX; 50 nM) almost abolished repolarization to ACh (n=4; P<0.01) and inhibited the maximum relaxation to ACh, {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 and authentic NO each by 30% (n=4–8; P<0.01). Application of ODQ (10 μM; 10 min) abolished the ChTX-insensitive responses to ACh, {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 and authentic NO (n=4 in each case; P<0.01
5. When the concentration of phenylephrine was reduced (to 0.3–0.5 μM) to ensure the level of smooth muscle contraction was the same as in the absence of potassium channel blocker, ChTX had no effect on the subsequent relaxation to SIN-1 (n=4; P>0.05). However, in the presence of tone induced by 1–3 μM phenylephrine (51.2±3.3 mN; n=4), ChTX significantly reduced relaxation to SIN-1 by nearly 50% (maximum relaxation 53.2±6.3%, n=4; P<0.01).
6. These data indicate that NO-evoked relaxation of the rabbit isolated carotid artery can be mediated by three distinct mechanisms: (a) a cyclic GMP-dependent, voltage-independent pathway, (b) cyclic GMP-mediated smooth muscle repolarization and (c) cyclic GMP-independent, ChTX-sensitive smooth muscle repolarization. Relaxation and repolarization to both authentic and endothelium-derived NO in this large conduit artery appear to be mediated by parallel cyclic GMP-dependent and -independent pathways. In contrast, relaxation to the NO-donors SIN-1 and SNAP appears to be mediated entirely via cyclic GMP-dependent mechanisms.

     

Acidosis induces relaxation mediated by nitric oxide and potassium channels in rat thoracic aorta

We investigated the mechanism by which extracellular acidification promotes relaxation in rat thoracic aorta. The relaxation response to HCl-induced extracellular acidification (7.4 to 6.5) was measured in aortic rings pre-contracted with phenylephrine (Phe, 10(-6) M) or KCl (45mM). The vascular reactivity experiments were performed in endothelium-intact and denuded rings, in the presence or absence of indomethacin (10(-5) M), L-NAME (10(-4) M), apamin (10(-6) M), and glibenclamide (10(-5) M). The effect of extracellular acidosis (pH 7.0 and 6.5) on nitric oxide (NO) production was evaluated in isolated endothelial cells loaded with diaminofluorescein-FM diacetate (DAF-FM DA, 5μM). The extracellular acidosis failed to induce any changes in the vascular tone of aortic rings pre-contracted with KCl, however, it caused endothelium-dependent and independent relaxation in rings pre-contracted with Phe. This acidosis induced-relaxation was inhibited by L-NAME, apamin, and glibenclamide, but not by indomethacin. The acidosis (pH 7.0 and 6.5) also promoted a time-dependent increase in the NO production by the isolated endothelial cells. These results suggest that extracellular acidosis promotes vasodilation mediated by NO, K(ATP) and SK(Ca), and maybe other K(+) channels in isolated rat thoracic aorta.     

Involvement of cyclic GMP and potassium channels in relaxation evoked by the nitric oxide donor, diethylamine NONOate, in the rat small isolated mesenteric artery

The relative functional importance of potassium channels and cGMP-dependent pathways in the relaxation of vascular smooth muscle to the novel nitric oxide donor, diethylamine NONOate (DEA NONOate), was investigated in a resistance artery. The contribution from cGMP-dependent signalling pathways was examined by exposing arteries to 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a selective inhibitor of soluble guanylyl cyclase, while the contribution through potassium channels was assessed with different sub-type-selective potassium channel blockers. DEA NONOate (3 nM-10 microM) evoked sustained relaxation in isolated segments of the rat small mesenteric artery contracted with phenylephrine (pEC50=6.7+/-0.2; n=11). The relaxation was attenuated significantly by either ODQ (10 microM; pEC50=5.8+/-0.4; n=7) or charybdotoxin (ChTX; 50 nM; pEC50=6.3+/-0.2; n=4), a peptide blocker of large conductance, calcium-activated potassium channels (BK(Ca)). The inhibitory effects of ODQ and ChTX were additive (pEC50=5.1+/-0.4; n=9). The selective inhibitor of BK(Ca) channels, iberiotoxin (IbTX; 30 nM), and 4-aminopyridine (4-AP; 1 mM), an inhibitor of voltage-gated potassium channels (Kv), failed to modify DEA NONOate-evoked relaxation. However, in the combined presence of both ODQ and either IbTX or 4-AP the relaxation was attenuated significantly (n=3). The blocker of ATP-modulated potassium channels (K(ATP)), glibenclamide (10 microM), and of small conductance calcium-activated potassium channels (SK(Ca)), apamin (30 nM), each failed to affect ODQ-sensitive or -resistant relaxations to DEA NONOate (n=3). In conclusion, relaxation to DEA NONOate in the rat isolated, small mesenteric artery can occur via both cGMP-dependent (ODQ-sensitive) and -independent (ODQ-resistant) mechanisms. However, the contribution made to relaxation by potassium channels appears to be unmasked following pharmacological attenuation of cGMP-dependent signalling pathways. The inhibitory action of ChTX suggests part of the cGMP-insensitive component involves the activation of potassium channels, a suggestion supported by the inhibitory actions of 4-AP and IbTX in the absence of cGMP.     

Contribution of NO,ATP-sensitive K+ channels and prostaglandins to adenosine receptor agonists-induced relaxation of the rat tail artery

The mechanism of relaxation in the rat tail artery induced by the adenosine A1 receptor-selective agonist N⁶-cyclohexyladenosine (CHA, 10 nM–300 μM) and the adenosine A1/A_2a receptor agonist 5’-N-ethylcarboxamidoadenosine (NECA, 10 nM–300 μM) has been characterized. To do this, we used α₁-receptor agonist phenylephrine to evoke contraction (10 μM), and inhibitors of nitric oxide synthase (L-NAME, 10 μM), ATP-sensitive K⁺ channels (glibenclamide, 10 μM) and prostaglandin synthesis (indomethacin, 10 μM). CHA and NECA induced relaxation of rat-tail artery by 80% and 70% in a concentration-dependent manner, respectively. The relaxation effect of NECA was completely abolished in the presence of L-NAME, while glibenclamide and indomethacin prevented CHA-induced relaxation of the rat tail artery by approximately 25% and 40%, respectively. Our results indicate that nonspecific effects such nitric oxide and prostaglandins release or the activation of potassium channels significantly contributed to the effects of CHA and NECA.     

Iron-sulphur cluster nitrosyls, a novel class of nitric oxide generator: mechanism of vasodilator action on rat isolated tail artery.

1. Two iron-sulphur cluster nitrosyls have been investigated as potential nitric oxide (NO.) donor drugs (A: tetranitrosyltetra-mu 3-sulphidotetrahedro-tetrairon; and B: heptanitrosyltri-mu 3-thioxotetraferrate(1-)). Both compounds are shown to dilate precontracted, internally-perfused rat tail arteries. 2. Bolus injections (10 microliters) of compound A or B generate two kinds of vasodilator response. Doses below a critical threshold concentration (DT) evoke transient (or T-type) responses, which resemble those seen with conventional nitrovasodilators. Doses > DT produce sustained (or S-type) responses, comprising an initial, rapid drop of pressure, followed by incomplete recovery, resulting in a plateau of reduced tone which can persist for several hours. 3. T- and S-type responses are attenuated by ferrohaemoglobin (Hb) and by methylene blue (MB), but not by inhibitors of endothelial NO. synthase. Addition of either Hb or MB to the internal perfusate can restore agonist-induced tone when administered during the plateau phase of an S-type response. Moreover, subsequent removal of Hb causes the artery to re-dilate fully. 4. We conclude that T- and S-type responses are both mediated by NO.. It is postulated that S-type responses represent the sum of two vasodilator components: a reversible component, superimposed upon a non-recoverable component. The former is attributed to free NO., preformed in solution at the time of injection; and the latter to NO. generated by gradual decomposition of a 'store' of iron-sulphur-nitrosyl complexes within the tissue. This hypothesis is supported by histochemical studies which show that both clusters accumulate in endothelial cells.     

Pharmacological investigation into the involvement of nitric oxide in K+-induced cortical spreading depression

Cortical spreading depression (CSD) is a transient, local disruption of cellular ionic homeostasis that propagates slowly across the cerebral cortex. As previous data have suggested a possible link between nitric oxide (NO) formation and CSD, we have examined whether CSD is suppressed by local inhibition of NO synthesis with 7-nitroindazole (7-NINA), a compound which may have a greater selectivity for the neuronal NO synthase isoform. Multifunctional microdialysis probes were implanted in the cortex of halothane-anaesthetised rats, and used for (1) elicitation of repetitive CSD by perfusion of 160 mM K+ through the probe, (2) recording of CSD as a negative shift of the extracellular direct current (DC) potential, and (3) perfusion of 7-NINA before and during CSD elicitation. Elicitation of CSD was moderately inhibited by 1 mM 7-NINA in the perfusion medium, as shown in one treated group (n=8) by a significant reduction of both number (from 5.1+/-0.4 to 3.6+/-0.4; P<0.05) and cumulative DC negativity (from 16.4+/-0.7 mV x min to 13.3+/-0.9 mV x min; P<0.01). However, effective concentrations of 7-NINA were at least 100-fold higher than its Ki for the target enzyme in vitro, the moderate inhibition of CSD by 7-NINA was not reversed by the NO precursor, L-arginine, and the amplitude of the K+-induced sustained DC potential negative shift was also reduced significantly by 7-NINA (from 27.9+/-0.9 mV to 23.9+/-1.2 mV; P<0.05). These data do not support the hypothesis that NO formation contributes to the elicitation of CSD by high extracellular K+. The finding that 7-NINA reduced the intensity of K+-induced depolarisation may be relevant to previous investigations that used this drug to examine the role of NO in the modulation of K+-induced neurotransmitter release.     

Kaempferol enhances endothelium-dependent relaxation in the porcine coronary artery through activation of large-conductance a2+-activated K+ channels

Background and Purpose

Kaempferol, a plant flavonoid present in normal human diet, can modulate vasomotor tone. The present study aimed to elucidate the signalling pathway through which this flavonoid enhanced relaxation of vascular smooth muscle.

Experimental Approach

The effect of kaempferol on the relaxation of porcine coronary arteries to endothelium-dependent (bradykinin) and -independent (sodium nitroprusside) relaxing agents was studied in an in vitro organ chamber setup. The whole-cell patch-clamp technique was used to determine the effect of kaempferol on potassium channels in porcine coronary artery smooth muscle cells (PCASMCs).

Key Results

At a concentration without direct effect on vascular tone, kaempferol (3 × 10⁻⁶ M) enhanced relaxations produced by bradykinin and sodium nitroprusside. The potentiation by kaempferol of the bradykinin-induced relaxation was not affected by N^ω-nitro-L-arginine methyl ester, an inhibitor of NO synthase (10⁻⁴ M) or TRAM-34 plus UCL 1684, inhibitors of intermediate- and small-conductance calcium-activated potassium channels, respectively (10⁻⁶ M each), but was abolished by tetraethylammonium chloride, a non-selective inhibitor of calcium-activated potassium channels (10⁻³ M), and iberiotoxin, a selective inhibitor of large-conductance calcium-activated potassium channel (K_Ca1.1; 10⁻⁷ M). Iberiotoxin also inhibited the potentiation by kaempferol of sodium nitroprusside-induced relaxations. Kaempferol stimulated an outward-rectifying current in PCASMCs, which was abolished by iberiotoxin.

Conclusions and Implications

The present results suggest that, in smooth muscle cells of the porcine coronary artery, kaempferol enhanced relaxations caused by endothelium-derived and exogenous NO as well as those due to endothelium-dependent hyperpolarization. This vascular effect of kaempferol involved the activation of K_Ca1.1 channels.