Vasoconstrictor stimulus determines the functional contribution of myoendothelial feedback to mesenteric arterial tone.

KEY POINTS: In isolated resistance arteries, endothelial modulation of vasoconstrictor responses to α1 -adrenoceptor agonists occurs via a process termed myoendothelial feedback: localized inositol trisphosphate (InsP3 )-dependent Ca2+ transients activate intermediate conductance Ca2+ -activated K+ (IKCa ) channels, hyperpolarizing the endothelial membrane potential to limit further reductions in vessel diameter. We demonstrate that IKCa channel-mediated myoendothelial feedback limits responses of isolated mesenteric arteries to noradrenaline and nerve stimulation, but not to the thromboxane A2 mimetic U46619 or to increases in intravascular pressure. In contrast, in the intact mesenteric bed, although responses to exogenous noradrenaline were limited by IKCa channel-mediated myoendothelial feedback, release of NO and activation of endothelial small conductance Ca2+ -activated K+ (SKCa ) channels in response to increases in shear stress appeared to be the primary mediators of endothelial modulation of vasoconstriction. We propose that (1) the functional contribution of myoendothelial feedback to arterial tone is determined by the nature of the vasoconstrictor stimulus, and (2) although IKCa channel-mediated myoendothelial feedback may contribute to local control of arterial diameter, in the intact vascular bed, increases in shear stress may be the major stimulus for engagement of the endothelium during vasoconstriction. ABSTRACT: Constriction of isolated resistance arteries in response to α1 -adrenoceptor agonists is limited by reciprocal engagement of inhibitory endothelial mechanisms via myoendothelial feedback. In the current model of feedback, agonist stimulation of smooth muscle cells results in localized InsP3 -dependent Ca2+ transients that activate endothelial IKCa channels. The subsequent hyperpolarization of the endothelial membrane potential then feeds back to the smooth muscle to limit further reductions in vessel diameter. We hypothesized that the functional contribution of InsP3 -IKCa channel-mediated myoendothelial feedback to limiting arterial diameter may be influenced by the nature of the vasoconstrictor stimulus. To test this hypothesis, we investigated the functional role of myoendothelial feedback in modulating responses of rat mesenteric resistance arteries to the adrenoceptor agonist noradrenaline, the thromboxane A2 mimetic U46619, increases in intravascular pressure and stimulation of perivascular sympathetic nerves. In isolated arteries, responses to noradrenaline and stimulation of sympathetic nerves, but not to U46619 and increases in intravascular pressure, were modulated by IKCa channel-dependent myoendothelial feedback. In the intact mesenteric bed perfused under conditions of constant flow, responses to exogenous noradrenaline were modulated by myoendothelial feedback, but shear stress-induced release of NO and activation of endothelial SKCa channels appeared to be the primary mediators of endothelial modulation of vasoconstriction to agonists and nerve stimulation. Thus, we propose that myoendothelial feedback may contribute to local control of diameter within arterial segments, but at the level of the intact vascular bed, increases in shear stress may be the major stimulus for engagement of the endothelium during vasoconstriction.

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.

Relaxation to authentic nitric oxide and SIN-1 in rat isolated mesenteric arteries: variable role for smooth muscle hyperpolarization.

Authentic nitric oxide (NO; 0.1 - 10 micromoles) caused transient, dose-dependent relaxation of phenylephrine-induced tone without changing membrane potential in mesenteric arteries. Larger doses, above 10 micromoles, did not evoke more relaxation (maximal relaxation to 150 micromoles NO in denuded arteries, 69+/-7%, n=8) but stimulated muscle hyperpolarization (maximum 19+/-3 mV, n=5). The soluble guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 microM), abolished relaxation to low doses of NO (n=4), but did not modify hyperpolarization with higher doses of NO (n=4). The potassium channel blocker charybdotoxin (ChTX; 50 nM) abolished hyperpolarization to high doses of NO and significantly reduced the maximal relaxation (to 43+/-6%, n=4; P<0.01). ODQ and ChTX together abolished tension and membrane potential change to all doses of NO (n=4). All relaxations to 3-morpholino-sydnonimine (SIN-1; 0.01 - 10 microM) were associated with hyperpolarization. When the endothelium was intact, ChTX inhibited hyperpolarization and relaxation to SIN-1 (n=5), while iberiotoxin (IbTX; 50 nM) or 4-aminopyridine (4-AP; 500 microM) reduced relaxation by 40% and 20%, respectively and by 80% in combination (n=6 in each case). In denuded arteries, relaxation to SIN-1 was unaffected by either ChTX or ODQ alone, but abolished by the inhibitors together (n=6). Alone, 4-AP did not alter relaxation, but in the presence of ODQ it reduced the maximal response by around 45% (n=6; P<0.01). 4-AP, ODQ and IbTX together inhibited relaxation to SIN-1 by 75% (n=6; P<0.01). Therefore, cyclic guanosine 3',5'-monophosphate (cyclic GMP)-independent smooth muscle hyperpolarization, possibly involving direct activation of calcium-activated and voltage-sensitive potassium channels, contributes to relaxation evoked by authentic NO and SIN-1. However, the importance of each pathway depends on the source of NO and with SIN-1 the relative contribution from each pathway is modified by the endothelium.

Investigation of the inhibitory effects of homocysteine and copper on nitric oxide-mediated relaxation of rat isolated aorta.

1. Elevated plasma levels of homocysteine (HC) and copper have both been associated with the development of inflammatory vascular diseases, such as atherosclerosis. In this study, the effects of a combination of HC and copper on nitric oxide (NO)-mediated relaxation of isolated rat aortic rings were investigated. 2. Exposure to HC (10-100 microM; 30 min) had no effect on relaxation to acetylcholine (ACh; 0.01-10 microM, n=4). Pre-incubation of aortic rings with a higher concentration of HC for an extended period (1 mM; 180 min) significantly inhibited endothelium-dependent relaxation (n=4), but this inhibition was prevented by the presence of the copper chelator bathocuprione (10 microM, 180 min, n=6). 3. Exposure to HC (100 microM) and copper (10-100 microM; 30 min) caused a copper concentration-dependent inhibition of endothelium-dependent relaxation (n=4). This inhibitory effect was reduced in the presence of either superoxide dismutase (SOD; 100 u ml(-1); n=4) or catalase (100 u ml(-1); n=4), and further reduced by the presence of both enzymes (n=5). 4. HC and copper (100 microM; 30 min) significantly inhibited endothelium-independent relaxation to glyceryl trinitrate (0.01-10 microM; n=8). In contrast, HC (1 mM), alone or in combination with copper (100 microM), did not inhibit relaxation to the endothelium-independent relaxant sodium nitroprusside (0.01-10 microM; n=4). 5. These data indicate that the presence of copper greatly enhances the inhibitory actions of HC on NO-mediated relaxation of isolated aortic rings. The reduction of inhibition by catalase and SOD indicates a possible role for copper-catalyzed generation of superoxide and hydrogen peroxide leading to an increased inactivation or decreased production of endothelium-derived NO.

Charybdotoxin and apamin block EDHF in rat mesenteric artery if selectively applied to the endothelium.

In rat mesenteric artery, endothelium-derived hyperpolarizing factor (EDHF) is blocked by a combination of apamin and charybdotoxin (ChTX). The site of action of these toxins has not been established. We compared the effects of ChTX and apamin applied selectively to the endothelium and to the smooth muscle. In isometrically mounted arteries, ACh (0.01-10 micrometers), in the presence of indomethacin (2.8 microM) and Nomega-nitro-L-arginine methyl ester (L-NAME) (100 microM), concentration dependently relaxed phenylephrine (PE)-stimulated tone (EC50 50 nM; n = 10). Apamin (50 nM) and ChTX (50 nM) abolished this relaxation (n = 5). In pressurized arteries, ACh (10 microM), applied intraluminally in the presence of indomethacin (2.8 microM) and L-NAME (100 microM), dilated both PE-stimulated (0.3-0.5 microM; n = 5) and myogenic tone (n = 3). Apamin (50 nM ) and ChTX (50 nM) applied intraluminally abolished ACh-induced dilatations. Bath superperfusion of apamin and ChTX did not affect ACh-induced dilatations of either PE-stimulated (n = 5) or myogenic tone (n = 3). This is the first demonstration that ChTX and apamin act selectively on the endothelium to block EDHF-mediated relaxation.

Interactions between endothelium-derived relaxing factors in the rat hepatic artery: focus on regulation of EDHF.

1. In rat isolated hepatic arteries contracted with phenylephrine, acetylcholine and the calcium ionophore A23187 each elicit endothelium-dependent relaxations, which involve both nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF). However, the contribution of prostanoids to these responses, and the potential interaction between EDHF and other endothelium-derived relaxing factors have not been examined. 2. In the presence of the NO synthase inhibitor N(G)-nitro-L-arginine (L-NOARG, 0.3 mM) and a mixture of charybdotoxin (0.3 microM) and apamin (0.3 microM), inhibitors of the target potassium (K) channel(s) for EDHF, acetylcholine and A23187 each induced a concentration-dependent and almost complete relaxation, which was abolished in the additional presence of indomethacin (10 microM). Thus, in addition to EDHF and NO, a relaxing factor(s) generated by cyclo-oxygenase (COX) contributes to endothelium-dependent relaxation in the rat hepatic artery. 3. The resting membrane potentials of endothelium-intact and endothelium-denuded vascular segments were -57 mV and -52 mV, respectively (P>0.05). In intact arteries, the resting membrane potential was not affected by L-NOARG plus indomethacin, but reduced to -47 mV in the presence of charybdotoxin plus apamin. Acetylcholine and A23187 (10 microM each) elicited a hyperpolarization of 13 mV and 15 mV, respectively. The hyperpolarization induced by these agents was not affected by L-NOARG plus indomethacin (12 mV and 14 mV, respectively), but reduced in the presence of charybdotoxin plus apamin (7 mV and 10 mV, respectively), and abolished in the combined presence of charybdotoxin, apamin and indomethacin. 4. The NO donor 3-morpholino-sydnonimine (SIN-1) induced a concentration-dependent relaxation, which was unaffected by charybdotoxin plus apamin, but abolished by the selective soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ, 10 microM). SIN-1 (10 microM) did not alter the resting membrane potential in endothelium-denuded vascular segments. 5. The COX-dependent relaxation induced by acetylcholine was abolished following exposure to 30 mM KCl, but unaffected by glibenclamide (10 microM). The prostacyclin analogue iloprost induced a concentration-dependent relaxation, which was also abolished in 30 mM KCl and unaffected by the combined treatment with glibenclamide, charybdotoxin and apamin. Iloprost (10 microM) induced a glibenclamide-resistant hyperpolarization (8 mV with and 9 mV without glibenclamide) in endothelium-denuded vascular segments. 6. Exposure to SIN-1 or iloprost did not affect the EDHF-mediated relaxation induced by acetylcholine (i.e. in the presence of L-NOARG and indomethacin). Replacement of L-NOARG with the NO scavenger oxyhaemoglobin (10 microM) or the soluble guanylate cyclase inhibitor ODQ (10 microM) or methylene blue (10 microM), which all significantly inhibited responses to endothelium-derived NO, did not affect the acetylcholine-induced relaxation in the presence of indomethacin, indicating that endogenous NO also does not suppress EDHF-mediated responses. 7. These results show that, in addition to EDHF and NO, an endothelium-derived hyperpolarizing factor(s) generated by COX contributes significantly to endothelium-dependent relaxation in the rat heptic artery. Neither this factor nor NO seems to regulate EDHF-mediated responses. Thus, EDHF does not serve simply as a 'back-up' system for NO and prostacyclin in this artery. However, whether EDHF modulates the NO and COX pathways remains to be determined.

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 microM) and A23187 (0.03-10 microM), and nitric oxide (NO), applied either as authentic NO (0.01-10 microM) or as the NO donors 3-morpholino-sydnonimine (SIN-1; 0.1-10 microM) and S-nitroso-N-acetylpenicillamine (SNAP; 0.1-10 microM), each evoked concentration-dependent relaxation in phenylephrine stimulated (1-3 microM; 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, 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 A23187 in the presence of 30 mM KCl were abolished by either the NO synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME; 100 microM) or the inhibitor of soluble guanylyl cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 microM; 10 min; n=4 in each case; P<0.01). 3. Exposure to ODQ significantly attenuated both repolarization and relaxation to ACh, 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 microM), iberiotoxin (100 nM) and apamin (50 nM), alone or in combination, had no significant effect on relaxation to ACh, 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, A23187 and authentic NO each by 30% (n=4-8; P<0.01). Application of ODQ (10 microM; 10 min) abolished the ChTX-insensitive responses to ACh, 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 microM) 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 microM 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.

Evidence that anandamide and EDHF act via different mechanisms in rat isolated mesenteric arteries.

The endogenous cannabinoid, anandamide, has been suggested as an endothelium-derived hyperpolarizing factor (EDHF). We found that anandamide-evoked relaxation in isolated segments of rat mesenteric artery was associated with smooth muscle hyperpolarization. However, although anandamide-evoked relaxation was inhibited by either charybdotoxin (ChTX) or iberiotoxin, inhibition of the relaxation to EDHF required a combination of ChTX and apamin. The relaxations induced by either anandamide or EDHF were not inhibited by the cannabinoid receptor (CB1) antagonist SRI41716A, or mimicked by selective CB1 agonists. Thus, anandamide appears to cause smooth muscle relaxation via a CB1 receptor-independent mechanism and cannabinoid receptor activation apparently does not contribute to EDHF-mediated relaxation in this resistance artery.

Effect of copper on nitric oxide synthase and guanylyl cyclase activity in the rat isolated aorta.

1. The potential role of copper (Cu2+) in modulating the activity of nitric oxide synthase (NOS) and guanylyl cyclase (GC) was investigated by use of diethyldithiocarbamic acid (DEDCA), a high affinity Cu2+ chelator. 2. DEDCA 100 microM inhibited sodium nitroprusside (SNP; 0.005-10 microM)-evoked relaxation of rat isolated aortic rings precontracted with 3 microM phenylephrine (PE). A lower concentration of DEDCA (10 microM) did not significantly attenuate SNP-evoked responses but did inhibit relaxation to the endothelium-dependent dilator, A23187 (0.01-10 microM). 3. The presence of 100 microM Cu2+, but not 100 microM Fe2+, alone enhanced A23187- and SNP-evoked relaxation of aortae precontracted with PE. 4. The inhibitory effect of DEDCA on SNP- and A23187-induced relaxation was reversed by equimolar concentrations of Cu2+ but not Fe2+, indicating that DEDCA does not act via removal of haem-iron from the NOS and GC complexes. 5. Superoxide dismutase (30 mu ml-1) was without effect on the inhibition of DEDCA relaxation induced by either SNP or A23187 in aortae precontracted with PE. 6. When assessed by radioimmunoassay, DEDCA inhibited SNP- and A23187-stimulated cyclic GMP formation with IC50 values of 0.5 microM and 50 microM, respectively. 7. These data demonstrate that Cu2+ plays a role in controlling NOS and GC activity in the rat aorta.

Nitric oxide is the mediator of both endothelium-dependent relaxation and hyperpolarization of the rabbit carotid artery.

It is controversial whether the endothelial cell release of nitric oxide (NO) or a different factor(s) accounts for endothelium-dependent hyperpolarization, because in many arteries endothelium-dependent relaxation and hyperpolarization resists inhibitors of NO synthase. The contribution of NO to acetylcholine-induced endothelium-dependent hyperpolarization and relaxation of the rabbit carotid artery was determined by measuring NO with electrochemical and chemiluminescence techniques. In the presence of phenylephrine to depolarize and contract the smooth muscle cells, acetylcholine caused concentration-dependent hyperpolarization and relaxation which were closely correlated to the release of NO. N(omega)-nitro-L-arginine methyl ester (30 microM) partially reduced the release of NO and caused a similar reduction in smooth muscle cell relaxation and hyperpolarization. To determine if the residual responses were mediated by another endothelium-derived mediator or NO released despite treatment with N(omega)-nitro-L-arginine methyl ester, N(omega)-nitro-L-arginine (300 microM) was added. The combined inhibitors further reduced, but did not eliminate, NO release, smooth muscle relaxation, and hyperpolarization. Hyperpolarization and relaxation to acetylcholine remained closely correlated with the release of NO in the presence of the inhibitors. In addition, the NO donor, SIN-1, caused hyperpolarization and relaxation which correlated with the concentrations of NO that it released. These studies indicate that (i) the release of NO by acetylcholine is only partially inhibited by these inhibitors of NO synthase when used even at high concentrations, and (ii) NO rather than another factor accounts fully for endothelium-dependent responses of the rabbit carotid artery.

Evidence that potassium channels make a major contribution to SIN-1-evoked relaxation of rat isolated mesenteric artery.

1. The NO donor 3-morpholino-sydnonimine (SIN-1; 0.01-10 microM) evoked concentration-dependent relaxation of rat isolated mesenteric arteries pre-constricted with phenylephrine (1-3 microM). The relaxation to SIN-1 was not significantly different between endothelium-intact or denuded arterial segments or segments in which basal nitric oxide (NO) synthesis was inhibited (n = 8; P > 0.05). In contrast, the membrane permeable analogue of guanosine 3':5'-cyclic monophosphate (cyclic GMP), 8-Br-cyclic GMP (0.01-1 mM), was much less effective in relaxing intact than denuded arterial segments or intact arterial segments pre-incubated with NO synthase blockers (n = 4; P < 0.01). 2. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 microM; 10 min) alone, did not alter SIN-1-evoked relaxation in any tissues (n = 5; P > 0.05). However, in parallel experiments, ODQ almost completely inhibited both basal and SIN-1-stimulated production of cyclic GMP in both the presence and absence of NO synthase blockers (n = 6; P < 0.01) indicating that full relaxation to SIN-1 can be achieved in the absence of an increase in cyclic GMP. 3. Exposure of endothelium-intact arterial segments to the potassium channel blocker charybdotoxin (50 nM; 10 min), significantly inhibited SIN-1-evoked relaxation, reducing the maximum response by around 90% (n = 5; P < 0.01). In contrast, in arterial segments in which either the endothelial cell layer had been removed or basal NO synthesis inhibited, relaxation to SIN-1 was not reduced in the presence of charybdotoxin (n = 6; P > 0.05). However, in the presence of NO synthase blockers and L-arginine (300 microM) together, charybdotoxin did significantly inhibit SIN-1-evoked relaxation to a similar extent as intact tissues (maximum response induced by around 80%; n = 4; P < 0.01). 4. Pre-incubation with apamin (30 nM; 10 min) or glibenclamide (10 microM; 10 min) did not alter SIN-1-evoked relaxation of phenylephrine-induced tone in any tissues (n = 4 and n = 6, respectively; P > 0.05). However, in the presence of either ODQ and apamin, or ODQ and glibenclamide, SIN-1-evoked relaxation was significantly attenuated in intact arterial segments and segments in which NO synthesis was blocked. 5. Exposure of intact arterial segments to charybdotoxin and apamin, in the presence of NO synthase blockers, also significantly inhibited SIN-1-evoked relaxation, reducing the maximum response by around 80% (n = 4; P < 0.01). 6. Addition of superoxide dismutase (SOD; 30 u ml-1), potentiated relaxations to SIN-1 in all tissues, but did not alter the effects of charybdotoxin and ODQ and SIN-1-evoked relaxation. 7. These data show that although relaxation to the NO-donor SIN-1 is not significantly different between endothelium-intact and denuded arterial segments, the mechanisms which mediate SIN-1-evoked relaxation in the rat isolated mesenteric artery appear to be modulated by the basal release of endothelium-derived NO. In the presence of an intact endothelial cell layer, the major mechanism for SIN-1-evoked relaxation appears to be the activation of charybdotoxin-sensitive potassium channels. In contrast, when basal NO synthesis is inhibited, SIN-1 appears to cause full relaxation by both the activation of a charybdotoxin-sensitive pathway and the stimulation of soluble guanylyl cyclase.

Influence of contractile agonists on the mechanism of endothelium-dependent relaxation in rat isolated mesenteric artery.

This study demonstrates directly that the relative contribution of nitric oxide (NO) and an NO synthase-independent repolarization to acetylcholine-evoked relaxation in rat isolated mesenteric resistance arteries is determined by the processes which mediate pre-contraction. Noradrenaline-induced contractions were reversed by acetylcholine via both NO and NO synthase-independent smooth muscle repolarization. In contrast, reversal of contractions to the thromboxane-mimetic, U46619, by acetylcholine was entirely mediated by the actions of NO, independently of a change in membrane potential.

Multiple pathways underlying endothelium-dependent relaxation in the rabbit isolated femoral artery.

1. In isolated segments of the rabbit femoral artery stimulated with noradrenaline, both acetylcholine (1 nM-10 microM) and the calcium ionophore A23187 (1 nM-100 microM) evoked endothelium-dependent smooth muscle relaxation and hyperpolarization while bradykinin (0.01-100 nM) had no effect. 2. The nitric oxide synthase inhibitors, NG-nitro-L-arginine (L-NOARG; 100 microM; 20 min) or NG-nitro-L-arginine methyl ester (L-NAME; 100 microM; 20 min) each abolished the hyperpolarization and the majority of the relaxation to acetylcholine (maximal response reduced from 96.8 +/- 2.3% to 2.0 +/- 1.4%). 3. The potassium channel blocker, glibenclamide (10 microM; 10 min) also abolished the change in membrane potential to acetylcholine but did not modify the smooth muscle relaxation. 4. In contrast, neither L-NAME nor glibenclamide modified the comparable responses of the femoral artery to A23187, which were also unaffected by the cyclo-oxygenase inhibitor, indomethacin (10 microM). 5. In artery segments stimulated with potassium chloride (25 mM), the maximal change in tension and membrane potential evoked by A23187 (100 microM) was significantly reduced from 95.0 +/- 4.5% and 23.0 +/- 2.0 mV to 69.0 +/- 10.1% and 12.0 +/- 1.5 mV, respectively. Under these conditions L-NAME further reduced the relaxation but not the accompanying hyperpolarization to A23187. 6. Endothelium-denuded arterial segments sandwiched with endothelium-intact 'donor' segments gave qualitatively similar relaxant responses to those described above for acetylcholine and A23187. 7. Exogenous nitric oxide (0.5-10 microM) stimulated a transient relaxation in pre-contracted artery segments, which at concentrations above 5 microM was accompanied by smooth muscle hyperpolarization(maximum 8.5 +/- 3.2 mV; n = 4). The hyperpolarization but not the relaxation to nitric oxide was abolished by either glibenclamide or 25 mM potassium.8. These data indicate that in the femoral artery, acetylcholine-induced relaxation can be attributed solely to the release of nitric oxide from the endothelium, which then stimulates relaxation independently of a change in smooth muscle membrane potential. In contrast, both the relaxation and hyperpolarization evoked by A23187 appear to be mediated predominantly by nitric oxide-independent pathways which appear to involve a diffusible factor released from the endothelium. The results suggest that this diffusible hyperpolarizing factor can be released from endothelial cells in the femoral artery by A23187 but not by acetylcholine.

Endothelium-dependent hyperpolarization: a role in the control of vascular tone.

Endothelial-dependent relaxation of vascular smooth muscle cells evoked by a number of agonists, including cholinomimetics and substance P, is often accompanied by an increase (repolarization and/or hyperpolarization) in the membrane potential. This change in membrane potential appears predominantly to reflect the action of an endothelial-derived hyperpolarizing factor (EDHF), which is distinct from NO (or endothelial-derived relaxing factor), and is discussed in this article by Chris Garland and colleagues. In large conducting arteries, EDHF may provide a secondary system to NO, which assumes primary importance in some disease states such as pulmonary hypertension and atherosclerosis. However, in small resistance arteries (100-300 microns), EDHF appears to be a major determinant of vascular calibre under normal conditions, and may therefore be of primary importance in the regulation of vascular resistance.

The relative importance of nitric oxide and nitric oxide-independent mechanisms in acetylcholine-evoked dilatation of the rat mesenteric bed.

1. The relative contribution of nitric oxide (NO) to acetylcholine-induced smooth muscle relaxation was investigated in the rat perfused mesenteric vasculature and in isolated segments of second, third and fourth order arterial branches. 2. The EC50 values and maximal relaxation to acetylcholine were not significantly different in the sequential arterial branches, being approximately 0.05 microM and 85%, respectively. 3. The NO synthase inhibitor L-NG-nitro-L-arginine methyl ester (L-NAME; 100 microM) reduced acetylcholine-evoked endothelium-dependent dilatation and relaxation in the perfused mesenteric bed and in isolated arterial segments. The maximum response to acetylcholine in both preparations was reduced by between 35% to 40% while the EC50 values were increased by 5-6 fold. L-NAME had no effect on basal smooth muscle tone in either case. 4. In contrast, endothelium-dependent dilatation of the perfused mesenteric bed evoked by A23187 (0.002-20 nmol), was unaffected by exposure to L-NAME. The EC50 values and maximal responses elicited by A23187 (20 nmol) before and after exposure to L-NAME were 0.96 +/- 0.5 nmol and 67.0 +/- 7.0% (n = 4), and 0.7 +/- 0.4 nmol and 70.0 +/- 5.0% (n = 4; P > 0.01), respectively. 5. Perfusion of the isolated mesenteric bed with raised K(+)-Krebs buffer (25 mM) had no effect on basal tone, but reduced the amplitude of both acetylcholine- and A23187-evoked dilatation. The maximum responses to acetylcholine (2 micromol) and A23187 (20 nmol) were reduced from 67.5 +/- 7.3% and 65.4+/-8.2% to 18.9 +/-11.0% (n=5; P<0.01) and 13.5 +/-12.0% (n=4; P<0.01), respectively.6. Exposure of the mesenteric bed to L-NAME in the presence of raised K+-Krebs further reduced the maximal response elicited by acetylcholine to only 8.9 +/- 2.8% (n =4; P< 0.01).7. These results indicate that acetylcholine-evoked vasodilatation of the rat mesenteric vasculature is mediated by both NO-dependent and -independent mechanisms. The relative contribution made by these mechanisms does not appear to differ in sequential branches of the mesenteric artery. In contrast,A23187-evoked vasodilatation appears to be mediated predominantly by a NO-independent mechanism which is sensitive to increases in the extracellular potassium concentration and may reflect the action of endothelium-derived hyperpolarizing factor (EDHF).

Smooth muscle hyperpolarization and relaxation to acetylcholine in the rabbit basilar artery.

Acetylcholine-evoked relaxation of noradrenaline-stimulated segments of the rabbit basilar artery was accompanied by a small, transient hyperpolarization of the smooth muscle cell membrane which was diminished by repeated exposure to the agonist. In the presence of glibenclamide (10 microM) or high concentrations of potassium chloride (65 mM), the acetylcholine-evoked smooth muscle hyperpolarization was abolished, whereas the relaxation response was unaffected. Nitric oxide (NO gas in solution; 0.5-15 microM) evoked dose-dependent relaxation in noradrenaline contracted arterial segments, but had no effect on the smooth muscle membrane potential, even at a saturated concentration (150 microM), which was 10 times higher than required to stimulate maximal relaxation. Additionally, NO-evoked relaxations were unaffected by glibenclamide (10 microM), but the responses were significantly attenuated in the presence of 65 mM potassium chloride. These data show that, as in the rabbit middle cerebral artery, acetylcholine-evoked hyperpolarization in the rabbit basilar artery is mediated by glibenclamide-sensitive potassium channels. However, in contrast to the middle cerebral artery and to other vessels such as the rat mesenteric artery, the change in smooth muscle membrane potential does not make an important contribution to the relaxation evoked either by this agonist or by NO.

Probucol and other antioxidants prevent the inhibition of endothelium-dependent relaxation by low density lipoproteins.

Hypercholesterolaemia and atherosclerosis impair responses to endothelium-derived nitric oxide (EDRF) in human and animal coronary arteries, a dysfunction that correlates with elevated low density lipoproteins (LDL). Previous studies show that native LDL immediately and reversibly inhibit acetylcholine-evoked EDRF responses in rabbit aortic ring precontracted with noradrenaline or serotonin whereas Cu(2+)-oxidised LDL (oxLDL) inhibit relaxations after 30 min with a potency that varies with the donor. We now show that antioxidants, probucol (10 microM) and ascorbic acid (100 microM) in vitro, prevent the inhibition by native LDL, indicating that this effect involves free radicals. As expected, the antioxidants had no influence on the inhibition by oxLDL. Superoxide dismutase appeared to have no effect on the inhibition by native or oxLDL. The oral administration of probucol to selected volunteers also prevented the inhibition of relaxation by their native LDL. These preparations showed a diminished susceptibility to oxidation and their oxLDL caused a markedly reduced and always reversible inhibition of relaxation compared to the potent and sometimes irreversible inhibition prior to administration of the drug. We conclude that antioxidants such as probucol reduce the formation of free radicals and the oxidative modification of LDL that lead to the impairment of EDRF responses and may prevent this same dysfunction in hypercholesterolaemia and atherosclerosis.

Differential effects of acetylcholine, nitric oxide and levcromakalim on smooth muscle membrane potential and tone in the rabbit basilar artery.

1. Endothelium-dependent hyperpolarization of smooth muscle cells in isolated, pre-contracted segments of rabbit basilar artery in response to acetylcholine (100 microM) was abolished in the presence of glibenclamide (10 microM). 2. Acetylcholine-evoked relaxation was unaffected by either glibenclamide or 65 mM potassium chloride, indicating that the change in membrane potential did not form an essential component of relaxation and that high concentrations of potassium did not inhibit the release or action of endothelium-derived relaxing factor in this vessel. 3. Saturated solutions of nitric oxide (NO) gas in solution (150 microM), which evoked maximal relaxation of arterial segments pre-contracted and depolarized by noradrenaline (10-100 microM), did not alter the membrane potential of either unstimulated or depolarized smooth muscle cells. 4. The potassium channel opener levcromakalim, evoked concentration-dependent relaxation and hyperpolarization in pre-constricted smooth muscle cells. The threshold concentrations for hyperpolarization and relaxation, the EC50 values and the maximally effective concentration of levcromakalim (around 30 nM, 150 nM and 10 microM, respectively) were not significantly different, and both components of the response were inhibited by glibenclamide (10 microM), indicating a close coupling between the two responses. 5. In the presence of 65 mM potassium chloride, the hyperpolarization to levcromakalim was abolished, while a small relaxation (25 +/- 4%) persisted, indicating an additional mechanism for relaxation to this agent. 6. These results show that different mechanisms underlie the relaxant action of potassium channel openers, NO and endothelium-derived factors in cerebral arteries and provide further evidence that in the basilar artery, in contrast to some other vessels, endothelium-dependent hyperpolarization to acetylcholine is not important for smooth muscle relaxation.

Electrophysiology of cerebral blood vessels.

In spite of the relatively large amount of in vitro and in vivo data indicating that, in a number of ways, cerebral arteries are pharmacologically different from peripheral arteries, the mechanisms responsible for these differences are far from clear. An understanding of these mechanisms is particularly important for a rational approach to the treatment of disorders of the cerebral circulation including migraine, hypertension and the responses of cerebral vessels to subarachnoid haemorrhage. This review outlines electrophysiological data which are available from cerebrovascular smooth muscle cells, including the possibility that inwardly-rectifying potassium channels, active at potentials close to the resting membrane potential, are intimately involved in the changes in smooth muscle tone which couple blood flow to regional changes in nerve cell activity. The membrane potential changes in response to perivascular nerve stimulation, noradrenaline, 5-hydroxytryptamine and endothelium-derived hyperpolarizing factor are also described, together with the underlying membrane mechanisms and their relationship to smooth muscle contraction and relaxation.

Oxidative modification of low-density lipoproteins and the inhibition of relaxations mediated by endothelium-derived nitric oxide in rabbit aorta.

1. The mechanism by which Cu(2+)-oxidized low-density lipoproteins (oxLDL) inhibit acetylcholine (ACh)-evoked relaxations mediated by endothelium-derived nitric oxide (EDRF) in rabbit aortic rings was investigated. The proposed role of lysophosphatidylcholine (LPC) in the inhibition was also studied. 2. The kinetics of lipid peroxidation of native low-density lipoproteins (LDL) from individual donors, as measured by changes in conjugated diene concentration, were related to the inhibitory effects of the resultant oxLDL. It was found that the more susceptible LDL was to oxidation, the greater the inhibition. 3. No correlation was found between the inhibitory effects of oxLDL and LPC content. 4. Synthetic 1-palmitoyl LPC produced an inhibition of ACh-induced relaxations and when added to precontracted rings evoked nitric oxide-mediated endothelium-dependent relaxation. This latter effect was not elicited by oxLDL. 5. Synthetic 1-palmitoyl (10 microM) had no effect on relaxations evoked by glyceryl trinitrate in endothelium-denuded aortic rings in contrast to the inhibition found previously for oxLDL. 6. Concentrations of oxLDL and phospholipase A2-treated LDL which inhibited relaxation contained very different LPC concentrations. Unlike oxLDL, the inhibitory effects of phospholipase A2-treated LDL preparations were independent of the donors and showed no lag period. 7. We suggest that there are differences in the mechanisms by which oxLDL and 1-palmitoyl LPC exert their inhibitory effects on relaxation. 8. The inhibition of relaxation by oxLDL (1-2 mg protein ml-1) was prevented by the presence of high-density lipoproteins (HDL; 1-2 mg protein ml-1).9. It is proposed that prevention of the inhibition of relaxation by HDL is consistent with the inhibitory factor(s) being lipophilic constituents of oxLDL. However, variations in the inhibitory effects of oxLDL preparations are not due to differences in their LPC content and factors other than LPC must contribute to the inhibition.