Novel endothelium-derived relaxing factors. Identification of factors and cellular targets

Nitric oxide (NO), together with prostacyclin (PGI2), mediates shear stress and endothelium-dependent vasodilator-mediated vasorelaxation. In the presence of inhibition of NO synthase (NOS) with nitroarginine analogues, such as of N(w)-nitro-L-arginine methyl ester (L-NAME) and N(w)-nitro-L-arginine (L-NNA), and indomethacin, to inhibit cyclooxygenase (COX) and the synthesis of PGI2, many blood vessels still respond with an endothelium-dependent relaxation to either chemical [i.e. acetylcholine (ACh)] or mechanical (shear stress) activation. This non-NO and non-PGI2 vasorelaxation appears to be mediated by hyperpolarization of the vascular smooth muscle cell (VSMC). Although NO can hyperpolarize VSMC, a novel mediator, the endothelium-derived hyperpolarizing factor (EDHF), which opens a VSMC K(+) channel(s) notably in resistance vessels, has been proposed. Little agreement exists as to the nature of this putative factor, but several candidate molecules have been proposed and evidence, notably from the microcirculation, suggests that endothelium-dependent hyperpolarization (EDH) may be mediated via low electrical resistance coupling via myoendothelial gap junctions. We describe a number of techniques that are being used to identify EDHF and present data that address the contribution of a small increase in extracellular K(+) as an EDHF.     

Functional evidence that K+ is the non-nitric oxide,non-prostanoid endothelium-derived relaxing factor in rat femoral arteries

The mechanisms of K(+)-induced relaxation and of acetylcholine (ACh)-stimulated, endothelium-dependent relaxation were assessed in rat femoral arteries mounted in a myograph. ACh-stimulated (1 nM-1 microM) relaxation of arteries precontracted with 1 microM noradrenaline was mostly resistant to the combination of indomethacin (INDO; 10 microM) and N(omega)-nitro-L-arginine (L-NNA, 100 microM). The remaining relaxation was abolished by 30 mM K(+) or ouabain (1 mM) and significantly reduced by 30 microM Ba(2+) or charybdotoxin (ChTx; 100 nM) plus apamin (100 nM). K(+)-induced relaxation effected by raising [K(+)](o) by 0.5-4 mM was endothelium-independent and inhibited by ouabain and Ba(2+). These results indicate that ACh-stimulated relaxations are effected mainly by a non-prostanoid, non-nitric oxide mechanism, presumably an endothelium-derived hyperpolarising factor (EDHF). Relaxations stimulated by EDHF and K(+) are both mediated by Na(+)-K(+) ATPase and inward rectifier potassium channels (K(IR)). This study provides further functional evidence that EDHF is K(+) derived from endothelial cells that relaxes arterial smooth muscle subsequent to activation of Na(+)-K(+) ATPase and K(IR).     

Chronic treatment of male rats with daidzein and 17 beta-oestradiol induces the contribution of EDHF to endothelium-dependent relaxation

1. We investigated the effect of chronic (7 days) treatment of male rats with the isoflavone daidzein (0.2 mg kg(-1) sc per day) or 17beta-oestradiol (0.1 mg kg(-1) sc per day) on the contribution of nitric oxide (NO), prostaglandins and endothelium-derived hyperpolarising factor (EDHF) to endothelium-dependent relaxation of isolated aortic rings. 2. The sensitivity and maximum relaxation to acetylcholine (ACh) were significantly greater in aortic rings from rats treated with daidzein or 17beta-oestradiol, in comparison to vehicle-treated rats. Inhibition of nitric oxide synthase with N-nitro-l-arginine (l-NOARG) abolished ACh-induced relaxation in the aortae from vehicle-treated rats, but only attenuated relaxation in aortae from daidzein or 17beta-oestradiol-treated rats. The presence of haemoglobin in addition to l-NOARG did not cause any further inhibition of relaxation. 3. The cyclooxygenase inhibitor indomethacin had no effect on endothelium-dependent relaxation in aortae from any treatment group. Charybdotoxin (ChTX), which blocks large-conductance calcium-activated potassium channels (BK(Ca)) and intermediate-conductance calcium-activated potassium channels (IK(Ca)), plus apamin, which blocks small-conductance calcium-activated potassium channels (SK(Ca)), but not iberiotoxin, which only blocks BK(Ca), attenuated endothelium-dependent relaxation of aortae from daidzein or 17beta-oestradiol-treated rats. Blockade of K(Ca) channels had no effect on the responses to ACh in aortae from vehicle-treated rats. In aortae from daidzein- or 17beta-oestradiol-treated rats, endothelium-dependent relaxation was also attenuated by inhibition of cytochrome P450 (CYP450) epoxygenase with 6-(2-propargylloxyphenyl)hexanoic acid (PPOH) or inhibition of K(IR) channels and Na(+)/K(+)-ATPase with barium and oubain, respectively. 4. This study demonstrates that endothelium-dependent relaxation of male rat aorta is normally entirely mediated by NO, whereas treatment with daidzein or 17beta-oestradiol stimulates a contribution from a non-NO, nonprostaglandin factor acting through the opening of SK(Ca) and IK(Ca) channels, and involving activation of Na/K-ATPase, K(IR) and CYP450 epoxygenase. This pattern of sensitivity to the tested inhibitors is consistent with the contribution of EDHF to relaxation. Thus, EDHF contributes to the enhanced endothelium-dependent relaxation that is observed after chronic treatment with the phytoestrogen daidzein or with 17beta-oestradiol.     

Effects of calcium dobesilate on the synthesis of endothelium-dependent relaxing factors in rabbit isolated aorta

1. Some cardiovascular disturbances which occur in diabetics are a consequence of alterations in vascular contractility as well as in endothelium-dependent relaxation.
2. Calcium dobesilate (DOBE) is a drug used in diabetic retinopathy and its mechanism of action is not yet understood.
3. The aim of this study was to investigate the effects of DOBE on synthesis and release of endothelium-dependent relaxing factor (EDRF) and endothelium-dependent hyperpolarizing factor (EDHF) in rabbit isolated aorta.
4. Endothelium-dependent relaxation induced by acetylcholine (ACh) (10⁻⁸–10⁻⁵ M) increased in the presence of DOBE 10⁻⁵ M only when vascular endothelium was kept intact.
5. N^G-nitro-L-arginine methyl ester (L-NAME; 10⁻⁸–10⁻⁴ M progressively decreased the enhancing effect of DOBE on endothelium-dependent relaxation whereas it was progressively increased by L-Arg.
6. DOBE 10⁻⁵ M increased in a non-significant manner endothelium-dependent relaxation induced by ACh when the arteries were incubated with both L-NAME 10⁻⁴ M and indomethacin 10⁻⁶ M.
7. DOBE (10⁻⁶ M and 10⁻⁵ M) was able to scavenge superoxide anion radicals generated by the hypoxanthine/xanthine oxidase reaction.
8. These results provide evidence that DOBE is able to affect the vascular disorders associated with diabetes mellitus since it enhances the synthesis of endothelium-dependent relaxing factors.

     

Mechanisms underlying the attenuation of endothelium-dependent vasodilatation in the mesenteric arterial bed of the streptozotocin-induced diabetic rat

Experiments were designed to investigate the mechanisms underlying the diabetes-related impairment of the vasodilatations of the perfused mesenteric arterial bed induced by acetylcholine (ACh) and K(+). In streptozotocin (STZ)-diabetic rats, the ACh-induced endothelium-dependent vasodilatation was attenuated. The dose-response curves for ACh in control and diabetic rats were each shifted to the right by N(G)-nitro-L-arginine (L-NOARG) and by isotonic high K(+) (60 mM). The ACh dose-response curves under isotonic high K(+) were not different between control and diabetic rats. We also examined the vasodilatation induced by K(+), which is a putative endothelium-derived hyperpolarizing factor (EDHF). The mesenteric vasodilatation induced by a single administration of K(+) was greatly impaired in STZ-induced diabetic rats. Treatment with charybdotoxin plus apamin abolished the ACh-induced vasodilatation but enhanced the K(+)-induced response in controls and diabetic rats. After pretreatment with ouabain plus BaCl(2), the ACh-induced vasodilatation was significantly impaired and the K(+)-induced relaxation was abolished in both control and diabetic rats. The impairment of the endothelium-dependent vasodilatation of the mesenteric arterial bed seen in STZ-induced diabetic rats may be largely due to a defective vascular response to EDHF. It is further suggested that K(+) is one of the endothelium-derived hyperpolarizing factors and that the vasodilatation response to K(+) is impaired in the mesenteric arterial bed from diabetic rats.     

Effects of chronic in vivo administration of nitroglycerine on ACh-induced endothelium-dependent relaxation in rabbit cerebral arteries

BACKGROUND AND PURPOSE: In the setting of nitrate tolerance, endothelium-dependent relaxation is reduced in several types of peripheral vessels. However, it is unknown whether chronic in vivo administration of nitroglycerine modulates such relaxation in cerebral arteries. EXPERIMENTAL APPROACH: Isometric force and smooth muscle cell membrane potential were measured in endothelium-intact strips from rabbit middle cerebral artery (MCA) and posterior cerebral artery (PCA). KEY RESULTS: ACh (0.1-10 microM) concentration-dependently induced endothelium-dependent relaxation during the contraction induced by histamine in both MCA and PCA. Chronic (10 days) in vivo administration of nitroglycerine reduced the ACh-induced relaxation in PCA but not in MCA, in the presence of the cyclooxygenase inhibitor diclofenac (3 microM). In the presence of the NO-synthase inhibitor N (omega)-nitro-L-arginine (L-NNA, 0.1 mM) plus diclofenac, in MCA from both nitroglycerine-untreated control and -treated rabbits, ACh (0.1-10 microM) induced a smooth muscle cell hyperpolarization and relaxation, and these were blocked by the small-conductance Ca(2+)-activated K(+)-channel inhibitor apamin (0.1 microM), but not by the large- and intermediate-conductance Ca(2+)-activated K(+)-channel inhibitor charybdotoxin (0.1 microM). In contrast, in PCA, ACh (<3 microM) induced neither hyperpolarization nor relaxation under these conditions, suggesting that the endothelium-derived relaxing factor is NO in PCA, whereas endothelium-derived hyperpolarizing factor (EDHF) plays a significant role in MCA. CONCLUSIONS AND IMPLICATIONS: It is suggested that in rabbit cerebral arteries, the function of the endothelium-derived relaxing factor NO and that of EDHF may be modulated differently by chronic in vivo administration of nitroglycerine.     

EDHF-mediated relaxation is impaired in fructose-fed rats.

Insulin resistance (IR) is associated with endothelial dysfunction. A defect in endothelium-dependent relaxation via outward potassium conductance has been observed in mesenteric arteries from IR rats. The purpose of this study was to assess whether this defect in endothelium-dependent relaxation was due to impaired endothelium-derived hyperpolarizing factor (EDHF) and to determine which specific potassium channel(s) are involved in relaxation. This was accomplished by using specific potassium channel inhibitors in the presence of nitric oxide synthase and cyclooxygenase inhibition. In addition, we sought to assess the function of smooth muscle cell adenosine triphosphate (ATP)-dependent potassium (K(ATP)) channels. Sprague-Dawley rats were randomized to control or IR. To determine EDHF-mediated relaxation, acetylcholine (ACh)-induced (10(-9)-10(-5) M) relaxation was measured (in vitro) in mesenteric arteries in the presence of indomethacin (10(-5) M) and N-nitro-L-arginine (L-NNA) (10(-4) M). Subsequently the combination of charybdotoxin (CTX) (0.1 microM) and apamin (0.5 microM) or glibenclamide (Glib) (10 microM) was added to the bath to inhibit KCa or K(ATP), respectively. In separate experiments, relaxation to pinacidil (10(-13)-10(-5) M), a K(ATP) activator, was assessed in vessels with intact endothelium, endothelium denuded, or with L-NNA. Maximal relaxation to ACh in the presence of L-NNA and indomethacin was 68+/-6% for control and 12+/-3% for IR (p<0.01). The addition of CTX + apamin almost abolished EDHF-mediated relaxation in control (Emax, 8+/-5% vs. 68+/-6%; p<0.01), whereas Glib had little affect. Neither CTX + apamin nor Glib had any affect on IR. Additionally, IR arteries were less sensitive to pinacidil than were controls (EC50, 1.5+/-0.9 microM vs. 5x10(-4)+/-3x10(-4) microM, respectively; p<0.01). Endothelial removal or L-NNA pretreatment of control arteries decreased the response to pinacidil similar to IR, whereas IR vessels were unaffected. EDHF-mediated relaxation is impaired in IR arteries. In addition, the K(Ca) channel appears to be imperative for activity of EDHF in rat small mesenteric arteries. Moreover, activation of K(ATP) channels by pinacidil is impaired in IR, and this appears to be a result of endothelial dysfunction.     

EDHF, NO and a prostanoid: hyperpolarization-dependent and -independent relaxation in guinea-pig arteries

The contribution of endothelium-derived hyperpolarizing factor (EDHF), nitric oxide (NO) and a prostanoid (PG) to endothelium-dependent hyperpolarization and relaxation were assessed in coronary and mammary arteries of guinea-pigs by integration of the responses evoked during discrete applications of acetylcholine (ACh). The results of this integration approach were compared with those using traditional peak analysis methods. N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 microM) and indomethacin (1 microM), alone or in combination, were without effect on peak hyperpolarizations or relaxations while they markedly reduced the integrated responses in both arteries. Integrated responses attributed to NO and PG were larger than those attributed to EDHF in the coronary artery (at 2 microM ACh, hyperpolarization (mV s): NO, 4200+/-91; PG, 5046+/-157; EDHF, 1532+/-94; relaxation (mN s mm(-1)): NO, 2488+/-122; PG, 2234+/-96; EDHF, 802+/-54). Integrated responses attributed to NO, PG and EDHF were similar in the mammary artery (at 2 microM ACh, hyperpolarization: NO, 347+/-69; PG, 217+/-49; EDHF, 310+/-63; relaxation: NO, 462+/-94; PG, 456+/-144; EDHF, 458+/-40). Gilbenclamide (1 microM) all but abolished the hyperpolarization attributable to NO and PG but not EDHF in both arteries allowing assessment of the role of the hyperpolarization in relaxation. Gilbenclamide was without effect on the integrated relaxation due to NO but significantly reduced the relaxation associated with PG in the two arteries. In conclusion, integration of the responses enabled a more complete assessment of the contribution of EDHF, NO and PG to endothelium-dependent responses, which were strikingly different in the two arteries. There is commonality in the role of hyperpolarization in relaxation in both arteries: EDHF-dependent relaxation is strongly dependent on hyperpolarization; hyperpolarization plays an important role in PG relaxation, whereas it has a small facilitatory role in NO-dependent relaxation.     

Role of potassium channels in endothelium-dependent relaxation resistant to nitroarginine in the rat hepatic artery.

1. In the presence of indomethacin (IM, 10 microM) and N omega-nitro-L- arginine (L-NOARG, 0.3 mM), acetylcholine (ACh) induces an endothelium-dependent smooth muscle hyperpolarization and relaxation in the rat isolated hepatic artery. The potassium (K) channel inhibitors, tetrabutylammonium (TBA, 1 mM) and to a lesser extent 4-aminopyridine (4-AP, 1 mM) inhibited the L-NOARG/IM-resistant relaxation induced by ACh, whereas apamin (0.1-0.3 microM), charybdotoxin (0.1-0.3 microM), iberiotoxin (0.1 microM) and dendrotoxin (0.1 microM) each had no effect. TBA also inhibited the relaxation induced by the receptor-independent endothelial cell activator, A23187. 2. When combined, apamin (0.1 microM) + charybdotoxin (0.1 microM), but not apamin (0.1 microM) + iberiotoxin (0.1 microM) or a triple combination of 4-AP (1 mM) + apamin (0.1 microM) + iberiotoxin (0.1 microM), inhibited the L-NOARG/IM-resistant relaxation induced by ACh. At a concentration of 0.3 microM, apamin + charybdotoxin completely inhibited the relaxation. This toxin combination also abolished the L-NOARG/ IM-resistant relaxation induced by A23187. 3. In the absence of L-NOARG, TBA (1 mM) inhibited the ACh-induced relaxation, whereas charybdotoxin (0.3 microM) + apamin (0.3 microM) had no effect, indicating that the toxin combination did not interfere with the L-arginine/NO pathway. 4. The gap junction inhibitors halothane (2 mM) and 1-heptanol (2 mM), or replacement of NaCl with sodium propionate did not affect the L-NOARG/IM-resistant relaxation induced by ACh. 5. Inhibition of Na+/K(+)-ATPase by ouabain (1 mM) had no effect on the L-NOARG/IM-resistant relaxation induced by ACh. Exposure to a K(+)-free Krebs solution, however, reduced the maximal relaxation by 13% without affecting the sensitivity to ACh. 6. The results suggest that the L-NOARG/IM-resistant relaxation induced by ACh in the rat hepatic artery is mediated by activation of K-channels sensitive to TBA and a combination of apamin + charybdotoxin. Chloride channels, Na+/K(+)-ATPase and gap junctions are probably not involved in the response. It is proposed that endothelial cell activation induces secretion of an endothelium-derived hyperpolarizing factor(s) (EDHF), distinct from NO and cyclo-oxygenase products, which activates more than one type of K-channel on the smooth muscle cells. Alternatively, a single type of K-channel, to which both apamin and charybdotoxin must bind for inhibition to occur, may be the target for EDHF.     

Characteristics of attenuated endothelium-dependent relaxation seen in rabbit intrapulmonary vein following chronic nitroglycerine administration

1 This study was undertaken to determine whether long-term in vivo administration of nitroglycerine (NTG) downregulates the endothelium-dependent relaxation induced by acetylcholine (ACh) in the rabbit intrapulmonary vein and, if so, whether the type 1 angiotensin II receptor (AT(1)R) blocker valsartan normalizes this downregulated relaxation. 2 In strips treated with the cyclooxygenase inhibitor diclofenac, ACh induced a relaxation only when the endothelium was intact. A small part of this ACh-induced relaxation was inhibited by coapplication of two Ca(2+)-activated K(+)-channel blockers (charybdotoxin (CTX)+apamin) and the greater part of the response was inhibited by the nitric-oxide-synthase inhibitor N(omega)-nitro-L-arginine (L-NNA). 3 The endothelium-dependent relaxation induced by ACh, but not the endothelium-independent relaxation induced by the nitric oxide donor NOC-7, was significantly reduced in NTG-treated rabbits (versus those in NTG-nontreated control rabbits). The attenuated relaxation was normalized by coapplication of valsartan with the NTG. 4 In the vascular wall, both the amount of localized angiotensin II and the production of superoxide anion were increased by in vivo NTG treatment. These variables were normalized by coapplication of valsartan with the NTG. 5 It is suggested that long-term in vivo administration of NTG downregulates the ACh-induced endothelium-dependent relaxation, mainly through an inhibition of endothelial nitric oxide production in the rabbit intrapulmonary vein. A possible role for AT(1)R is proposed in the mechanism underlying this effect.     

Hypertension and the absence of EDHF-mediated responses favour endothelium-dependent contractions in renal arteries of the rat

Background and purpose:Experiments were designed to determine the modulation by nitric oxide (NO) and endothelium-dependent hyperpolarizations (EDHF-mediated responses) of endothelium-dependent contractions in renal arteries of normotensive and hypertensive rats.Experimental approach:Rings, with or without endothelium, of renal arteries of 8-month-old Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) were suspended in myographs for isometric force recording.Key results:ACh evoked relaxations in preparations contracted with phenylephrine. L-NAME (inhibitor of NOS) attenuated (WKY) or abolished (SHR) these relaxations. TRAM-34 plus UCL 1684 (inhibitors of EDHF-mediated responses) did not decrease the relaxation, except in rings of WKY when L-NAME was also present. High concentrations of ACh caused a secondary increase in tension, augmented in rings of WKY by L-NAME or TRAM-34 plus UCL 1684. The increase in tension was prevented by indomethacin. Under baseline tension, ACh induced endothelium-dependent contractions, prevented by indomethacin (COX inhibitor) or terutroban (TP receptor antagonist). The calculated endothelium-dependent contractions were larger in rings of SHR compared with those of WKY. In preparations of SHR, the contractions were augmented by L-NAME in the presence of SC19220 (EP-1 receptor antagonist). In arteries of WKY, the endothelium-dependent contractions were augmented by TRAM-34 plus UCL 1684. The responses were reduced by SC19220.Conclusions and implications:In the renal artery of the rat, EDCF-mediated contractions are augmented by hypertension. The endothelium-dependent contractions are facilitated by NOS inhibition (in the presence of an EP-1 receptor antagonist) and by the withdrawal of EDHF-mediated responses.British Journal of Pharmacology (2008) 155, 217-226; doi:10.1038/bjp.2008.256; published online 23 June 2008.     

Mechanical stretch reveals different components of endothelial-mediated vascular tone in rat aortic strips

1. Since the role of mechanical stretches in vascular tone regulation is poorly understood, we studied how stretch can influence endothelial tone. 2. Isometric contractions of isolated rat aortic helical strips were recorded. The resting tension was set at 0.7 g, 1.2 g or 2.5 g. Endothelium-preserved strips were precontracted with either phenylephrine or prostaglandin F(2 alpha) (PGF(2 alpha)). 3. In control conditions, acetylcholine (ACh) dose-dependently relaxed phenylephrine-precontracted strips independently of resting tension. 4. At 0.7 g resting tension, nitric oxide synthase (NOS) inhibitors did not reduce ACh-induced relaxation, while either a guanylyl cyclase inhibitor or a NO trapping agent prevented it. At 1.2 g and 2.5 g resting tensions, NOS inhibitors shifted the ACh dose-response curve to the right. 5. After preincubation with indomethacin (5 microM) or ibuprofen (10 and 100 microM), at 0.7 g and 1.2 g resting tensions, ACh induced an endothelium-dependent, dose-dependent contraction. ACh (10(-6) M) increased the contraction up to two times greater the phenylephrine-induced one. Lipoxygenase inhibitors prevented it. At high stretch, the ACh vasorelaxant effect was marginally influenced by cyclooxygenase (COX) inhibition. Similar results were obtained when aortic strips were precontracted with PGF(2 alpha). 6. Our data indicate that when resting tension is low, ACh mobilizes a stored NO pool that, synergistically with COX-derived metabolites, can relax precontracted strips. COX inhibition up-regulates the lipoxygenase metabolic pathway, accounting for the ACh contractile effect. At an intermediate resting tension, NO production is present, but COX inhibition reveals a lipoxygenase-dependent, ACh-induced contraction. At high resting tension, NO synthesis predominates and COX metabolites influence ACh-induced relaxation marginally.     

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.     

Persistence of EDHF pathway and impairment of the nitric oxide pathway after chronic mercury chloride exposure in rats: mechanisms of endothelial dysfunction

Chronic mercury exposure impairs vascular function, leading to the depression of endothelium-dependent vasodilatation. Loss of the nitric oxide (NO) pathway has been implicated, but little is known about effects on other endothelial mediators. This study investigated the mechanisms of endothelial dysfunction in rats subjected to chronic mercury chloride exposure. The endothelium-dependent relaxation of rat aorta evoked by acetylcholine (ACh) and isoproterenol was impaired in a dose-dependent manner by chronic mercury chloride exposure. Endothelium-independent responses to sodium nitroprusside (SNP) were not affected by chronic mercury chloride exposure. In healthy vessels, ACh-induced relaxation was inhibited by L-N-nitroarginine methyl ester (L-NAME; 10(-4) M) and partially by glybenclamide (10(-5) M), indicating the involvement of NO and endothelium-derived hyperpolarizing factor (EDHF). In vessels from mercury-exposed rats, responses to ACh were insensitive to L-NAME but were significantly reduced by glybenclamide, indicating selective loss of NO-mediated relaxation. In vessels from mercury-exposed rats, responses to ACh were partially restored after treatment with the antioxidant, superoxide dismutase (SOD) and catalase, this effect was not seen when aorta from exposed group was incubated with L-NAME along with SOD and catalase indicating selective loss of NO-mediated vasodilatation and with no affect the EDHF-mediated component of relaxation. The results imply that chronic mercury exposure selectively impairs the NO pathway as a consequence of oxidative stress, while EDHF is able to maintain endothelium-dependent relaxation at a reduced level.     

Type 1 diabetes and hypercholesterolaemia reveal the contribution of endothelium-derived hyperpolarizing factor to endothelium-dependent relaxation of the rat aorta

1. The present study evaluated the effect of diabetes, hypercholesterolaemia and their combination on the contribution of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) to relaxation of rat isolated aortic rings and the potential contribution of oxidant stress to the disturbance of endothelial function. 2. Thoracic aortic rings from control, diabetic, hypercholesterolaemic and diabetic plus hypercholesterolaemic rats were suspended in organ baths for tension recording. Generation of superoxide by the aorta was measured using lucigenin-enhanced chemiluminescence. 3. The maximal response to acetylcholine (ACh) was significantly reduced in diabetic or hypercholesterolaemic rats compared with control rats. In rats with diabetes plus hypercholesterolaemia, both the sensitivity and maximal response to ACh was impaired. In control rats, the response to ACh was abolished by the NO synthase inhibitor N(G)-nitro-L-arginine (L-NNA) or inhibition of soluble guanylate cyclase with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). In contrast, in rats with diabetes, hypercholesterolaemia or both, relaxation to ACh was resistant to inhibition by L-NNA or ODQ, but abolished by additional inhibition of K(Ca) channels with charybdotoxin plus apamin. 4. The generation of superoxide was not significantly enhanced in aortic rings from either diabetic or hypercholesterolaemic rats, but was significantly increased in aortic rings from rats with diabetes plus hypercholesterolaemia. 5. These results suggest that when diabetes and hypercholesterolaemia impair endothelium-dependent relaxation, due to a diminished contribution from NO, a compensatory contribution of EDHF to endothelium-dependent relaxation of the aorta is revealed. The attenuation of NO-mediated relaxation, at least in the presence of both diabetes and hypercholesterolaemia, is associated with enhanced superoxide generation.     

Temocapril, an angiotensin converting enzyme inhibitor, protects against diabetes-induced endothelial dysfunction

The effect of chronic treatment with the angiotensin converting enzyme inhibitor, temocapril, on prevention of endothelial dysfunction was evaluated in an experimental model of diabetes mellitus. Endothelium-dependent relaxation to acetylcholine was impaired while endothelium-independent relaxation to nitroglycerin was unaltered in diabetic aortic ring segments. Treatment of diabetic animals with temocapril prevented the impaired endothelium-dependent relaxation without altering responses to nitroglycerin. Acetylcholine-induced relaxation was largely due to nitric oxide (NO)-mediated relaxation; however, a small but significant portion of relaxation in aortic rings from temocapril-treated diabetic rats was resistant to inhibition by the nitric oxide synthase (NOS) inhibitor, L-nitroarginine.     

Acetylcholine-induced vasodilation may depend entirely upon NO in the femoral artery of young piglets

1 To characterize agonist-induced relaxation in femoral artery rings from young piglets, we compared the effect of a NOS-inhibitor N(omega)-nitro-L-arginine (L-NOARG), an NO-inactivator oxyhaemoglobin (HbO) and a soluble guanyl cyclase(sGC)-inhibitor 1H-[1,2,4]Oxadiazolo-[4,3,-alpha]quinoxalin-1-one (ODQ) on acetylcholine(ACh)-induced relaxation. The involvement of K(+) channel activation was studied on relaxations induced by ACh, the two NO donors sodium nitroprusside (SNP) and diethylamine (DEA) NONOate, and the cell membrane permeable guanosine 3'5' cyclic monophosphate (cGMP) analogue 8-Br-cGMP. 2 Full reversal of phenylephrine-mediated precontraction was induced by ACh (1 nM-1 microM) (pD(2) 8.2+/-0.01 and R(max) 98.7+/-0.3%). L-NOARG (100 microM) partly inhibited relaxation (pD(2) 7.4+/-0.02 and R(max) 49.6+/-0.8%). The L-NOARG/indomethacin(IM)-resistant response displayed characteristics typical for endothelium-derived hyperpolarizing factor (EDHF), being sensitive to a combination of the K(+) channel blockers charybdotoxin (CTX) (0.1 microM) and apamin (0.3 microM). 3 ODQ (10 microM) abolished relaxations induced by ACh and SNP. L-NOARG/IM-resistant relaxations to ACh were abolished by HbO (20 microM). 4 Ouabain (1 microM) significantly inhibited ACh-induced L-NOARG/IM-resistant relaxations and relaxations induced by SNP (10 microM) and 8-Br-cGMP (0.1 mM). A combination of ouabain and Ba(2+) (30 microM) almost abolished L-NOARG/IM-resistant ACh-induced relaxation (R(max) 7.7+/-2.5% vs 23.4+/-6.4%, with and without Ba(2+), respectively, P<0.05). 5 The present study demonstrates that in femoral artery rings from young piglets, despite an L-NOARG/IM-resistant component sensitive to K(+) channel blockade with CTX and apamin, ACh-induced relaxation is abolished by sGC-inhibition or a combination of L-NOARG and HbO. These findings suggest that relaxation can be fully explained by the NO/cGMP pathway.     

Thromboxane A2 inhibition of SKCa after NO synthase block in rat middle cerebral artery

BACKGROUND AND PURPOSE: NO/prostanoid independent, EDHF-mediated hyperpolarization and dilation in rat middle cerebral arteries is mediated solely by endothelial cell IK(Ca). However, when the NO-pathway is also active, both SK(Ca) and IK(Ca) contribute to EDHF responses. As the SK(Ca) component can be inhibited by stimulation of thromboxane A(2) (TxA(2)) TP receptors and NO has the potential ability to inhibit thromboxane synthesis, we investigated whether TxA(2) might explain loss of functional input from SK(Ca) during NOS inhibition in cerebral arteries. EXPERIMENTAL APPROACH: Rat middle cerebral arteries were mounted in a wire myograph. Endothelium-dependent responses to the PAR2 agonist, SLIGRL were assessed as simultaneous changes in smooth muscle membrane potential and tension. KEY RESULTS: Responses were obtained in the presence of L-NAME as appropriate. Inhibition of TP receptors with either ICI 192,605 or SQ 29,548, did not affect EDHF mediated hyperpolarization and relaxation, but in their presence neither TRAM-34 nor apamin (to block IK(Ca) and SK(Ca) respectively) individually affected the EDHF response. However, in combination they virtually abolished it. Similar effects were obtained in the presence of the thromboxane synthase inhibitor, furegrelate, which additionally revealed an iberiotoxin-sensitive residual EDHF hyperpolarization and relaxation in the combined presence of TRAM-34 and apamin. CONCLUSIONS AND IMPLICATIONS: In the rat middle cerebral artery, inhibition of NOS leads to a loss of the SK(Ca) component of EDHF responses. Either antagonism of TP receptors or block of thromboxane synthase restores an input through SK(Ca). These data indicate that NO normally enables SK(Ca) activity in rat middle cerebral arteries.     

Critical role of gap junctions in endothelium-dependent hyperpolarization in rat mesenteric arteries

1. Acetylcholine (ACh) evokes endothelium-dependent hyperpolarization in arterial cells, presumably through endothelium-derived hyperpolarizing factor (EDHF). The identity of EDHF is still elusive; however, several recent studies suggest the possible involvement of myoendothelial gap junctions in the EDHF response. 2. To elucidate the role of gap junctions in endothelium-dependent hyperpolarization, we examined the effects of the gap junction inhibitors 18 alpha-glycyrrhetinic acid (18 alpha-GA; 10(-4) mol/L) and carbenoxolone (3 x 10(-4) mol/L), a water-soluble form of 18 beta-GA, on hyperpolarization and relaxation to ACh in rat proximal and distal mesenteric arteries. Experiments were performed in the presence of indomethacin (10(-5) mol/L) and N(G)-nitro-L-arginine (10(-4) mol/L). 3. In both proximal and distal mesenteric arteries, ACh-induced hyperpolarization and relaxation were partially inhibited by 18 alpha-GA and abolished by carbenoxolone. 4. Endothelium-independent hyperpolarization to levcromakalim, an ATP-sensitive K+ channel opener, were unaffected by 18 alpha-GA or carbenoxolone in both arteries. 5. Relaxations to levcromakalim were unaffected by 18 alpha-GA, but were inhibited somewhat by carbenoxolone in proximal mesenteric arteries. 6. These findings suggest that myoendothelial gap junctions play a critical role in EDHF-mediated responses in both proximal and distal mesenteric arteries of the rat.     

Potassium does not mimic EDHF in rat mesenteric arteries

1. K(+) has been proposed to be EDHF in small arteries. We compared ACh-stimulated, EDHF-mediated dilatation/relaxation with raised [K(+)](o) in rat mesenteric arteries. 2. In pressurized arteries, ACh (10 microM) dilated all arteries. Raising [K(+)](o) from 5.88 to 10. 58 mM only dilated 30% of arteries. Ba(2+) (30 microM) did not affect dilatation to ACh, but abolished 40% of dilatations to raised [K(+)](o). 3. If [K(+)](o) was lowered to 1.18 mM, restoring [K(+)](o) to 5.88 mM produced dilatation which was depressed by Ba(2+) or ouabain (1 mM). Combined application of Ba(2+) and ouabain abolished dilatation. In 1.18 mM K(+), dilatation to ACh was depressed by ouabain, but not by Ba(2+). Combined application of Ba(2+) and ouabain depressed dilatation further. Gap junction inhibitors (Gap-27; 300 microM and 18-alpha-glycyrrhetinic acid; 100 microM) also depressed dilatation to ACh. 4. In arteries mounted isometrically, ACh (1 microM) relaxed endothelium intact (+E), but not endothelium denuded (-E) arteries. Raising [K(+)](o) from 5.9 - 10.9 mM failed to relax all arteries. When [K(+)](o) was lowered to 1 mM, raising [K(+)](o) to 6 mM produced relaxation. In -E arteries, relaxation was unaffected by Ba(2+) but abolished by ouabain. In +E arteries, Ba(2+) depressed and ouabain abolished relaxation. In +E arteries, with 1 mM K(+), ACh relaxation was depressed by ouabain but not Ba(2+). The combined application of Ba(2+) and ouabain further depressed relaxation. 5. In summary, both EDHF and raised [K(+)](o) dilate/relax rat mesenteric arteries, though sensitivities to barium and ouabain differ. K(+) may be a relaxing factor in this tissue, but its characteristics differ from EDHF. Gap junction inhibitors depress EDHF, implying an important role for myo-endothelial gap junctions.