Endothelial dysfunction and vascular disease - a 30th anniversary update.

The endothelium can evoke relaxations of the underlying vascular smooth muscle, by releasing vasodilator substances. The best-characterized endothelium-derived relaxing factor (EDRF) is nitric oxide (NO) which activates soluble guanylyl cyclase in the vascular smooth muscle cells, with the production of cyclic guanosine monophosphate (cGMP) initiating relaxation. The endothelial cells also evoke hyperpolarization of the cell membrane of vascular smooth muscle (endothelium-dependent hyperpolarizations, EDH-mediated responses). As regards the latter, hydrogen peroxide (H2 O2 ) now appears to play a dominant role. Endothelium-dependent relaxations involve both pertussis toxin-sensitive Gi (e.g. responses to α2 -adrenergic agonists, serotonin, and thrombin) and pertussis toxin-insensitive Gq (e.g. adenosine diphosphate and bradykinin) coupling proteins. New stimulators (e.g. insulin, adiponectin) of the release of EDRFs have emerged. In recent years, evidence has also accumulated, confirming that the release of NO by the endothelial cell can chronically be upregulated (e.g. by oestrogens, exercise and dietary factors) and downregulated (e.g. oxidative stress, smoking, pollution and oxidized low-density lipoproteins) and that it is reduced with ageing and in the course of vascular disease (e.g. diabetes and hypertension). Arteries covered with regenerated endothelium (e.g. following angioplasty) selectively lose the pertussis toxin-sensitive pathway for NO release which favours vasospasm, thrombosis, penetration of macrophages, cellular growth and the inflammatory reaction leading to atherosclerosis. In addition to the release of NO (and EDH, in particular those due to H2 O2 ), endothelial cells also can evoke contraction of the underlying vascular smooth muscle cells by releasing endothelium-derived contracting factors. Recent evidence confirms that most endothelium-dependent acute increases in contractile force are due to the formation of vasoconstrictor prostanoids (endoperoxides and prostacyclin) which activate TP receptors of the vascular smooth muscle cells and that prostacyclin plays a key role in such responses. Endothelium-dependent contractions are exacerbated when the production of nitric oxide is impaired (e.g. by oxidative stress, ageing, spontaneous hypertension and diabetes). They contribute to the blunting of endothelium-dependent vasodilatations in aged subjects and essential hypertensive and diabetic patients. In addition, recent data confirm that the release of endothelin-1 can contribute to endothelial dysfunction and that the peptide appears to be an important contributor to vascular dysfunction. Finally, it has become clear that nitric oxide itself, under certain conditions (e.g. hypoxia), can cause biased activation of soluble guanylyl cyclase leading to the production of cyclic inosine monophosphate (cIMP) rather than cGMP and hence causes contraction rather than relaxation of the underlying vascular smooth muscle.

Endothelial dysfunction and vascular disease.

The endothelium can evoke relaxations (dilatations) of the underlying vascular smooth muscle, by releasing vasodilator substances. The best characterized endothelium-derived relaxing factor (EDRF) is nitric oxide (NO). The endothelial cells also evoke hyperpolarization of the cell membrane of vascular smooth muscle (endothelium-dependent hyperpolarizations, EDHF-mediated responses). Endothelium-dependent relaxations involve both pertussis toxin-sensitive G(i) (e.g. responses to serotonin and thrombin) and pertussis toxin-insensitive G(q) (e.g. adenosine diphosphate and bradykinin) coupling proteins. The release of NO by the endothelial cell can be up-regulated (e.g. by oestrogens, exercise and dietary factors) and down-regulated (e.g. oxidative stress, smoking and oxidized low-density lipoproteins). It is reduced in the course of vascular disease (e.g. diabetes and hypertension). Arteries covered with regenerated endothelium (e.g. following angioplasty) selectively loose the pertussis toxin-sensitive pathway for NO release which favours vasospasm, thrombosis, penetration of macrophages, cellular growth and the inflammatory reaction leading to atherosclerosis. In addition to the release of NO (and causing endothelium-dependent hyperpolarizations), endothelial cells also can evoke contraction (constriction) of the underlying vascular smooth muscle cells by releasing endothelium-derived contracting factor (EDCF). Most endothelium-dependent acute increases in contractile force are due to the formation of vasoconstrictor prostanoids (endoperoxides and prostacyclin) which activate TP receptors of the vascular smooth muscle cells. EDCF-mediated responses are exacerbated when the production of NO is impaired (e.g. by oxidative stress, ageing, spontaneous hypertension and diabetes). They contribute to the blunting of endothelium-dependent vasodilatations in aged subjects and essential hypertensive patients.

K+-induced hyperpolarization in rat mesenteric artery: identification, localization and role of Na+/K+-ATPases.

1. Mechanisms underlying K(+)-induced hyperpolarizations in the presence and absence of phenylephrine were investigated in endothelium-denuded rat mesenteric arteries (for all mean values, n=4). 2. Myocyte resting membrane potential (m.p.) was -58.8+/-0.8 mV. Application of 5 mM KCl produced similar hyperpolarizations in the absence (17.6+/-0.7 mV) or presence (15.8+/-1.0 mV) of 500 nM ouabain. In the presence of ouabain +30 microM barium, hyperpolarization to 5 mM KCl was essentially abolished. 3. In the presence of 10 microM phenylephrine (m.p. -33.7+/-3 mV), repolarization to 5 mM KCl did not occur in the presence or absence of 4-aminopyridine but was restored (-26.9+/-1.8 mV) on addition of iberiotoxin (100 nM). Under these conditions the K+-induced repolarization was insensitive to barium (30 microM) but abolished by 500 nM ouabain alone. 4. In the presence of phenylephrine + iberiotoxin the hyperpolarization to 5 mM K(+) was inhibited in the additional presence of 300 nM levcromakalim, an action which was reversed by 10 microM glibenclamide. 5. RT-PCR, Western blotting and immunohistochemical techniques collectively showed the presence of alpha(1)-, alpha(2)- and alpha(3)-subunits of Na(+)/K(+)-ATPase in the myocytes. 6. In K(+)-free solution, re-introduction of K(+) (to 4.6 mM) hyperpolarized myocytes by 20.9+/-0.5 mV, an effect unchanged by 500 nM ouabain but abolished by 500 microM ouabain. 7. We conclude that under basal conditions, Na(+)/K(+)-ATPases containing alpha(2)- and/or alpha(3)-subunits are partially responsible for the observed K(+)-induced effects. The opening of myocyte K(+) channels (by levcromakalim or phenylephrine) creates a 'K(+) cloud' around the cells which fully activates Na(+)/K(+)-ATPase and thereby abolishes further responses to [K(+)](o) elevation.

Suppression of K(+)-induced hyperpolarization by phenylephrine in rat mesenteric artery: relevance to studies of endothelium-derived hyperpolarizing factor.

In intact mesenteric arteries, increasing [K(+)]o by 5 mM hyperpolarized both endothelial and smooth muscle cells. Subsequent exposure to 10 microM phenylephrine depolarized both cell types which were then repolarized by a 5 mM increase in [K(+)]o. In endothelium-denuded vessels, increasing [K(+)]o by 5 mM hyperpolarized the smooth muscle but K(+) had no effect after depolarization by 10 microM phenylephrine. On subsequent exposure to iberiotoxin plus 4-aminopyridine, the repolarizing action of 5 mM K(+) was restored. In endothelium-intact vessels exposed to phenylephrine, pretreatment with a gap junction inhibitor (gap 27) reduced K(+)-mediated smooth muscle repolarization without affecting the endothelial cell response. It is concluded that phenylephrine-induced efflux of K(+) via smooth muscle K(+) channels produces a local increase in [K(+)]o which impairs repolarization to added K(+). Thus, studies involving vessels precontracted with agonists which increase [K(+)]o maximize the role of gap junctions and minimize any contribution to the EDHF pathway from endothelium-derived K(+).

Further investigations into the endothelium-dependent hyperpolarizing effects of bradykinin and substance P in porcine coronary artery.

In porcine coronary arteries, smooth muscle hyperpolarizations produced by the nitric oxide donor, NOR-1, and the prostacyclin analogue, iloprost, were compared with those induced by substance P and bradykinin and attributed to the endothelium-derived hyperpolarizing factor (EDHF). In the presence of 300 microM L-nitroarginine and 10 microM indomethacin, iloprost-induced hyperpolarizations were partially inhibited by 10 microM glibenclamide whereas those to NOR-1, substance P and bradykinin were unaffected. Hyperpolarizations produced by maximally-effective concentrations of NOR-1 and NS1619 were identical (to -65 mV). They were significantly less than those generated by either substance P or bradykinin (to approximately -80 mV) and were abolished by iberiotoxin 100 nM, a concentration which had essentially no effect on responses to substance P or bradykinin. Incubation of segments of intact arteries for 16 - 22 h in bicarbonate-buffered Krebs solution had little effect on EDHF responses to substance P or bradykinin. In contrast, after incubation for this period of time in HEPES-buffered Tyrode solution or Krebs containing 10 mM HEPES the EDHF response to substance P was abolished and that to bradykinin was markedly reduced. The residual bradykinin-induced hyperpolarization following incubation in Tyrode solution was inhibited by iberiotoxin and by 10 microM 17-octadecynoic acid. We conclude that substance P activates only the EDHF pathway in the presence of nitric oxide synthase and cyclo-oxygenase inhibitors. Incubation in HEPES-buffered Tyrode solution abolishes the EDHF responses to substance P and bradykinin to reveal an additional hyperpolarizing mechanism, associated with the opening of K(+) channels, activated only by bradykinin.

Vascular endothelial growth factor and the in vivo increase in plasma extravasation in the hamster cheek pouch.

1. The purpose of this study in the hamster cheek pouch was to determine whether or not vascular endothelial growth factor (VEGF) induced changes in plasma extravasation and if so, the mechanism(s) involved. 2. The cheek pouch microcirculatory bed of the anaesthetized hamster was directly observed under microscope and the number of vascular leakage sites, as shown by fluorescein isothiocyanate (FITC-dextran, 150 kD) extravasation, was counted. Drugs and VEGF were applied topically. VEGF from 0.05 to 0.5 microg ml(-1) (1.2 to 12 nM) produced a dose-dependent increase in the number of microvascular leakage sites from virtually none in basal conditions to up to 250 in some pouches. The effects of VEGF (0.1 microg ml(-1) or 2.4 nM) were blocked in a concentration-dependent manner by the non-specific heparin growth factor antagonist TBC-1635 (0.1, 1 and 3microM). The placenta growth factor (PlGF-1: 0.1 and 0.5 microg ml(-1) or 3.4 and 17 nM) did not increase plasma extravasation, per se, but abolished the effects of VEGF (2.4 nM). 3. The increases in microvascular leakage produced by VEGF (2.4 nM) were partially but significantly (P<0.05) inhibited by genistein (5 and 10 microM, up to 33% inhibition), LY 294002 (30 microM, 41%), bisindolylmaleimide (1 microM, 65%) and virtually abolished by indomethacin (3 microM, 88%) and L-nitro-arginine (10 microM, 95%), these drugs being inhibitors of tyrosine kinase, phosphatidylinositol-3-kinase, protein kinase C, cyclo-oxygenase and nitric oxide synthase respectively. None of these inhibitors, at the concentration tested, induced alone an increase in plasma extravasation. 4. These results indicate that the VEGF-induced plasma extravasation may involve the stimulation of VEGF-R2 (Flk-1/KDR) and the activation of phosphatidylinositol-3-kinase and protein kinase C. The production of both nitric oxide and prostaglandin is required to observe an increase in vascular leakage.

An endothelium-derived hyperpolarizing factor distinct from NO and prostacyclin is a major endothelium-dependent vasodilator in resistance vessels of wild-type and endothelial NO synthase knockout mice.

In addition to nitric oxide (NO) and prostacyclin (PGI(2)), the endothelium generates the endothelium-derived hyperpolarizing factor (EDHF). We set out to determine whether an EDHF-like response can be detected in wild-type (WT) and endothelial NO synthase knockout mice (eNOS -/-) mice. Vasodilator responses to endothelium-dependent agonists were determined in vivo and in vitro. In vivo, bradykinin induced a pronounced, dose-dependent decrease in mean arterial pressure (MAP) which did not differ between WT and eNOS -/- mice and was unaffected by treatment with N(omega)-nitro-l-arginine methyl ester and diclofenac. In the saline-perfused hindlimb of WT and eNOS -/- mice, marked N(omega)-nitro-l-arginine (l-NA, 300 micromol/liter)- and diclofenac-insensitive vasodilations in response to both bradykinin and acetylcholine (ACh) were observed, which were more pronounced than the agonist-induced vasodilation in the hindlimb of WT in the absence of l-NA. This endothelium-dependent, NO/PGI(2)-independent vasodilatation was sensitive to KCl (40 mM) and to the combination of apamin and charybdotoxin. Gap junction inhibitors (18alpha-glycyrrhetinic acid, octanol, heptanol) and CB-1 cannabinoid-receptor agonists (Delta(9)-tetrahydrocannabinol, HU210) impaired EDHF-mediated vasodilation, whereas inhibition of cytochrome P450 enzymes, soluble guanylyl cyclase, or adenosine receptors had no effect on EDHF-mediated responses. These results demonstrate that in murine resistance vessels the predominant agonist-induced endothelium-dependent vasodilation in vivo and in vitro is not mediated by NO, PGI(2), or a cytochrome P450 metabolite, but by an EDHF-like principle that requires functional gap junctions.

Role of endothelial cell hyperpolarization in EDHF-mediated responses in the guinea-pig carotid artery.

1. Experiments were performed to identify the potassium channels involved in the acetylcholine-induced endothelium-dependent hyperpolarization of the guinea-pig internal carotid artery. Smooth muscle and endothelial cell membrane potentials were recorded in isolated arteries with intracellular microelectrodes. Potassium currents were recorded in freshly-dissociated smooth muscle cells using patch clamp techniques. 2. In single myocytes, iberiotoxin (0.1 microM)-, charybdotoxin (0.1 microM)-, apamin (0.5 microM)- and 4-aminopyridine (5 mM)-sensitive potassium currents were identified indicating the presence of large- and small-conductance calcium-sensitive potassium channels (BK(Ca) and SK(Ca)) as well as voltage-dependent potassium channels (K(V)). Charybdotoxin and iberiotoxin inhibited the same population of BK(Ca) but a conductance specifically sensitive to the combination of charybdotoxin plus apamin could not be detected. 4-aminopyridine (0. 1 - 25 mM) induced a concentration-dependent inhibition of K(V) without affecting the iberiotoxin- or the apamin-sensitive currents. 3. In isolated arteries, both the endothelium-dependent hyperpolarization of smooth muscle and the hyperpolarization of endothelial cells induced by acetylcholine or by substance P were inhibited by 5 mM 4-aminopyridine. 4. These results indicate that in the vascular smooth muscle cells of the guinea-pig carotid artery, a conductance specifically sensitive to the combination of charybdotoxin plus apamin could not be detected, comforting the hypothesis that the combination of these two toxins should act on the endothelial cells. Furthermore, the inhibition by 4-aminopyridine of both smooth muscle and endothelial hyperpolarizations, suggests that in order to observe an endothelium-dependent hyperpolarization of the vascular smooth muscle cells, the activation of endothelial potassium channels is likely to be required.

Role of gap junctions and EETs in endothelium-dependent hyperpolarization of porcine coronary artery.

1. The effects of endothelium-derived hyperpolarizing factor (EDHF: elicited using substance P or bradykinin) were compared with those of 11,12-EET in pig coronary artery. Smooth muscle cells were usually impaled with microelectrodes through the adventitial surface. 2. Substance P (100 nM) and 11,12-EET (11,12-epoxyeicosatrienoic acid; 3 microM) hyperpolarized endothelial cells in intact arteries. These actions were unaffected by 100 nM iberiotoxin but were abolished by charybdotoxin plus apamin (each 100 nM). 3. Substance P (100 nM) and bradykinin (30 nM) hyperpolarized intact artery smooth muscle; Substance P had no effect after endothelium removal. 11,12-EET hyperpolarized de-endothelialized vessels by 12.6+/-0.3 mV, an effect abolished by 100 nM iberiotoxin. 4. 11,12-EET hyperpolarized intact arteries by 18.6+/-0.8 mV, an action reduced by iberiotoxin, which was ineffective against substance P. Hyperpolarizations to 11, 12-EET and substance P were partially inhibited by 100 nM charybdotoxin and abolished by further addition of 100 nM apamin. 5. 30 microM barium plus 500 nM ouabain depolarized intact artery smooth muscle but responses to substance P and bradykinin were unchanged. 500 microM gap 27 markedly reduced hyperpolarizations to substance P and bradykinin which were abolished in the additional presence of barium plus ouabain. 6. Substance P-induced hyperpolarizations of smooth muscle cells immediately below the internal elastic lamina were unaffected by gap 27, even in the presence of barium plus ouabain. 7. In pig coronary artery, 11,12-EET is not EDHF. Smooth muscle hyperpolarizations attributed to 'EDHF' are initiated by endothelial cell hyperpolarization involving charybdotoxin- (but not iberiotoxin) and apamin-sensitive K(+) channels. This may spread electrotonically via myoendothelial gap junctions but the involvement of an unknown endothelial factor cannot be excluded.

Endothelin-1 pathway in human alveolar epithelial cell line A549 and human umbilical vein endothelial cells.

AIM: This study was designed to characterize the endothelin pathway in an immortalized human adenocarcinoma-derived alveolar epithelial cell line (A549) and human umbilical vein endothelial cell line (HUVEC). METHODS: The release of ET-1 and big-ET-1 was measured in the incubation medium of both cell lines. The expression of mRNAs coding for the endothelin isoforms (hppET-1, -2, -3), the endothelin converting enzymes (hECE-1a, b, c, and d) and the hETA and hETB receptors was investigated using RT-PCR. The expression of ECE-1 mRNA in various human tissues and in A549 cells was investigated by Northern blot analysis and the subcellular localization of ECE-1 in A549 cells was investigated by immunoblotting using a polyclonal antibody. RESULTS: Under control conditions, HUVEC release both ET-1 and big-ET-1 (ratio 5 to 1) while in A549 cells the big-ET-1 levels were below the threshold of detection. The release of these two peptides was minimally affected by various inhibitors of peptidases. However, in both cell lines phosphoramidon produced a concentration-dependent inhibition of ET-1 release and an enhanced accumulation of big-ET-1. Both HUVEC and A549 cells express the mRNAs for ppET-1, ET-A, and ET-B receptor subtypes and ECE-1 (isoforms ECE-1b, c and/or d). In addition, in HUVEC the mRNAs for ppET-2 and for the isoform ECE-1a were also detected. In A549 cells, ECE-1 had a preferential subcellular localization in the membrane fraction but was not detected in the cytosol. CONCLUSION: Both A549 and HUVEC produce and release endothelin-1 through a specific enzymatic pathway, whether or not ECE-1 is the only enzyme involved remains to be determined. A549 might be used as a screening assay for drug discovery such as for inhibitors of endothelin-1 release.

Further investigation of endothelium-derived hyperpolarizing factor (EDHF) in rat hepatic artery: studies using 1-EBIO and ouabain.

1. The characteristics of endothelium-dependent hyperpolarization in rat hepatic artery have been further investigated in the presence of inhibitors of cyclo-oxygenase and nitric oxide synthase. 2. Using sharp micro-electrodes, the smooth muscle hyperpolarization induced by acetylcholine, KCl or 1-ethyl-2-benzimidazolinone (1-EBIO) in intact hepatic arteries was abolished by 30 micronM barium plus 500 nM ouabain. 3. In vessels without endothelium, the smooth muscle hyperpolarization induced by KCl was not reduced by 30 micronM barium alone. However, in the presence of barium the effects of KCl were partially inhibited by 100 nM ouabain and essentially abolished by 500 nM ouabain. 4. Using sharp micro-electrodes, the hyperpolarization of both the smooth muscle and the endothelium induced by 1-EBIO or by acetylcholine was unaffected by 100 nM iberiotoxin. However, in the presence of 100 nM charybdotoxin, the effects of 1-EBIO were abolished whereas those of acetylcholine were only partially reduced. The hyperpolarization induced by levcromakalim was unaffected by either charybdotoxin or iberiotoxin. 5 Under whole-cell patch-clamp recording conditions, 1-EBIO induced a voltage-insensitive, charybdotoxin-sensitive K+ current in cultured endothelial cells but was without effect on K+ currents in smooth muscle cells isolated from hepatic arteries. 6 It is concluded that the endothelium-dependent hyperpolarization of smooth muscle induced by either acetylcholine or by 1-EBIO in rat hepatic artery is initially associated with the opening of endothelial calcium-sensitive K+-channels insensitive to iberiotoxin. The resulting accumulation of K+ in the myoendothelial space activates an isoform of Na+/K+-ATPase which is sensitive to low concentrations of ouabain.

NPY receptor subtype in the rabbit isolated ileum.

1. The purpose of this work was to verify the hypothesis that the rabbit ileum is a selective preparation for the NPY Y5 receptor by using new selective antagonists recently synthesized. Spontaneous contractions of the rabbit isolated ileum were recorded and binding experiments were performed in cells expressing the human NPY Y1, Y2, Y4 or Y5 receptor subtype. 2. NPY analogues produced a concentration-dependent transient inhibition of the spontaneous contractions of the rabbit ileum with the following order of potency hPP > rPP > PYY > or = [Leu31,-Pro34]-NPY > NPY >> NPY13-36. Pre-exposure to rPP, PYY, [Leu31,Pro34]-NPY or NPY (but not NPY13-36) inhibited the effect of subsequent administration of hPP suggesting cross-desensitization of the preparation. The apparent affinity of the various agonists studied was correlated to the affinity reported for the human Y4 receptor subtype (and to a lesser extent for the rat Y4 subtype) but not to the affinity for the Y5 receptor subtype. 3. BIBO 3304, a selective NPY Y1 receptor antagonist, and CGP 71683A, a selective NPY Y5 receptor antagonist, did not affect the response to hPP. JCF 109, another NPY Y5 receptor antagonist, produced an inhibition of the response to hPP but only at the highest dose tested (10 microM) which also, by itself, produced intrinsic inhibitory effects. 4. 1229U91, a non-selective ligand for Y1, Y2, Y4 and Y5 receptors with high affinity toward the Y1 and Y4 receptor subtypes, produced a concentration-dependent transient inhibition of the spontaneous contractions of the rabbit ileum and a dose-dependent inhibition of the response to hPP (apparent pKB: 7.2). 5. These results suggest that in the rabbit ileum, the NPY receptor involved in the inhibition of the spontaneous contractile activity is a NPY Y4 receptor subtype.

Epoxyeicosatrienoic acids, potassium channel blockers and endothelium-dependent hyperpolarization in the guinea-pig carotid artery.

1. Using intracellular microelectrodes, we investigated the effects of 17-octadecynoic acid (17-ODYA) on the endothelium-dependent hyperpolarization induced by acetylcholine in the guinea-pig isolated internal carotid artery with endothelium. 2. In the presence of Nomega-nitro-L-arginine (L-NOARG, 100 microM) and indomethacin (5 microM) to inhibit nitric oxide synthase and cyclo-oxygenase, acetylcholine (1 microM) evoked an endothelium-dependent hyperpolarization which averaged -16.4 mV starting from a resting membrane potential of -56.8 mV. There was a negative correlation between the amplitude of the hyperpolarization and the absolute values of the resting membrane potential. 3. The acetylcholine-induced endothelium-dependent hyperpolarization was not altered by charybdotoxin (0.1 microM) or iberiotoxin (30 nM). It was partially but significantly reduced by apamin (0.5 microM) to -12.8+/-1.2 mV (n=10) or the combination of apamin plus iberiotoxin (-14.3+/-3.4mV, n=4). However, the combination of charybdotoxin and apamin abolished the hyperpolarization and under these conditions, acetylcholine evoked a depolarization (+ 7.1+/-3.7 mV, n = 8). 4. 17-ODYA (10 microM) produced a significant hyperpolarization of the resting membrane potential which averaged -59.6 mV and a partial but significant inhibition of the acetylcholine-induced endothelium-dependent hyperpolarization (-10.9 mV). 5. Apamin did not modify the effects of 17-ODYA but in the presence of charybdotoxin or iberiotoxin, 17-ODYA no longer influenced the resting membrane potential or the acetylcholine-induced hyperpolarization. 6. When compared to solvent (ethanol, 1% v/v), epoxyeicosatrienoic acids (EpETrEs) (5,6-, 8,9-, 11,12- and 14,15-EpETrE, 3 microM) did not affect the cell membrane potential and did not relax the guinea-pig isolated internal carotid artery. 7. These results indicate that, in the guinea-pig internal carotid artery, the involvement of metabolites of arachidonic acid through the cytochrome P450 pathway in endothelium-dependent hyperpolarization is unlikely. Furthermore, the hyperpolarization mediated by the endothelium-derived hyperpolarizing factor (EDHF) is probably not due to the opening of BK(Ca) channels.

Relaxation of canine coronary artery to electrical stimulation: limited role of free radicals.

Electrical stimulation induces tetrodotoxin-insensitive relaxation of the canine coronary artery. The present study was designed to verify whether this relaxation involves the production of oxygen-derived free radicals. Isolated rings of canine coronary arteries were suspended for isometric tension recording in organ chambers filled with Krebs-Ringer bicarbonate solution. They were stimulated electrically (9 V, 3 Hz, 2 ms for 2 min) by means of two platinum electrodes during contractions evoked by various vasoactive agonists. Under control conditions, electrical stimulation caused rapid, reversible relaxations. Superoxide dismutase in association with catalase or mannitol, sodium ascorbate, dimethyl sulfoxide, and glutathione did not inhibit the relaxation caused by stimulation applied for only 2 min; neither did the removal of chloride ions from the salt solution nor the association of Cl-free solution in the presence of mannitol, superoxide dismutase, and catalase. Prolonging the electrical stimulation (9 V, 3 Hz, 2 ms) for up to 20 min produced a secondary relaxation. This second phase was inhibited by sodium ascorbate. These experiments indicate that the rapid relaxation induced by short-lasting electrical stimulation is probably not due to the generation of oxygen-derived free radicals. However, prolonged stimulation causes the production of such radicals, which then evoke irreversible inhibition of the vascular smooth muscle of the canine coronary artery.