Acrolein induces vasodilatation of rodent mesenteric bed via an EDHF-dependent mechanism

Acrolein is generated endogenously during lipid peroxidation and inflammation and is an environmental pollutant. Protein adducts of acrolein are detected in atherosclerotic plaques and neurons of patients with Alzheimer's disease. To understand vascular effects of acrolein exposure, we studied acrolein vasoreactivity in perfused rodent mesenteric bed. Acrolein induced endothelium-dependent vasodilatation that was more robust and more sensitive than dilation induced by 4-hydroxy-trans-2-nonenal, trans-2-hexenal, or propionaldehyde. Acrolein-induced vasodilatation was mediated by K(+)-sensitive components, e.g., it was abolished in 0 [K(+)](o) buffer or in 3 mM tetrabutylammonium, inhibited 75% in 50 microM ouabain, and inhibited 64% in 20 mM K(+) buffer. Moreover, combined treatment with the Ca(2+)-activated K(+) channel inhibitors 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34, 100 nM) and apamin (5 microM) significantly reduced vasodilatation without altering sensitivity to acrolein. However, acrolein-induced % dilation was unaffected by l-NAME or indomethacin pretreatment indicating mechanistic independence of NO and prostaglandins. Moreover, acrolein induced vasodilatation in cirazoline-precontracted mesenteric bed of eNOS-null mice confirming eNOS independence. Pretreatment with 6-(2-propargyloxyphenyl) hexanoic acid (PPOH 50 microM), an epoxygenase inhibitor, or the superoxide dismutase mimetic Tempol (100 microM) significantly attenuated acrolein-induced vasodilatation. Collectively, these data indicate that acrolein stimulates mesenteric bed vasodilatation due to endothelium-derived signal(s) that is K(+)-, ouabain-, PPOH-, and Tempol-sensitive, and thus, a likely endothelium-derived hyperpolarizing factor (EDHF). These data indicate that low level acrolein exposure associated with vascular oxidative stress or inflammation stimulates vasodilatation via EDHF release in medium-sized arteries--a novel function.     

Openers of calcium-activated potassium channels and endothelium-dependent hyperpolarizations in the guinea pig carotid artery

This study was designed to determine whether putative openers of calcium-activated potassium channels of small and/or intermediate conductance (SK_Ca and IK_Ca) induce vascular smooth muscle hyperpolarizations and to identify the underlying mechanisms. The membrane potential of guinea pig carotid artery smooth muscle cells was recorded with intracellular microelectrodes in the presence of N ^ω-nitro-l-arginine and indomethacin. Acetylcholine and NS-309 produced endothelium-dependent hyperpolarizations. The effects of acetylcholine were partially and significantly inhibited by apamin. The combinations of charybdotoxin plus apamin and TRAM-34 plus apamin markedly and significantly reduced these hyperpolarizations. 1-ethyl-2-benzimidazolinone (1-EBIO) induced hyperpolarizations that were unaffected by TRAM-34 but partially inhibited by charybdotoxin, apamin, TRAM-34 plus apamin, and charybdotoxin plus apamin. Riluzole produced only marginal hyperpolarizations. Therefore, in the guinea pig carotid artery, endothelium-dependent hyperpolarization to acetylcholine involves the activation of both SK_Ca and IK_Ca, with a predominant role for the former channel. 1-EBIO is a non-selective and weak opener of SK_Ca, while riluzole is virtually ineffective. By contrast, NS-309 is a reasonably potent and selective opener of both SK_Ca and IK_Ca, and this compound mimics the endothelium-dependent hyperpolarizations to acetylcholine.     

Role of SK(Ca) and IK(Ca) in endothelium-dependent hyperpolarizations of the guinea-pig isolated carotid artery

1. This study was designed to determine whether the endothelium-dependent hyperpolarizations evoked by acetylcholine in guinea-pig carotid artery involve a cytochrome P450 metabolite and whether they are linked to the activation of two distinct populations of endothelial K(Ca) channels, SK(Ca) and IK(Ca.) 2. The membrane potential was recorded in the vascular smooth muscle cells of the guinea-pig isolated carotid artery. All the experiments were performed in the presence of N(omega)-L-nitro arginine (100 microM) and indomethacin (5 microM). 3. Under control conditions (Ca(2+): 2.5 mM), acetylcholine (10 nM to 10 muM) induced a concentration- and endothelium-dependent hyperpolarization of the vascular smooth muscle cells. Two structurally different specific blockers of SK(Ca), apamin (0.5 microM) or UCL 1684 (10 microM), produced a partial but significant inhibition of the hyperpolarization evoked by acetylcholine whereas charybdotoxin (0.1 microM) and TRAM-34 (10 microM), a nonpeptidic and specific blocker of IK(Ca), were ineffective. In contrast, the combinations of apamin plus charybdotoxin, apamin plus TRAM-34 (10 microM) or UCL 1684 (10 microM) plus TRAM-34 (10 microM) virtually abolished the acetylcholine-induced hyperpolarization. 4. In the presence of a combination of apamin and a subeffective dose of TRAM-34 (5 microM), the residual hyperpolarization produced by acetylcholine was not inhibited further by the addition of either an epoxyeicosatrienoic acid antagonist, 14,15-EEZE (10 microM) or the specific blocker of BK(Ca), iberiotoxin (0.1 microM). 5. In presence of 0.5 mM Ca(2+), the hyperpolarization in response to acetylcholine (1 microM) was significantly lower than in 2.5 mM Ca(2+). The EDHF-mediated responses became predominantly sensitive to charybdotoxin or TRAM-34 but resistant to apamin. 6. This investigation shows that the production of a cytochrome P450 metabolite, and the subsequent activation of BK(Ca), is unlikely to contribute to the EDHF-mediated responses in the guinea-pig carotid artery. Furthermore, the EDHF-mediated response involves the activation of both endothelial IK(Ca) and SK(Ca) channels, the activation of either one being able to produce a true hyperpolarization.     

Flow-induced enhancement of vasoconstriction and blockade of endothelium-derived hyperpolarizing factor (EDHF) by ascorbate in the rat mesentery

BACKGROUND AND PURPOSE: We previously reported that ascorbate inhibits flow- and agonist-induced, EDHF-mediated vasodilatation in the bovine ciliary circulation. This study examined whether ascorbate had similar actions in the rat mesenteric vasculature. EXPERIMENTAL APPROACH: The effects of ascorbate were examined both in rat second order mesenteric arterial rings suspended in a static wire myograph and the rat mesentery perfused at different rates of flow. KEY RESULTS: Ascorbate (50 microM) had no effect on U46619-induced tone or acetylcholine-induced, EDHF-mediated vasodilatation in either rings of mesenteric artery or the perfused mesentery at rates of flow below 10 ml min(-1). At higher rates of flow, ascorbate produced two distinct effects in the rat mesentery: a rapid and maintained enhancement of vasoconstrictor tone and a slow (max at 3 h) inhibition of acetylcholine-induced, EDHF-mediated vasodilatation. The enhancement of vasoconstrictor tone appeared to be due to inhibition of flow-induced EDHF-like activity, since it was endothelium-dependent, but could be elicited during blockade of nitric oxide synthase and cyclooxygenase. Despite this, the classical inhibitors of EDHF, apamin and charybdotoxin, failed to affect the ascorbate-induced enhancement of tone, although they inhibited acetylcholine-induced vasodilatation. CONCLUSIONS AND IMPLICATIONS: Ascorbate inhibits both flow- and agonist-induced EDHF in the rat mesentery. The strikingly different timecourses of these two effects, together with their differential sensitivity to apamin and charybdotoxin, suggest that the flow- and agonist-induced EDHFs in the rat mesenteric vasculature may either be different entities or operate by different mechanisms.     

Role of calcium-activated potassium channels in impaired acetylcholine vasodilatory responses in diabetic rats

Muscarinic agonists produce endothelium-dependent vasodilatation in the presence of nitric oxide synthase (NOS) inhibition. The importance of this mechanism was assessed in the methoxamine-preconstricted perfused mesenteric vascular bed (MVB) of streptozotocin diabetic Sprague-Dawley rats. At 9 weeks of age, male rats were treated with streptozotocin (55 mg/kg in citrate buffer) or with citrate buffer alone. The superior mesenteric artery was cannulated and the MVB was detached from its intestinal borders. Concentration-response curves to acetylcholine were determined in the presence and in the absence of indomethacin, tetrabutylammonium (a calcium-activated potassium channel blocker), high extracellular potassium, or NOS inhibition with Nomega-nitro-l-arginine and l-NG-nitro-l-arginine. There was a rightward shift in the concentration-response curve with an increase in median inhibitory concentration (p < 0.05) and a reduction in acetylcholine IMAX (p < 0.05) values in 14-week streptozotocin rats. The ability of NOS inhibition to attenuate vasodilatation was reduced in the 14-week streptozotocin group relative to the 2-week streptozotocin treatment group (p < 0.05). However, the ability of tetrabutylammonium to block acetylcholine-mediated vasodilatation remained consistent in streptozotocin rats at both stages. The results demonstrate that an alternate pathway involving calcium-activated potassium channels may compensate for diminished nitric oxide bioactivity. This effect is contingent on the duration of diabetes. This study provides insight into the development and progression of altered diabetic vascular responses.     

The contribution of d-tubocurarine-sensitive and apamin-sensitive K-channels to EDHF-mediated relaxation of mesenteric arteries from eNOS-/- mice

The nature of the potassium channels involved in determining endothelium-derived hyperpolarizing factor-mediated relaxation was investigated in first-order small mesenteric arteries from male endothelial nitric oxide synthase (eNOS-/-)-knockout and control (+/+) mice. Acetylcholine-induced endothelium-dependent relaxation of small mesenteric arteries of eNOS-/- was resistant to N-nitro-L-arginine and indomethacin and the guanylyl cyclase inhibitor, 1H-(1,2,4) oxadiazolo (4,3-a) quinoxalin-1-one. Apamin and the combination of apamin and iberiotoxin or apamin and charybdotoxin induced a transient endothelium-dependent contraction of small mesenteric arteries from both eNOS-/- and +/+ mice. Acetylcholine-induced relaxation in eNOS-/- mice was unaffected by charybdotoxin or apamin alone but significantly inhibited by the combination of these agents. However, the combination of scyllatoxin and iberiotoxin did not mimic the inhibitory effect of the apamin/charybdotoxin combination. Tubocurarine alone completely blocked acetylcholine-induced relaxation in eNOS-/- mice. Single channel analysis of myocytes from small mesenteric arterioles revealed a large conductance calcium-activated potassium channel that was sensitive to iberiotoxin, charybdotoxin, and tetraethylammonium. Tubocurarine blocked this channel from the cytosolic side but not when applied extracellularly. Solutions of nitric oxide (NO) gas also relaxed small mesenteric arteries that had been contracted with cirazoline in a concentration-dependent manner, and the sensitivity to NO was reduced by iberiotoxin and the combination of apamin, scyllatoxin, or tubocurarine with charybdotoxin but not by apamin, charybdotoxin, scyllatoxin, or tubocurarine alone. These data indicate that acetylcholine-induced endothelium-derived hyperpolarizing factor-mediated relaxation in small mesenteric arteries from eNOS-/- involved the activation of tubocurarine and apamin-/charybdotoxin-sensitive K-channels. In eNOS+/+ mice, the acetylcholine-induced response was primarily mediated by NO and was sensitive to iberiotoxin and the combination of apamin and charybdotoxin.     

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.     

Selective blockade of endothelial Ca2+-activated small- and intermediate-conductance K+-channels suppresses EDHF-mediated vasodilation

1. Activation of Ca(2+)-activated K(+)-channels (K(Ca)) has been suggested to play a key role in endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilation. However, due to the low selectivity of commonly used K(Ca)-channel blockers it is still elusive which endothelial K(Ca)-subtypes mediate hyperpolarization and thus initiate EDHF-mediated vasodilation. 2. Using the non-cytochrome P450 blocking clotrimazole-derivatives, 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) and 2-(2-chlorophenyl)-2,2-diphenylacetonitrile (TRAM-39) as highly selective IK1-inhibitors, we investigated the role of the intermediate-conductance K(Ca) (rIK1) in endothelial hyperpolarization and EDHF-mediated vasodilation. 3. Expression and function of rIK1 and small-conductance K(Ca) (rSK3) were demonstrated in situ in single endothelial cells of rat carotid arteries (CA). rIK1-currents were blocked by TRAM-34 or TRAM-39, while rSK3 was blocked by apamin. In current-clamp experiments, endothelial hyperpolarization in response to acetylcholine was abolished by the combination of apamin and TRAM-34. 4. In phenylephrine-preconstricted CA, acetylcholine-induced NO and prostacyclin-independent vasodilation was almost completely blocked by ChTX, CLT, TRAM-34, or TRAM-39 in combination with the SK3-blocker apamin. Apamin, TRAM-34, and CLT alone or sulphaphenzole, a blocker of the cytochrome P450 isoform 2C9, were ineffective in blocking the EDHF-response. 5. In experiments without blocking NO and prostacyclin synthesis, the combined blockade of SK3 and IK1 reduced endothelium-dependent vasodilation. 6. In conclusion, the use of selective IK1-inhibitors together with the SK3-blocker apamin revealed that activation of both K(Ca), rIK1 and rSK3 is crucial in mediating endothelial hyperpolarization and generation of the EDHF-signal while the cytochrome P450 pathway seems to play a minor or no role in rat CA.     

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.     

Impairment of both nitric oxide-mediated and EDHF-type relaxation in small mesenteric arteries from rats with streptozotocin-induced diabetes

BACKGROUND AND PURPOSE

To investigate whether diabetes affects either or both nitric oxide (NO)-mediated and endothelium-derived hyperpolarizing factor (EDHF)-type relaxation in endothelium-dependent relaxation of mesenteric arteries from streptozotocin-induced diabetic rats.

EXPERIMENTAL APPROACH

Wire myography was employed to examine endothelial function of mesenteric arteries. Superoxide levels were measured by L-012 and lucigenin-enhanced chemiluminescence. Western blotting was used to quantify protein expression levels.

KEY RESULTS

Superoxide levels were significantly increased in diabetic mesenteric arteries compared with normal arteries. Diabetes significantly reduced the sensitivity to the endothelium-dependent relaxant, acetylcholine (ACh) in mesenteric arteries. When the contribution of NO to relaxation was abolished by N-nitro-L-arginine (L-NNA) + a soluble guanylate cyclase inhibitor (ODQ), the sensitivity to ACh was significantly decreased in the diabetic arteries compared with normal arteries, indicating an impaired EDHF-type relaxation despite increased expression of intermediate- and small-conductance calcium-activated potassium channels. Conversely, when the contribution of EDHF was inhibited with TRAM-34 + apamin + iberiotoxin, maximum relaxations to ACh were significantly decreased in diabetic compared with normal arteries, suggesting that the contribution of NO was also impaired by diabetes. Basal levels of NO release, indicated by contraction to L-NNA, were also significantly decreased in diabetic arteries. Western blot analysis demonstrated that diabetic arteries had an increased expression of Nox2, decreased pSer⁴⁷³Akt and a reduced proportion of endothelial NO synthase (eNOS) expressed as a dimer, indicating uncoupling.

CONCLUSION AND IMPLICATIONS

The contribution of both NO and EDHF-type relaxations was impaired in diabetes and was caused by increased oxidative stress, decreased pSer⁴⁷³Akt and/or eNOS uncoupling.     

Endothelium-dependent and BRL 34915-induced vasodilatation in rat isolated perfused mesenteric arteries: role of G-proteins, K+ and calcium channels

1. In the isolated perfused, noradrenaline (NA)-constricted mesenteric arteries of the rat, acetylcholine (0.003-1 nmol), histamine (0.01-10 nmol) and the calcium ionophore A23187 (0.01-1 nmol), caused endothelium-dependent vasodilatation while the vasodilatation by the K+ channel activator BRL 34915 (0.1-1 nmol) was independent of endothelium. 2. The guanylate cyclase inhibitor, methylene blue at 10 microM did not inhibit the action of any of the vasodilators but at 50 microM reduced the vasodilator effect of acetylcholine (ACh), histamine and A23187. 3. Infusion of ouabain or perfusion with K(+)-free or excess K+ (50 mM) Krebs solution reduced the vasodilator effect of ACh, histamine and A23187, suggesting the action of these agents involves, at least in part, activation of Na+/K(+)-ATPase. The vasodilator effect of BRL 34915 was not affected by ouabain, but abolished during perfusion with Krebs solution containing excess K+ or depleted of K+. 4. Five structurally distinct K+ channel blockers (apamin, crude scorpion venom, procaine, quinidine and tetraethylammonium) attenuated the vasodilator effect of ACh, histamine and A23187. The K+ channel blockers, except apamin and crude scorpion venom, also inhibited the vasodilatation produced by BRL 34915. 5. The vasodilator effect of ACh, histamine or A23187 was not altered in mesenteric vessels of pertussis toxin-treated rats, suggesting that the K+ channels associated with the endothelium-dependent vasodilator effect of these agents are either not coupled to G-proteins or are coupled to G-proteins that are insensitive to pertussis toxin.(ABSTRACT TRUNCATED AT 250 WORDS)     

K(Ca) 3.1 channels maintain endothelium-dependent vasodilatation in isolated perfused kidneys of spontaneously hypertensive rats after chronic inhibition of NOS

BACKGROUND AND PURPOSE

The purpose of the study was to investigate renal endothelium-dependent vasodilatation in a model of severe hypertension associated with kidney injury.

EXPERIMENTAL APPROACH

Changes in perfusion pressure were measured in isolated, perfused kidneys taken from 18-week-old Wistar–Kyoto rat (WKY), spontaneously hypertensive rats (SHR) and SHR treated for 2 weeks with N^ω-nitro-L-arginine methyl ester in the drinking water (L-NAME-treated SHR, 6 mg·kg⁻¹·day⁻¹).

KEY RESULTS

Acetylcholine caused similar dose-dependent renal dilatation in the three groups. In vitro administration of indomethacin did not alter the vasodilatation, while the addition of N^w-nitro-L-arginine (L-NA) produced a differential inhibition of the vasodilatation, (inhibition in WKY > SHR > L-NAME-treated SHR). Further addition of ODQ, an inhibitor of soluble guanylyl cyclase, abolished the responses to sodium nitroprusside but did not affect the vasodilatation to acetylcholine. However, the addition of TRAM-34 (or charybdotoxin) inhibitors of Ca²⁺-activated K⁺ channels of intermediate conductance (K_Ca3.1), blocked the vasodilatation to acetylcholine, while apamin, an inhibitor of Ca²⁺-activated K⁺ channels of small conductance (K_Ca2.3), was ineffective. Dilatation induced by an opener of K_Ca3.1/K_Ca2.3 channels, NS-309, was also blocked by TRAM-34, but not by apamin. The magnitude and duration of NS-309-induced vasodilatation and the renal expression of mRNA for K_Ca3.1, but not K_Ca2.3, channels followed the same ranking order (WKY < SHR < L-NAME-treated SHR).

CONCLUSIONS AND IMPLICATIONS

In SHR kidneys, an EDHF-mediated response, involving activation of K_Ca3.1 channels, contributed to the mechanism of endothelium-dependent vasodilatation. In kidneys from L-NAME-treated SHR, up-regulation of this pathway fully compensated for the decrease in NO availability.     

Dominant role of an endothelium-derived hyperpolarizing factor (EDHF)-like vasodilator in the ciliary vascular bed of the bovine isolated perfused eye

1. The roles of the endothelium-derived nitric oxide, prostacyclin and endothelium-derived hyperpolarizing factor (EDHF) in mediating vasodilator responses to acetylcholine and bradykinin were assessed in the ciliary vascular bed of the bovine isolated perfused eye preparation. 2. Vasodilatation to acetylcholine or bradykinin was unaffected by the nitric oxide synthase inhibitor, L-NAME (100 microM), or the cyclo-oxygenase inhibitor, flurbiprofen (30 microM), but was virtually abolished following treatment with a high concentration of KCl (30 mM), or by damaging the endothelium with the detergent, CHAPS (0.3%, 2 min). 3. Acetylcholine-induced vasodilatation was unaffected by glibenclamide (10 microM), an inhibitor of ATP-sensitive K(+) channels (K(+)(ATP)), but was significantly attenuated by TEA (10 mM), a non-selective inhibitor of K(+) channels. 4. The small conductance calcium-sensitive K(+) channel (SK(+)(Ca)) inhibitor, apamin (100 nM), and the large conductance calcium-sensitive K(+) channel (BK(+)(Ca)) inhibitor, iberiotoxin (50 nM), had no significant effect on acetylcholine-induced vasodilatation. In contrast, the intermediate (IK(+)(Ca))/large conductance calcium-sensitive K(+) channel inhibitor, charybdotoxin (50 nM), powerfully blocked these vasodilator responses, and uncovered a vasoconstrictor response. 5. The combination of apamin (100 nM) with a sub-threshold concentration of charybdotoxin (10 nM) significantly attenuated acetylcholine-induced vasodilatation, but the combination of apamin (100 nM) with iberiotoxin (50 nM) had no effect. 6. In conclusion, blockade by a high concentration of KCl, by charybdotoxin, or by the combination of apamin with a sub-threshold concentration of charybdotoxin, strongly suggests that vasodilatation in the bovine isolated perfused eye is mediated by an EDHF.     

Different modulation by Ca2+-activated K+ channel blockers and herbimycin of acetylcholine- and flow-evoked vasodilatation in rat mesenteric small arteries

1. The present study addressed whether endothelium-dependent vasodilatation evoked by acetylcholine and flow are mediated by the same mechanisms in isolated rat mesenteric small arteries, suspended in a pressure myograph for the measurement of internal diameter. 2. In pressurized arterial segments contracted with U46619 in the presence of indomethacin, shear stress generated by the flow evoked relaxation. Thus, in endothelium-intact segments low (5.1+/-0.6 dyn cm(-2)) and high (19+/-2 dyn cm(-2)) shear stress evoked vasodilatations that were reduced by, respectively, 68+/-11 and 68+/-8% (P<0.05, n=7) by endothelial cell removal. Acetylcholine (0.01-1 microM) evoked concentration-dependent vasodilatation that was abolished by endothelial cell removal. 3. Incubation with indomethacin alone did not change acetylcholine and shear stress-evoked vasodilatation, while the combination of indomethacin with the nitric oxide (NO) synthase inhibitor, N(G),N(G)-asymmetric dimethyl-L-arginine (ADMA 1 mM), reduced low and high shear stress-evoked vasodilatation with, respectively, 52+/-15 and 58+/-10% (P<0.05, n=9), but it did not change acetylcholine-evoked vasodilatation. 4. Inhibition of Ca(2+)-activated K(+) channels with a combination of apamin (0.5 microM) and charybdotoxin (ChTX) (0.1 microM) did not change shear stress- and acetylcholine-evoked vasodilatation. In the presence of indomethacin and ADMA, the combination of apamin (0.5 microM) and ChTx (0.1 microM) increased contraction induced by U46619, but these blockers did not change the vasodilatation evoked by shear stress. In contrast, acetylcholine-evoked vasodilatation was abolished by the combination of apamin and charybdotoxin. 5. In the presence of indomethacin, the tyrosine kinase inhibitor, herbimycin A (1 microM), inhibited low and high shear stress-evoked vasodilatation with, respectively, 32+/-12 and 68+/-14% (P<0.05, n=8), but it did not change vasodilatation induced by acetylcholine. In the presence of indomethacin and ADMA, herbimycin A neither changed shear stress nor acetylcholine-evoked vasodilatation. 6. The present study suggests that Ca(2+)-activated K(+) channels sensitive for the combination of apamin and ChTx are involved in acetylcholine-evoked, mainly non-NO nonprostanoid factor-mediated, vasodilatation, while an Src tyrosine kinase plays a role for flow-evoked NO-mediated vasodilatation in rat mesenteric small arteries.     

Guanethidine effects on the guinea pig vas deferens are antagonized by the blockers of calcium-activated potassium conductance, apamin, methylene blue, and quinine

The blocking effects of guanethidine on electrically induced, neurally mediated, contractions of the guinea pig vas deferens in vitro could be markedly antagonized by the bee venom polypeptide apamin (20-60 nM), by 0.1 mM methylene blue, and (less regularly) by 0.1-0.15 mM quinine, three substances known to inhibit calcium-activated potassium conductance in a variety of cells. Guanethidine (20 microM) was also found to inhibit (by 88%) the release of [3H]norepinephrine induced by electrical stimulation (20-Hz, 2-msec, biphasic pulses of supramaximal voltage). Such inhibition was decreased to 39% when 20 nM apamin was present together with guanethidine, thus showing that the effect of this polypeptide is presynaptic. On the basis of these findings, we suggest that guanethidine may block adrenergic neurons by activating their calcium-activated potassium conductance, presumably by releasing intracellular calcium.     

Inhibition of EDHF by two new combinations of K+-channel inhibitors in rat isolated mesenteric arteries

It is widely established that in rat mesenteric arteries, endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation evoked by acetylcholine is abolished by a combination of charybdotoxin plus apamin. 4-Aminopyridine, an inhibitor of voltage-gated (Kv) K(+)-channels, in combination with apamin had moderate effects on the EDHF-mediated relaxation. Maurotoxin (MTX), an inhibitor of Kv and intermediate-conductance Ca(2+)-activated K(+)-channels (IK), had no effect on EDHF-mediated relaxation. However, MTX in combination with apamin completely abolished EDHF-mediated relaxation and endothelial cell hyperpolarization. The selective IK inhibitor 2-(2-chlorophenyl)-2,2-diphenyl acetonitrile (TRAM-39) had no significant effect on EDHF-mediated relaxation. EDHF-mediated vasorelaxation and hyperpolarization was abolished by a combination of TRAM-39 and apamin. These data demonstrate two new combinations of K(+)-channel inhibitors for the investigation of EDHF. Furthermore, by using TRAM-39, a potent selective inhibitor of IK channels, we provide the first direct evidence that abolition of EDHF requires the simultaneous presence of intermediate- and small-conductance Ca(2+)-activated K(+)-channel inhibitors.     

Calcium-activated potassium channel and connexin expression in small mesenteric arteries from eNOS-deficient (eNOS-/-) and eNOS-expressing (eNOS+/+) mice

Endothelium-derived hyperpolarizing factor (EDHF), notably in the microcirculation, plays an important role in the regulation of vascular tone. The cellular events that mediate EDHF are critically dependent, in a vessel dependent manner, on small conductance calcium-activated potassium channels (SK) and intermediate conductance calcium-activated potassium channels (IK) as well as the presence of the gap junction connexins 37, 40, and 43. We hypothesized that the expression levels of SK, IK, as well as vascular connexins, notably 37, 40 and 43 but, potentially, connexin 45, would show correlation with the contribution of EDHF to acetylcholine-mediated vasodilatation as well as, in the absence of endothelial-derived NO, higher expression levels in eNOS^−/− mice. Wire myograph studies were performed to confirm the contribution of EDHF to endothelium-dependent relaxation in 1st, 2nd and 3rd order small mesenteric arteries from C57BL/6J eNOS-expressing (eNOS^+/+) and eNOS-deficient C57BL/6J (eNOS^−/−) mice. Small mesenteric arteries, as well as the branch points between 1st and 2nd and 2nd and 3rd order vessels, were analysed for the expression of mRNA for SK1, SK2, SK3, IK and large conductance calcium-activated potassium channels (BK) and comparable studies were performed for connexins 37, 40, 43 and 45. Although the contribution of EDHF to endothelium-dependent relaxation was significantly greater in the 3rd order vessels from the eNOS^+/+ the real-time (RT) polymerase chain reaction (PCR) data showed no differences for the expression levels of mRNA for any of the channel subtypes or the connexins within the small mesenteric arteries from either the eNOS^+/+ or eNOS^−/− mice, nor, based on RT PCR analysis, were there differences in expression of the potassium channels studied in the branch points versus 1st, 2nd or 3rd order vessels. These data suggest that neither the gene expression of calcium-activated potassium channels nor vascular connexins are modulated by NO; however, their functional contribution to endothelium-dependent relaxation may be modulated by other physiological parameters.     

The effects of perhexiline on the rat coronary vasculature

The predominant site and mechanism(s) of perhexiline-induced coronary vasodilatation were investigated in the rat heart. Perhexiline was more potent in the Langendorff perfused heart than in the left anterior descending coronary artery (EC₅₀; 0.27 μM, confidence limits 0.19–0.39: 2.7 μM, 2.0–3.4, respectively). Selective endothelial inactivation with Triton X-100 in the perfused heart, reduced the response to perhexiline 1 μM (76+8% to 30+3% of control). 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) 3 μM, N-nitro- -arginine 100 μM, or a combination of the latter with indomethacin 10 μM, had no significant effect on responses to perhexiline in the perfused heart. Unlike bradykinin-induced vasodilatation, responses to perhexiline were not inhibited by tetrabutylammonium 1 mM, or charybdotoxin 20 nM. SKF525A 5 μM inhibited both perhexiline and bradykinin responses, while apamin 1 μM and glibenclamide 3 μM inhibited neither. Perhexiline exerts partially endothelium-dependent coronary vasodilator effects in the rat, predominantly on small coronary arteries, which appear to be independent of nitric oxide (NO), prostacyclin and the endothelium-derived hyperpolarising factor (EDHF) released by bradykinin.     

1-Ethyl-2-benzimidazolinone stimulates endothelial K(Ca) channels and nitric oxide formation in rat mesenteric vessels.

Hyperpolarization of most blood vessels occurs by the opening of K(Ca) channels. 1-Ethyl-2-benzimidazolinone (1-EBIO) is a direct activator of K(Ca) channels in epithelial cells and is potentially valuable for studying cellular hyperpolarization. This study reports the effects of 1-EBIO on isolated rat mesenteric beds perfused with normal (4.7 mM), or high (20 or 80 mM) K+ physiological salt solution (PSS) and constricted with an alpha1-adrenoceptor agonist, cirazoline (0.3-1 microM). Arterial perfusion pressures were decreased by 1-EBIO (0.1-30 nmol) in a dose- and endothelium-dependent manner. Infusion of penitrem A (100 nM), a maxi-K+ channel blocker, or apamin (0.5 microM), a small-conductance (SK(Ca)) K+ channel blocker, produced significant increases in cirazoline-mediated tone (mm Hg): 103.3 +/- 8.7 (control) vs. 156.3 +/- 14.3 (penitrem A); or 93.0 +/- 15.8 (control) vs. 114.0 +/- 15.4 (apamin). 1-EBIO relaxations were attenuated by penitrem A, while apamin, dendrotoxin (50 nM; a Kv channel antagonist), or ouabain (100 microM; a sodium pump blocker) failed to alter the responses. I-EBIO-mediated relaxations decreased significantly with increasing extracellular [K+]: relaxations to 30 nmol were 89.3% +/- 3.2% (4.7 mM K+, normal PSS) vs. 59.5% +/- 3.4% and 19.0% +/- 3.9% for 20 and 80 mM K+ PSS, respectively. Nomega-nitro-L-arginine-methyl ester (L-NAME; 100 microM), and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 microM), selective inhibitors of nitric oxide synthase, and nitric oxide-sensitive guanylate cyclase, respectively, abolished 1-EBIO relaxations in vessels perfused with 20 or 80 mM K+ PSS. We conclude that: (1) maxi-K+ and SK(Ca) channels are present in rat mesenteric arterial vessels and actively contribute to vascular tone, (2) vasodilator action of 1-EBIO involves the opening of endothelial maxi-K+ channels and nitric oxide synthesis.     

Multiple mechanisms of vascular smooth muscle relaxation by the activation of proteinase-activated receptor 2 in mouse mesenteric arterioles.

1. Activation of PAR2 in second-order mesenteric arteriole (MA) rings from C57BL/6J, NOS3 (-/-) and PAR2 (-/-) mice was assessed for the contributions of NO, cyclo-oxygenases, guanylyl cyclase, adenylyl cyclase, and of K(+) channel activation to vascular smooth muscle relaxation. 2. PAR2 agonist, SLIGRL-NH(2) (0.1 to 30 microM), induced relaxation of cirazoline-precontracted MA from C57BL/6J and NOS3 (-/-), but not PAR2 (-/-) mice. Maximal relaxation (E(max)) was partially reduced by a combination of L-(G)N-nitroarginine methyl ester (L-NAME), 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and indomethacin. An ODQ/L-NAME/indomethacin resistant relaxation was also caused by trypsin (30 nM) in PAR2 (+/+), but not in PAR2 (-/-) mice. Relaxation was endothelium-dependent and inhibited by either 30 mM KCl-precontraction, or pretreatment with apamin, charybdotoxin, and their combination; iberiotoxin did not substitute for charybdotoxin nor did scyllatoxin substitute fully for apamin. 3. Tetraethylammonium (TEA), glibenclamide, tetrodotoxin, 17-octadecynoic acid, carboxy-2-phenyl-4,4,5,5,-tetramethyl-imidazoline-1-oxyl-3-oxide, SQ22536, carbenoxolone, arachidonyl trifluoromethyl ketone, 7-nitroindazole, N-(3-(aminomethyl)benzyl)acetamidine (1400W), N-(2-cyclohexyloxy-4-nitrophenyl)-methanesulfonamide (NS-398) and propanolol did not inhibit relaxation. 4-aminopyridine significantly increased the potency of SLIGRL-NH(2). A combination of 30 microM BaCl(2) and 10 microM ouabain significantly reduced the potency for relaxation, and in the presence of L-NAME, ODQ and indomethacin, E(max) was reduced. 4. We conclude PAR2-mediated relaxation of mouse MA utilizes multiple mechanisms that are both NO-cGMP-dependent, and -independent. The data are also consistent with a role for endothelium-dependent hyperpolarization of vascular smooth muscle that involves the activation of an apamin/charybdotoxin-sensitive K(+) channel(s) and, in part, may be mediated by K(+).