Contribution Of Endothelium-derived Hyperpolarizing Factor Research Articles

P716Role of EDRFs in aortic and mesenteric arterial function of the UC Davis Type 2 Diabetic Mellitus (UCD-T2DM) male rats

Abstract Several studies have shown vascular dysfunction in diabetic rats, however, there is no data on the vascular function of UC Davis type 2 diabetes Mellitus (UCD-T2DM) rats. UCD-T2DM is a novel but validated model of type 2 diabetes (T2D) which more closely resembles the pathophysiology of diabetes in humans. The aim of this study was to determine whether aortic or mesenteric arterial function were altered in UCD-T2DM male rats. Specifically, we determined the responses to endothelium-dependent vasodilator and vasoconstrictor in this model. We also studied the relative contribution of endothelium-derived relaxing factors (EDRFs) in modulating vascular reactivity of aorta or mesenteric artery (MA) in UCD-T2DM male rats. Endothelium-dependent vasodilation (EDV) to acetylcholine (ACh) was measured in rat aorta or MA pre-contracted with phenylephrine (PE). Particularly, EDV to ACh were measured before and after pretreatment with indomethacin (indo, cyclooxygenase inhibitor), ODQ (soluble guanylate cyclase inhibitor), L-NNA (nitric oxide synthase (NOS) inhibitor), TRAM-34 (IKca blocker), or Apamin (SKca blocker). Constrictor response curves to PE were also generated in both aorta and MA. Furthermore, the protein expression of IK channels were determined using Western blotting. We demonstrated that EDV to ACh was impaired in MA, while potentiated in the aortic rings of UCD-T2DM rats. In diabetic MA, the importance of endothelium-derived hyperpolarizing factor (EDHF) in ACh-induced relaxation was decreased, while the contribution of nitric oxide (NO) was enhanced. On the other hand, in diabetic aorta, the relative contribution of EDHF in relaxation responses to ACh was significantly enhanced (as measured by ACh response which was insensitive to indo, ODQ and L-NNA). The incubation of aorta or MA with TRAM-34 blunted the relaxation responses to ACh in both control and diabetic groups. However, the inhibitory effects of TRAM 34 on ACh responses were more prominent in arteries taken from UCD-T2DM groups compared to those in controls. By contrast, ACh responses were not affected following incubation of aorta or MA with Apamin in either control or diabetic groups suggesting that SKca channels plays no role in EDHF-mediated relaxation in either vascular beds. Accordingly, the IKca expression level was significantly higher in aortic rings of diabetic group than in the controls. The responsiveness to PE was significantly enhanced in the aortic rings of diabetic group, whereas in MA, diabetes did not alter the contractile responses to PE. These data suggest that in MA of UCD-T2DM rats, the predisposition to vascular injury may be due to a shift away from EDHF, initially the major vasodilatory factor, toward a greater reliance on NO. However, in aorta, despite elevated contractile responses, ACh responses were enhanced. Increased contribution of NO-independent factors specifically IKca channel plays a role in potentiated responses to ACh in this model. Acknowledgement/Funding NIHLBI

Histamine-Induced Dilation of Isolated Porcine Retinal Arterioles: Role of Endothelium-Derived Hyperpolarizing Factor.

Although endothelium-dependent nitric oxide (NO)-mediated dilation of retinal arterioles has been well described, the role of endothelium-derived hyperpolarizing factor (EDHF) in the retinal arteriolar response remains unclear. In the current study, we examined the contribution of EDHF to the retinal arteriolar dilation to the inflammatory agent histamine and investigated the signaling mechanisms underlying this vasomotor activity. Porcine retinal arterioles were isolated, cannulated, and pressurized without flow for functional study by using video microscopic techniques. The immunohistochemical staining was performed to determine histamine receptor subtypes. Histamine (0.1-30 μM) produced concentration-dependent dilation of retinal arterioles in a manner sensitive to H1- and H2-receptor antagonists chlorpheniramine and famotidine, respectively. Histamine-induced vasodilation was almost abolished after endothelial removal. In the intact vessels, vasodilation to histamine was partially inhibited by the inhibitors of cyclooxygenase (indomethacin), NO synthase (NG-nitro-L-arginine methyl ester, L-NAME), or Ca2+ -activated K+ (KCa) channels (apamin plus charybdotoxin). Combination of the above inhibitors abolished histamine-induced vasodilation. Residual vasodilation in the presence of indomethacin and L-NAME was further reduced by the cytochrome P450 enzyme inhibitor sulfaphenazole but not by the gap junction inhibitor carbenoxolone or the hydrogen peroxide scavenger catalase. Immunohistochemical signals for H1- and H2-receptor expression were found only in the endothelium. The endothelium plays an essential role in the dilation of porcine retinal arterioles to histamine via H1- and H2-receptor activation. The EDHF derived from cytochrome P450 contributed in part to this vasodilation via KCa channel activation, in addition to the endothelial release of NO and prostanoids.

Mechanisms of endothelial dysfunction in resistance arteries from patients with end-stage renal disease.

The study focuses on the mechanisms of endothelial dysfunction in the uremic milieu. Subcutaneous resistance arteries from 35 end-stage renal disease (ESRD) patients and 28 matched controls were studied ex-vivo. Basal and receptor-dependent effects of endothelium-derived factors, expression of endothelial NO synthase (eNOS), prerequisites for myoendothelial gap junctions (MEGJ), and associations between endothelium-dependent responses and plasma levels of endothelial dysfunction markers were assessed. The contribution of endothelium-derived hyperpolarizing factor (EDHF) to endothelium-dependent relaxation was impaired in uremic arteries after stimulation with bradykinin, but not acetylcholine, reflecting the agonist-specific differences. Diminished vasodilator influences of the endothelium on basal tone and enhanced plasma levels of asymmetrical dimethyl L-arginine (ADMA) suggest impairment in NO-mediated regulation of uremic arteries. eNOS expression and contribution of MEGJs to EDHF type responses were unaltered. Plasma levels of ADMA were negatively associated with endothelium-dependent responses in uremic arteries. Preserved responses of smooth muscle to pinacidil and NO-donor indicate alterations within the endothelium and tolerance of vasodilator mechanisms to the uremic retention products at the level of smooth muscle. We conclude that both EDHF and NO pathways that control resistance artery tone are impaired in the uremic milieu. For the first time, we validate the alterations in EDHF type responses linked to kinin receptors in ESRD patients. The association between plasma ADMA concentrations and endothelial function in uremic resistance vasculature may have diagnostic and future therapeutic implications.

Upregulation of endothelium-derived hyperpolarizing factor compensates for the loss of nitric oxide in mesenteric arteries of dahl salt-sensitive hypertensive rats

This study was designed to determine whether a high-salt diet would alter endothelial function and, if so, the relative contributions of endothelium-derived hyperpolarizing factor (EDHF) and nitric oxide (NO) to any changes in vasomotor responses. Male Dahl salt-sensitive (DS) rats were given either a high-salt diet (8% NaCl, DS-H) or a low-salt diet (0.4% NaCl, DS-L) for 6 weeks. Membrane potentials and contractile responses were recorded from the isolated superior mesenteric arteries. After salt loading, DS-H developed hypertension, while DS-L remained normotensive. No difference was found in acetylcholine (ACh)-induced, endothelium-dependent relaxation between the groups. However, after treatment with indomethacin and NO synthase inhibitor, EDHF-like relaxation was significantly greater in DS-H than in DS-L. In contrast, NO-mediated relaxation was significantly smaller in DS-Hthan in DS-L. Iberiotoxin (IbTx), a specific blocker of large-conductance calcium-dependent potassium (BKCa) channels, attenuated EDHF-like relaxation in DS-H but not in DS-L. IbTx marginally inhibited EDHF-mediated hyperpolarization only in DS-H. Endothelium-independent relaxation in response to sodium nitroprusside or levcromakalim was similar in both groups. In conclusion, EDHF-like relaxation is upregulated through the activation of BKCa channels in the mesenteric arteries of DS-H. As the overall relaxation in response to ACh did not differ between the groups, the upregulation of EDHF appears to compensate for the loss of NO in the mesenteric arteries of DS-H.

Leptin and the regulation of endothelial function in physiological and pathological conditions

Obesity and the accompanying metabolic syndrome are among the most important causes of cardiovascular pathologies associated with endothelial dysfunction, such as arterial hypertension and atherosclerosis. This detrimental effect of obesity is mediated, in part, by excessive production of the adipose tissue hormone leptin. Under physiological conditions leptin induces endothelium-dependent vasorelaxation by stimulating nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF). Leptin activates endothelial NO synthase (eNOS) through a mechanism involving AMP-activated protein kinase (AMPK) and protein kinase B/Akt, which phosphorylates eNOS at Ser(1177) , increasing its activity. Under pathological conditions, such as obesity and metabolic syndrome, the NO-mediated vasodilatory effect of leptin is impaired. Resistance to the acute NO-mimetic effect of leptin is accounted for by chronic hyperleptinaemia and may result from different mechanisms, such as downregulation of leptin receptors, increased levels of circulating C-reactive protein, oxidative stress and overexpression of suppressor of cytokine signalling-3. In short-lasting obesity, impaired leptin-induced NO production is compensated by EDHF; however, in advanced metabolic syndrome, the contribution of EDHF to the haemodynamic effect of leptin becomes inefficient. Resistance to the vasodilatory effects of leptin may contribute to the development of arterial hypertension owing to unopposed stimulation of the sympathetic nervous system by this hormone.

3′,4′-Dihydroxyflavonol Reduces Superoxide and Improves Nitric Oxide Function in Diabetic Rat Mesenteric Arteries

Background3',4'-Dihydroxyflavonol (DiOHF) is an effective antioxidant that acutely preserves nitric oxide (NO) activity in the presence of elevated reactive oxygen species (ROS). We hypothesized that DiOHF treatment (7 days, 1 mg/kg per day s.c.) would improve relaxation in mesenteric arteries from diabetic rats where endothelial dysfunction is associated with elevated oxidant stress.Methodology/Principal FindingsIn mesenteric arteries from diabetic rats there was an increase in ROS, measured by L-012 and 2',7'-dichlorodihydrofluorescein diacetate fluorescence. NADPH oxidase-derived superoxide levels, assayed by lucigenin chemiluminescence, were also significantly increased in diabetic mesenteric arteries (diabetes, 4892±946 counts/mg versus normal 2486±344 counts/mg, n = 7–10, p<0.01) associated with an increase in Nox2 expression but DiOHF (2094±300 counts/mg, n = 10, p<0.001) reversed that effect. Acetylcholine (ACh)-induced relaxation of mesenteric arteries was assessed using wire myography (pEC50 = 7.94±0.13 n = 12). Diabetes significantly reduced the sensitivity to ACh and treatment with DiOHF prevented endothelial dysfunction (pEC50, diabetic 6.86±0.12 versus diabetic+DiOHF, 7.49±0.13, n = 11, p<0.01). The contribution of NO versus endothelium-derived hyperpolarizing factor (EDHF) to ACh-induced relaxation was assessed by evaluating responses in the presence of TRAM-34+apamin+iberiotoxin or N-nitro-L-arginine+ODQ respectively. Diabetes impaired the contribution of both NO (maximum relaxation, Rmax diabetic 24±7 versus normal, 68±10, n = 9–10, p<0.01) and EDHF (pEC50, diabetic 6.63±0.15 versus normal, 7.14±0.12, n = 10–11, p<0.01) to endothelium-dependent relaxation. DiOHF treatment did not significantly affect the EDHF contribution but enhanced NO-mediated relaxation (Rmax 69±6, n = 11, p<0.01). Western blotting demonstrated that diabetes also decreased expression and increased uncoupling of endothelial NO synthase (eNOS). Treatment of the diabetic rats with DiOHF significantly reduced vascular ROS and restored NO-mediated endothelium-dependent relaxation. Treatment of the diabetic rats with DiOHF also increased eNOS expression, both in total and as a dimer.Conclusions/SignificanceDiOHF improves NO activity in diabetes by reducing Nox2-dependent superoxide production and preventing eNOS uncoupling to improve endothelial function.

Endothelial Dysfunction in Diabetes

Type 2 diabetes is characterized by a two- to fourfold increased risk of cardiovascular disease. This is generally attributed to the adverse effects of hyperglycemia and oxidative stress on vascular biology. It has also been shown that patients with prediabetic conditions, such as impaired fasting glucose and impaired glucose tolerance, are at increased risk of cardiovascular disease as well (1). This result suggests that abnormalities in carbohydrate metabolism form a continuum that progressively worsens cardiovascular health; the first step of the adverse sequence of events that leads to the atherosclerotic process is thought to be endothelial dysfunction (2). Vascular endothelial cells play a major role in maintaining cardiovascular homeostasis. In addition to providing a physical barrier between the vessel wall and lumen, the endothelium secretes a number of mediators that regulate platelet aggregation, coagulation, fibrinolysis, and vascular tone. The term “endothelial dysfunction” refers to a condition in which the endothelium loses its physiological properties: the tendency to promote vasodilation, fibrinolysis, and anti-aggregation. Endothelial cells secrete several mediators that can alternatively mediate either vasoconstriction, such as endothelin-1 and thromboxane A2, or vasodilation, such as nitric oxide (NO), prostacyclin, and endothelium-derived hyperpolarizing factor. NO is the major contributor to endothelium-dependent relaxation in conduit arteries, whereas the contribution of endothelium-derived hyperpolarizing factor predominates in smaller resistance vessels. In patients with diabetes, endothelial dysfunction appears to be a consistent finding; indeed, there is general agreement that hyperglycemia and diabetes lead to an impairment of NO production and activity. The endothelium has a limited intrinsic capacity of self-repair, being built up by terminally differentiated cells with a low proliferative potential. That is why endothelial repair is accomplished through the contribution of circulating cells, namely endothelial progenitor cells (EPCs), in physiological and pathological conditions. In this review, we will outline the mechanisms of endothelial dysfunction …

New Molecular Mechanisms for Cardiovascular Disease: Contribution of Endothelium-Derived Hyperpolarizing Factor in the Regulation of Vasoconstriction in Peripheral Resistance Arteries

Endothelium regulates vascular tone via release of endothelium-derived relaxing factors (EDRF) including nitric oxide (NO), prostaglandin I₂ (PGI₂), and endothelium-derived hyperpolarizing factor (EDHF). The mesenteric vascular bed produces vascular resistance to develop blood pressure and regulate tissue blood flow that plays an important role in maintenance of systemic blood pressure. There is now strong evidence that in these small resistance arteries, EDHF plays a major role in the response to vasoactive substances and regulation of vascular tone. Pharmacological analysis to investigate the role of the vascular endothelium in the regulation of α₁-adrenoceptor agonist (methoxamine)-induced vasoconstriction in rat mesenteric vascular beds showed that vasoconstriction induced by continuous perfusion of methoxamine (7 µM), but not high KCl (60 mM), time-dependently decreased to 20% of the initial constriction. The time-dependent reduction of methoxamine-induced vasoconstriction was inhibited by endothelium removal, inhibitor of EDHF (30 mM KCl, K+-channel blockers), and gap-junction inhibitor, but not NO synthase inhibitor and cyclooxygenase inhibitor and ageing. These results suggest that vascular endothelium counteracts to normalize excess vasoconstriction of the mesenteric resistance arteries by releasing EDHF, which is associated with activation of multiple K+-channels and gap junction involvement and markedly decreases with ageing.

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

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. 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. 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 pSer⁴⁷³ Akt and a reduced proportion of endothelial NO synthase (eNOS) expressed as a dimer, indicating uncoupling. The contribution of both NO and EDHF-type relaxations was impaired in diabetes and was caused by increased oxidative stress, decreased pSer⁴⁷³ Akt and/or eNOS uncoupling.

Gender differences in the effects of streptozotocin-induced diabetes on parasympathetic vasodilatation in the rat submandibular gland

Objectives Submandibular vasodilatory responses are impaired in male streptozotocin-diabetic rats. However, the effects of diabetes on submandibular vascular reactivity in female rats have not been examined. The purpose of this study was to determine whether there are gender differences in the effects of diabetes on parasympathetic vasodilatation in the rat submandibular gland. Methods Diabetes was induced using streptozotocin, and vascular responses (calculated as the % increase in submandibular vascular conductance) to parasympathetic stimulation (1–10 Hz) were measured using laser-Doppler flowmetry. To estimate the relative contributions of nitric oxide (NO), prostacyclin (PGI 2) and endothelium-derived hyperpolarizing factor (EDHF), vascular conductance was measured before and after inhibition of cyclooxygenase (COX) and NO synthase (NOS). Results Frequency-dependent increases in blood flow were observed in both male and female rats, but the contribution of EDHF was greater in females than in males. Further, PGI 2 appeared to play a role only in males. Vasodilatory responses were diminished in all diabetic animals, and when compared with their respective controls the degree of impairment was similar in males and females. However, in diabetic males inhibition of COX and NOS had little or no effect, whereas inhibition of NO, but not COX, resulted in a further significant decrease in vascular responses in diabetic females. Conclusions Parasympathetic vasodilatation in the rat submandibular gland is diminished equally in diabetic males and females. However, in males diabetes predominantly impairs PGI 2- and NO-dependent vasodilatation, whereas in females the contribution of EDHF-mediated pathways are affected and NO-dependent vasodilatation is preferentially maintained.

056 The differential contribution of nitric oxide and endothelium-derived hyperpolarising factor to resting and stimulated vasodilator tone in healthy black and white individuals

Whether impaired endothelial function in individuals of black ethnicity compared to whites is due to alterations in nitric oxide (NO) or endothelium-derived hyperpolarizing factor (EDHF) activity is unknown. We hypothesised that there is a differential racial contribution of these two agonists to vasodilator tone. <h3>Methods</h3> In 45 white and 36 black healthy subjects, we measured forearm blood flow (FBF) using strain gauge plethysmography at rest and after infusions of bradykinin (BK, 100, 200, 400 ng/min), acetylcholine (ACH 7.5, 15, 30 μg/min) and sodium nitroprusside (1.6 and 3.2 μg/min). Measurements were repeated after NO blockade with L-NMMA, calcium-dependent potassium channel blockade with tetraethylammonium (TEA, inhibiting EDHF activity), and combined blockade. <h3>Results</h3> Both L-NMMA and TEA reduced resting FBF in both groups indicating similar contribution of NO and EDHF to resting tone; whites vs blacks with L-NMMA: −22% and −18%, p=0.01; with TEA: −23% and −24%, p=0.001; with L-NMMA+TEA: −42% and −35%, p=0.001, respectively, compared to baseline. BK-mediated vasodilation was greater in whites than blacks (p=0.04). L-NMMA attenuated BK responses in whites (−30%, p=0.0005) but not in blacks (−18%, p=0.11), indicating a greater contribution of NO in whites. TEA reduced blood flow (23% and 22% in whites and blacks, respectively, p&lt;0.001), indicating similar contribution of EDHF in both groups. ACH-mediated vasodilation was greater in whites than blacks (p=0.001). TEA did not change flow in either group, but L-NMMA attenuated ACH-mediated dilation only in whites and not in blacks (−18%, p&lt;0.01 vs −2%, p=NS), indicating a reduced NO in blacks. Sodium nitroprusside-mediated dilation was greater in whites than blacks (p=0.006), indicating reduced sensitivity of the smooth muscle cells to NO in blacks. <h3>Conclusions</h3> Although NO and EDHF contribute equally to resting blood flow, there is reduced availability and sensitivity to NO during pharmacologic stimulation even in healthy blacks compared to whites. In contrast, EDHF activity is preserved in blacks. Preserved EDHF but reduced NO activity in blacks may underlie the increased risk of hypertension and cardiovascular disease in black individuals, and may have therapeutic implications that need to be explored.

057 The role of cytochrome p450 metabolites as an endothelium-derived hyperpolarising factor in the regulation of vascular tone in vivo in the forearm vasculature of healthy and diabetic individuals

Background Epoxyeicosatrienoic acids are arachidonic-acid-derived products of cytochrome P450 (CYP450) epoxygenases. Endothelium-derived hyperpolarizing factor (EDHF) stimulates calcium dependent potassium channels by CYP450 metabolites that can be inhibited with fluconazole (FLU). The role of EDHF in impaired endothelial function remains undefined. We hypothesised a differential contribution of EDHF in diabetics (DM) compared to healthy subjects. Methods In 16 healthy and 14 DM subjects, we measured forearm blood flow (FBF) with venous occlusion strain gauge plethysmography at rest and after endothelium-dependent and -independent vasodilation with acetylcholine (7.5, 15, 30 μg/min) or bradykinin (100, 200, 400 ng/min) and sodium nitroprusside (1.6 and 3.2 μg/min), respectively. Measurements were repeated after NO blockade with LNG monomethyl arginine (L-NMMA, 8 μmol/min), CYP450 blockade with fluconazole (0.4–1.6 μmol/min) and combined NO and CYP450 blockade. Results L-NMMA reduced resting FBF by −1.75 mls/min (p Conclusions NO is the major contributor to resting vasodilator tone in healthy subjects, whereas both NO and hyperpolarisation via CYP450 metabolites contribute to resting tone in DM. However, in the absence of NO in healthy subjects CYP450 metabolites were observed to have an important contribution suggesting an upregulation of EDHF activity via CYP450 metabolites when there is a decreased NO bioavailability. CYP450 metabolites do not contribute to pharmacologically stimulated vasodilation in either DM or healthy subjects, but whether other EDHFs play a role needs to be studied.

Endothelium‐derived hyperpolarizing factor as an in vivo back‐up mechanism in the cutaneous microcirculation in old mice

There is now strong evidence that an endothelium-derived hyperpolarizing factor (EDHF), other than nitric oxide (NO) or prostaglandin (PG), exists for dilating arteries and arterioles. In vitro studies on isolated vessels pointed out a role for EDHF as a back-up mechanism when the NO pathway is impaired, but there was a lack of in vivo studies showing a functional role for EDHF. Ageing has pronounced effects on vascular function and particularly on endothelium-dependent relaxation, providing a novel situation in which to assess the contributions of EDHF. The purpose of the present study was thus to determine if, in vivo, there was a functional role for EDHF as a back-up mechanism in the cutaneous microcirculation in the ageing process. We investigated in vivo the contribution of each endothelial factor (NO, PG and EDHF) in the cutaneous vasodilatation induced by iontophoretic delivery of acetylcholine and local pressure application in young adult (6-7 months) and old (22-25 months) mice, using pharmacological inhibitors. The cutaneous vasodilator responses induced by acetylcholine and local pressure application were dependent upon NO and PG pathways in young adult mice, whereas they were EDHF-dependent in old mice. EDHF appears to serve as a back-up mechanism when ageing reaches pathological states in terms of the ability for NO and PG to relax cutaneous microvessels, allowing for persistent cutaneous vasodilatator responses in old mice. However, as a back-up mechanism, EDHF did not completely restore cutaneous vasodilatation, since endothelial responses were reduced in old mice compared to young adult mice.

In vivo measurement of flow-mediated vasodilation in living rats using high-resolution ultrasound

In humans, endothelial vasodilator function serves as a surrogate marker for cardiovascular health and is measured as changes in conduit artery diameter after temporary ischemia [flow-mediated dilation (FMD)]. Here we present an FMD-related approach to study femoral artery (FA) vasodilation in anesthetized rats. Diameter and Doppler flow were monitored in the FA. Using high-resolution ultrasound (35 MHz) and automated analysis software, we detected dose-dependent vasodilation using established endothelium-independent [intravenous nitroglycerin EC(50) = 3.3 x 10(-6) mol/l, peak 21Delta% (SD 4)] and endothelium-dependent [intra-arterial acetylcholine EC(50) = 1.3 x 10(-6) mol/l, peak 27Delta% (SD 4)] pharmacological vasodilators. Wall shear stress induced by intra-aortic injection of adenosine and infusion of saline at increasing rates (1.5-4.5 ml/min) led to vasodilation at 1 to 2 min. Transient hindlimb ischemia by common iliac occlusion (5 min) led to reactive hyperemia with flow velocity and wall shear stress increase and was followed by FA dilation [16Delta% (SD 2)], the latter of which was completely abolished by nitric oxide synthase (NOS) inhibition with N(G)-monomethyl-L-arginine [1Delta% (SD 2)]. FMD was significantly reduced in adult 20-24-wk-old animals compared with 9- to 10-wk-old animals, consistent with age-dependent endothelial dysfunction [16Delta% (SD 3) vs. 10Delta% (SD 3), P < 0.05]. Whereas FMD was completely NOS dependent in 9- to 10-wk-old animals, NOS-dependent mechanisms accounted for only half of the FMD in 20-24-wk-old animals, with the remainder being blocked by charybdotoxin and apamin, suggesting a contribution of endothelium-derived hyperpolarizing factor. To our knowledge, this is the first integrative physiological model to reproducibly study FMD of conduit arteries in living rats.

Endothelium-derived hyperpolarizing factor in preeclampsia: heterogeneous contribution, mechanisms, and morphological prerequisites

We hypothesized that in preeclampsia (PE), contribution of endothelium-derived hyperpolarizing factor (EDHF) and the mechanism/s of its action differ from that in normal pregnancy (NP). We aimed to assess endothelial function and morphology in arteries from NP and PE with particular focus on EDHF. Arteries ( approximately 200 mum) were dissected from subcutaneous fat biopsies obtained from women undergoing cesarean section. With the use of wire myography, responses to the endothelium-dependent agonist bradykinin (BK) were determined before and after inhibition of pathways relevant to EDHF activity. The overall responses to BK in arteries from PE (n = 13) and NP (n = 17) were similar. However, in PE, EDHF-mediated relaxation was reduced (P < 0.05). All women within the PE group were divided into two subgroups: with more (group 1) or less (group 2) than 50% reduction of EDHF-typed responses after 18-alpha-glycyrrhetinic acid (an inhibitor of myoendothelial gap junctions, MEGJs). The division showed that 1) MEGJs are principally involved when the EDHF contribution is reduced; and 2) when the EDHF contribution is similar to that in NP, the H(2)O(2) and/or cytochrome P-450 epoxygenase products of arachidonic acid (AA), along with MEGJs, confer EDHF-mediated relaxation. In contrast, MEGJs were the main pathway for EDHF in NP. The abundant presence of MEGJs in arteries from NP but deficiency of them in PE was observed using transmission electron microscopy. We conclude that PE is associated with heterogeneous contribution of EDHF, and the mechanism behind EDHF-typed responses is mediated either by MEGJs alone or in combination with H(2)O(2) or cytochrome P-450 epoxygenase metabolites of AA.

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.

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.

Implications for Endothelium-Derived Hyperpolarizing Factor (EDHF) in Womens Cardiovascular Health

Several clinical conditions related to women's cardiovascular health strongly correlates with endothelial dysfunction, which is conventionall y associated with alterations in synthesis, release or bioavailabili ty of endothelium- derived nitric oxide (NO). Current review pays attention to rapidly growing evidence about the importance of Endothelium-Derived Hyperpolarizing Factor (EDHF). EDHF-mediated action is pertinent to resistance circulation where EDHF overcomes NO contribution, while in large conduit vessels endothelium-dependent dilatation is predominantly conferred by NO. This indicates that changes in synthesis, release, or pharmacological manipulation of EDHF is of critical importance in the maintenance of organ perfusion, peripheral resistance and blood pressure, the disturbances in which distinctively predispose the development of cardiovascula r disorders. This review describes current knowledge about EDHF, including nature and characterization of its action, alterations in the mechanisms of EDHF contribution to endothelium-dependent relaxation with particular focus on preeclampsia, gender differences and cardiovascular complications after menopause. The distinction in the relative contribution of NO versus EDHF and estrogen-related regulation of EDHF-mediated responses are highlighted. The indications that EDHF-mediated response accounts for different chemical mediator or electrical transmission depending on species, vascular bed and healthy or diseased condition are discussed paying attention to women's cardiovascular health and future therapeutic implications.

Effects of insulin on the acetylcholine-induced hyperpolarization in the guinea pig mesenteric arterioles

Background: Insulin induces endothelium-dependent vasodilatation, which may be casually related to the insulin resistance and hypertension. Endothelium-derived nitric oxide (NO) is the most important mechanism of insulin-induced vasodilatation, and a possible contribution of endothelium-derived hyperpolarizing factor (EDHF) is also considered. Attempts were made to observe the effects of insulin on acetylcholine (ACh)-induced hyperpolarization in the submucosal arteriole of the guinea pig ileum, the objective being to investigate possible involvement of EDHF in the actions of insulin. Methods: Conventional microelectrode techniques were applied to measure the membrane potential of smooth muscle cells in the submucosal arteriole. EDHF-induced hyperpolarization was elicited by ACh in the presence of both N ω-nitro- l-arginine ( l-NNA) (100 μM) and diclofenac (1 μM). Results: The resting membrane potential was −70.9 mV, and Ba 2+ (0.5 mM) depolarized the membrane to −33.0 mV. Insulin (10 μU/ml to 100 mU/ml) did not change the membrane potential in the absence or presence of Ba 2+. In the presence of Ba 2+, ACh (3 μM) hyperpolarized the membrane with two components, an initial large hyperpolarization followed by a slow and small one. Low concentration of insulin (100 μU/ml) did not alter the ACh-induced hyperpolarization. High concentration of insulin (100 mU/ml) shortened the time required to reach the peak amplitude and tended to increase the peak amplitude of the ACh-induced hyperpolarization. Conclusions: The data show that insulin enhances the ACh-induced hyperpolarization in the submucosal arterioles of the guinea pig ileum. The results suggested that EDHF also accounts for one of the endothelial factors involved in the insulin-induced vasodilatation.

Role of endothelium-derived hyperpolarizing factor in phenylephrine-induced oscillatory vasomotion in rat small mesenteric artery.

In small mesenteric arteries, endothelium-derived hyperpolarizing factor (EDHF) in addition to endothelium-derived relaxing factors (EDRFs) including NO plays an important role in acetylcholine-induced vasodilation. It has been reported that EDRFs play an important role in alpha(1)-adrenoceptor agonist-induced oscillatory vasomotion and in limiting vasoconstrictor response to the agonists; however, contribution of EDHF to the alpha(1)-agonist-induced oscillation is unknown. Rat small mesenteric arteries were isolated and cannulated at each end with a glass micropipette. The vessels were immersed in a bath (37 degrees C) containing physiologic saline solution. Changes in vessel diameter were measured using an optical density video detection system. Denudation of the endothelium and inhibition of NO synthesis caused a leftward shift in the concentration-response relation for phenylephrine in the mesenteric arteries, whereas inhibition of cyclooxygenase by indomethacin had no effect. Blockade of Ca2+-activated K+ (K(Ca)) channels by charybdotoxin and apamin caused a further leftward shift in the concentration-response relation in the vessels pretreated with Nomega-nitro-l-arginine methylester and indomethacin. Phenylephrine at concentrations higher than 10(-6) m caused endothelium-dependent oscillatory vasomotion, which was reduced but not abolished after combined inhibition of the cyclooxygenase and NO synthase pathways. However, the K(Ca) channel blockers completely abolished the remaining component of oscillation. Endothelially-derived NO is an important modulator of sustained agonist-induced vasoconstriction. NO, as well as endothelially-derived cyclooxygenase products and EDHF, also contribute significantly to phenylephrine-induced oscillatory vasomotion.