EDHF-mediated Responses Research Articles

Combination Therapy With Olmesartan and Azelnidipine Improves EDHF-Mediated Responses in Diabetic Apolipoprotein E-Deficient Mice

The endothelium modulates vascular tone by synthesizing and releasing several vasodilating factors, including vasodilator prostaglandins, nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF). In the present study, we examined whether an angiotensin-receptor blocker, a calcium-channel blocker or their combination improved EDHF-mediated responses in diabetic apolipoprotein E-deficient (ApoE(-/-)) mice. We used male C57BL/6N (control) and streptozocin-induced diabetic ApoE(-/-) mice. The diabetic ApoE(-/-) mice were administered oral vehicle (untreated), olmesartan (OLM, 30 mgxkg(-1)xday(-1)), azelnidipine (AZL, 10 mgxkg(-1)xday(-1)), their combination (OLM + AZL), or hydralazine (HYD 5 mgxkg(-1)xday(-1)) for 5 weeks. In the untreated group, systolic blood pressure was significantly higher and both EDHF-mediated relaxation and endothelium-dependent hyperpolarization were markedly reduced as compared with the control group. Although EDHF-mediated relaxation was not significantly improved in the HYD, OLM and AZL groups, it was significantly improved in the OLM + AZL group, as was also the case with phosphorylation of Akt and endothelial NO synthase (eNOS). In contrast, the endothelium-independent relaxation response to sodium nitroprusside or NS-1619 (a direct opener of K(Ca) channels) was unaltered in any group. OLM + AZL may improve the severely impaired EDHF-mediated responses in diabetic ApoE(-/-) mice, in which activation of the endothelial Akt - eNOS pathway may be involved.

EDHF: an update

The endothelium controls vascular tone not only by releasing NO and prostacyclin, but also by other pathways causing hyperpolarization of the underlying smooth muscle cells. This characteristic was at the origin of the term 'endothelium-derived hyperpolarizing factor' (EDHF). However, this acronym includes different mechanisms. Arachidonic acid metabolites derived from the cyclo-oxygenases, lipoxygenases and cytochrome P450 pathways, H(2)O(2), CO, H(2)S and various peptides can be released by endothelial cells. These factors activate different families of K(+) channels and hyperpolarization of the vascular smooth muscle cells contribute to the mechanisms leading to their relaxation. Additionally, another pathway associated with the hyperpolarization of both endothelial and vascular smooth muscle cells contributes also to endothelium-dependent relaxations (EDHF-mediated responses). These responses involve an increase in the intracellular Ca(2+) concentration of the endothelial cells, followed by the opening of SK(Ca) and IK(Ca) channels (small and intermediate conductance Ca(2+)-activated K(+) channels respectively). These channels have a distinct subcellular distribution: SK(Ca) are widely distributed over the plasma membrane, whereas IK(Ca) are preferentially expressed in the endothelial projections toward the smooth muscle cells. Following SK(Ca) activation, smooth muscle hyperpolarization is preferentially evoked by electrical coupling through myoendothelial gap junctions, whereas, following IK(Ca) activation, K(+) efflux can activate smooth muscle Kir2.1 and/or Na(+)/K(+)-ATPase. EDHF-mediated responses are altered by aging and various pathologies. Therapeutic interventions can restore these responses, suggesting that the improvement in the EDHF pathway contributes to their beneficial effect. A better characterization of EDHF-mediated responses should allow the determination of whether or not new drugable targets can be identified for the treatment of cardiovascular diseases.

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.

NON-VOIDING CONTRACTIONS CAUSED BY BLADDER OUTLET OBSTRUCTION MAY BE ASSOCIATED WITH UPREGULATION OF CYCLOOXYGENASE-2 IN THE RAT URETHRA

Endothelial dysfunction is considered as a major risk factor of cardiovascular complications of type I and type II diabetes. Our previous studies have demonstrated that endothelial dysfunction in the small mesenteric arteries from 12–16 week old type II diabetic mice was associated with decreased bio-availability of nitric oxide whereas endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation was preserved. The objective of the present study was to characterize EDHF-mediated relaxations of small mesenteric arteries from db/db mice. A depolarizing concentration of KCl or tetraethylammonium (TEA, 10 mM) significantly inhibited the EDHF-mediated relaxation to acetylcholine and bradykinin in small mesenteric arteries from both db/+ and db/db mice. Charybdotoxin or iberiotoxin alone and a combination of ouabain and barium significantly reduced the maximal relaxation to acetylcholine in small mesenteric arteries from db/db mice and charybdotoxin or iberiotoxin either alone or in combination with apamin reduced the sensitivity to the EDHF-mediated component of the relaxation response to bradykinin. 17-octadecynoic acid, but not catalase, significantly reduced the sensitivity to EDHF-mediated responses to bradykinin in db/db mice; 17-octadecynoic acid had no effect on acetylcholine-mediated relaxations. No differences were, however, detected for mRNA expression levels of calcium-activated potassium channels or connexins 37, 40, 43 and 45. Collectively, these data suggest that bradykinin-induced, EDHF-dependent relaxation in small mesenteric arteries from db/db mice is mediated via cytochrome P450 product that activates the large conductance calcium-activated potassium (BKCa or Slo) channel, whereas the acetylcholine-induced, EDHF-mediated relaxation involves neither cytochrome P450 product nor hydrogen peroxide.

C014 Endothelial dysfunction in isolated perfused kidneys of L-NAME-treated spontaneous hypertensive rats

In hypertensive humans, the development of end-stage renal disease is often associated with an endothelial dysfunction attributed to a decrease in the bioavailability of nitric oxide (NO). An early endothelial dysfunction is also reported in most animal models of hypertension and profound alterations in renal function are observed in aged hypertensive animals. As kidneys play an important role in blood pressure regulation, the purpose of the present study was to determine whether a chronic treatment with a NO synthase inhibitor, L-NAME (Nω- nitro-L-arginine methyl ester) accelerates the renal dysfunction in younger adult spontaneously hypertensive rats (SHR). Indeed, in this model, the alterations in cardiac function mimic those observed in aging SHR. Male SHR were subjected to chronic L-NAME-treatment (6 mg/ kg/day) for two weeks. At 19 weeks, the kidneys were isolated, perfused with oxygenated physiological salt solution and changes in renal perfusion pressure continuously recorded. During a sustained constriction caused by methoxamine, bolus injections of acetylcholine and of the NO donor sodium nitroprusside caused dose-dependent dilatations in both groups of SHR. The responses to acetylcholine were not affected by the L-NAME treatment while those to sodium nitroprusside were significantly augmented. In the two groups, indomethacin, a cyclooxygenase inhibitor, did not affect the vasodilatation caused by acetylcholine. However, an acute treatment with another NO synthase inhibitor, L-nitroarginine, produced a major inhibition of the acetylcholine response in the SHR while it did not affect it in the kidney of SHR chronically treated with L-NAME. In the presence of indomethacin plus L-nitro-arginine, TRAM-34, an inhibitor of Ca2+-activated K+ channels of intermediate conductance, IK(Ca), blocked the responses to acetylcholine in both groups of SHR. These data show that in SHR, the acetylcholine-induced vasodilatation involves both NO release and an endothelium-derived hyperpolarizing factor (EDHF)-mediated component while in the L-NAME-treated SHR, the NO-component is abolished and the EDHF-mediated component becomes the major contributor to this vasodilatation. In the kidney of L-NAME-treated SHR, EDHF-mediated responses are a compensatory mechanism that sustain endothelium-dependent dilatations when NO activity is impaired. The responses involve IK(Ca), activation and must be further characterized.

Calcium‐activated potassium channels and endothelial dysfunction: therapeutic options?

The three subtypes of calcium-activated potassium channels (K(Ca)) of large, intermediate and small conductance (BK(Ca), IK(Ca) and SK(Ca)) are present in the vascular wall. In healthy arteries, BK(Ca) channels are preferentially expressed in vascular smooth muscle cells, while IK(Ca) and SK(Ca) are preferentially located in endothelial cells. The activation of endothelial IK(Ca) and SK(Ca) contributes to nitric oxide (NO) generation and is required to elicit endothelium-dependent hyperpolarizations. In the latter responses, the hyperpolarization of the smooth muscle cells is evoked either via electrical coupling through myo-endothelial gap junctions or by potassium ions, which by accumulating in the intercellular space activate the inwardly rectifying potassium channel Kir2.1 and/or the Na(+)/K(+)-ATPase. Additionally, endothelium-derived factors such as cytochrome P450-derived epoxyeicosatrienoic acids and under some circumstances NO, prostacyclin, lipoxygenase products and hydrogen peroxide (H(2)O(2)) hyperpolarize and relax the underlying smooth muscle cells by activating BK(Ca). In contrast, cytochrome P450-derived 20-hydroxyeicosatetraenoic acid and various endothelium-derived contracting factors inhibit BK(Ca). Aging and cardiovascular diseases are associated with endothelial dysfunctions that can involve a decrease in NO bioavailability, alterations of EDHF-mediated responses and/or enhanced production of endothelium-derived contracting factors. Because potassium channels are involved in these endothelium-dependent responses, activation of endothelial and/or smooth muscle K(Ca) could prevent the occurrence of endothelial dysfunction. Therefore, direct activators of these potassium channels or compounds that regulate their activity or their expression may be of some therapeutic interest. Conversely, blockers of IK(Ca) may prevent restenosis and that of BK(Ca) channels sepsis-dependent hypotension.

Roles of Endothelial Oxidases in Endothelium-derived Hyperpolarizing Factor Responses in Mice

The endothelium synthesizes and releases several vasodilator substances, including prostacyclin, nitric oxide (NO), and endothelium-derived hyperpolarizing factor (EDHF). We have demonstrated that endothelium-derived hydrogen peroxide (H2O2) is an EDHF in animals and humans and that superoxide anions derived from endothelial nitric oxide synthases (NOSs) system are an important precursor for EDHF/H2O2 in mice. There are several intracellular sources of superoxide anions other than NOSs, including NAD(P)H oxidase, xanthine oxidase, lipoxygenase, and mitochondrial electron transport chain. In this study, we examined the possible role of endothelial oxidases other than NOSs in the EDHF-mediated responses. In angiotensin II-infused mice, both EDHF-mediated relaxations and hyperpolarizations to acetylcholine were significantly reduced, nitric oxide-mediated relaxations were rather enhanced, and vascular smooth muscle responses were preserved. Antihypertensive treatment normalized blood pressure but failed to improve EDHF-mediated responses in those mice. Acute inhibition of endothelial oxidases other than NOSs, including NAD(P)H oxidase, xanthine oxidase, lipoxygenase, or mitochondrial electron transport chain, had no inhibitory effects on EDHF-mediated responses. Furthermore, in p47phox-knockout mice, EDHF-mediated responses were unaltered. These results suggest that endothelial oxidases other than NOSs are not involved in EDHF/H2O2 responses in mice, suggesting a specific link between endothelial NOSs system and EDHF responses under physiological conditions.

Angiotensin II-Induced Hypertension Is Associated with a Selective Inhibition of Endothelium-Derived Hyperpolarizing Factor-Mediated Responses in the Rat Mesenteric Artery

Hypertension has been shown to be associated with impaired endothelium-derived hyperpolarizing factor (EDHF)-mediated arterial relaxation and hyperpolarization. Treatments of hypertensive rats with inhibitors of the renin-angiotensin system have been shown to restore both EDHF-mediated responses and the expression of connexins involved in the intercellular transfer of the hyperpolarization in mesenteric arteries. The present study was designed to determine whether chronic treatment of rats with angiotensin II impairs EDHF-mediated responses and the expression of connexins in the mesenteric arterial wall. Male Wistar rats were treated with angiotensin II (0.4 mg/kg/day) for 21 days using osmotic minipumps. Arterial pressure was measured by tail-cuff plethysmography. Contractile responses and membrane potential were measured in isolated mesenteric arteries. The expression of the three connexins (Cxs), Cx37, Cx40, and Cx43, was quantified in segments of mesenteric arteries by immunohistochemistry and quantitative real-time reverse transcriptase-polymerase chain reaction. Angiotensin II administration increased the mean systolic blood pressure. EDHF-mediated relaxation and hyperpolarization to acetylcholine and red wine polyphenols were significantly impaired in mesenteric arteries from angiotensin II-treated rats in comparison with control animals, whereas nitric oxide-mediated relaxation was unaltered. The expression of connexins Cx37, Cx40, and Cx43 was significantly decreased in the mesenteric artery from angiotensin II-treated rats. These findings indicate that angiotensin II-induced hypertension is associated with a selective impairment of EDHF-mediated relaxation and hyperpolarization in the rat mesenteric artery. The inhibition of EDHF-mediated responses is due, at least in part, to a decreased expression of connexins Cx37, Cx40, and Cx43 in the arterial wall.

Abstract 3629: Crucial Role of Nitric Oxide Synthases System in Endothelium-Dependent Hyperpolarization in Mice

We have previously demonstrated that endothelium-derived hydrogen peroxide (H 2 O 2 ) is an endothelium-derived hyperpolarizing factor (EDHF) in mouse and human mesenteric arteries and porcine coronary microvessels. We also have demonstrated that endothelial NO synthase (eNOS) is a major source of EDHF/H 2 O 2 , where Cu,Zn-superoxide dismutase (SOD) plays an important role to dismutate eNOS-derived superoxide anions to EDHF/H 2 O 2 in animals and humans. However, the mechanism for the endothelial production of H 2 O 2 as an endogenous EDHF remains to be elucidated. Indeed, some EDHF-mediated responses still remain in singly eNOS −/− mice and the remaining responses are also sensitive to catalase that dismutates H 2 O 2 to form water and oxygen. It is widely known that 3 NOS isoforms (neuronal, inducible, and endothelial) compensate each other. In this study, we examined the effects of genetic disruption of all NOS isoforms (n/i/eNOS −/− ) on EDHF responses in mice. We examined the contribution of the whole NOS system to EDHF-mediated responses in eNOS −/− , n/eNOS −/− and n/i/eNOS −/− mice that we have recently generated. Isometric tensions and membrane potentials were recorded by organ chamber experiments and microelectrode technique, respectively. EDHF-mediated relaxations and hyperporalizations in response to acetylcholine of mesenteric arteries were progressively reduced as the number of disrupted NOS genes increased (n=6 each), whereas vascular smooth muscle functions were preserved (n=6 each). Expressions of endothelial NOS isoforms in the NOSs −/− mice were compensated by NOS gene that had not been disrupted (n=5 each). Laser confocal microscopic examination demonstrated that endothelial H 2 O 2 and superoxide production was absent in n/i/eNOS −/− mice (n=3–5), whereas antihypertensive treatment with hydralazine failed to improve the EDHF-mediated responses (n= 4). Involvement of NOS uncoupling was ruled out as modulation of BH 4 synthesis had no effects (n=6–7) and BH 4 /BH 2 ratio (an index of BH 4 bioavailability) was preserved (n=4). These results provide the first direct evidence that EDHF-mediated responses are totally dependent on endothelial NOSs system in mouse mesenteric arteries.

Endothelium-derived hyperpolarizing factor (EDHF) mediates endothelium-dependent vasodilator effects of aqueous extracts from Eucommia ulmoides Oliv. leaves in rat mesenteric resistance arteries.

The vascular effects of an aqueous extract prepared from the leaves of Eucommia ulmoides Oliv. (ELE), a medicinal herb commonly used in antihypertensive herbal prescriptions in China, were investigated in rat mesenteric resistance arteries. The mesenteric vascular bed was perfused with Krebs solution and the perfusion pressure was measured with a pressure transducer. In preparations with an intact endothelium and precontracted with 7 microM methoxamine, perfusion of ELE (107102 mg/ml for 15 min) caused a concentration-dependent vasodilatation, which was abolished by chemical removal of the endothelium. The ELE-induced vasodilatation was inhibited by neither indomethacin (INDO, a cyclooxygenase inhibitor) nor NG-nitro-L-arginine-methyl ester (L-NAME, a nitric oxide inhibitor). The ELE-induced vasodilatation was significantly inhibited by tetraethylammonium (TEA, a K channel blocker) and 18alpha-glycyrrhetinic acid (18alpha-GA, a gap-junction inhibitor), and abolished by high K-containing Krebs' solution. Atropine (a muscarinic acetylcholine receptor antagonist) significantly inhibited the vasodilatation induced by ELE at high concentrations. These results suggest that the ELE-induced vasodilatation is endothelium-dependent but nitric oxide (NO)- and prostaglandin I2 (PGI2)-independent, and is mainly mediated by the endothelium-derived hyperpolarizing factor (EDHF) in the mesenteric resistance arteries. Furthermore, the ELE-induced EDHF-mediated response involves the activation of K-channels and gap junctions.

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

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

Crucial role of nitric oxide synthases system in endothelium-dependent hyperpolarization in mice

The endothelium plays an important role in maintaining vascular homeostasis by synthesizing and releasing several relaxing factors, such as prostacyclin, nitric oxide (NO), and endothelium-derived hyperpolarizing factor (EDHF). We have previously demonstrated in animals and humans that endothelium-derived hydrogen peroxide (H2O2) is an EDHF that is produced in part by endothelial NO synthase (eNOS). In this study, we show that genetic disruption of all three NOS isoforms (neuronal [nNOS], inducible [iNOS], and endothelial [eNOS]) abolishes EDHF responses in mice. The contribution of the NOS system to EDHF-mediated responses was examined in eNOS−/−, n/eNOS−/−, and n/i/eNOS−/− mice. EDHF-mediated relaxation and hyperpolarization in response to acetylcholine of mesenteric arteries were progressively reduced as the number of disrupted NOS genes increased, whereas vascular smooth muscle function was preserved. Loss of eNOS expression alone was compensated for by other NOS genes, and endothelial cell production of H2O2 and EDHF-mediated responses were completely absent in n/i/eNOS−/− mice, even after antihypertensive treatment with hydralazine. NOS uncoupling was not involved, as modulation of tetrahydrobiopterin (BH4) synthesis had no effect on EDHF-mediated relaxation, and the BH4/dihydrobiopterin (BH2) ratio was comparable in mesenteric arteries and the aorta. These results provide the first evidence that EDHF-mediated responses are dependent on the NOSs system in mouse mesenteric arteries.

Role of cytochrome P450-dependent transient receptor potential V4 activation in flow-induced vasodilatation

Fluid shear stress elicits endothelium-dependent vasodilatation via nitric oxide and prostacyclin-dependent and -independent mechanisms. The latter includes the opening of Ca(2+)-operated potassium channels by cytochrome P450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) leading to endothelial hyperpolarization. We previously reported that EETs activate the transient receptor potential (TRP) V4 channel in vascular endothelial cells and that Ca(2+) influx in these cells in response to mechanical stimuli is dependent on the activation of CYP epoxygenases. We therefore hypothesized that the TRPV4 channel is involved in the flow-induced vasodilatation attributed to the endothelium-derived hyperpolarizing factor (EDHF). In the presence of N(omega)-nitro-l-arginine methyl ester and diclofenac, precontracted mouse carotid arteries displayed a considerable vasodilatation in response to step-wise increases in luminal flow. The EDHF-mediated, flow-induced vasodilatation could be inhibited by the epoxygenase inhibitor MS-PPOH, was abolished after down-regulation of CYP epoxygenases in tissue culture, and could be restored by viral expression of CYP2C9 in the endothelium. The TRPV4-channel inhibitor ruthenium red (RuR) inhibited the EDHF-mediated flow response, but the combination of MS-PPOH and RuR had no further effect. RuR also inhibited the response in CYP2C9-overexpressing vessels. Moreover, TRPV4-deficient mice demonstrated a blunted EDHF-mediated response to increases in luminal flow in comparison to their wild-type littermates, and the addition of MS-PPOH was without effect in these mice (up to 38 +/- 3% in TRPV4(-/-) vs. 57 +/- 6% in TRPV4(+/+), P < 0.01). In cultured human endothelial cells, exposure to fluid shear stress induced the translocation of the TRPV4 channel from a perinuclear localization to the cell membrane. We conclude that the TRPV4 channel is involved in flow-induced, endothelium-dependent vasodilatation of murine carotid arteries. Moreover, the activation of the TRPV4 channel by flow requires an active CYP epoxygenase and the translocation of the channel to the cell membrane.

The Potential of Soluble Epoxide Hydrolase Inhibition in the Treatment of Cardiac Hypertrophy

The Potential of Soluble Epoxide Hydrolase Inhibition in the Treatment of Cardiac Hypertrophy

The EDHF Story

See related article, pages 1247–1255 The dilator function of the vascular endothelium is critical for optimal tissue perfusion, and this is brought into stark reality in a number of diseases, such as hypertension, diabetes, and preeclampsia, in which endothelium-dependent vasodilator function is impaired. Prostacyclin and nitric oxide (NO) were the earliest identified endothelium-dependent vasodilators, but it was soon found that in many vascular beds, particularly in resistance vessels, vasorelaxation persisted when production of these autacoids was suppressed. This residual vasorelaxation was invariably associated with hyperpolarization of the vascular smooth muscle and the entity was dubbed “endothelium-derived hyperpolarizing factor” (EDHF).1 From the outset, the identity of EDHF has been the subject of vigorous debate. Nonetheless, that the “EDHF effect” is blocked by agents that target calcium-activated K+ channels is among the few aspects of EDHF on which there is apparent consensus. There was also the debate on the significance, or otherwise, of EDHF. Is it just the poor cousin of NO, stepping into the breech when the latter is compromised, especially under conditions of enhanced oxidative stress? Then a beacon of light shone out from Japan, with the demonstration that EDHF assumed greater importance as vessel diameter decreased.2 This was a key observation, supporting the importance of EDHF in the region of the vascular system where resistance is primarily determined. This was hotly followed by the revelation that EDHF may be targeted in diseases, diabetes,3–5 hypertension,6 and apolipoprotein E-deficient mice.7 Thus, EDHF, suggested to provide vasodilation when NO was compromised, was also under attack in disease. Despite its Cinderella status, investigation into the nature of EDHF has continued with breakneck intensity, with the conviction that …

C‐type natriuretic peptide and endothelium‐dependent hyperpolarization in the guinea‐pig carotid artery

C-type natriuretic peptide (CNP) has been proposed to make a fundamental contribution in arterial endothelium-dependent hyperpolarization to acetylcholine. The present study was designed to address this hypothesis in the guinea-pig carotid artery. The membrane potential of vascular smooth muscle cells was recorded in isolated arteries with intracellular microelectrodes. Acetylcholine induced endothelium-dependent hyperpolarizations in the presence or absence of N (G)-nitro-L-arginine, indomethacin and/or thiorphan, inhibitors of NO-synthases, cyclooxygenases or neutral endopeptidase, respectively. Acetycholine hyperpolarized smooth muscle cells in resting arteries and produced repolarizations in phenylephrine-stimulated arteries. CNP produced hyperpolarizations with variable amplitude. They were observed only in the presence of inhibitors of NO-synthases and cyclooxygenases and were endothelium-independent, maintained in phenylephrine-depolarized carotid arteries, and not affected by the additional presence of thiorphan. In arteries with endothelium, the hyperpolarizations produced by CNP were always significantly smaller than those induced by acetylcholine. Upon repeated administration, a significant tachyphylaxis of the hyperpolarizing effect of CNP was observed, while consecutive administration of acetycholine produced sustained responses. The hyperpolarizations evoked by acetylcholine were abolished by the combination of apamin plus charybdotoxin, but unaffected by glibenclamide or tertiapin. In contrast, CNP-induced hyperpolarizations were abolished by glibenclamide and unaffected by the combination of apamin plus charybdotoxin. In the isolated carotid artery of the guinea-pig, CNP activates K(ATP) and is a weak hyperpolarizing agent. In this artery, the contribution of CNP to EDHF-mediated responses is unlikely.

Endothelin and Aged Blood Vessels

Age is one of the most important risk factors for cardiovascular disease. For example, a 30-year-old male smoker with untreated hypertension, diabetes, dyslipidemia, and a strong family history of premature coronary heart disease (CHD) would have a 10-year coronary heart disease event rate of only 16% (“intermediate risk”) by Framingham criteria; however, given exactly the same risk profile for a 60-year-old, the risk climbs to >50%. Aging, particularly sedentary aging, leads not only to the development of atherosclerosis but also to ventricular and arterial stiffening.1 This reduces functional capacity and contributes to chronic diseases of the elderly, such as systolic hypertension or heart failure with a preserved ejection fraction. In the search for the underlying mechanism for this effect of sedentary aging, one common feature that has been identified universally in older men is an increased baseline vascular tone,2 which has both hemodynamic and metabolic consequences.3 Although this age-related increase in vascular tone is mediated in part by a chronically elevated sympathetic α-adrenergic vasoconstriction,2 endothelial function also plays a critical role in vascular stability and the regulation of vasomotor function. The endothelium regulates vascular tone through the release of dilator and constrictor substances. Endothelin (ET)-1 is the most potent endothelium-derived constricting factor and influences peripheral vascular tone by interacting with ETA and ETB receptors on smooth muscle cells and the endothelium.4 Several animal studies have examined the profound effects of vascular aging on the ET-1 pathway and support a central role for ET-1 to explain the age-related increase in vascular tone.5–7 In the present issue of Hypertension , Van Guilder et al8 present new and important information regarding the role of aging and physical fitness on the vasomotor effects of ET-1. Using a cross-sectional design in a small but carefully studied …

Reduction of NO- and EDHF-Mediated Vasodilatation in Hypertension: Role of Asymmetric Dimethylarginine

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthase (NOS), and endothelial dysfunction is related to the elevation of ADMA level in hypertension. Besides the NO-mediated pathway, the endothelium-derived hyperpolarizing factor (EDHF)-mediated pathway is involved in endothelial dysfunction. The aims of the present study were to evaluate the changes of endothelium-dependent dilatation of arteries in hypertension and the role of ADMA in NO- and EDHF-mediated vasodilatation. The great omental arteries were isolated from essential hypertensive and normotensive patients, and mesenteric arteries were isolated from spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. NO-, EDHF-, and prostaglandin (PGI2)-mediated endothelium-dependent vasodilatation were measured, and plasma concentrations of ADMA were determined in rats. Cultured endothelial cells were treated with ADMA (1–10 μM) for 48 h, and the mRNA and protein level of small-conductance Ca2+-activated K+ channel 3 (SK3), which has been thought to be a key mediator of EDHF, was determined. Both NO- and EDHF-mediated endothelium-dependent responses were decreased in the great omental arteries of hypertensive patients and mesenteric arteries of SHR. Plasma levels of ADMA were significantly increased in SHR. In cultured endothelial cells, the expressions of SK3 mRNA and protein were concentration-dependently down-regulated in the presence of ADMA. The present study suggests that the inhibitory effect of ADMA on endothelial function not only involves NO-mediated endothelium-dependent vasodilatation but also the EDHF-mediated pathways in hypertensive animals and humans, and that ADMA can down-regulate the expression of SK3 channels in endothelial cells.

The oestrogen receptor β contributes to sex related differences in endothelial function of murine small arteries via EDHF

Sex related differences in cardiovascular function have been reported in oestrogen receptor beta knockout (ERbetaKO) mice. In this study we examined the role of endothelium-derived hyperpolarizing factor (EDHF) in differences in small artery endothelial function between ERbetaKO and wild-type (WT) mice. Small femoral arteries were isolated from ERbetaKO and WT mice and mounted on a wire myograph. Concentration-response curves to ACh were compared before and after incubation with inhibitors of nitric oxide (NO) and prostacyclin (PGI2) synthesis. Comparison of the expression of the principal vascular connexins (Cx37, 40 and 43), implicated in EDHF-mediated dilatation were undertaken by immunohistochemistry. Vascular ultrastructure was studied by transmission and scanning electron microscopy. ACh-induced relaxation of arteries (< 200 microm internal diameter) was greater in WT females versus males and was attributable to a greater EDHF component of relaxation. This sex difference was absent in ERbetaKO mice. Arteries from ERbetaKO males (but not females) were more sensitive to ACh compared to WT. The pharmacological evidence and morphological prerequisite for involvement of gap junctions in EDHF-mediated responses was confirmed in male arteries. The absence of ERbeta had no influence on expression of main Cx subtypes within vascular wall or on ultrastructure and morphology of the endothelium. The data suggest that in WT male mice, ERbeta reduces EDHF-mediated relaxation through gap junction communication.

Vascular Gap Junctions in Hypertension

The proteins that form the gap junctions (connexins) are widely expressed in organs that are central to the development of hypertension: endocrine organs, kidney, brain, heart, and vasculature (Figure 1). Surprisingly, there is little information on the modification of connexins in hypertension in any of these organs except the vasculature, the subject of our review, but it would be hoped that this lack of information might spur research on these organs. Figure 1. Possible roles for the connexins in the control of blood pressure. A, Electron micrograph of a longitudinal view of an arteriole. Vascular connexins can play multiple roles in regulating function and vasomotor tone. Cell–cell communication, both radial and longitudinal, links the cells of the arteriolar wall. B, Most every organ involved in the control of blood pressure abundantly expresses ≥1 of the connexins. Essentially no experimental evidence exists regarding a possible link between connexins and hypertension in nonvascular tissues. Augmented vasomotor tone typically plays a key role in the development of hypertension, and tone depends on cell–cell communication established by paracrine molecules and, in addition, gap junctions. Paracrine-based linkages between cells of the vasculature are well known, and the possible roles of such linkages in the genesis of hypertension have been extensively explored. Much less is known of the roles of gap junctional communication in establishing vasomotor tone and of the effects of modification of gap junctions on hypertension. The gap junctions are formed by joining 2 hexameric assemblies of connexin protein monomers, that is, hemichannels (1 in Figure 2A). In the vasculature, assays of message and protein show that the hemichannels can be assembled from combinations of 4 connexins, named according to their apparent molecular weight: Cx37, Cx40, Cx43, and Cx45. Two hemichannels are linked to form a gap junction, which can provide both homocellular …