Identity Of Endothelium-derived Hyperpolarizing Factor Research Articles

The Significance of Endothelium-Derived Hyperpolarizing Factor in the Human Circulation

Although nitric oxide (NO) is recognized as the primary vasodilator derived from vascular endothelium in regulating the vascular tone, another factor, i.e. the endothelium-derived hyperpolarizing factor (EDHF), has recently gained much attention and has been demonstrated to participate in vasodilatation in various blood vessels from different species, despite its unidentified nature. Most of the studies were conducted in animals and the knowledge of this factor in the human vasculature is relatively limited. This review attempts to address the relevance of EDHF-mediated function in humans with the possible identity of EDHF and mechanisms involved. We consider the human vasculature where EDHF involvement has been documented including the systemic, coronary, and visceral (gastrointestinal, renal and reproductive) circulation. In these vascular systems, EDHF plays a role under physiological conditions either as another mechanism or as the "back-up" for NO. Furthermore, the contribution of EDHF changes under certain physiological conditions, such as ageing and pregnancy. In addition, altered EDHF function has been suggested in various pathological conditions including heart diseases, atherosclerosis, hypertension, diabetes, eclampsia, glaucoma, chronic renal failure, erectile dysfunction and ischemia-reperfusion period during open heart surgery. Pharmacological agents such as potassium channel openers or cytochrome P450 metabolites have been used to either protect or recover EDHF-dependent mechanisms. To further develop new therapeutic strategies that target EDHF, a better understanding is essential with regard to the function of EDHF under pathophysiological conditions in humans. Furthermore, the interaction between NO and EDHF as well as their relative contributions in various conditions are critical.

Endothelium-derived hyperpolarizing factor in human internal mammary artery is 11,12-epoxyeicosatrienoic acid and causes relaxation by activating smooth muscle BK(Ca) channels.

Left internal mammary arteries (LIMAs) synthesize endothelium-derived hyperpolarizing factor (EDHF), a short-lived K(+) channel activator that persists after inhibition of nitric oxide (NO) and prostaglandin synthesis. EDHF hyperpolarizes and relaxes smooth muscle cells (SMCs). The identity of EDHF in humans is unknown. We hypothesized that EDHF (1) is 11,12-epoxyeicosatrienoic acid (11,12-EET); (2) is generated by cytochrome P450-2C, CYP450-2C; and (3) causes relaxation by opening SMC large-conductance Ca(2+)-activated K(+) channels (BK(Ca)). The identity of EDHF and its mechanism of action were assessed in 120 distal human LIMAs and 20 saphenous veins (SVs) obtained during CABG. The predominant EET synthesized by LIMAs is 11,12-EET. Relaxations to exogenous 11,12-EET and endogenous EDHF are of similar magnitudes. Inhibition of EET synthesis by chemically distinct CYP450 inhibitors (17-octadecynoic acid, N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide), or a selective EET antagonist (4,15-epoxyeicosa-5(Z)-enoic acid) impairs EDHF relaxation. 11,12-EET activates a BK(Ca) current and hyperpolarizes LIMA SMCs. Inhibitors of BK(Ca) but not inward-rectifier or small-conductance K(Ca) channels abolish relaxation to endogenous EDHF and exogenous 11,12-EET. BK(Ca) and CYP450-2C mRNA and proteins are more abundant in LIMAs than in SVs, perhaps explaining the lack of EDHF activity of the SV. Laser capture microdissection and quantitative RT-PCR demonstrate that BK(Ca) channels are primarily in vascular SMCs, whereas the CYP450-2C enzyme is present in both the endothelium and SMCs. In human LIMAs, EDHF is 11,12-EET produced by an EDHF synthase CYP450-2C and accounting for approximately 40% of net endothelial relaxation. 11,12-EET causes relaxation by activating SMC BK(Ca) channels.

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

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

Effect of NO on EDHF response in rat middle cerebral arteries.

Whereas the actual identity of endothelium-derived hyperpolarizing factor (EDHF) is still not certain, it involves a process requiring the endothelium and eliciting hyperpolarization and relaxation of smooth muscle. It is neither nitric oxide (NO) nor prostacyclin, and its presence has been demonstrated in a variety of vessels. Recent studies in peripheral vessels report that EDHF-mediated dilations were either attenuated or blocked by NO. Studies presented here demonstrate that NO does not block EDHF-mediated dilations in cerebral vessels. Rat middle cerebral arteries were cannulated, pressurized, and luminally perfused. EDHF-mediated dilations were elicited by the luminal application of ATP in the presence of N(G)-nitro-L-arginine methyl ester (L-NAME) and indomethacin (inhibitors of NO synthase and cyclooxygenase, respectively). These dilations persisted when S-nitroso-N-acetylpenicillamine, an NO donor, was added exogenously in the presence of L-NAME, or when endogenous NO was present but its cGMP actions were blocked by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of guanylate cyclase. These findings demonstrate that the EDHF response is not suppressed by NO in cerebral vessels and suggests a role for EDHF during normal physiological conditions.

Role of Gap-Junctions in Endothelium-Dependent Hyperpolarization in Rat Small and Large Mesenteric Arteries

P39 In arteries, acetylcholine (ACh) releases endothelium-derived hyperpolarizing factor (EDHF) which is distinct from nitric oxide. The identity of EDHF is still elusive; however several recent studies suggests the possible involvement of myoendothelial gap-junctions in small arteries. To elucidate the involvement of gap-junctions in EDHF responses, we examined the effects of 18α-glycyrrhetinic acid (18α-GA, 10 -4 mol/L), a gap junction inhibitor, on endothelium-dependent hyperpolarization and relaxation to ACh in rat small and large mesenteric arteries. Experiments were perfomed in the presence of indomethacin (10 -5 mol/L) and NG-nitro-L-arginine (10 -4 mol/L) . In large arteries, ACh (10 -6 mol/L)-induced hyperpolarization measured from intimal side, was attenuated by the 18α-GA (12.4±1.2 vs. 8.4±0.8mV in the absence and presence of 18α-GA, p -7 mol/L), ACh-induced relaxation was also attenuated (maximal relaxation, 71.5±6.3 vs. 50.5±6.4%, p + -channels, did not differ in the absence and presence of 18α-GA (16.7±0.6 vs. 16.3±1.5mV, ns, n=8 and n=6 respectively). These findings suggest that myoendothelial gap-junctions partly contribute to the EDHF-mediated responses both in rat small and large mesenteric arteries.

Role of Gap-Junctions in Endothelium-Dependent Hyperpolarization in Rat Small and Large Mesenteric Arteries

P39 In arteries, acetylcholine (ACh) releases endothelium-derived hyperpolarizing factor (EDHF) which is distinct from nitric oxide. The identity of EDHF is still elusive; however several recent studies suggests the possible involvement of myoendothelial gap-junctions in small arteries. To elucidate the involvement of gap-junctions in EDHF responses, we examined the effects of 18α-glycyrrhetinic acid (18α-GA, 10 -4 mol/L), a gap junction inhibitor, on endothelium-dependent hyperpolarization and relaxation to ACh in rat small and large mesenteric arteries. Experiments were perfomed in the presence of indomethacin (10 -5 mol/L) and NG-nitro-L-arginine (10 -4 mol/L) . In large arteries, ACh (10 -6 mol/L)-induced hyperpolarization measured from intimal side, was attenuated by the 18α-GA (12.4±1.2 vs. 8.4±0.8mV in the absence and presence of 18α-GA, p<0.05, n=7). In rings pre-contracted with norepinephrine (5×10 -7 mol/L), ACh-induced relaxation was also attenuated (maximal relaxation, 71.5±6.3 vs. 50.5±6.4%, p<0.05, n=8 ). In small arteries, ACh-induced hyperpolarization, measured from either adventitial side or intimal side was attenuated by 18α-GA (adventitial side, 12.3±1.2 vs. 4.6±0.7mV, p<0.05, n=8; intimal side, 8.0±0.7 vs. 5.0±0.7mV, p<0.05, n=4). On the other hand, smooth muscle hyperpolarization to levcromakalim, a direct activator of ATP-sensitive K + -channels, did not differ in the absence and presence of 18α-GA (16.7±0.6 vs. 16.3±1.5mV, ns, n=8 and n=6 respectively). These findings suggest that myoendothelial gap-junctions partly contribute to the EDHF-mediated responses both in rat small and large mesenteric arteries.

Hyperpolarizing Factors

There is now overwhelming evidence for factors, other than nitric oxide (NO), that mediate endothelium-dependent vasodilation by hyperpolarizing the underlying smooth muscle via activation of Ca 2+-activated K + channels. Although the identity of endothelium-derived hyperpolarizing factor (EDHF) remains to be established, cytochrome P450 (CYP)-dependent metabolites of arachidonic acid (AA), namely, the epoxides, fulfill several of the criteria required for consideration as putative mediators of endothelium-dependent hyperpolarization. They are produced by the endothelium, released in response to vasoactive hormones, and elicit vasorelaxation via stimulation of Ca 2+-activated K + channels. Our studies in the rat indicate that, of the epoxides, 5,6-epoxyeicosatrienoic acid (5,6-EET) is the most likely mediator of NO-independent, but CYP-dependent coronary vasodilation in response to bradykinin. Studies in the rat kidney, however, support the existence of additional EDHFs as acetylcholine also exhibits NO-independent vasodilation that is unaffected by CYP inhibitors in concentrations that attenuate responses to bradykinin. In some blood vessels, NO may tonically suppress the expression of CYP-dependent EDHF. In the event of impaired NO synthesis, therefore, a CYP-dependent vasodilator mechanism may serve as a backup to a primary NO-dependent mechanism, although they may act in concert. In other vessels, particularly microvessels, an EDHF may constitute the major vasodilator mechanism for hormones and other physiological stimuli. EDHFs appear to be important regulators of vascular tone; alterations in this system can be demonstrated in hypertension and diabetes, conditions associated with altered endothelium-dependent vasodilator responsiveness.

An Endogenous Cannabinoid as an Endothelium-Derived Vasorelaxant

Since the identification of nitric oxide (NO) as an important mediator of endothelium-dependent relaxation, it has become clear that there is an additional endothelial relaxant factor, termed the endothelium-derived hyperpolarizing factor (EDHF). The identity of EDHF has remained elusive, but it is thought to be an arachidonic acid metabolite. We now report that EDHF-mediated relaxations in the rat mesenteric arterial bed are blocked by a highly selective cannabinoid receptor antagonist, SR141716A, consistent with EDHF being a cannabinoid-like substance. Furthermore, in conscious rats, the NO-independent depressor and regional vasodilator effects of bradykinin were inhibited by SR141716A. The relaxations in the isolated mesentery were accompanied by the accumulation of an arachidonic acid metabolite, which co-eluted on TLC separation with arachidonoylethanolamide (anandamide), an endogenous cannabinoid derived from arachidonate. We further report that anandamide is a potent vasorelaxant in the mesentery, acting via a hyperpolarizing mechanism. These findings suggest that an endogenous cannabinoid is an endothelium-derived vasorelaxant, which may be EDHF.