EDHF-mediated Hyperpolarization Research Articles

Difference in Endothelium-Derived Hyperpolarizing Factor–Mediated Hyperpolarization and Nitric Oxide Release Between Human Internal Mammary Artery and Saphenous Vein

Background —The greater nitric oxide (NO) release that occurs in the internal mammary artery (IMA) when compared with the saphenous vein (SV) has been suggested by more endothelium-dependent relaxation in the IMA or measured by bioassay; however, no direct measurement of NO- or endothelium-derived hyperpolarizing factor (EDHF)–mediated hyperpolarization has been reported. The present study measured such hyperpolarization, as well as NO release, in these vessels. Methods and Results —IMA (n=46) and SV (n=61) segments taken from patients undergoing coronary surgery were studied in the organ chamber. Hyperpolarization (by intracellular glass microelectrode) and NO release (by NO-sensitive electrode) in response to acetylcholine and bradykinin, with and without incubation with N G -nitro- l -arginine, indomethacin, and oxyhemoglobin, were measured. The resting membrane potential of the smooth muscle cells from the IMA (58±0.8 mV; n=15) was higher than that in those from the SV (−62±0.9 mV; n=23; P =0.0001). The EDHF-mediated hyperpolarization induced by acetylcholine (10 −5 mol/L: −9.4±1.5 mV in IMA, n=10, versus −4.5±1.0 mV in SV, n=17; P <0.01) and bradykinin (10 −7 mol/L: −10.9±1.5 mV in IMA, n=8, versus −5.1±0.5 mV in SV, n=8; P <0.01) and the basal release of NO (16.8±1.6 nmol/L in IMA, n=13, versus 9.9±2.8 nmol/L in SV, n=13; P <0.001) were significantly greater in the IMA than in the SV. The duration of acetylcholine- and bradykinin-induced NO release was longer in the IMA than in the SV. Conclusions —The basal release of NO and EDHF-mediated hyperpolarization were significantly greater in the IMA than in the SV. In addition, the duration of the stimulated release of NO was longer in the IMA than in the SV. These differences may contribute to the superior long-term patency of IMA grafts.

Angiotensin-converting enzyme inhibitor prevents age-related endothelial dysfunction.

Vascular relaxation via endothelium-derived hyperpolarizing factor (EDHF) declines in association with aging and also with hypertension, and antihypertensive treatment improves the endothelial dysfunction connected with hypertension. We tested whether the angiotensin-converting enzyme inhibitor improves EDHF-mediated responses in normotensive rats, with special reference to the age-related process. Wistar-Kyoto rats (WKY) were treated with either 20 mg. kg(-1). d(-1) enalapril (WKY-E group) or a combination of 50 mg. kg(-1). d(-1) hydralazine and 7.5 mg. kg(-1). d(-1) hydrochlorothiazide (WKY-H group) from 9 to 12 months of age. Twelve-month-old WKY (WKY-O) and 3-month-old WKY (WKY-Y) served as controls (n=6 to 10 in each group). The 2 treatments lowered systolic blood pressure comparably. EDHF-mediated hyperpolarization to acetylcholine (ACh) in mesenteric arteries was significantly improved in WKY-E, but not in WKY-H, compared with WKY-O, and the hyperpolarization in WKY-E was comparable to that in WKY-Y (hyperpolarization to 10(-)(5) mol/L ACh in the presence of norepinephrine: WKY-O, -14+/-2 mV; WKY-E, -22+/-3 mV; WKY-H, -15+/-2 mV; and WKY-Y, -28+/-0 mV). EDHF-mediated relaxation, as assessed by relaxation to ACh in norepinephrine-precontracted rings in the presence of indomethacin and NO synthase inhibitor, was also significantly improved in WKY-E, but not in WKY-H, to a level comparable to that in WKY-Y (maximum relaxation: WKY-O, 45+/-6%; WKY-E, 63+/-8%; WKY-H, 43+/-4%; and WKY-Y, 72+/-4%). Hyperpolarization and relaxation to levcromakalim, an ATP-sensitive K(+) channel opener, were similar in all groups. These findings suggest that the angiotensin-converting enzyme inhibitor prevents the age-related decline in EDHF-mediated hyperpolarization and relaxation in normotensive rats, presumably through an inhibition of the renin-angiotensin system.

Renin-Angiotensin System Blockade Improves Endothelial Dysfunction in Hypertension

Angiotensin-converting enzyme (ACE) inhibitor improves the impaired hyperpolarization and relaxation to acetylcholine (ACh) via endothelium-derived hyperpolarizing factor (EDHF) in arteries of spontaneously hypertensive rats (SHR). We tested whether the angiotensin type 1 (AT(1)) receptor antagonist also improves EDHF-mediated responses and whether the combined AT(1) receptor blockade and ACE inhibition exert any additional effects. SHR were treated with either AT(1) receptor antagonist TCV-116 (5 mg. kg(-1). d(-1)) (SHR-T), enalapril (40 mg. kg(-1). d(-1)) (SHR-E), or their combination (SHR-T&E) from 8 to 11 months of age. Age-matched, untreated SHR (SHR-C) and Wistar Kyoto (WKY) rats served as controls (n=8 to 12 in each group). Three treatments lowered blood pressure comparably. EDHF-mediated hyperpolarization to ACh in mesenteric arteries in the absence or presence of norepinephrine was significantly improved in all treated SHR. In addition, the hyperpolarization in the presence of norepinephrine was significantly greater in SHR-T&E than in SHR-E (ACh 10(-5) mol/L with norepinephrine: SHR-C -7; SHR-T -19; SHR-E -15; SHR-T&E -22; WKY -14 mV). EDHF-mediated relaxation, assessed in the presence of indomethacin and N:(G)-nitro-L-arginine, was markedly improved in all treated SHR. Hyperpolarization and relaxation to levcromakalim, a direct opener of ATP-sensitive K(+)-channel, were similar in all groups. These findings suggest that AT(1) receptor antagonists are as effective as ACE inhibitors in improving EDHF-mediated responses in SHR. The beneficial effects of the combined AT(1) receptor blockade and ACE inhibition appears to be for the most part similar to those of each intervention.

Invited Commentary

Invited Commentary

Nifedipine increases cytochrome P4502C expression and endothelium-derived hyperpolarizing factor-mediated responses in coronary arteries.

In addition to NO and prostacyclin, endothelial cells release a factor that elicits vasodilatation by hyperpolarizing the underlying vascular smooth muscle cells. In some vascular beds, this so-called endothelium-derived hyperpolarizing factor (EDHF) displays the characteristics of a cytochrome P450 (CYP)-derived arachidonic acid metabolite, such as an epoxyeicosatrienoic acid. Native porcine and cultured human coronary artery endothelial cells were screened for CYP epoxygenases, and CYP2B, CYP2C, and CYP2J were detected with reverse transcription-polymerase chain reaction. The CYP inducer beta-naphthoflavone and the Ca(2+) antagonist nifedipine significantly increased CYP2C mRNA but did not change the expression of CYP2J or CYP2B. To determine the relationship between CYP2C expression and EDHF production in native endothelial cells, we incubated porcine coronary arteries with nifedipine. Nifedipine enhanced endothelial CYP2C protein expression, as well as the generation of 11,12-epoxyeicosatrienoic acid. In organ bath experiments, pretreatment with nifedipine enhanced bradykinin-induced, EDHF-mediated relaxations as well as the concomitant hyperpolarization of smooth muscle cells. The specific CYP2C9 inhibitor sulfaphenazole, on the other hand, significantly attenuated EDHF-mediated hyperpolarization and relaxation. These results demonstrate that in porcine coronary arteries, the elevated expression of a CYP epoxygenase, homologous to CYP2C8/9, is associated with enhanced EDHF-mediated hyperpolarization in response to bradykinin. Therefore, we propose that an isozyme of CYP2C is the most likely candidate for the CYP-dependent EDHF synthase in porcine coronary arteries.

Cytochrome P450 2C is an EDHF synthase in coronary arteries.

In most arterial beds a significant endothelium-dependent dilation to various stimuli persists even after inhibition of nitric oxide synthase and cyclo-oxygenase. This dilator response is preceded by an endothelium-dependent hyperpolarization of vascular smooth muscle cells, which is sensitive to a combination of the calcium-dependent potassium-channel inhibitors charybdotoxin and apamin, and is assumed to be mediated by an unidentified endothelium-derived hyperpolarizing factor (EDHF). Here we show that the induction of cytochrome P450 (CYP) 2C8/34 in native porcine coronary artery endothelial cells by beta-naphthoflavone enhances the formation of 11,12-epoxyeicosatrienoic acid, as well as EDHF-mediated hyperpolarization and relaxation. Transfection of coronary arteries with CYP 2C8/34 antisense oligonucleotides results in decreased levels of CYP 2C and attenuates EDHF-mediated vascular responses. Thus, a CYP-epoxygenase product is an essential component of EDHF-mediated relaxation in the porcine coronary artery, and CYP 2C8/34 fulfils the criteria for the coronary EDHF synthase.

Inhibition of the production of endothelium-derived hyperpolarizing factor by cannabinoid receptor agonists.

1. The endogenous cannabinoid, anandamide, has been reported to induce an 'endothelium-derived hyperpolarizing factor (EDHF)-like' relaxation in vitro. We therefore investigated the effects of cannabinoid CB1 receptor agonists; HU 210, delta9-tetrahydrocannabinol (delta9-THC) and anandamide, and a CB1 antagonist/inverse agonist, SR 141716A, on nitric oxide (NO) and EDHF-mediated relaxation in precontracted rings of porcine coronary, rabbit carotid and mesenteric arteries. 2. In rings of mesenteric artery HU 210 and delta9-THC induced endothelium- and cyclo-oxygenase-independent relaxations which were sensitive to SR 141716A. Anandamide (0.03-30 microM) induced a slowly developing, endothelium-independent relaxation which was abolished by diclofenac and was therefore mediated by cyclo-oxygenase product(s). None of the CB1 agonists tested affected the tone of precontracted rings of rabbit carotid or porcine coronary artery. 3. In endothelium-intact segments, HU 210, delta9-THC and anandamide did not affect NO-mediated responses but under conditions of continuous NO synthase/cyclo-oxygenase blockade, significantly inhibited acetylcholine and bradykinin-induced relaxations which are attributed to the production of EDHF. The effects of HU 210 and delta9-THC were not observed when experiments were performed in the presence of SR 141716A suggesting the involvement of the CB1 receptor. 4. In a patch clamp bioassay of EDHF production, HU 210 decreased the EDHF-mediated hyperpolarization of detector smooth muscle cells when applied to the donor segment but was without effect on the membrane potential of detector cells. The inhibition of EDHF production was unrelated to alterations in Ca2+ -signalling or cytochrome P450 activity. 5. These results suggest that the activation of endothelial CB1 receptors appears to be negatively coupled to the production of EDHF.

EDHF-mediated hyperpolarization and its vasorelaxant mechanisms

EDHF-mediated hyperpolarization and its vasorelaxant mechanisms

Antihypertensive treatment improves endothelium-dependent hyperpolarization in the mesenteric artery of spontaneously hypertensive rats.

The vascular endothelium releases endothelium-derived hyperpolarizing factor (EDHF). The mesenteric arteries of 6- to 8-month-old spontaneously hypertensive rats (SHRs) exhibit an impairment of the hyperpolarization induced by acetylcholine via EDHF. We determined whether antihypertensive treatment can improve EDHF-mediated responses in SHRs. Beginning at age 8 to 9 months, the animals were treated with either enalapril (40 mg x kg(-1) x d(-1)) (SHR-Es) or a combination of hydralazine (25 mg x kg(-1) x d(-1)) and hydrochlorothiazide (7.5 mg x kg(-1) x d(-1)) (SHR-Hs) for 3 months. The control groups were age-matched SHRs (SHR-Cs) and Wistar Kyoto rats (WKYs). The two treatments lowered the blood pressure to comparable extents. The acetylcholine-induced hyperpolarization in the mesenteric artery of treated SHRs improved to a level comparable to that in WKYs (acetylcholine 10(-5) mol/L with norepinephrine 10(-5) mol/L: SHR-E, -14.4 +/- 1.8; SHR-H, -12.0 +/- 1.3; SHR-C, -7.2 +/- 1.2; and WKY, -13.3 +/- 2.3 mV). EDHF-mediated relaxation, as assessed by relaxation to acetylcholine resistant to N(G)-nitro-L-arginine in norepinephrine-contracted rings, was markedly improved in treated SHRs (maximal relaxation: SHR-E, 79.3+/-3.2%; SHR-H, 47.4+/-8.6%; SHR-C, 4.8+/-2.4%; and WKY, 45.1+/-6.0%). When the rings were contracted with 77 mmol/L KCl to eliminate EDHF response, no difference was found in relaxation to acetylcholine among the four groups. Similarly, the hyperpolarization and relaxation to levcromakalim, a K+ channel opener, were comparable among the groups. Antihypertensive treatment improved EDHF-mediated hyperpolarization and relaxation in the mesenteric artery in SHRs, whereas NO-mediated relaxation did not appear to be modulated by drug therapy. Thus, alterations in the EDHF system may play a pivotal role in endothelial dysfunction and its improvement with drug therapy in SHRs.

Endothelium-derived hyperpolarizing factor, but not nitric oxide, is reversibly inhibited by brefeldin A.

The subcellular localization of the enzymes synthesizing endothelium-derived vasodilator autacoids has been proposed to play a role in determining the ability of endothelial cells to enhance autacoid production in response to stimulation. We therefore investigated the effects of brefeldin A-induced disruption of the Golgi apparatus and Golgi-plasma membrane trafficking on the production of nitric oxide (NO), prostacyclin, and the endothelium-derived hyperpolarizing factor (EDHF) by native and cultured endothelial cells. In porcine coronary artery segments, brefeldin A (35 micromol/L, 90 minutes) did not affect relaxations to sodium nitroprusside or the K+ channel opener cromakalim but elicited a rightward shift in the concentration-response curve to bradykinin without altering the maximum vasodilator response (Rmax). Brefeldin A failed to attenuate the bradykinin-induced, NO-mediated relaxation under depolarizing conditions but inhibited the bradykinin response under conditions of combined cyclooxygenase/NO synthase blockade, suggesting that this agent selectively interferes with the production of EDHF. Indeed, incubation of porcine coronary arteries with brefeldin A, which did not affect the bradykinin-induced accumulation of either cyclic GMP or 6-keto-prostaglandin F1alpha, markedly and reversibly attenuated the EDHF-mediated hyperpolarization of detector smooth muscle cells in a patch-clamp bioassay system. The microtubule destabilizer nocodazole also affected both the EC50 and Rmax to bradykinin in porcine coronary arteries. Since EDHF is thought to be a cytochrome P450-derived metabolite of arachidonic acid and both brefeldin A and nocodazole are known to interfere with the targeting of cytochrome P450 from the Golgi apparatus to the plasma membrane, it is conceivable that brefeldin A inhibits EDHF formation by preventing the targeting of the EDHF-synthesizing enzymes to the plasma membrane.

HYPERPOLARIZATION AND RELAXATION MEDIATED BY ENDOTHELIUM-DERIVED FACTORS AND ABNORMALITIES IN HYPERTENSIVE ANIMALS

The membrane hyperpolarization produced by endothelium-derived hyperpolarizing factor (EDHF) is one of the components in the endothelium-dependent relaxations. Different subtype of endothelium receptor is involved in the release of EDHF from that for nitric oxide. Although EDHF has not been identified, it may be related to cytochrome P450. The responses mediated by EDHF have been studied by observation of membrane potential or relaxation which is resistant to inhibitors of nitric oxide synthase and cyclooxygenase. The responses are dependent on potassium gradient across cell membrane, therefore, the response is mediated by potassium channels. The EDHF-mediated hyperpolarization or relaxation is sensitive to charybdotoxin plus apamin, inhibitors of calcium activated potassium channels. The EDHF-mediated response is more remarkable in small resistant arteries than large arteries, therefore, EDHF may play an important role in the regulation of vascular resistance. The EDHF-mediated response is decreased in hypertensive or aged animals. Understanding of EDHF-mediated response will promote development of a new strategy for the treatment of pathophysiological conditions.

Hyperkalemia alters EDHF-mediated hyperpolarization and relaxation in coronary arteries.

Hyperkalemic solutions are widely used to preserve organs for transplantation and for cardiac surgery. The present study was designed to test the hypothesis that hyperkalemia may alter endothelial function through a non-nitric oxide (NO) pathway, since preliminary studies have shown that the NO pathway may not be affected. Porcine coronary artery rings were studied in organ chambers. After incubation with 20 or 50 mM K+ for 1 h, the indomethacin- and NG-nitro-L-arginine+ (L-NNA)-resistant relaxation induced by A23187 or bradykinin, which could be further inhibited by tetraethylammonium but not glibenclamide, was significantly reduced. Incubation with hyperkalemia also significantly increased the concentration eliciting 50% of the maximal response to A23187 and bradykinin. A23187-induced hyperpolarization of the membrane potential was significantly reduced by hyperkalemic incubation. However, 1-h incubation with hyperkalemia does not affect the endothelial Ca2+ concentration. We conclude that exposure to hyperkalemia reduces the indomethacin- and L-NNA-resistant endothelium-dependent relaxation and endothelium-dependent hyperpolarization. This reduction in the relaxation and hyperpolarization is related to the endothelium-derived hyperpolarizing factor by affecting its effect on the smooth muscle cell, probably through partially depolarizing the membrane, and the Ca2(+)- activated K+ channels rather than by affecting its biosynthesis and/or release in the endothelial cell. Our study may suggest a new mechanism for coronary dysfunction after exposure to hyperkalemic cardioplegia and organ preservation solutions.