Endothelium-derived Hyperpolarizing Factor Response Research Articles

Sex Differences in Sepsis‐Induced Nitric Oxide and EDHF Signaling Pathways in Rat Pulmonary Artery

Sepsis still remains a major cause of hospital infection related deaths in the US. Pulmonary manifestation of sepsis is characterized by pulmonary hypertension, pulmonary edema and refractory hypoxemia. Pulmonary hypertension is associated with abnormal regulation of vascular tone in sepsis. Evidence exists that males are more susceptible to sepsis than females. The objective of present study was to elucidate the influence of gender on endothelial function, particularly with reference to nitric oxide (NO) and endothelium‐derived hyperpolarizing factor (EDHF) signaling pathways in pulmonary artery of septic rats. Sepsis was induced in rats by cecal ligation and puncture (CLP). To determine the effect of sepsis‐induced alteration in the NO and EDHF signaling pathways in both sexes, arterial relaxation was measured using isometric tension experiments. Results demonstrate that in control animals the potency but not the amplitude of the response to the vasoconstrictor, phenylephrine, was significantly greater in the pulmonary arteries from female than male rats. Sepsis caused a significant reduction in the efficacy of phenylephrine in pulmonary arteries from either sex. In controls, acetylcholine (ACh) was more potent in dilating pulmonary arteries from female than male rats and sepsis identically inhibited ACh‐induced relaxations in both male and female animals. Sepsis completely abolished the EDHF response in pulmonary arterial rings from both sexes. Pre‐treatment with L‐NAME (eNOS inhibitor) and indomethacin (prostacyclin inhibitor) significantly increased the basal tone of pulmonary arterial rings from female rats and was significantly reduced in sepsis. However, L‐NAME and indomethacin did not alter the basal tone of pulmonary artery from both control and septic male rats. Real time PCR analysis of mRNA expression revealed that sepsis decreased IKCa, SKCa1, and SKCa2 mRNA expression levels in female rats and IKCa and SKCa2 mRNA expression levels in male rats. SKCa1 SKCa1 mRNA expression levels did not change in male rats while sepsis had no effect on SKCa3 mRNA expression levels in both sexes. In conclusion, the results of the present study suggest that sepsis‐induced alteration in the reactivity of pulmonary artery is gender dependent. This can be attributed to a decrease in NO but predominantly EDHF‐mediated vasorelaxation resulting from a significant decrease in expression of endothelium localized potassium channels.

NO synthase inhibition attenuates EDHF-mediated relaxation induced by TRPV4 channel agonist GSK1016790A in the rat pulmonary artery: Role of TxA2.

The aim of the present study was to observe the concomitant activation of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) pathways by TRPV4 channel agonist GSK1016790A in the rat pulmonary artery and explore the mechanism by which NO synthase inhibition attenuates EDHF-mediated relaxation in endothelium-intact rat pulmonary artery. Tension experiments were conducted on the pulmonary artery from male Wistar rats. TRPV4 channel agonist GSK1016790A (GSK) caused concentration-dependent relaxation (Emax 86.9±4.6%; pD2 8.7±0.24) of the endothelium-intact rat pulmonary artery. Combined presence of apamin and TRAM-34 significantly attenuated the relaxation (Emax 61.1±6.0%) to GSK. l-NAME (100μM) significantly attenuated (8.2±2.9%) the relaxation response to GSK that was resistant to apamin plus TRAM-34. However, presence of ICI192605 or furegrelate alongwith l-NAME revealed the GSK-mediated EDHF-response (Emax of 28.5±5.2%; Emax 24.5±4.3%) in this vessel, respectively. Further, these two TxA2 modulators (ICI/furegrelate) alongwith l-NAME had no effect on SNP-induced endothelium-independent relaxation in comparison to l-NAME alone. This EDHF-mediated relaxation was sensitive to inhibition by K(+) channel blockers apamin and TRAM-34 or 60mMK(+) depolarizing solution. Further, combined presence of apamin and TRAM-34 in U46619 pre-contracted pulmonary arterial rings significantly reduced the maximal relaxation (Emax 71.6±6.9%) elicited by GSK, but had no effect on the pD2 (8.1±0.03) of the TRPV4 channel agonist in comparison to controls (Emax, 92.4±4.3% and pD2, 8.3±0.06). The present study suggests that NO and EDHF are released concomitantly and NO synthase inhibition attenuates GSK-induced EDHF response through thromboxane pathway in the rat pulmonary artery.

β1‐Adrenoceptor stimulation suppresses endothelial IKCa‐channel hyperpolarization and associated dilatation in resistance arteries

In small arteries, small conductance Ca²⁺-activated K⁺ channels (SK(Ca)) and intermediate conductance Ca²⁺-activated K⁺ channels (IK(Ca)) restricted to the vascular endothelium generate hyperpolarization that underpins the NO- and PGI₂-independent, endothelium-derived hyperpolarizing factor response that is the predominate endothelial mechanism for vasodilatation. As neuronal IK(Ca) channels can be negatively regulated by PKA, we investigated whether β-adrenoceptor stimulation, which signals through cAMP/PKA, might influence endothelial cell hyperpolarization and as a result modify the associated vasodilatation. Rat isolated small mesenteric arteries were pressurized to measure vasodilatation and endothelial cell [Ca²⁺]i , mounted in a wire myograph to measure smooth muscle membrane potential or dispersed into endothelial cell sheets for membrane potential recording. Intraluminal perfusion of β-adrenoceptor agonists inhibited endothelium-dependent dilatation to ACh (1 nM-10 μM) without modifying the associated changes in endothelial cell [Ca²⁺]i . The inhibitory effect of β-adrenoceptor agonists was mimicked by direct activation of adenylyl cyclase with forskolin, blocked by the β-adrenoceptor antagonists propranolol (non-selective), atenolol (β₁) or the PKA inhibitor KT-5720, but remained unaffected by ICI 118 551 (β₂) or glibenclamide (ATP-sensitive K⁺ channels channel blocker). Endothelium-dependent hyperpolarization to ACh was also inhibited by β-adrenoceptor stimulation in both intact arteries and in endothelial cells sheets. Blocking IK(Ca) {with 1 μM 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34)}, but not SK(Ca) (50 nM apamin) channels prevented β-adrenoceptor agonists from suppressing either hyperpolarization or vasodilatation to ACh. In resistance arteries, endothelial cell β₁-adrenoceptors link to inhibit endothelium-dependent hyperpolarization and the resulting vasodilatation to ACh. This effect appears to reflect inhibition of endothelial IK(Ca) channels and may be one consequence of raised circulating catecholamines.

The Intermediate Conductance Calcium-activated Potassium Channel KCa3.1 Regulates Vascular Smooth Muscle Cell Proliferation via Controlling Calcium-dependent Signaling

The intermediate conductance calcium-activated potassium channel KCa3.1 contributes to a variety of cell activation processes in pathologies such as inflammation, carcinogenesis, and vascular remodeling. We examined the electrophysiological and transcriptional mechanisms by which KCa3.1 regulates vascular smooth muscle cell (VSMC) proliferation. Platelet-derived growth factor-BB (PDGF)-induced proliferation of human coronary artery VSMCs was attenuated by lowering intracellular Ca(2+) concentration ([Ca(2+)]i) and was enhanced by elevating [Ca(2+)]i. KCa3.1 blockade or knockdown inhibited proliferation by suppressing the rise in [Ca(2+)]i and attenuating the expression of phosphorylated cAMP-response element-binding protein (CREB), c-Fos, and neuron-derived orphan receptor-1 (NOR-1). This antiproliferative effect was abolished by elevating [Ca(2+)]i. KCa3.1 overexpression induced VSMC proliferation, and potentiated PDGF-induced proliferation, by inducing CREB phosphorylation, c-Fos, and NOR-1. Pharmacological stimulation of KCa3.1 unexpectedly suppressed proliferation by abolishing the expression and activity of KCa3.1 and PDGF β-receptors and inhibiting the rise in [Ca(2+)]i. The stimulation also attenuated the levels of phosphorylated CREB, c-Fos, and cyclin expression. After KCa3.1 blockade, the characteristic round shape of VSMCs expressing high l-caldesmon and low calponin-1 (dedifferentiation state) was maintained, whereas KCa3.1 stimulation induced a spindle-shaped cellular appearance, with low l-caldesmon and high calponin-1. In conclusion, KCa3.1 plays an important role in VSMC proliferation via controlling Ca(2+)-dependent signaling pathways, and its modulation may therefore constitute a new therapeutic target for cell proliferative diseases such as atherosclerosis.

The effect of streptozotocin-induced diabetes on the EDHF-type relaxation and cardiac function in rats

The endothelium-derived hyperpolarizing factor (EDHF) response is a critical for the functioning of small blood vessels. We investigated the effect of streptozotocin-induced diabetes on the EDHF response and its possible role in the regulation of cardiac function. The vasorelaxant response to ACh- or NS309- (direct opener endothelial small- (SKCa)- and intermediate-conductance (IKCa) calcium-activated potassium channels; main components of EDHF response) were measured in pressurized mesenteric arteries (diameter 300–350μm). The response to 1μM ACh was reduced in diabetes (84.8±2.8% control vs 22.5±5.8% diabetics; n⩾8; P<0.001). NS309 (1μM) relaxations were also decreased in diabetic arteries (78.5±8.7% control vs 32.1±5.8% diabetics; n⩾5; P<0.001). SKCa and IKCa-mediated EDHF relaxations in response ACh or NS309 were also significantly reduced by diabetes. Ruthenium red, RuR, a blocker of TRP channels, strongly depress the response to ACh and NS309 in control and diabetic arteries. RuR decreased SKCa and IKCa-mediated EDHF vasodilatation in response to NS309 but not to ACh. An elevation in systolic blood pressure was observed in diabetic animals. ECG recording of control hearts showed shortening of PR interval. RuR reduced PR interval and R wave amplitude in diabetic hearts. In conclusion, the reduced EDHF-type relaxations in STZ-induced diabetes is due impairment of KCa channels function. TRP channels possibly contribute to EDHF vasodilatation via direct opening of endothelial KCa. It is possible that EDHF and TRP channels contribute to the regulation of cardiac function and therefore can be considered as therapeutic targets to improve cardiovascular complications of diabetes.

EDHF: spreading the influence of the endothelium.

Our view of the endothelium was transformed around 30 years ago, from one of an inert barrier to that of a key endocrine organ central to cardiovascular function. This dramatic change followed the discoveries that endothelial cells (ECs) elaborate the vasodilators prostacyclin and nitric oxide. The key to these discoveries was the use of the quintessentially pharmacological technique of bioassay. Bioassay also revealed endothelium-derived hyperpolarizing factor (EDHF), particularly important in small arteries and influencing blood pressure and flow distribution. The basic idea of EDHF as a diffusible factor causing smooth muscle hyperpolarization (and thus vasodilatation) has evolved into one of a complex pathway activated by endothelial Ca(2+) opening two Ca(2+) -sensitive K(+) -channels, K(Ca)2.3 and K(Ca)3.1. Combined application of apamin and charybdotoxin blocked EDHF responses, revealing the critical role of these channels as iberiotoxin was unable to substitute for charybdotoxin. We showed these channels are arranged in endothelial microdomains, particularly within projections towards the adjacent smooth muscle, and close to interendothelial gap junctions. Activation of K(Ca) channels hyperpolarizes ECs, and K(+) efflux through them can act as a diffusible 'EDHF' stimulating Na(+) /K(+) -ATPase and inwardly rectifying K-channels. In parallel, hyperpolarizing current can spread from the endothelium to the smooth muscle through myoendothelial gap junctions upon endothelial projections. The resulting radial hyperpolarization mobilized by EDHF is complemented by spread of hyperpolarization along arteries and arterioles, effecting distant dilatation dependent on the endothelium. So the complexity of the endothelium still continues to amaze and, as knowledge evolves, provides considerable potential for novel approaches to modulate blood pressure.

Potentiation of EDHF-mediated relaxation by chloride channel blockers

To investigate the involvement of Cl⁻ channels in endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation in rat mesenteric arteries. Cl⁻ channel and K(ir) channel activities were studied using whole-cell patch clamping in rat mesenteric arterial smooth muscle cells. Isometric tension of arterial rings was measured in organ chambers. The volume-activated Cl⁻ current in rat mesenteric arterial smooth muscle cells was abolished by Cl⁻ channel blockers NPPB or DIDS. The EDHF-mediated vasorelaxation was potentiated by NPPB and DIDS. The EDHF response was diminished by a combination of apamin and charybdotoxin, which agreed with the hypothesis that EDHF response involves the release of K(+) via the Ca²(+)-activated K(+) channels in endothelial cells. The elevation of K(+) concentration in bathing solution from 1.2 mmol/L to 11.2 mmol/L induced an arterial relaxation, which was abolished by the combination of BaCl₂ and ouabain. It is consistent to the hypothesis that K(+) activates K(+)/Na(+)-ATPase and inward rectifier K(+) (K(ir)) channels, leading to the hyperpolarization and relaxation of vascular smooth muscle. The K(+)-induced relaxation was augmented by NPPB, DIDS, or withdrawal of Cl⁻ from the bathing solution, which could be reversed by BaCl₂, but not ouabain. The potentiating effect of Cl⁻ channel blockers on K(+)-induced relaxation was probably due to the interaction between Cl⁻ channels and K(ir) channels. Moreover, the K(+)-induced relaxation was potentiated when the arteries were incubated in hyperosmotic solution, which is known to inhibit volume-activated Cl⁻ channels. The inhibition of Cl⁻ channels, particularly the volume-activated Cl⁻ channels, may potentiate the EDHF-induced vasorelaxation through the K(ir) channels.

Effects of interleukin‐6 treatment on neurovascular function, nerve perfusion and vascular endothelium in diabetic rats

Interleukin-6 (IL-6), a member of the neuropoietic cytokine family, participates in neural development and has neurotrophic activity. Recent research has also indicated actions to improve vasa nervorum function in diabetes. Both these facets are potentially relevant for treatment of diabetic neuropathy. The aim of this study was to determine whether IL-6 treatment corrected changes in neurovascular function in streptozotocin-induced diabetic rats. After 1 month of diabetes, rats were given IL-6 for 1 month. The rats were subjected to sensory testing and measurements of nerve conduction velocities and nerve blood flow by hydrogen clearance microelectrode polarography. Further groups were used to study responses of the isolated gastric fundus and renal artery. Results were statistically analysed using ANOVA and post hoc tests. Diabetic rats showed mechanical hyperalgesia, thermal hyperalgesia, and tactile allodynia. The former was unaffected by IL-6 treatment, whereas the latter two measures were corrected. Immunohistochemical staining of dorsal root ganglia for IL-6 did not reveal any changes with diabetes or treatment. The results showed that 22 and 17.4% slowing of sciatic motor and saphenous sensory nerve conduction velocities, respectively, with diabetes were improved by IL-6. Sciatic endoneurial perfusion was halved by diabetes and corrected by IL-6. A 40.6% diabetic deficit in maximal non-adrenergic, non-cholinergic relaxation of gastric fundus to nerve stimulation was unaffected by IL-6. Renal artery endothelium-dependent relaxation was halved by diabetes, the endothelium-derived hyperpolarizing factor (EDHF) component being severely attenuated. IL-6 did not affect nitric oxide-mediated vasorelaxation, but markedly improved EDHF responses. IL-6 improved aspects of small and large nerve fibre and vascular endothelium dysfunction in diabetic rats. The functional benefits related to increased nerve blood flow via an EDHF mechanism, and IL-6 could have therapeutic potential in diabetic neuropathy and vasculopathy, which should be further evaluated.

Recycling of the Ca2+-activated K+ Channel, KCa2.3, Is Dependent upon RME-1, Rab35/EPI64C, and an N-terminal Domain

Regulation of the number of Ca(2+)-activated K(+) channels at the endothelial cell surface contributes to control of the endothelium-derived hyperpolarizing factor response, although this process is poorly understood. To address the fate of plasma membrane-localized KCa2.3, we utilized an extracellular epitope-tagged channel in combination with fluorescence and biotinylation techniques in both human embryonic kidney cells and the human microvascular endothelial cell line, HMEC-1. KCa2.3 was internalized from the plasma membrane and degraded with a time constant of 18 h. Cell surface biotinylation demonstrated that KCa2.3 was rapidly endocytosed and recycled back to the plasma membrane. Consistent with recycling, expression of a dominant negative (DN) RME-1 or Rab35 as well as wild type EPI64C, the Rab35 GTPase-activating protein, resulted in accumulation of KCa2.3 in an intracellular compartment. Expression of DN RME-1, DN Rab35, or wild type EPI64C resulted in a decrease in steady-state plasma membrane expression. Knockdown of EPI64C increased cell surface expression of KCa2.3. Furthermore, the effect of EPI64C was dependent upon its GTPase-activating proteins activity. Co-immunoprecipitation studies confirmed an association between KCa2.3 and both Rab35 and RME-1. In contrast to KCa2.3, KCa3.1 was rapidly endocytosed and degraded in an RME-1 and Rab35-independent manner. A series of N-terminal deletions identified a 12-amino acid region, Gly(206)-Pro(217), as being required for the rapid recycling of KCa2.3. Deletion of Gly(206)-Pro(217) had no effect on the association of KCa2.3 with Rab35 but significantly decreased the association with RME-1. These represent the first studies elucidating the mechanisms by which KCa2.3 is maintained at the plasma membrane.

Compromised vascular endothelial cell SKCa activity: a fundamental aspect of hypertension?

Smooth muscle hyperpolarization originating in the endothelium and commonly referred to as the EDHF (endothelium-derived hyperpolarizing factor) response provides a very significant drive to vasodilatation particularly in small resistance arteries. Together with other endothelium-dependent dilator pathways 'EDHF' hyperpolarization is compromised by cardiovascular disease, including hypertension. However, although attenuated vascular hyperpolarization has been described in animal models of hypertension, the underlying mechanisms are not fully understood. In the current issue of the British Journal of Pharmacology, Weston et al. combine classic pharmacological approaches with electrophysiological and molecular techniques to suggest that attenuated endothelium-dependent hyperpolarization (and as a consequence vasodilatation) reflects major disruption of pathways associated with the activation of endothelial small conductance Ca(2+)-activated K-channels (SK(Ca)) in mesenteric arteries from spontaneously hypertensive rats. In addition to reductions in SK(Ca) and K(IR) proteins, changes in caveolin-1 isomers were also detected, possibly indicating channel realignment within plasmalemmal structures.

Resistance Artery Adaptation to Pregnancy Counteracts the Vasoconstricting Influence of Plasma From Normal Pregnant Women

Using a rat model, we investigated the effects of circulating factors in pregnancy on cerebrovascular and systemic vascular function by comparing myogenic reactivity, tone, and endothelial vasodilator production of the posterior cerebral artery (PCA) and mesenteric artery (MA) of nonpregnant (NP) animals perfused with nonpregnant and pregnant human plasma. Arteries from late pregnant (LP) animals were then perfused similarly to evaluate a potential adaptive effect of pregnancy on vessel function. A 3-hour exposure to pregnant plasma caused increased myogenic reactivity and tone in vessels from NP animals and produced a decreased endothelium-derived hyperpolarizing factor response in NP PCAs, findings that were not seen with MAs. The increased reactivity and tone noted in NP vessels was abolished when pregnant plasma was perfused through LP arteries, suggesting these vessels adapt during pregnancy to the vasoconstricting influence of pregnant plasma.

Twenty-Four-Hour Exposure to Altered Blood Flow Modifies Endothelial Ca2+-Activated K+ Channels in Rat Mesenteric Arteries

We tested the hypothesis that changes in arterial blood flow modify the function of endothelial Ca2+-activated K+ channels [calcium-activated K+ channel (K(Ca)), small-conductance calcium-activated K+ channel (SK3), and intermediate calcium-activated K+ channel (IK1)] before arterial structural remodeling. In rats, mesenteric arteries were exposed to increased [+90%, high flow (HF)] or reduced blood flow [-90%, low flow (LF)] and analyzed 24 h later. There were no detectable changes in arterial structure or in expression level of endothelial nitric-oxide synthase, SK3, or IK1. Arterial relaxing responses to acetylcholine and 3-oxime-6,7-dichlore-1H-indole-2,3-dione (NS309; activator of SK3 and IK1) were measured in the absence and presence of endothelium, NO, and prostanoid blockers, and 6,12,19,20,25,26-hexahydro-5,27:13,18:21,24-trietheno-11,7-metheno-7H-dibenzo [b,n] [1,5,12,16]tetraazacyclotricosine-5,13-diium dibromide (UCL 1684; inhibitor of SK3) or 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34; inhibitor of IK1). In LF arteries, endothelium-dependent relaxation was markedly reduced, due to a reduction in the endothelium-derived hyperpolarizing factor (EDHF) response. In HF arteries, the balance between the NO/prostanoid versus EDHF response was unaltered. However, the contribution of IK1 to the EDHF response was enhanced, as indicated by a larger effect of TRAM-34 and a larger residual NS309-induced relaxation in the presence of UCL 1684. Reduction of blood flow selectively blunts EDHF relaxation in resistance arteries through inhibition of the function of K(Ca) channels. An increase in blood flow leads to a more prominent role of IK1 channels in this relaxation.

Essential role of nitric oxide in sepsis-induced impairment of endothelium-derived hyperpolarizing factor-mediated relaxation in rat pulmonary artery

Both endothelial nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) are important vasodilators in pulmonary circulation. Sepsis is known to impair endothelium-dependent dilation in the pulmonary vasculature, but the mechanisms are incompletely understood. We have examined the relative contribution of EDHF/NO to the attenuated endothelium-dependent relaxation of pulmonary artery in sepsis, and the role of inducible nitric oxide synthase (iNOS)-derived NO in this mechanism. Sepsis was induced in male adult Wistar rats by caecal ligation and puncture. At 18h after surgery, left and right branches of pulmonary arteries were isolated for tension recording, NO/cyclic guanosine monophosphate (cGMP) measurements, mRNA and protein expressions. Despite a marked decrease in the arterial endothelial nitric oxide synthase (eNOS) mRNA and phosphorylated-eNOS (p-eNOS) protein expressions in sepsis, endothelium-dependent relaxation to acetylcholine (ACh) mediated by NO, acetylcholine-stimulated NO release and tissue cGMP levels were moderately inhibited. Sepsis however abolished the N(G)-Nitro-l-arginine methyl ester (L-NAME)/indomethacin-resistant arterial relaxation (EDHF response) to acetylcholine in this vessel. In vitro treatment of the arterial rings from septic rats with 1400W, a selective inhibitor of iNOS restored the EDHF response, but had no effect on the acetylcholine-induced relaxation mediated by endothelial NO. The functional role of iNOS-derived NO in impairing EDHF-mediated relaxation was coincident with an increased basal NO production, iNOS mRNA and protein expressions in the rat pulmonary artery. In conclusion, the loss of the EDHF response may be primarily responsible for the endothelial dysfunction in sepsis, and its restoration by a selective iNOS inhibitor may improve pulmonary vasodilation.

Atorvastatin Restores the Impaired Vascular Endothelium-dependent Relaxations Mediated by Nitric Oxide and Endothelium-derived Hyperpolarizing Factors but Not Hypotension in Sepsis

Sepsis has been reported to impair endothelium-dependent vasodilations mediated by nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF). Although some studies demonstrate that statins can improve NO-mediated response in septic animals, little is known about its effect on the EDHF response. The present study examined the effects of atorvastatin pretreatment on sepsis-induced endothelial dysfunctions and hypotension in rats. Eighteen hours after the induction of sepsis by cecal ligation and puncture, thoracic aorta and second generation pulmonary arteries were isolated to examine acetylcholine-induced endothelium-dependent dilations mediated by NO and EDHF, respectively. The messenger RNA (mRNA) expression for endothelial NO synthase (eNOS) and inducible NO synthase (iNOS) was done by real-time polymerase chain reaction. NO was measured as nitrate/nitrite release using Griess method. Mean arterial pressure was measured by the invasive method. Sepsis significantly decreased (26%) the relaxation response to acetylcholine in the rat aorta. It also markedly inhibited the eNOS mRNA expression and acetylcholine-stimulated NO release in this vessel. Pretreatment of the rats with atorvastatin (10 mg/kg, orally) 48, 24, and 2 hours before induction of sepsis preserved acetylcholine-induced relaxation, eNOS mRNA expression, acetylcholine-stimulated NO release, and attenuated increase in the inducible NO synthase mRNA expression and basal NO production in the aorta. The maximal EDHF response mediated by acetylcholine was 25.30% +/- 3.00% in the pulmonary artery. Sepsis abolished this response but atorvastatin restored it (22.55% +/- 2.50%). Atorvastatin, however, failed to prevent sepsis-induced hypotension. These results suggest that atorvastatin can restore impaired endothelium-dependent vasodilations mediated by NO and EDHF but not hypotension in sepsis.

Impairment of calcium-activated potassium channels in endothelium-derived hyperpolarizing factor responses in spontaneously hypertensive rats

Impairment of calcium-activated potassium channels in endothelium-derived hyperpolarizing factor responses in spontaneously hypertensive rats

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.

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.

Cilostazol improves endothelial dysfunction by increasing endothelium-derived hyperpolarizing factor response in mesenteric arteries from Type 2 diabetic rats

Diabetes mellitus impairs endothelial function, an effect that can be considered a hallmark of the development of cardiovascular diseases in diabetics. Cilostazol, a selective phosphodiesterase 3 inhibitor, is currently used to treat patients with diabetic vascular complications. However, the effects of cilostazol on responses mediated by endothelium-derived relaxing [in particular, nitric oxide (NO) and hyperpolarizing factors (EDHF)] and contracting factors remain unclear. Here, we hypothesized that cilostazol could improve endothelial dysfunctions in mesenteric arteries isolated from type 2 diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Using cilostazol-treated (100 mg/kg/day for 4 weeks) or -untreated OLETF and control (Long Evans Tokushima Otsuka) rats, we examined the acetylcholine-induced endothelium-dependent responses and the cell-permeant cyclic adenosine monophosphate (cAMP) analog-induced relaxations in the superior mesenteric artery. We also determined blood parameters in these animals. In OLETF rats, chronic treatment with cilostazol reduced the blood levels of triglyceride, non-esterified fatty acids, and leptin, and increased antioxidant capacity, but did not alter the blood glucose or insulin levels. In studies on mesenteric arteries from cilostazol-treated OLETF animals, the cilostazol treatment improved: (a) the acetylcholine-induced EDHF-mediated relaxation and (b) the cAMP-mediated relaxation. However, cilostazol did not alter the NO-mediated relaxation or the endothelium-derived contracting factor-mediated contraction. These results suggest that cilostazol improves endothelial functions in OLETF mesenteric arteries by increasing EDHF signaling, and that it normalizes some metabolic abnormalities in OLETF rats. On that basis, cilostazol may prove to be a potent drug for the clinical treatment of diabetic vasculopathy.

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.

Sex differences in vascular function: implication of endothelium-derived hyperpolarizing factor

The vascular endothelium plays a crucial role in the regulation of vascular homeostasis by controlling vascular tone, coagulation, and inflammatory responses. These actions are exerted by endothelial factors including nitric oxide, prostacyclin, and endothelium-derived hyperpolarizing factor (EDHF). The greater incidence of cardiovascular disease (CVD) in men and postmenopausal women compared with premenopausal women implies a vasoprotective phenotype of females, which may be influenced by sex hormones. These hormones, particularly estrogen, have modulatory effects on the endothelium and circulating cells that have been implicated in vascular inflammation and in the development of CVD. EDHF seems to be the predominant endothelial factor in the resistance vasculature of females and this mediator could afford the beneficial cardiovascular risk profile observed in premenopausal woman. In this review, we discuss sex differences in EDHF biology and how sex hormones can modulate EDHF responses. We also review the implication of sex hormone-dependent regulation of EDHF in inflammatory processes, platelet function, and repair after vascular damage, each of which have a critical role in several aspects of the pathogenesis of CVD.