Altered myogenic constriction and endothelium-derived hyperpolarizing factor-mediated relaxation in small mesenteric arteries of hypertensive subtotally nephrectomized rats

OBJECTIVES: Chronic renal failure (CRF) is associated with altered systemic arterial tone and hypertension. Myogenic constriction and endothelium-derived hyperpolarizing factor (EDHF)-dependent relaxation represent major vasoregulatory mechanisms in small systemic arteries. Elevated myogenic response and impaired EDHF might participate in the development of essential hypertension; however, their role in CRF-related hypertension is unknown. We investigated whether myogenic response and EDHF are altered in subtotally nephrectomized (sNX) rats and whether these changes are modifiable by chronic treatment with angiotensin-converting enzyme (ACE) inhibitor. METHODS: In a pressure arteriograph, myogenic constriction and EDHF-mediated relaxation were evaluated in small mesenteric arteries isolated from male Wistar rats 15 weeks after either sham operation (n = 7) (SHAM), sNX (n = 12) or sNX followed by 9 weeks of treatment with lisinopril (sNX + LIS, 2.5 mg/kg, n = 13). RESULTS: Surprisingly, myogenic response was reduced in hypertensive CRF rats (maximal myogenic tone: 37 +/- 2 and 18 +/- 4%, P < 0.01; peak myogenic index: -0.80 +/- 0.08 and -0.40 +/- 0.12%/mmHg, P < 0.05 in SHAM and sNX respectively). At the same time EDHF-mediated relaxation was also impaired (maximal response: 92 +/- 2 and 77 +/- 5%, P < 0.01; pD2: 6.5 +/- 0.1 and 5.9 +/- 0.1, P < 0.05). Both myogenic response and EDHF were inversely related to the severity of renal failure and restored by treatment with lisinopril to levels found in SHAM animals. CONCLUSION: Major constrictive (myogenic) and dilatory (EDHF) mechanisms of small systemic arteries are impaired in hypertensive CRF rats. These alterations do not seem to participate in the development of hypertension, being rather directly related to the severity of renal impairment. Both systemic vascular changes might be restored by renoprotective treatment with ACE inhibitor.     

Identification of a cytochrome P450 2C9-derived endothelium-derived hyperpolarizing factor in essential hypertensive patients.

OBJECTIVES: We assessed the role of cytochrome P450 2C9 (CYP 2C9)-derived endothelium-derived hyperpolarizing factor (EDHF) in the forearm microcirculation of essential hypertensive patients (EH) by utilizing sulfaphenazole (SUL), a selective CYP 2C9 inhibitor. BACKGROUND: In EH patients, EDHF acts as a compensatory pathway when nitric oxide (NO) availability is reduced. Cytochrome P450 2C9 is a possible source of EDHF. METHODS: In 36 healthy subjects (normotensive [NT]) and 32 hypertensive patients (HT), we studied forearm blood flow (strain-gauge plethysmography) changes induced by intraarterial acetylcholine (ACH) and bradykinin (BDK), repeated during N(G)-monomethyl-L-arginine (L-NMMA) (100 mug/100 ml/min) or SUL (0.03 mg/100 ml/min). In HT, the effect of SUL on ACH and BDK was repeated during vitamin C (8 mg/100 ml/min). Sodium nitroprusside (SNP) was utilized as control. RESULTS: In NT, vasodilation to ACH and BDK was blunted by L-NMMA and not changed by SUL. In contrast, in HT responses to ACH and BDK, reduced compared with NT, were resistant to L-NMMA. In these patients, SUL blunted vasodilation to ACH and to a greater extent the response to BDK. When retested with vitamin C, SUL was no longer effective on both endothelial agonists. In 2 final groups of normotensive control subjects, vasodilation to ACH or BDK residual to cyclooxygenase and L-NMMA blockade was further inhibited by simultaneous SUL infusion. Response to SNP, similar between NT and HT, was unaffected by SUL. CONCLUSIONS: Cytochrome P450 epoxygenase-derived EDHF acts as a partial compensatory mechanism to sustain endothelium-dependent vasodilation in HT, particularly the BDK-mediated response, when NO activity is impaired because of oxidative stress.     

Hyperhomocysteinemia impairs endothelium-derived hyperpolarizing factor-mediated vasorelaxation in transgenic cystathionine beta synthase-deficient mice

Hyperhomocysteinemia (HHcy) is associated with endothelial dysfunction (ED), but the mechanism is largely unknown. In this study, we investigated the role and mechanism of HHcy-induced ED in microvasculature in our newly established mouse model of severe HHcy (plasma total homocysteine, 169.5 μM). We found that severe HHcy impaired nitric oxide (NO)- and endothelium-derived hyperpolarizing factor (EDHF)-mediated, endothelium-dependent relaxations of small mesenteric arteries (SMAs). Endothelium-independent and prostacyclin-mediated endothelium-dependent relaxations were not changed. A nonselective Ca(2+)-activated potassium channel (K(Ca)) inhibitor completely blocked EDHF-mediated relaxation. Selective blockers for small-conductance K(Ca) (SK) or intermediate-conductance K(Ca) (IK) failed to inhibit EDHF-mediated relaxation in HHcy mice. HHcy increased the levels of SK3 and IK1 protein, superoxide (O(2)(-)), and 3-nitrotyrosine in the endothelium of SMAs. Preincubation with antioxidants and peroxynitrite (ONOO(-)) inhibitors improved endothelium-dependent and EDHF-mediated relaxations and decreased O(2)(-) production in SMAs from HHcy mice. Further, EDHF-mediated relaxation was inhibited by ONOO(-) and prevented by catalase in the control mice. Finally, L-homocysteine stimulated O(2)(-) production, which was reversed by antioxidants, and increased SK/IK protein levels and tyrosine nitration in cultured human cardiac microvascular endothelial cells. Our results suggest that HHcy impairs EDHF relaxation in SMAs by inhibiting SK/IK activities via oxidation- and tyrosine nitration-related mechanisms.     

Incidence of myoendothelial gap junctions in the proximal and distal mesenteric arteries of the rat is suggestive of a role in endothelium-derived hyperpolarizing factor-mediated responses

Although the chemical nature of endothelium-derived hyperpolarizing factor (EDHF) remains elusive, electrophysiological evidence exists for electrical communication between smooth muscle cells and endothelial cells suggesting that electrotonic propagation of hyperpolarization may explain the failure to identify a single chemical factor as EDHF. Anatomical evidence for myoendothelial gap junctions, or the sites of electrical coupling, is, however, rare. In the present study, serial-section electron microscopy and reconstruction techniques have been used to examine the incidence of myoendothelial gap junctions in the proximal and distal mesenteric arteries of the rat where EDHF responses have been reported to vary. Myoendothelial gap junctions were found to be very small in the mesenteric arteries, the majority being <100 nm in diameter. In addition, they were significantly more common in the distal compared with the proximal regions of this arterial bed. Pentalaminar gap junctions between adjacent endothelial cells were much larger and were common in both proximal and distal mesenteric arteries. These latter junctions were frequently found near the myoendothelial gap junctions. These results provide the first evidence for the presence of sites for electrical communication between endothelial cells and smooth muscle cells in the mesenteric vascular bed. Furthermore, the relative incidence of these sites suggests that there may be a relationship between the activity of EDHF and the presence of myoendothelial gap junctions.     

Endothelium-derived hydrogen peroxide accounts for the enhancing effect of an angiotensin-converting enzyme inhibitor on endothelium-derived hyperpolarizing factor-mediated responses in mice

Background- We have recently identified that endothelium-derived hydrogen peroxide (H2O2) is an endothelium-derived hyperpolarizing factor (EDHF) in animals and humans, for which endothelial nitric oxide synthase (eNOS) is an important source. Angiotensin-converting enzyme (ACE) inhibitors are known to enhance EDHF-mediated responses. In this study, we examined whether endothelium-derived H2O2 accounts for the enhancing effect of an ACE inhibitor on EDHF-mediated responses and, if so, what mechanism is involved. METHODS AND RESULTS: Control and eNOS-/- mice were maintained with or without temocapril (10 mg/kg per day orally) for 4 weeks, and isometric tensions and membrane potentials of mesenteric arteries were recorded. In control mice, temocapril treatment significantly enhanced EDHF-mediated relaxations and hyperpolarizations to acetylcholine (n=8 each). Catalase, a specific scavenger of H2O2, abolished the beneficial effects of temocapril, although it did not affect endothelium-independent relaxations to sodium nitroprusside or NS1619, a direct opener of K(Ca) channels (n=6 each). Western blot analysis demonstrated that the temocapril treatment significantly upregulated the expression of eNOS. By contrast, this enhancing effect of temocapril was absent in eNOS-/- mice (n=6). CONCLUSIONS: These results indicate that endothelium-derived H2O2 accounts for the enhancing effect of temocapril on EDHF-mediated responses caused in part by eNOS upregulation, further supporting our H2O2 theory.     

Substance P dilates epicardial coronary arteries and increases coronary blood flow in humans

The effects of intracoronary infusions of substance P in conscious humans on epicardial vessel diameter and coronary sinus oxygen saturation were investigated in 13 patients who had angiographically normal coronary arteries. The dose of substance P ranged from 2.8 to 89.6 pmol/min, starting at 2.8 pmol/min and rising by doubling increments. All infusions were performed for 2-minute periods. Epicardial vessel diameter was measured by a computerized analysis system (CAAS) of the angiogram performed at the end of each infusion period. Coronary sinus oxygen saturation (CSO2) was measured continuously by a fiber-optic oximeter catheter in the coronary sinus. A reduction of arterial pressure, indicative of a systemic effect of substance P, occurred only when infusions were 44.8 pmol/min or more. At considerably smaller dosages, a dose-dependent increase in the left anterior descending artery diameter was seen with substance P; maximal dilation occurred at a dosage of 11.2 pmol/min. The percent increase in vessel diameter for this dosage was 29.4 +/- 19.8% (mean +/- SD) at the distal site of analysis and 16.7 +/- 10% at the proximal site of analysis. CSO2 rose in a dose-dependent manner and was maximal at a dosage of 11.2 pmol/min, which caused a 14.6 +/- 7.2% O2 rise above the preinfusion saturation of 43.5 +/- 11.1% O2. As the heart rate-blood pressure double product showed no change, it is argued that these changes in CSO2 probably reflect rises in coronary flow that were seen with dosages of substance P without systemic effect. In three patients, preconstriction induced by ergonovine was reversed by substance P infusions, which produced a degree of dilation equivalent to that of isosorbide dinitrate. We conclude that substance P at very small dosages, which are without peripheral effects, causes in vivo epicardial coronary artery dilatation as well as resistive vessel dilatation in humans.     

Role of endothelium-derived relaxing factor and prostaglandins in responses of coronary arteries to thromboxane in vivo

We examined the relative contribution of endothelial and vascular smooth muscle-derived prostaglandins and endothelium-derived relaxing factor in modulating both the large coronary artery and resistance vessel responses to thromboxane in vivo. Vascular responses to the thromboxane analogue U46619 were measured in four separate experimental protocols: 1) The vascular responses were measured in the presence and absence of intact endothelium to examine the role of endothelium-derived vasodilators. 2) Responses were measured in the presence of intact endothelium before and after inhibition of cyclooxygenase with indomethacin to examine the role of endothelial and vascular smooth muscle-derived prostaglandins. 3) Responses were measured after endothelial removal before and after indomethacin to examine the role of vascular smooth muscle-derived prostaglandins. 4) Responses were measured after indomethacin and before and after removal of endothelium to examine the role of endothelium-derived relaxing factor. In anesthetized dogs (n = 41) that underwent constant pressure perfusion of the left anterior descending coronary artery (LAD), LAD diameter was measured with sonomicrometer crystals, and coronary flow was measured with an electromagnetic flow probe. Intracoronary infusion of U46619 (0.01-1.0 microgram/min) produced a dose-dependent constriction of LAD. Constriction of the LAD was augmented after endothelial removal, after indomethacin treatment in both the presence and absence of endothelium, and after removal of the endothelium in the presence of indomethacin. Inhibition of prostaglandin synthesis had the greatest effect of augmenting constriction of LAD to thromboxane. Coronary flow was decreased by U46619 only in the presence of indomethacin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of endothelial cell KCa3.1 channels during endothelium-derived hyperpolarizing factor signaling in mesenteric resistance arteries

Arterial hyperpolarization to acetylcholine (ACh) reflects coactivation of K(Ca)3.1 (IK(Ca)) channels and K(Ca)2.3 (SK(Ca)) channels in the endothelium that transfers through myoendothelial gap junctions and diffusible factor(s) to affect smooth muscle relaxation (endothelium-derived hyperpolarizing factor [EDHF] response). However, ACh can differentially activate K(Ca)3.1 and K(Ca)2.3 channels, and we investigated the mechanisms responsible in rat mesenteric arteries. K(Ca)3.1 channel input to EDHF hyperpolarization was enhanced by reducing external [Ca(2+)](o) but blocked either with forskolin to activate protein kinase A or by limiting smooth muscle [Ca(2+)](i) increases stimulated by phenylephrine depolarization. Imaging [Ca(2+)](i) within the endothelial cell projections forming myoendothelial gap junctions revealed increases in cytoplasmic [Ca(2+)](i) during endothelial stimulation with ACh that were unaffected by simultaneous increases in muscle [Ca(2+)](i) evoked by phenylephrine. If gap junctions were uncoupled, K(Ca)3.1 channels became the predominant input to EDHF hyperpolarization, and relaxation was inhibited with ouabain, implicating a crucial link through Na(+)/K(+)-ATPase. There was no evidence for an equivalent link through K(Ca)2.3 channels nor between these channels and the putative EDHF pathway involving natriuretic peptide receptor-C. Reconstruction of confocal z-stack images from pressurized arteries revealed K(Ca)2.3 immunostain at endothelial cell borders, including endothelial cell projections, whereas K(Ca)3.1 channels and Na(+)/K(+)-ATPase alpha(2)/alpha(3) subunits were highly concentrated in endothelial cell projections and adjacent to myoendothelial gap junctions. Thus, extracellular [Ca(2+)](o) appears to modify K(Ca)3.1 channel activity through a protein kinase A-dependent mechanism independent of changes in endothelial [Ca(2+)](i). The resulting hyperpolarization links to arterial relaxation largely through Na(+)/K(+)-ATPase, possibly reflecting K(+) acting as an EDHF. In contrast, K(Ca)2.3 hyperpolarization appears mainly to affect relaxation through myoendothelial gap junctions. Overall, these data suggest that K(+) and myoendothelial coupling evoke EDHF-mediated relaxation through distinct, definable pathways.     

Endothelial G protein beta-subunits trigger nitric oxide-but not endothelium-derived hyperpolarizing factor-dependent dilation in rabbit resistance arteries

A single subtype of heterotrimeric G protein-coupled receptor controls both nitric oxide (NO) (sensitive to L-arginine analogues) and endothelium-derived hyperpolarizing factor (EDHF) (sensitive to high-external K(+) and apamine) production by the vascular endothelium leading to dilation. We hypothesized that alpha- and betagamma-subunits of the G protein serve as distinct intermediates to produce NO and EDHF. In pressurized resistance arteries, selective pinocytotic endothelial incorporation of specific antibodies (Abs) directed against alpha(q/11)-subunits abolished acetylcholine (Ach)-mediated dilation but failed to influence oxymetazoline (Oxy, alpha(2)-adrenergic receptor agonist)-induced dilation. In contrast, alpha(i1-2)-subunit Abs prevented Oxy- but not Ach-induced dilation. Thus, as expected, endothelial muscarinic and alpha(2)-adrenoceptors couple to G(q) protein and G(i) proteins, respectively. beta-subunit Abs reduced both Ach- and Oxy-induced dilation. The beta-subunit Abs abolished the nitro-L-arginine (L-NNA)-sensitive component but did not impair the high-external K(+)-sensitive component of the dilation induced by Ach and Oxy. Thus, G protein beta-subunits primarily accounted for NO production. Neutralization of Hsp90 and inhibition of the phospholipase C by U73122 (1 micromol/L) or intracellular Ca(2+) buffering with BAPTA-AM (10 micromol/L) sharply reduced NO-dependent but not K(+)-sensitive dilation. In conclusion, mobilization of the G protein beta-subunit is pivotal to NO-dependent dilation triggered through muscarinic and alpha(2)-adrenergic receptors. In contrast, receptor-operated EDHF-dependent dilation was insensitive to beta-subunit Abs. Although not directly activating the NO pathway, alpha-subunit activation is an absolute prerequisite for receptor-operated endothelium-dependent dilation of resistance arteries.     

Impaired endothelium-derived hyperpolarizing factor-mediated dilations and increased blood pressure in mice deficient of the intermediate-conductance Ca2+-activated K+ channel

The endothelium plays a key role in the control of vascular tone and alteration in endothelial cell function contributes to several cardiovascular disease states. Endothelium-dependent dilation is mediated by NO, prostacyclin, and an endothelium-derived hyperpolarizing factor (EDHF). EDHF signaling is thought to be initiated by activation of endothelial Ca(2+)-activated K(+) channels (K(Ca)), leading to hyperpolarization of the endothelium and subsequently to hyperpolarization and relaxation of vascular smooth muscle. In the present study, we tested the functional role of the endothelial intermediate-conductance K(Ca) (IK(Ca)/K(Ca)3.1) in endothelial hyperpolarization, in EDHF-mediated dilation, and in the control of arterial pressure by targeted deletion of K(Ca)3.1. K(Ca)3.1-deficient mice (K(Ca)3.1(-/-)) were generated by conventional gene-targeting strategies. Endothelial K(Ca) currents and EDHF-mediated dilations were characterized by patch-clamp analysis, myography and intravital microscopy. Disruption of the K(Ca)3.1 gene abolished endothelial K(Ca)3.1 currents and significantly diminished overall current through K(Ca) channels. As a consequence, endothelial and smooth muscle hyperpolarization in response to acetylcholine was reduced in K(Ca)3.1(-/-) mice. Acetylcholine-induced dilations were impaired in the carotid artery and in resistance vessels because of a substantial reduction of EDHF-mediated dilation in K(Ca)3.1(-/-) mice. Moreover, the loss of K(Ca)3.1 led to a significant increase in arterial blood pressure and to mild left ventricular hypertrophy. These results indicate that the endothelial K(Ca)3.1 is a fundamental determinant of endothelial hyperpolarization and EDHF signaling and, thereby, a crucial determinant in the control of vascular tone and overall circulatory regulation.     

Expression of cytochrome P4502E1 gene in hepatocellular carcinoma

AIM: To investigate cytochrome P4502E1 (CYP2E1) gene expression in occurrence and progression of hepatocellular carcinoma (HCC). METHODS: The human liver arrayed library was spotted onto the nylon membranes to make cDNA array. Hybridization of cDNA array was performed with labeled probes synthesized from RNA isolated from HCC and adjacent liver tissues. Sprague-Dawley rats were administrated diethylnitrosamine (DENA) to induce HCC. CYP2E1 expression was detected by the method of RT-PCR and Northern blot analysis. RESULTS: CYP2E1 was found by cDNA array hybridization to express differently between HCC and liver tissues. CYP2E1 only expressed in liver, but did not express in HCC tissues and expressed lowly in cirrhotic tissues. In the progression of cirrhosis and HCC, the expression level of CYP2E1 was gradually decreased and hardly detected until the late stage of HCC. CONCLUSION: Using arrayed library to make cDNA arrays is an effective method to find differential expression genes. CYP2E1 is a unique gene expressing in liver but did not express in HCC. CYP2E1 expression descended along with the initiation and progression of HCC, which is noteworthy further investigations in its significance in the development of HCC.     

Cytochrome P450 2C expression and EDHF-mediated relaxation in porcine coronary arteries is increased by cortisol

OBJECTIVES/METHODS: In addition to nitric oxide (NO) and prostacyclin, endothelium-dependent dilation is mediated by the endothelium-derived hyperpolarizing factor (EDHF) which, in the coronary circulation, has been characterised as a metabolite of arachidonic acid synthesised by an cytochrome P450 (CYP) epoxygenase homologous to CYP 2C8/9. As the promotor regions of CYP 2C8 and 2C9 contain consensus sequences for glucocorticoid response elements, we determined the effect of cortisol on EDHF-mediated relaxations as well as on the expression of CYP 2C in isolated segments of porcine coronary artery. RESULTS: Bradykinin-induced NO-mediated relaxation of KCl-constricted arterial rings was slightly attenuated following exposure to cortisol. However, EDHF-mediated relaxations of U46619-constricted arterial rings assessed in the presence of the cyclo-oxygenase inhibitor diclofenac and the NO synthase inhibitor N(omega)nitro-L-arginine (0.3 mM), were significantly enhanced (maximum relaxation: 66+/-7%, P<0.05 vs. control rings: 36+/-8%). Cortisol treatment (0.1 microM, 24 h) did not affect the endothelium-independent relaxation elicited by sodium nitroprusside and acute incubation with cortisol (0.1 microM, 30 min) did not alter either NO- or EDHF-mediated responses. The expression of CYP 2C (quantified by RT-PCR, Western blot analysis and confocal microscopy) was enhanced in porcine coronary endothelial cells following incubation with cortisol for 18-24 h. CONCLUSIONS: These results demonstrate the concomitant upregulation of EDHF-mediated relaxations and CYP 2C expression by long-term treatment with cortisol. These observations support the concept that an epoxygenase homologous to CYP 2C8/9 plays a crucial role in the generation of EDHF-mediated responses in the coronary endothelium.     

Endothelium-derived hyperpolarizing factor synthase (Cytochrome P450 2C9) is a functionally significant source of reactive oxygen species in coronary arteries

In the porcine coronary artery, a cytochrome P450 (CYP) isozyme homologous to CYP 2C8/9 has been identified as an endothelium-derived hyperpolarizing factor (EDHF) synthase. As some CYP enzymes are reported to generate reactive oxygen species (ROS), we hypothesized that the coronary EDHF synthase may modulate vascular homeostasis by the simultaneous production of ROS and epoxyeicosatrienoic acids. In bradykinin-stimulated coronary arteries, antisense oligonucleotides against CYP 2C almost abolished EDHF-mediated responses but potentiated nitric oxide (NO)-mediated relaxation. The selective CYP 2C9 inhibitor sulfaphenazole and the superoxide anion (O(2-)) scavengers Tiron and nordihydroguaretic acid also induced a leftward shift in the NO-mediated concentration-relaxation curve to bradykinin. CYP activity and O(2-) production, determined in microsomes prepared from cells overexpressing CYP 2C9, were almost completely inhibited by sulfaphenazole. Sulfaphenazole did not alter the activity of either CYP 2C8, the leukocyte NADPH oxidase, or xanthine oxidase. ROS generation in coronary artery rings, visualized using either ethidium or dichlorofluorescein fluorescence, was detected under basal conditions. The endothelial signal was attenuated by CYP 2C antisense treatment as well as by sulfaphenazole. In isolated coronary endothelial cells, bradykinin elicited a sulfaphenazole-sensitive increase in ROS production. Although 11,12 epoxyeicosatrienoic acid attenuated the activity of nuclear factor-kappaB in cultured human endothelial cells, nuclear factor-kappaB activity was enhanced after the induction or overexpression of CYP 2C9, as was the expression of vascular cell adhesion molecule-1. These results suggest that a CYP isozyme homologous to CYP 2C9 is a physiologically relevant generator of ROS in coronary endothelial cells and modulates both vascular tone and homeostasis.     

Hydrogen peroxide is an endothelium-derived hyperpolarizing factor in animals and humans

The endothelium plays an important role in maintaining vascular homeostasis by synthesizing and releasing several vasodilating substances, including vasodilator prostaglandins, nitric oxide (NO), and endothelium-derived hyperpolarizing factor (EDHF). Since the first report for the existence of EDHF, several substances/mechanisms have been proposed for the nature of EDHF, including epoxyeicosatrienoic acids (metabolites of arachidonic P450 epoxygenase pathway), K ions, and electrical communications through myoendothelial gap junctions. We have recently demonstrated that endothelium-derived hydrogen peroxide (H(2)O(2)) is an EDHF in mouse and human mesenteric arteries and in porcine coronary microvessels. For the synthesis of H(2)O(2) as an EDHF, endothelial Cu,Zn-superoxide dismutase plays an important role in mesenteric arteries of mice and humans. We also have demonstrated that EDHF-mediated responses are attenuated by several arteriosclerotic risk factors, including diabetes mellitus and hyperlipidemia and their combination in particular. Recent studies have indicated that endothelium-derived H(2)O(2) plays an important protective role in coronary autoregulation and myocardial ischemia/reperfusion injury in vivo. Indeed, our H(2)O(2)/EDHF theory demonstrates that endothelium-derived H(2)O(2), another reactive oxygen species in addition to NO, plays an important role as a redox signaling molecule to cause vasodilatation as well as cardioprotection. In this review, we summarize our knowledge on H(2)O(2)/EDHF regarding its identification, mechanisms of synthesis, and clinical implications.     

Rapid endothelial cell-selective loading of connexin 40 antibody blocks endothelium-derived hyperpolarizing factor dilation in rat small mesenteric arteries

In resistance arteries, spread of hyperpolarization from the endothelium to the adjacent smooth muscle is suggested to be a crucial component of dilation resulting from endothelium-derived hyperpolarizing factor (EDHF). To probe the role of endothelial gap junctions in EDHF-mediated dilation, we developed a method, which was originally used to load membrane impermeant molecules into cells in culture, to load connexin (Cx)-specific inhibitory molecules rapidly (approximately 15 minutes) into endothelial cells within isolated, pressurized mesenteric arteries of the rat. Validation was achieved by luminally loading cell-impermeant fluorescent dyes selectively into virtually all the arterial endothelial cells, without affecting either tissue morphology or function. The endothelial monolayer served as an effective barrier, preventing macromolecules from entering the underlying smooth muscle cells. Using this technique, endothelial cell loading either with antibodies to the intracellular carboxyl-terminal region of Cx40 (residues 340 to 358) or mimetic peptide for the cytoplasmic loop (Cx40; residues 130 to 140) each markedly depressed EDHF-mediated dilation. In contrast, multiple antibodies directed against different intracellular regions of Cx37 and Cx43, and mimetic peptide for the intracellular loop region of Cx37, were each without effect. Furthermore, simultaneous intra- and extraluminal incubation of pressurized arteries with inhibitory peptides targeted against extracellular regions of endothelial cell Cxs (43Gap 26, 40Gap 27, and (37,43)Gap 27; 300 micromol/L each) for 2 hours also failed to modify the EDHF response. High-resolution immunohistochemistry localized Cx40 to the end of endothelial cell projections at myoendothelial gap junctions. These data directly demonstrate a critical role for Cx40 in EDHF-mediated dilation of rat mesenteric arteries.