Myoendothelial gap junction frequency does not account for sex differences in EDHF responses in rat MCA

Previous findings from our laboratory have shown that dilations to endothelium-derived hyperpolarizing factor (EDHF) in rat middle cerebral artery (MCA) are less in females compared to males. Myoendothelial gap junctions (MEGJs) appear to mediate the transfer of hyperpolarization between endothelium and smooth muscle in males. In the present study, we hypothesized that MEGJs are the site along the EDHF pathway which is compromised in female rat MCA. Membrane potential in endothelium was measured using the voltage-sensitive dye di-8-ANEPPS and in smooth muscle using intracellular glass microelectrodes in the presence of l-NAME (3x10(-5 )M) and indomethacin (10(-5 )M). Electron microscopy was used to assess MEGJ characteristics. In endothelial cells, the di-8-ANEPPS fluorescence ratio change to 10(-5 )M UTP was similar in males (-2.9+/-0.5%) and females (-3.2+/-0.2%), indicating comparable degrees of endothelial cell hyperpolarization. However, smooth muscle cell hyperpolarization to 10(-5 )M UTP was significantly attenuated in females (0 mV hyperpolarization; -31+/-1.5 mV resting) compared to males (8 mV hyperpolarization; -28+/-1.7 mV resting). Ultrastructural evidence suggested that MEGJ frequency and area of contact were comparable between males and females. Taken together, our data suggest that in rat MCA, MEGJ frequency does not account for the reduced EDHF responses observed in females compared to males. We conclude that reduced myoendothelial coupling and/or homocellular coupling within the media may account for these differences.     

Myoendothelial gap junctions may provide the pathway for EDHF in mouse mesenteric artery

Endothelium-dependent hyperpolarization of vascular smooth muscle provides a major pathway for relaxation in resistance arteries. This can occur due to direct electrical coupling via myoendothelial gap junctions (MEGJs) and/or the release of factors (EDHF). Here we provide evidence for the existence of functional MEGJs in the same, defined branches of BALB/C mouse mesenteric arteries which show robust EDHF-mediated smooth muscle relaxation. Cyclopiazonic acid (CPA, 10 microM) was used to stimulate EDHF in arteries mounted under isometric conditions and constricted with phenylephrine. Simultaneous measurement of smooth muscle membrane potential and tension demonstrated that CPA caused a hyperpolarization of around 10 mV, reversing the depolarization to phenylephrine by 94% and the associated constriction by 66%. The relaxation to CPA was endothelium dependent, associated with the opening of Ca2+-activated K channels, and only in part due to the release of nitric oxide (NO). In the presence of the NO synthase inhibitor, L-NAME (100 microM), the relaxation to CPA could be almost completely inhibited with the putative gap junction uncoupler, carbenoxolone (100 microM). Inhibition of the synthesis of prostaglandins or metabolites of arachidonic acid had no effect under the same conditions, and small rises in exogenous K+ failed to evoke consistent or marked smooth muscle relaxation, arguing against a role for these molecules and ions as EDHF. Serial section electron microscopy revealed a high incidence of MEGJs, which was correlated with heterocellular dye coupling. Taken together, these functional and morphological data from a defined mouse resistance artery suggest that the EDHF response in this vessel may be explained by extensive heterocellular coupling through MEGJs, enabling spread of hyperpolarizing current.     

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.     

cAMP facilitates EDHF-type relaxations in conduit arteries by enhancing electrotonic conduction via gap junctions

We have investigated the role of cAMP in NO- and prostanoid-independent relaxations that are widely attributed to an endothelium-derived hyperpolarizing factor (EDHF). Under control conditions EDHF-type relaxations evoked by acetylcholine (ACh) in rabbit iliac arteries were transient, but in the presence of the cAMP phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) or the cell permeant cAMP analog 8-bromo-cAMP, relaxations became sustained with their maxima potentiated approximately 2-fold. Relaxation was associated with transient approximately 1.5-fold elevations in smooth muscle cAMP levels with both mechanical and nucleotide responses being abolished by interrupting gap junctional communication with the connexin-mimetic peptide Gap 27 and by endothelial denudation. However, IBMX induced a sustained endothelium-independent approximately 2-fold rise in cAMP levels, which was not further amplified by ACh, suggesting that the contribution of cAMP to the EDHF phenomenon is permissive. After selective loading of the endothelium with calcein AM, direct transfer of dye from the endothelium to the media was enhanced by IBMX or 8-bromo-cAMP, but not by 8-bromo-cGMP, whereas Gap 27 promoted sequestration within the intima. ACh-induced hyperpolarizations of subintimal smooth muscle in arterial strips with intact endothelium were abolished by Gap 27 and the adenylyl cyclase inhibitor 2',5'-dideoxyadenosine but were unaffected by IBMX. By contrast, in strips partially denuded of endothelium, IBMX enhanced the transmission of hyperpolarization from the endothelium to remote smooth muscle cells. These findings support the hypothesis that endothelial hyperpolarization underpins the EDHF phenomenon, with cAMP governing subsequent electrotonic signaling via both myoendothelial and homocellular smooth muscle gap junctions.     

Electrical coupling between endothelial cells and smooth muscle cells in hamster feed arteries: role in vasomotor control

Endothelial cells (ECs) govern smooth muscle cell (SMC) tone via the release of paracrine factors (eg, NO and metabolites of arachidonic acid). We tested the hypothesis that ECs can promote SMC relaxation or contraction via direct electrical coupling. Vessels (resting diameter, 57+/-3 microm; length, 4 mm) were isolated, cannulated, and pressurized (75 mm Hg; 37 degrees C). Two microelectrodes were used to simultaneously impale 2 cells (ECs or SMCs) in the vessel wall separated by 500 microm. Impalements of one EC and one SMC (n=26) displayed equivalent membrane potentials at rest, during spontaneous oscillations, and during hyperpolarization and vasodilation to acetylcholine. Injection of -0.8 nA into an EC caused hyperpolarization ( approximately 5 mV) and relaxation of SMCs (dilation, approximately 5 microm) along the vessel segment. In a reciprocal manner, +0.8 nA caused depolarization ( approximately 2 mV) of SMCs with constriction ( approximately 2 microm). Current injection into SMCs while recording from ECs produced similar results. We conclude that ECs and SMCs are electrically coupled to each other in these vessels, such that electrical signals conducted along the endothelium can be directly transmitted to the surrounding smooth muscle to evoke vasomotor responses.     

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.     

Heterogeneity of endothelium-dependent responses to acetylcholine in canine femoral arteries and veins. Separation of the role played by endothelial and smooth muscle cells

The purpose of this study was to determine whether heterogeneity in endothelium-dependent responses to acetylcholine between canine blood vessels of different anatomical origin reflects variations in endothelial function or in responsiveness of vascular smooth muscle cells. Experiments were conducted in a bioassay system, where segments of femoral artery or vein with endothelium were perfused intraluminally and the perfusate used to superfuse rings of femoral arteries or veins without endothelium. Indomethacin was present in all experiments to prevent the synthesis of prostanoids. The blood vessels were contracted by phenylephrine. Measurement of wall tension in both the perfused segment and bioassay ring allowed simultaneous detection of endothelium-derived relaxing factor(s) released abluminally (segment) and intraluminally (ring). Intraluminal infusion of acetylcholine (ACh) induced relaxations in the perfused artery but not in vein segments. During arterial superfusion ACh induced relaxation in femoral arterial rings but contraction in venous rings. After treatment with atropine the arterial perfusate evoked relaxations in venous rings. Infusion of ACh through the femoral vein evoked only moderate relaxations in arterial rings. These data demonstrate that depressed endothelium-dependent relaxation to ACh in femoral veins compared to femoral arteries is due to a masking effect of the direct stimulating action of ACh and decreased release of the same mediator or the release of a different relaxing factor from venous endothelium.     

Distinct roles for protease-activated receptors 1 and 2 in vasomotor modulation in rat superior mesenteric artery

OBJECTIVE: Protease-activated receptors (PARs) 1 and 2 are expressed in various blood vessels including rat aorta, modulating vascular tone. We investigated the roles of PAR-1 and PAR-2 in vasomotor modulation in rat superior mesenteric artery. METHODS AND RESULTS: Effects of the PAR-2-activating peptide Ser-Leu-Ile-Gly-Arg-Leu-amide (SLIGRL-amide) and the PAR-1-activating peptide Thr-Phe-Leu-Leu-Arg-amide (TFLLR-amide) on isometric tension were examined in isolated rat superior mesenteric artery or aorta. Both SLIGRL-amide and TFLLR-amide caused relaxation in the precontracted rat aortic rings. The latter peptide, but not the former, produced contraction in the resting rings. NG-nitro-L-arginine methyl ester (L-NAME), but not apamin/charybdotoxin known to block the endothelium-derived hyperpolarizing factor (EDHF) pathway, abolished the relaxation and facilitated the contraction. In the precontracted rat superior mesenteric artery, SLIGRL-amide, but not TFLLR-amide, elicited endothelium-dependent relaxation, which was only partially inhibited by L-NAME with and without indomethacin. The residual relaxation was abolished by apamin/charybdotoxin. Carbenoxolone, a gap junction inhibitor, significantly attenuated the SLIGRL-amide-evoked, EDHF-dependent relaxation, although neither 17-octadecynoic acid, a P450 epoxygenase inhibitor, nor catalase, a hydrogen peroxide scavenger, revealed inhibitory effects. The residual response resistant to carbenoxolone was unaffected by ouabain/BaCl2. In the resting artery, TFLLR-amide, but not SLIGRL-amide, produced only slight contraction, which was dramatically facilitated by combination of L-NAME and apamin/charybdotoxin or by removal of the endothelium. CONCLUSIONS: Our data suggest that, in rat superior mesenteric artery, endothelial PAR-2, upon activation, causes relaxation via both NO and EDHF pathways, and that activation of muscular PAR-1 exhibits potential contractile activity that is largely masked by NO and EDHFs pathways triggered by endothelial PAR-1. Gap junctions might be involved in the EDHF mechanisms in this artery.     

DCEBIO-mediated dilations are attenuated in the female rat middle cerebral artery

BACKGROUND: Unlike in peripheral vessels, the endothelium-derived hyperpolarizing factor (EDHF)-mediated component to P2Y(2) receptor-mediated dilations is significantly attenuated in the middle cerebral artery (MCA) of female rats compared to male rats. One aspect to the EDHF phenomenon is activation of the intermediate calcium-sensitive potassium (IK(Ca)) channels located on the endothelium. In an attempt to pinpoint the site along the EDHF pathway that is compromised in females, we tested the hypothesis that direct activation of IK(Ca) channels with DCEBIO would elicit attenuated hyperpolarization in the endothelium and smooth muscle of females compared to males. METHODS: Inhibitors of nitric oxide synthase and cyclooxygenase were present throughout all experiments. Vessel diameter changes were assessed in pressurized and luminally perfused MCAs. Membrane potential changes in the endothelium and smooth muscle were measured using the perforated patch clamp method and sharp electrodes, respectively. RESULTS AND CONCLUSIONS: The maximum vasodilation to 3 x 10(-4)M DCEBIO was significantly reduced in females (37 +/- 9%) compared to intact males (70 +/- 4%). Endothelial cell hyperpolarization to DCEBIO was similar in both males and females. Smooth muscle cell hyperpolarization was attenuated in females (2 +/- 1 mV) compared to males (15 +/- 3 mV). Taken together, our data suggest that the transfer of hyperpolarization from the endothelium to the smooth muscle is impeded in the female rat MCA.     

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.     

Endothelial cell pathway for conduction of hyperpolarization and vasodilation along hamster feed artery

Acetylcholine (ACh) evokes the conduction of vasodilation along resistance microvessels. However, it is not known which cell layer (endothelium or smooth muscle) serves as the conduction pathway. In isolated, cannulated feed arteries ( approximately 70 microm in diameter at 75 mm Hg; length approximately 4 mm) of the hamster retractor muscle, we tested the hypothesis that endothelial cells provide the pathway for conduction. Microiontophoresis of ACh (500 ms, 500 nA) onto the distal end of a feed artery evoked hyperpolarization (-13+/-2 mV) of both cell layers with vasodilation (15+/-1 microm) along the entire vessel. To selectively damage endothelial cells (confirmed by loss of vasodilation to ACh and labeling of disrupted cells with propidium iodide), an air bubble was perfused through a portion of the vessel lumen, or a 70-kDa fluorescein-conjugated dextran (FCD) was illuminated within a segment (300 microm) of the lumen. After endothelial cell damage, hyperpolarization and vasodilation conducted up to, but not through, the treated segment. To selectively damage smooth muscle cells (confirmed by loss of vasoconstriction to phenylephrine and labeling with propidium iodide), FCD was perifused around the vessel and illuminated. Vasodilation and hyperpolarization conducted past the disrupted smooth muscle cells without attenuation. We conclude that endothelial cells provide the pathway for conducting hyperpolarization and vasodilation along feed arteries in response to ACh.     

Endothelium-Derived Hyperpolarizing Factor: Characterization as a Cytochrome P450 1A-Linked Metabolite of Arachidonic Acid in Perfused Rat Mesenteric Prearteriolar Bed

The isolatedperfused rat mesenteric bed releases endothelium-derived hyperpolarizing factor (EDHF) in response to acetylcholine (ACh) or histamine. I propose that EDHF released in the mesenteric vascular bed is a cytochrome P450 (CYP)-linked, arachidonate metabolite. In the presence of nitro-l-arginine methyl ester (L-NAME) and indomethacin, injections of ACh (0.001 to 10 nmol) or histamine (0.1 to 1,000 nmol) elicited transient, dose-dependent dilation of cirazoline (an α₁-adrenoceptor selective agonist) preconstricted mesenteric beds. The L-NAME-resistant responses to ACh or histamine were insensitive to tetrodotoxin (1 μmol/L), thus negating its neuronal origin, but were profoundly attenuated by a K⁺ channel inhibitor tetrabutylammonium (0.5 mmol/L). 7-Ethoxyresorufin (a selective and competitive blocker of CYP 1A isozyme) blunted ACh and histamine mediated EDHF responses but did not alter vasodilation initiated through K⁺ channel activation by either cromakalim or NS-1619, or through the nitric oxide-cGMP pathway (sodium nitroprusside). Clotrimazole, an imidazole that inhibits CYP by binding to the heme moiety, attenuated ACh, histamine, and cromakalim but not sodium nitroprusside-mediated vasodilator responses. Other CYP isozyme selective inhibitors, such as metyrapone (CYP 2B), 7-pentoxyresorufin (CYP 2B1), sulfaphenazole (CYP 2C/3A), and 17-octadecynoic acid (4A-linked ω-hydroxylase inhibitor), did not alter ACh or histamine-induced EDHF response. The EDHF-mediated dilations initiated by ACh and histamine, as well as K_ATP activation by cromakalim, were blocked by mepacrine, a nonselective phospholipase A₂ inhibitor. Mepacrine did not alter K_Ca activation by compound NS-1619. I conclude that 1) the isolated perfused rat mesenteric prearteriolar bed releases in response to ACh and histamine, an EDHF that causes vasodilation through K⁺ channel activation; 2) the EDHF is most likely a CYP-derived arachidonate product; 3) CYP 1A is well suited as the isozyme responsible for EDHF production in this vascular bed; and 4) PLA₂ appears to mediate the release of the precursor arachidonic acid.     

Distinct hyperpolarizing and relaxant roles for gap junctions and endothelium-derived H2O2 in NO-independent relaxations of rabbit arteries

We have compared the contributions of gap junctional communication and chemical signaling via H2O2 to NO-independent relaxations evoked by the Ca2+ ionophore A23187 and acetylcholine (ACh) in rabbit ilio-femoral arteries. Immunostaining confirmed the presence of connexins (Cxs) 37 and 40 in the endothelium and Cxs 40 and 43 in smooth muscle. Maximal endothelium-dependent subintimal smooth muscle hyperpolarizations evoked by A23187 and ACh were equivalent (approximately 20 mV) and almost abolished by an inhibitory peptide combination targeted against Cxs 37, 40, and 43. However, maximal NO-independent relaxations evoked by A23187 were unaffected by such peptides, whereas those evoked by ACh were depressed by approximately 70%. By contrast, the enzyme catalase, which destroys H2O2, attenuated A23187-induced relaxations over a broad range of concentrations, but only minimally depressed the maximum response to ACh. Catalase did not affect A23187- or ACh-evoked hyperpolarizations. After loading with an H2O2-sensitive probe, A23187 caused a marked increase in endothelial fluorescence that correlated temporally with relaxation, whereas only a weak delayed increase was observed with ACh. In arteries without endothelium, the H2O2-generating system xanthine/xanthine oxidase induced a catalase-sensitive relaxation that mimicked the gap junction-independent response to A23187 as it was maximally equivalent to approximately 80% of induced tone, but associated with a smooth muscle hyperpolarization <5 mV. We conclude that myoendothelial gap junctions underpin smooth muscle hyperpolarizations evoked by A23187 and ACh, but that A23187-induced relaxation is dominated by extracellular release of H2O2. Endothelium-derived H2O2 may thus be regarded as a relaxing factor, but not a hyperpolarizing factor, in rabbit arteries.     

Heterogeneity of endothelium-dependent vasorelaxation in conductance and resistance arteries from Lyon normotensive and hypertensive rats

OBJECTIVES: The nature of endothelial factors in response to acetylcholine (ACh) was investigated in conductance and resistance arteries from Lyon normotensive (LN) and Lyon hypertensive (LH) rats. Differences in endothelial function between the two strains were evaluated. METHODS AND DESIGN: Relaxations to ACh were studied in the aorta and small mesenteric arteries (SMA). The relative contribution of nitric oxide (NO), prostanoids and endothelial-derived hyperpolarizing factor (EDHF) was assessed using appropriate inhibitors. Western blot of endothelial NO synthase was achieved. The membrane potential of smooth muscle cells was assessed using microelectrodes. RESULTS: In LN rats, endothelium-dependent relaxation to ACh involved exclusively NO in the aorta, whereas both NO and EDHF were implicated in SMA. In the latter, relaxation was almost entirely prevented by blockade of either the NO or EDHF pathway, although ACh was still able to produce hyperpolarization in the presence of NO synthase and cyclooxygenase inhibitors. In LH rats, relaxation to ACh was unchanged in SMA but moderately depressed in the aorta, despite unchanged endothelial NO synthase protein expression and sensitivity to NO. In addition, indomethacin, but not a selective cyclooxygenase-2 inhibitor, significantly reduced ACh relaxations in the aorta from LH rats but not from LN rats. CONCLUSIONS: These results document differential endothelial function in a conductance and in resistance arteries from LN rats and LH rats. They show that simultaneous participation of NO and EDHF is required to promote relaxation in SMA from both strains, whereas NO alone accounts for relaxation in aorta from LN rats. In LH rats, aortic relaxation induced by ACh is slightly decreased despite the involvement of vasodilator products from cyclooxygenase-1.     

Cell morphological changes in venous remodeling induced by arteriovenous grafting in rat limb

Vascular remodeling induced in rat limb by arteriovenous (AV) shunting was investigated by evaluating changes in vascular diameter and cell morphology. In Wistar rats, a vein graft was implanted in situ in the hind limb. Flow-rate in the grafted vein was assessed by measuring flow in the common femoral artery using an ultrasonic flowmeter. Nuclei and actin filaments of the venous wall were stained with propidium iodine and phalloidine-FITC, and the samples were observed using confocal laser microscopy. The grafted veins became circular in cross-section with increase in diameter during two weeks after AV shunting. Owing to the increase in diameter, the estimated wall shear stress was not increased so much as the flow-rate. The confocal laser microscopic observation showed that endothelial cells (ECs) and smooth muscle cells (SMCs) in the grafted veins were either aligned well (2 out of 8 samples), or ECs were denudated and SMCs were disrupted (in 6 out of 8 samples). The cell density of ECs was unchanged from the control level. In conclusion, the grafted vein was remodeled with morphological changes in ECs and SMCs during 2 weeks after AV shunting.     

Myoendothelial coupling is unidirectional in guinea pig spiral modiolar arteries

Gap junctions (GJs) facilitate communication and promote transfer of signaling molecules or current between adjacent cells in various organs to coordinate cellular activity. In arteries, homocellular GJs are present between adjacent smooth muscle cells (SMCs) and between adjacent endothelial cells (ECs), whilst many arteries also exhibit heterocellular GJs between SMCs and ECs. To test the hypothesis that there is differential cell coupling in guinea pig spiral modiolar arteries (SMA), we used intracellular recording technique to record cellular activities simultaneously in ECs or SMCs in acutely isolated guinea pig SMA preparations. Cell types were identified by injection of a fluorescent dye, propidium iodide (PI), through recording microelectrodes. Stable intracellular recordings were made in 120 cells among which 61 were identified as SMCs and 28 as ECs. Dual intracellular recordings were conducted to detect the coexistence of the two distinct levels of resting potential (RP) and to estimate the intensity of electrical coupling between two cells by a current pulse of up to 0.5-1.5 nA. The electrotonic potential was detected not only in the current-injected cell, but also in the majority of non-injected cells. The electrical coupling ratios (ECRs) of homocellular cells were not significant (P>0.05) (0.084±0.032 (n=6) and 0.069±0.031 (n=7) for EC-EC and SMC-SMC pairs, respectively). By contrast, the ECRs of heterocellular cells were significantly different when a current pulse (1.5 nA, 2s) was injected into EC and SMC respectively (0.072±0.025 for EC; 0.003±0.001 for SMC, n=5, P<0.01). The putative gap junction blocker 18β-glycyrrhetinic acid significantly attenuated electrical coupling in both homocellular and heterocellular forms. The results suggest that homocellular GJs within SMCs or ECs are well coordinated but myoendothelial couplings between ECs and SMCs are unidirectional.     

Intact endothelium-dependent dilation and conducted responses in resistance vessels of hypercholesterolemic mice in vivo

Atherosclerosis and hyperlipidemia impair endothelium-dependent nitric oxide (NO)-mediated dilations in conducting arteries. In addition to NO, the endothelium releases an endothelium-derived hyperpolarizing factor (EDHF) in response to acetylcholine (ACh), which is particularly important in microvessels and initiates a dilation that conducts along the vessel through gap junctional communication. The expression of connexins is, however, altered by hypercholesterolemia. Therefore, we studied endothelium-dependent dilations and their conduction in murine hypercholesterolemic models. Dilations were assessed by intravital microscopy in arterioles with a diameter of approximately 35 microm in ApoE and LDL receptor (LDLR(-/-))-deficient mice after superfusion or locally confined application of ACh. ACh induced comparable concentration-dependent dilations in wild-type, LDLR(-/-), and ApoE(-/-) mice fed a normal or high-cholesterol diet, however EC(50) was slightly higher in ApoE(-/-) mice. Furthermore, the NO donor sodium-nitroprusside dilated arterioles to a similar extent (approximately 60%). Locally initiated ACh dilations (approximately 68%) conducted up to a distance of 1,100 microm without significant attenuation even under severe hypercholesterolemic conditions. Since ACh dilation in the arterioles of mice is mainly mediated via EDHF, we conclude that hypercholesterolemia does not alter EDHF release and efficacy. This conclusion is confirmed by an intact conducted response since EDHF is a prerequisite for this response. The intact conduction also suggests that gap-junctional communication is functionally preserved in these models.     

Mediation of EDHF-induced reduction of smooth muscle [Ca(2+)](i) and arteriolar dilation by K(+) channels, 5,6-EET, and gap junctions

OBJECTIVE: To characterize the role of K(+) channels, the cytochrome P-450 (CYP) metabolite 5,6-EET, and gap junctions in modulation of arteriolar myogenic tone by a non-nitric oxide nonprostaglandin mediator, termed "endothelium-dependent hyperpolarizing factor" (EDHF), released to acetylcholine (ACh) in skeletal muscle arterioles. METHODS: In isolated rat gracilis arterioles, simultaneous changes in smooth muscle (aSM) [Ca(2+)](i) (assessed by changes in fura-2 ratiometric signal, R(Ca)) and diameter were measured in response to ACh in the presence of indomethacin and L-NAME. RESULTS: ACh, the K(ATP) channel opener pinacidil, and the Ca(2+) channel inhibitor verapamil elicited comparable decreases in aSM [Ca(2+)](i) (max.: -32 +/- 3%, 29 +/- 3%, and -30 +/- 3%, respectively) and arteriolar dilations (max.: 90 +/- 4%, 96 +/- 2%, and 95 +/- 2%, respectively). ACh-induced responses were inhibited by KCl-depolarization, K(Ca) channel blockers (TEA, charybdotoxin), or gap junction inhibitors (18alpha-glycyrrhetinic acid, hyperosmolar sucrose). The K(ATP) channel inhibitor glibenclamide, the K(IR) channel inhibitor barium chloride, or the CYP inhibitor 17-octadecynoic acid (ODYA) were without effect. The putative EDHF analogue 5,6-EET elicited constrictions in the presence of the endothelium that could be prevented by indomethacin or a TxA(2) receptor antagonist, whereas in the absence of the endothelium, EDHF elicited only small, charybdotoxin-insensitive decreases in aSM R(Ca) and dilations (max.: -8 +/- 2% and 27 +/- 4%, respectively). CONCLUSIONS: In skeletal muscle arterioles, EDHF 1) substantially and rapidly reduces myogenic tone by decreasing aSM [Ca(2+)](i) via opening K(Ca) channels, 2) it is unlikely to be 5,6-EET or other CYP metabolites, but 3) requires functional gap junctions.     

人脐动脉内皮细胞和平滑肌细胞体外培养鉴定及缝隙连接通讯功能测定

目的:应用双光子激光扫描共聚焦显微镜鉴定体外培养的脐动脉内皮细胞(ECs)和平滑肌细胞(SMCs),应用荧光光漂白恢复技术(FRAP)测定血管ECs、SMCs之间的缝隙连接通讯(GJIC)功能。方法:人脐动脉ECs、SMCs分离培养,Ⅷ因子和SMα-actin相关抗原鉴定ECs和SMCs,应用FRAP技术测定血管内皮细胞、平滑肌细胞之间的GJIC功能,记录实时成像结果,应用动态比(M)计算漂白区域内标记荧光的分子中动态分子的比例。结果:第一组ECs和SMCs单独培养,选择漂白细胞与周围至少3个同种细胞相连接,SMCs被漂白后平均M值为31.79±5.69;ECs被漂白后平均M值为23.43±2.11;第二组ECs和SMCs混合培养,选择ECs和SMCs独立相连的2个细胞,SMCs被漂白后平均M值为14.47±3.28,ECs被漂白后平均M值为6.41±0.80。结论:FRAP实时动态恢复曲线可直接观察荧光恢复强度及速度,参照FRAP恢复曲线,M值可做为组间GJIC比较相对定量的可靠指标,通过检测证实ECs和SMCs之间存在GJIC,且荧光由ECs向SMCs方向的传递大于由SMCs向ECs方向的传递。     

Myoendothelial coupling is not prominent in arterioles within the mouse cremaster microcirculation in vivo

A smooth muscle hyperpolarization is essential for endothelium-dependent hyperpolarizing factor-mediated dilations. It is debated whether the hyperpolarization is induced by a factor (endothelium-derived hyperpolarizing factor) and/or is attributable to direct current transfer from the endothelium via myoendothelial gap junctions. Here, we measured membrane potential in endothelial cells (EC) and smooth muscle cells (SMC) in vivo at rest and during acetylcholine (ACh) application in the cremaster microcirculation of mice using sharp microelectrodes before and after application of specific blockers of Ca2+-dependent K+ channels (K(Ca)). Moreover, diameter changes in response to ACh were studied. Membrane potential at rest was lower in EC than SMC (-46.6+/-1.0 versus -36.5+/-1.0mV, P<0.05). Bolus application of ACh induced robust hyperpolarizations in EC and SMC, but the amplitude (11.1+/-0.9 versus 5.1+/-0.9mV, P<0.05) and duration of the response (10.7+/-0.8 versus 7.5+/-1.0s, P<0.05) were larger in EC. Blockers of large conductance K(Ca) (charybdotoxin or iberiotoxin) abrogated ACh-induced hyperpolarizations in SMC but did not alter endothelial hyperpolarizations. In contrast, apamin, a blocker of small conductance K(Ca) abolished ACh-induced hyperpolarizations in EC and had only small effects on SMC. ACh-induced dilations were strongly attenuated by iberiotoxin but only slightly by apamin. We conclude that myoendothelial coupling in arterioles in vivo in the murine cremaster is weak, as EC and SMC behaved electrically different. Small conductance K(Ca) mediate endothelial hyperpolarization in response to ACh, whereas large conductance K(Ca) are important in SMC. Because tight myoendothelial coupling was found in vitro in previous studies, we suggest that it is differentially regulated between vascular beds and/or by mechanisms acting in vivo.