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(Circulation Research. 2005;97:781.)
© 2005 American Heart Association, Inc.

Integrative Physiology

Myoendothelial Coupling Is Not Prominent in Arterioles Within the Mouse Cremaster Microcirculation In Vivo

Daniel Siegl, Michael Koeppen, Stephanie E. Wölfle, Ulrich Pohl, Cor de Wit

From the Physiologisches Institut (S.E.W., C.d.W.), Universität Lübeck; and the Physiologisches Institut (D.S., M.K., U.P.), Ludwig-Maximilians-Universität München, Germany.

Correspondence to Dr Cor de Wit, Physiologisches Institut Universität Lübeck Ratzeburger Allee 160 23538 Lübeck, Germany. E-mail dewit{at}uni-luebeck.de

	Abstract

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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.

Key Words: microcirculation • endothelium-dependent hyperpolarizing factor • myoendothelial coupling • gap junctions • acetylcholine-induced hyperpolarization

	Introduction

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In addition to NO- and prostaglandin-mediated endothelium-dependent dilation, endothelium-dependent hyperpolarizing factor (EDHF)-mediated dilation characterized by a hyperpolarization of smooth muscle cells (SMC) resistant to NO synthase and cyclooxygenase inhibition is considered a key mechanism of endothelial control of vascular tone.^1,2 This pathway is of special importance in resistance vessels and can be elicited by various endothelial stimuli including acetylcholine (ACh) and bradykinin. Whereas the mechanisms of action of NO and prostaglandins are well characterized, this is less clear for the EDHF-mediated dilation. There is evidence that multiple transduction pathways are involved, and thus different substances have been proposed to act as EDHFs. A common feature of all pathways proposed so far is the hyperpolarization of endothelial cells (EC) as an early step in response to stimulation and receptor activation. This endothelial hyperpolarization is evoked most likely through the activation of small (SK_Ca) and/or intermediate (IK_Ca) conductance Ca²⁺-dependent K⁺ channels.^3–5 It has been proposed that this endothelial hyperpolarization increases interstitial K⁺ concentration, which activates the Na⁺/K⁺–ATPase and inward rectifier K⁺ channels in SMC, and thus K⁺ ions represent an EDHF.^6,7 Others have presented evidence that endothelial epoxyeicosatrienoic acid (EET) formation is necessary for EDHF-type dilations to occur, and accordingly cytochrome p450 epoxygenases have been proposed to act as EDHF synthases.⁸ In a number of vessel types from different species, EETs were identified as EDHFs.^9–13 However, in many vessels, including those of the murine microcirculation, EDHF-type dilations were not sensitive to substances that interfere with the p450 monooxygenase pathway.^14,15 Additionally, other factors have been proposed to represent EDHF, including hydrogen peroxide¹⁶ and C-type natriuretic peptide.¹⁷ All of these EDHFs are supposed to diffuse from EC to the adjacent SMC. However, recent electrophysiological experiments have suggested that EDHF is not a factor. Rather it was proposed that EDHF-mediated dilations are induced by electrotonic spread of current from hyperpolarized EC to SMC via myoendothelial gap junctions.^18–20 Depending on the vessel studied, morphological evidence for myoendothelial gap junctions has been presented.^20–23 The intercellular channels are formed by connexin proteins, and the involvement of these channels in EDHF-type relaxations has been inferred from the abrogation of dilations after incubation of vessels with gap junction–inhibiting peptides, which interfere with channel formation.^24–26 Although these findings suggest a role for myoendothelial coupling, they do not imply that it is actually current that is transferred from EC to SMC, but second messengers could also pass through the intercellular channels, eg, cAMP. However, direct measurement of membrane potential in isolated vessels have demonstrated that EC and SMC are well coupled,²⁷ exhibit similar resting membrane potentials, and hyperpolarize synchronously and indistinguishably in response to ACh stimulation arguing in favor of a tight electrical coupling between the vascular cell layers.^18,19 A direct charge transfer might be of special importance in small resistance arterioles, as smooth muscle consists of only 1 or 2 layers, which enables EC directly to control the membrane potential of SMC more effectively than in larger vessels. However, in the skeletal muscle microcirculation of hamsters studied in vivo, EC and SMC behave electrically different,²⁸ and it is therefore questionable whether data obtained in isolated vessels reflect the in vivo situation. In the present study, we examined myoendothelial coupling in the murine cremaster microcirculation in vivo by measuring membrane potentials of EC and SMC at resting conditions and during stimulation with ACh. To identify the type of K⁺ channel mediating the hyperpolarization, specific blockers of Ca²⁺-dependent K⁺ channels (K_Ca) were applied. Moreover, we studied diameter responses on ACh in the presence of these blockers. We found that EC and SMC behave distinctly with respect to their membrane potential at rest and during ACh stimulation, which argues against a strong, direct myoendothelial coupling in vivo.

	Materials and Methods

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Animal Preparation and Experimental Setup
Experiments were performed in 4- to 7-month-old male mice (C57BL/6), in accordance with the German animal protection law. Mice were anesthetized by an IP injection of droperidol (20 mg/kg), fentanyl (0.1 mg/kg), and midazolam (2 mg/kg), followed by continuous infusion via a catheter placed in the jugular vein. The animals were tracheotomized to ensure airway patency, and esophageal temperature was maintained at 37°C by conductive heat. The cremaster muscle was prepared for intravital microscopy on a custom-made stage, as described,²⁹ and continuously superfused with a bicarbonate-buffered saline solution (pH 7.35 to 7.45, 35°C) of the following composition (in mmol/L): 143 Na⁺, 6 K⁺, 2.5 Ca²⁺, 1.2 Mg²⁺, 128 Cl^–, 25 HCO₃^–, 1.2 SO₄^2–, and 1.2 H₂PO₄^–. For membrane potential recordings, 2 to 4 arterioles with a diameter between 30 and 60 µm were carefully dissected free from the surrounding tissue over a distance of 5 mm using a stereo microscope (Leitz, Wetzlar, Germany). Thereafter, the animal was transferred onto a microscope stage (ZEISS, Axioskop FS, Göttingen, Germany), positioned on a movable platform (Luigs & Neumann, Ratingen, Germany) and a vibration-isolated table. A glass microelectrode (80 to 120 M, filled with 3 mol/L KCl containing 5% carboxyfluorescein in the tip), connected to a membrane potential amplifier (SEC 1L, NPI Advanced Electronics, Tamm, Germany), was positioned vertical to the vessel axis and advanced using an electronic micromanipulator equipped with a piezo stepper (PM 20H, Samwoo Scientific Co, Seoul, South Korea). An Ag/AgCl pellet positioned in the superfusion solution served as the reference electrode. The successful impalement of the cell was verified by a sharp deflection of the potential recording, a low resistance over the cell membrane measured by the application of a test pulse, and a stable potential for at least 30 s. The cell type being impaled was analyzed following each recording by the pattern of cell labeling with carboxyfluorescein, which enabled the identification of the cell by its orientation relative to the vessel axis. Data were collected with a computer based monitoring system (XmAD) at a sampling rate of 1000 Hz and stored for later analysis. In a subset of experiments, arteriolar diameter changes on ACh were assessed in the cremaster microcirculation as described.¹⁵ In each animal, 7 to 12 arterioles were monitored, microscopic images projected on a charge-coupled video camera, and displayed on a monitor and recorded on videotape for later measurement of luminal diameters. All mice were euthanized by an overdose of anesthesia at the end of the experimental protocol.

Experimental Protocols
Membrane Potential Measurement
After a postoperative stabilization period of 30 minutes, measurements of membrane potential were started. On successful cell penetration and recording of the resting membrane potential for 30 s, the vessel was stimulated by agonist application via a glass micropipette with a tip of 1 to 3 µm positioned in close proximity to the arteriolar wall. The stimulation pipette was usually placed at a distance between 50 and 600 µm from the measurement electrode. Acetylcholine (10 mmol/L) or sodium-nitroprusside (SNP) (10 mmol/L) was applied by pressure ejection (150 kPa) for increasing periods of time (from 10 to 1000 ms) to achieve increasing local concentrations. Before application of the next pressure pulse, membrane potential was allowed to return to baseline level. Potential recordings from EC were generally more stable over time as compared with smooth muscle cells. Recordings that were not stable for more than 2 minutes were excluded from data analysis. Typically, measurements lasted for 5 minutes before the pipette dislodged spontaneously or the protocol was finished. In general, we were able to obtain 4 to 10 recordings from a single experiment during a 4-hour period in 2 to 4 arterioles. In a subgroup of experiments, after obtaining recordings in nontreated preparations, blockers of K_Ca were applied onto the arteriole under investigation. This was done using a second glass pipette with a larger tip opening (5 to 10 µm) filled with charybdotoxin (ChTx), iberiotoxin (IbTx), or apamin (Apa) (100 µmol/L) and repeated pressure ejections (150 kPa, 300 ms, 10 to 20 times). Thereafter, vascular cells were again impaled and a similar protocol used. The likelihood to impale EC was higher, but recordings from both cell types were obtained before and after treatment in the same, equally treated animals.

Measurement of Diameter Changes
The cremaster was superfused continuously with the cyclooxygenase inhibitor indomethacin (3 µmol/L) and in a subset of experiments the NO synthase inhibitor N-nitro-L-arginine (L-NA) (30 µmol/L) 30 minutes before the protocol was started and throughout the experiment. Arteriolar diameters were measured shortly before and during the local superfusion of ACh (1, 3, 10 µmol/L). Increasing concentrations were applied consecutively, with a recovery period of 5 minutes between washout and application of the next concentration or drug. During this recovery period, the arterioles regained their baseline diameter. The same protocol was then repeated after application of IbTx and/or Apa (0.1 µmol/L) to the superfusion solution for 20 minutes. Additionally, responses on application of SNP (3, 10 µmol/L) were studied. The maximal diameter of the arterioles was obtained during superfusion of a combination of different vasodilators (adenosine [100 µmol/L], SNP [100 µmol/L], and ACh [100 µmol/L]) at the end of the experimental protocol.

Statistics and Calculations
Vascular tone is given as the quotient of the resting diameter of the vessel divided by its maximum. Changes of the inner diameter of the vessels were normalized to the maximal possible constriction or dilation according to the relationship: percentage of maximal response=(D_Tr–D_Co)/(D_M–D_Co)x100, where D_Tr is the diameter observed after treatment and D_Co is the control diameter before treatment. D_M is (for dilator responses) the diameter at maximal dilation or (for constrictions) the minimal luminal diameter (0). Comparisons within groups were performed using paired t tests, and, for multiple comparisons, probability values were corrected according to Bonferroni. Data between groups were compared by analysis of variance followed by post hoc analysis of the means, with P<0.05 considered significant. Data are presented as mean±SEM.

	Results

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Resting Membrane Potential and Dye Coupling
Electrophysiological data were obtained in arterioles with a diameter between 40 and 60 µm, ie, second-order branching vessels in 41 animals. Carboxyfluorescein diffused into the cell during the measuring period and stained the cell bodies, allowing identification of the cell under study. However, some cells remained nonstained, and these measurements were excluded from the data analysis (9/205 cells). Cells orientated parallel to the vessel were identified as EC, whereas vascular smooth muscle cells (SMC) appeared to be wrapped circularly around the vessel (Figure 1). The dye did not spread from EC to adjacent EC or SMC in most cases even after a prolonged period of time (up to 10 minutes). Dye spreading was only observed in a limited number of EC (<5% of all EC), and this staining of adjacent cells was only very weak. Likewise, after staining and measuring SMC, no other cells were labeled with dye (Figure 1). The resting membrane potential of EC in the blood perfused vessel was –46.6±1.0 mV (119 cells in 40 animals). In contrast, SMC were more depolarized and their resting potential was –36.5±1.0 mV (77 cells in 31 animals). Figure 2 shows the frequency distribution of the membrane potentials measured in EC and SMC.

View larger version (95K): [in this window] [in a new window]   Figure 1. Intracellular labeling of EC and SMC. During intracellular recording carboxyfluorescein diffused from the microelectrode into the cell and stained the cell bodies. Cells were identified using fluorescence microscopy (bottom images) after the measuring period according to shape and orientation with respect to the vessel. Upper images show superimposed bright field view. A, EC orientated parallel to the vessel wall. B, SMC perpendicular to the vessel wall appeared to be wrapped around the vessel. Image was taken after 2 separate impalements of SMC showing 2 stained cells apart from each other. Note that adjacent cells remained unstained showing lack of dye coupling.

View larger version (18K): [in this window] [in a new window]   Figure 2. Frequency distribution of the resting membrane potential of EC (top) and SMC (bottom) in blood-perfused arterioles in the microcirculation. Data are normally distributed and fitted curve is shown. In each panel, a box plot with median and percentiles (box: 25th, 75th; whiskers: 10th, 90th; circles: 5th, 95th) is depicted. SMC were significantly more depolarized than EC (ANOVA). Data were obtained in 41 animals and represent measurements obtained in 119 EC and 77 SMC. The likelihood to penetrate EC and to obtain a stable measurement was generally higher.

ACh-Induced Hyperpolarization and Effect of Blockers of K_Ca
The local application of ACh induced robust hyperpolarizations in EC and SMC. Figure 3 shows a representative example of a measurement of the membrane potential in an EC and SMC, respectively. Each bolus application of ACh induced a hyperpolarization, which increased in amplitude and duration in EC with increasing pulse duration leading to higher, but unknown local ACh concentrations. This increment was less pronounced in SMC (Figure 3). Because the local ACh concentration after a pressure-pulse is difficult to predict in different experiments, the data were divided in 4 groups of stimulation according to pulse duration. With a pressure pulse between 10 and 50 ms (32±1 ms), EC hyperpolarized from –47.9±0.8 to –56.2±0.9 mV, between 51 and 200 ms (120±5 ms) from –49.7±0.9 to –59.6±0.8 mV, between 201 and 700 ms (457±11 ms) from –46.8±1.0 to –57.3±1.1 mV, and >700 ms (1250±67 ms) from –45.4±1.4 to –57.3±1.4 mV. The duration of the pressure pulses were not different in SMC, which hyperpolarized from –39.3±2.4 to –43.5±2.0 mV at 31±4 ms, from –40.6±1.6 to –45.7±1.9 mV at 137±10 ms, from –35.3±1.8 to –40.1±2.1 mV at 458±17 ms, and from –39.3±3.5 to –44.3±3.2 mV at 1333±167 ms of stimulation duration. The amplitudes of membrane-potential changes with different pulse durations are depicted in Figure 4. The difference with increasing stimulus duration seen in an individual EC is reflected in the summary data as a significantly larger amplitude in the group with the longest pressure pulse as compared with the group with the shortest stimulation duration. In SMC, an increase in amplitude with the stimulation duration was not found. However, the response duration was enhanced with increasing stimulation duration in EC, an effect that was also found in SMC. Most interestingly, amplitude and duration were enhanced in EC as compared with SMC (Figure 4), showing a distinct response in each cell type. In 3 animals, responses in EC were also measured >1 mm upstream of the ACh stimulation site. Also at this distance (1210±20 µm), a significant hyperpolarization was observed in EC on remote stimulation with ACh (by –3.7±0.3 mV at 89±20 ms and by –5.3±0.2 mV at 1318±245 ms). The NO-donor SNP applied by a pressure pulse at the site of recording did not induce changes of membrane potential in SMC (–30.6±2.0 to –30.1±2.0 mV, n=14) but resulted in a small hyperpolarization in EC (–39.6±1.5 to –40.2±1.5 mV, n=33, P<0.05).

View larger version (21K): [in this window] [in a new window]   Figure 3. Hyperpolarization in EC and SMC in response to brief stimulation with ACh. Recordings illustrate representative changes of the membrane potential in EC (top) and SMC (bottom). Arrows indicate delivery of ACh stimulus. The local ACh concentration was increased by extending the pressure pulse as indicated by triangle (stimulation time varied from 0.01 to 1 s; not to scale). Note the differences in amplitude and duration of hyperpolarizations in EC and SMC.

View larger version (14K): [in this window] [in a new window]   Figure 4. Amplitude and duration of ACh-induced hyperpolarizations in EC and SMC. With increasing local ACh concentrations achieved by extending the duration of stimulation, the amplitude of the hyperpolarization (top) increased in EC (P<0.05 at longest vs shortest stimulation time) but not in SMC. The duration of the response (bottom) increased in EC and SMC significantly with higher local ACh concentrations. Importantly, amplitude and response duration was significantly lower in SMC vs EC (*P<0.05).

To study the type of K⁺ channels involved in the ACh-induced hyperpolarization, blockers of K_Ca were applied locally onto the vessel wall. After ChTx, EC hyperpolarization remained unaffected. EC hyperpolarized before ChTx from –47.8±2.5 to –57.6±2.8 mV (P<0.05, n=13 in 6 experiments) at 319±57 ms ACh pressure pulse and after ChTx from –54.6±2.7 to –62.8±3.0 mV (P<0.05, n=14 in 6 experiments) at a stimulus duration of 315±69 ms. Likewise, the duration of the response in EC was not altered (before: 6.6±0.9 s; after: 5.7±0.7 s; P=0.43). SMC hyperpolarized from –45.0±4.4 to –52.7±3.0 mV (P<0.05; n=7 in 6 experiments) in this group under control conditions, but, in contrast to EC, the ACh-induced hyperpolarization was completely abrogated after ChTx (from –35.2±2.3 to –36.6±2.1 mV, P=0.13, n=8; Figure 5). The specific blocker of large conductance K_Ca (BK_Ca), IbTx, produced similar results: the hyperpolarization of EC in response to ACh was unaffected, whereas the hyperpolarization in SMC was abrogated after IbTx (Figures 5 and 6). After local treatment with Apa, a blocker of SK_Ca, a reverse pattern of inhibition was observed. EC hyperpolarized on ACh from –42.8±2.2 to –49.2±2.7 mV (P<0.05, n=10) in this group under control conditions, and this hyperpolarization was blocked after Apa (from –41.6±2.2 to –42.9±2.1 mV, P=0.10, n=16, Figure 5). In contrast to EC, the maximal amplitude of the small hyperpolarization in SMC was unaffected by Apa (Figure 6, n=5), although the onset of the hyperpolarization was delayed (Figure 5). Of all antagonists, only ChTx affected the resting membrane potential in vascular cells. SMC were significantly depolarized after ChTx (from –45.6±4.3 to –34.6±1.6 mV), but no significant change was found in EC (before: –50.3±2.3; after –54.7±2.5 mV; P=0.21). IbTx did not change the membrane potential of EC (–51.3±3.9 versus –54.5±2.9 mV) or SMC (–37.5±2.7 versus –33.8±2.3 mV), and also Apa was without effect (EC: –43.6±2.4 versus –42.0±2.3 mV; SMC –34.4±4.2 versus –35.9±0.8 mV). However, it has to be kept in mind that these measurements were not performed continuously during drug application, and because of the variation in resting membrane potentials, small changes in response to the blockers cannot be verified.

View larger version (22K): [in this window] [in a new window]   Figure 5. ACh-induced hyperpolarization in EC and SMC after brief stimulation in the presence of KCa-channel blockers. In EC (top) and SMC (bottom), short bolus application of ACh induced a transient hyperpolarization in untreated preparations (, control). The blockade of KCa had divergent effects in EC and SMC. Endothelial hyperpolarizations remained unchanged after ChTx () or IbTx (), but were abrogated by Apa (). In contrast, ChTx or IbTx blocked the hyperpolarization in SMC. The responses in SMC after Apa were delayed and the onset varied. Data represent measurements from 5 or 6 animals in each group. In each animal, values from EC and SMC were obtained.

View larger version (13K): [in this window] [in a new window]   Figure 6. Maximal amplitudes of ACh-induced hyperpolarizations and effect of blockers of KCa channels. The maximal amplitude of ACh-induced hyperpolarization in EC was unchanged after blocking BKCa with IbTx but nearly abolished after the blockade of SKCa with Apa. In contrast, the hyperpolarization was abrogated in SMC after IbTx but not altered by Apa. However, responses in SMC were delayed in the presence of Apa (see Figure 5). Digits in columns indicate numbers of cells measured. Data were obtained in 6 (IbTx) and 5 (Apa) animals. Significant differences between control and treatment group (ANOVA) are indicated (*P<0.05).

Effect of K⁺-Channel Blockers on ACh-Induced Dilations
In a different group of mice, the effect of the K_Ca-channel blockers on ACh-induced diameter changes was studied. The maximal diameter of the arterioles studied was 23±1 µm. Superfusion of ACh induced a concentration-dependent dilation in the presence of L-NA and indomethacin (30 and 3 µmol/L). This dilation was attenuated after the application of IbTx (0.1 µmol/L, Figure 7). IbTx reduced the ACh-induced dilation also in the absence of L-NA to a similar amount, and the subsequent addition of L-NA (30 µmol/L) had no additional inhibitory effect (Figure 7). In vessels treated with L-NA and indomethacin, Apa (0.1 µmol/L) also attenuated the ACh-induced dilations but to a lesser extent than IbTx. However, the subsequent addition of IbTx induced a further reduction of the ACh-induced responses to a level that was not different from that observed in vessels treated only with IbTx (Figure 7). The dilation induced by the NO-donor SNP (10 µmol/L) remained unaffected by IbTx and/or Apa (control: 79±3%; IbTx: 78±3%; Apa: 84±2%; Apa and IbTx: 79±2%). Likewise, smaller dilations induced by SNP (3 µmol/L), which were comparable to the dilation induced by 3 µmol/L ACh, were not attenuated in the presence of IbTx (56±5 versus 57±5%; n=32 arterioles in 3 experiments). However, IbTx induced a significant constriction in resting vessels in the absence of L-NA (–8±3%, P<0.05), and this constriction was also observed in vessels treated with L-NA (–10±4%, P<0.05).

View larger version (19K): [in this window] [in a new window]   Figure 7. Effect of blockers of KCa channels on ACh-induced diameter changes. Top, ACh-induced a concentration-dependent dilation in the presence of the blocker of NO synthase (L-NA, 30 µmol/L, ), which was significantly attenuated after additional blockade of BKCa by IbTx (0.1 µmol/L, ; n=72 arterioles in 7 experiments). Blockade of SKCa by Apa (Apa, 0.1 µmol/L, ; n=88 arterioles in 8 experiments) also reduced ACh-induced dilations, however to a lesser degree than IbTx. The additional application of IbTx on Apa-treated arterioles induced a further significant reduction of ACh-induced dilations (; n=41 arterioles in 4 experiments) to similar levels as after IbTx alone. Bottom, The ACh-induced concentration-dependent dilation () was reduced to a similar degree by IbTx (0.1 µmol/L, ; n=42 arterioles in 4 experiments) in the absence of L-NA. The additional blockade of NO synthase did not further attenuate the dilation (L-NA, 30 µmol/L, ). All experiments were performed in the continuous presence of indomethacin (3 µmol/L). *P<0.05, control vs treatment group; #P<0.05, Apa vs Apa+IbTx; paired t tests.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The membrane potential is an important determinant of vascular tone especially in arterioles and is transmitted between vascular cells either directly via myoendothelial gap junctions or indirectly via a diffusible factor (EDHF). We propose that myoendothelial coupling and direct charge transfer is not prominent in arterioles in vivo. This conclusion is supported by 3 key observations of this study, each of them showing that endothelial and smooth muscle cells behave electrically different. Firstly, EC and SMC exhibited different resting membrane potentials. Secondly, ACh elicited quantitatively different hyperpolarizations in EC and SMC. Thirdly, and most importantly, our data demonstrate that different K_Ca are involved in inducing the hyperpolarization in response to ACh in these cells. Specifically, SK_Ca mediate the hyperpolarization in EC, whereas BK_Ca are activated in smooth muscle cells in response to ACh stimulation. The activation of both channels contributes to the mechanical ACh-response, however, BK_Ca channels appear to be more important.

The importance of hyperpolarizations in eliciting dilations in response to endothelium-dependent agonists (eg, ACh) has been extensively documented.^1,2 Aside from various factors that have been implicated to be released from EC, myoendothelial coupling via gap junctions has been proposed to transfer the hyperpolarization between EC and SMC directly.^18–20 However, the evidence for this was obtained mostly in isolated vessels and it is unclear whether such communication is also found in vivo. The data from the present study suggest that electrical myoendothelial coupling is not prominent in arterioles of the murine skeletal muscle in vivo because the resting membrane potential in EC and SMC was significantly different. Whereas the resting potential of SMC was in a similar range as reported for cheek pouch arterioles of the hamster in vivo,²⁸ EC exhibited a more negative potential. However, the comparison of resting membrane potentials of vascular cells is hindered by the fact that values reported from different vessels and preparations obtained in vitro are substantially different. If a tight myoendothelial coupling exists, the 2 cell layers should exhibit similar membrane potentials, a finding that is very common in vessels in vitro.¹⁸

In response to ACh stimulation, we found a robust hyperpolarization in both cell types. This hyperpolarization was larger in amplitude and in duration in EC than SMC, again demonstrating a different behavior that is in contrast to hyperpolarizations obtained in vitro, which are reported to be indistinguishable from each other.¹⁸ However, an attenuation of the amplitude in SMC might be attributable to an ohmic resistor represented by myoendothelial gap junctions and solely this observation does not exclude current transfer. To our knowledge, only 1 study has addressed this question in vivo, and these authors also observed slightly different responses in EC and SMC in the hamster microcirculation.²⁸ Further proof for a lack of tight myoendothelial coupling in vivo was added by studying conducted dilations during selective destruction of the endothelial or smooth muscle layer.³⁰ The contrasting results from work done in vivo versus in vitro is surprising and requires explanation. As vessels studied in vitro are generally larger than arterioles studied in the microcirculation in vivo, it is difficult to decide whether differences in myoendothelial coupling are solely attributable to this fact, eg, different vessel sizes. Clearly, the presence of myoendothelial junctions varies with the type and size of vessel studied.^21,31 The measurement of membrane potential required the careful dissection of the arterioles from the surrounding skeletal muscle. This inevitable artifact and the impalement of the cell might have resulted in an uncoupling of the 2 cell layers. However, the fact that EC responded to ACh stimulation of the arteriole at a remote, distant site argues against a sole uncoupling of the impaled cell from its neighbors. Moreover, the lack of tight myoendothelial coupling might be specific for this vascular bed (cremaster muscle), as most in vitro data demonstrating strong myoendothelial coupling were obtained in vessels from other tissues. Alternatively, in vivo, a mechanism is active that regulates communication between the cell layers. Such a mechanism might involve flow or shear stress, as membrane potential measurements in vitro are usually performed in the absence of flow. As outlined before, resting membrane potentials, especially of EC, were more hyperpolarized in vivo, and simply this fact might affect myoendothelial communication. Other candidates for the regulation of (myoendothelial) gap junctions are the second messengers cAMP³² or EETs.³³ Whatever the exact nature of the mechanism is, we propose that in vivo, a tight control of the SMC membrane potential by direct current transfer from EC is prevented.

We used selective blockers to identify the K⁺ channels that are activated to induce the hyperpolarizations on ACh. Apa, a specific blocker of SK_Ca, completely prevented the hyperpolarization in EC. In contrast, hyperpolarization in SMC was sensitive to ChTx, which blocks IK_Ca and BK_Ca, but was also sensitive to the more specific blocker of BK_Ca, IbTx. These latter blockers did not alter the endothelial hyperpolarizations, suggesting that SMC hyperpolarization was not transmitted from EC via gap junctions but was attributable to the activation of BK_Ca by an unknown diffusible factor. Activation of this channel has been attributed to EETs in other vessels.¹ However, in a previous study, we were not able to attenuate ACh dilations with blockers of the cytochrome P450 pathway.¹⁵ The factor that activates BK_Ca thus remains elusive. However, the present study suggests that myoendothelial junctions are not involved and that BK_Ca acts as an important mediator of the mechanical response by inducing SMC hyperpolarization. The efficacy of IbTx to block hyperpolarizations in SMC was mirrored by its effect on mechanical responses as IbTx strongly reduced ACh-dilations. IbTx also reduced the vessels resting diameter suggesting a continuous dilator effect by activation of these channels. Nevertheless, we were not able to find a significant depolarization in response to IbTx, whereas ChTx did induce a depolarization in SMC. However, it has to be kept in mind that the membrane potential was not measured continuously during application of these blockers, but single cells were impaled before and after drug application. This might leave subtle changes of the membrane potential undetected because of the variation of the membrane potential of the cells.

The important contribution of SK_Ca in EC to the resting membrane potential and vascular tone has been demonstrated previously in an elegant model using genetic alteration of channel expression.³⁴ In this study, we demonstrated the importance of this channel for the endothelial hyperpolarization elicited by ACh. However, Apa only attenuated the EDHF-type dilation in the cremaster arterioles, and its effect was smaller as compared with the effect of IbTx (Figure 7). Most importantly, the blocking potency of Apa and IbTx was not independent as IbTx was equally effective with or without Apa. These findings suggest that the activation of SK_Ca is contributing to the mechanical response and serves to activate an EDHF-type dilation, which is in line with many previous reports.

In summary, we conclude that myoendothelial coupling is not dominant in vivo, as shown by measuring the membrane potential. This is in contrast to data obtained in vitro. The mechanism that downregulates myoendothelial coupling in vivo remains obscure and requires further study. We propose that flow or shear stress might be involved and possibly include NO. Although the regulation of cellular coupling is still poorly understood, it appears to be physiologically important and might explain differences obtained in vivo versus in vitro. From a physiological perspective, a fine-tuned regulation of membrane potential and the possibility to modify it between EC and SMC seems plausible and supports specific cellular functions in the microcirculation. However, this leaves the question as to the mechanisms and possible factors by which EC act to change the membrane potential of SMC still open.

	Acknowledgments

 
This work was supported by the Deutsche Forschungsgemeinschaft (WI 2071/1-1) and the Friedrich-Baur Stiftung. We thank Dr Tania Szado for helpful discussions.

	Footnotes

 
Original received March 14, 2005; resubmission received August 4, 2005; accepted September 1, 2005.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Busse R, Edwards G, Feletou M, Fleming I, Vanhoutte PM, Weston AH. EDHF: bringing the concepts together. Trends Pharmacol Sci. 2002; 23: 374–380.[CrossRef][Medline] [Order article via Infotrieve]
2. Griffith TM. Endothelium-dependent smooth muscle hyperpolarization: do gap junctions provide a unifying hypothesis? Br J Pharmacol. 2004; 141: 881–903.[CrossRef][Medline] [Order article via Infotrieve]
3. Quignard JF, Feletou M, Edwards G, Duhault J, Weston AH, Vanhoutte PM. Role of endothelial cell hyperpolarization in EDHF-mediated responses in the guinea-pig carotid artery. Br J Pharmacol. 2000; 129: 1103–1112.[CrossRef][Medline] [Order article via Infotrieve]
4. Eichler I, Wibawa J, Grgic I, Knorr A, Brakemeier S, Pries AR, Hoyer J, Kohler R. Selective blockade of endothelial Ca²⁺-activated small- and intermediate-conductance K⁺-channels suppresses EDHF-mediated vasodilation. Br J Pharmacol. 2003; 138: 594–601.[CrossRef][Medline] [Order article via Infotrieve]
5. Bychkov R, Burnham MP, Richards GR, Edwards G, Weston AH, Feletou M, Vanhoutte PM. Characterization of a charybdotoxin-sensitive intermediate conductance Ca²⁺-activated K⁺ channel in porcine coronary endothelium: relevance to EDHF. Br J Pharmacol. 2002; 137: 1346–1354.[CrossRef][Medline] [Order article via Infotrieve]
6. Edwards G, Dora KA, Gardener MJ, Garland CJ, Weston AH. K⁺ is an endothelium-derived hyperpolarizing factor in rat arteries. Nature. 1998; 396: 269–272.[CrossRef][Medline] [Order article via Infotrieve]
7. Weston AH, Richards GR, Burnham MP, Feletou M, Vanhoutte PM, Edwards G. K⁺-induced hyperpolarization in rat mesenteric artery: identification, localization and role of Na⁺/K⁺-ATPases. Br J Pharmacol. 2002; 136: 918–926.[CrossRef][Medline] [Order article via Infotrieve]
8. Fisslthaler B, Popp R, Kiss L, Potente M, Harder DR, Fleming I, Busse R. Cytochrome P4502C is an EDHF synthase in coronary arteries. Nature. 1999; 401: 493–497.[CrossRef][Medline] [Order article via Infotrieve]
9. Nishikawa Y, Stepp DW, Chilian WM. In vivo location and mechanism of EDHF-mediated vasodilation in canine coronary microcirculation. Am J Physiol. 1999; 277: H1252–H1259.[Medline] [Order article via Infotrieve]
10. Bolz SS, Fisslthaler B, Pieperhoff S, de Wit C, Fleming I, Busse R, Pohl U. Antisense oligonucleotides against cytochrome P450 2C8 attenuate EDHF-mediated Ca²⁺ changes and dilation in isolated resistance arteries. FASEB J. 2000; 14: 255–260.[Abstract/Free Full Text]
11. de Wit C, Esser N, Lehr HA, Bolz SS, Pohl U. Pentobarbital sensitive EDHF comediates ACh-induced arteriolar dilation in the hamster microcirculation. Am J Physiol. 1999; 276: H1527–H1534.[Medline] [Order article via Infotrieve]
12. Huang A, Sun D, Smith CJ, Connetta JA, Shesely EG, Koller A, Kaley G. In eNOS knockout mice skeletal muscle arteriolar dilation to acetylcholine is mediated by EDHF. Am J Physiol Heart Circ Physiol. 2000; 278: H762–H768.[Abstract/Free Full Text]
13. Gauthier KM, Deeter C, Krishna UM, Reddy YK, Bondlela M, Falck JR, Campbell WB. 14,15-Epoxyeicosa-5(Z)-enoic acid: a selective epoxyeicosatrienoic acid antagonist that inhibits endothelium-dependent hyperpolarization and relaxation in coronary arteries. Circ Res. 2002; 90: 1028–1036.[Abstract/Free Full Text]
14. Brandes RP, Schmitz-Winnenthal FH, Feletou M, Gödecke A, Huang PL, Vanhoutte PM, Fleming I, Busse R. An endothelium-derived hyperpolarizing factor distinct from NO and prostacyclin is a major endothelium-dependent vasodilator in resistance vessels of wild-type and endothelial NO synthase knockout mice. Proc Natl Acad Sci U S A. 2000; 97: 9747–9752.[Abstract/Free Full Text]
15. Koeppen M, Feil R, Siegl D, Feil S, Hofmann F, Pohl U, de Wit C. cGMP-dependent protein kinase mediates NO- but not acetylcholine-induced dilations in resistance vessels in vivo. Hypertension. 2004; 44: 952–955.[Abstract/Free Full Text]
16. Matoba T, Shimokawa H, Nakashima M, Hirakawa Y, Mukai Y, Hirano K, Kanaide H, Takeshita A. Hydrogen peroxide is an endothelium-derived hyperpolarizing factor in mice. J Clin Invest. 2000; 106: 1521–1530.[Medline] [Order article via Infotrieve]
17. Chauhan SD, Nilsson H, Ahluwalia A, Hobbs AJ. Release of C-type natriuretic peptide accounts for the biological activity of endothelium-derived hyperpolarizing factor. Proc Natl Acad Sci U S A. 2003; 100: 1426–1431.[Abstract/Free Full Text]
18. Emerson GG, Segal SS. Electrical coupling between endothelial cells and smooth muscle cells in hamster feed arteries. Role in vasomotor control. Circ Res. 2000; 87: 474–479.[Abstract/Free Full Text]
19. Coleman HA, Tare M, Parkington HC. EDHF is not K⁺ but may be due to spread of current from the endothelium in guinea pig arterioles. Am J Physiol Heart Circ Physiol. 2001; 280: H2478–H2483.[Abstract/Free Full Text]
20. Sandow SL, Tare M, Coleman HA, Hill CE, Parkington HC. Involvement of myoendothelial gap junctions in the actions of endothelium-derived hyperpolarizing factor. Circ Res. 2002; 90: 1108–1113.[Abstract/Free Full Text]
21. Sandow SL, Hill CE. 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. Circ Res. 2000; 86: 341–346.[Abstract/Free Full Text]
22. Sandow SL, Looft-Wilson R, Doran B, Grayson TH, Segal SS, Hill CE. Expression of homocellular and heterocellular gap junctions in hamster arterioles and feed arteries. Cardiovasc Res. 2003; 60: 643–653.[Abstract/Free Full Text]
23. Dora KA, Sandow SL, Gallagher NT, Takano H, Rummery NM, Hill CE, Garland CJ. Myoendothelial gap junctions may provide the pathway for EDHF in mouse mesenteric artery. J Vasc Res. 2003; 40: 480–490.[CrossRef][Medline] [Order article via Infotrieve]
24. Chaytor AT, Martin PEM, Edwards DH, Griffith TM. Gap junctional communication underpins EDHF-type relaxations evoked by ACh in the rat hepatic artery. Am J Physiol Heart Circ Physiol. 2001; 280: H2441–H2450.[Abstract/Free Full Text]
25. Hutcheson IR, Chaytor AT, Evans WH, Griffith TM. Nitric oxide independent relaxations to acetylcholine and A23187 involve different routes of heterocellular communication. Role of gap junctions and phospholipase A(2). Circ Res. 1999; 84: 53–63.[Abstract/Free Full Text]
26. Chaytor AT, Evans WH, Griffith TM. Central role of heterocellular gap junctional communication in endothelium-dependent relaxations of rabbit arteries. J Physiol. 1998; 508: 561–573.[Abstract/Free Full Text]
27. Yamamoto Y, Klemm MF, Edwards FR, Suzuki H. Intercellular electrical communication among smooth muscle and endothelial cells in guinea-pig mesenteric arterioles. J Physiol. 2001; 535: 181–195.[Abstract/Free Full Text]
28. Welsh DG, Segal SS. Endothelial and smooth muscle cell conduction in arterioles controlling blood flow. Am J Physiol. 1998; 274: H178–H186.[Medline] [Order article via Infotrieve]
29. de Wit C, Roos F, Bolz SS, Kirchhoff S, Krüger O, Willecke K, Pohl U. Impaired conduction of vasodilation along arterioles in connexin40 deficient mice. Circ Res. 2000; 86: 649–655.[Abstract/Free Full Text]
30. Budel S, Bartlett IS, Segal SS. Homocellular conduction along endothelium and smooth muscle of arterioles in hamster cheek pouch: unmasking an NO wave. Circ Res. 2003; 93: 61–68.[Abstract/Free Full Text]
31. Sandow SL, Goto K, Rummery NM, Hill CE. Developmental changes in myoendothelial gap junction mediated vasodilator activity in the rat saphenous artery. J Physiol. 2004; 556: 875–886.[Abstract/Free Full Text]
32. Griffith TM, Chaytor AT, Taylor HJ, Giddings BD, Edwards DH. cAMP facilitates EDHF-type relaxations in conduit arteries by enhancing electrotonic conduction via gap junctions. Proc Natl Acad Sci U S A. 2002; 99: 6392–6397.[Abstract/Free Full Text]
33. Popp R, Brandes RP, Ott G, Busse R, Fleming I. Dynamic modulation of interendothelial gap junctional communication by 11,12-epoxyeicosatrienoic acid. Circ Res. 2002; 90: 800–806.[Abstract/Free Full Text]
34. Taylor MS, Bonev AD, Gross TP, Eckman DM, Brayden JE, Bond CT, Adelman JP, Nelson MT. Altered expression of small-conductance Ca²⁺-activated K⁺ (SK3) channels modulates arterial tone and blood pressure. Circ Res. 2003; 93: 124–131.[Abstract/Free Full Text]

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