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Inward rectifying potassium channels facilitate cell-to-cell communication in hamster retractor muscle feed arteries

¹Smooth Muscle Research Group and the Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta, Canada; ²Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan; and ³Department of Electrical and Computer Engineering, University of Calgary, Calgary, Alberta, Canada

Submitted 28 February 2006 ; accepted in final form 30 March 2006

	ABSTRACT

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This study examined whether inward rectifying K⁺ (K_IR) channels facilitate cell-to-cell communication along skeletal muscle resistance arteries. With the use of feed arteries from the hamster retractor muscle, experiments examined whether K_IR channels were functionally expressed and whether channel blockade attenuated the conduction of acetylcholine-induced vasodilation, an index of cell-to-cell communication. Consistent with K_IR channel expression, this study observed the following: 1) a sustained Ba²⁺-sensitive, K⁺-induced dilation in preconstricted arteries; 2) a Ba²⁺-sensitive inwardly rectifying K⁺ current in arterial smooth muscle cells; and 3) K_IR2.1 and K_IR2.2 expression in the smooth muscle layer of these arteries. It was subsequently shown that the discrete application of acetylcholine elicits a vasodilation that conducts with limited decay along the feed artery wall. In the presence of 100 µM Ba²⁺, the local and conducted response to acetylcholine was attenuated, a finding consistent with a role for K_IR in facilitating cell-to-cell communication. A computational model of vascular communication accurately predicted these observations. Control experiments revealed that in contrast to Ba²⁺, ATP-sensitive- and large-conductance Ca²⁺ activated-K⁺ channel inhibitors had no effect on the local or conducted vasodilatory response to acetylcholine. We conclude that smooth muscle K_IR channels play a key role in facilitating cell-to-cell communication along skeletal muscle resistance arteries. We attribute this facilitation to the intrinsic property of negative slope conductance, a biophysical feature common to K_IR2.1- and 2.2-containing channels, which enables them to increase their activity as a cell hyperpolarizes.

gap junctions; intercellular conduction; K⁺ channels; skeletal muscle arteries

Cell-to-cell communication is typically assessed in vascular studies by applying vasoactive agents to a small portion of a resistance artery (30, 32, 33). This discrete application initiates a localized change in smooth muscle or endothelial cell membrane potential that, with the aid of gap junctions, conducts to neighboring vascular cells (5, 6, 33, 34). The extent to which the electrical or the corresponding vasomotor response conducts longitudinally reveals the nature of vascular communication. With this approach, microcirculatory investigations (5, 6, 32) have reported a range of vascular behaviors, including the ability of the endothelium to initiate hyperpolarizations that conduct with little or no electronic decay. This absence of decay has fostered the view that there is an ionic conductance within the vessel wall that facilitates the conduction of a hyperpolarizing phenomenon (4, 11).

For facilitation to occur in a resistance artery, a hyperpolarizing response must, in a voltage-dependent manner, augment an outward K⁺ conductance or inhibit a depolarizing inward current. Whereas a variety of ionic currents are present in resistance arteries (11, 21), one of the few conductances of which the biophysical properties fit the preceding criteria is the inward rectifying K⁺ (K_IR) current. K_IR channels are tetrameric structures, which in vascular tissue, are composed of K_IR2.x subunits, including K_IR2.1 and K_IR2.2 (1, 13). When expressed in human embryonic kidney cells, these subunits give rise to macroscopic currents of which the outward component paradoxically increases as a cell hyperpolarizes toward the K⁺ equilibrium potential (E_K) (2, 28). This so called "negative slope conductance" arises from the known ability of intracellular Mg²⁺-polyamines to block K_IR2.1- and K_IR2.2-containing channels in a voltage-dependent manner (8, 18).

Given the intrinsic biophysical property of negative slope conductance, this study sought to ascertain whether K_IR channels facilitate cell-to-cell communication along skeletal muscle resistance arteries. With the use of a full range of methodologies, initial experiments confirmed the expression of K_IR2.1- and K_IR2.2-containing channels in the smooth muscle cell layer of hamster retractor muscle feed arteries. Consistent with K_IR facilitating cell-to-cell communication, micromolar Ba²⁺ attenuated the conduction of acetylcholine-induced dilation along the arterial wall. Computational modeling accurately predicted this attenuation along with the ability of Ba²⁺ to attenuate the local and direct vasodilatory effects of acetylcholine. Other K⁺ channel inhibitors did not attenuate conduction, a finding that indicates a unique role for K_IR in the facilitation process. We propose that by facilitating cell-to-cell communication, K_IR channels could augment the dynamic range of blood flow responses in skeletal muscle.

	METHODS

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Animal handling procedures were approved by the Animal Care and Use Committees at the University of Calgary or Michigan State University. Briefly, male golden Syrian hamsters (10–12 wk of age) were euthanized with pentobarbital sodium (90 mg/kg ip) or CO₂ asphyxiation, and an incision was made through the skin. Retractor muscles with attached feed arteries were rapidly removed and placed in ice-cold (pH = 7.4) phosphate- [whole vessel experiments (19)] or HEPES- [electrophysiology experiments (12)]-buffered solution. Feed arteries were then dissected free from fat cells, connective tissue, and muscle tissue.

Vessel myography. An isolated feed artery was placed in an arteriograph and cannulated with glass micropipettes. The arteriograph was then positioned on an inverted microscope where the vessel was slowly pressurized to 60 mmHg (in vivo pressure) and superfused with physiological saline solution (PSS; 5% CO₂, 21% O₂; 36°C) containing the following (in mM): 119 NaCl, 4.7 KCl, 1.7 KH₂PO₄, 1.2 MgSO₄, 1.6 CaCl₂, 5 glucose, and 20 NaHCO₃. All vessels developed myogenic tone (response range 20–40 µm) during the 60-min equilibration period. After equilibration, vessel tone was further increased by 1) elevating intravascular pressure (80 mmHg); or 2) superfusing the preparation with 0.1–1 µM phenylephrine. This was undertaken to strongly depolarize feed arteries and thus enhance the voltage-dependent block of K_IR channels by intracellular Mg²⁺ and polyamines (8, 18). Before experimentation, contractile status was ascertained by briefly superfusing the feed arteries with 60 mM KCl and 1 µM acetylcholine. Arterial diameter was monitored by using a x10 objective and an automated diameter tracking system (IonOptix, Milton, MA).

Preconstricted feed arteries were exposed to one of two experimental protocols. First, to ascertain the functional presence of K_IR channels, preconstricted feed arteries were superfused with elevated K⁺ PSS (15.4 mM) and then exposed to an increasing concentration of Ba²⁺ (1–300 µM). Second, to monitor cell-to-cell communication, a micropipette filled with acetylcholine (200 mM) was positioned with the tip adjacent to the one end of a feed artery (Fig. 1). With the use of iontophoresis (0.25–5.0 s pulse; ejection current 0.5 µA; retain current 0.2 µA), acetylcholine was discretely applied to preconstricted arteries in the absence and presence of Ba²⁺ (30 or 100 µM), glybenclamide [10 µM; an ATP-sensitive K⁺ (K_ATP) channel inhibitor (23)] or glybenclamide ± iberiotoxin [50 nM; a large-conductance Ca²⁺-activated K⁺ (BK) channel inhibitor (21)]. Peak dilatory responses were evaluated 0-, 900-, and 1,800-µm distal to the site of agent application.

View larger version (6K): [in this window] [in a new window]   Fig. 1. Diagram illustrating measurement of cell-to-cell communication. A feed artery is cannulated, and a stimulus pipette containing acetylcholine (0.2 M) is placed close to the vessel wall. Acetylcholine is liberated from the pipette by microiontophoresis (500 nA ejection current, 0.25–5.0 s duration), and arterial diameter is measured every 900 µm along the longitudinal axis of the artery.

Macroscopic K⁺ currents were measured by using conventional whole cell patch clamp. Briefly, an aliquot of cells was placed in the recording chamber and allowed to settle before being superfused with a HEPES-buffered solution (22°C, pH = 7.4) containing (in mM) 140 NaCl, 5 KCl, 1 MgCl₂, 2 CaCl₂, 10 HEPES, and 10 glucose. Fire-polished patch pipettes (tip resistance 2–4 m) were backfilled with solution (pH = 7–7.2) containing (in mM) 100 K-aspartate, 43 KCl, 1 MgCl₂, 10 HEPES, 10 glucose, and 0.5 EGTA. These pipettes were gently lowered onto a smooth muscle cell, and whole cell electrical access (<15 M) was achieved by applying a sharp pulse of negative pressure. Smooth muscle cells were held at –60 mV with the aid of an Axopatch 200A patch-clamp amplifier coupled to a personal computer running Clampex software. Whole cell currents were filtered at 1 kHz, sampled at 5 kHz, and then normalized to cell capacitance (18 ± 1 pF; n = 20).

To monitor the Ba²⁺-sensitive K_IR current, smooth muscle cells were first superfused with solutions containing 100 mM K⁺ (equimolar substitution of KCl for NaCl). Voltage was then stepped to –140 mV for 200 ms and then ramped to +20 mV at a rate of 0.8 mV/ms. The currents from three trials (5 s between trails) were subsequently averaged. Ba²⁺ sensitivity was assessed by applying the preceding protocol to cells superfused with solutions containing an increasing concentration of this K_IR channel inhibitor for 2–3 min.

Western blotting. Feed arteries from four hamsters were placed in 100 µl of lysis buffer (pH 7.4) containing (in mM) 150 NaCl, 1 CaCl₂, 1 MgCl₂, 10 HEPES, 0.5% Tween, 10% mammalian protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Samples were mechanically disrupted, exposed to three freeze-thaw cycles, and then centrifuged (10 min; 13,000 rpm). Supernatant was placed in a clean tube, assayed for total protein, and stored at –20°C for up to 1 wk. Samples were prepared for electrophoresis by placing 15 µl of supernatant in 5 µl of 4x sample buffer, and 2 µl of DTT. After heating (10 min; 90°C) was completed, 20 mg of protein were loaded to run on a 10% polyacrylamide gel. Protein was transferred to a polyvinylidene difluoride membrane, blocked for 2 h (5% nonfat milk in Tris-buffered saline), and incubated overnight (4°C) with a primary antibody (K_IR2.1 or K_IR2.2; 1:200 dilution; Sigma-Aldrich) diluted in milk-Tris-buffered saline. The PVDF membrane was subsequently washed and incubated with a horseradish peroxidase-conjugated secondary antibody (1:1,000; Jackson Laboratories) diluted in milk-Tris-buffered saline (1 h; 20–22°C). Washing was repeated and the blot developed using chemiluminescent substrate (Pierce Biochemicals, Rockford, IL).

Immunohistochemistry. Feed arteries were arranged in cyroprotective media and rapidly frozen in liquid nitrogen. Tissue blocks were mounted in a cryostat and vessels cut in cross section (8–10 µm thickness). Cross sections were air-dried on poly-L-lysine-coated slides and washed with Tris-buffered saline (pH 7.4) containing 50 mM Tris, 0.9% NaCl, 0.1% BSA, and 0.01% Triton X. Sections were lightly fixed in 2% paraformaldehyde-Tris-buffered saline (20 min) and incubated (4 h) with a quench solution containing 100 mM Tris, 1.8% NaCl, 5% goat serum, 2% BSA, and 0.2% Triton X. Sections were subsequently incubated overnight (4°C) with a polyclonal (K_IR2.1 or K_IR2.2; 1:200) and a monoclonal (eNOS; 1:2,500; Chemicon, Ternecula, CA) antibody diluted in quench solution. Slides were then washed with Tris-buffered saline and incubated for 4 h (20–22°C) with an anti-rabbit-Cy3 and an anti-mouse-Cy5 IgG-specific secondary antibody. After further washing was completed, slides were air-dried and mounted in antifade media (Molecular Probes, Eugene, OR). Arterial cross sections were viewed and photographed by using a Zeiss fluorescent microscope coupled to a x63 oil immersion lens.

Computational modeling. To complement our experimental approach, a computational model developed by Diep et al. (3) determined whether the elimination of a smooth muscle K_IR-like current could attenuate vascular communication. Computational theory and base parameters are identical to the original publication with two notable exceptions. First, a second layer of smooth muscle cells was added to our virtual artery, a change consistent with the electron microscopy findings of Sandow et al. (27). Second, the nonlinear resistor representing the smooth muscle ionic conductance was divided into two components, one for K_IR and the second for all other conductances. This study assumed that K_IR was minimally active at –40 mV (0 pAs), maximally active at –60 mV (1.5 pAs), and reversed at –80 mV. A sigmoidal and exponential function fixed the data points between –40 to –60 mV and –60 to –80 mV, respectively. At 1.5 pAs, peak outward current would be below the resolution limit of whole cell patch-clamp electrophysiology. The nonlinear resistor representing all other smooth muscle conductances was comparable to Diep et al. (3) except that its relative magnitude (below –40 mV) was increased by 20%. Consequently, net inward current at –60 mV was 1.5 pAs greater than our previous publication (3). To initiate current flow in our virtual artery, hyperpolarizing current was injected into one arterial segment of endothelium (50 µm in length and consisting of 48 endothelial cells) for 400 ms. Voltage responses were subsequently monitored along the entire length of the virtual artery (2,200 µm).

Statistical analysis. Data are expressed as means ± SE and n indicates feed artery number. Repeated measures ANOVAs compared feed artery responses in the absence and presence of Ba²⁺ or glybenclamide ± iberiotoxin. Where appropriate, a Bonferroni comparison was used for post hoc analysis. P values 0.05 were considered statistically significant.

Chemicals. Buffer chemicals, acetylcholine, phenylephrine, BaCl₂, iberiotoxin, and glybenclamide were purchased from Sigma-Aldrich. All other reagents are listed with source.

	RESULTS

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K_IR channel expression in retractor muscle feed arteries. The functional expression of K_IR channels was assessed in initial experiments by examining the responsiveness of preconstricted feed arteries to elevated extracellular [K⁺]. Consistent with their presence, feed arteries dilated in a sustained and near-maximal manner as extracellular [K⁺] was elevated from 5.4 to 15.4 mM (Fig. 2). In this dilated, and presumably hyperpolarized state, the application of micromolar Ba²⁺ [a K_IR channel inhibitor (23)] initiated feed artery constriction. This increase in tone was concentration dependent with 300 µM Ba²⁺ required to restore feed artery diameter to control levels. The method of vessel preconstriction had no qualitative influence on feed artery responsiveness to elevated extracellular K⁺ or Ba²⁺ (Fig. 2).

View larger version (16K): [in this window] [in a new window]   Fig. 2. Inward rectifying K+ (KIR) channels are functionally present in retractor muscle feed arteries. A: effects of elevated K+ (5.4–15.4 mM) and Ba2+ on feed arteries preconstricted to 80 mmHg intravascular pressure (n = 8; 78 ± 4 and 114 ± 8 µm for preconstricted and maximal diameters, respectively). B: effects of elevated K+ (5.4–15.4 mM) and Ba2+ on feed arteries preconstricted to 60 mmHg intravascular pressure plus 0.1–1 µM phenylephrine (n = 6; 62 ± 6 and 109 ± 9 µm for preconstricted and maximal diameters, respectively). *Significant difference from 0 mM Ba2+.

View larger version (9K): [in this window] [in a new window]   Fig. 3. Ba2+-sensitive KIR current in smooth muscle cells isolated from the retractor muscle feed artery. A: representative whole cell current recording in a high K+ (100 mM) bath solution ±Ba2+ (300 µM). B: Ba2+-subtracted current from A. C: Ba2+ induces a concentration-dependent inhibition of KIR current (IC50 at –140 mV = 3.7 µM).

View larger version (35K): [in this window] [in a new window]   Fig. 4. KIR2.1 and KIR2.2 expression in retractor muscle feed arteries. A: Western blot analysis of KIR2.1 and KIR2.2 in whole feed arteries extracted from four hamsters. B: immunohistochemical analysis of feed artery cross sections. Sections were labeled for KIR2.1 or KIR2.2 (pseudocolored in red) and endothelial nitric oxide synthase (eNOS) (pseudocolored in green). Staining was absent in cross sections where primary KIR antibody was preabsorbed with antigenic peptide. Bottom cross sections were not labeled for eNOS.

View larger version (13K): [in this window] [in a new window]   Fig. 5. KIR channels facilitate conduction of endothelium-dependent vasodilation along arteries preconstricted to intravascular pressure. Feed arteries preconstricted to 80 mmHg intravascular pressure were stimulated with an acetylcholine pipette while vessel diameter was monitored 0-, 900-, and 1,800-µm distal to the point of agent application. Note that Ba2+ superfusion attenuated dilation at local site of acetylcholine application (see Fig. 7 for details). Local responses were, however, matched by increasing the acetylcholine pulse duration by two- to threefold in the presence of Ba2+. A: vasodilatory responses in the absence and presence of 30 µM Ba2+ (n = 6; 60 ± 5, 56 ± 5, and 108 ± 6 µm for preconstricted, preconstricted + Ba2+, and maximal diameters, respectively). B: vasodilatory responses in the absence and presence of 100 µM Ba2+ (n = 6; 67 ± 7, 60 ± 5, and 129 ± 2 µm for preconstricted, preconstricted + Ba2+, and maximal diameters, respectively). C: effects of Ba2+ on conduction decay. Conduction decay was calculated as change in vasodilation between the 900- and 1,800-µm measurement sites. *Significant difference from control.

View larger version (14K): [in this window] [in a new window]   Fig. 6. KIR channels facilitate conduction of endothelium-dependent vasodilation along arteries preconstricted to intravascular pressure and phenylephrine. Feed arteries preconstricted to 60 mmHg intravascular pressure and 0.1–1 µM phenylephrine were stimulated with an acetylcholine pipette while vessel diameter was monitored 0-, 900-, and 1,800-µm distal to the point of agent application. Note that Ba2+ superfusion attenuated dilation at the local site of acetylcholine application (see Fig. 7 for details). Local responses were, however, matched by increasing the acetylcholine pulse duration by two- to threefold in the presence of Ba2+. A: vasodilatory responses in the absence and presence of 30 µM Ba2+ (n = 6; 45 ± 6, 36 ± 5, and 97 ± 6 µm for preconstricted, preconstricted + Ba2+ and maximal diameters, respectively). B: vasodilatory responses in the absence and presence of 100 µM Ba2+ (n = 6; 50 ± 3, 38 ± 2, and 103 ± 7 µm for preconstricted, preconstricted + Ba2+, and maximal diameters, respectively). C: effects of Ba2+ on conduction decay. Conduction decay was calculated as change in vasodilation between the 900- and 1,800-µm measurement sites. *Significant difference from control.

View larger version (11K): [in this window] [in a new window]   Fig. 7. Ba2+ attenuates feed arteries dilation at the local site of acetylcholine application. In contrast to Figs. 5 and 6, an identical acetylcholine pulse was applied via micropipette to preconstricted feed arteries under control conditions or in the presence of 30 or 100 µM Ba2+. Vasomotor responses were monitored at the local site of agent application. A: feed arteries were preconstricted to 80 mmHg intravascular pressure (n = 6; diameter values in Fig. 5). B: feed arteries were preconstricted to 60 mmHg intravascular pressure + 0.1–1 µM phenylephrine (n = 6; diameter values in Fig. 6). *Significant difference from control.

View larger version (14K): [in this window] [in a new window]   Fig. 8. ATP-sensitive K+ (KATP) and large conductance Ca2+ (BKCa) channels do not facilitate conducted vasodilation. Feed arteries preconstricted to 60 mmHg intravascular pressure + 0.1–1 µM phenylephrine were stimulated with an acetylcholine pipette while vessel diameter was monitored 0-, 900-, and 1,800-µm distal to the point of agent application. Experiments were performed in the absence and presence of glybenclamide (10 µM) ± iberiotoxin (50 nM). A: effects of glybenclamide (n = 6; 49 ± 2, 48 ± 2, 106 ± 2 µm for preconstricted, preconstricted + glybenclamide, and maximal diameters, respectively) on the conduction of acetylcholine-induced vasodilation. B: effects of glybenclamide and iberiotoxin (n = 5; 47 ± 2, 38 ± 2, and 106 ± 2 µm for preconstricted, preconstricted + glybenclamide-iberiotoxin, and maximal diameters, respectively). C: conduction decay was calculated as change in vasodilation between 900- and 1,800-µm sites.

View larger version (16K): [in this window] [in a new window]   Fig. 9. Computational modeling predicts that smooth muscle KIR channels facilitate the conduction of endothelium-dependent hyperpolarization. Simulations: 35 or 85 pAs of hyperpolarizing current was injected for 400 ms into each of 48 endothelial cells located within one 50-µm segment of endothelium (general location denoted by arrow). Voltage responses (VM) were monitored in outer smooth muscle cell layer in the presence and absence of a smooth muscle KIR-like current. A and B: smooth muscle VM response at 400 ms as color mapped along the vessel wall (A) or represented in a two-dimensional voltage plot (B). Note that in the absence of KIR, current injection per endothelial cell had to be increased to elicit a comparable response in smooth muscle cells overlying the stimulus site. C: current injection was maintained at 35 pAs per endothelial cell, and VM response (at 400 ms) was monitored in the smooth muscle cells overlying stimulus site.

	DISCUSSION

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Cell-to-cell communication in small skeletal muscle arteries. Cell-to-cell communication is typically assessed in small skeletal muscle arteries by applying vasoactive agents to a small portion of the vessel (30, 32, 33). This discrete application initiates local changes in endothelial or smooth muscle membrane potentials, which, with the aid of gap junctions, spread to neighboring vascular cells (5, 6, 33). With this approach, the nature of cell-to-cell communication is revealed by the extent to which the electrical or the corresponding vasomotor response conducts longitudinally along the arterial wall. Past studies have observed that hyperpolarizing responses initiated in endothelial cells conduct robustly along most skeletal muscle resistance vessels (5, 6, 32, 33). In contrast, agents that depolarize and constrict smooth muscle cells elicit a decidedly varied response, ranging from little to strong conduction (14, 15, 32, 35). This variability in cell-to-cell communication has been a source of active inquiry with recent computational work (3) indicating that it predictably reflects 1) the known differences in gap junctional resistivity among vascular cells, and 2) the varied orientation of endothelial and smooth muscle cells within the vessel wall.

Given that endothelium-initiated hyperpolarizations often conduct along skeletal muscle arteries with little electrotonic decay, past studies (4, 11) have suggested that an ion channel may be playing an active role in facilitating cell-to-cell communication. For facilitation to occur, a conducting hyperpolarization must, in a voltage-dependent manner, augment an outward conductance or inhibit a depolarizing inward current. Although a range of ionic conductances are expressed in resistance arteries (11, 21), K_IR is one of the few of which the biophysical properties could facilitate the conduction of endothelium-initiated hyperpolarization. K_IR channels are composed of four -subunits, and past molecular studies have shown that K_IR2.1 and K_IR2.2 are prominent in vascular tissue (1, 13). In expression systems, these K_IR subunits form homo- or heteromultimeric channels in which outward current over a physiological voltage range increases with hyperpolarization (2, 28). This augmentation is referred to as "negative slope conductance," and it arises from the ability of intracellular Mg²⁺ polyamines to block these channels in a voltage-dependent manner (8, 18).

K_IR expression in retractor muscle feed arteries. Initial experiments focused on confirming the presence of K_IR in retractor muscle feed arteries. Characteristic of arteries expressing this K⁺ conductance, elevated extracellular K⁺ (from 5.4 to 15.4 mM) elicited a sustained and near-maximal dilation (Fig. 2). In this dilated-hyperpolarized state where K_IR is the dominant conductance setting resting membrane potential, the K_IR channel inhibitor Ba²⁺ elicited a progressive concentration-dependent constriction. Consistent with these functional observations, patch-clamp electrophysiology revealed a Ba²⁺-sensitive K_IR current in feed arterial smooth muscle cells (Fig. 3). The IC₅₀ of Ba²⁺ was subtly higher than that for rat arterial smooth muscle cells (1, 22, 25), a finding indicative perhaps of a species-dependent difference in the affinity of barium for K_IR2.1 or K_IR2.2. At physiological membrane potential, this value will be substantively higher given the known voltage dependency of Ba²⁺ block (1, 28). Western analysis confirmed the expression of K_IR2.1 and K_IR2.2 (Fig. 4A) in whole feed arteries, whereas immunohistochemistry analysis revealed their presence in the smooth muscle layer (Fig. 4B). K_IR subunit expression was not apparent in the endothelium, although these findings should be interpreted cautiously given that the thin cross-sectional profile of endothelial cells makes identification of a modestly expressed protein difficult.

Facilitation of cell-to-cell communication. To examine whether K_IR channels actively facilitate cell-to-cell communication, isolated retractor muscle feed arteries were strongly preconstricted and exposed to a brief acetylcholine pulse to elicit a vasodilation that conducts. In theory, strong preconstriction should depolarize smooth muscle cells and minimize K_IR activity through the blocking effects of intracellular Mg²⁺ polyamines (8, 18). Under this basal condition, K_IR activity would be expected to rise as an endothelium-dependent hyperpolarization spreads to smooth muscle cells via myoendothelial gap junctions. Such an increase would promote smooth muscle cell hyperpolarization and limit the voltage differential between the two cell types. This, in turn, would minimize radial charge loss to the smooth muscle cell layer and facilitate charge conduction along the endothelium. Consistent with the theory, our findings demonstrate that the discrete application of acetylcholine elicits a robust conducted response in which vasodilatory decay was enhanced by Ba²⁺ (Figs. 5 and 6). This enhanced decay was particularly evident at 100 µM Ba²⁺, a concentration that 1) constricts feed arteries when K_IR activity is intrinsically high (Fig. 2), and 2) should substantively inhibit K_IR channels even at depolarized physiological voltages (Fig. 3). A similar attenuation was absent in feed arteries treated with K_ATP and B_K channel inhibitors (Fig. 8). Whereas this study is the first to reveal a role for K_IR in facilitating cell-to-cell communication, it is not the first to address this question (7, 24). Indeed, similar to this study where decay was measured over a length of vessel distal to the point of agent application, Goto et al. (7) noted that while Ba²⁺ altered the ability of acetylcholine to initiate hyperpolarization, it did not consistently enhance vasomotor or electronic decay. Species or tissue differences in the type and number of expressed K_IR could partially account for this discrepancy. Furthermore, the Ba²⁺ concentration (30 µM) employed by Goto et al. (7) may not have blocked a sufficient portion of K_IR channels to augment decay. We highlight this issue given that in this study, 30 µM Ba²⁺ only partially blocked sustained K⁺-induced dilation (Fig. 2) and did not significantly augment conduction decay (Figs. 5 and 6).

In addition to the preceding experiments, a supplemental approach was undertaken to reinforce the importance of K_IR channels in facilitating cell-to-cell communication. Using a computational model based on the properties of resistance arteries (3), we explored on a theoretical level whether electrical conduction could be attenuated by eliminating a K_IR-like current from the smooth muscle ionic representation. Our K_IR-like current retained the intrinsic property of "negative slope conductance" and as such it was designed to be minimally and maximally active at –40 mV and –60 mV, respectively. Peak outward current was set to 1.5 pAs, a subtle value generally below the detection limit of whole cell patch-clamp electrophysiology. Consistent with our vasomotor observations, simulations revealed that the elimination of the smooth muscle K_IR current did indeed enhance the decay of electrical conduction (Fig. 9, A and B). Interestingly, in the absence of K_IR, more current had to be injected per endothelial cell to elicit a comparable hyperpolarization in the overlying smooth muscle cells. If, however, the electrical stimulus was maintained (Fig. 9C), hyperpolarization in the overlying smooth muscle cells was reduced. These latter predictions are surprisingly consistent with our experimental observations and the need to lengthen the acetylcholine pulse duration during Ba²⁺ superfusion to elicit a local vasomotor response that was comparable with control data (Figs. 5–7).

Functional implications. Blood flow to skeletal muscle is controlled by a network of resistance arteries linked in series and parallel with one another. Under high metabolic demand, these resistance arteries dilate in a coordinated manner to dramatically increase tissue blood flow (20, 30, 31). This coordinated dilation is intriguing in the retractor muscle where feed arteries lie external to the tissue. As such, these important resistors are not directly exposed to metabolic stimuli and thus rely on other mechanisms to initiate dilation. Using a light-dye ablation technique to focally damage endothelial cells, Segal and Jacobs (31) reported that cell-to-cell communication likely plays a key role in coordinating proximal vessel dilation. In detail, it was proposed that feed artery dilation arises as vasoactive stimuli produced by contracting muscle fibers initiate a hyperpolarization that conducts from distal arterioles into proximal feed arteries (31). For this mechanism to be effective, hyperpolarization must conduct over significant distances and across branch points with little electrotonic decay. It is enticing to suggest that K_IR channels with their intrinsic property of negative slope conductance play a role in limiting this electrotonic decay and thus promote proximal vessel dilation and muscle blood flow.

In summary, this study employed a range of experimental approaches to reveal the importance of K_IR channels in facilitating cell-to-cell communication along retractor muscle feed arteries. We attribute this facilitation to the intrinsic property of negative slope conductance, a biophysical feature common to K_IR2.1- and K_IR2.2-containing channels, which enables these integral membrane proteins to increase their activity as a cell hyperpolarizes. Facilitation could enable vasoactive stimuli produced by skeletal muscle fibers to more effectively dilate resistance arteries that lie external to the tissue. This would likely augment the dynamic range of blood flow responses to skeletal muscle.

	GRANTS

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This work was supported by a Grant-In-Aid from the Heart and Stroke Foundation of Canada (D. G. Welsh), the Natural Science and Engineering Research Council of Canada (E. J. Vigmond), and National Heart, Lung, and Blood Institute Grant HL-32469 (W. F. Jackson). D. G. Welsh is a senior scholar with the Heart and Stroke Foundation of Canada and the Alberta Heritage Foundation for Medical Research and holds a Canada Research Chair in Gap Junctional Communication. E. J. Vigmond is a scholar with the Alberta Ingenuity Fund.

	ACKNOWLEDGMENTS

 
The authors are grateful to Hai Kim Diep and Kevin D. Luykenaar for assistance.

	FOOTNOTES

 

Address for reprint requests and other correspondence: D. G. Welsh, Dept. of Physiology and Biophysics, HM-86, Heritage Medical Research Bldg., 3330 Hospital Dr., NW, Calgary, Alberta, Canada, T2N-4N1 (e-mail: dwelsh{at}ucalgary.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

	REFERENCES

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1. Bradley KK, Jaggar JH, Bonev AD, Heppner TJ, Flynn ER, Nelson MT, and Horowitz B. Kir2.1 encodes the inward rectifier potassium channel in rat arterial smooth muscle cells. J Physiol 515: 639–651, 1999.[Abstract/Free Full Text]
2. Dhamoon AS, Pandit SV, Sarmast F, Parisian KR, Guha P, Li Y, Bagwe S, Taffet SM, and Anumonwo JM. Unique Kir2x properties determine regional and species differences in the cardiac inward rectifier K⁺ current. Circ Res 94: 1332–1339, 2004.[Abstract/Free Full Text]
3. Diep HK, Vigmond EJ, Segal SS, and Welsh DG. Defining electrical communication in skeletal muscle resistance arteries: a computational approach. J Physiol 568: 267–281, 2005.[Abstract/Free Full Text]
4. Emerson GG, Neild TO, and Segal SS. Conduction of hyperpolarization along hamster feed arteries: augmentation by acetylcholine. Am J Physiol Heart Circ Physiol 283: H102–H109, 2002.[Abstract/Free Full Text]
5. Emerson GG and Segal SS. Electrical coupling between endothelial cells and smooth muscle cells in hamster feed arteries: role in vasomotor control. Circ Res 87: 474–479, 2000.[Abstract/Free Full Text]
6. Emerson GG and Segal SS. Endothelial cell pathway for conduction of hyperpolarization and vasodilation along hamster feed artery. Circ Res 86: 94–100, 2000.[Abstract/Free Full Text]
7. Goto K, Rummery NK, Grayson TH, and Hill CE. Attenuation of conducted vasodilatation in rat mesenteric arteries during hypertension: role of inwardly rectifying potassium channels. J Physiol 561: 215–231, 2004.[Abstract/Free Full Text]
8. Guo D, Ramu Y, Klem AM, and Lu Z. Mechanism of rectification in inward-rectifier K⁺ channels. J Gen Physiol 121: 261–275, 2003.[Abstract/Free Full Text]
9. Gustafsson F, Mikkelsen HB, Arensbak B, Thuneberg L, Neve S, Jensen LJ, and Holstein-Rathlou NH. Expression of connexin 37, 40 and 43 in rat mesenteric arterioles and resistance arteries. Histochem Cell Biol 119: 139–148, 2003.[Web of Science][Medline]
10. Hill CE, Rummery N, Hickey H, and Sandow SL. Heterogeneity in the distribution of vascular gap junctions and connexins: implications for function. Clin Exp Pharmacol Physiol 29: 620–625, 2002.[CrossRef][Web of Science][Medline]
11. Jackson WF. Potassium channels in the peripheral microcirculation. Microcirculation 12: 113–127, 2005.[Web of Science][Medline]
12. Jackson WF, Huebner JM, and Rusch NJ. Enzymatic isolation and characterization of single vascular smooth muscle cells from cremasteric arterioles. Microcirculation 4: 35–50, 1997.[Web of Science][Medline]
13. Karkanis T, Li S, Pickering JG, and Sims SM. Plasticity of K_IR channels in human smooth muscle cells from internal thoracic artery. Am J Physiol Heart Circ Physiol 284: H2325–H2334, 2003.[Abstract/Free Full Text]
14. Kumer SC, Damon DN, and Duling BR. Patterns of conducted vasomotor response in the mouse. Microvasc Res 59: 310–315, 2000.[CrossRef][Web of Science][Medline]
15. Kurjiaka DT, Bender SB, Nye DD, Wiehler WB, and Welsh DG. Hypertension attenuates cell-to-cell communication in hamster retractor muscle feed arteries. Am J Physiol Heart Circ Physiol 288: H861–H870, 2005.[Abstract/Free Full Text]
16. Li X and Simard JM. Connexin45 gap junction channels in rat cerebral vascular smooth muscle cells. Am J Physiol Heart Circ Physiol 281: H1890–H1898, 2001.[Abstract/Free Full Text]
17. Little TL, Beyer EC, and Duling BR. Connexin 43 and connexin 40 gap junctional proteins are present in arteriolar smooth muscle and endothelium in vivo. Am J Physiol Heart Circ Physiol 268: H729–H739, 1995.[Abstract/Free Full Text]
18. Lu Z. Mechanism of rectification in inward-rectifier K+ channels. Annu Rev Physiol 66: 103–129, 2004.[CrossRef][Web of Science][Medline]
19. Luykenaar KD, Brett SE, Wu BN, Wiehler WB, and Welsh DG. Pyrimidine nucleotides suppress K_DR currents and depolarize rat cerebral arteries by activating Rho kinase. Am J Physiol Heart Circ Physiol 286: H1088–H1100, 2004.[Abstract/Free Full Text]
20. Murrant CL and Sarelius IH. Local and remote arteriolar dilations initiated by skeletal muscle contraction. Am J Physiol Heart Circ Physiol 279: H2285–H2294, 2000.[Abstract/Free Full Text]
21. Nelson MT and Quayle JM. Physiological roles and properties of potassium channels in arterial smooth muscle. Am J Physiol Cell Physiol 268: C799–C822, 1995.[Abstract/Free Full Text]
22. Quayle JM, McCarron JG, Brayden JE, and Nelson MT. Inward rectifier K⁺ currents in smooth muscle cells from rat resistance- sized cerebral arteries. Am J Physiol Cell Physiol 265: C1363–C1370, 1993.[Abstract/Free Full Text]
23. Quayle JM, Nelson MT, and Standen NB. ATP-sensitive and inwardly rectifying potassium channels in smooth muscle. Physiol Rev 77: 1165–1232, 1997.[Abstract/Free Full Text]
24. Rivers RJ, Hein TW, Zhang C, and Kuo L. Activation of barium-sensitive inward rectifier potassium channels mediates remote dilation of coronary arterioles. Circulation 104: 1749–1753, 2001.[Abstract/Free Full Text]
25. Robertson BE, Bonev AD, and Nelson MT. Inward rectifier K⁺ currents in smooth muscle cells from rat coronary arteries: block by Mg²⁺, Ca²⁺, and Ba²⁺. Am J Physiol Heart Circ Physiol 271: H696–H705, 1996.[Abstract/Free Full Text]
26. Sandow SL and 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 86: 341–346, 2000.[Abstract/Free Full Text]
27. Sandow SL, Looft-Wilson R, Doran B, Grayson TH, Segal SS, and Hill CE. Expression of homocellular and heterocellular gap junctions in hamster arterioles and feed arteries. Cardiovasc Res 60: 643–653, 2003.[Abstract/Free Full Text]
28. Schram G, Pourrier M, Wang Z, White M, and Nattel S. Barium block of Kir2 and human cardiac inward rectifier currents: evidence for subunit-heteromeric contribution to native currents. Cardiovasc Res 59: 328–338, 2003.[Abstract/Free Full Text]
29. Segal SS and Duling BR. Communication between feed arteries and microvessels in hamster striated muscle: segmental vascular responses are functionally coordinated. Circ Res 59: 283–290, 1986.[Abstract/Free Full Text]
30. Segal SS and Duling BR. Flow control among microvessels coordinated by intercellular conduction. Science 234: 868–870, 1986.[Abstract/Free Full Text]
31. Segal SS and Jacobs TL. Role for endothelial cell conduction in ascending vasodilatation and exercise hyperaemia in hamster skeletal muscle. J Physiol 536: 937–946, 2001.[Abstract/Free Full Text]
32. Segal SS, Welsh DG, and Kurjiaka DT. Spread of vasodilatation and vasoconstriction along feed arteries and arterioles of hamster skeletal muscle. J Physiol 516: 283–291, 1999.[Abstract/Free Full Text]
33. Welsh DG and Segal SS. Endothelial and smooth muscle cell conduction in arterioles controlling blood flow. Am J Physiol Heart Circ Physiol 274: H178–H186, 1998.[Abstract/Free Full Text]
34. Xia J and Duling BR. Electromechanical coupling and the conducted vasomotor response. Am J Physiol Heart Circ Physiol 269: H2022–H2030, 1995.[Abstract/Free Full Text]
35. Yashiro Y and Duling BR. Integrated Ca(2+) signaling between smooth muscle and endothelium of resistance vessels. Circ Res 87: 1048–1054, 2000.[Abstract/Free Full Text]

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