Tuning Electrical Conduction Along Endothelial Tubes of Resistance Arteries Through Ca2+-Activated K+ ChannelsNovelty and Significance
Rationale: Electrical conduction through gap junction channels between endothelial cells of resistance vessels is integral to blood flow control. Small and intermediate-conductance Ca2+-activated K+ channels (SKCa/IKCa) initiate electrical signals in endothelial cells, but it is unknown whether SKCa/IKCa activation alters signal transmission along the endothelium.
Objective: We tested the hypothesis that SKCa/IKCa activity regulates electrical conduction along the endothelium of resistance vessels.
Methods and Results: Freshly isolated endothelial cell tubes (60 μm wide; 1–3 mm long; cell length, ≈35 μm) from mouse skeletal muscle feed (superior epigastric) arteries were studied using dual intracellular microelectrodes. Current was injected (±0.1–3 nA) at site 1 while recording membrane potential (Vm) at site 2 (separation distance=50–2000 μm). SKCa/IKCa activation (NS309, 1 μmol/L) reduced the change in Vm along endothelial cell tubes by ≥50% and shortened the electrical length constant (λ) from 1380 to 850 μm (P<0.05) while intercellular dye transfer (propidium iodide) was maintained. Activating SKCa/IKCa with acetylcholine or SKA-31 also reduced electrical conduction. These effects of SKCa/IKCa activation persisted when hyperpolarization (>30 mV) was prevented with 60 mmol/L [K+]o. Conversely, blocking SKCa/IKCa (apamin+charybdotoxin) depolarized cells by ≈10 mV and enhanced electrical conduction (ie, changes in Vm) by ≈30% (P<0.05).
Conclusions: These findings illustrate a novel role for SKCa/IKCa activity in tuning electrical conduction along the endothelium of resistance vessels by governing signal dissipation through changes in membrane resistance. Voltage-insensitive ion channels can thereby tune intercellular electrical signaling independent from gap junction channels.
- conducted vasodilation
- gap junctions
- potassium channels
- ion channels
- membrane potential
- vascular endothelium
Electrical conduction through gap junction channels (GJCs) coordinates vasomotor responses in vascular resistance networks.1–3 Through axial orientation and robust expression of GJCs, endothelial cells (ECs) serve as the primary pathway for cell-to-cell conduction along the vessel wall,4–6 with electrical signals transmitted to smooth muscle cells (SMCs) via myoendothelial GJCs.3,7–9 Once initiated, hyperpolarization (and vasodilation) can travel along the resistance vasculature for millimeters.10–13 As shown in exercising skeletal muscle, vasodilation originating downstream within the microcirculation can ascend the resistance network via conduction along the endothelium to encompass feed arteries upstream and thereby increase total blood flowing into the active muscle.14
In accord with the cable properties of electrical conduction, the distance that signals travel along the endothelium is determined by the length constant (λ=rm/ra)1/2, where rm=membrane resistance and ra=axial resistance to current flow.15,16 With negligible resistance of cytoplasm, intercellular ra is determined primarily by GJCs.8,16 Previous studies have focused on alterations of conducted vasodilation through manipulating the functional integrity of GJCs5,14,17 or the expression profile of their connexin subunits.18–21 Alternatively, changes in rm may also provide a mechanism for governing electrical conduction independent of manipulating GJCs. In turn, changes in rm should reflect the activation of ion channels in the plasma membrane,15,16 which otherwise insulates the cell interior from the extracellular milieu. Remarkably, the effect of governing rm through ion channel activation has not been studied in light of electrical conduction along the endothelium of resistance vessels.
The small (KCa 2.3; KCNN3)-and intermediate (KCa 3.1; KCNN4)-conductance Ca2+-activated K+ channels (SKCa/IKCa) are abundantly expressed in EC membranes.22–24 On activation of SKCa/IKCa the efflux of K+ promotes hyperpolarization.22,25 Although this response may promote vasodilation and tissue perfusion,24 a reduction in rm along the endothelium could dissipate electrical signals and thereby compromise electrical conduction and impair ascending vasodilation. Given the prominent role of SKCa/IKCa in the initiation of EC hyperpolarization,9,24 we questioned whether activation of these ion channels affects electrical conduction along the endothelium.
Understanding how the activation of SKCa/IKCa may govern electrical conduction along the endothelium of intact vessels is limited by myoendothelial coupling to SMCs, perivascular nerve activity, and circulating vasoactive agents along with the shear stress exerted by blood flow. Further, the initiation of an electrical signal to trigger conduction has typically consisted of an agonist (eg, acetylcholine [ACh]4,10,26) or electric field stimulation27,28 via micropipettes. However, such “local” stimuli can activate multiple cells simultaneously, with uncertainty in the precise origin and intensity of the actual stimulus. To avoid such confounding influences, we have freshly isolated EC tubes from feed arteries of mouse abdominal skeletal muscle.29,30 Intact EC tubes from these resistance vessels can exceed 3 mm in length, enabling the efficacy of electrical conduction along the endothelium to be evaluated. Using sharp intracellular microelectrodes, current microinjection into a single EC alters membrane potential (Vm) independent of agonists or ion channel activation. By simultaneously recording Vm at defined distances from the site of current injection, we tested the hypothesis that SKCa/IKCa activity can “tune” electrical conduction along the endothelium.
Findings reported in the present study are the first to show that activating SKCa/IKCa with either pharmacological agents (NS309, SKA-31) or a physiological agonist (ACh) impairs electrical conduction along the endothelium of a resistance artery. Concomitantly, intercellular coupling through GJCs remains intact as confirmed by robust dye transfer between ECs. We further demonstrate that blocking constitutively open SKCa/IKCa enhances electrical conduction. Thus, altering rm via opening and closing plasma membrane ion channels effectively tunes the conduction of electrical signals along the endothelium.
An expanded Methods section is available in the Online Data Supplement.
Tissue Preparation and Intracellular Recording
Procedures were approved by the Institutional Animal Care and Use Committee and performed in accord with the National Research Council's Guide for the Care and Use of Laboratory Animals (8th edition, 2011). Male C57BL/6 mice (bred at the University of Missouri; age, 3–6 months; n=45) were anesthetized (pentobarbital; 60 mg/kg intraperitoneal injection). Abdominal muscles were removed bilaterally; feed arteries (superior epigastric) were dissected free from surrounding tissue. Using gentle trituration after mild enzymatic digestion, SMCs were dissociated to produce intact EC tubes (width: 60 μm, length, 1–3 mm).29,30 Experiments were performed at 32°C and completed within 3 hours.29,30
A freshly isolated EC tube was secured at each end in a tissue bath on the stage of an inverted microscope and superfused continuously with physiological salt solution (composition in mmol/L: 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid, 10 glucose). A pair of intracellular microelectrodes (borosilicate glass; tip resistance, 150–200 MΩ) coupled to independent amplifiers were inserted into ECs along the midline of the tube with a separation distance of 50 to 2000 μm. With individual ECs ≈35 μm long,29 50-μm separation enabled “local” responses to be recorded from site 2 as close to the signal origin (site 1) as possible while ensuring that microelectrodes were in separate (adjacent) cells.30 Successful dual impalements were indicated by correspondence of electrical events between microelectrodes.
Figure 1 illustrates the experimental approach and summarizes the analyses used. To study electrical conduction over distance (Figures 2 and 3), impalement at site 1 was maintained while the microelectrode for recording Vm at site 2 was repositioned at the next distance (order randomized across experiments). As standard procedure for evaluating interventions, separation distance was maintained at 500 μm (Figures 4, 5, 6, 7, and 8)7,30 corresponding to ≈15 ECs placed end-to-end. Current was microinjected (±0.1–3 nA; 2-second pulse) at site 1 while recording Vm at site 2 under control conditions, during activation of SKCa/IKCa with NS309, SKA-31, or ACh and during inhibition of SKCa/IKCa with apamin+charybdotoxin (Ap+ChTX). Entire EC tubes were exposed by adding agents to the superfusion solution and Vm was confirmed to be the same between microelectrodes to ensure that the entire EC tube was isopotential before current injections.
One EC tube was studied per mouse. Analyses included resting Vm (mV) under control conditions; change in Vm (Δ mV)=peak response Vm minus preceding baseline Vm; conduction amplitude (CA, mV/nA)=Vm at site 2/current injected at site 1; fraction of control CA=CA during treatment/preceding control CA; conduction efficiency=CA at each distance/CA at 50 μm separation; length constant (λ)=distance over which the electrical signal decayed to 37% (1/e) of the “local” value. Data were analyzed using analysis of variance with Bonferroni post hoc comparisons, regression analyses, and paired t tests. Differences were accepted as statistically significant with P<0.05. Summary data are presented as mean±SE. Values for n refer to the number of EC tubes studied under respective conditions.
Effects of SKCa/IKCa Activation on Electrical Conduction Over Distance
Resting Vm was −26±2 mV at sites 1 and 2 independent of separation distance (n=11). As shown in Figure 2, ΔVm at site 2 increased in direct proportion to current injected irrespective of polarity. The amplitude of ΔVm2 for each current decreased when separation distance between microelectrodes was increased from 500 to 1500 μm (Figure 2A through 2C), yet the I-V relationship remained linear (Figure 2C). At 500-μm separation, the slope of the I-V relationship through ±0.1 to 3 nA was 7.5±0.5 mV/nA. When calculated for −1 nA, CA (500 μm) was not different (7.7±0.5 mV/nA; n=11), substantiating −1 nA as a standard reference.
The ΔVm (−37 mV) in response to 1 μmol/L NS309 corresponds to the peak hyperpolarization obtained with ACh (3 μmol/L).30 At each distance, opening SKCa/IKCa with 1 μmol/L NS309 reduced CA (Figure 2A and 2B), yet the I-V relationship remained linear (Figure 2D). Thus, NS309 decreased CA at 500 μm to nearly the same extent as did increasing distance to 1500 μm under control conditions (Figure 2C). After continuous paired recordings (control, NS309) at each distance, washout of NS309 (10–15 minutes) restored control Vm and CA (eg, CA at 500 μm: control, 7.7±0.4 mV/nA; NS309 washout, 7.9±0.4 mV/nA, n=16), confirming the reversibility of SKCa/IKCa activation. The next separation distance was evaluated in the same manner.
During current injections, NS309 (1 μmol/L) reduced ΔVm2 at the “local” site by half and decreased λ by ≈40% (control: 1380±80 μm; NS309: 850±60 μm; Figure 3A, P<0.05). By normalizing CA at each separation distance to respective “local” values, conduction efficiency was also reduced at each distance (P<0.05; Figure 3B). The injection current required for the same local change in Vm obtained with −1 nA under control conditions increased to −2 nA with 1 μmol/L NS309, consistent with a corresponding decrease in rm. When local hyperpolarization was thereby matched between conditions, the decay in ΔVm2 over distance was again greater (P<0.05) with NS309 (Figure 3C and Online Table I). Thus, activating SKCa/IKCa impaired electrical conduction irrespective of ΔVm at the site of current injection.
For the following experiments, current was injected at site 1 while Vm was recorded at site 2 with 500-μm separation maintained between microelectrodes.7,30 The stability of impalements enabled multiple treatments to be evaluated during continuous (paired) recordings. Responses were evaluated through the full range of current microinjection, with data presented for −1 nA.
Progressive Inhibition of Electrical Conduction during Graded Activation of SKCa/IKCa
A question central to these experiments was: Can electrical conduction be “tuned” according to the level of SKCa/IKCa activation? Current was injected at site 1 and Vm recorded at site 2 (500 μm) before and during increments in SKCa/IKCa activation with recovery between each treatment. Hyperpolarization began at 100 nmol/L NS309 and increased with [NS309] (Figure 4A; P<0.05); maximal response at 10 μmol/L corresponded to Vm=−81±1 mV. Impairment of CA (P<0.05) began at 300 nmol/L NS309 and CA decreased as [NS309] increased with conduction abolished at 10 μmol/L (Figure 4B and 4C). At 1 μmol/L, NS309 reduced CA by half (Figure 4B and 4C). In separate experiments, using SKA-31 to activate SKCa/IKCa24 produced effects similar to NS309 though with attenuated potency and efficacy (compare Figure 4D through 4F with Figure 4A through 4C). Nevertheless, for both pharmacological agents, a “threshold” Vm associated with significant reduction in CA occurred at ≈−40 mV (Figure 4A and 4D). These findings indicate that the efficacy of electrical conduction along the endothelium can be tuned according to the level of SKCa/IKCa activation.
Maintenance of Dye Transfer During Inhibition of Electrical Conduction
Impairment of electrical conduction during SKCa/IKCa activation is interpreted to reflect greater current dissipation along the endothelium. Thus, an essential question is: Does intercellular coupling persist when electrical conduction is inhibited? We used dye transfer as a functional qualitative index of GJC patency between neighboring ECs.4,7,10,27,30 An EC tube was exposed to the NS309 concentration that inhibited conduction (10 μmol/L) beginning 5 minutes before and throughout recordings. With 0.1% propidium iodide dye (MW=668.4 Da) in the microelectrode, fluorescent images were acquired after 30 minutes of continuous recording. Under control conditions (Figure 5A) and during hyperpolarization (to −81±4 mV) with inhibition of electrical conduction (CA [500 μm]: 0.3±0.1 mV/nA), dye microinjected into one EC readily spread to neighboring ECs (Figure 5B; n=3). Thus, dye transfer during maximal activation of SKCa/IKCa with NS309 was qualitatively similar to control, confirming that intercellular coupling through GJCs remained patent during absence of electrical conduction. For reference, in EC tubes exposed to carbenoxolone (100 μmol/L) or β-glycyrrhetinic acid (40 μmol/L) to block GJCs, dye transfer was inhibited reversibly along with electrical conduction.30
Inhibition of Electrical Conduction Independent of Hyperpolarization
The activation of SKCa/IKCa consistently hyperpolarized ECs, raising the question: Does loss of electrical conduction during SKCa/IKCa activation depend on hyperpolarization? According to the Nernst relationship, raising [K+]o from 5 (control) to 60 mmol/L (equimolar replacement of NaCl with KCl) reduces EK from −89 mV to −23 mV and should thereby “clamp” Vm near control (Vm=−23±1 mV, n=7) and prevent hyperpolarization during K+ channel activation. To test this effect, current was injected and Vm recorded before and during superfusion with either NS309 (1 μmol/L), 60 mmol/L [K+]o, or both together. Despite lack of hyperpolarization, the decrease in CA (500 μm) during NS309+60 mmol/L [K+]o was not different from that during NS309 alone (Figure 6A and 6B). However, with 60 mmol/L [K+]o alone, the I-V relationship deviated (P<0.05) from linearity when negative current exceeded −1.5 nA (Figure 6C), attributable to the corresponding reduction in EK. By evaluating Vm2 responses to −1 nA, comparisons between conditions were maintained within the linear range of the I-V relationship. Treatment with NS309 consistently reduced CA (500 μm) by half (Figure 6D), confirming that inhibition of electrical conduction during SKCa/IKCa activation was independent of a change in Vm.
Inhibition of Conduction by ACh
To determine whether a physiological agonist that activates SKCa/IKCa exerts similar effects, EC tubes were superfused with ACh (3 μmol/L).30 During peak hyperpolarization, responses at site 2 decreased by ≈70% versus control (Figure 7). Exposure to ACh+60 mmol/L [K+]o confirmed that ACh inhibited CA (500 μm) irrespective of hyperpolarization (Online Figure I). Thus, activating SKCa/IKCa with a classic vasodilator also impaired electrical conduction irrespective of hyperpolarization.
Enhanced Electrical Conduction With SKCa/IKCa Blockade
Progressive activation of SKCa/IKCa impaired electrical conduction in a graded manner (Figure 4). Thus, if a subpopulation of SKCa/IKCa was open at rest, then inhibition of these channels should enhance electrical conduction. This prediction was tested by injecting current and recording Vm before and during exposure to Ap+ChTX. Exposure to these SKCa/IKCa blockers depolarized ECs (control: −28±2 mV; Ap+ChTX: −18±2 mV; n=6; P<0.05) and increased CA (500 μm) by ≈30% (P<0.05; Figure 8). Further, the relationship between ΔVm at site 2 (500 μm) and current injected at site 1 remained linear (with greater slope) through the entire range of current injection (R2≥0.99; n=6).
Negligible Role for KATP or BKCa Channels
If manipulating SKCa/IKCa activity alters electrical conduction, then complementary effects would be expected for other K+ channels. We tested for such an effect of ATP-regulated K+ channels (KATP) using levcromakalim (10 μmol/L; n=5). However, this KATP opener22,25 had no significant effect on resting Vm (−27±1 mV) or CA (500 μm; control, 8.4±1.1, levcromakalim: 8.6±1.1 mV/nA). Complementary experiments investigating a role for the large-conductance Ca2+ (and voltage)-sensitive K+ channel (BKCa) using the activator NS161922,25 (10 or 30 μmol/L; n=4) were also without effect on resting Vm (−27±2 mV) or CA (500 μm; control, 8.7±1.3, NS1619: 9.3±1.1 mV/nA). These findings indicate that neither KATP nor BKCa are functionally expressed in EC tubes.
Negligible Role for Nitric Oxide
Endothelial cell hyperpolarization may also promote nitric oxide (NO) synthesis.31,32 We therefore tested whether inhibition of NO synthase altered the effect of SKCa/IKCa activation on electrical conduction. Irrespective of treatment with the inhibitor Nω-nitro-l-arginine (100 μmol/L), exposure to NS309 (1 μmol/L) or to ACh (3 μmol/L) suppressed CA (500 μm) to the same extent (Online Figure II).
This study has revealed the ability of membrane ion channel activation to tune the efficacy of electrical conduction along the endothelium of resistance vessels. Using intact EC tubes freshly isolated from feed arteries of mouse skeletal muscle, findings illustrate that activation of SKCa/IKCa impairs axial signal transmission. This effect is explained by a fall in membrane resistance (rm) that dissipates charge as current flows from cell to cell along the endothelium. During intracellular microinjection of propidium iodide, robust dye transfer between neighboring ECs confirmed that cell-cell coupling through GJCs was maintained in the absence of electrical conduction. Thus, irrespective of SMCs or of changing cell-cell coupling through GJCs, dynamic changes in rm can profoundly alter the efficacy of electrical signal transmission along the endothelium of resistance vessels. Remarkably, such tuning of electrical conduction was governed by ion channels that are insensitive to voltage.
The Nature of Electrical Conduction Along EC Tubes
Conduction amplitude was defined as the slope of the I-V relationship with responses routinely evaluated at −1 nA, which falls within the linear range throughout our experiments. At a standard reference distance of 500 μm, our values of CA (≈8 mV/nA; Figure 2C and 2D and Figure 3A) compare favorably with values (≈7 mV per 0.8 nA) calculated for the conduction of hyperpolarization along resistance arteries of hamster skeletal muscle.7 Moreover, hyperpolarizing and depolarizing signals conducted with similar efficacy along EC tubes as shown by the linear (ie, ohmic) changes in Vm throughout the range of currents injected (±0.1–3 nA) at each distance (Figure 2). Such behavior indicates low functional expression of voltage-sensitive ion channels and is consistent with the lack of effect of NS1619 on Vm or CA. In turn, the deviation from linearity observed during hyperpolarization with >1.5 nA in the presence of 60 mmol/L [K+]o (Figure 6C) can be explained by the reduction in EK. In contrast to the present findings, depolarizing currents injected into ECs (or SMCs) of feed arteries from hamsters evoked less than half of the absolute change in Vm than did hyperpolarizing current of equal intensity.7 Such deviation observed for intact vessels (versus the linearity of responses for EC tubes) may be attributed to the influence of SMCs through myoendothelial coupling;7,9 for example, through voltage-gated K+ channels (Kv) that may not otherwise be present in ECs.25
Whereas activating SKCa/IKCa with NS309 (1 μmol/L) reduced CA by 50% or more at each distance (Figure 3A), comparing absolute values does not resolve changes in the nature of electrical conduction because the local response to current injection (ie, change in Vm) is reduced. Expressing responses at each distance relative to the local response provides a normalized index of “conduction efficiency” and illustrates significantly greater decay in electrical conduction during SKCa/IKCa activation when compared with control (Figure 3B). However, it is important to evaluate the effect of SKCa/IKCa activation when conduction is initiated from a similar local change in Vm. We therefore compared responses to −2 nA current during NS309 with responses to −1 nA under control conditions. With local responses (ΔVm) not different between conditions, the decay in Vm over distance remained significantly greater during SKCa/IKCa activation (Figure 3C and Online Table I).
Along the entire distance (2000 μm) studied, responses decayed by 4.4% per 100 μm. This value is consistent with ≈5% per 100 μm reported for the decay of electrical conduction along the endothelium of retinal arterioles.33 Further, the consistency of such behavior suggests that the biophysical properties of EC tubes determined here can be applied to the endothelium of vessels in other resistance networks. Indeed, λ determined in the present study for electrical conduction (≈1.4 mm; Figure 3) is consistent with control values reported for intact guinea pig arterioles (≈1.1–1.6 mm)15 and hamster retractor feed arteries (1.2 mm).16 A limitation of the present study is the lack of SMCs, which precludes resolution of how SKCa/IKCa activation may influence electrical signaling through myoendothelial GJCs. Nevertheless, even with SMCs present in earlier studies,15,16,33 the similarity in values for λ across preparations and laboratories supports the role of the endothelium as the primary cellular pathway for axial current flow. In turn, minimal charge loss through myoendothelial GJCs is explained by the high input resistance of SMCs.8
Tuning Electrical Conduction Independent of GJC Regulation
Electrical conduction along the endothelium is promoted by high rm preventing signal dissipation and low ra through GJCs between neighboring cells. Whereas GJCs have been a focus for regulating vascular conduction,5,17–21 the present study demonstrates that electrical conduction along the endothelium can be progressively inhibited by graded activation of SKCa/IKCa (Figure 4). This interpretation is consistent with the robust expression and ionic conductance of both KCa2.3 (SKCa) and KCa3.1 (IKCa) channels estimated from patch-clamp studies of native ECs freshly isolated from mouse aortae23 and the established role of SKCa/IKCa in governing EC hyperpolarization.9,22,25 From a typical resting Vm of ≈−25 mV, incremental activation of SKCa/IKCa with NS309 approached EK and coincided with loss of conduction (Figure 4A). In a reciprocal manner, blocking SKCa/IKCa with Ap+ChTX depolarized EC tubes by ≈10 mV, with a ≈30% increase in CA (500 μm; Figure 8). This modest effect of SKCa/IKCa inhibition compared with SKCa/IKCa activation on electrical conduction can be explained by a low percentage of the total SKCa/IKCa in EC membranes being open under control conditions.
The SKCa/IKCa opener SKA-31 had less potency and reduced efficacy compared with NS309 (Figure 4A and 4D). Nevertheless, the apparent threshold of channel activation required to significantly reduce CA corresponded to a Vm of ≈−40 mV for both agents (0.3 μmol/L NS309, 3 μmol/L SKA-31; Figure 4). Hyperpolarization may also promote the production of NO,31,32 which may in turn alter rm34 or cell-to-cell coupling through acting on GJCs.35 Nevertheless, the inhibition of electrical conduction by NS309 was maintained when hyperpolarization was prevented (Figure 6) and when NO synthesis was inhibited (Online Figure II). Importantly, and similar to the direct activation of SKCa/IKCa by NS309 and SKA-31, CA (500 μm) was reduced ≈70% during indirect activation of these channels by ACh (Figure 7). Further, as seen with NS309, preventing hyperpolarization to ACh (Online Figure I) or inhibiting NO synthesis (Online Figure II) did not alter the ability of ACh to suppress electrical conduction. Thus, the modulation of rm (and tuning of electrical conduction) along the endothelium may reflect a more generalized response to physiological agonists that signal through G protein–coupled receptors,36 irrespective of NO.
The present findings support the hypothesis that opening ion channels in the plasma membrane impairs electrical conduction along the endothelium. As dye transfer remained intact during exposure to NS309 when conduction was abolished (Figure 4), loss of conduction cannot be attributed to closure of GJCs. In turn, despite the lack of effect for levcromakalim or NS1619, we infer that the activation or inhibition of other ion channels that are functionally expressed in membranes of electrically coupled cells can have effects consistent with those shown here for SKCa/IKCa. Consistent with this inference are recent findings from rat mesenteric arteries, where spreading vasodilation in response to ACh (or isoproterenol) was enhanced when K+ channels (BKCa and Kv) were inhibited in SMCs that were electrically coupled to ECs.37 Hyperpolarization can activate other K+ channels (eg, inward rectifying, KIR) to increase Vm and enhance electrical conduction.11,38 Nevertheless, it is unlikely that such a role would be manifest in EC tubes, given the linearity of the I-V relationship (Figure 2) and a resting Vm (≈−25 mV) above that associated with the negative slope conductance of KIR.25 Further, given the same decrease in CA to NS309 or ACh when hyperpolarization was prevented (Figure 6D and Online Figure I, respectively), it appears unlikely that that activation of KIR contributes to electrical conduction along the endothelium. However, this conclusion does not preclude a role for KIR expressed in SMCs to contribute to conducted vasodilation along intact vessels.11,38
Summary and Conclusions
Electrical signaling underlies the correspondence between changes in Vm and diameter along the resistance vasculature.6,7,10 Previous studies have focused on the role of intercellular coupling through GJCs in determining the efficacy of electrical conduction,1–3,8 particularly as it applies to conducted vasodilation along arterioles and ascending vasodilation during exercise.2,14 Paradigms for studying conducted responses have typically involved stimulating a vessel segment (or the tissue in which vessels are embedded) while monitoring vasomotor responses at sites remote from the region of stimulation.14,18,19,21,37,39,40 However, there is ambiguity in defining the number of cells activated, the precise stimulus intensity at the signal origin, and the specific role(s) of respective cell layers. In the present study, the endothelium of resistance arteries was isolated as an intact tube to eliminate the influence of blood flow or surrounding cells and tissue. Intracellular microelectrodes enabled electrical conduction to be initiated from a point source (single cell) within the electrical syncytium using prescribed current pulses while monitoring Vm at defined distances. In turn, selective activation (or inhibition) of SKCa/IKCa along the endothelium resolved how electrical conduction can be tuned through changes in membrane resistance while individual cells remain coupled to each other through GJCs.
This study presents 2 novel findings. First, electrical conduction can be finely tuned through the activity of voltage-insensitive membrane ion channels. Thus, our experiments are the first to demonstrate an integral role for SKCa/IKCa activation in governing EC signaling that is distinct from their established role of initiating EC hyperpolarization9,24 (Figure 9). Second, SKCa/IKCa can define the spatial domain of electrical signaling along the endothelium (Figure 9). The SKCa/IKCa have gained recognition as therapeutic targets in light of their ability to initiate hyperpolarization and promote vasodilation to increase tissue blood flow.24,41,42 The present findings imply that selectively targeting ion channels for therapeutic intervention should account for integrated effects throughout resistance networks as well as local effects on vasomotor tone.
Sources of Funding
This work was supported by National Institutes of Health grants R37-HL041026, R01-HL086483, and F32-HL110701.
Dr William F. Jackson provided helpful comments during the initial stages of this study.
In February 2012, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.77 days.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.111.262592/-/DC1.
Non-standard Abbreviations and Acronyms
- large-conductance Ca2+-activated K+ channels
- conduction amplitude
- endothelial cell
- gap junction channels
- small- and intermediate-conductance Ca2+-activated K+ channels
- ATP-regulated K+ channels
- inward-rectifying K+ channels
- voltage-gated K+ channels
- nitric oxide
- 6,7-dichloro-1H-indole-2,3-dione 3-oxime
- smooth muscle cell
- membrane potential
- Received December 9, 2011.
- Revision received March 26, 2012.
- Accepted March 29, 2012.
- © 2012 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
The endothelium conducts electrical signals along resistance vessels to coordinate relaxation of smooth muscle cells.
Gap junctions provide a low-resistance pathway for current to flow between endothelial cells.
Small and intermediate Ca2+-activated K+ channels (SKCa/IKCa) are highly expressed in endothelial cells and initiate hyperpolarization.
What New Information Does This Article Contribute?
Electrical conduction of hyperpolarization and depolarization along the endothelium produce equivalent (but opposite) changes in membrane potential.
Irrespective of gap junctions or charge polarity, opening voltage-insensitive ion channels dissipates electrical signals along the endothelium.
Activation of SKCa/IKCa channels effectively “tunes” the ability of the endothelium to conduct electrical signals.
The endothelium is recognized as the principal pathway for initiating vasodilation through SKCa/IKCa activation and for the conduction of hyperpolarization in resistance networks. In regulating conducted vasodilation, attention has focused on the role and manipulation of intercellular gap junctions to alter the resistance to current flow between cells. In contrast, we studied whether altering the electrical resistance of endothelial plasma membranes via activation or blockade of ion channels can regulate electrical conduction. Our new findings demonstrate that irrespective of charge polarity, electrical conduction along the endothelium is impaired during SKCa/IKCa activation and is enhanced by SKCa/IKCa blockade. At the same time, neighboring cells remain well coupled to each other through gap junctions. We suggest that impaired tissue blood flow during endothelial dysfunction may reflect enhanced ion channel activation (ie, “leaky” plasma membranes) that impairs coordinated control of vascular resistance. Therapeutic interventions designed to improve tissue blood flow by altering ion channel activation should take into account the effects on electrical conduction in resistance networks.