Tuning electrical conduction along endothelial tubes of resistance arteries through Ca(2+)-activated K(+) channels

Abstract

Rationale: Electrical conduction through gap junction channels between endothelial cells of resistance vessels is integral to blood flow control. Small and intermediate-conductance Ca(2+)-activated K(+) channels (SK(Ca)/IK(Ca)) initiate electrical signals in endothelial cells, but it is unknown whether SK(Ca)/IK(Ca) activation alters signal transmission along the endothelium.

Objective: We tested the hypothesis that SK(Ca)/IK(Ca) 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 (V(m)) at site 2 (separation distance=50-2000 μm). SK(Ca)/IK(Ca) activation (NS309, 1 μmol/L) reduced the change in V(m) 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 SK(Ca)/IK(Ca) with acetylcholine or SKA-31 also reduced electrical conduction. These effects of SK(Ca)/IK(Ca) activation persisted when hyperpolarization (>30 mV) was prevented with 60 mmol/L [K(+)](o). Conversely, blocking SK(Ca)/IK(Ca) (apamin+charybdotoxin) depolarized cells by ≈10 mV and enhanced electrical conduction (ie, changes in V(m)) by ≈30% (P<0.05).

Conclusions: These findings illustrate a novel role for SK(Ca)/IK(Ca) 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.

Figure 1. Experimental design

Inset at top: dual intracellular electrodes positioned in an EC tube (Differential Interference Contrast image). Site 1 and Site 2 shown within ovals correspond to cartoon below. Current (±0.1 – 3.0 nA, 2s pulses) was injected at Site 1 while V_m was recorded with separation distances between microelectrodes of 50–2000 μm. To determine the role of SK_Ca/IK_Ca in tuning electrical conduction, SK_Ca/IK_Ca on all cells were activated (NS309, SKA-31, ACh) or inhibited (apamin + charybdotoxin) by addition of respective agents to the superfusion solution. With no change in axial resistance (r_a) to current flow through gap junction channels (GJC), changes in conduction amplitude (CA; ΔV_m at Site 2/current injected at Site 1; mV/nA), reflect changes in membrane resistance (r_m) associated with activation or inhibition of SK_Ca/IK_Ca. Data in Figs. 3, 4, 6–8 reflect CA responses to −1 nA current injection (note that −ΔV_m/−1 nA = positive CA value). The Fraction of Control CA (Figs. 4C, F and 8C) was defined as CA during treatment/CA under control conditions at the same 500 μm site. Conduction Efficiency (Fig. 3B) was defined as (CA at X μm) / (CA at 50 μm), where X= 50–2000 μm.

Figure 2. Effect of distance and SK_Ca/IK_Ca activation on electrical conduction

At defined separation distances between intracellular microelectrodes, continuous (paired) recordings at Site 2 during current microinjection at Site 1 were obtained under Control conditions and during NS309 treatment to activate SK_Ca/IK_Ca. After each set of paired recordings, washout of NS309 (10–15 min) restored Control V_m before recording at the next distance. A, Representative V_m recording at Site 2 during ±0.1–3 nA microinjected at Site 1 (distance = 500 μm) before and during NS309. From Control V_m of ~−25 mV, NS309 hyperpolarized ECs to ~−60 mV and decreased V_m2 responses. B, As in A with separation distance = 1500 μm. Note reduction in Control V_m2 responses compared to A followed by loss of V_m2 responses during NS309. C, Summary data illustrating the effect of microelectrode separation distance on the change in V_m at Site 2 (ΔV_m2) under Control conditions with 500 μm (black line) or 1500 μm (grey line) separation. With reduced slope at greater distance, ΔV_m2 responses remained linear through the full range of current injected (R² = 1). With −1 nA current, absolute ΔV_m2 decreased from 7.7 ± 0.6 mV at 500 μm to 3.8 ± 0.3 mV at 1500 μm. D, With separation maintained at 500 μm for the same EC tubes in C, activation of SK_Ca/IK_Ca with NS309 (1 μmol/L) reduced CA to 3.1 ± 0.3 mV. Summary data in C and D are means ± S.E.; n = 11. Note: Panels C and D include (with permission: British Journal of Pharmacology © 2011) control data from 8 experiments presented in Figure 2B,C of Behringer et al.

Figure 3. Effect of SK_Ca/IK_Ca activation on spatial decay of electrical conduction

Summary data (means ± S.E.) illustrating electrical conduction versus distance between intracellular microelectrodes before and during treatment with NS309 (1 μmol/L). At each distance, continuous (paired) recordings were obtained under Control conditions and during NS309. A, For Conduction Amplitude (−1 nA microinjected at Site 1), NS309 reduced the local response by half and λ by ~40% (Control: 1380±80 μm; NS309: 850±60 μm). B, Conduction Efficiency = data from A normalized to respective values at 50 μm before and during NS309; note greater decay with NS309. C, With current microinjection adjusted to produce the same local ΔV_m (Control: −1 nA; NS309: −2 nA), the ΔV_m2 (= resting V_m - peak response V_m) with distance indicates greater decay of hyperpolarization with NS309. For these experiments, n = 11 at 50 – 1,500 μm; n=7 at 2,000 μm). *Control significantly different from NS309, P < 0.05 Note: Panel A includes (with permission: British Journal of Pharmacology © 2011) control data from 8 experiments presented in Figure 2B,C of Behringer et al.

Figure 4. Effect of progressive activation of SK_Ca/IK_Ca on electrical conduction

Summary data (means ± S.E.) for continuous recordings at Site 2 located 500 μm from current microinjection (−1 nA) at Site 1. A, resting V_m with increasing [NS309]; C indicates Control. B, Conduction Amplitude as a function of [NS309]; C indicates Control. C, As in B indicating effect of SK_Ca/IK_Ca activation with NS309 relative to Control (Fraction of Control CA = CA during respective [NS309] / Control CA). D, as in A for SKA-31. E, as in B for SKA-31. F, as in C for SKA-31. *Significantly different from Control, P < 0.05, n = 6–8 in each panel. Note reduced potency and efficacy for SKA-31 versus NS309.

Figure 5. Intercellular dye transfer through GJCs is maintained during SK_Ca/IK_Ca activation with NS309

Propidium iodide dye (0.1% in 2M KCl) was included in microelectrode during intracellular recording to evaluate intercellular coupling through gap junction channels. Arrows indicate impaled cell in each panel and recordings lasted 30 min. A, Dye transfer during Control conditions. B, As in A with NS309 (10 μmol/L) introduced 5 min prior to cell penetration. With 500 μm separation between microelectrodes, cells hyperpolarized to −81 ± 1 mV and electrical conduction was abolished (Figs. 4A–4C) yet robust dye transfer to surrounding cells was maintained. Images are representative of n=3 experiments.

Figure 6. Impaired electrical conduction during SK_Ca/IK_Ca activation is independent of hyperpolarization

Continuous (paired) recordings of V_m at Site 2 (V_m2) located 500 μm from current microinjections at Site 1. A, V_m2 during ±0.1–3 nA before and during SK_Ca/IK_Ca activation with NS309 (1 μmol/L). During NS309, note hyperpolarization (to ~−60 mV) and decrease in V_m2 responses. B, As in A before and during treatment with NS309 + 60 mmol/L [K⁺]_o to prevent hyperpolarization to NS309. Note decrease in V_m2 responses during NS309 similar to findings in A. Transient hyperpolarization near end of recording (“↓”) attributable to slower washout of NS309 versus KCl. a, b and c correspond to small vertical arrows in A and B showing individual recordings of V_m2 during −1 nA injected at Site 1 for (a) control (ΔV_m = −6 mV), (b) NS309 (ΔV_m = −3 mV), and (c) NS309 + 60 mmol/L [K⁺]_o (ΔV_m = −3 mV; note lack of hyperpolarization to NS309 with reduced hyperpolarization to −1 nA current). C, Summary data for ΔV_m2 responses to full range of current injection (± 0.1–3 nA) during Control and during 60 mmol/L [K⁺]_o. Note deviation from linearity for ΔV 2 responses to >−1.5 nA during 60 mmol/L [K⁺]_o. *Significantly different from Control, P < 0.05. D, Summary data (means ± S.E.) for CA at Site 2 to −1 nA current injection at Site 1 during Control, during 60 mmol/L [K⁺]_o, during NS309 (1 μmol/L), and during NS309 (1 μmol/L) + 60 mmol/L [K⁺]_o. *Significantly different from Control (P < 0.05). +Significantly different from 60 mmol/L [K⁺]_o, P < 0.05. Summary data (means ± S.E.) in C and D are for n = 7.

Figure 7. Acetylcholine impairs electrical conduction

Data are from continuous (paired) recordings of V_m at Site 2 located 500 μm from current microinjection (−1 nA). A, Representative recording illustrating responses to ±0.1–3 nA before and to ±1, 2, and 3 nA during ACh (3 μmol/L). The V_m during ACh was −67 ± 5 mV (n=6). B, Summary data (means ± S.E.; n=6) for Conduction Amplitude (CA). Note decrease in CA during ACh. Hyperpolarization to −2 nA during ACh (−3.9 ± 0.8 mV, n=6) was significantly less (P < 0.05) than hyperpolarization to −1 nA under Control conditions (−7.1 ± 0.5 mV, n=6). *Significantly different from Control, P < 0.05. Following washout of ACh, CA returned to 7.2 ± 0.5 mV/nA (n=6).

Figure 8. Enhanced electrical conduction during SK_Ca/IK_Ca blockade

All data are from continuous (paired) recordings 500 μm from the site of current microinjection. A, Representative recording indicating V_m2 responses to ±0.1–3 nA microinjections before and during SK_Ca/IK_Ca block with Ap (300 nmol/L) + ChTX (100 nmol/L). Note depolarization (~10 mV) and increases in V_m2 responses during Ap + ChTX. a and b correspond to arrows in A and illustrate V_m2 responses to −1 nA injected at Site 1 during Control and subsequent exposure to Ap + ChTX: ΔV_m2 was −8 mV in a and −10 mV in b. Records are from EC tube with resting V_m = −34 mV to illustrate depolarization and increased CA associated with blocking SK_Ca/IK_Ca. B, Summary data (means ± S.E.) for Control and with Ap + ChTX. C, Data from B expressed as Fraction of Control Conduction Amplitude (= CA during Ap + ChTX /Control CA) to illustrate relative effect of SK_Ca/IK_Ca inhibition. *Significantly different from Control, P < 0.05 (n = 6).

Figure 9. Role for SK_Ca/IK_Ca in tuning electrical conduction along EC tubes

Cartoon illustrating biophysical determinants of electrical signaling along neighboring cells in an EC tube. A, Electrical current (+/-) microinjected into one cell spreads to neighboring cells through gap junction channels (GJC). B, Activation of SK_Ca/IK_Ca (e.g., with NS309, SKA-31 or ACh; Figs. 3, 4, 7) increases current leak through the plasma membrane (i.e., r_m decreases), thus less current is available to spread to neighboring cells through GJC. C, Blocking SK_Ca/IK_Ca (e.g., with apamin + charybdotoxin; Fig. 8) reduces current leak across the plasma membrane (i.e., r_m increases) thus more current is available to spread to neighboring cells through GJC. Axial resistance to current flow between cells through GJC (r_a) is assumed to remain constant. With respective agents superfused over the entire EC tube, SK_Ca/IK_Ca channels were either opened (e.g. via NS309; B) or closed (Ap + ChTX; C) along the entire EC tube.