Membrane potential governs calcium influx into microvascular endothelium: integral role for muscarinic receptor activation

Abstract

In resistance arteries, coupling a rise of intracellular calcium concentration ([Ca(2+)]i) to endothelial cell hyperpolarization underlies smooth muscle cell relaxation and vasodilatation, thereby increasing tissue blood flow and oxygen delivery. A controversy persists as to whether changes in membrane potential (V(m)) alter endothelial cell [Ca(2+)]i. We tested the hypothesis that V(m) governs [Ca(2+)]i in endothelium of resistance arteries by performing Fura-2 photometry while recording and controlling V(m) of intact endothelial tubes freshly isolated from superior epigastric arteries of C57BL/6 mice. Under resting conditions, [Ca(2+)]i did not change when V(m) shifted from baseline (∼-40 mV) via exposure to 10 μM NS309 (hyperpolarization to ∼-80 mV), via equilibration with 145 mm [K(+)]o (depolarization to ∼-5 mV), or during intracellular current injection (±0.5 to 5 nA, 20 s pulses) while V(m) changed linearly between ∼-80 mV and +10 mV. In contrast, during the plateau (i.e. Ca(2+) influx) phase of the [Ca(2+)]i response to approximately half-maximal stimulation with 100 nm ACh (∼EC50), [Ca(2+)]i increased as V(m) hyperpolarized below -40 mV and decreased as V(m) depolarized above -40 mV. The magnitude of [Ca(2+)]i reduction during depolarizing current injections correlated with the amplitude of the plateau [Ca(2+)]i response to ACh. The effect of hyperpolarization on [Ca(2+)]i was abolished following removal of extracellular Ca(2+), was enhanced subtly by raising extracellular [Ca(2+)] from 2 mm to 10 mm and was reduced by half in endothelium of TRPV4(-/-) mice. Thus, during submaximal activation of muscarinic receptors, V(m) can modulate Ca(2+) entry through the plasma membrane in accord with the electrochemical driving force.

Figure 1

Diagram of simultaneous [Ca²⁺]_i and V_m measurements in an endothelial tube A window 140 μm × 50 μm (encompassing ∼50 ECs) monitored [Ca²⁺]_i using Fura‐2 dye photometry. For simultaneous measurements, the window was located adjacent to a microelectrode recording V _m (right) located 300 μm from a microelectrode injecting current (−5 to +5 nA; left) to control (i.e. clamp) V _m at a designated level.

Figure 2

Effects of 10 μm NS309 and 145 mm [K⁺]_o on V_m and [Ca²⁺]_i before and during stimulation with ACh A, simultaneous V _m (top) and F ₃₄₀/F ₃₈₀ (bottom) recordings during 100 nm ACh under control conditions. B, as in A with 10 μm NS309 pretreatment (2 min) to activate SK_Ca/IK_Ca preceding ACh. Top: note hyperpolarization to ∼80 mV with NS309; ACh increased V _m to ∼−85 mV. Bottom: note lack of F ₃₄₀/F ₃₈₀ response until ACh is introduced. C, as in A with 145 mm [K⁺]_o pretreatment for 2 min. Depolarization to ∼−5 mV had no effect on F ₃₄₀/F ₃₈₀ until the addition of ACh. Note diminished F ₃₄₀/F ₃₈₀ during plateau phase versus control (compare with A). Arrow indicates a transient secondary rise in F ₃₄₀/F ₃₈₀ ∼2 min after initial peak response to ACh. Short horizontal bars above F ₃₄₀/F ₃₈₀ traces (bottom panels of A–C) indicate periods of data acquisition for Rest, Peak and Plateau (90 s after Peak) values, respectively; note difference in time scales. D, summary of F ₃₄₀/F ₃₈₀ at corresponding V _m before (Rest) and during ACh (Peak and Plateau) for paired experiments before and during NS309 (n = 10 endothelial tubes from 10 mice). E, as in D for paired experiments during ACh alone and during ACh with 145 mm [K⁺]_o (n = 9 endothelial tubes from 9 mice). *P < 0.05, NS309 or 145 mm KCl versus respective Control values.

Figure 3

Changing V_m with current injection does not alter resting [Ca²⁺]_i A, simultaneous V _m (top) and F ₃₄₀/F ₃₈₀ (bottom) recordings during control conditions at rest (V _m, −35 ± 3 mV; F ₃₄₀/F ₃₈₀, 0.69 ± 0.02) and during current injection (−5 to +5 nA; 20 s pulses at arrowheads). While V _m (top) responded in a stepwise manner F ₃₄₀/F ₃₈₀ (bottom) did not change. B, linear regression of V _m versus injected current (r ^{2 =} 0.990 ± 0.002). C, summary data illustrating stability of F ₃₄₀/F ₃₈₀ throughout range of V _m during respective levels of current injection (shown near data points for reference). Arrows in B and C indicate resting V _m and F ₃₄₀/F ₃₈₀, respectively. Summary data binned according to level of current injection for n = 7 endothelial tubes from 6 mice.

Figure 4

Changing V_m with intracellular current injection alters [Ca²⁺]_i only during stimulation with ACh A, simultaneous recordings of V _m (top) and F ₃₄₀/F ₃₈₀ (bottom) with −3 nA current pulses before and during 100 nm ACh. Note lack of effect of hyperpolarization on [Ca²⁺]_i until ACh is introduced. B, summary of F ₃₄₀/F ₃₈₀ values at designated values of V _m throughout levels of current injection (shown near data points) prior to ACh. Resting F ₃₄₀/F ₃₈₀ was not altered from control (arrow) while V _m ranged from −82 ± 2 mV to 4 ± 3 mV. C, summary of F ₃₄₀/F ₃₈₀ values at designated values of V _m throughout range of current injection during plateau of [Ca²⁺]_i response to ACh; one current level studied during each ACh stimulation. *P < 0.05, F ₃₄₀/F ₃₈₀ during current injection versus zero current (arrow; elevated F ₃₄₀/F ₃₈₀ versus B due to ACh). Summary data in B and C binned according to level of current injection for n = 6–7 endothelial tubes from 6–7 mice.

Figure 5

[Ca²⁺]_i changes with V_m during stimulation with ACh A, simultaneous V _m (top) and F ₃₄₀/F ₃₈₀ (bottom) recordings during current injection (−5 to +5 nA; 20 s pulses at arrowheads) during 100 nm ACh. B, summary of F ₃₄₀/F ₃₈₀ values for corresponding values of V _m at rest (arrow) and during 100 nm ACh; data binned according to level of current injection (shown near data points). This relationship is sigmoidal (R ² = 0.974 ± 0.005) with V _m at half‐maximal F ₃₄₀/F ₃₈₀ = −44 ± 2 mV. Note increase in F ₃₄₀/F ₃₈₀ with magnitude of hyperpolarization and decrease in F ₃₄₀/F ₃₈₀ (to baseline levels) as V _m approximates 0 mV. Summary data represent n = 30 endothelial tubes from 25 mice. *P < 0.05, F ₃₄₀/F ₃₈₀ during current injection versus F ₃₄₀/F ₃₈₀ without current injection (arrow).

Figure 6

Plateau [Ca²⁺]_i responses to ACh correlate with [Ca²⁺]_i responses to depolarization A, scatter plot illustrating magnitude of ∆F ₃₄₀/F ₃₈₀ during negative current injections of −1, −3 and −5 nA during the plateau ∆F ₃₄₀/F ₃₈₀ response to 100 nm ACh (Y‐axis) versus the plateau ∆F ₃₄₀/F ₃₈₀ response (from resting baseline) to 100 nm ACh (X‐axis). Note lack of correlation (R ² < 0.035). B, as in A during positive current injections of +1, +3 and +5 nA. Note negative correlations between the decreased [Ca²⁺]_i during depolarization and the plateau [Ca²⁺]_i response to ACh (R ² > 0.55). Individual data points correspond to summary data in Fig. 5 B.

Figure 7

[Ca²⁺]_i responses to changing V_m during stimulation with ACh require extracellular Ca²⁺ A, simultaneous V _m (top) and F ₃₄₀/F ₃₈₀ (bottom) recordings during current injection (−5 to +5 nA; 20 s pulses at arrowheads) during 100 nm ACh in Ca²⁺‐free PSS. Note absence of sustained elevation (i.e. plateau phase) in [Ca²⁺]_i response. B, overlay of summary data for F ₃₄₀/F ₃₈₀ values at corresponding levels of V _m (binned according to level of current injection) during 100 nm ACh with 2 mm [Ca²⁺]_o and with Ca²⁺‐free PSS. Arrows indicate V _m and F ₃₄₀/F ₃₈₀ under respective conditions with zero current. Summary data are paired experiments for n = 8 endothelial tubes from 8 mice. *P < 0.05, F ₃₄₀/F ₃₈₀ during current injection versus F ₃₄₀/F ₃₈₀ with zero current.

Figure 8

Increasing [Ca²⁺]_o increases [Ca²⁺]_i with maximal hyperpolarization during stimulation with ACh A, simultaneous V _m (top) and F ₃₄₀/F ₃₈₀ (bottom) recordings during current injection (−5 to +5 nA; 20 s pulses at arrowheads) during 100 nm ACh; [Ca²⁺]_o = 10 mm. B, overlay of summary data for F ₃₄₀/F ₃₈₀ values at corresponding levels of V _m (binned according to level of current injection). Arrows indicate V _m and F ₃₄₀/F ₃₈₀ during plateau response to ACh with zero current. Summary data are paired experiments for n = 10 endothelial tubes from 10 mice. *P < 0.05, F ₃₄₀/F ₃₈₀ during 10 mm [Ca²⁺]_o versus 2 mm [Ca²⁺]_o.

Figure 9

Lack of TRPV4 expression attenuates Ca²⁺ influx with hyperpolarization during stimulation with ACh A, simultaneous V _m (top) and F ₃₄₀/F ₃₈₀ (bottom) recordings in the presence of 100 nm ACh during current injection (alternating 20 s pulses at arrowheads) in endothelial tube from a TRPV4^−/− mouse. B, summary data for F ₃₄₀/F ₃₈₀ at corresponding V _m (binned according to level of current injection) during 100 nm ACh in endothelial tubes from TRPV4^−/− mice. *P < 0.05, F ₃₄₀/F ₃₈₀ during current injection versus no current injection (arrow). Data represent 8 endothelial tubes from 5 TRPV4^−/− mice. C, overlay of summary data from B with data from wild‐type (C57BL/6) mice (from Fig. 5 B) at corresponding levels of V _m and current injection (all experiments with [Ca²⁺]_o = 2 mm). Arrows indicate values with zero current. *P < 0.05, F ₃₄₀/F ₃₈₀ of wild‐type versus TRPV4^−/− at given level of current injection.