Elementary Ca2+ signals through endothelial TRPV4 channels regulate vascular function

Abstract

Major features of the transcellular signaling mechanism responsible for endothelium-dependent regulation of vascular smooth muscle tone are unresolved. We identified local calcium (Ca(2+)) signals ("sparklets") in the vascular endothelium of resistance arteries that represent Ca(2+) influx through single TRPV4 cation channels. Gating of individual TRPV4 channels within a four-channel cluster was cooperative, with activation of as few as three channels per cell causing maximal dilation through activation of endothelial cell intermediate (IK)- and small (SK)-conductance, Ca(2+)-sensitive potassium (K(+)) channels. Endothelial-dependent muscarinic receptor signaling also acted largely through TRPV4 sparklet-mediated stimulation of IK and SK channels to promote vasodilation. These results support the concept that Ca(2+) influx through single TRPV4 channels is leveraged by the amplifier effect of cooperative channel gating and the high Ca(2+) sensitivity of IK and SK channels to cause vasodilation.

Fig. 1

GSK-induced Ca²⁺ signals represent Ca²⁺ influx through plasmalemmal TRPV4 channels. (A) Ca²⁺ imaging of an en face preparation of third-order mesenteric arteries from a GCaMP2-expressing mouse, showing changes in the activity of local, IP₃R-independent Ca²⁺ signals recorded over time (traces, below) from regions of interest (1.7 μm²), denoted by boxes in images (above). An EC in the field is outlined by red dashes (above, left). Scale bar: 10 μm. Bottom left: Localized events (arrows) detected in the absence of IP₃R-mediated signaling; bottom right: localized events after addition of GSK (10 nM). (B) Top panels: Pseudocolor overlay images showing all IP₃R-independent Ca²⁺ signaling events detected in the absence or presence of GSK (10 nM) in a single field over a 94-s interval. Scale bar: 10 μm. Bottom panel and trace: Pseudo–line-scan image and associated trace recorded from a single site over the same interval. Scale bar: 2 s. Calibration bar at right indicates intensity of signals (F/F₀; fluorescence F divided by baseline fluorescence F₀). (C) Representative traces illustrating differences in the kinetic properties of IP₃R-mediated Ca²⁺ pulsars (left) and TRPV4-mediated Ca²⁺ events (right) from the same field of view in the presence of 10 nM GSK (without CPA). (D) Increased numbers of sites per 2 min per field and activity per field (right; n = 4 to 6 arteries) in the presence of GSK (10 nM), 4α-PDD (5 μM), or 11,12-EET (1 μM). (E) Inhibition of GSK-induced increases in activity per site and activity per field by HC (1 μM; n = 5) and RuR (5 μM; n = 3). Error bars (C and D), SEMs.

Fig. 2

TRPV4 Ca²⁺ sparklets as unitary events that exhibit cooperative activation. (A) A fit of multiple Gaussians to all-points histograms showing evenly spaced ΔF/F₀ levels of 0.19 at 2 mM extracellular Ca²⁺ and 0.29 at 10 mM Ca²⁺. (B) Effect of decreasing Ca²⁺ electrochemical gradient by membrane depolarization with 100 mM K⁺ on Ca²⁺ signals. (C) Exponential distribution of the durations of GSK (10 nM)–induced sparklets with Fluo-4 and 10 μM EGTA-AM. Sampling rate, 50 to 60 images/s. τ, time constant. (D) Cooperative channel gating demonstrated by deviation of the frequencies of second, third, and fourth levels (measured as open probabilities, P_O) from a binomial distribution predictive of independent events. Same conditions as in (C).

Fig. 3

Activation of IK and SK channels and induction of EDHF-dependent vasodilation by Ca²⁺ influx through TRPV4 channels. (A) Inhibition of GSK (10 nM)–induced outward current by 300 nM ChTx (n = 5 cells) or 300 nM apamin (n = 5 cells). (B) Densities of IK and SK channel currents in cells treated with GSK (10 nM) or in cells dialyzed with 3 μM Ca²⁺ (n = 8 cells). Conv. W.C., conventional whole-cell configuration; Perf. Patch, perforated-patch configuration. (C) Top trace: GSK (3 nM)–induced vasodilation of pressurized (80 mmHg) mesenteric arteries; middle trace: effects of 100 μM L-NNA + 10 μM indomethacin (Indo; n = 5), or 1 μM paxilline (n = 3) on GSK-induced dilations; bottom trace: effects of L-NNA and 30 μM CPA (n = 5) on GSK (10 nM)–induced dilations; bar graph (left): absence of effects of the TRPV4 antagonists HC (1 μM; n = 4) and RuR (5 μM; n = 4), and TRPV4 knockout (n = 3) on dilations induced by the IK and SK agonist NS309 (1 μM); bar graph (right): quantification of effects of CPA + L-NNA (n = 5), L-NNA + Indo (n = 5), paxilline (n = 3), endothelium removal (n = 5), RuR (n = 5), HC (n = 5), and TRPV4 knockout (n = 4) on GSK (10 nM)–induced dilations. (D) Vasodilation in response to GSK (3, 10, 30 nM) in the presence or absence of 200 nM ChTx (n = 7), or ChTx + 300 nM apamin (Apa). Effects on dilation [bar graphs in (C) and (D)] determined relative to initial tone [26 ± 1% (SEM), n = 10], defined as the percentage decrease in arterial diameter to pressure (80 mmHg) relative to the diameter in external Ca²⁺-free solution. Error bars (B to D), SEMs.

Fig. 4

Effects of muscarinic receptor activation on global Ca²⁺ and TRPV4 sparklets. (A) Global Ca²⁺, shown as fractional fluorescence from outlined whole cells treated with CCh (10 μM) with and without CPA (30 μM). (B) GSK (3 nM)–induced sparklets in the presence or absence of 5 μM ACh (left) and 1 μM HC (right; n = 5). (C) Effects of 200 nM ChTx + 300 nM apamin (Apa; n = 5), 100 μM L-NNA (n = 6), L-NNA + HC (n = 5), and ChTx + Apa + L-NNA (n = 4) on dilations in response to the muscarinic agonist CCh (1 μM). Error bars (B and C), SEMs. Except where indicated by a bracket (Student’s t test), P-values are for comparison to control (one-way analysis of variance).