Flow-Dependent Modulation of Endothelial Ca2+ Dynamics by Small Conductance Ca2+-Activated K+ Channels in Mouse Carotid Arteries

Abstract

Background: Small conductance Ca²⁺ activated K⁺ channels (K_Ca2.3) are important regulators of vascular function. They provide Ca²⁺-dependent hyperpolarization of the endothelial membrane potential, promoting agonist-induced vasodilation. Another important mechanism of influence may occur through positive feedback regulation of endothelial Ca²⁺ signals, likely via amplification of influx through membrane cation channels. K_Ca2.3 channels have recently been implicated in flow-mediated dilation of the arterial vasculature and may contribute to the crucial homeostatic role of shear stress in preventing vascular wall remodeling and progressive vascular disease (i.e., atherosclerosis). The impact of K_Ca2.3 channels on endothelial Ca²⁺ signaling under physiologically relevant shear stress conditions remains unknown.

Methods: In the current study, we employ mice expressing an endothelium-specific Ca²⁺ fluorophore (cdh5-GCaMP8) to characterize the K_Ca2.3 channel influence on the dynamic Ca²⁺ signaling profile along the arterial endothelium in the presence and absence of shear-stress.

Results: Our data indicate K_Ca2.3 channels have a minimal influence on basal Ca²⁺ signaling in the carotid artery endothelium in the absence of flow, but they contribute substantially to amplification of Ca²⁺ dynamics in the presence of flow and their influence can be augmented through exogenous positive modulation.

Conclusions: The findings suggest a pivotal role for K_Ca2.3 channels in adjusting the profile of homeostatic dynamic Ca²⁺ signals along the arterial intima under flow.

Keywords: Ca2+ activated K+ channels; calcium; endothelium; shear stress.

Figure 1

Characterization of dynamic Ca²⁺ signaling in cdh5-GCaMP8 mouse carotid artery endothelium. (A) Open carotid arteries were mounted in a flow chamber for fluorescence confocal imaging of the vascular endothelium under no-flow or flow conditions (red arrows). Images show isolation and tracking of basal dynamic Ca²⁺ signals from image sequences using S8 (under no-flow conditions). The three dimensional plot shows extrapolation of events in the field (x,y) over time (t). (B) Quantification of the basal Ca²⁺ signaling profile based on event frequency, amplitude, duration, and maximal area (18 arteries from 14 animals). (C) Characterization of Ca²⁺ dynamics in response to flow (10 dyn/cm²). (D) Summary of Ca²⁺ dynamics under flow (n = 4).

Figure 1

Characterization of dynamic Ca²⁺ signaling in cdh5-GCaMP8 mouse carotid artery endothelium. (A) Open carotid arteries were mounted in a flow chamber for fluorescence confocal imaging of the vascular endothelium under no-flow or flow conditions (red arrows). Images show isolation and tracking of basal dynamic Ca²⁺ signals from image sequences using S8 (under no-flow conditions). The three dimensional plot shows extrapolation of events in the field (x,y) over time (t). (B) Quantification of the basal Ca²⁺ signaling profile based on event frequency, amplitude, duration, and maximal area (18 arteries from 14 animals). (C) Characterization of Ca²⁺ dynamics in response to flow (10 dyn/cm²). (D) Summary of Ca²⁺ dynamics under flow (n = 4).

Figure 2

Effect of K_Ca2.3 inhibition on endothelial Ca²⁺ dynamics under no-flow conditions. (A) Panels show binary masks and time-extrapolated plots of Ca²⁺ events before and after addition of apamin (0.5 µM) for 10 min. (B) Summary of Ca²⁺ dynamics before and after apamin (n = 5).

Figure 3

Effect of K_Ca2.3 inhibition on endothelial Ca²⁺ dynamics under flow conditions. (A) Panels show binary masks and time-extrapolated plots of Ca²⁺ events at 10 dyn/cm² shear stress before and after addition of apamin (0.5 µM) for 10 min. (B) Summary of Ca²⁺ dynamics before and after apamin (n = 5).

Figure 4

Effect of K_Ca2.3 stimulation on endothelial Ca²⁺ dynamics under no-flow and flow conditions. (A) Panels show binary masks and time-extrapolated plots of Ca²⁺ events before and after addition of CyPPA (10 µM) for 10 min. (B) Summary of Ca²⁺ dynamics before and after CyPPA (n = 5). (C) Panels show binary masks and time-extrapolated plots of Ca²⁺ events at 5 dyn/cm² shear stress before and after addition of CyPPA (10 µM) for 10 min. (D) Summary of Ca2+ dynamics before and after CyPPA (n = 4).

Figure 4

Effect of K_Ca2.3 stimulation on endothelial Ca²⁺ dynamics under no-flow and flow conditions. (A) Panels show binary masks and time-extrapolated plots of Ca²⁺ events before and after addition of CyPPA (10 µM) for 10 min. (B) Summary of Ca²⁺ dynamics before and after CyPPA (n = 5). (C) Panels show binary masks and time-extrapolated plots of Ca²⁺ events at 5 dyn/cm² shear stress before and after addition of CyPPA (10 µM) for 10 min. (D) Summary of Ca2+ dynamics before and after CyPPA (n = 4).