Recruitment of dynamic endothelial Ca2+ signals by the TRPA1 channel activator AITC in rat cerebral arteries

Abstract

Objective: Stimulation of endothelial TRP channels, specifically TRPA1, promotes vasodilation of cerebral arteries through activation of Ca2+ -dependent effectors along the myoendothelial interface. However, presumed TRPA1-triggered endothelial Ca2+ signals have not been described. We investigated whether TRPA1 activation induces specific spatial and temporal changes in Ca2+ signals along the intima that correlates with incremental vasodilation.

Methods: Confocal imaging, immunofluorescence staining, and custom image analysis were employed.

Results: We found that endothelial cells of rat cerebral arteries exhibit widespread basal Ca2+ dynamics (44 ± 6 events/minute from 26 ± 3 distinct sites in a 3.6 × 10(4) μm2 field). The TRPA1 activator AITC increased Ca2+ signals in a concentration-dependent manner, soliciting new events at distinct sites. Origination of these new events corresponded spatially with TRPA1 densities in IEL holes, and the events were prevented by the TRPA1 inhibitor HC-030031. Concentration-dependent expansion of Ca2+ events in response to AITC correlated precisely with dilation of pressurized cerebral arteries (p = 0.93 by F-test). Correspondingly, AITC caused rapid endothelium-dependent suppression of asynchronous Ca2+ waves in subintimal smooth muscle.

Conclusions: Our findings indicate that factors that stimulate TRPA1 channels expand Ca2+ signal-effector coupling at discrete sites along the endothelium to evoke graded cerebral artery vasodilation.

Figure 1

Basal Ca²⁺ dynamics in rat cerebral artery endothelium. A. 2-minute accumulate image from a continuous time series showing Ca²⁺-dependent fluorescence measured in endothelial cells of opened Fluo-4 AM-loaded cerebral arteries. Lower panel is a pseudo-color intensity projection of the same basal Ca²⁺ transients. Scale bar is 20 μm. See also Movie S1. B. Recordings obtained from four separate sites corresponding to arrows in (A); 5-pixel diameter regions of interest (ROIs). C. Recordings of basal events in autodetected ROIs in the absence or presence of Ca²⁺ free buffer (5 minutes), cyclopiazonic acid (CPA; 25 μM for 15 minutes), or the combination of xestospongin C (Xest C; 30 μM) and 2-APB (80 μM) for 40 minutes. Time controls at 5 and 50 min were not significantly different (Fig S1). D. Summary data (n = 5–7 per group; * p<0.01).

Figure 2

Properties of basal Ca²⁺ events in rat cerebral artery endothelium. Histograms show distributions of specific event parameters: amplitude, duration, spatial spread and frequency per site. Data were compiled from 10 animals.

Figure 3

Effect of the TRPA1 channel activator AITC on cerebral artery endothelial Ca²⁺ signals. A. Recordings showing the changes in detected Ca²⁺ dynamics in response to different concentrations of AITC. Inset shows continuous tracings from three distinct sites before and after AITC (15 μM) addition (arrow). B. Panels are two-minute accumulates of Ca²⁺ dynamics (black ellipses depict signals above background threshold) recorded in a representative experiment before and after AITC addition. The merged panel is a composite where red shows events before AITC, green shows events after AITC, and yellow shows sites where events occurred both before and after AITC. The bar graph summarizes the total number of Ca²⁺ events and distinct sites detected at each AITC concentration (n = 5; * and ** p < 0.05 vs. corresponding 0 μM AITC; each AITC concentration was assessed in a separate vessel). C. Scatter plots show distributions of amplitude, duration and spatial spread of events occurring in sampled endothelial fields (n= 3–4) at each AITC concentration (*p < 0.05 vs. 0 μM AITC, nonparametric Kruskal-Wallis and Dunn's tests).

Figure 4

Analysis of single-site Ca²⁺ dynamics in AITC-stimulated cerebral artery endothelium. Ca²⁺ -dependent fluorescence was measured in open cerebral artery preparations before and after AITC. A. Plots show the effect of AITC (0, 15, 30 and 60 μM) on various Ca²⁺ event parameters only at sites that exhibited basal Ca²⁺ dynamics before AITC addition. Each point is the net change for 1–3 separate experiments at each concentration (3–4 animals per group). No significant change in single-site frequency, amplitude, duration or spatial spread (p > 0.05, one-way ANOVA) was indicated at the concentrations tested. B. Effect of pre-existing basal activity on the magnitude of AITC-induced Ca²⁺ increase at a given site. For each site, the total Ca²⁺ signal (AUC; area under curve for all events at that site) after 30 μM AITC is plotted as a function of total Ca²⁺ signal before AITC (quantified 2 minutes before and 2 minutes after AITC). In bar graph, mean Ca²⁺ AUC values are displayed separately for sites that exhibited one or more basal Ca²⁺ events prior to AITC addition and sites exhibiting no basal Ca²⁺ events prior to AITC. Net Ca²⁺ increases were significantly larger at sites that did not discharge prior to AITC exposure (p < 0.001, data composite of 4 animals).

Figure 5

Specific role of TRPA1 in the AITC-stimulated endothelial Ca²⁺ signals. A. Immunofluorescence images of the intima-media interface in opened rat cerebral arteries showing a merged composite as well as separate images of the internal elastic lamina (IEL; green), TRPA1 (red), and nuclei (blue) of endothelial cells (horizontal) and subintimal smooth muscle cells (vertical). A vessel branch is seen on the right side. Scale bar is 20 μm. Below: Panels show an expanded view of the region circumscribed by the white box in (A); distinct TRPA1 puncta (red) are emphasized by black arrows and Ca²⁺-dependent fluorescence (white) is shown (30-second accumulate) before and after addition of 30 μM AITC. Ca²⁺ was measured before fixation and immunostaining. Bottom panels show a time series of a single event site, noting the initiation and spread of a Ca²⁺ event from a singular TRPA1 puncta. B. Recordings show AITC-stimulated endothelial Ca²⁺ signals measured in the absence or presence of the TRPA1-selective inhibitor HC-030031 (10 μM for 10 min). C. Data summarized in the bar graph show the relative inhibition of AITC-induced Ca²⁺ events by 10 μM HC-030031, Ca²⁺-free solution, or 25 μM CPA (n = 4–7; * p<0.05).

Figure 6

Correlation of AITC-induced endothelial Ca²⁺ events and cerebral artery dilation. Left panel shows AITC-induced increases in cerebral artery diameter measured in pressurized isolated arteries [18]. Right panel shows relative AITC-induced increases in endothelial Ca²⁺ signals measured in open cerebral arteries. Nonlinear regression curves were generated for diameter changes (solid line) as well as changes in Ca²⁺; Ca²⁺ sites and total events occurring at these sites (dotted lines). Changes in average Ca²⁺ within the entire endothelial field are plotted as open circles. Points are means of at least three observations from a total of 14 animals.

Figure 7

Endothelium dependence of AITC-induced inhibition of asynchronous cerebral artery smooth muscle Ca²⁺ waves. Panels show Ca²⁺-dependent fluorescence measured in open cerebral arteries either in the presence of intact endothelium (A) or following removal of the endothelium (B). In these preparations, distinct subintimal smooth muscle cells (yellow arrows) were monitored before and after addition of 30 μM AITC. Recordings from multiple smooth muscle cells are shown (data representative of six vessels from two animals). For A and B, insets show time-course images of single smooth muscle cells and the corresponding Ca²⁺ recordings from indicated regions of interest (circles) before and after AITC addition (black arrows). ECs; Endothelial cells.