AngII increases TRPV4 sparklet activity through the local activation of PKC. (A) Representative [Ca²⁺]_i records from a TRPV4 sparklet site in an arterial myocyte exposed to AngII (100 nM) or AngII plus Gö6976 (100 nM). Broken red lines mark the amplitude of elementary and multichannel TRPV4 sparklets; the solid line shows the baseline. (B) Scatter plot of TRPV4 sparklet activities under the experimental conditions shown in A. The red lines are the median values (*, P < 0.05; n = 17; Mann-Whitney test). (C) Confocal images of tsA-201 cells expressing opto-α₁AR (i), opto-α₁AR + PKCα (ii), or TRPV4 + opto-α₁AR + PKCα (iii). (i) Image of a tsA-201 showing opto-α₁AR-YFP membrane localization. The inset shows the fluorescence intensity along the yellow dashed line. Inset bars: (horizontal) 2 µm; (vertical) 10 AU. (ii and iii) Ca²⁺ imaging of tsA-201 cells expressing opto-α₁AR + PKC (ii) or TRPV4 + opto-α₁AR + PKC (iii), and loaded with the Ca²⁺ indicator Rhod-2. The traces in ii and iii show the time course of [Ca²⁺]_i in sites 1 and 2. The gray rectangles on the traces show when opto-α₁AR was stimulated with 474 nm light in sites 1 and 2. Bars in traces: (horizontal) 20 s; (vertical) 1,000 AU. (D) Representative TRPV4 whole-cell currents of tsA-201 cells coexpressing TRPV4 channels, PKCα, and opto-α₁AR before (black line) and after (red line) cell-wide opto-α₁AR activation (60 s with 474 nm light). (E) Bar plot of the amplitude of TRPV4 currents at −80 mV and +80 mV before and after opto-α₁AR activation. Data are means ± SEM (error bars; *, P < 0.05; n = 6; one-tailed Student’s t test).