Fig. 1. Myogenic- and phenylephrine-induced vasoconstriction with associated RSK2 and RLC₂₀ phosphorylation in WT and Rsk2KO mesenteric arteries measured by pressurization and Western blotting.

(A) Scheme showing the phosphorylation cascade for activation of RSK2. (B) Cartoon showing the apparatus for controlled luminal pressurization of mouse mesenteric artery cascades to obtain sufficient material for biochemical analysis of phosphorylation events. (C) Typical traces showing myogenic constriction of WT and Rsk2^KO mesenteric arteries in response to increases in intraluminal pressure in Ca²⁺-containing and Ca²⁺-free Krebs solution. (D) Summary of myogenic responses of Rsk2^KO and WT arteries at 40, 60, 80, and 100 mmHg. P < 0.001, P < 0.01, P < 0.01, and P < 0.01, respectively, determined by two-way analysis of variance (ANOVA). Rsk2^KO: n = 3 mice and 6 arteries; WT: n = 3 mice and 6 arteries. (E) Phenylephrine (PE) concentration responses of Rsk2^KO and WT third- and fourth-order mesenteric arteries pressurized to 80 mmHg. ***P = 0.005 determined by two-way ANOVA. Rsk2^KO: n = 3 mice and 7 arteries; WT: n = 3 mice and 6 arteries. EC₅₀ values for phenylephrine-induced contractions (bar graph) were not significantly different. P values were determined by two-tailed homoscedastic Student’s t test. (F) Time course of RSK2 phosphorylation (normalized to total RSK2) at the MEK/ERK Thr⁵⁷⁷ site and at the PDK Ser²²⁷ site following a pressure step from 20 to 80 mmHg in WT arteries. Data are means ± SEM; n = 6 mice and 6 arteries for each time point for Ser²²⁷ and 5 mice and 5 arteries for each time point for Thr⁵⁷⁷. *P < 0.05 for each time point compared to the corresponding control phosphorylation for Ser²²⁷; ^#P < 0.05 for each time point compared to the corresponding control phosphorylation for Thr⁵⁷⁷, two-tailed homoscedastic Student’s t test. (G) Time course of RLC₂₀ Ser¹⁹ phosphorylation following a pressure step from 20 to 80 mmHg in WT and Rsk2^KO artery arcades. Data are means ± SEM; n = 11 WT mice and 11 arteries for each time point and n = 4 Rsk2^KO mice and 4 arteries for each time point. **P < 0.01 for each time point after an increase in pressure, compared to Ser¹⁹ phosphorylation in 0-mmHg pressure WT arteries; ^#P < 0.05 for each time point compared to Ser¹⁹ phosphorylation in 0-mmHg pressure Rsk2^KO arteries, two-tailed homoscedastic Student’s t test.