Focal adhesion kinase activation is involved in contractile stimulation-induced detrusor muscle contraction in mice

Abstract

Recent studies suggested smooth muscle contraction may involve mechanisms besides the myosin regulatory light chain (MLC) phosphorylation-induced actomyosin crossbridge cycling. This study aims to determine if focal adhesion kinase (FAK) activation is involved in mouse detrusor muscle contraction. The mouse detrusor muscle strips were preincubated with PF-573228 (2 μM), latrunculin B (1 μM), or the same volume of vehicle (DMSO) for 30 min. The contractile responses to KCl (90 mM), electrical field stimulation (EFS, 2-32 Hz), or carbachol (CCh, 10^-7.5-10^-4.5 M) were measured. In a separate experiment, the phosphorylated FAK (p-FAK) and MLC (p-MLC) levels were measured in the detrusor strips stimulated with CCh (10 μM) after incubation with PF-573228 or vehicle (DMSO) compared to those with vehicle incubation but without CCh stimulation. KCl-induced contractile responses decreased significantly after incubation with PF-573228 or latrunculin B compared to the corresponding vehicle-treated strips (p < 0.0001). The contractile responses induced by EFS were markedly inhibited by preincubation with PF-573228 at 8, 16, and 32 Hz (p < 0.05) or latrunculin B at 16 and 32 Hz (p < 0.01). Following the application of PF-573228 or Latrunculin B, CCh-induced dose-response contractions were lower than the corresponding vehicle group (p = 0.0021 and 0.0003, respectively). Western blot examination showed that CCh stimulation enhanced the expression of p-FAK and p-MLC, while preincubation with PF-573228 prevented the increase of p-FAK but not p-MLC. In conclusion, FAK activation involves tension development induced by contractile stimulation in the mouse detrusor muscle. This effect is likely caused by promoting actin polymerization rather than elevating MLC phosphorylation.

Keywords: Actin polymerization; Bladder; Contractility; Detrusor muscle; FAK.

Figure 1.

Effects of PF-573228 on KCl- (A, n=5), EFS- (B, n=6), or CCh-induced (C, n=7) contractions of mouse detrusor muscle strips. In an organ bath, contractions were induced 30 min after the addition of PF-573228 (2 μM) or an equivalent amount of vehicle (DMSO). The peak contractions were compared. Values are expressed as a percentage of the maximal response of each muscle to 120 mM KCl. The scatter plot graphs in A and B show individual data points, means, and SD error bars. n = the number of independent mice in each experimental condition. ^# p < 0.05, *p < 0.01, Statistically significant difference from vehicle-treated detrusor strips. Each point in C represents the mean with SD.

Figure 2.

Western blot analysis of CCh-induced FAK and MLC phosphorylation and the effects of PF-573228 on CCh-induced FAK and MLC phosphorylation in mouse detrusor muscle strips (A). α-tubulin is used as the loading control. Each lane represents a sample obtained from an individual mouse. After densitometric analysis of blots using ImageJ, the ratio of the relative level of phosphorylated FAK (B) or MLC (C) to total FAK or MLC is plotted for each experimental condition. ^# p<0.05, * p<0.01 One-way ANOVA followed by Tukey post-test, n = 4. n = the number of independent mice in each experimental condition.

Figure 3.

Effects of Latrunculin B on KCl- (A, n=5), EFS- (B, n=5), or CCh-induced (C, n=7) contractions of mouse detrusor muscle strips. In an organ bath, contractions were induced 30 min after the addition of Latrunculin B (1 μM) or an equivalent amount of vehicle (DMSO). The peak contractions were compared. Values are expressed as a percentage of the maximal response of each muscle to 120 mM KCl. The scatter plot graphs in A and B show individual data points, means, and SD error bars. n = the number of independent mice in each experimental condition. *p < 0.01, Statistically significant difference from vehicle-treated detrusor strips. Each point in C represents the mean with SD.