Regulation of vascular smooth muscle contraction through Calcium‐dependent Calcium sensitization

Abstract

We used Bay‐K‐8644 (Bay‐K), a calcium channel agonist, to examine basal and calcium‐stimulated levels of [Ca2+]i (Ca), force, myosin light chain phosphorylation (MLCp) and myosin phosphatase targeting subunit phosphorylation (MYPT1p) in the absence (control) and presence of inhibitors of ROCK (H‐1152) and MLCK (wortmannin). Also used was staurosporine (St), which inhibits ROCK, MLC kinase (MLCK) and integrin‐linked kinase (ILK) at 1μM and ZIP kinase (ZIPK) at 10µM. Bay‐K induced increases in Ca, MYPT1p‐T853, MLCp and force and not MYPT1p‐T696. A Ca‐free solution inhibited the increases in MYPT1‐pT853, MLCp and force. Wortmannin inhibited the increases in MLCp and force and not MYPT1p‐T853. H‐1152 inhibited the increases in MYPT1p‐T853, MLCp and force but not Ca. These data suggest that Bay‐K caused Ca sensitization by activating ROCK. H‐1152 (1 µM) reduced the basal level of MYPT1‐pT853 but not MLCp. Wortmannin and TFP did not reduce basal MYPT1p‐T853 nor basal MLCp. St (1µM) reduced basal MYPT1‐pT696, MYPT1‐pT853 and MLCp. These data support a model in which constitutive ILK provides basal MLCp through MYPT1p‐T696 induced MLCP inhibition, and increases in Ca can further increase MLCp by activating MLCK and inhibiting MLCP via activation of ROCK and increases in MYPT1‐T853. Constitutive ROCK activity and MYPT1‐pT853 do not appear to play a role in basal MLCp. Support: R01HL061320