A temporal Ca^{2 þ} desensitization of myosin light chain kinase in phasic smooth muscles induced by CaMKKb/PP2A pathways

Cell signaling pathways regulating myosin regulatory light chain (LC20) phosphorylation contribute to determining contractile responses in smooth muscles. Following excitation and contraction, phasic smooth muscles, such as the digestive tract and urinary bladder, undergo relaxation due to a decline of cellular Ca^{2 þ} concentration and decreased Ca^{2 þ} sensitivity of LC20 phosphorylation, named Ca^{2 þ} desensitization. Here, we determined the mechanisms underlying the temporal Ca^{2 þ} desensitization of LC20 phosphorylation in phasic smooth muscles using permeabilized strips of the mouse ileum and urinary bladder. Upon stimulation with pCa6.0 at 20C, contraction and LC20 phosphorylation peaked within 30 s and then declined to about 50% of the peak force at 2 min after stimulation. During the relaxation phase after the contraction, LC20 kinase [myosin light chain kinase (MLCK)] was inactivated, but no fluctuation in LC20 phosphatase activity occurred, suggesting that MLCK inactivation is a cause of the Ca^{2 þ}-induced Ca^{2 þ} desensitization of LC20 phosphorylation. MLCK inactivation was associated with phosphorylation at the calmodulin-binding domain of the kinase. Treatment with STO-609 and TIM-063 antagonists for Ca^{2 þ} / calmodulin (CaM)-dependent protein kinase kinase-b (CaMKKb) attenuated both the phasic response of the contraction and MLCK phosphorylation, whereas neither CaM kinase II, AMP-activated protein kinase, nor p21-activated kinase induced MLCK inactivation in phasic smooth muscles. Conversely, protein phosphatase 2A inhibition amplified the phasic response. Signaling pathways through CaMKKb and protein phosphatase 2A may contribute to regulating the phasic response of smooth muscle contraction.