The importance of Rho-associated kinase-induced Ca2+ sensitization as a component of electromechanical and pharmacomechanical coupling in rat ureteric smooth muscle

Abstract

Ureteric peristalsis, which occurs via alternating contraction and relaxation of ureteric smooth muscle, ensures the unidirectional flow of urine from the kidney to the bladder. Understanding of the molecular mechanisms underlying ureteric excitation-contraction coupling, however, is limited. To address these knowledge deficits, and in particular to test the hypothesis that Ca2+ sensitization via activation of the RhoA/Rho-associated kinase (ROK) pathway plays an important role in ureteric smooth muscle contraction, we carried out a thorough characterization of the electrical activity, Ca2+ signaling, MYPT1 (myosin targeting subunit of myosin light chain phosphatase, MLCP) and myosin regulatory light chain (LC20) phosphorylation, and force responses to membrane depolarization induced by KCl (electromechanical coupling) and carbachol (CCh) (pharmacomechanical coupling). The effects of ROK inhibition on these parameters were investigated. We conclude that the tonic, but not the phasic component of KCl- or CCh-induced ureteric smooth muscle contraction is highly dependent on ROK-catalyzed phosphorylation of MYPT1 at T855, leading to inhibition of MLCP and increased LC20 phosphorylation.

Cross-talk between Rho-associated Kinase and Cyclic Nucleotide-dependent Kinase Signaling Pathways in the Regulation of Smooth Muscle Myosin Light Chain Phosphatase

“…Thr 697 phosphorylation inhibits MLCP activity by a competitive inhibition mechanism, as MYPT1 phosphorylated at Thr 697 becomes a substrate for PP1c␦ (17). Phosphorylation at Thr 855 has been shown to cause dissociation of MYPT1 from phosphorylated LC 20 , resulting in an apparent decrease in MLCP activity (18), and has also been shown to inhibit MLCP activity without dissociation (16). Additional studies have described intracellular translocations of MYPT1 following Thr 697 phosphorylation (19).…”

“…This phenomenon, commonly called Ca 2ϩ sensitization, involves an increase in the MLCK:MLCP activity ratio (3), which is achieved primarily through the activities of Ca 2ϩ -independent kinases that can directly phosphorylate LC 20 (5)(6)(7)(8) and/or inhibit MLCP activity (9 -11). MLCP is composed of three subunits: a 38-kDa catalytic subunit (PP1c␦), a 115-130-kDa myosin targeting subunit (MYPT1), and an additional 20-kDa subunit (M20).…”

“…[2][3][4]. The extent of phosphorylation of LC 20 at Ser 19 is primarily dependent on the relative activities of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). Although MLCK is a Ca 2ϩ /calmodulin-dependent protein kinase, MLCP activity can be regulated independently of changes in cytosolic free [Ca 2ϩ ] ([Ca 2ϩ ] i ).…”

“…blood vessels and gastrointestinal tract (1). Contractile force is driven by the phosphorylation status of Ser 19 of the 20-kDa myosin regulatory light chain (LC 20 ), 4 which facilitates formation of the actomyosin complex and cross-bridge cycling (reviewed in Refs. [2][3][4].…”

“…Relaxation occurs as [Ca 2ϩ ] i is restored via re-uptake into the sarcoplasmic reticulum and extrusion to the extracellular space. The decrease in [Ca 2ϩ ] i leads to inactivation of MLCK and dephosphorylation of LC 20 by MLCP (3).…”