Modulation of smooth muscle tonus in the lower urinary tract: interplay of myosin light‐chain kinase (MLCK) and MLC phosphatase (MLCP)
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What's known on the subject? and What does the study add? MLCK and MLCP play key roles in regulating muscle tones in the bladder and urethra. Bladder has higher MLCK and lower MLCP activities relative to the urethra, providing evidence at the molecular level for the concept of the bladder being phasic while the urethra being tonic at their respective default states. OBJECTIVE • To assess and compare the expression and activity of myosin light-chain kinase (MLCK) and MLC phosphatase (MLCP) in rat bladder and urethra. MATERIALS AND METHODS • Bladder and urethral smooth muscles were obtained from 2-month-old female Sprague-Dawley rats. They were analysed by real-time polymerase chain reaction for the mRNA expression of MLCK and myosin phosphatase-targeting subunit of protein phosphatase type 1 (MYPT1, a subunit of MLCP). • Levels of MLCK and MYPT1 mRNA expression were determined as a ratio to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). • The tissues were also analysed by Western blotting for MLCK and MYPT1 protein expression as a ratio to the expression of β-actin. • A two-step enzymatic activity assay using phosphorylated and dephosphorylated smooth muscle myosin was used to assess MLCK and MLCP activity. RESULTS • MLCK mRNA expression was higher in the bladder than in the urethra [mean (sd) ratio to GAPDH: 0.26 (0.17) vs 0.14 (0.12); P= 0.09]. • MYPT1 mRNA expression was significantly higher in the bladder than in the urethra [mean (sd) ratio to GAPDH: 2.31 (1.04) vs 0.56 (0.36); P= 0.001]. • Expression of both MLCK and MYPT1 protein was significantly higher in the bladder compared with the urethra [mean (sd) ratio to β-actin: 1.63 (0.25) vs 0.91 (0.29) and 0.97 (0.10) vs 0.37 (0.29), respectively; both P < 0.001]. • Enzymatic assay identified significantly greater MLCK activity in the bladder than in the urethra. While, MLCP activity was lower in the bladder than in the urethra. CONCLUSION • In healthy young female rats, MLCK activity is higher and MLCP activity is lower in the bladder relative to the urethra. These differences probably play a role in modulating the functional differences between bladder and urethral smooth muscle tone.Keywords:
Myosin-light-chain phosphatase
Smooth muscle tissue
The interaction of myosin and actin constitutes the basic mechanism of muscle contractile activity. In smooth muscle cells (SMCs) including vascular smooth muscle cells this interaction is predominantly regulated by the phosphorylation of the regulatory light chain (RLC) of myosin. The RLC is phosphorylated by myosin light chain kinase (MLCK) and dephosphorylated by myosin light chain phosphatase (MLCP). Therefore, the contractility of SMCs is determined by the relative ratio of the activity of MLCK vs. that of MLCP. During a contractile response, MLCK is activated by calmodulin-bound Ca2+. Meanwhile, the activity of MLCP is suppressed by protein kinase C (PKC) through the 17-kDa PKC-potentiated inhibitor protein (CPI-17) and by Rho kinase (ROCK). The varied activities of these major signaling pathways endow SMCs with different contractile profiles such as phasic and tonic contractions to meet the diversified physiological requirements.
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Contractility
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Dephosphorylation
Meromyosin
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Myosin-light-chain phosphatase
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For many years the simple view was held that contractile force in smooth muscle was proportional to cytosolic Ca2+ concentrations ([Ca2+]i). With the discovery that phosphorylation of myosin light chain by Ca2+/calmodulin-dependent myosin light chain kinase initiated contraction, regulation of the contractile elements developed more complex properties. Molecular and biochemical investigations have identified important domains of myosin light chain kinase: light chain binding sites, catalytic core, pseudosubstrate prototope, and calmodulin-binding domain. New protein phosphatase inhibitors such as okadaic acid and calyculin A should help in the identification of the physiologically important phosphatase and potential modes of regulation. The proposal of an attached, dephosphorylated myosin cross bridge (latch bridge) that can maintain force has evoked considerable controversy about the detailed functions of the myosin phosphorylation system. The latch bridge has been defined by a model based on physiological properties but has not been identified biochemically. Thin-filament proteins have been proposed as secondary sites of regulation of contractile elements, but additional studies are needed to establish physiological roles. Changes in the Ca2+ sensitivity of smooth muscle contractile elements with different modes of cellular stimulation may be related to inactivation of myosin light chain kinase or activation of protein phosphatase activities. Thus, contractile elements in smooth muscle cells are not dependent solely on [Ca2+]i but use additional regulatory mechanisms. The immediate challenge is to define their relative importance and to describe molecular-biochemical properties that provide insights into proposed physiological functions.
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Smooth muscle tissue
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Myosin-light-chain phosphatase
Contractility
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The myosin holoenzyme is a multimeric protein complex consisting of heavy chains and light chains. Myosin light chains are calmodulin family members which are crucially involved in the mechanoenzymatic function of the myosin holoenzyme. This review examines the diversity of light chains within the myosin superfamily, discusses interactions between the light chain and the myosin heavy chain as well as regulatory and structural functions of the light chain as a subunit of the myosin holoenzyme. It covers aspects of the myosin light chain in the localization of the myosin holoenzyme, protein-protein interactions and light chain binding to non-myosin binding partners. Finally, this review challenges the dogma that myosin regulatory and essential light chain exclusively associate with conventional myosin heavy chains while unconventional myosin heavy chains usually associate with calmodulin.
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Ca 2+ /calmodulin (CaM)-dependent phosphorylation of myosin regulatory light chain (RLC) in smooth muscle by myosin light chain kinase (MLCK) and dephosphorylation by myosin light chain phosphatase (MLCP) are subject to modulatory cascades that influence the sensitivity of RLC phosphorylation and hence contraction to intracellular Ca 2+ concentration ([Ca 2+ ] i ). We designed a CaM-sensor MLCK containing smooth muscle MLCK fused to two fluorescent proteins linked by the MLCK CaM-binding sequence to measure kinase activation in vivo and expressed it specifically in mouse smooth muscle. In phasic bladder muscle, there was greater RLC phosphorylation and force relative to MLCK activation and [Ca 2+ ] i with carbachol (CCh) compared with KCl treatment, consistent with agonist-dependent inhibition of MLCP. The dependence of force on MLCK activity was nonlinear such that at higher concentrations of CCh, force increased with no change in the net 20% activation of MLCK. A significant but smaller amount of MLCK activation was found during the sustained contractile phase. MLCP inhibition may occur through RhoA/Rho-kinase and/or PKC with phosphorylation of myosin phosphatase targeting subunit-1 (MYPT1) and PKC-potentiated phosphatase inhibitor (CPI-17), respectively. CCh treatment, but not KCl, resulted in MYPT1 and CPI-17 phosphorylation. Both Y27632 (Rho-kinase inhibitor) and calphostin C (PKC inhibitor) reduced CCh-dependent force, RLC phosphorylation, and phosphorylation of MYPT1 (Thr694) without changing MLCK activation. Calphostin C, but not Y27632, also reduced CCh-induced phosphorylation of CPI-17. CCh concentration responses showed that phosphorylation of CPI-17 was more sensitive than MYPT1. Thus the onset of agonist-induced contraction in phasic smooth muscle results from the rapid and coordinated activation of MLCK with hierarchical inhibition of MLCP by CPI-17 and MYPT1 phosphorylation.
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Calphostin C
Dephosphorylation
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Myosin light chain phosphatase with its regulatory subunit, myosin phosphatase target subunit 1 (MYPT1) modulates Ca2+-dependent phosphorylation of myosin light chain by myosin light chain kinase, which is essential for smooth muscle contraction. The role of MYPT1 in vascular smooth muscle was investigated in adult MYPT1 smooth muscle specific knock-out mice. MYPT1 deletion enhanced phosphorylation of myosin regulatory light chain and contractile force in isolated mesenteric arteries treated with KCl and various vascular agonists. The contractile responses of arteries from knock-out mice to norepinephrine were inhibited by Rho-associated kinase (ROCK) and protein kinase C inhibitors and were associated with inhibition of phosphorylation of the myosin light chain phosphatase inhibitor CPI-17. Additionally, stimulation of the NO/cGMP/protein kinase G (PKG) signaling pathway still resulted in relaxation of MYPT1-deficient mesenteric arteries, indicating phosphorylation of MYPT1 by PKG is not a major contributor to the relaxation response. Thus, MYPT1 enhances myosin light chain phosphatase activity sufficient for blood pressure maintenance. Rho-associated kinase phosphorylation of CPI-17 plays a significant role in enhancing vascular contractile responses, whereas phosphorylation of MYPT1 in the NO/cGMP/PKG signaling module is not necessary for relaxation. Myosin light chain phosphatase with its regulatory subunit, myosin phosphatase target subunit 1 (MYPT1) modulates Ca2+-dependent phosphorylation of myosin light chain by myosin light chain kinase, which is essential for smooth muscle contraction. The role of MYPT1 in vascular smooth muscle was investigated in adult MYPT1 smooth muscle specific knock-out mice. MYPT1 deletion enhanced phosphorylation of myosin regulatory light chain and contractile force in isolated mesenteric arteries treated with KCl and various vascular agonists. The contractile responses of arteries from knock-out mice to norepinephrine were inhibited by Rho-associated kinase (ROCK) and protein kinase C inhibitors and were associated with inhibition of phosphorylation of the myosin light chain phosphatase inhibitor CPI-17. Additionally, stimulation of the NO/cGMP/protein kinase G (PKG) signaling pathway still resulted in relaxation of MYPT1-deficient mesenteric arteries, indicating phosphorylation of MYPT1 by PKG is not a major contributor to the relaxation response. Thus, MYPT1 enhances myosin light chain phosphatase activity sufficient for blood pressure maintenance. Rho-associated kinase phosphorylation of CPI-17 plays a significant role in enhancing vascular contractile responses, whereas phosphorylation of MYPT1 in the NO/cGMP/PKG signaling module is not necessary for relaxation.
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Myosin-light-chain phosphatase
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