Muscarinic stimulation does not induce rhoA/ROCK-mediated Ca 2+ sensitization of the contractile element in chicken gizzard smooth muscle

Anabuki, Jun; Hori, Masaru; Hayakawa, Kou; Akahane, Satoshi; Ozaki, Hiroshi; Karaki, Hideaki

doi:10.1007/s004240000425

Cited by 21 publications

(22 citation statements)

References 32 publications

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“…In embryonic smooth muscle, our results demonstrating a dose dependence of the inhibition of GTP␥S-induced force enhancement by the Rho-kinase inhibitor Y-27632 (Fig. 3) are similar to those of others (8,24,26). The magnitude of force enhancement was not affected by inhibition of MLCK or by inhibition of PKC with a concentration of staurosporine known to inhibit contractions of smooth muscle produced by phorbol esters (18).…”

Section: Force Versus Casupporting

confidence: 71%

“…It is unclear why the results with GTP␥S in the adult gizzard are different in our study and that of Anabuki et al (24), but it could be because of the skinning techniques (25). In addition, Anabuki et al (24) showed a modest GTP␥S-induced force enhancement at only one [Ca 2ϩ ] level, and it may be that GTP␥S at other [Ca 2ϩ ] levels would not have produced a significant increase in force. Our data suggest that the diversity in the magnitude of agonist-induced force enhancement in smooth muscle tissues (8, 24) may be partially because of the relative expression of M133/M130, or splice-in/out MYPT isoforms.…”

Section: Force Versus Cacontrasting

confidence: 51%

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Agonist-induced Force Enhancement

Richards

Ogut

Brozovich

2002

Journal of Biological Chemistry

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The magnitude of agonist-induced Ca 2؉ sensitization of force is tissue-dependent, but an explanation for this diversity is unknown. Ca 2؉ sensitization is thought to involve a G-protein-mediated inhibition of myosin light chain phosphatase activity by phosphorylation of the myosin-targeting subunit (MYPT). The MYPT has two isoforms that differ by a central insert, which lies near this phosphorylation site. Expression of MYPT isoforms is both developmentally regulated and tissue-specific. We hypothesized that the presence or absence of the central insert determines the magnitude of agonist-induced Ca 2؉ sensitization. Throughout development, the chicken aorta exclusively expresses the splice-in MYPT isoform, and guanosine 5-O-(thiotriphosphate) (GTP␥S) produces a significant force enhancement. Early during development, the chicken gizzard expresses the splice-in MYPT isoform, and GTP␥S produced a Ca 2؉ sensitization. In the gizzard coincident with the shift in expression from the splice-in to splice-out MYPT isoform, GTP␥S no longer produced force enhancement. In addition, adenosine 5-O-(thiotriphosphate) (ATP␥S) phosphorylated only adult gizzard tissue, the only tissue that did not demonstrate a Ca 2؉ sensitization. These results suggest that the relative expression of splice-in/ splice-out MYPT isoforms determines the magnitude of agonist-induced force enhancement and that MYPT phosphorylation is not required for Ca 2؉ sensitization.The importance of Ca 2ϩ -calmodulin-dependent myosin light chain kinase (MLCK) 1 for smooth muscle activation has been well described (1). However, recent evidence suggests that regulation of MLC phosphatase activity is physiologically important for force regulation in smooth muscle (reviewed in Refs. 2-6). This regulation of MLC phosphatase activity can produce a Ca 2ϩ sensitization, or an increase in force at a constant [Ca 2ϩ ], and a Ca 2ϩ desensitization, or a reduction in force at a constant [Ca 2ϩ ]. Agonist stimulation produces a Ca 2ϩ sensitization of the contractile filaments (7). The mechanism for the agonist-induced Ca 2ϩ sensitization, or force enhancement, is thought to depend on a G-protein-mediated inhibition of MLC phosphatase activity resulting in an increase in MLC 20 phosphorylation (8). The MLC phosphatase is a trimeric protein consisting of a 38-kDa catalytic subunit, a 20-kDa subunit of unknown function, and a large (110 -133 kDa) myosin-targeting subunit (MYPT) (reviewed in Ref. 3)). G-protein-induced inhibition of MLC phosphatase activity has been suggested to be regulated by phosphorylation of the MYPT (9), possibly by either Rhokinase (10, 11) or Zip-like kinase (12). However, direct activation of the G-proteins (8), as well as agonist stimulation (7), does not produce the same magnitude of force enhancement in all smooth muscle tissues, and the mechanism to explain this diversity has yet to be elucidated.The MYPT has two isoforms that are produced by alternative splicing of a central exon (13). The alternatively spliced segment lies near a phosphoryl...

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Section: Force Versus Casupporting

confidence: 71%

Section: Force Versus Cacontrasting

confidence: 51%

Agonist-induced Force Enhancement

Richards

Ogut

Brozovich

2002

Journal of Biological Chemistry

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“…This signaling pathway probably requires an intact cell membrane and thus would not be present in permeabilized and/or skinned preparations, explaining the absence of an effect of Rho-kinase inhibition on Ca 2ϩ activation of skinned smooth muscle strips. 18,19 However, Ca 2ϩ activation of Rho is consistent with the observations that in permeabilized vascular smooth muscle, Ca 2ϩ induces a translocation of Rho to the membrane 20 and the inhibition of force produced by depolarization when Rho is inactivated. 21 It has been demonstrated that the magnitude of the change in MLC 20 phosphorylation produced by depolarization is less than that produced by agonist stimulation for any given level of intracellular Ca 2ϩ .…”

supporting

confidence: 74%

Rho Signaling

Brozovich

2003

Circulation Research

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F orce development in smooth muscle is dependent on Ca 2ϩ /calmodulin (Ca 2ϩ /CaM) activation of myosin light chain kinase (MLCK) and the subsequent phosphorylation of the regulatory myosin light chain (MLC 20 ). 1 However, when smooth muscle is stimulated with agonists 2 or by direct activation of the G-proteins, 3-6 a higher force is developed for a given [Ca 2ϩ ] than for depolarization. Although the mechanism for this Ca 2ϩ sensitization of the contractile filaments is still the subject of investigation, evidence suggests that the signaling pathway may involve either the activation of Rho (for reviews see [7][8][9] ) and/or protein kinase C (PKC), 10,11 although a pathway involving Rho and subsequent activation of Rho-kinase is more widely accepted. Agonist stimulation of smooth muscle can be divided into two phases: an initial rapid, transient increase in Ca 2ϩ and MLC 20 phosphorylation, and a subsequent phase of a sustained force despite lower levels of Ca 2ϩ and MLC 20 phosphorylation. 12 There is evidence that the sustained phase of force maintenance is dependent on the activation of Rho. 13 Although uncertainty exists regarding the mechanism by which receptor-mediated activation of the G-proteins leads to force enhancement, it is generally accepted that membrane depolarization leads to an increase in intracellular [Ca 2ϩ ] and a Ca 2ϩ -dependent activation of MLCK to increase MLC 20 phosphorylation and force. Thus, it has become dogma that depolarization leads to only an increase in Ca 2ϩ and a subsequent activation of MLCK, without the activation of additional signaling pathways. Challenging these generally accepted principles is recent evidence that Rho-kinase inhibition almost completely eliminates the sustained portion of the phasic force response to 60 mmol/L KCl depolarization in rat tail artery without affecting the Ca 2ϩ transient. 14 The reduction in force was accompanied by a decrease in MLC 20 phosphorylation. These results suggest that Rho-kinase is activated during depolarization of smooth muscle, and its activation leads to an inhibition of MLC phosphatase activity.In this issue of Circulation Research, Sakurada et al 15 extend and further delineate the mechanism by which inhibition of Rho-kinase depresses force during depolarization. During both agonist and KCl depolarization of rabbit aortic smooth muscle, these investigators demonstrated that the GTP-bound active form of Rho increases in a concentrationdependent manner and the inhibition of Rho-kinase with either Y27632 or HA1077 reduced force and MLC 20 phosphorylation with similar concentration dependencies. These results suggest not only that Rho is activated during depolarization but that its activation increases force. Additionally, the group demonstrated that activation of Rho during KCl depolarization requires transmembrane Ca 2ϩ flux and results in phosphorylation of the myosin binding subunit (MYPT1) of MLC phosphatase at Thr 695 . MYPT1 phosphorylation at this site has been previously demonstrated to inhibit MLC phosphata...

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“…In the ileum, ROK blockade by Y-27632 and HA-1077 leaves the phasic component nearly intact and abolishes the tonic component (185), whereas in the bladder, both components are greatly diminished (79). Although GTP␥S induces ROK activation and Ca 2ϩ sensitization in chicken gizzard smooth muscle, muscarinic receptor stimulation does not cause ROK-dependent Ca 2ϩ sensitization in this muscle type (6). However, Y-27632 reduces stomach antrum motility in conscious rats (197) and stomach fundus contraction induced by muscarinic receptor activation in vitro (156).…”

Section: Rok and Pkc Participate In Elevating Ca 2؉ Sensitivity Of Mamentioning

confidence: 81%

Regulation of smooth muscle calcium sensitivity: KCl as a calcium-sensitizing stimulus

Ratz

Berg

Urban

et al. 2005

American Journal of Physiology-Cell Physiology

236

188

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KCl has long been used as a convenient stimulus to bypass G protein-coupled receptors (GPCR) and activate smooth muscle by a highly reproducible and relatively "simple" mechanism involving activation of voltage-operated Ca2+ channels that leads to increases in cytosolic free Ca2+ ([Ca2+]i), Ca2+-calmodulin-dependent myosin light chain (MLC) kinase activation, MLC phosphorylation and contraction. This KCl-induced stimulus-response coupling mechanism is a standard tool-set used in comparative studies to explore more complex mechanisms generated by activation of GPCRs. One area where this approach has been especially productive is in studies designed to understand Ca2+ sensitization, the relationship between [Ca2+]i and force produced by GPCR agonists. Studies done in the late 1980s demonstrated that a unique relationship between stimulus-induced [Ca2+]i and force does not exist: for a given increase in [Ca2+]i, GPCR activation can produce greater force than KCl, and relaxant agents can produce the opposite effect to cause Ca2+ desensitization. Such changes in Ca2+ sensitivity are now known to involve multiple cell signaling strategies, including translocation of proteins from cytosol to plasma membrane, and activation of enzymes, including RhoA kinase and protein kinase C. However, recent studies show that KCl can also cause Ca2+ sensitization involving translocation and activation of RhoA kinase. Rather than complicating the Ca2+ sensitivity story, this surprising finding is already providing novel insights into mechanisms regulating Ca2+ sensitivity of smooth muscle contraction. KCl as a "simple" stimulus promises to remain a standard tool for smooth muscle cell physiologists, whose focus is to understand mechanisms regulating Ca2+ sensitivity.

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Muscarinic stimulation does not induce rhoA/ROCK-mediated Ca 2+ sensitization of the contractile element in chicken gizzard smooth muscle

Cited by 21 publications

References 32 publications

Agonist-induced Force Enhancement

Agonist-induced Force Enhancement

Rho Signaling

Regulation of smooth muscle calcium sensitivity: KCl as a calcium-sensitizing stimulus

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