Phosphorylation of the myosin phosphatase targeting subunit and CPI‐17 during Ca<sup>2+</sup> Sensitization in Rabbit Smooth Muscle

Kitazawa, Takio; Eto, Masumi; Woodsome, Terence P.; Khalequzzaman, -

doi:10.1113/jphysiol.2002.029306

Cited by 211 publications

(280 citation statements)

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“…8), but the addition of carbachol (within 30 s) immediately phosphorylated CPI-17 at Thr 38 , and the phosphorylation levels were well maintained for over 10 min during the carbachol stimulation. These results are consistent with previous results (24,53) obtained in other smooth muscles and suggest that CPI-17 phosphorylation may play a pivotal role in the carbachol-induced Ca 2ϩ sensitization in rat ileum. Furthermore, in the ileal smooth muscle, Y-27632 inhibited the carbachol-induced increase in CPI-17 phosphorylation by ϳ30% (Fig.…”

Section: Discussionsupporting

confidence: 83%

“…Incubation of the tissue for 5-15 min with carbachol significantly increased MYPT-1 phosphorylation, but its extent was very small (less than 20%). These results coincide with those of the most recent studies in the rabbit portal vein and vas deferens (24) and the rabbit femoral artery (53). Therefore, we concluded that MYPT-1 phosphorylation does not play an essential role in the Ca 2ϩ sensitization induced by receptor stimulation in rat ileum.…”

Section: Discussionsupporting

confidence: 81%

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Chronic Treatment with Interleukin-1β Attenuates Contractions by Decreasing the Activities of CPI-17 and MYPT-1 in Intestinal Smooth Muscle

Ohama

Hori

Sato

et al. 2003

Journal of Biological Chemistry

113

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Interleukin-1␤ (IL-1␤) is a proinflammatory cytokine that plays a central role in inflammatory bowel disease (IBD). In order to elucidate the mechanism of motility disorders frequently observed in IBD, we investigated the long term effects of IL-1␤ on rat ileal smooth muscle contractility by using an organ culture system. When ileal smooth muscle strips were cultured with IL-1␤ (10 ng/ml), contractions elicited by high K ؉ and carbachol were inhibited in a time-dependent manner. IL-1␤ more strongly inhibited the carbachol-induced contractions than high K ؉ with decreasing myosin light chain phosphorylation. In the ␣-toxin-permeabilized ileal muscle, carbachol with GTP or guanosine 5 -3-O-(thio)triphosphate increased the Ca 2؉ sensitivity of contractile elements, and this G protein-coupled Ca 2؉ sensitization was significantly reduced in the IL-1␤-treated ileum. Among the functional proteins involved in the smooth muscle Ca 2؉ sensitization, CPI-17 expression was significantly reduced after the culture with IL-1␤, whereas the expressions of RhoA, ROCK-I, ROCK-II, MYPT-1, myosin light chain kinase, and myosin phosphatase (PP1) were unchanged. The phosphorylation level of CPI-17 by carbachol was low in accordance with the decrease in CPI-17 expression due to IL-1␤ treatment. In contrast, constitutively phosphorylated MYPT-1 was also decreased in the IL-1␤-treated muscles. These results suggest that long term treatment with IL-1␤ decreases either CPI-17 expression or MYPT-1 phosphorylation, which may result in an increase in myosin phosphatase activity to reduce force generation. Based on these findings, we consider IL-1␤ to be an important mediator of gastrointestinal motility disorders in IBD, and CPI-17 and MYPT-1 are key molecules in the decreased smooth muscle contractility due to IL-1␤.

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Section: Discussionsupporting

confidence: 83%

Section: Discussionsupporting

confidence: 81%

Chronic Treatment with Interleukin-1β Attenuates Contractions by Decreasing the Activities of CPI-17 and MYPT-1 in Intestinal Smooth Muscle

Ohama

Hori

Sato

et al. 2003

Journal of Biological Chemistry

113

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“…Several kinases purified from smooth muscles, such as PKC␣͞␦, ZIPkinase, and integrin-linked kinase, activate CPI-17 by phosphorylation at Thr-38 (15)(16)(17). Phosphorylation of CPI-17 at Thr-38 in smooth muscle cells occurs in response to various agonists, such as histamine, endothelin-1, and angiotensin II, in parallel with induction of myosin phosphorylation and contraction (18,19). On the other hand, phosphorylation of CPI-17 is reversed during vasodilation induced by nitric oxide production (20).…”

mentioning

confidence: 99%

Phosphoprotein inhibitor CPI-17 specificity depends on allosteric regulation of protein phosphatase-1 by regulatory subunits

Eto

Kitazawa

Brautigan

2004

Proc. Natl. Acad. Sci. U.S.A.

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Inhibition of myosin phosphatase is critical for agonist-induced contractility of vascular smooth muscle. The protein CPI-17 is a phosphorylation-dependent inhibitor of myosin phosphatase and, in response to agonists, Thr-38 is phosphorylated by protein kinase C, producing a >1,000-fold increase in inhibitory potency. Here, we addressed how CPI-17 could selectively inhibit myosin phosphatase among other protein phosphatase-1 (PP1) holoenzymes. PP1 in cell lysates was separated by sequential affinity chromatography into at least two fractions, one bound specifically to thiophospho-CPI-17, and another bound specifically to inhibitor-2. The MYPT1 regulatory subunit of myosin phosphatase was concentrated only in the fraction bound to thiophospho-CPI-17. This binding was eliminated by addition of excess microcystin-LR to the lysate, showing that binding at the active site of PP1 is required. Phospho-CPI-17 failed to inhibit glycogen-bound PP1 from skeletal muscle, composed primarily of PP1 with the striated muscle glycogentargeting subunit (G M) regulatory subunit. Phospho-CPI-17 was dephosphorylated during assay of glycogen-bound PP1, not MYPT1-associated PP1, even though these two holoenzymes have the same PP1 catalytic subunit. Phosphorylation of CPI-17 in rabbit arteries was enhanced by calyculin A but not okadaic acid or fostriecin, consistent with PP1-mediated dephosphorylation. We propose that CPI-17 binds at the PP1 active site where it is dephosphorylated, but association of MYPT1 with PP1C allosterically retards this hydrolysis, resulting in formation of a complex of MYPT1⅐PP1C⅐P-CPI-17, leading to an increase in smooth muscle contraction.

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“…Agonist-induced Ca 2+ sensitization is due to inhibition of MLCP activity resulting in an increased ratio of MLCK/ MLCP activity [21]. It has been suggested that two different pathways lead to MLCP inhibition, which operate through Rho and PKC, respectively [5,6,11,14,18]. Activation of G q/11 -coupled M 3 receptors, via stimulating phospholipase C (PLC) activity, produces the second messenger diacylglycerol (DAG) which activates PKC, and this causes phosphorylation of CPI-17 (PKC-activated 17 kD inhibitor protein) to inhibit MLCP activity [9,11].…”

Section: Discussionmentioning

confidence: 99%

“…It has been suggested that two different pathways lead to MLCP inhibition, which operate through Rho and PKC, respectively [5,6,11,14,18]. Activation of G q/11 -coupled M 3 receptors, via stimulating phospholipase C (PLC) activity, produces the second messenger diacylglycerol (DAG) which activates PKC, and this causes phosphorylation of CPI-17 (PKC-activated 17 kD inhibitor protein) to inhibit MLCP activity [9,11]. Although the small G-protein Rho is activated by G 12/13 -type G-proteins, recent studies have suggested that it is activated by G q/11 -type Gproteins as well [1,32].…”

Section: Discussionmentioning

confidence: 99%

Muscarinic Receptor Subtypes Mediating Ca2+ Sensitization of Intestinal Smooth Muscle Contraction: Studies with Receptor Knockout Mice

Suguro

Matsuyama

Tanahashi

et al. 2010

The Journal of Veterinary Medical Science

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ABSTRACT. In the present study, we have characterized muscarinic receptor subtypes that mediate carbachol-induced Ca 2+ sensitization of contraction in intestinal smooth muscle, using mutant mice lacking M 2 or M 3 muscarinic receptors or both receptor subtypes. In -toxinpermeabilized muscle strips from wild-type (WT) mice, isometric tension responses to Ca 2+ applied cumulatively (pCa 7.0-5.0) were increased when the muscarinic agonist carbachol (100 M) was added to the medium, as judged from shifts of pCa-tension curves in both 50% effective concentration (EC 50 ) and maximum response (E max ) of pCa-tension curve. In preparations from M 2 -knockout (KO) mice, pCa-tension curves were also shifted by carbachol (100 M), and the extents of the EC 50 and E max changes resembled those observed in preparations from WT mice. In preparations from M 3 -KO or M 2 /M 3 -double KO mice, however, no significant changes in pCa-tension curves were obtained after carbachol application. ] c to increase the MLCK/ MLCP activity ratio, resulting in enhanced contractions compared to those without receptor activation [21].In intestinal smooth muscles, acetylcholine and its derivatives such as carbachol, activate muscarinic receptors, inducing Ca 2+ sensitization as well as cytosolic Ca 2+ mobilization and contraction [6,29]. Among the five muscarinic receptor subtypes (M 1 -M 5 ), the M 2 and M 3 are predominantly expressed in intestinal smooth muscles [2]. By using muscarinic receptor antagonists with limited subtype selectivity, Murthy et al. [14] suggested that acetylcholineinduced Rho kinase activation and PKC activation in rabbit intestinal smooth muscles is mediated by the M 3 subtype, leading to Ca 2+ sensitization. However, such pharmacological studies often remain inconclusive because of the poor subtype selectivity of available muscarinic antagonists. For example, pharmacological studies suggested that carbacholevoked contractions are exclusively mediated by M 3 receptors, whereas recent studies using muscarinic receptor knockout mice implicated both M 3 and M 2 receptors in this response [30,31].Therefore, in the present study, using M 2 -knockout (KO) or M 3 -KO or M 2 /M 3 -double KO mice and wild type (WT) mice, we examined the activity of carbachol in inducing Ca 2+ sensitization in -toxin-permeabilized smooth muscles of the small intestine. In general, M 2 and M 3 muscarinic receptors are coupled preferentially with G i/o -and G q/11 -type G-proteins, respectively. We also examined whether the carbacholinduced Ca 2+ sensitization was affected by YM-254890, a selective inhibitor of G q/11 -type G-protein activity [23,26].

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Phosphorylation of the myosin phosphatase targeting subunit and CPI‐17 during Ca²⁺ Sensitization in Rabbit Smooth Muscle

Cited by 211 publications

References 44 publications

Chronic Treatment with Interleukin-1β Attenuates Contractions by Decreasing the Activities of CPI-17 and MYPT-1 in Intestinal Smooth Muscle

Chronic Treatment with Interleukin-1β Attenuates Contractions by Decreasing the Activities of CPI-17 and MYPT-1 in Intestinal Smooth Muscle

Phosphoprotein inhibitor CPI-17 specificity depends on allosteric regulation of protein phosphatase-1 by regulatory subunits

Muscarinic Receptor Subtypes Mediating Ca2+ Sensitization of Intestinal Smooth Muscle Contraction: Studies with Receptor Knockout Mice

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Phosphorylation of the myosin phosphatase targeting subunit and CPI‐17 during Ca2+ Sensitization in Rabbit Smooth Muscle

Cited by 211 publications

References 44 publications

Chronic Treatment with Interleukin-1β Attenuates Contractions by Decreasing the Activities of CPI-17 and MYPT-1 in Intestinal Smooth Muscle

Chronic Treatment with Interleukin-1β Attenuates Contractions by Decreasing the Activities of CPI-17 and MYPT-1 in Intestinal Smooth Muscle

Phosphoprotein inhibitor CPI-17 specificity depends on allosteric regulation of protein phosphatase-1 by regulatory subunits

Muscarinic Receptor Subtypes Mediating Ca2+ Sensitization of Intestinal Smooth Muscle Contraction: Studies with Receptor Knockout Mice

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Phosphorylation of the myosin phosphatase targeting subunit and CPI‐17 during Ca²⁺ Sensitization in Rabbit Smooth Muscle