Calcium Sensitization in Human Esophageal Muscle: Role for RhoA Kinase in Maintenance of Lower Esophageal Sphincter Tone

Sims, Stephen M.; Chrones, Tom; Preiksaitis, Harold G.

doi:10.1124/jpet.108.140806

Cited by 21 publications

(33 citation statements)

References 39 publications

(80 reference statements)

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“…Several lines of evidence support the predominance of the RhoA/ROCK pathway in the IAS as follows: The maximal decrease in the IAS tone is significantly higher following the ROCK inhibition with Y 27632 (80%) compared with the PKC inhibition with Gö 6850 and calphostin C (27 and 19%, respectively). These data are similar to the previous data in the rat IAS (22,23,28) and intact human and cat lower esophageal sphincter smooth muscle (15,21,31). Clamping experiments performed in the present studies further show that, following maximal decrease in the IAS tone with ROCK inhibition, PKC inhibition produces a limited effect.…”

Section: Discussionsupporting

confidence: 81%

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RhoA/ROCK pathway is the major molecular determinant of basal tone in intact human internal anal sphincter

Rattan

Singh

2012

American Journal of Physiology-Gastrointestinal and Liver Physi

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Rattan S, Singh J. RhoA/ROCK pathway is the major molecular determinant of basal tone in intact human internal anal sphincter. Am J Physiol Gastrointest Liver Physiol 302: G664 -G675, 2012. First published January 12, 2012; doi:10.1152/ajpgi.00430.2011.-The knowledge of molecular control mechanisms underlying the basal tone in the intact human internal anal sphincter (IAS) is critical for the pathophysiology and rational therapy for a number of debilitating rectoanal motility disorders. We determined the role of RhoA/ROCK and PKC pathways by comparing the effects of ROCK-and PKCselective inhibitors Y 27632 and Gö 6850 (10 Ϫ8 to 10 Ϫ4 M), respectively, on the basal tone in the IAS vs. the rectal smooth muscle (RSM). Western blot studies were performed to determine the levels of RhoA/ROCK II, PKC-␣, MYPT1, CPI-17, and MLC20 in the unphosphorylated and phosphorylated forms, in the IAS vs. RSM. Confocal microscopic studies validated the membrane distribution of ROCK II. Finally, to confirm a direct relationship, we examined the enzymatic activities and changes in the basal IAS tone and p-MYPT1, p-CPI-17, and p-MLC20, before and after Y 27632 and Gö 6850. Data show higher levels of RhoA/ROCK II and related downstream signal transduction proteins in the IAS vs. RSM. In addition, data show a significant correlation between the active RhoA/ROCK levels, ROCK enzymatic activity, downstream proteins, and basal IAS tone, before and after ROCK inhibitor. From these data we conclude 1) RhoA/ ROCK and downstream signaling are constitutively active in the IAS, and this pathway (in contrast with PKC) is the critical determinant of the basal tone in intact human IAS; and 2) RhoA and ROCK are potential therapeutic targets for a number of rectoanal motility disorders for which currently there is no satisfactory treatment. smooth muscle tone; Rho kinase; rectoanal incontinence ALTHOUGH MULTIFACTORIAL, the basal tone in the IAS plays an important role in rectoanal incontinence (3,24). It has been shown before that the basal tone in the IAS in different species including humans is primarily myogenic in nature (2,5,7,17,25), because of the characteristic properties of the IAS smooth muscle cells (SMCs) (7,23,25). Knowledge of molecular determinants of basal tone in the IAS is critical for treating a number of rectoanal motility disorders such as fecal incontinence, Hirschsprung's disease, recurrent anal fissures, and hemorrhoids (3,17).Mechanisms underlying the agonists-induced sustained contraction of intestinal smooth muscle involve prolonged myosin light chain (MLC 20 ) phosphorylation (p-MLC 20 ), the phosphorylation of cytoskeleton filaments and associated proteins, increase in Ca 2ϩ influx, and Ca 2ϩ sensitization (19). It is well known that RhoA/ROCK and PKC play a significant role in this regard. In the phasic contraction, p-MLC 20 is rapidly dephosphorylated via myosin light chain phosphatase (MLCP). In the sustained contraction, on the other hand, this dephosphorylation is prevented or delayed primarily via MLCP inhibition by...

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Section: Discussionsupporting

confidence: 81%

“…The present data show a predominant role of RhoA/ROCK over PKC, similar to that of intact IAS in other animals (11) and other spontaneously tonic intact smooth muscle of the LES in humans and animals (15,21,31). These data are, however, Ϫ5 M cause significant decreases in the IAS tone (*P Ͻ 0.05; n ϭ 4).…”

Section: Gö Göcontrasting

confidence: 49%

RhoA/ROCK pathway is the major molecular determinant of basal tone in intact human internal anal sphincter

Rattan

Singh

2012

American Journal of Physiology-Gastrointestinal and Liver Physi

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“…In contrast to the basal tone in human and cat lower esophageal sphincter, ACh-and EFS-induced phasic contraction in the esophageal body is primarily mediated by PKC (12,25,37). Similar conclusions were drawn by the inhibition of carbachol-induced contraction of urinary bladder by PKC inhibitor (7).…”

Section: Discussionsupporting

confidence: 64%

“…[In contrast to the phasic activity in RSM, a number of studies have shown that basal tone in the lower esophageal sphincter and IAS in humans and animals is mediated primarily via the RhoA/ROCK pathway (25,30,32,37).] The present data also show selectivity of the effects of PKC and ROCK inhibitors on the respective pathways.…”

Section: Discussioncontrasting

confidence: 38%

Role of PKC and RhoA/ROCK pathways in the spontaneous phasic activity in the rectal smooth muscle

Singh

Rattan

2013

American Journal of Physiology-Gastrointestinal and Liver Physi

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The role of PKC and RhoA/ROCK pathways in the phasic activities in the rectal smooth muscles (RSM) in the basal state is not known. We examined this issue by determining the effects of PKC inhibitors (calphostin C and Gö-6850) and a ROCK inhibitor (Y-27632) on the slow-rate (~3/min) and fast-rate (~25/min) phasic activities. We also examined the corresponding signal transduction cascades and the PKC and ROCK enzymatic activities in the RSM in the basal state. PKC inhibition with calphostin C and Gö-6850 (10(-5) M) caused a significant decrease (~25%) in slow-rate (but not fast-rate) phasic activity (monitored by frequency and amplitude of contractions) of the RSM. Conversely, ROCK inhibition with Y-27632 (10(-5) M) caused a significant decrease not only in slow-rate, but also fast-rate, phasic activity caused by ROCK inhibition in the RSM. Western blot analysis revealed that the PKC inhibition-induced decrease in RSM phasic activity was associated with decreases in PKCα translocation, phosphorylated (Thr(38)) PKC-potentiated inhibitor (CPI-17), and phosphorylated (Thr(18)/Ser(19)) 20-kDa myosin regulatory light chain. Conversely, decreases in the phasic activity in the RSM by ROCK inhibition were accompanied by the additional decrease in phosphorylated (Thr(696)) myosin phosphatase target subunit 1. Data show that while PKC and RhoA/ROCK pathways play a significant role in slow-rate high-amplitude spontaneous phasic activity, only the RhoA/ROCK pathway primarily mediates fast-rate low-amplitude phasic activity, in the RSM. Such knowledge is important in the understanding of the pathophysiology of large intestinal motility disorders. Relative contributions of the PKC vs. the RhoA/ROCK pathway in the phasic activity remain to be determined.

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“…In the LES, ROCK inhibitors have been shown to produce full relaxation of the basal tone in human and various animal species, while PKC inhibitors have only limited effects on LES basal tone. 17,80 These studies suggest that a mechanism of Ca 2+ sensitization mediated by the RhoA/ROCK pathway plays an important role in the basal tone in the LES, although MYPT1 and CPI-17 phosphorylation in the LES has not been measured. Interestingly, the proton pump inhibitor pantoprazole inhibited MYPT1 T853 phosphorylation evoked by CCh in gastric and LES tissue, suggesting that it may also be a ROCK inhibitor, although it had no effect on constitutive MYPT1 or CPI-17 phosphorylation.…”

Section: Cpi-17 and Mypt1 Are Constitutively Phosphorylated In Gastromentioning

confidence: 99%

Calcium Sensitization Mechanisms in Gastrointestinal Smooth Muscles

Perrino

2016

J Neurogastroenterol Motil

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An increase in intracellular Ca2+ is the primary trigger of contraction of gastrointestinal (GI) smooth muscles. However, increasing the Ca 2+ sensitivity of the myofilaments by elevating myosin light chain phosphorylation also plays an essential role. Inhibiting myosin light chain phosphatase activity with protein kinase C-potentiated phosphatase inhibitor protein-17 kDa (CPI-17) and myosin phosphatase targeting subunit 1 (MYPT1) phosphorylation is considered to be the primary mechanism underlying myofilament Ca 2+ sensitization. The relative importance of Ca 2+ sensitization mechanisms to the diverse patterns of GI motility is likely related to the varied functional roles of GI smooth muscles. Increases in CPI-17 and MYPT1 phosphorylation in response to agonist stimulation regulate myosin light chain phosphatase activity in phasic, tonic, and sphincteric GI smooth muscles. Recent evidence suggests that MYPT1 phosphorylation may also contribute to force generation by reorganization of the actin cytoskeleton. The mechanisms responsible for maintaining constitutive CPI-17 and MYPT1 phosphorylation in GI smooth muscles are still largely unknown. The characteristics of the cell-types comprising the neuroeffector junction lead to fundamental differences between the effects of exogenous agonists and endogenous neurotransmitters on Ca 2+ sensitization mechanisms. The contribution of various cell-types within the tunica muscularis to the motor responses of GI organs to neurotransmission must be considered when determining the mechanisms by which Ca 2+ sensitization pathways are activated. The signaling pathways regulating Ca 2+ sensitization may provide novel therapeutic strategies for controlling GI motility. This article will provide an overview of the current understanding of the biochemical basis for the regulation of Ca

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Calcium Sensitization in Human Esophageal Muscle: Role for RhoA Kinase in Maintenance of Lower Esophageal Sphincter Tone

Cited by 21 publications

References 39 publications

RhoA/ROCK pathway is the major molecular determinant of basal tone in intact human internal anal sphincter

RhoA/ROCK pathway is the major molecular determinant of basal tone in intact human internal anal sphincter

Role of PKC and RhoA/ROCK pathways in the spontaneous phasic activity in the rectal smooth muscle

Calcium Sensitization Mechanisms in Gastrointestinal Smooth Muscles

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