Rho Kinase as a Novel Molecular Therapeutic Target for Hypertensive Internal Anal Sphincter

Rattan, Satish; Godoy, Márcio A. F. de; Patel, Chirag

doi:10.1053/j.gastro.2006.03.043

Cited by 41 publications

(62 citation statements)

References 44 publications

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“…Male Sprague-Dawley rats (300 -350 g) were euthanized by decapitation and internal anal sphincter (IAS) smooth muscle strips were prepared in oxygenated modified Krebs' physiological solution (KPS) as described before (41). The composition of KPS was as follows (in mM): 118.07 NaCl, 4.69 KCl, 2.52 CaCl 2, 1.16 MgSO4, 1.01 NaH2PO4, 25 NaHCO3, and 11.10 glucose.…”

Section: Methodsmentioning

confidence: 99%

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Immunoglobulins from scleroderma patients inhibit the muscarinic receptor activation in internal anal sphincter smooth muscle cells

Singh¹,

Mehendiratta²,

Galdo

et al. 2009

American Journal of Physiology-Gastrointestinal and Liver Physi

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Section: Methodsmentioning

confidence: 99%

“…SMCs from the circular smooth muscle layer of the rat IAS were isolated as described before (27,41). Briefly, the IAS smooth muscle tissues prepared above were cut into one or two cubes and incubated in oxygenated KPS containing 0.1% collagenase and 0.01% soybean trypsin inhibitor at 37°C for two successive 1-h periods.…”

Section: Methodsmentioning

confidence: 99%

Immunoglobulins from scleroderma patients inhibit the muscarinic receptor activation in internal anal sphincter smooth muscle cells

Singh¹,

Mehendiratta²,

Galdo

et al. 2009

American Journal of Physiology-Gastrointestinal and Liver Physi

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“…These pathways do so primarily via the phosphorylation of regulatory binding subunit of MLCP (MYPT1), the catalytic subunit (PP1c␦) of MLCP, and protein kinase C-potentiated inhibitor or phosphoprotein for myosin phosphatase (CPI-17), the endogenous inhibitory protein of MLCP (8,14,19,26). Our previous studies in rats have shown that, in the basal state under similar concentrations of Ca 2ϩ , the IAS develops steady tone whereas the rectal smooth muscle (RSM) does not (28).…”

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confidence: 99%

“…Additionally, the animal data suggest a critical role of RhoA/ROCK for the basal IAS tone at the intracellular level (22,23,28) compared with the phasic activities in the RSM and anococcygeus (ASM) smooth muscles. According to those studies, the IAS has characteristically higher levels of p-MYPT1, CPI-17, p-CPI-17, and p-MLC 20 .…”

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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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“…Proteins involved in the Rho-signaling cascade are significantly elevated in a variety of cancers [39][40][41][42][43], and pharmacological inhibition of ROCK activity shows preclinical efficacy in the treatment of in vivo malignancies including prostate, breast, glioma, melanoma, and human papillomavirus infected tumors [25,[44][45][46][47]. Given that ROCK signaling regulates both solid tumor progression and angiogenesis, we sought to examine its role in angiosarcoma solid tumors which are formed extensively by dysfunctional, aberrantly proliferating endothelial cells.…”

Section: Discussionmentioning

confidence: 99%

Rock1 & 2 Perform Overlapping and Unique Roles in Angiogenesis and Angiosarcoma Tumor Progression

Montalvo¹,

Spencer²,

Hackathorn³

et al. 2012

CMM

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Abstract:The serine/threonine protein kinase paralogs ROCK1 & 2 have been implicated as essential modulators of angiogenesis; however their paralog-specific roles in endothelial function are unknown. shRNA knockdown of ROCK1 or 2 in endothelial cells resulted in a significant disruption of in vitro capillary network formation, cell polarization, and cell migration compared to cells harboring non-targeting control shRNA plasmids. Knockdowns led to alterations in cytoskeletal dynamics due to ROCK1 & 2-mediated reductions in actin isoform expression, and ROCK2-specific reduction in myosin phosphatase and cofilin phosphorylation. Knockdowns enhanced cell survival and led to ROCK1 & 2-mediated reduction in caspase 6 and 9 cleavage, and a ROCK2-specific reduction in caspase 3 cleavage. Microarray analysis of ROCK knockdown lines revealed overlapping and unique control of global transcription by the paralogs, and a reduction in the transcriptional regulation of just under 50% of VEGF responsive genes. Finally, paralog knockdown in xenograft angiosarcoma tumors resulted in a significant reduction in tumor formation. Our data reveals that ROCK1 & 2 exhibit overlapping and unique roles in normal and dysfunctional endothelial cells, that alterations in cytoskeletal dynamics are capable of overriding mitogen activated transcription, and that therapeutic targeting of ROCK signaling may have profound impacts for targeting angiogenesis.

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Rho Kinase as a Novel Molecular Therapeutic Target for Hypertensive Internal Anal Sphincter

Cited by 41 publications

References 44 publications

Immunoglobulins from scleroderma patients inhibit the muscarinic receptor activation in internal anal sphincter smooth muscle cells

Immunoglobulins from scleroderma patients inhibit the muscarinic receptor activation in internal anal sphincter smooth muscle cells

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

Rock1 & 2 Perform Overlapping and Unique Roles in Angiogenesis and Angiosarcoma Tumor Progression

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