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

Abstract

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...

Calcium Sensitization Mechanisms in Gastrointestinal Smooth Muscles

“…93,94 ROCK and MYPT1 phosphorylation contribute substantially to the constitutive tonic contracted state of the IAS. 95 ROCK inhibition decreases the basal tone, and reduces the phosphorylation levels of MYPT1 T697, CPI-17 T38, and MLC S19 in rat IAS smooth muscles. 48,95 With human IAS smooth muscles, it has been reported that PKC inhibitors significantly decrease T38 phosphorylation, with modest effects on IAS smooth muscle tone, while ROCK inhibition produces significantly greater relaxation of IAS smooth muscle cells than PKC inhibition.…”

“…48,95 With human IAS smooth muscles, it has been reported that PKC inhibitors significantly decrease T38 phosphorylation, with modest effects on IAS smooth muscle tone, while ROCK inhibition produces significantly greater relaxation of IAS smooth muscle cells than PKC inhibition. 95,96 However, it has also been reported that the PKC activator PDBu relaxes opossum IAS. 97 A variety of non-cholinergic agonists have been reported to increase CPI-17 and MYPT1 phosphorylation and activate myofilament Ca 2+ sensitization.…”

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

“…Recently, significant data in intact IAS of humans and animals demonstrated that the predominant molecular regulatory mechanism for IAS tone is the RhoA/ROCK pathway over the PKC pathway (26, 30, 32). However, little is known about the patterns of rectal smooth muscle (RSM) contractions and regulatory mechanisms of the RSM, especially in the basal state.A good starting point would be analysis of the contractile patterns in the phasic activity in the RSM in the basal state (29,30,32). In the gastrointestinal tract smooth muscle, spontaneous contractions vary from 3 min Ϫ1 (in the stomach) to 8 -12 min Ϫ1 (in the small and large intestine) (33).…”

“…These factors are impairment of basal internal anal sphincter (IAS) tone and external anal sphincter, puborectalis, and colorectal smooth muscle compliance. Recently, significant data in intact IAS of humans and animals demonstrated that the predominant molecular regulatory mechanism for IAS tone is the RhoA/ROCK pathway over the PKC pathway (26,30,32). However, little is known about the patterns of rectal smooth muscle (RSM) contractions and regulatory mechanisms of the RSM, especially in the basal state.…”

“…A good starting point would be analysis of the contractile patterns in the phasic activity in the RSM in the basal state (29,30,32). In the gastrointestinal tract smooth muscle, spontaneous contractions vary from 3 min Ϫ1 (in the stomach) to 8 -12 min Ϫ1 (in the small and large intestine) (33).…”