Nature of extracellular signal that triggers RhoA/ROCK activation for the basal internal anal sphincter tone in humans

Abstract

The extracellular signal that triggers activation of rho-associated kinase (RhoA/ROCK), the major molecular determinant of basal internal anal sphincter (IAS) smooth muscle tone, is not known. Using human IAS tissues, we identified the presence of the biosynthetic machineries for angiotensin II (ANG II), thromboxane A2 (TXA2), and prostaglandin F2α (PGF2α). These end products of the renin-angiotensin system (RAS) (ANG II) and arachidonic acid (TXA2 and PGF2α) pathways and their effects in human IAS vs. rectal smooth muscle (RSM) were studied. A multipronged approach utilizing immunocytochemistry, Western blot analyses, and force measurements was implemented. Additionally, in a systematic analysis of the effects of respective inhibitors along different steps of biosynthesis and those of antagonists, their end products were evaluated either individually or in combination. To further describe the molecular mechanism for the IAS tone via these pathways, we monitored RhoA/ROCK activation and its signal transduction cascade. Data showed characteristically higher expression of biosynthetic machineries of RAS and AA pathways in the IAS compared with the RSM. Additionally, specific inhibition of the arachidonic acid (AA) pathway caused ~80% decrease in the IAS tone, whereas that of RAS lead to ~20% decrease. Signal transduction studies revealed that the end products of both AA and RAS pathways cause increase in the IAS tone via activation of RhoA/ROCK. Both AA and RAS (via the release of their end products TXA2, PGF2α, and ANG II, respectively), provide extracellular signals which activate RhoA/ROCK for the maintenance of the basal tone in human IAS.

Ca2+/calmodulin/MLCK pathway initiates, and RhoA/ROCK maintains, the internal anal sphincter smooth muscle tone

“…These reconstructs were shown to have functional and molecular properties similar to the intact IAS and demonstrated that the basal tone is dependent on RhoA/ROCK. Altogether, these data suggest that the sphincteric tone is critically dependent on RhoA/ROCK that may be either constitutively active or involve GPCR activation via autocrine control (6,32). In support of these concepts, recent studies by Drs.…”

Role of microRNAs in gastrointestinal smooth muscle fibrosis and dysfunction: novel molecular perspectives on the pathophysiology and therapeutic targeting

“…The SMs that form the sphincters (tonic SMs) have a unique contractile mechanism resulting in a higher basal tone compared with phasic SMs. It is well established that the major molecular determinant of basal tone in sphincteric GI SMs, such as internal anal sphincter (IAS), is a RhoA-associated kinase (RhoA)/Rho kinase (ROCK) pathway within the specialized SMCs (105)(106)(107)(108).…”

Activation of angiotensin II type 1 receptors and contractile activity in human sigmoid colon in vitro

“…Ang II effects are mediated by specific receptors, the Ang II type 1 (AT 1 ) and the Ang II type 2 (AT 2 ) receptors, both members of the G-protein-coupled receptor family (De Gasparo et al 2000, Jackson 2001. The presence of Ang II receptors and the other RAS components at various levels along the GI tract has been found in animal preparations , De Godoy et al 2004, De Godoy & Rattan 2005, Wang et al 2005, Ewert et al 2006, Mastropaolo et al 2013a) and also in humans (Hirasawa et al 2002, Ewert et al 2006, Casselbrant et al 2007, Spak et al 2008, Rattan et al 2015, suggesting their potential physiological action. Indeed, some studies have shown that Ang II induces contractile responses either indirectly, via activation of neural AT 1 receptors, or directly, via activation of AT 1 receptors located on smooth muscle cells in different GI tracts of various species, including humans (Hawcock & Barnes 1993, Ewert et al 2006, Casselbrant et al 2007, Spak et al 2008).…”

“…, Rattan et al . ), suggesting their potential physiological action. Indeed, some studies have shown that Ang II induces contractile responses either indirectly, via activation of neural AT 1 receptors, or directly, via activation of AT 1 receptors located on smooth muscle cells in different GI tracts of various species, including humans (Hawcock & Barnes , Ewert et al .…”