RhoA/Rho-Kinase: Pathophysiological and Therapeutic Implications in Gastrointestinal Smooth Muscle Tone and Relaxation

Background

Major molecular pathways affecting gastrointestinal (GI) smooth muscle function include Ca²⁺/calmodulin (CaM)/myosin light chain kinase (MLCK), the protein kinase C (PKC)/CPI-17, and RhoA/Rho-kinase (ROCK)/myosin light chain phosphatase (MLCP).¹ Among these, RhoA/ROCK is emerging as an important mediator of smooth muscle contraction and dysfunction that cannot be explained on the basis of previously known bioenergetics.^1–3 The role of RhoA/ROCK in the pathophysiology and therapy has been investigated extensively and reviewed in organs systems other than the GI tract.^4–8 This mini-review provides current information on the role of RhoA/Rho-kinase in the pathophysiology and potential therapy for the following disorders: diffuse esophageal spasm (DES), achalasia, gastroesophageal reflux disease (GERD), gastroparesis, pylorospasm, biliary dyskinesia, adynamic ileus, colonic inertia, Hirschsprung’s disease, Ogilvie’s syndrome, recurrent anal fissures (RAF), hemorrhoids, rectoanal incontinence (Table 1).

Table 1.

Implications of the Role of RhoA/ROCK in the Pathophysiology and Therapeutic Target in Different Organs of the Gastrointestinal Tract

Organ	Disorders
Esophageal Body	Diffuse esophageal spasm
Lower Esophageal Sphincter (LES)	Achalasia Gastroesophageal reflux disease
Stomach	Gastroparesis Pylorospasm
Gallbladder	Biliary dyskinesia
Small Intestine	Adynamic ileus
Colon	Colonic inertia Hirschsprung’s disease Ogilvie’s syndrome
Internal Anal Sphincter (IAS)	Recurrent anal fissures Hemorrhoids Rectoanal incontinence

The functional integration of the enteric nervous system (ENS) in conjunction with the central nervous system (CNS), smooth muscle, and perhaps interstitial cells of Cajal (ICC), is responsible for GI motility involving systematic contraction and relaxation of the smooth muscle. However, certain smooth muscle contractions or tone may occur independent of the innervation and are myogenic in nature.⁹ Contraction and relaxation of the GI smooth muscle can be elicited also pharmacologically. Despite significant investigations, including the use of animal models, the exact nature of the molecular mechanisms underlying the pathophysiology of GI smooth muscle contraction and relaxation are not known. GI smooth muscles exhibiting spontaneous myogenic basal tone, such as the lower esophageal sphincter (LES) and internal anal sphincter (IAS), provide an opportunity to examine specific molecular pathways for contraction and relaxation in the absence of any stimulus. ^9–12 Side-by-side comparison of functionally diverse yet adjoining smooth muscles (phasic vs. tonic), e.g. LES vs. the esophageal body and IAS vs. rectal smooth muscle (RSM) in the basal state, provides an excellent platform for the elucidation of these pathways (particularly RhoA/ROCK), in human health and disease. This may lead to an important target for the rational therapy for the corresponding complex and debilitating conditions, characterized by either hypo- or hypertonic states.^13–17

Signal Transduction in GI Smooth Muscle Contractility

The mechanism of signal transduction in smooth muscle cells (SMC) involves three types of membrane-bound proteins: a membrane-spanning receptor, a GTP-binding protein that couples to the receptor, and effector enzymes capable of generating intracellular regulatory pathways.¹ Stimulatory ligands for these GI smooth muscle receptors come from several sources. Excitatory motor neurons of the ENS release acetylcholine (ACh) and tachykinins including substance P, neurokinin A, norepinephrine and neuropeptide Y.^18,19 Ligands from SMC include Angiotensin II,^12,20,21 eicosanoids,^22–24 sphingosine-1-phosphate (S1P), lysophosphatidic acid (LPA), and endocannabinoids.^25,26 Receptor ligands may also originate from adventitious cells and via circulation.^18,19 A vast range of receptors reside on the surface of GI SMC, which may activate Gα_12,13-proteins resulting in the activation of RhoA/ROCK (Figure 1).

Figure 1.

Schematic representation of the major pathways involved in excitation-contraction, and relaxation coupling in GI smooth muscle. RhoA/ROCK may be activated either by G-protein coupled receptor activation or independent of it. The complex of RhoA.GDI.GDP usually reflects inactive state of RhoA. It transforms via RhoGEFs into an active RhoA.GDI.GTP complex formation. GEFs catalyze the exchange of GDP for GTP on RhoA. RhoGAP drives the reaction in the reverse direction. Activated RhoA/ROCK leads to the phosphorylation of MLC₂₀, and subsequent contraction via these different pathways. First, there is inhibition of MLCP through its regulatory subunit MYPT1. Second, there is phosphorylation of CPI-17 and subsequent inhibition of MLCP via its catalytic subunit PP1c. Finally there is MLCK-like activity. Shown on the left side, RhoA/ROCK may either activate PKC directly or via PLD1/PA/DAG. There is growing evidence in different smooth muscle systems that PKC may lie upstream of RhoA/ROCK pathway (denoted by bidirectional arrow). In contrast with the multiple pathways for the phosphorylation of MLC₂₀ as in the case of RhoA/ROCK, the major target for the PKC is CPI-17. Interestingly, phosphorylation of CPI-17 is not limited to RhoA/ROCK and PKC, but other kinases, such as ZIP kinase, and ILK can mediate this event.

Green arrows and substrates indicate stimulation, red lines and substrates indicate inhibition.

* = hypothetical sites of upregulation in the case to spontaneously tonic smooth muscle (e.g. sphincteric) or during the pathologically hypercontractility state of different GI smooth muscles. Conversely, these sites may be inherently downregulated in the case of phasic smooth muscle in the basal state or during the pathologically hypocontractility state; #, hypothetical sites of inherent downregulation of the tonic smooth muscle and upregulation of the phasic smooth muscle in the basal state.

Abbreviations: ROCK, Rho kinase; Rho.GDI, guanine exchange dissociation inhibitor; Rho.GEF, GDT-GTP exchange factor; GAP, GTPase-activating protein; ZIPK, zipper-interacting kinase; ILK, integrin-linked kinase

RhoA Activity

The Rho family comprises twenty members distributed into eight subfamilies: Rho, Rnd, RhoD/F, RhoH, Rac, Cdc42, RhoU/V and RhoBTB.²⁷ The Rho subfamily includes the RhoA, RhoB, and RhoC isoforms, of which RhoA is the most studied. RhoA/ROCK is of specific interest in the regulation of smooth muscle contractility and relaxation.^1,28 At least 21 effectors of RhoA have been identified including Rho-kinase,²⁹ phospholipase D1 (PLD1), Rhotekin³⁰, and Rhophilin.³¹

Activation of RhoA depends upon its binding to the guanosine nucleotides, guanosine triphosphate (GTP) or guanosine diphosphate (GDP). Rho proteins possess a nucleotide binding pocket with an extremely high affinity for both GTP and GDP, and function as a binary signaling switch by cycling between the active GTP-bound form located in the membrane and the inactive GDP-bound cytosolic form. Three general classes of regulators of Rho protein signaling have been identified: guanine nucleotide exchange factor (GEFs), GTPase-activating proteins (GAPs), and guanine nucleotide dissociation inhibitors (GDIs) (Figure 1).³²

GEFs control the release of GDP from the Rho protein, transiently resulting in nucleotide-free Rho. The relative intracellular abundance of GTP, relative to GDP ensures that it is predominantly GTP that fills the nucleotide binding pocket. Thus GEFs facilitate Rho activation.^33,34 Conversely, GAPs regulate Rho inactivation.³⁵ The balance between GEF and GAP activity determines the guanine nucleotide status of Rho, thereby regulating its activity. GDI proteins form a large complex with Rho to prevent diffusion within the plasma membrane and into the cytosol, thus acting as an anchor and allowing for very specific spatial control of Rho activation.³²

The lipid modification (geranyl-geranylation) of RhoA allows its interaction with the membrane suggesting that such an association is a requirement for Ca²⁺ sensitization of smooth muscle.³⁶ This is supported by the observation that Ca²⁺ sensitizing agonists and GTPγS cause translocation of RhoA/ROCK to the plasma membrane,^37,38 whereas inactivation of RhoA by the bacterial C3 exoenzyme inhibits this translocation, and Ca²⁺ sensitization.³⁸

Rho-Kinase (ROCK)

ROCK (a cytoplasmic serine/threonine-specific kinase) translocates to the plasma membrane where it binds GTP-bound RhoA, leading to autophosphorylation and activation.^29,34,39 ROCK has two isoforms, ROCKI (ROKβ) and ROCKII (ROKα), expressed in smooth muscle.⁴⁰ ROCKII is the most common isoform involved in smooth muscle contraction.^2,41 via the prevention of naturally occurring dephosphorylation of phosphorylated-MLC₂₀ (p-MLC₂₀).^1,42–44 After interaction with actin and myosin leading to an initial contraction, p-MLC₂₀ undergoes rapid dephosphorylation by MLCP, thereby terminating the contraction. For sustained contraction, therefore, it is critical that this dephosphorylation is prevented, which can occur via PKC or RhoA/ROCK. RhoA/ROCK plays a major role in this regard.^45–47 ROCKII prevents dephosphorylation of p-MLC₂₀ or increases its phosphorylation through three different manners (Figure 1): inhibition of the MYPT1 subunit of MLCP by its phosphorylation;^28,34,48,49 phosphorylation of CPI-17 at the threonine-38 residue (p^Thr38-CPI-17), which inhibits PP1c of MLCP;^28,50,51 and MLCK-like activity.^52,53

Role of RhoA/ROCK/MYPT1/CPI-17 in Gastrointestinal Smooth Muscle Function

The consensus pathway for the maintenance of tone of GI smooth muscle involves receptor stimulation causing RhoA/ROCK activation to inhibit MLCP, resulting in maintained p-MLC₂₀, and sustained muscle contraction (Figure 1).³ The IAS and LES are prototypical tonic GI smooth muscles in that they exhibit spontaneous tone in the absence of any stimulus. Higher levels of RhoA/ROCK and the corresponding signal machinery (RhoA/ROCK, p-MYPT1, CPI-17, p-CPI-17 and p-MLC₂₀) have been observed in the IAS compared to predominantly phasic RSM.¹⁵ Also those studies¹⁵ demonstrated the order in reverse for the levels of MYPT1 in the basal state, lower in the IAS. Additionally, the mechanism responsible for smooth muscle tone has been shown to be intrinsic to the characteristically different SMC.^16,54 Consistent with the animal data for the IAS, it has been shown recently that the RhoA/ROCK pathway is critical in the maintenance of spontaneous LES tone in humans.^13,55 Besides its role in the sustained contraction, a number of studies have shown that the active relaxation of the smooth muscle is mediated via inhibition of RhoA/ROCK signal transduction pathways.^1,42,56,57

RhoA/ROCK Signal Transduction Machinery is Upregulated in the Spontaneously Active Basal Tone

Based on agonist responses, the smooth muscle responses have been classified arbitrarily as either tonic (displaying a protracted contraction) or phasic (contraction lasting less than one minute).^{1,3,55,58–61} Sphincters of the GI tract represent truly tonic smooth muscles because they spontaneously develop tone which is largely myogenic.^11–13,15 These tonic tissues have characteristically higher levels of CPI-17 and lower levels of MYPT1. CPI-17 is an endogenous inhibitory protein for MLCP.^62,63 Collectively, the net effect is subdued MLCP leading to higher levels of p-MLC₂₀ and maintained tone.^14–16 While upregulation of RhoA/ROCK is critical for the basal IAS tone, we speculate its further upregulation in the pathophysiologically hypertonic state,¹⁷ and (Rattan et al. unpublished data). From basic research studies, we speculate renin-angiotensin system (RAS)^20,64 and cyclooxygenase especially COX-1 products (thromboxanes and prostaglandin F_2α)^23,65 via the activation of specific membrane receptors activate RhoA/ROCK for a part of the basal tone in the IAS. Conceivably, therefore, RAS and COX-1 via RhoA/ROCK^14,66 may serve as important therapeutic targets for different motility disorders in humans.^17,21,66,67

RhoA/ROCK as Novel Therapeutic Targets for Gastrointestinal Dysmotility

RhoA/ROCK and Inhibitors

ROCK inhibitors, such as Y-27632 and fasudil (HA-1077) have been evaluated across a number of organ systems,^4–8,68 while other compounds are being actively pursued.⁶⁹ Y-27632’s mechanism of inhibition of ROCK appears to be by competing with ATP for its binding to the kinase.⁷⁰ Additionally, phosphorylation of CPI-17 is diminished by pretreatment with either Y-27632 or GF109203x (PKC inhibitor), suggesting a crosstalk between multiple kinases in different smooth muscle systems.^51,71 Recent studies systematically compared the selectivity vs. potency of available ROCK inhibitors (Y-27632, HA-1077, H-1152, and ROCK inhibitor II), in the IAS vs. RSM.⁷² These studies using the same smooth muscle strips, determined the relationship between the basal smooth muscle tone, and the enzymatic activities of ROCK, PKC and MLCK. A comparison of IC₅₀ values reveals a general agreement of the data with the existing literature in different systems (Table 2).

Table 2.

Alphabetical Listing of Potency Comparison of Different ROCK Inhibitors

ROCK Inhibitor	Selectivity	Potency (from different systems)	Potency in IAS vs. Rectal Smooth musclec
a Fasudil (HA-1077)	ROCK I & ROCK II	IC50b = 10.7 μmols/L	IAS IC50 = 1.82 μmols/L RSM IC50 = 4.57 μmols/L
GSK-269962A	ROCK I	ROCK I IC50 = 0.0016 μmols/L ROCK II IC50 = 0.006 μmols/L
Hydroxy Fasudil (HA-1100)	ROCK I & ROCK II	IC50 = 1.8 μmols/L
Rho Kinase Inhibitor (H-1152 or H-1152P)	ROCK I & ROCK II	IC50 = 0.0118 μmols/L	IAS IC50 = 0.0079 μmols/L RSM IC50 = 2.51 μmols/L
Rho Kinase Inhibitor II	ROCK I & ROCK II	IC50 = 0.2 μmols/L	IAS IC50 = 2.95 μmols/L RSM IC50 = 15.4 μmols/L
Rho Kinase Inhibitor III Rockout	ROCK I & ROCK II	IC50 = 25.0 μmols/L
Rho Kinase Inhibitor IV	ROCK II	IC50 = 0.0118 μmols/L
SB-772077-B	ROCK I & ROCK II	ROCK I IC50 = 0.0056 μmols/L
SR-3677	ROCK II	ROCK I IC50 = 0.056 μmols/L ROCK II IC50 = 0.0072 μmols/L
SLx-2119	ROCK II	ROCK I IC50 = 24.0 μmols/L ROCK II IC50 = 0.105 μmols/L
Y-27632	ROCK I & ROCK II	IC50 = 5.0 μmols/L	IAS IC50 = 0.436 μmols/L RSM IC50 = 13.4 μmols/L

^aThere are a number of ongoing clinical trials on ROCK inhibitors especially Fasudil for different medical conditions e.g. hypertension, asthma, and cerebral vasospasm.⁶⁸ However, to our knowledge, there are no such trials for GI dysmotility.

^bIC₅₀, the concentration of the inhibitor that causes 50% decrease in the ROCK activity.

^cThe IC₅₀s are compared in the internal anal sphincter (IAS) vs. the rectal smooth muscle (RSM). The IAS is spontaneously tonic smooth muscle while the RSM is primarily phasic with a small tonic component.

Specific Details of RhoA/ROCK in Different Regions of the GI Tract

Esophagus and Lower Esophageal Sphincter (LES)

ROCK inhibitors inhibit both the basal tone in the LES and the increased tone caused by Angiotensin II in humans, cats, opossum and rat.^55,73–75 In human studies, ROCK inhibitors Y-27632 and HA-1077 cause complete relaxation of the LES while the PKC inhibitors calphostin C and chelerythrine have minimal effect.¹³ Additionally, ROCK inhibitors decrease nerve-evoked “on” and “off”-esophageal contractions, responsible for the peristalsis. The exact significance of RhoA/ROCK in esophageal peristalsis is not known because studies have been limited to a single esophageal site. While activated ROCK inhibits, inactivated ROCK (by the ROCK inhibitors) may unleash MLCP, thereby facilitating relaxation. Therefore, premature unleashing of MLCP following ROCK inhibitors may not allow the development of full esophageal contraction. Targeted relaxation of the LES and EB contraction by ROCK inhibitors may provide a rational therapy in achalasia and DES.

Stomach

There is growing evidence that the RhoA/ROCK pathway has strong implications in the basal and agonist-stimulated gastric smooth muscle activity. In reconstituted smooth muscle fibers from the cultured guinea pig gastric SMCs, norepinephrine-induced contraction was found to be completely abolished by an intracellular Ca²⁺ chelator, papaverine, an MLCK inhibitor, and the ROCK inhibitor Y-27632.⁷⁶ The role of RhoA/ROCK vs. PKC pathways was examined in rabbit gastric smooth muscle tissue and the SMC. Phenylephrine and LPA-induced contraction may involve both pathways.^54,77,78 However, it was not possible to determine their relative contribution because p^Thr695-MYPT1 masked p^Thr695-MYPT1, primarily responsible for RhoA/ROCK-mediated SMC contraction. On the contrary, in mice and rats gastric smooth muscle contraction in the basal state or in response to carbachol, KCl, LPA, and field stimulation is predominantly mediated via ROCK activation.^79–81 One novel study⁸² showed that under physiological conditions, the ROCK inhibitor Y-27632 causes a precipitous decrease in the basal gastric contractility and motility index in conscious rats.

Gallbladder

Although there are significant data on the role of different regulators in gallbladder motility, the molecular mechanisms that govern gallbladder motility are not known.⁸³ Extensive studies via molecular biology and pharmacological analyses in human gallbladder reveal that RhoA/ROCK may provide the sole molecular pathway for the spontaneous and stimulated tone.⁸⁴ Similar data were obtained in the guinea pig and sheep gallbladder.^85–87

Small Intestine

Luminal contents of the small intestine move in an aboral direction in a highly regulated process that can result in clinical symptoms when disordered.⁸⁸ Only a few medications (but with considerable limitations) are available for small intestinal motility disorders: octreotide, erythromycin, and newer agents with activity targeted to the 5-HT₃ and 5-HT₄ receptors can result in decreased or increased intestinal motility.⁸⁹

In the guinea-pig, a sustained component of intestinal smooth muscle contraction in response to carbachol, LPA, and ACh (but not K⁺ depolarization) has been shown to be ROCK-dependent.^90–93 Otto et al.⁹³ identified another staurosporine-sensitive kinase (distinct from ROCK and PKC) which induces a large increase in the Ca²⁺ sensitivity via thiophosphorylation of MYPT1. Borman et al. in the rabbit ileum identified this kinase to be zipper-interacting protein kinase (ZIPK).⁹⁴ Follow up studies by Ihara et al.⁹⁵ in rat ileal longitudinal smooth muscle identified yet another pathway: integrin-linked kinase (ILK) for microcystin (phosphatase inhibitor)-induced contraction of smooth muscle. ILK has been shown previously to be involved in phasic esophageal smooth muscle contraction.⁷³ Presently, the relative contribution of these kinases in relation to RhoA/ROCK in the physiology and pathophysiology of the small intestine is not known. Hersch et al.⁹⁶ determined the relative contribution of different pathways in SMC from the longitudinal vs. circular muscle layers of rabbit small intestine in response to endothelin. The contractile effect consisted of initial and sustained components. While the initial component was shown to be clearly MLCK-mediated, the latter sustained component was RhoA/ROCK rather than PKC-mediated. Different studies using rat ileal smooth muscle^97,98 reached a similar conclusion.

Colon

In circular SMC from rabbit colon, Patil et al. showed inhibition both of ACh-induced sustained contraction and of p-MLC₂₀ by the ROCK and PKC inhibitor Y-27632.^99,100 The authors suggested independent roles of RhoA and PKC pathways, subsequently converging on MLCP inhibition.¹⁰⁰ However, recent observations that PDBu-activated contraction was attenuated by the ROCK inhibitor suggests that PKC lies upstream of ROCK (Rattan et al. unpublished data). Signal transduction for the basal and agonist-induced contraction and relaxation may be region specific. For example, in the longitudinal smooth muscle of rat proximal colon, the RhoA/ROCK pathway alone mediates Ca²⁺-sensitization coupled to the membrane muscarinic receptors, while in the distal colon, both PKC and RhoA/ROCK mediate similar responses.¹⁰¹ Later studies by the same group¹⁰², however, showed that the differential role of ROCK vs. PKC in the rat distal colon was applicable in the longitudinal but not in the circular smooth muscle.

Internal Anal Sphincter (IAS) and Rectum

The ROCK inhibitors (Y-27632 and H-1152) were found to be more than 30-fold potent in decreasing the IAS tone than RSM phasic activity.⁷² These effects correlated most closely with a decrease in ROCK activity without modifying PKC and MLCK activities. ROCK inhibitor II and HA-1077 were both found to be least potent and least selective. Therefore, selection of the ROCK inhibitor is critical in the specific therapeutic modality. Further in-depth studies in isolated SMC¹⁶ from the IAS showed characteristically upregulated RhoA/ROCK signal transduction machinery as compared with the RSM. Additionally, direct injection of active ROCK caused further shortening of the SMC, which was selectively blocked by Y-27632. Conversely, active PKC-induced shortening of the IAS SMC was blocked not only by the PKC but also by the ROCK inhibitor. Collectively, the data suggest a role of RhoA/ROCK as molecular determinant in the basal tone. On the contrary, studies by Bitar et al. suggest a major contribution of PKC in the basal IAS tone.^103,104 These authors reached a similar conclusion using reconstructs from human IAS SMC.¹⁰⁵ In these studies, the effect of de-differentiation of the tonic SMC during the long-term culture is not known. Interestingly, recent data in tonic smooth muscles suggest that PKC may work partly via ROCK activation.^13,16,77,106

RhoA/ROCK and Protein Prenyltransferase Inhibitors

RhoA undergoes post-translational prenylation to allow translocation to the membrane and functional activation.^107,108 Prenyltransferase inhibitors have been shown to selectively disrupt downstream signaling pathways of Rho GTPases.¹⁰⁹ Recent studies¹¹⁰ reported a decrease in the levels of basal IAS tone and membranous prenylated-RhoA in the IAS before and after the administration of prenylation inhibitor GGTI-297, which was reversible by geranylgeranyl pyrophosphate. These data suggest that RhoA prenylation is critical for RhoA translocation and for the RhoA/ROCK-mediated Ca²⁺ sensitization for smooth muscle tone.^37,59 These observations provide another avenue to explore the role of agents such as GGTI-297 and statins in the specific cases of GI hypermotility.^111,112

Pathophysiological and Therapeutic Implications of RhoA/ROCK in the Gastrointestinal Disease

Up- or downregulation of RhoA/ROCK may contribute to different gastrointestinal motility disorders, which may, in turn, potentially be treated by ROCK inhibitors or activators, respectively. There are possible avenues for the topical application of ROCK inhibitors to minimize systemic effects,^17,113 which may have utility in relieving the IAS spasm associated with RAF to allow subsequent healing. Critical clinical data are needed to determine the exact role of RhoA/ROCK in the pathophysiology and therapeutic management of certain debilitating GI motility dysfunctions characterized by smooth muscle hypersensitivity: achalasia, DES, abnormal gastric motility, pylorospasm, irritable bowel syndrome, biliary dyskinesia, Hirschsprung’s disease, RAF, hemorrhoids, megacolon, and certain forms of severe constipation; or smooth muscle hyposensitivity: GERD, gastroparesis, adynamic ileus, colonic inertia, and certain forms of rectoanal incontinence characterized by hypotensive IAS.

One of the most popular therapeutic approaches in treating GI smooth muscle hypermotility is botulinum toxin A (BTX-A).^17,114,115 This works primarily via the inhibition of release of ACh either at the end plate of the skeletal muscle or in cholinergic parasympathetic or sympathetic neurons of the smooth muscle.^116,117 A derivative of BTX, C3 exoenzyme is known to inhibit RhoA via ADP ribosylation.^118–121 Therefore, BTX may not only decrease the hyperactivity of skeletal muscle but also of GI smooth muscle.^16,34,101 In order to avoid the nonselective side effects following RhoA/ROCK inhibition, there is a need for region-specific and topically active inhibitors. Alternatively, tissue-specific silencing of the RhoA/ROCK gene using small interfering RNA is another potential approach.⁴¹ Hypomotility disorders may be difficult as there are no specific activators of RhoA/ROCK. Therefore, one possible consideration is the molecular introduction of constitutively active RhoA/ROCK.

Abbreviations used in this Paper

DES

diffuse esophageal spasm

IC₅₀

the concentration of the inhibitor that cause 50% decrease in ROCK activity

IAS

internal anal sphincter

LPA

lysophosphatidic acid

MLC

myosin light chain

MLCP

MLC-phosphatase

MYPT1

myosin binding (regulatory or inhibitory) subunit of MLCP

PPIc

catalytic subunit of MLCP

PKC

protein kinase C

CPI-17

protein kinase C potentiated inhibitory

RAF

recurrent anal fissures

ROCK

Rho kinase

ROCKII or ROCKα

(primary isoform of Rho kinase involved in the smooth muscle contraction)

RSM

rectal smooth muscle

SMC

smooth muscle cells

Y-27632

(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, (Rho kinase inhibitor)

Rho.GEF

GDT-GTP exchange factor

GAPs

GTPase-activating proteins

GDIs

guanine nucleotide dissociation inhibitors