Role of rho kinase in the functional and dysfunctional tonic smooth muscles

Abstract

Tonic smooth muscles play pivotal roles in the pathophysiology of debilitating diseases of the gastrointestinal and cardiovascular systems. Tonic smooth muscles differ from phasic smooth muscles in the ability to spontaneously develop myogenic tone. This ability has been primarily attributed to the local production of specific neurohumoral substances that may work in conjunction with calcium sensitization via signal transduction events associated with the Ras homolog gene family, member A (RhoA)/Rho-associated, coiled-coil containing protein kinase 2 (ROCK II) pathways. In this article, we discuss the molecular pathways involved in the myogenic properties of tonic smooth muscles, particularly the contribution of protein kinase C versus the RhoA/ROCK II pathway in the genesis of basal tone, pathophysiology, and novel therapeutic approaches for certain gastrointestinal and cardiovascular diseases. Emerging evidence suggests that manipulation of RhoA/ROCK II activity through inhibitors or silencing of RNA interface techniques could represent a new therapeutic approach for various gastrointestinal and cardiovascular diseases.

Myogenic properties of tonic smooth muscles

Smooth muscles can be broadly classified according to the contractile patterns: phasic and tonic. Phasic smooth muscles contract transiently in response to neural stimulation and neurohumoral substances such as angiotensins and prostanoids. By contrast, tonic smooth muscles develop and sustain myogenic basal tone in the absence of an external stimulation. The term ‘myogenic’ implies that the stimulus for tone development and maintenance originates in the muscle itself, and its response is carried out via specialized properties of the smooth muscle cells (SMC) [1–4]. Classic examples of tonic smooth muscles in gastrointestinal (GI) tract include the internal anal sphincter (IAS) and the lower esophageal sphincter (LES). Such smooth muscles maintain tone and relax in response to nonadrenergic noncholinergic inhibitory neurotransmission, allowing the passage of food or waste products [5–7]. In the cardiovascular system, small arteries are the most well-established examples of tonic smooth muscles, and they play crucial roles in a number of physiologically important functions such as the establishment of basal vascular tone and autoregulation of blood flow [8,9]. The purpose of this article is to synthesize information on the cellular mechanisms underlying the myogenic properties in tonic smooth muscles and their role in the pathophysiology of debilitating diseases such as rectoanal incontinence, esophageal reflux, and achalasia, Hirschsprung’s disease (HPD), recurrent anal fissures, and hemorrhoids (examples of hypo- and hypertensive sphincteric smooth muscles, respectively), and cardiovascular hypertension. We will also discuss potential novel therapeutic approaches towards those diseases based on the molecular mechanisms underlying myogenic tone.

Role of the Ca²⁺/calmodulin pathway in the smooth muscle motor response

The motor response in SMCs is controlled by the sliding of myosin and actin filaments over each other. This process requires chemical energy, which is provided by the hydrolysis of ATP (Figure 1). Myosin utilizes ATP for molecular conformational changes in its own structure to facilitate its attachment and interaction with actin filaments, leading to the formation of crossbridges. Myosin attachment happens through protruding globular heads in the myosin filaments that interact with actin filaments. These heads tilt and drag along the actin filament to produce movement. The crossbridges then release the actin filament and adopt their original conformation, resulting in relaxation. This process is known as crossbridge cycling and is considered to be common to all smooth muscles [10].

Figure 1.

Cross-bridge cycling in smooth muscle contraction. A. An initial rise in [Ca²⁺]_i accompanied with increase in p-MLC₂₀ via calmodulin-dependent MLCK activation causes activation of myosin ATPase and the formation of the ATP-myosin complex. B. ATP binding to protruding head in myosin structure causes a conformation change. C. As the ATP is hydrolyzed to ADP + P, the protruding head assumes an “erectile” position. D. When the P leaves myosin, the protruding head interacts with actin. E. These heads tilt and drag along the actin filament to produce movement. As ADP leaves the protruding head, the head tilts back towards the original conformation and drags along the actin filament to produce movement or smooth muscle contraction. Whether this phosphorylated cross-bridges cycling and contraction is very brief or sustained is determined by the balance between the forces that initiates the contraction (Ca²⁺/calmodulin/MLCK/p-MLC₂₀) and dephosphorylation of p-MLC₂₀ by MLCP. For the sustained contraction, it is important that dephosphorylation is inhibited by MLCP inhibition (via p-MYPT1) which is primarily mediated via RhoA/ROCK II or PKC activation. This has been explained further in the following figures.

Myosin activation is prerequisite for the crossbridge cycling. The protruding heads in the myosin filaments contain heavy chains and light chains. Phosphorylation of the 20 kDa myosin light chains (MLC₂₀) is primarily responsible for the activation of myosin to initiate the smooth muscle contraction. The enzyme responsible for the initiation of MLC₂₀ phosphorylation is called myosin light-chain kinase (MLCK), the activity of which depends largely on free intracellular Ca²⁺ ([Ca²⁺]_i). Once SMCs are stimulated to contract via activation of G-protein-coupled transmembrane receptors (GPCRs) by specific agonists or otherwise as described below, there is an increase in [Ca²⁺]_i via Ca²⁺ influx, release from the membrane or the intracellular storage organelles such as endoplasmic reticula and mitochondria. Following the increase in [Ca²⁺]_i, Ca²⁺ binds to calmodulin (CaM) to form a Ca²⁺-CaM complex. This complex phosphorylates MLCK to induce the phosphorylation of myosin light chain (p-MLC₂₀) followed by the onset of the smooth muscle contraction [10–12]. p-MLC₂₀ is immediately dephosphorylated by myosin light chain phosphatase (MLCP), thus terminating the contraction, and the smooth muscle comes back to its original position, as is the case in the phasic contraction (Figure 2).

Figure 2.

Maintenance of initiated cross bridge-cycling (by Ca²⁺/MLCK/phosphorylated-MLC₂₀ or p-MLC₂₀) in the sustained smooth muscle contraction or the basal myogenic tone depends on the mechanisms that inhibit MLCP. (Reaction that represents lack or low level of p-MLC₂₀ points towards the left, and the one for the higher levels of p-MLC₂₀ points towards right). MLCK drives the reaction towards right, and MLCP will change the direction to the left. Likewise, MLCP inhibition either by ROCK II or PKC will drive the reaction to the right. In this regard, as discussed in the text and in the Figure 4 legend below, RhoA/ROCK pathway is predominant as compared with the PKC in the basal myogenic tone. In addition, as indicated, there appears to be a cross-talk between these pathways. In the phasic smooth muscles such as that of the esophageal body and the ASM in the basal state, MLCP may be unleashed (because of the subdued inhibitory RhoA/ROCK or PKC pathways), thus keeping these smooth muscles totally relaxed in the absence of any agonist or stimulus. In such phasic smooth muscles however, in response to an appropriate agonist or stimulus, the tissues respond to a phasic or transient contraction following an increase in p-MLC₂₀ which is dephosphorylated immediately (in a matter of a few sec., depending upon the stimulus), by the leftward reaction by MLCP, returning it to its original relaxed state.

Although p-MLC₂₀ via MLCK is the main signaling event in initiating SMC contractions, MLCP phosphorylation, certain endogenous kinases as further described below, inhibit MLCP, thus prolonging the SMC contraction as is the case in the sustained contraction or in the basal tone. MLCP is composed of three subunits: i). A regulatory 110 to 130 kDa subunit anchors MLCP to phosphorylated MLC₂₀, termed as myosin-targeting subunit of MLCP (MYPT1); ii) a 37 to 38 kDa catalytic subunit (type 1 serine/threonine phosphatase, PP1c); and iii) a 20 kDa subunit (with unknown function). The targeting subunit is called the myosin phosphatase target subunit (MYPT). There are several MYPT isoforms but MYPT1 is the major subunit expressed in smooth muscles that regulates the enzymatic activity of MLCP by targeting and phosphorylating the catalytic subunit PP1c. Therefore, the kinases that modulate the PP1c and MYPT1 activities will regulate MLCP activity (and thus the smooth muscle contraction). In this regard, protein kinase C (PKC) and Rho-associated, coiled-coil containing protein kinase 2 (ROCK II) have been reported to be the primary regulators of MLCP activity [10–13]. Different mechanisms could contribute to the inhibition of MLCP activity such as: the alteration of the heterotrimeric structure of MLCP, the phosphorylation of MYPT1 at a specific site, and by the inhibitory protein CPI-17 (17 kDa PKC-potentiated inhibitory protein of PP1c). Other kinases that might also phosphorylate MYPT1 at the inhibitory site and lead to increase in p-MLC20 include Zip-like kinase, Zip kinase, myotonic dystrophy protein kinase [14].

The RhoA/ROCK pathway

RhoA belongs to a family of small GTPases that include RhoA, RhoB, and RhoC isoforms, of which RhoA is the best understood because of its important roles in the smooth muscle motility. RhoA might have high-affinity binding to guanosine trisphosphate (GTP) as well as guanosine diphosphate (GDP). However, the active form of RhoA is bound to GTP located in the cell membrane, whereas the GDP-bound form is inactive and located in the cytoplasm. Switching between the GDP/GTP-bound forms is controlled by specific regulators called guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and guanine nucleotide dissociation inhibitors (GDIs). GEFs facilitate RhoA-GTP binding, whereas GAPs facilitate RhoA-GDP binding. GDIs control RhoA diffusion between the membrane and the cytosol by forming a large complex with RhoA and allowing for specific geographic control of RhoA activation [15,16]. Therefore, RhoA activation and inactivation may be initiated by the activation of RhoA-specific GEFs and RhoA-specific GAPs, respectively [17–19]. Therefore, one of the logical mechanisms for the sustained RhoA/Rho-kinase activation will be inhibition of RhoA-specific GAP activity via phosphorylation of p190A RhoGAP (shown to be at Ser¹¹⁵⁰) in vitro and in vivo [17].

Additional studies have shown that the activation and inactivation of RhoA-specific GEFs and RhoA-specific GAPs may be dependent on tyrosine phosphatase SHP2 [18]. These molecular processes responsible for RhoA/ROCK activation may be linked to activation of GPCRs (via G_12/13 family of heterotrimeric G-proteins) by Ang II and prostanoids [18,20,21]. They might also be GPCR-independent [22] or by triggered by depolarization [23]. It has been established that RhoA/ROCK is key molecule in G-protein-mediated Ca²⁺ sensitization for smooth muscle contraction [19,24]. (It has been recognized that the sphincteric SMC may be distinctly characterized by the presence of biosynthetic machinery for and high levels of Ang II [25–27] and prostanoids [28–32] with the high levels ongoing, spontaneous, spike-like action potentials with more positive resting membrane potential [33]). Additional studies in the colon and IAS have shown that phosphorylation of heat shock protein (HSP27, a member of small HSP family) [34,35], and RhoA prenylation [36,37] may also play important roles in RhoA activation.

ROCK is a cytoplasmic serine/threonine-specific kinase that serves as an effector of RhoA. Upon RhoA activation, ROCK migrates to the cell membrane and interacts with RhoA-GTP, resulting in autophosphorylation and activation [15,19,38–40]. ROCK I and ROCK II are the two ROCK isoforms expressed in SMCs; ROCK II, nevertheless, is most implicated in contraction [12,41–43]. The mechanism of ROCK II-mediated smooth muscle contraction is primarily based on the inhibition of MLC₂₀ dephosphorylation (causing increase in p-MLC₂₀) via phosphorylation of p^Thr696, or ^Thr803-MYPT1 subunit of MLCP; p^Thr38-CPI-17 and consequent inhibition of PP1c of MLCP; and MLCK-like activity (Figure 3) [16,24,41,44–51].

Figure 3.

Molecular pathways involved in smooth muscle contraction. Activation of GPCRs (or a mechanism independent of GPCR activation) increases the intracellular concentrations of Ca²⁺ ([Ca²⁺]_i) (either via Ca²⁺ influx, or release from the membrane or the intracellular storage organelles such as endoplasmic reticulum (ER) or mitochondria) leading to formation of Ca²⁺/CaM complex and activation of MLCK. MLCK phosphorylates MLC_20, resulting in SMC contraction. Conversely, MLCP dephosphorylates MLC₂₀ resulting in SMC relaxation. GPCR activation might also induce RhoA to bind to GTP, a reaction catalyzed by RhoGEF and reversed by RhoGAP. RhoAGTP activates ROCK II, which inhibits MLCP either directly or via phosphorylating CPI-17. In addition, ROCK II, as indicated, may also cause increase in p-MLC₂₀ via MLCK-like action. In the tonic smooth muscles, constitutively active RhoA/ROCK appears to be responsible for the sustained inhibition of MLCP that maintains higher levels of p-MLC₂₀.

Role of PKC vs. RhoA/ROCK in the maintenance of basal tone

Both PKC and ROCK have been implicated in the inhibition of MLCP activity via phosphorylation of CPI-17 at threonine-38 (Thr³⁸) residue (p^Thr38-CPI-17 or simply p-CPI-17). CPI-17 is an endogenous inhibitory protein of the catalytic subunit of MLCP [15,49,52–54]. p-CPI-17 is ~7,000-fold more potent than nonphosphorylated CPI-17. Phosphorylation of CPI-17 by ROCK has also been suggested by in vivo studies [15,55,56]. In these studies, increase in p-CPI-17 was specifically inhibited by the ROCK inhibitors. In some of these studies, however, the possibility of cross-talk between PKC and RhoA/ROCK pathways may not be completely ruled out.

Earlier studies testing the effects of selective inhibition of PKC in the LES and IAS suggested PKC to be the primary molecular mechanism in the basal tone of the gastrointestinal smooth muscles [2,57,58]. Recent studies however, aimed at assessing the relative contribution of ROCK vs. PKC show that ROCK-mediated inhibition of MLCP is primarily responsible for the basal tone in the LES of humans and animals, and for the IAS of various animals [2,11,13,59,60]. The role of these pathways however, in the intact human IAS remains to be determined.

To examine the molecular bases for the myogenic tone in the IAS, recent multipronged studies using force measurements and molecular biology were focused on the role of RhoA/ROCK in the rat IAS vs. the adjoining smooth muscles of rectum (RSM; that has a mixture of tonic and phasic activities) and the anococcygeus (ASM; a purely phasic smooth muscle) [11,61–63]. The studies also compared cellular distribution of RhoA/ROCK, levels of RhoA-GTP (the active form of RhoA), RhoA-Rho guanine nucleotide dissociation inhibitor (GDI) complex formation, levels of phosphorylated MYPT1 (at threonine 696; p^Thr696-MYPT1), under different experimental conditions, including inhibition of RhoA/ROCK. Levels of RhoA/ROCK were found to be higher at the cell membrane in the IAS SMCs compared with those from the RSM and ASM. C3 exoenzyme (RhoA inhibitor) and Y27632 (ROCK inhibitor) caused concentration-dependent relaxations in the IAS SMCs. In addition, active ROCK-II (primary isoform of ROCK in the SMC contraction) caused further shortening in the IAS SMCs. C3 exoenzyme increased RhoA-RhoGDI binding and reduced the levels of RhoA-GTP and p^Thr696-MYPT1. Y27632 attenuated PKC-induced contractions in IAS SMC. Conversely, a PKC inhibitor (Gö 6850, which only causes a partial relaxation of the SMC) had no significant effect on ROCK-II-induced contractions. Further experiments showed the highest levels of RhoA, RhoA-GTP, ROCK-II, MLC₂₀, p-MYPT1, and p-MLC₂₀ in the IAS vs. RSM and ASM SMCs. The trend was the reverse with the levels of inactive RhoA (GDP-RhoA-RhoGDI complex) and MYPT1 [62].

Attenuation of PKC-induced contraction of the IAS SMC by not only the PKC inhibitor but also the selective blockade of active ROCK II by a ROCK inhibitor suggests that PKC pathway is partially mediated via ROCK activation [62]. In agreement with this, it has been shown that vascular smooth muscle contractions caused by PKC are mediated primarily via ROCK activation [15,64]. These conclusions were based on the use of ROCK inhibitors, and also via the use of dominant-negative ROCK and pseudosubstrates peptides as PKC inhibitors [65], and via ezrin, radixin and moesin-binding (ERM) phosphorprotein (EBP50) depletion by siRNA [66]. (It is well known that ERM proteins serve as ROCK substrates). Furthermore, it has been shown recently that ROCK mediates PKC-dependent apoptosis in prostate cancer cells [67]. In addition, recent studies in humans have shown that ROCK inhibitors cause a concentration-dependent decrease in the basal tone of the LES, causing near obliteration of the tone in the maximal effective concentrations. These studies also show that PKC inhibition only had a limited effect [59]. Together, these data suggest that ROCK and PKC pathways may lie in series, PKC being upstream of RhoA/ROCK. In this process (although not fully proven), in the basal tone of the IAS, RhoA/ROCK regulate the higher levels of p-MLC₂₀ and the basal tone via p-MYPT1 and p-CPI-17 while effect of PKC may be limited to p-CPI-17 [15,39,40,58,59,62]. However, direct effect of RhoA/ROCK in the p-MLC₂₀ via MLCK-like effect in intact smooth muscles remains to be determined.

Therapeutic potential of RhoA/ROCK inhibitors in GI diseases

Prototypes of tonic tissues, the LES and IAS play major roles in the pathophysiology of a number of the gastrointestinal motility disorders. The LES maintains tone in the basal state and relaxes during swallowing to allow passage of food. Similarly, the IAS maintains spontaneous tone and relaxes to allow passage of processed food in response to the rectoanal inhibitory reflex that is initiated by rectal distension caused by the stool. Significant changes in the basal tone of the IAS and LES have been directly associated with the pathophysiology of debilitating diseases such as rectoanal incontinence, certain forms of constipation, recurrent anal fissures, hemorrhoids, Hirschsprung’s disease (HPD), achalasia, and gastroesophageal reflux disease[4,6,7,68–76]. Therefore, agents able to restitute the hypo- or hypertonic states of the IAS or LES, particularly those acting on the RhoA/ROCK pathway, have novel and significant therapeutic potentials (Figure 4) [59,60,62].

Figure 4.

A model to explain the role of the constitutively active RhoA/ROCK II pathway in the basal state of the LES and IAS tone (normal), and in the pathophysiology of the hypertensive and hypotensive states associated with the corresponding motility disorders. A. In the basal state, RhoA/ROCK II displaces the equilibrium between MLCK and MLCP activities towards higher levels of p-MLC₂₀ and maintained cross-bridge interactions with actin. B. In the hypertensive state, upregulation of the RhoA/ROCK II pathway via further displacement of the MLCK/MLCP equilibrium may lead to the still higher levels of p-MLC₂₀ [106–108]. RhoA and ROCK II are targets for potential therapeutic interventions via traditional molecules such as C₃ exozyme and Y27632 or via novel therapies such as selective siRNAs. C. In the hypotensive state, downregulation of the RhoA/ROCK II might displace the MLCK/MLCP equilibrium towards lower levels of p-MLC₂₀ resulting in the hypotensive tonic smooth muscles [109–112]. In that case, the tone may be improved by the agonists (e.g. angiotensins and prostanoids) via RhoA/ROCK activation. Although, PKC also has the potential of inhibiting MLCP as explained above, its role in these tonic smooth muscles (as described above based on the humans and animal data in the LES and IAS), may be limited. In addition, as indicated, there appears to be a cross-talk between the RhoA/ROCK and PKC pathways. Exact relative contribution of these pathways and the nature of interaction between them remain to be determined.

Higher levels of RhoA/ROCK II (along with the higher levels of p-MLC₂₀₎ have been reported at the SMC level in the IAS compared with the adjoining RSM, and the ASM [61,62]. In addition, introduction of active ROCK-II into the IAS SMCs causes a concentration-dependent increase in the spontaneous contractions of the IAS SMCs, which is attenuated by the ROCK inhibitor Y27632 [62]. These results are in line with a transgenic mice study showing that inhibition of RhoA/ROCK II lowers the local levels of p-MLC₂₀, increases the basal length of IAS SMCs and inhibits tone development in the isolated strips of the IAS [77]. The studies also show that inhibition of the RhoA/ROCK II pathway correlates with upregulation of H-ras, which has been implicated in the pathogenesis of certain colorectal cancers, and rectoanal incontinence associated with the lower intraluminal pressures in the IAS [78,79]. However, the role for RhoA/ROCK II upregulation in the pathophysiology of the spontaneously hypertensive IAS via modulation of the signal transduction pathways for the locally-produced angiotensin II (Ang II) and cyclooxygenase (COX) products (thromboxanes and prostaglandins) [25,26,28,29], remains to be determined. In different systems, these agents are known to activate GPCRs and stimulate the RhoA/ROCK signaling pathway (Figure 4) [2,18,20,80].

In the LES, ROCK inhibitors have been shown to attenuate the basal tone and its increase caused by Ang II and prostanoids in various animal species [2,57,81]. Additional studies in the human LES [59] have shown that ROCK inhibitors (Y27632 and HA-1077) produce full relaxation, whereas PKC inhibitors (calphostin C and chelerythrine) only have limited effects. These studies concluded that a mechanism of Ca²⁺ sensitization mediated by the RhoA/ROCK pathway plays an important role in the basal tone in the human LES. Interestingly, mice lacking lsc/p115-GEF developed hypertension of the LES and showed impaired relaxation to inhibitory stimuli [82]. Native Lsc/p115-GEF has been related to potential reduction in RhoA activity because of its high affinity for RhoA devoid of bound nucleotide. The hypothesis is that GTP would be less able to displace the exchange factor and potentially slow the overall rate of GDP to GTP cycling, thus reducing RhoA activity [83].

Therapeutic potential of RhoA/ROCK inhibitors in cardiovascular diseases

As in gastrointestinal smooth muscles, the contractile state of vascular SMC (VSMC) depends on the levels of p-MLC₂₀ determined by the balance between the activities of Ca²⁺/CaM/MLCK-dependent (plus Ca²⁺-independent) and the MLCP pathways [19,38,43]. A number of neurohumoral agonists (e.g. Ang II and prostanoids) are known to modulate these pathways via GPCRs that lead not only to increases in the [Ca²⁺]_i but also stimulate RhoA/ROCK. Thus, as outlined above, the inhibition of MLCP via p-MYPT1 and/or p-CPI-17 by ROCK leads to increased p-MLC₂₀ and enhanced VSMC contractility [38–40,50,84]. In addition, modulation of VSMC contractility via ROCK is of particular importance during tonic contractions in different vascular beds, including the pulmonary artery, mesenteric artery and portal vein. Another important role for ROCK has been shown to be the maintenance of myogenic tone in small arteries [38–40,85,86]. Therefore, the RhoA/ROCK pathway and its interventions have important roles in the pathophysiology and therapy of cardiovascular hypertension resulting from increased peripheral vascular resistance attributed to increased contractility of VSMC. Studies show that the ROCK inhibitors such as Y27632 lower blood pressure in spontaneously hypertensive (SHR) and deoxycorticosterone-acetate (DOCA)/salt-treated and renal hypertensive rats [39]. Interestingly, in the therapeutic doses, Y27632 does not cause a significant decrease in blood pressure in normotensive animals [39,87].

Studies in isolated vascular segments confirm an association of RhoA/ROCK in the pathophysiology of cardiovascular hypertension. In mesenteric and cerebral arteries from SHR, the relaxation induced by treatment with Y27632 is markedly higher as compared with the control Wistar-Kyoto (WKY) rats [88,89]. By comparison, the selective PKC inhibitors calphostin C and Ro 31-8220 had little or no effect on arterial diameter. Vasodilator responses to Y27632 were found to be independent of PKC. In two models of chronic hypertension in SHR and rats treated with N-nitro-L-arginine methyl ester (nitric oxide synthase or NOS inhibitor), Y27632 elicited cerebral vasodilation significantly greater than in normotensive WKY rats. This indicates that the chronically hypertensive state (and not genetic factors) contributes to the increased responses to ROCK inhibition. In the same experimental models, PKC inhibition had no significant effect on arterial diameter in chronically hypertensive rats. These data suggest that ROCK, but not PKC, contributes to chronic hypertension [89]. Similar results have also been shown for mesenteric arteries from DOCA/salt-treated rats [90]. Direct evidence for increased activity of RhoA in hypertensive states has also been found in stroke-prone SHR, DOCA/salt- and renal hypertensive rats [91–93].

Therapeutic potential of RhoA/ROCK silencing with small interfering RNA (siRNA)

The main advantage of an siRNA approach over conventional inhibitors is that the conventional inhibitors such as Y27632 do not exhibit specificity for the ROCK isoforms. In addition, siRNAs are potentially ~1,000-fold more potent than the conventional ROCK inhibitors [13,41,94–100].

Because the pathophysiology of many diseases is based on the over expression of certain genes, and intense research on gene function has allowed the identification of many putative target genes, approaches based on siRNA are emerging as particularly attractive therapeutic strategies. siRNA is highly versatile since saran molecules can be easily designed to specifically target virtually any gene because of their dependence on complementary base-pair interactions. The siRNA approach that silences a specific gene at the post-transcriptional and post-translation levels comprises the introduction or local generation of double-stranded RNA (dsRNA) molecules specifically designed to match the mRNA sequences coding a protein of interest [94]. Once in the cell, the dsRNA interacts with an RNase III family enzyme named dicer. The dicer fragments RNA into 21–23 bp segments called small-interfering RNA (or siRNA). The siRNA then attaches to a multi-protein complex of endonucleases (RNA-induced silencing complex, RISC), unwinds, pairs with, and fragments the target mRNA and, consequently, inhibits the synthesis of specifically targeted protein.

Regarding siRNA delivery in vivo, there are several studies showing that it can be successfully done. The direct siRNA administration to the eye has been used to target VEGF after laser-induced choroidal neovascularization, and to induce alterations of synaptic function through down-regulation of amyloid precursor protein (APP)/amyloid precursor-like protein 2 after intra-ocular injection of siRNA for APP in rodent models [101,102]. There are also studies describing the downregulation of target genes relevant in depression (serotonin transporter), or hyperanalgesia (pain-related cation channel P2X3) after intrathecal/intraventricular siRNA administration in rodent models [103,104]. Noteworthy, one of the studies showed a significant reduction of P2X3 protein expressed in dorsal root ganglia or translocated into the dorsal horn of the spinal cord, and a blockade of pathophysiological pain response and relief from neuropathic pain [104]. Another study using intranasal application of siRNAs targeting RSV or PIV with or without transfection reagents showed that the siRNA therapy protected mice from respiratory syncytial virus (RSV) and parainfluenza virus (PIV) infections [105].

There are several studies describing the effects of RhoA/ROCK siRNA in vitro. In a study assessing the role of the ROCK isoforms (ROCK I and ROCK II) in aortic VSMC contractility, siRNA specific for the ROCK isoforms was introduced into cultured VSMCs [41]. VSMC contraction induced by lysophosphatidic acid (LPA) was used to measure cellular force production. When LPA was applied, control cells (transfected with scrambled siRNA) underwent a gradual contraction that was abolished by pre-treatment with the ROCK inhibitor Y27632. These results support the notion that VSMC contractility was mediated by ROCK. ROCK II siRNA-silenced cells contracted significantly less than scrambled control and ROCK I siRNA-silenced cells. These results were accompanied by significant reductions in p-MLC₂₀ and suggest prominent role of ROCK II in the pathophysiology and a possible role for ROCK silencing via ROCK II-specific siRNA in the treatment of cardiovascular hypertension [41]. Comparable results have been reported in pulmonary arteries showing that ROCK II siRNA-induced specific and significant decrease in the VSMC motility [95]. However, additional studies using in vivo animal models are necessary to assess the therapeutic potentials of ROCK II siRNA.

The effects of RhoA/ROCK II silencing in the GI tract have also been reported. A study designed to evaluate the effects of COX and RhoA silencing with selective siRNAs in colon carcinoma cells established an association of COX type II and RhoA in decreasing the cellular motility via RhoA/ROCK pathway [96]. Besides, preliminary results from ongoing studies in our laboratory (where topical application of ROCK II siRNA to the anal region restituted the hypertensive IAS and the output of fecal pellets in rats), the effects of such approaches in the functional and dysfunctional GI smooth muscle proper, remain to be determined.

Because of the original reports of RNA interference (RNAi) in cells from a range of species [97–99], there has been increasing interest in harnessing this endogenous mechanism as a novel approach to human therapy. From a drug discovery perspective, siRNAs have distinct advantages over conventional drug therapies; these include higher selectivity, potency, increased number of potential leads, reduced time for lead development, and simpler scalable processes than conventional therapeutics) [100]. Nevertheless, several major obstacles, such as detailed in vivo data, for example, lack of data for the safety and efficacy in humans, and the development of efficacious delivery systems, need to be overcome before the introduction of RNAi therapy i humans.

Concluding remarks

Basal tone in the smooth muscles of the gastrointestinal sphincters (typified by the LES and IAS) and those of certain blood vessels of the cardiovascular system provide true representations of the sustained contraction in the absence of any exogenous stimulus or agonist. The basal tone in such smooth muscles is primarily myogenic. However, the molecular mechanisms underlying the myogenic control in relation to the external triggers (perhaps produced within the cells) and other modulatory neurohumoral stimuli are poorly understood. Such information on the molecular control mechanisms and the associated signal transduction cascade is extremely vital for our advanced understanding of the pathophysiology, and novel and specifically targeted therapy of a number of debilitating disorders. Because of the lack of this knowledge, presently, the therapeutic avenues for such abnormalities are limited to temporary symptomatic approaches that are marred with a number of side effects. In this regard, RhoA/ROCK is emerging as an important pathway that may explain the molecular mechanism underlying the pathophysiology of such disorders, and serve as specific molecular target for the safer and more potent therapy. In addition, because of the factors outlined above, exploration of RhoA/ROCK siRNA approaches with specific distinct advantages in treating such disorders, deserve special consideration.