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 can 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 vs 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.

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.

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.

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.

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 [–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 [–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.