The tone of pulmonary smooth muscle: ROK and Rho music?

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Abstract
In normal lungs, minor aspiration or mucus plugging can cause local underventilation and hypoxia, which increases tone in pulmonary arterial smooth muscle, resulting in local vasoconstriction, diversion of blood flow to better-ventilated lung regions, preservation of normal ventilation-perfusion matching, and maintenance of systemic arterial oxygen tension. In diseased lungs, hypoxia and other vasoconstrictor influences are typically diffuse. As a result, vascular smooth muscle tone is increased throughout the lung, leading to pulmonary hypertension, right ventricular failure, and increased morbidity and mortality. In normal airways, the function of smooth muscle is not so clear ([56][1], [72][2]). Indeed, it has been suggested that airway smooth muscle, like wisdom teeth and the appendix, is vestigial and provides potential for problems but no benefits ([56][1]). True or not, the importance of airway smooth muscle tone in diseases such as asthma cannot be denied. It is now widely accepted ([39][3]) that an increase in smooth muscle tone is triggered by an increase in cytosolic Ca2+ concentration ([Ca2+]c), which can result from 1) release of intracellular Ca2+ stored in sarcoplasmic reticulum (SR) through channels in the SR membrane known as inositol trisphosphate receptors (IP3R) and ryanodine receptors (RyR) and/or 2) influx of extracellular Ca2+ through sarcolemmal receptor-, store-, or voltage-operated Ca2+ channels (ROCC, SOCC, VOCC) ([Fig 1][4]). At increased concentration, Ca2+ binds to calmodulin (CaM), and Ca2+/CaM then activates myosin light chain kinase (MLCK), which phosphorylates the regulatory myosin light chain (MLC) located near the head structure of the myosin crossbridge that binds to actin ([38][5]). The result is a conformational change that allows actin to stimulate myosin ATPase activity, crossbridges to cycle, and actin to slide past myosin ([11][6], [85][7]). Because actin-myosin filaments are anchored to the cytoskeleton and plasma membrane at structures known as dense bodies and dense plaques, the myocyte contracts ([75][8]). This sequence of events predicts that smooth muscle tone should be proportional to [Ca2+]c. By the 1980s, however, it was apparent that a number of G protein-dependent agonists could increase tone independently of increases in myocyte [Ca2+]c ([22][9], [28][10], [43][11], [57][12], [61][13], [65][14]), suggesting that tone depended not only on [Ca2+]c but also on the sensitivity of the contractile apparatus to [Ca2+]c. In 1989, Somlyo et al. ([76][15]) proposed that Ca2+ sensitivity could be increased by inhibition of the phosphatase (MLCP) responsible for dephosphorylating phosphorylated MLC (P-MLC). As shown in [Fig. 1][4], inhibition of MLCP would increase [P-MLC] and therefore the force of contraction at any given rate of MLC phosphorylation. This hypothesis was quickly confirmed ([42][16], [44][17], [46][18]) and the responsible phosphatase identified ([73][19]); however, the signaling pathway linking receptor activation to MLCP inhibition remained unknown. In 1992, Ridley and Hall ([66][20]) alerted the scientific community to the importance of a small monomeric GTPase known as Rho, which caused formation of stress fibers and focal adhesions when injected into serum-starved 3T3 cells. Serum or growth factors had the same effects, and these could be inhibited by blockade of endogenous Rho function, indicating that Rho was an essential component of the transduction pathway linking growth factor stimulation to assembly of stress fibers and focal adhesions. Subsequent studies from a number of laboratories demonstrated that Rho was also expressed in smooth muscle and activated by a wide variety of contractile agonists ([77][21]). Rho was shown to increase Ca2+ sensitivity and [P-MLC] in intact smooth muscle; however, these effects did not occur in myocytes extensively permeabilized with Triton X-100, suggesting that activity required interaction of Rho with some component of plasma membrane ([25][22], [30][23]). One possibility was Rho kinase (ROK), a serine/threonine protein kinase known to be one of Rho's major targets ([37][24]). In 1996, Kimura et al. ([40][25]) showed that Rho-activated ROK did indeed phosphorylate and inhibit MLCP. In 1997, Gong et al. ([24][26]) confirmed that translocation of Rho to plasma membrane was necessary for its enhancing effects on Ca2+ sensitivity. Demonstrations that responses to contractile agonists were blocked by inhibitors of Rho ([21][27], [25][22], [50][28], [63][29]) or ROK ([20][30], [83][31], [91][32]) indicated that Rho/ROK signaling was a major effector of agonist-induced Ca2+ sensitization in smooth muscle ([77][21]). To date, 20 Rho proteins have been identified in humans and divided into five subfamilies based on structural and functional similarities: Rho-like, Rac-like, Cdc42-like, Rnd, and RhoBTB ([10][33], [15][34]). The three members of the Rho-like subfamily (RhoA, RhoB, and RhoC) contribute to contractility and stress fiber formation ([10][33]). Rho and the other monomeric GTPases act as molecular switches that are “on” when bound to GTP, permitting recognition and activation of target proteins, and “off” when bound to GDP. As shown in [Fig. 1][4], Rho is turned on by guanine nucleotide exchange factors (GEFs), which exchange bound GDP for GTP, and turned off by GTPase-activating proteins (GAPs), which promote hydrolysis of bound GTP to GDP. Currently, the number of proteins identified as GEFs and GAPs exceeds 70 and 80, respectively. In addition to these switching mechanisms, Rho activity is regulated by GTPase-dissociation inhibitors (GDIs). GDIs bind a large portion of the cell's inactive Rho-GDP and block activation by preventing nucleotide exchange and attachment of Rho's geranyl-geranylated COOH-terminal tail to plasma membrane ([10][33], [77][21]). Many agonists interacting with receptors linked to heterotrimeric G proteins are thought to activate...

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