Division of Pulmonary and Critical Care Medicine, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21224
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, 72). 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). True or not, the importance of airway smooth muscle tone in diseases such as asthma cannot be denied.
It is now widely accepted (39) 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). 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). The result is a conformational change that allows actin to stimulate myosin ATPase activity, crossbridges to cycle, and actin to slide past myosin (11, 85). 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).
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In 1992, Ridley and Hall (66) 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). 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, 30). One possibility was Rho kinase (ROK), a serine/threonine protein kinase known to be one of Rho's major targets (37). In 1996, Kimura et al. (40) showed that Rho-activated ROK did indeed phosphorylate and inhibit MLCP. In 1997, Gong et al. (24) 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, 25, 50, 63) or ROK (20, 83, 91) indicated that Rho/ROK signaling was a major effector of agonist-induced Ca2+ sensitization in smooth muscle (77).
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, 15). The three members of the Rho-like subfamily (RhoA, RhoB, and RhoC) contribute to contractility and stress fiber formation (10). 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, 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, 77). Many agonists interacting with receptors linked to heterotrimeric G proteins are thought to activate Rho via GEFs and/or tyrosine kinases (Fig. 1) (10, 77). Rho can be inhibited by C3 exotransferase (C3) or epidermal cell differentiation inhibitor, protein toxins produced by Clostridium botulinum and Escherichia coli, respectively, which ADP-ribosylate Rho to prevent nucleotide exchange by GEFs (7, 78).
ROK exists in two isoforms: ROK (also known as ROCK2) and ROK
(also known as ROCK1). ROK's catalytic site is near its NH2 terminus, whereas its Rho binding site is at the COOH-terminal portion of its central coiled-coil domain. Binding of Rho-GTP to ROK causes a conformational change in the kinase, resulting in an activating autophosphorylation (77). In addition to inhibiting MLCP directly, ROK can inhibit MLCP indirectly by phosphorylating a 17-kDa protein known as CPI-17 (C kinase-potentiated phosphatase inhibitor) (16, 17), which then phosphorylates and inhibits MLCP (41, 45). As its name implies, CPI-17 can also be phosphorylated by protein kinase C (PKC) (49, 89) as well as other kinases (29). These reactions may provide ROK-independent pathways for Ca2+ sensitization. ROK can also phosphorylate MLC directly (3, 47); however, the physiological significance of this reaction remains unclear (77). ROK can be inhibited pharmacologically by Y-27632 (34) and HA-1077 (70), also known as fasudil.
This issue of the American Journal of Physiology-Lung Cellular and Molecular Physiology contains six call for papers original articles evaluating the role of Rho/ROK signaling in regulation of tone in pulmonary smooth muscle. Two of these papers (14, 54) deal with airway smooth muscle and four (8, 18, 33, 58) with vascular smooth muscle. Airway smooth muscle plays pivotal roles in lung diseases such as asthma. The complicated links between the airway inflammation and hyperresponsiveness that characterize asthma are not yet well understood (19); however, mediators of inflammation, such as TNF-, can potentiate contractile responses of airway smooth muscle to endogenous agonists such as histamine and leukotrienes (74). Moreover, expression of Rho protein was increased in both cultured airway smooth muscle cells treated with TNF-
(32) and airways of rats made hyperresponsive by repeated exposure to antigen (12). The paper by Mercier et al. (54) reports that 5-oxo-6,8,11,14-eicosatetraenoic acid, a proinflammatory product of the 5-lipoxygenase pathway of arachidonic acid metabolism, contracts isolated guinea pig airways through release of thromboxane A2 and secondary activation of ROK. More studies like this are needed to clarify the role of Rho/ROK signaling as a link between airway inflammation and smooth muscle tone.
Currently, the mainstay of asthma therapy is inhalation of 2-selective adrenergic agonists, such as albuterol and salmeterol, which relax smooth muscle via activation of adenylate cyclase, resulting in cAMP-dependent activation of protein kinase A (PKA) and decreases in [Ca2+]c and/or Ca2+ sensitivity (36, 48). The paper by Endou et al. (14) explores interactions between cAMP/PKA and Rho/ROK signaling in the regulation of airway smooth muscle tone. One of their important conclusions is that cAMP/PKA can reverse Ca2+ sensitization mediated by PKC, but not Ca2+ sensitization mediated by Rho/ROK. This suggests that inhibition of Rho/ROK signaling might provide an important therapeutic alternative to
2-agonists, whose effects can be limited after prolonged intensive use due to receptor uncoupling, internalization, or phosphorylation, a phenomenon known as "beta desensitization" (36).
Generation of smooth muscle tone requires not only contraction of actin-myosin filaments but also physical linkage of these filaments to plasma membrane and extracellular matrix. Most investigators have assumed that these linkages are relatively static and play no role in acute contractile responses; however, recent work in airway smooth muscle indicates otherwise. Stimulation with contractile agonists increased the ratio of filamentous actin (F-actin) to globular actin, indicating actin polymerization (31, 53). Inhibitors of actin polymerization, such as latrunculin and cytochalasin, prevented agonist-induced contraction but not MLC phosphorylation (26, 53), suggesting that actin polymerization was necessary for contraction, but did not act through stimulation of actin-myosin interaction. Vinculin and talin, proteins that link F-actin to integrins in the dense plaques at the plasma membrane, localized to plasma membrane during contractile stimulation (62). Depletion or inhibition of paxillin, a protein that regulates assembly of focal adhesions, prevented agonist-induced contraction and recruitment of vinculin to plasma membrane but did not affect the increases in [Ca2+]c or [P-MLC] (62). Observations such as these indicate that the structural proteins of the cytoskeleton and its connections to plasma membrane and extracellular matrix are dynamically regulated and play essential roles in contractile responses (26).
For some reason, the cytoskeleton of pulmonary vascular smooth muscle cells has been largely ignored. The paper by Boer et al. (8) begins to correct this deficiency and suggests that cytoskeletal reorganization plays a key role in the response of pulmonary arterial smooth muscle to endotoxin. In vitro exposure of pulmonary arteries to endotoxin for 20 h increased vascular compliance and caused cytoskeletal disassembly in smooth muscle. These effects were reproduced by cytochalasin (which prevents actin polymerization) or Y-27632 (a ROK inhibitor) and inhibited by lysophosphatidic acid (a Rho activator), suggesting that downregulation of Rho/ROK signaling was required for the response. How Rho/ROK may have caused cytoskeletal disassembly is not known. Figure 1 shows two of many possibilities. Downregulation of Rho/ROK activity could decrease the activating phosphorylation of LIM kinase (LIMK) by ROK and, secondarily, the inactivating phosphorylation of cofilin by LIMK (52). Alternatively, decreased Rho-GTP activity could act independently of ROK by decreasing activity of mDia, a mammalian homolog of the Drosophila protein "diaphanous" that belongs to the formin family of proteins (59). Decreased mDia activity would then lead to inactivation of profilin. Because profilin and cofilin promote actin polymerization and depolymerization, respectively, the result would be disassembly of F-actin (64, 81). Although Boer et al. (8) did not test responses to contractile agonists in their arteries, it is not hard to imagine that they would be depressed, as were agonist-induced contractions of airway smooth muscle treated with cytochalasin (26, 53). Recent revelations about the structure and function of the smooth muscle cytoskeleton (26, 27, 75, 86) emphasize that the roles played by structural proteins in generation of smooth muscle tone as well as regulation of these roles by Rho/ROK and other signaling pathways deserve much more investigative attention than they have received.
The classic stimulus for pulmonary vasoconstriction is hypoxia. Y-27632 inhibited acute hypoxic pulmonary vasoconstriction (HPV) in isolated rat lungs and pulmonary arteries (67, 87). Acute hypoxia decreased MLCP activity and increased ROK activity, phosphorylation of MLC and MLCP, and tone in rat pulmonary arterial smooth muscle (87, 88). These responses were blocked by Y-27632. The increases in ROK activity and [P-MLC] were also blocked by C3. Together, these results indicate that Rho/ROK signaling in pulmonary arterial smooth muscle is required for the acute pulmonary vascular response to hypoxia.
In this issue of AJP-Lung, Fagan et al. (18) and Nagaoka et al. (58) evaluate the contribution of Rho/ROK signaling to chronic hypoxic pulmonary hypertension. Y-27632 delivered continuously by subcutaneous osmotic pump reduced pulmonary hypertension and vascular remodeling in mice exposed to hypoxia for 2 wk (18). Acute administration of Y-27632 or HA-1077 reduced the elevations of baseline pulmonary arterial pressure and vascular resistance in chronically hypoxic rats and in isolated lungs from these rats and blocked hypoxic enhancement of pulmonary vasoconstrictor responses to KCl in isolated lungs and pulmonary arteries (58). These results suggest that Rho/ROK signaling is required for the chronic as well as the acute pulmonary vascular response to hypoxia and may act by increasing myocyte Ca2+ sensitivity. Observations that the ROK inhibitor HA-1077 improved mortality, right ventricular systolic pressure and hypertrophy, and pulmonary vascular histopathology in rats treated with monocrotaline (2) suggest that Rho/ROK signaling may play a similar role in other forms of pulmonary hypertension.
In contrast, Sauzeau et al. (71) reported that Ca2+ sensitivity and contractile responses to KCl were not altered in pulmonary arteries from chronically hypoxic rats and that Rho expression, GTPS-induced enhancement of Ca2+ sensitivity, and contractile responses to endothelin-1, norepinephrine, and U-46619 were reduced. The authors concluded that chronic hypoxia prevented myocyte Ca2+ sensitization by downregulating Rho/ROK signaling. It is difficult to reconcile these results and conclusions with those of Nagaoka et al. (58) because right ventricular hypertrophy and contraction of isolated pulmonary arteries to KCl are the only outcomes the studies have in common, and both are different. A recent study of fetal and newborn pigs suggests that effects of hypoxia on stress fiber formation and activation of Rho and ROK can vary, depending on age and the origin of the myocytes within the vessel wall (6). These and other as yet unknown sources of variability may explain the difference. As usual, resolution will require additional studies and more measurements, in this case including but not limited to confirmation of inhibitor specificity and determination of Rho, ROK, MLCK, and MLCP activities in small distal pulmonary arteries, where most of the action is.
One of the most striking results reported by Nagaoka et al. (58) is the immediate reduction of pulmonary vascular resistance to nearly normal levels after acute intravenous administration of Y-27632 to rats exposed to hypoxia for 34 wk. Whatever Y-27632's mechanism of action, the rapidity of this effect can only be explained by a reduction in vasomotor tone. Its surprising magnitude invalidates the generally held notion that chronic hypoxic pulmonary hypertension results mainly from an anatomic reduction in vascular cross-sectional area due to remodeling. Rather, it must be concluded that, although remodeling may be necessary for pulmonary hypertension in this model, it is not sufficient. Whether this is true in other species or forms of pulmonary hypertension or in pulmonary hypertension of longer duration remains to be determined. It is also possible, but not so easily demonstrated, that tone is necessary but not sufficient for chronic hypoxic pulmonary hypertension. In this case, alterations of pulmonary vascular resistance would have to be viewed as resulting from the interaction of tone and structure, whose effects could amplify or attenuate one another. This possibility deserves thorough investigation.
HPV is thought to divert blood flow from poorly ventilated hypoxic lung regions to well-ventilated normoxic lung regions; however, since poorly ventilated lung regions are also acidotic and hypercapnic, local vasomotor responses could result from changes in pH and PCO2 as well as PO2. Many studies report that acidosis either increases or has no effect on HPV (9, 79). These findings contrast with the vasodilatory effects of acidosis in systemic vessels (1). The study by Hyvelin et al. (33) tests the hypothesis that effects of acidosis are different in pulmonary and systemic arteries because Rho/ROK signaling is resistant to acidosis and plays a greater role in pulmonary arteries. In support of this hypothesis, phenylephrine caused greater activation of Rho, and Y-27632 caused greater relaxation of phenylephrine-induced contractions, in pulmonary arteries than in aorta. Moreover, acidosis did not affect relaxations induced by Y-27632 in permeabilized arterial smooth muscle in which [Ca2+]c was maintained constant. It is difficult to understand how a system of proteins as complex as the Rho/ROK signaling pathway could be unaffected by pH; therefore, it may be that activation of Rho/ROK signaling limits changes of intracellular pH in pulmonary arterial myocytes by stimulating Na-H exchange. Such stimulation has been demonstrated in other cell types and may be mediated by Rho/ROK-dependent phosphorylation of MLC and reorganization of the cytoskeleton (60, 80, 82).
In 1994, Yanagisawa and Okada (90) suggested that depolarizing extracellular concentrations of KCl increased Ca2+ sensitivity in vascular smooth muscle. Three of the call for papers in this issue of AJP-Lung (18, 33, 58) report the interesting finding that Y-27632 relaxed pulmonary arterial contractions induced by KCl. This effect of Y-27632 has been recently reported for other smooth muscles (4, 5, 23, 55, 68, 69, 84), where KCl-induced increases in [P-MLC] were also inhibited, but KCl-induced increases in [Ca2+]c were unaltered. If KCl acts independently of receptors, as commonly assumed, these results suggest that depolarization, [Ca2+]c or some related variable increased Ca2+ sensitivity through activation of Rho/ROK.
Acceptance of this conclusion requires that several alternative explanations be ruled out. For example, the inhibitory effect of Y-27632 on KCl-induced contractions could be due to nonspecific relaxant effects on smooth muscle. Arguing against this possibility is the finding that Y-27632 did not inhibit contractile responses induced by the phosphatase inhibitor calyculin A (5). In addition, the concentrations of Y-27632 required to inhibit KCl-induced contractions were similar to those required to inhibit contractions induced by receptor-linked agonists (5, 68, 69). A second possible explanation is that depolarization caused release of agonists from nerve endings in the tissue, which then stimulated receptor-linked activation of Rho/ROK in smooth muscle; however, inhibition of KCl-induced contractions by Y-27632 persisted in tissues treated with blockers of common neurotransmitters (5, 55, 69). A third possibility is that Y-27632 inhibited KCl responses not by blocking activation of Rho/ROK but by abolishing constitutive basal Rho/ROK activity, thereby activating MLCP sufficiently to prevent increases in [P-MLC] at any level of MLCK activity (Fig. 1). Against this possibility are recent observations that KCl increased Rho-GTP concentration (69) and caused translocation of ROK to plasma membrane (84) in vascular smooth muscle, indicating activation of Rho/ROK signaling. Moreover, the increases in Rho-GTP and tone induced by KCl could be prevented by removal of extracellular Ca2+ or antagonists of VOCCs and CaM (69).
Together, these results indicate that Rho/ROK, like MLCK, can be activated by Ca2+/CaM in a receptor-independent manner. The mechanisms of this activation are unknown but could involve Ca2+/CaM-dependent increases in synthesis of Rho-GTP (69) or movement of ROK to sarcolemma (84) (Fig. 1). Furthermore, it is not clear that activation of Rho/ROK by KCl would be manifested only by [P-MLC]-dependent crossbridge cycling in actin-myosin filaments. Conceivably, the inhibitory effect of the ROK antagonist Y-27632 on KCl-induced contractions could be explained by cytoskeletal disassembly, disconnection of contractile filaments from plasma membrane, and decreased force of contraction at any [P-MLC] (35, 68). Conversely, Rho/ROK-mediated enhancement of cytoskeletal connections might explain why ML-9, an MLCK antagonist that should block MLC phosphorylation and therefore limit alteration of [P-MLC] by MLCP, did not completely inhibit KCl-induced contractions in isolated pulmonary arteries (58, 92). The role of cytoskeletal connections as well as crossbridge cycling, in the generation of smooth muscle tone, needs to be clarified.
Finally, to complete this complex circle, recent evidence suggests that Rho/ROK signaling may affect [Ca2+]c as well as Ca2+ sensitivity. In tracheal smooth muscle, Y-27632 blocked nifedipine-insensitive noncapacitative increases in [Ca2+]c and tension induced by methacholine or histamine (35). In systemic arterial smooth muscle, Y-27632 blocked depolarization and nimodipine-insensitive increases in [Ca2+]c, tension, and Ba2+ influx induced by norepinephrine but did not alter increases in [Ca2+]c induced by thapsigargin or caffeine (23). In cerebral arterial smooth muscle, Y-27632 inhibited contraction, depolarization, increases in [Ca2+]c, and decreases in delayed-rectifier K+ current (Kdr) induced by UTP (51). Assuming that Y-27632 is a specific antagonist of ROK (13), these observations indicate that Rho/ROK signaling may alter activity of sarcolemmal receptor-operated cation and/or Kdr channels, resulting in Ca2+ influx through ROCCs, depolarization, and secondary Ca2+ influx through VOCCs. More work is needed to confirm these possibilities and to examine whether Rho/ROK signaling affects other determinants of [Ca2+]c, such as IP3R, RyR, and Ca2+ pumps at the SR and plasma membrane.
These are but a few of the issues raised by this interesting group of papers, which focus on a single monomeric GTPase and one of its many targets. The universe of GTPase signal cascades is rapidly expanding with the investigation of noncontractile cells, which has been underway for some time. In these cells, Rho family GTPases signal a wide variety of functions, including migration, phagocytosis, proliferation, secretion, and maintenance of cell shape. Interest is now turning toward contractile cells and, as these papers illustrate, pulmonary smooth muscle is an arena of particular promise.
FOOTNOTES
Address for reprint requests and other correspondence: J. T. Sylvester, Division of Pulmonary and Critical Care Medicine, The Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224 (E-mail: jsylv{at}jhmi.edu)
* With apologies to Chuck Berry.
REFERENCES