doi: 10.1161/01.RES.0000131755.49084.04
© 2004 American Heart Association, Inc.
Editorials |
Cold-Induced Vasoconstriction
A Waltz Pairing Rho-Kinase Signaling and 
2-Adrenergic Receptor Translocation
From the Department of Physiology, Medical College of Georgia, Augusta, Ga.
Correspondence to Cleber E. Teixeira, Department of Physiology, Medical College of Georgia, 1120 Fifteenth Street, CA-3101, Augusta, GA 30912-3000. E-mail cteixeira{at}mail.mcg.edu
Key Words: vascular smooth muscle • Rho-kinase • microtubules • 2-adenoreceptors
Advances made over the past few years have made us appreciate the critical roles of Rho-associated kinase (Rho-kinase/ROCK) in a bewildering variety of cellular and biological responses. Rho-kinase represents a downstream target for the low–molecular-weight G-protein, Rho, and is believed to transmit signals to cytoskeletal and regulatory proteins. In recent years, enormous progress has been made in understanding the role of this signaling pathway in regulating cellular movements, cell growth, and cell–cell interactions. Rho-kinase has been implicated in many cellular processes, such as stress fiber and focal adhesion formation, smooth muscle contraction, neurite retraction, and cell migration.1,2
In addition to Ca2+-dependent increases in myosin light chain (MLC) phosphorylation for vascular contraction to occur, the concept of a Ca2+-sensitizing mechanism for the promotion of the phosphorylated state of MLC has become evident.3 Extensive studies demonstrate that the sustained contractile force in vascular smooth muscle preparations generated on agonist stimulation does not parallel intracellular Ca2+ levels. On exposure to an agonist, Ca2+ concentration in the cytosol peaks and then quickly drops to near-resting levels despite the maintenance of contractile force. Further, tonic force generation in many vascular preparations persists in Ca2+-free media, further implicating an increase in Ca2+ sensitivity of the contractile apparatus, or the activation of a Ca2+-independent mechanism.4,5 This phenomenon has been termed agonist-induced force enhancement, or Ca2+ sensitization, and several mechanisms have been proposed.3,6 Although the signaling pathway(s) for Ca2+ sensitization have not been fully elucidated, the most proposed mechanism converges on an inhibition of MLC phosphatase activity through the Rho/Rho-kinase–linked pathway.3
In this issue of Circulation Research, Bailey et al7 provide compelling evidence that, under cooling conditions, the Rho/Rho-kinase signaling pathway is activated to mediate cold-induced constriction in cutaneous arteries by favoring the translocation of 2-adrenoceptors (2-AR) from the Golgi complex to the plasma membrane as well as by increasing the Ca2+ sensitivity of the contractile proteins. There are 3 basic sets of observations in this study. First, it is clearly shown that moderate cooling (28°C) increased the constriction of the mouse tail artery evoked by the 2-AR agonist UK 14,304. Using pharmacological inhibitors of Rho-kinase, such as fasudil, Y-27632 and H-1152, the constriction evoked by the agonist was inhibited, an effect not observed at 37°C. Second, the authors demonstrate that moderate cooling causes a time-dependent activation of Rho in vascular smooth muscle cells cultured from human cutaneous arterioles. In permeabilized arteries, they present evidence that moderate cooling, but not activation of 2-AR by UK 14,304, increases the sensitivity to Ca2+. Third, using HEK 293 cells transiently transfected with HA-tagged 2-AR as well as RNA interference to decrease expression of ROCK-I, the authors show that moderate cooling-induced increases in the localization of 2-AR in the cell surface is efficiently prevented by Rho-kinase inhibition.
Constriction of cutaneous blood vessels in response to cooling is a protective physiological response that acts to reduce loss of body heat.8 As noted by the authors, the direct constrictor response to cold is mediated at the cellular level by the rapid and selective augmentation of 2-AR activity.9–11 Specifically, it has been shown that increased activity of 2C-AR mediates the augmented vasoconstriction to cooling in mouse arterial microvessels.12 Cooling from 37°C to 28°C for brief intervals is sufficient to uncover 2C-AR–mediated vasoconstriction,12 because 2C-AR are localized to intracellular compartments in rat fibroblasts at 37°C.13 These observations might implicate the 2C-AR as a putative thermosensor in the vessel wall, which would be consistent with the original notion of the 2C-AR as a "silent receptor."14 However, the authors demonstrate that a component of the Rho-kinase signaling pathway, rather than the 2-AR itself, appears to be the thermosensitive switch, because the latter do not respond directly to cold. These observations suggest that not only is Rho activated during moderate cooling but also does its activation trigger Rho-kinase stimulation to increase force. Undoubtedly, the most intriguing finding was the identification of Rho-kinase as a mediator of 2-AR translocation in conditions of low temperatures.
It is apparent that much work needs to be performed to define the physiological significance of this mechanism. Experimentally, cold temperatures are known to depolymerize microtubules, and many pharmacological agents can also shift the microtubule equilibrium. Paclitaxel (Taxol) promotes and stabilizes polymerized microtubules by binding to specific sites on the tubulin polymer, preventing the dissociation of tubulin.15 Other agents such as nocodazole, colchicine, and vinblastine favor the depolymerized state of the microtubule either by binding to the polymer, inducing a destabilizing conformational change, or by binding to free /ß tubulin dimers, preventing their hydrolysis onto the microtubule. Physiologically, many cellular proteins are known to regulate the dynamic state of the microtubule polymer, and, in turn, the cytosolic network regulates the localization of numerous proteins in the cell.16–19 As outlined by Gunderson and Cook, some functional models explaining the means by which the microtubule system may regulate various cellular processes include sequestering and release, delivery, and scaffolding.18 Potentially via such mechanisms, the microtubule network is involved in the regulation of a variety of cellular processes, including protein attachment, vesicular trafficking and transport, cellular motility, intracellular signal transduction, and regulation of cell division.
The study by Bailey et al, the current article in focus,7 lifts the veil on the role of RhoA in the cellular response to moderate cooling. A scenario may be imagined, therefore, in which mature and functional receptors are stored in the Golgi compartments in readiness for an appropriate translocation stimulus.
Various studies have found the stimulation of focal adhesion and stress fiber formation on depolymerization of the microtubule network to be blocked by the inactivation of RhoA.20–22 Additionally, microtubule-depolymerizing agents were shown to increase the activity of RhoA in in vitro assays, providing evidence to suggest that microtubule depolymerization may lead to the activation of RhoA signaling.23,24 In non-neuronal cells, Rho family GTPases have been known to be implicated in microtubule dynamics and in microfilament organization.25,26 Furthermore, several lines of evidence indicate that neurite retraction occurs when Rho and Rho-kinase are activated in neuroblastoma cells, which is accompanied by the suppression of assembly of microtubules and intermediate filaments.27 Because the regulation of microtubules and intermediate filaments is thought to be necessary to accomplish the shape change or movement for the cells in response to the signals, it is conceivable that Rho and Rho-kinase modulate microtubules and intermediate filaments as well as microfilaments. Further studies are necessary to understand the relationship between the Rho signaling pathway and cytoskeleton, including microfilaments, microtubules, and intermediate filaments. Membrane trafficking is intimately involved in many of the functions that are controlled by Rho family members, including the establishment of cell polarity and the control of cell proliferation and motility. It is therefore likely that the roles of Rho GTPases in these processes are mediated, at least in part, via their modulation of trafficking events.
One mechanism by which microtubule depolymerization may activate downstream proteins is termed "sequestering and release."18 If RhoA is localized on the tubulin polymer, it is possible that in accordance with this mechanism, the depolymerization of the microtubule may release bound RhoA from the polymer into the cytosol, enabling its interaction with various downstream targets. However, contrarily, studies have found inactive RhoA to be primarily cytosolic.28 A more likely scenario is that a promoter of small G-protein activity, such as a guanine nucleotide exchange factor (GEF), may instead be "sequestered" and "released" by microtubule depolymerization, and, in turn, lead to RhoA activation.18,29 Evidence exists supporting the localization of various Rho-GEFs on the microtubule network.29–32 Rho-GEFs enable RhoA activation by promoting the dissociation of GDP from RhoA, thus favoring the subsequent binding of GTP as opposed to GDP.33,34 Microtubules may bind or "sequester" Rho-GEFs, thus inhibiting their interaction with RhoA. On microtubule depolymerization, a GEF may be freed or "released," enabling its interaction with RhoA, thus catalyzing the activation of the small G-protein (based on "sequestration and release")Figure).18,29
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Nevertheless, the study by Bailey et al provides an important piece to this puzzle. Because 2C-AR translocation from the Golgi complex to the plasma membrane represents a key step in the adrenergically mediated contractile effect elicited by cold, an attractive feature was the use of a live-cell labeling and silencing RNA techniques to investigate the role of Rho/Rho-kinase in the 2C-AR trafficking. In experiments performed in HEK 293 cells transiently transfected with HA-tagged 2C-AR, the authors confirmed that moderate cooling increases the localization of 2C-AR to the cell surface. This occurred without affecting cellular expression of the receptor. When Rho-kinase was inhibited by fasudil, cold-induced translocation of the 2C-AR was prevented, whereas the inhibitor had no effect on the cell surface expression of the receptor at 37°C. In subsequent experiments, a role for Rho-kinase in cold-induced translocation of 2C-AR was successfully confirmed by using RNA interference to decrease expression of the kinase in HEK 293 cells. Silencing RNA (siRNA) oligoduplexes targeted to ROCK-I not only decreased the expression of the kinase but also again prevented translocation of 2C-AR elicited by cold.
Also important was the availability and use of specific inhibitors of Rho-kinase, such as fasudil, Y-27632, and H-1152. The correlations that were reported by Bailey et al support their conclusion that physiological mechanisms in response to cold appear to be tightly mediated by Rho-kinase in a process that likely involves microtubule depolymerization. This does not come as a surprise because of the differential localization of Rho family members to distinct membrane compartments. We expect that future studies will identify additional trafficking functions for some of the less well-characterized members of the Rho family. A considerable challenge we now face is the dissection of the downstream signaling events that mediate the roles of Rho proteins in trafficking processes. Although the precise role of the actin cytoskeleton in membrane trafficking is still not understood, Rho GTPases are ideally placed to serve as coordinators of actin dynamics and trafficking events. The recent identification of a large number of components of the trafficking machinery acting as downstream effectors of Rho GTPases indicates that cytoskeletal reorganization is definitely not the entire story. Understanding these mechanisms in the integrated biology of smooth muscle remains an important issue.
Footnotes
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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