Role of Rho Kinase

Rho-associated coiled coil-forming serine/threonine kinase (Rho-kinase, also known as Rho-associated kinase) has recently been identified as a pivotal mediator of Ca2+ sensitization and vascular smooth muscle contraction (50). Since the first isolation of this kinase (23), it has become apparent that, upon activation by the binding of the small monomeric G-protein RhoA, Rho-kinase inhibits MLCP resulting in Ca2+-independent contraction of vascular smooth muscle. Originally, the inhibition of MLCP was thought to be via a direct action ofRho-kinase upon the myosin binding subunit ofMLCP (18). However, recent evidence suggests that, as with PKC, Rho-kinase can also inhibit MLCP via phosphorylation and activation of CPI-17 (19, 20, 28). Therefore, phosphorylation of CPI-17 and inhibition of MLCP may be a convergent mechanism through which both PKC and Rho-kinase elicit an increase in myofilament Ca2+-sensitivity although the effect of PKC may be transient, at least with respect to agonist-induced Ca2+ sensitization (15). Rho-kinase is also known to directly phosphorylate MLC20 in vitro, but this mechanism does not appear to occur in intact arteries (14).

Subsequent to the initial identification of a role for Ca2+-sensitization in HPV (34) and the exclusion ofPKC from this process in rat isolated arteries, there was a hiatus in research directed at this mechanism until the "specific" Rho-kinase inhibitor, Y-27632 (52), became widely available to examine the possible role of Rho-kinase in this process. Indeed, the availability of Y-27632 has resulted in an explosion of reports in the literature implicating Rho-kinase in a diverse number of intracellular pathways in both smooth muscle and non-muscle cell types (50).

The first evidence that Rho-kinase may be involved in HPV, and specifically in the Ca2+ sensitization-mediated development of sustained HPV, came from studies of isolated arteries and in situ perfused lungs of the rat (36). It was found that Y-27632 preferentially inhibited sustained HPV, whilst having a minimal effect upon the transient contraction (Fig. 4). Indeed, the small attenuation ofthe transient phase of HPV may be due to the effect of Y-27632 upon the sustained portion of the response. As mentioned previously, the transient response is superimposed on the more-slowly developing sustained phase. Upon selective inhibition of the transient phase by sub-micromolar concentrations of the trivalent cation La3+, it is apparent that the sustained phase of HPV in isolated arteries is immediate in onset (37), similar to HPV in perfused lungs and in vivo. Inhibition of this response by Y-27632 would therefore produce an apparent reduction in the transient phase without necessarily directly affecting the mechanisms responsible for the contraction. An important aspect of this study was that Y-27632 abolished the monophasic rise in pulmonary artery pressure in the perfused lung, whereas only the sustained phase was inhibited in small isolated pulmonary arteries. This observation would appear to question the physiological relevance of the large transient response to HPV in vitro, and raises the possibility that it may be, at least in part, an artifact of the experimental conditions employed to elicit HPV in isolated arteries. However, a similar transient phase of HPV, although markedly smaller, has been observed in vivo when the induction of hypoxia is rapid (38, 58).

Hypoxia

Hypoxia

Figure 4. Y-27632 inhibits HPV in rat isolated arteries and perfused lungs. A: The effect of the Rho-kinase inhibitor Y-27632 (3 |xM) on HPV in rat isolated arteries. At this concentration, Y-27632 inhibited the sustained phase of HPV, whereas the inhibitory effect upon the transient phase was less marked (Data = mean ± SEM of 9 control and 7 Y-27632-treated arteries). B: An experimental record from an in situ perfused rat lung. Y-27632 (600 nM) abolished HPV in this preparation.

Figure 4. Y-27632 inhibits HPV in rat isolated arteries and perfused lungs. A: The effect of the Rho-kinase inhibitor Y-27632 (3 |xM) on HPV in rat isolated arteries. At this concentration, Y-27632 inhibited the sustained phase of HPV, whereas the inhibitory effect upon the transient phase was less marked (Data = mean ± SEM of 9 control and 7 Y-27632-treated arteries). B: An experimental record from an in situ perfused rat lung. Y-27632 (600 nM) abolished HPV in this preparation.

The inhibition of HPV by Y-27632 in rat isolated intrapulmonary arteries, in situ perfused rat lungs (35) and perfused mouse lungs (Fagan K, personal communication) are consistent with a pivotal role for Rho-kinase-mediated Ca2+-sensitization in the mechanism of sustained HPV. It is interesting that pretreatment of perfused rat lungs with Y-27632 completely prevents the hypoxic response. This raises the question of whether activation of Rho-kinase is involved in initiating HPV, or whether a basal level of Rho-kinase activity and suppression ofMLCP activity, are necessary for the hypoxia-induced Ca2+ signal, and associated activation of MLCK, to increase MLC20 phosphorylation and elicit contraction. Moreover, as with all new pharmacological tools, the direct extrapolation of the effects of Y-27632 to Rho-kinase inhibition must be undertaken with caution and direct evidence for the activation of Rho-kinase during the response in question is important. This evidence was recently provided by Dr. R. Rhoades' laboratory (55). These investigators reported that hypoxia-induced activation of Rho-kinase was associated with a concomitant increase in MLC20 phosphorylation in cultured rat intrapulmonary artery smooth muscle cells maintained in 10% fetal bovine serum (54). In an elegant series of experiments, Wang and co-workers provided strong evidence that hypoxia can activate Rho-kinase, in a RhoA-dependent manner, and that inhibition of either RhoA (via application of exoenzyme C3) or Rho-kinase (via incubation of Y-27632) attenuated the increase in MLC20 phosphorylation in response to hypoxia. Furthermore, Y-27632 inhibited sustained HPV in rat isolated pulmonary arteries. These experiments, therefore, provided the first biochemical evidence for a role of Rho-kinase in HPV. However, this study also raised an important issue with respect to the role of the endothelium in sustained HPV (discussed in section 6).

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