Rho Rhokinase in Ca2 Sensitization and Pulmonary Vasoconstriction

VSMC tone is regulated by phosphorylation (causing contraction) and dephosphorylation (causing relaxation) of the 20-kDa regulatory MLC. Phosphorylation of MLC is catalyzed by Ca2+/calmodulin-dependent MLC kinase (MLCK) and dephosphorylation by Ca2+-independent MLC phosphatase (MLCP) that is targeted to myosin by its regulatory MBS. Thus, the balance in activities of MLCK and MLCP regulates contraction. At a given level of cytosolic Ca2+, the activity of both enzymes can be modulated by second messenger-mediated pathways to change MLC phosphorylation and force, i.e., to change theCa2+ sensitivity of contraction. There are multiple mechanisms of Ca2+ sensitization, but two major pathways lead to inhibition of MLCP and increased phosphorylation of MLC: one by Rho-kinase-mediated phosphorylation of the MBS, and the other by PKC-mediated phosphorylation and activation of the 17-kDa MLCP-inhibitor protein CPI-17 (61). CPI-17 is also phosphorylated by Rho-kinase, and recent studies suggest this pathway is a more physiologically significant mechanism of Ca2+ sensitization of vasoconstriction than is the Rho-kinase-mediated phosphorylation of MBS (43). The opposing roles of MLCK and MLCP in regulation of MLC phosphorylation and contraction, and the pathway for promotion of contraction by Rho-kinase-mediated inhibition of MLCP, are illustrated in Figure 2.

Because sensitization can augment vasoconstriction, it follows that desensitization can cause vasodilation. In addition to inducing VSMC relaxation by decreasing cytosolic [Ca2+] and MLCK activity, the NO -* soluble guanylate protein kinase (cGK) pathway also decreases Ca2+ sensitivity (4). cGMP-induced Ca2+ desensitization has been attributed to cGK-mediated phosphorylation and inhibition of RhoA, and a resultant increase in MLCP activity. However, NO/cGMP-induced vasorelaxation appears to involve only a transient increase in MLCP activity, and sustained relaxation may be due to other cGMP-mediated mechanisms that are not related to dephosphorylation of MLC, such as phosphorylation of telokin or HSP20 (13). Vasodilation by cAMP similarly involves diverse mechanisms (4), including inhibition of Rho/Rho-kinase signaling and increased activity of MLCP (51, 54).

tICa1

CaI+/CaM

MLCK

Agonist

Y-27632 & fasudil

Relaxation t

p-MLC Contraction

- GTP-RhoA

I f Rho-kinase

p-CPl-17

MLCP

Figure 2. Diagram of the relationship between Ca2+ signaling and Rho/Rho-kinase-mediated Ca2t sensitization in the regulation of VSMC tone. It is generally believed that increased cytosolic [Ca2+] and Ca27calmodulin (CaM)-dependent activation of MLCK initiate MLC phosphorylation and contraction. However, it is becoming increasingly clear that sustained contraction, even in face of decreasing cytosolic [Ca2+] is due to activation of RhoA and Rho-kinase-mediated inhibition of MLCP which maintains, or even promotes, MLC phosphorylation. The shaded area illustrates the signaling pathway by which GTP-RhoA-dependent stimulation of Rho-kinase leads to phosphorylation of the myosin binding subunit (p-MBS) and/or the inhibitory protein CPI-17 (p-CPI-17) that then inhibits MLCP and promotes MLC phosphorylation and contraction. The Rho-kinase blockers Y-27632 and fasudil inhibit this pathway leading to increased MLCP activity, dephosphorylation of MLC, and relaxation.

Several agonists elicit Ca2+ sensitization-associated contractions in pulmonary arteries that are attenuated by the Rho-kinase inhibitor Y-27632 (8, 26, 27). As reviewed in Chapter 7, Rho/Rho-kinase-mediated Ca2+ sensitization also plays a major role in the mechanism of HPV. Y-27632 both prevents and reverses HPV in rat pulmonary arteries and perfused lungs, and it has been suggested that activation of Rho-kinase is essential to the sustained hypoxic response (50). Acute hypoxia causes an early and sustained phosphorylation of MLC in rat pulmonary VSMC in culture, and the sustained response is inhibited by the Clostridium botulinum toxin C3 exoenzyme (which ADP-ribosylates and inactivates Rho) and by Y-27632 (66). It was suggested that HPV is initiated by Ca2+-induced stimulation of MLCK, and the sustained response is due to Rho/Rho-kinase-mediated inhibition of MLCP. While Rho/Rho-kinase signaling clearly regulates acute pulmonary vasoreactivity, the role of Rho/Rho-kinase-mediated Ca2+ sensitization in the increased vascular tone and vasoreactivity of hypoxic hypertensive lungs is uncertain. As discussed below in Sections 9 and

10, we have begun to investigate if and by what mechanisms Rho/Rho-kinase -mediated Ca2+ sensitization of vasoconstriction is increased in hypoxic hypertensive pulmonary arteries.

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