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Recent data have shown Ca2+-dependent activation of Rho-kinase by sustained depolarization of arterial smooth muscle. Visceral smooth muscles, however, contract phasically in response to action potentials and it is unclear whether Ca2+-dependent or -independent Rho-kinase activation occurs. We have therefore investigated this, under physiologically relevant conditions, in intact ureter. Action potentials, ionic currents, Ca2+ transients, myosin light chain (MLC) phosphorylation and phasic contraction evoked by action potentials in guinea-pig and rat ureter were investigated. In rat, but not guinea-pig ureter, three Rho-kinase inhibitors, Y-27632, HA-1077 and H-1152, significantly decreased phasic contractions and Ca2+ transients. Voltage- and current-clamp data showed that Rho-kinase inhibition reduced the plateau component of the action potential, inhibited Ca2+-channels and, indirectly, Ca2+-activated Cl− channels. The Ca2+ channel agonist Bay K8644 could reverse these effects. The K+ channel blocker TEA could also reverse the inhibitory effect of Y-27632 on the action potential and Ca2+ transient. Ca2+ transients and inward current, activated by carbachol-induced sarcoplasmic reticulum Ca2+release, were not affected by Rho-kinase inhibition. Rho-kinase inhibition produced a Ca2+-independent increase in the relaxation rate of contraction, associated with acceleration of MLC dephosphorylation, which was sensitive to calyculin A. These data show for the first time that: (1) Rho-kinase has major effects on Ca2+ signalling associated with the action potential, (2) this effect is species dependent and (3) Rho-kinase controls relaxation of phasic contraction of myogenic origin. Thus Rho-kinase can modulate phasic smooth muscle in the absence of agonist, and the mechanisms are both Ca2+-dependent, involving ion channels, and Ca2+-independent, involving MLC phosphorylation activity.
In smooth muscle, force can be modulated in Ca2+-dependent and Ca2+-independent mechanisms. Ca2+-independent pathways alter the activity of myosin light chain kinase (MLCK) and/or phosphatase (MLCP), and thus cause Ca2+ sensitization by changing the relation between [Ca2+] and force (Morgan & Morgan, 1984; Somlyo & Somlyo, 2000, 2003). In some smooth muscles, inhibition of MLCP is important in Ca2+ sensitization (Somlyo & Somlyo, 2003). The small GTPase, Rho-A, stimulates Rho-associated kinase (Rho-kinase), which phosphorylates the regulatory subunit of MLCP. Phosphorylation inhibits MLCP activity and thus potentiates force. Identification of this pathway has been aided by the use of relatively selective blockers of Rho-kinase, Y-27632, HA-1077 and H-1152 (Uehata et al. 1997; Fu et al. 1998; Yoshi et al. 1999; Sward et al. 2000; Somlyo & Somlyo, 2000; Ikenoya et al. 2002; Sasaki et al. 2002; Burdyga et al. 2003).
Until recently, Rho and Rho-kinase activation were considered to occur as a consequence of agonist binding. Recently, however, this assumption has been challenged. Mita et al. (2002) used high K+ to depolarize the caudal artery and found the contraction to be Rho-kinase sensitive and associated with inhibition of MLCP. Subsequent studies have confirmed this observation and shown the activation of Rho-kinase (Buyukafsar et al. 2003; Sakamoto et al. 2003; Asano & Nomura, 2003; Ghisdal et al. 2003; Urban et al. 2003). Sakurada et al. (2003) have shown that the activation of Rho and Rho-kinase by depolarization is Ca2+-dependent in vascular smooth muscle. Thus it appears that Rho and Rho-kinase are activated by two pathways, one Ca2+-independent and one Ca2+-dependent.
If some of the effects of Rho-kinase are Ca2+-dependent and activated by depolarization, the question of whether Rho-kinase can affect excitability and thus [Ca2+]i becomes important. There are a few reports of ion channels in a variety of tissues being regulated by Rho-kinase, e.g. anion channels (Nilius et al. 1999) and K+ channels (Cachero et al. 1998; Storey et al. 2002; Jones, 2003; Luykenaar et al. 2004). Effects of Rho-kinase on ionic currents in excitable tissues would have important effects on the action potential and [Ca2+]i. Although not frequent, some studies of Rho-kinase inhibition in smooth muscle have reported a fall in intracellular [Ca2+] ([Ca2+]i) (Takizawa et al. 1993; Ito et al. 2002; Maeda et al. 2003; Ghisdal et al. 2003). Thus it seems reasonable to ask if Rho-kinase inhibition is associated with decreased Ca2+ entry, and directly measure both [Ca2+]i and the Ca2+ current.
In the studies reviewed above, the smooth muscle under study is most often vascular. The tonic activity of blood vessels is very different from the phasic activity exhibited by visceral smooth muscle, including the fact that electrical activity is more important for phasic activity, and a rapid dephosphorylation of myosin light chains (MLCs) also occurs (Himpens et al. 1988; Gong et al. 1992; Murahashi et al. 1999; Murahashi et al. 1999). Thus it seems reasonable to hypothesize that the effects of Rho-kinase inhibition may differ in the two muscle types. There have been few studies of this in phasic smooth muscle, but the effects of Rho-kinase inhibition appear less marked compared to tonic smooth muscle (Kupittayanant et al. 2001; Kitazawa et al. 2003; Sward et al. 2003; Hashitani et al. 2004).
We have therefore investigated the role of Rho-kinase in electromechanical coupling in the ureter, i.e. a phasic smooth muscle, under myogenic action potential control. We have attempted to answer the following questions: (1) does Rho-kinase inhibition affect the action potential, Ca2+ signalling and phasic contractions? (2) does Rho-kinase affect ion channels? (3) is sarcoplasmic reticulum (SR) Ca2+ release targeted by Rho-kinase? (4) is Rho-kinase involved in the control of temporal relationships between MLC phosphorylation and force? and (5) are there Ca2+-dependent as well as Ca2+-independent actions of Rho-kinase in the ureter?
In rat ureter we found significant effects of Rho-kinase inhibition on Ca2+ signalling and force, as well as on the duration of the action potential. We also found that Rho-kinase targets at least two components of electromechanical coupling in ureteric smooth muscle, i.e. the activity of MLCP and ion channels, to modulate contractility.