RhoA kinase and protein kinase C participate in regulation of rabbit stomach fundus smooth muscle contraction.

The degree to which the RhoA kinase (ROK) blockers, Y-27632 (1 μM) and HA-1077 (10 μM), and the PKC blocker, GF-109203X (1 μM), reduced force produced by carbachol, a muscarinic receptor agonist, and phenylephrine, an α-adrenoceptor agonist, was examined in rabbit stomach fundus smooth muscle. When examining the effect on cumulative carbachol concentration-response curves (CRCs), ROK and PKC blockers shifted the potency (EC50) to the right but did not reduce the maximum response. In a single-dose carbachol protocol using moderate (∼EC50) and maximum carbachol concentrations, Y-27632 and HA-1077 reduced peak force, but GF-109203X had no effect. By contrast, all three agents inhibited the carbachol contractions of rabbit bladder (detrusor) smooth muscle. Compared to carbachol, phenylephrine produced a weaker maximum response that was not inhibited by phentolamine, atropine nor capsaicin but was inhibited by Y-27632 , HA-1077 and GF-109203X. In detrusor, classical down-regulation occurred, but in fundus, up-regulation of responsiveness occurred. This up-regulation in fundus may have been a post-receptor event, because a KCl-induced contraction produced after a carbachol CRC was stronger than one produced before the carbachol stimulus. In conclusion, these data suggest that ROK plays a critical role in the regulation of rabbit fundus smooth muscle contraction, which is distinct from chicken gizzard smooth muscle, where ROK is reported to exist but to not play a role in muscarinic receptor-induced contraction. Additional unique findings are that PKC participates in phenylephrine- but not carbachol-induced contraction in fundus, that carbachol does not activate identical subcellular signalling systems in fundus and detrusor, and that fundus, unlike detrusor, responds to carbachol stimulation with post-receptor up-regulation of contraction. Keywords: Signal transduction, stomach fundus, detrusor, smooth muscle, Ca2+-sensitization, RhoA kinase inhibitors Introduction Several reports published just over a decade ago revealed that smooth muscle contractile proteins could be ‘sensitized' to Ca2+ (Nishimura et al., 1988; Fujiwara et al., 1989; Kitazawa et al., 1989). In a review, Karaki (1989) described Ca2+ sensitization as a mechanism by which receptor agonists produce greater increases in force for a given increase in [Ca2+]i than a stimulus such as KCl. This model remains valid, except that recent reports indicate that KCl can also modulate the degree of Ca2+ sensitivity (Yanagisawa & Okada, 1994; Ratz, 1995; Mita et al., 2002), although to a lesser degree (Ratz, 1999). The precise mechanisms that cause Ca2+ sensitization are still being investigated. However, in general terms, Ca2+ sensitization may be due to an alteration in the sensitivity of myosin light chain (MLC) phosphorylation to Ca2+, principally through inhibition of MLC phosphatase by RhoA kinase (ROK) and protein kinase C (PKC), and to thin filament regulation (Hori & Karaki, 1998; Somlyo & Somlyo, 2000). Y-27632 and HA-1077 have recently proven invaluable in identification of the relative role that Ca2+ sensitization plays in regulation of contraction of intact smooth muscles of different organ systems. In permeabilized guinea-pig ileum, the ROK inhibitors, Y-27632 and HA-1077, inhibit GTPγS-induced Ca2+ sensitized contraction with IC50 values of, respectively, 1.7 μM and 2.7 μM (Sward et al., 2000). In arterial tissue, Y-27632 is reported to inhibit tonic contraction with an IC50 value of 0.7 μM (Uehata et al., 1997). These values are similar to Ki values reported for inhibition of ROK activity by these agents (Uehata et al., 1997; Davies et al., 2000; Sward et al., 2000), and a recent thorough study documenting the specificity of 28 commercially available kinase inhibitors indicates that Y-27632 and HA-1077 are highly selective for inhibition of ROK when used at appropriate concentrations (Davies et al., 2000). To date, Y-27632 or HA-1077 is reported to inhibit vascular (Uehata et al., 1997), respiratory (Chiba et al., 1999), ileal (Sward et al., 2000), bladder (Jezior et al., 2001) and myometrial (Kupittayanant et al., 2001) smooth muscle contractions. However, the degree to which these agents inhibit, and thus, the extent by which ROK participates in contractions appears to differ in different smooth muscle types. For example, 10 μM Y-27632 only modestly reduces spontaneous human myometrial contractions (Kupittayanant et al., 2001), but abolishes tonic α-adrenoceptor-induced contraction of rabbit aorta (Uehata et al., 1997). Muscarinic receptor stimulation induces a biphasic contraction of ileum, bladder and chicken gizzard consisting of a rapid, peak contraction (phasic component) followed by a tonic component that is weaker than the peak response. ROK blockade by Y-27632 or HA-1077 abolishes the tonic component but leaves the phasic component nearly intact in ileum (Sward et al., 2000), while in bladder, both components are greatly diminished (Jezior et al., 2001). Interestingly, despite the finding that GTPγS induces ROK activation and Ca2+ sensitization in chicken gizzard smooth muscle, muscarinic receptor stimulation does not cause ROK-dependent Ca2+ sensitization in this muscle type (Anabuki et al., 2000). However, Y-27632 does reduce gastric antrum motility in conscious rats (Tomomasa et al., 2000). The present study was designed to determine whether the selective ROK inhibitors, Y-27632 and HA-1077, inhibit contractions produced in isolated rabbit stomach fundus smooth muscle. For a comparison, the ability of GF-109203X, an inhibitor of conventional and novel PKC isotypes (Gailly et al., 1997), to inhibit fundus contraction was examined, and the effects of each agent on muscarinic receptor-induced contraction of rabbit bladder wall (detrusor) smooth muscle was tested.