Agonist-stimulated contraction of smooth muscle, including that of the uterus, involves the integration of many signal transducing events linking receptor occupation at the plasma membrane with varied intracellular effectors and eventual alteration of the activation state of the myofilaments in the cell cytoplasm. Precise co-ordination of these myometrial signalling events at term is paramount to ensuring efficient and powerful contractile activity necessary for expeditious expulsion of the fetus. A comprehension of the complex intracellular mechanisms of action of uterotonic agents is thus essential not only to our understanding of parturition but also for the management of situations involving dysfunctional uterine performance including, for example, those associated with pre-term labour, non-labouring uteri and post-partum haemorrhage. As such, in this paper we review recent studies that shed light on many of the mechanisms contributing to the efficient coupling of extracellular contractile stimuli and intracellular effectors in uterine smooth muscle. Special consideration is given to the processes of receptor-coupled recruitment to the plasma membrane of intracellular proteins important for agonist-induced alterations in Ca 2+ sensitivity of contraction such as rhoA and ROK (rho-associated kinase); evidence for the involvement of caveolins, proteins integral to plasma membranous caveolae, in this signal transduction cascade is discussed. (MLC 20 ;Word et al. 1991;Taggart et al. 1997). This increased phosphorylation of MLC 20 allows for enhanced actomyosin Mg-ATPase activity and subsequent contractility. Smooth muscle relaxation is preceded by dephosphorylation of MLC 20 by a myosin phosphatase (MLCP; Khromov et al. 1995) which has been cloned from uterine tissue (Johnson et al. 1997). However, a prominent additional mechanism whereby agonists can stimulate smooth muscle contractility is by sensitising the myofilaments to the activating [Ca 2+ ] i . This occurs in both intact (Taggart & Wray, 1998) and permeabilised (Izumi et al. 1996;Somlyo & Somlyo, 1998;Taggart et al. 1999) Receptor activation of uterine smooth muscle
ABSTRACT. The role of rhoA/rho-associated kinase (ROK) signaling pathways in agonist-induced contraction of the rat myometrium was investigated. We measured the [Ca 2+ ] i -force relationship, phosphorylation of myosin regulatory light chains (MLC 20 /calmodulin-dependent enzyme myosin light chain kinase and subsequent phosphorylation of the regulatory myosin light chains (MLC 20 ) [2,3]. This increased phosphorylation of MLC 20 enhances actomyosin ATPase activity and uterine force production. Smooth muscle relaxation is preceded by dephosphorylation of MLC 20 by myosin light chain phosphatase (MLCP) [4]. In addition, it has been shown that an increase in the Ca 2+ -sensitivity of the smooth muscle myofilaments contributes to the contraction induced by Ca 2+ -mobilizing agonists [1,5,6]. Recent studies in other smooth muscles have indicated that both rhoA and rhoA-associated kinase (ROK) are involved in this mechanism [7]. ROK is able to regulate the phosphorylation of MLC 20 by the inactivation of myosin light chain phosphatase (MLCP) through the phosphorylation of myosin binding subunit [8].The spontaneous contractions of the myometrium are augmented by a variety of uterotonic agents playing an important role at parturition. These agonists have been reported to increase myometrial contraction, not only by increasing [Ca 2+ ] i , but also by increasing the Ca 2+ sensitivity of myometrial force production through a receptor-coupled, G-protein-mediated mechanism [5,9] that has been suggested, in part at least, to be mediated via activation of ROK [6,10]. Interestingly, in myometria from both rat and humans the mRNA expressions of rhoA and ROK in the pregnant myometrium were increased in comparison to those in the non-pregnant myometrium [11,12]. Our preliminary data has suggested that agonist-induced Ca 2+ -sensitization of force of single permeabilized uterine cells can be attenuated by inhibition of ROK [10]. We have also previously demonstrated in single isolated myometrial cells that rhoA and ROKα translocate from the cytosol to the membrane after carbachol stimulation [13] supportive of the hypothesis that membranous relocalization of rhoA/ROK is necessary for agonist-induced Ca 2+ -sensitization to occur [7,10,13]. Although the above reports are suggestive of an involvement of rhoA and/or ROK in the Ca 2+ sensitization phenomenon, the extent of the putative role of rhoA and ROK in the contractile regulation of pregnant rat myometrium has not been fully elucidated. Therefore, in this study we have further examined the role of ROK-mediated pathways in regulating myometrial contractile function. This has involved comparing the effects of ROK inhibition with Y-27632 on (i) the relationships between [Ca 2+ ] i and force and MLC 20 phosphorylation and force in intact myometrial tissue and (ii) the agonist-induced Ca 2+ sensitivity of contraction in single permeabilized cells.
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