Expression of CPI‐17 and myosin phosphatase correlates with Ca2+ sensitivity of protein kinase C‐induced contraction in rabbit smooth muscle

Various smooth muscles have unique contractile characteristics, such as the degree of Ca2+ sensitivity induced by physiological and pharmacological agents. Here we evaluated six different rabbit smooth muscle tissues for protein kinase C (PKC)-induced Ca2+ sensitization. We also examined the expression levels of myosin light chain phosphatase (MLCP), the MLCP inhibitor phosphoprotein CPI-17, and the thin filament regulator h-calponin. Immunohistochemical and Western blot analyses indicated that CPI-17 was found primarily in smooth muscle, although expression varied among different tissues. Vascular muscles contained more CPI-17 than visceral muscles, with further distinction existing between tonic and phasic subtypes. For example, the tonic femoral artery possessed approximately 8 times the cellular CPI-17 concentration of the phasic vas deferens. In contrast to CPI-17 expression patterns, phasic muscles contained more MLCP myosin-targeting subunit than tonic tissues. Calponin expression was not statistically different. Addition of phorbol ester to α-toxin-permeabilized smooth muscle caused an increase in contraction and phosphorylation of both CPI-17 and myosin light chain (MLC) at submaximal [Ca2+]i. These responses were several-fold greater in femoral artery as compared to vas deferens. We conclude that the expression ratio of CPI-17 to MLCP correlates with the Ca2+ sensitivities of contraction induced by a PKC activator. PKC stimulation of arterial smooth muscle with a high CPI-17 and low MLCP expression generated greater force and MLC phosphorylation than stimulation of visceral muscle with a relatively low CPI-17 and high MLCP content. This implicates CPI-17 inhibition of MLCP as an important component in modulating vascular muscle tone. Smooth muscle is heterogeneous in both structure and function. Based on their electrophysiological and mechanical characteristics, smooth muscles can be broadly classified into tonic and phasic types (Somlyo & Somlyo, 1968). Tonic muscles such as the aorta, pulmonary and femoral arteries, along with the trachea, usually respond to excitatory agonists with a graded depolarization. Prolonged depolarization induced by high K+ typically evokes a slowly developing sustained contraction in tonic muscles. In contrast, phasic muscles, which include the portal vein, ileum, bladder and vas deferens, react to excitatory agonists by generating a spike-like action potential. In addition, depolarization with high K+ elicits an initial phasic contraction, followed by a decline to a low steady-state level (Himpens et al. 1989). The contractile diversities among different smooth muscles appear to be attributed, at least in part, to variations in cellular protein expression rather than to different time courses in [Ca2+]i (Himpens et al. 1988). Myosin heavy chain (see review by Adelstein & Sellers, 1996) and light chain isoforms (see Barany & Barany, 1996) are specifically distributed among smooth muscle tissues and the composition of these isoforms determines myosin motor activity. Moreover, variable expression of other regulatory contractile proteins possibly contributes to the differentiation of smooth muscle function (Khalil, 1992; North et al. 1994; Szymanski et al. 1998; Dirksen et al. 2000). The primary mechanism that regulates actomyosin ATPase and contraction in all vertebrate smooth muscle is reversible phosphorylation of the 20 kDa myosin light chain (MLC) by MLC kinase (MLCK) and MLC phosphatase (MLCP) (Hartshorne,1987; Kamm & Stull, 1989). MLCK is a Ca2+-calmodulin-dependent kinase that is stimulated by an increase in [Ca2+]i. Although Ca2+ is the major contractile messenger for all types of smooth muscle, the Ca2+ sensitivity of MLC phosphorylation and contraction is an equally important factor for the regulation of smooth muscle contraction (see Somlyo & Somlyo, 1994). Tonic muscles (pulmonary and femoral arteries) have a 3-fold greater calcium sensitivity of MLC phosphorylation and contraction as compared to phasic muscles (portal vein and ileum) (Kitazawa et al. 1991a). The higher Ca2+ sensitivity of tonic muscle contraction is explained by a 4-fold lower dephosphorylation rate of MLC in tonic muscle, as compared to phasic muscle. This is evident even in phasic tissues with a higher MLCK content, suggesting that in situ, the primary determinant of smooth muscle Ca2+ sensitivity is MLCP rather than MLCK activity (Gong et al. 1992). It is well documented that G protein-coupled receptor activation increases the Ca2+ sensitivity of MLC phosphorylation and contraction in all types of smooth muscle through the inhibition of MLCP (Kitazawa et al. 1991b; Kubota et al. 1992; see Somlyo & Somlyo, 1994). MLCP is a heterotrimeric enzyme with a 38 kDa type-1 phosphatase catalytic subunit δ-isoform (PP1C), a 110-130 kDa myosin-targeting subunit (MYPT1) and a 20-21 kDa subunit of unknown function (see Hartshorne et al. 1998). Two major pathways have been proposed for the regulation of MLCP. First, RhoA-activated kinase (Rho-kinase) phosphorylates Ser-696 of MYPT1, which inhibits the phosphatase activity (Kimura et al. 1996; Feng et al. 1999; Kawano et al. 1999; see Somlyo & Somlyo, 2000). In smooth muscle cells, this phosphorylation is enhanced by stimulation with the G protein activator GTPγS (Trinkle-Mulcahy et al. 1995; Sward et al. 2000; Nagumo et al. 2000). In addition to MYPT1 phosphorylation, a second pathway involves an inhibitor protein for MLCP, called CPI-17, which was first isolated from pig aorta smooth muscle (Eto et al. 1995) and is only expressed in smooth muscle (Eto et al. 1997, 1999). Phosphorylation of CPI-17 at Thr-38 by PKC converts this protein to a potent inhibitor of the catalytic activity of not only PP1C, but also the MLCP holoenzyme (Eto et al. 1997; Senba et al. 1999) and MLCP anchored to myofibrils in situ (Li et al. 1998; Eto et al. 2000). The PKC δ-isoform has been isolated as a dominant CPI-17 kinase from pig aorta (Eto et al. 2001), and a recombinant PKC α-isoform can much more rapidly phosphorylate Thr-38 of CPI-17 as compared to calponin, caldesmon, MLC and myosin (Kitazawa et al. 1999). Furthermore, selective depletion of CPI-17 by skinning of smooth muscle cells diminishes PKC-induced Ca2+ sensitization of femoral artery strips and the response can be reconstituted by addition of PKC and CPI-17 together, but not by PKC alone (Kitazawa et al. 1999). These results indicate that CPI-17 is at least a key component in PKC-induced MLCP inhibition and calcium sensitization. It has recently been demonstrated that stimulation of the femoral artery by agonists, GTPγS and PKC activator induces Thr-38 phosphorylation of CPI-17 and contractile Ca2+ sensitization (Kitazawa et al. 2000). A PKC inhibitor (GF109203X) and a Rho-kinase inhibitor (Y27632) partially inhibited the CPI-17 phosphorylation and contraction induced by histamine, suggesting that possibly Rho-kinase and PKC phosphorylate CPI-17 in response to histamine signalling (Kitazawa et al. 2000; Koyama et al. 2000). In summary, these results imply that the CPI-17-MLCP signalling pathway may be as significant as the Rho-kinase-MLCP pathway in G protein-coupled agonist-induced Ca2+ sensitization of smooth muscle contraction. CPI-17 expression is almost exclusively confined to smooth muscle tissues, and we believe that it is an endogenous mediator for the PKC signalling pathway that induces contractile Ca2+ sensitization. Yet between different smooth muscle tissues (aorta vs. urinary bladder), differential expression of CPI-17 is suggested by Northern and Western blot analyses (Eto et al. 1997, 1999). In preliminary experiments at submaximal [Ca2+] (Woodsome & Kitazawa, 2000), we observed that in the phasic vas deferens and urinary bladder, phorbol ester-induced contractions were several times smaller than those caused by GTPγS. In contrast, the tonic femoral artery displayed equal responses to both phorbol 12,13-dibutyrate (PDBu) and GTPγS (Masuo et al. 1994). Therefore we hypothesized that the variation in PDBu-induced Ca2+ sensitization may possibly be due to differential expression of CPI-17 and the phospho-CPI-17 target MLCP. In this study, we used immunoblotting techniques to measure the tissue-specific expression and concentration of CPI-17, MLCP and h-calponin in six different rabbit smooth muscle tissues, including vascular tonic or phasic and visceral tonic or phasic sub-phenotypes. These results were then compared to the contractile response and MLC phosphorylation induced by PKC or G protein activation at a given level of Ca2+ in the various smooth muscles. The preliminary results were presented at the February 2000 Biophysical Society Meeting in New Orleans (Woodsome & Kitazawa, 2000).