Is myosin phosphatase regulated in vivo by inhibitor‐1? Evidence from inhibitor‐1 knockout mice

The Ca2+ sensitivity of smooth muscle contractility is modulated via regulation of phosphatase activity. Protein phosphatase inhibitor-1 (I-1) is the classic type-1 phosphatase inhibitor, but its presence and role in cAMP-dependent protein kinase (PKA) modulation of smooth muscle is unclear. To address the relevance of I-1 in vivo, we investigated smooth muscle function in a mouse model lacking the I-1 protein (I-1(−/−) mice). Significant amounts of I-1 protein were detected in the wild-type (WT) mouse aorta and could be phosphorylated by PKA, as indicated by 32P-labelled aortic extracts from WT mice. Despite the significant presence of I-1 in WT aorta, phenylephrine and KCl concentration- isometric force relations in the presence or absence of the PKA pathway activator isoproterenol (isoprenaline) were unchanged compared to I-1(−/−) aorta. cGMP-dependent protein kinase (PKG) relaxation pathways were also not different. Consistent with these findings, dephosphorylation rates of the 20 kDa myosin light chains (MLC20), measured in aortic extracts, were nearly identical between WT and I-1(−/−) mice. In the portal vein, I-1 protein ablation was associated with a significant (P < 0.05) rightward shift in the EC50 of isoproterenol relaxation (EC50 = 10.4 ± 1.4 nm) compared to the WT value (EC50= 3.5 ± 0.2 nm). Contraction in response to acetylcholine as well as Ca2+ sensitivity were similar between WT and I-1(−/−) aorta. Despite the prevalence of I-1 and its activation by PKA in the aorta, I-1 does not appear to play a significant role in contractile or relaxant responses to any pharmacomechanical or electromechanical agonists used. I-1 may play a role as a fine-tuning mechanism involved in regulating portal vein responsiveness to β-adrenergic agonists. Regulation of phosphoprotein phosphatases has been shown to be a major component in the Ca2+ sensitization of smooth muscle contraction (Somlyo & Somlyo, 2000). The ∼19 kDa phosphatase inhibitor-1 (I-1) protein is the classic regulator of type-1 phosphatase activity (Oliver & Shenolikar, 1998) and has been shown to be present in smooth muscle (Elbrecht et al. 1990). However, studies of the relevance of I-1 in regulating smooth muscle myosin phosphatase activity in vitro and, consequently, smooth muscle function have provided conflicting results (Alessi et al. 1992; Tokui et al. 1996). These data may be due to the inability of in vitro systems to recapitulate the complex regulatory mechanisms acting on the phosphatase, including its regulation by kinases and/or targeting subunits. Thus, an in vivo approach is essential to determine the physiological effects of the inhibitor-1 protein. To address this issue, we used the recently developed I-1 knockout (I-1(−/−)) mouse to delimit the role of I-1 in both tonic and phasic smooth muscle contractility. A major mechanism involved in regulating smooth muscle contractility is the phosphorylation of serine-19 on the 20 kDa myosin light chains (MLC20) (de Lanerolle & Paul, 1991; Hartshorne et al. 1998), resulting in the activation of myosin-actin crossbridge cycling and contraction. The phosphorylation status of MLC20, and consequently contraction, is controlled by the dynamic balance between the activities of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). MLCK has been well characterized (Gallagher et al. 1997) and its activation is Ca2+ dependent, involving a Ca2+-calmodulin complex. It is now clear that MLCP is also regulated and this regulation has been suggested to involve the cytosolic I-1 (Somlyo et al. 1989). MLCP is a heterotrimer consisting of a ∼37 kDa catalytic subunit (PP1c), a ∼20 kDa subunit of unknown function, and a ∼110–130 kDa myosin phosphatase targeting subunit (MYPT1) (Shirazi et al. 1994; Hartshorne et al. 1998). Interaction of PP1c with MYPT1 confers selectivity of PP1c towards the myosin molecule (Alessi et al. 1992) and enhances PP1c activity (Shirazi et al. 1994; Ichikawa et al. 1996). As in other tissues, the targeting subunit may play a direct role in regulating PP1c activity. For instance, in striated muscle, phosphorylation of the glycogen targeting subunit, RGL, results in dissociation of PP1c, and inactivation of the phosphatase by I-1 (Hubbard & Cohen, 1989). By analogy, phosphorylation of MYPT1 by protein kinase C (PKC) may perform a similar function in regulating the interactions between MYPT1 and PP1c (Feng et al. 1999; Toth et al. 2000). Although the physiological significance of altered MLCP regulation is not known with certainty, myosin phosphatase activity probably plays a paramount role in certain pathological states (Solaro, 2000). I-1 is an endogenous smooth muscle phosphatase inhibitor that is inactive when dephosphorylated. However, when activated by PKA (Cohen, 1989) or PKG (Hemmings et al. 1984; Tokui et al. 1996) phosphorylated I-1 specifically inhibits type-1 phosphatase activity. Regulation of myosin phosphatase activity has also been suggested as an important mechanism in the Ca2+-sensitizing properties of agents such as cGMP (Lee et al. 1997). Activation of I-1 by PKG suggests a possible role for I-1 in this effect, but this remains unconfirmed in vivo. In vitro experiments using the purified myosin phosphatase holoenzyme have suggested that the MLCP is relatively insensitive to I-1 (Alessi et al. 1992). Contrary to these results, activated (phosphorylated) I-1 has been shown to inhibit myosin phosphatase activity (Mitsui et al. 1992). This finding is supported by contractility experiments, performed in β-escin-skinned smooth muscle cells (Tokui et al. 1996), which demonstrated that phosphorylated I-1 enhanced contraction at submaximal [Ca2+], suggesting that I-1 may play a role in regulating the Ca2+ sensitivity of smooth muscle phosphatase activity in vivo. These results seemingly lead to an apparent paradox, since β-adrenergic receptor stimulation, which results in phosphorylation of I-1, is generally associated with smooth muscle relaxation. Oliver & Shenolikar (1998) suggest that I-1 phosphorylation may decrease type-1 phosphatase activity against the inhibitory phosphorylation site on MLCK (Conti & Adelstein, 1981). This would decrease myosin phosphorylation, and provides a path for I-1 phosphorylation in β-adrenergic receptor-mediated relaxation of smooth muscle. However, evidence to support such a conclusion is lacking. Thus, the role of I-1 in the regulation of smooth muscle contractility in vivo is not known with certainty. The potential importance of I-1 in regulating smooth muscle phosphatase activity is underscored by two other mechanisms known to regulate the myosin phosphatase. These involve the phosphorylation of MLCP by Rho-kinase (Fujita et al. 1995; Kimura et al. 1996) or inhibition of MLCP by the ∼17 kDa PKC-activated protein phosphatase inhibitor CPI-17 (Li et al. 1998), each representing unique mechanisms for inhibiting myosin phosphatase activity in vivo (Somlyo & Somlyo, 2000). These studies suggest that phosphatase regulation via endogenous inhibitor proteins, such as I-1, may represent novel and potentially important participants in smooth muscle function. To assess the role of I-1 in smooth muscle contractility, we have examined contractility in tonic (aorta) and phasic (portal vein) muscle from WT and I-1(−/−) mice, in response to a variety of electromechanical and pharmacomechanical agonists. Our data suggest that I-1 ablation does not affect contractility in aorta, but may play a subtle role in regulating β-adrenergic receptor responsiveness in the portal vein.