Myosin light chain phosphatase (MLCP) plays a pivotal role in smooth muscle contraction by regulating Ca2؉ sensitivity of myosin light chain phosphorylation. A smooth muscle phosphoprotein called CPI-17 specifically and potently inhibits MLCP in vitro and in situ and is activated when phosphorylated at Thr-38, which increases its inhibitory potency 1000-fold. We produced a phosphospecific antibody for this site in CPI-17 and used it to study in situ phosphorylation of endogenous CPI-17 in arterial smooth muscle in response to agonist stimulation. In the intact femoral artery, CPI-17 phosphorylation was negligible at the resting state and was not increased during contraction induced by K ؉ depolarization. The Ca 2؉ -sensitizing agonists histamine and phenylephrine induced nearly equivalent contractions, but histamine generated significantly higher levels of CPI-17 phosphorylation. In ␣-toxin-permeabilized strips at pCa 6.7, contractile force and CPI-17 phosphorylation were proportional in response to histamine, guanosine 5-O-(␥-thiotriphosphate), and histamine plus guanyl-5-yl thiophosphate, implying that histamine increased CPI-17 phosphorylation through activation of G proteins. Inhibitors of Rho-kinase (Y27632) and protein kinase C (PKC; GF109203X) reduced contraction and CPI-17 phosphorylation in parallel, suggesting that CPI-17 functions downstream of Rho kinases and PKC. The results show that agonists such as histamine signal through phosphorylation of CPI-17 to produce Ca 2؉ sensitization of smooth muscle contraction.

Myosin phosphatase (MLCP) plays a critical regulatory role in the Ca2+ sensitivity of myosin phosphorylation and smooth muscle contraction. It has been suggested that phosphorylation at Thr695 of the MLCP regulatory subunit (MYPT1) and at Thr38 of the MLCP inhibitor protein CPI‐17 results in inhibition of MLCP activity. We have previously demonstrated that CPI‐17 Thr38 phosphorylation plays an important role in G‐protein‐mediated inhibition of MLCP in tonic arterial smooth muscle. Here, we attempted to evaluate the function of MYPT1 in phasic rabbit portal vein (PV) and vas deferens (VD) smooth muscles. Using site‐ and phospho‐specific antibodies, phosphorylation of MYPT1 Thr695 and CPI‐17 Thr38 was examined along with MYPT1 Thr850, which is a non‐inhibitory Rho‐kinase site. We found that both CPI‐17 Thr38 and MYPT1 Thr850 were phosphorylated in response to agonists or GTPγS concurrently with contraction and myosin phosphorylation in α‐toxin‐permeabilized PV tissues. In contrast, phosphorylation of MYPT1 Thr695 did not increase. Comparable results were also obtained in both permeabilized and intact VD. The Rho‐kinase inhibitor Y‐27632 and the protein kinase C (PKC) inhibitor GF109203X suppressed phosphorylation of MYPT1 Thr850 and CPI‐17 Thr38, respectively, in intact VD while MYPT1 Thr695 phosphorylation was insensitive to both inhibitors. These results indicate that phosphorylation of MYPT1 Thr695 is independent of stimulation of G‐proteins, Rho‐kinase or PKC. In the phasic PV, phosphorylation of CPI‐17 Thr38 may contribute towards inhibition of MLCP while the phasic visceral VD, which has a low CPI‐17 concentration, probably utilizes other Ca2+ sensitizing mechanisms for inhibiting MLCP besides phosphorylation of MYPT1 and CPI‐17.

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.

Phosphorylation of myosin light chain (MLC) and contraction of differentiated smooth muscle cells in vascular walls are regulated by Ca2+-dependent activation of MLC kinase, and by Rho-kinase- or protein-kinases-C-dependent inhibition of MLC phosphatase (MLCP). We examined regulatory pathways for MLC kinase and MLCP in cultured vascular smooth muscle cells (VSMCs), and for isometric force generation of VSMCs reconstituted in collagen fibers. Protein levels of RhoA, Rho-kinase and MYPT1 (a regulatory subunit of MLCP) were upregulated in cultured VSMCs, whereas a MLCP inhibitor protein, CPI-17, was downregulated. Endothelin-1 evoked a steady rise in levels of Ca2+, MLC phosphorylation and the contractile force of VSMCs, whereas angiotensin-II induced transient signals. Also, Thr853 phosphorylation of MYPT1 occurred in response to stimuli, but neither agonist induced phosphorylation of MYPT1 at Thr696. Unlike fresh aortic tissues, removal of Ca2+ or addition of voltage-dependent Ca2+-channel blocker did not inhibit contractions of reconstituted VSMC fibers induced by agonists or even high concentrations of extracellular K+ ions. Inhibitors of Ins(1,4,5)P3-receptor and Rho-kinase antagonized agonist-induced or high-K+-induced contraction in both reconstituted fibers and fresh tissues. These results indicate that both Ins(1,4,5)P3-induced Ca2+ release and Rho-kinase-induced MYPT1 phosphorylation at Thr853 play pivotal roles in MLC phosphorylation of cultured VSMCs where either Ca2+-influx or CPI-17-MLCP signaling is downregulated.