Actions Downstream of Cyclic GMP/Protein Kinase G Can Reverse Protein Kinase C-mediated Phosphorylation of CPI-17 and Ca2+Sensitization in Smooth Muscle

Abstract

Ca2؉ sensitivity of smooth muscle contraction is modulated by several systems converging on myosin light chain phosphatase (MLCP). Rho-Rho kinase is considered to inhibit MLCP via phosphorylation, whereas protein kinase C (PKC) induced sensitization has been shown to be dependent on phosphorylation of the inhibitory protein CPI-17. We have explored the interaction of cGMP-dependent protein kinase (PKG) with Ca 2؉ sensitization pathways using permeabilized mouse smooth muscle. Three conditions giving ϳ50% of maximal active force were compared in small intestinal preparations: 1) Ca 2؉ -activated unsensitized muscle (pCa 5.9 with Rho kinase inhibitor Y27632); 2) Rho-Rho kinase-sensitized muscle (pCa 6.1 with guanosine 5-3-O-(thio)triphosphate); and 3) PKC-sensitized muscle (pCa 6.0 with Y27632 and PKC activator phorbol 12,13-dibutyrate). 8-Br-cGMP relaxed the sensitized muscles but had marginal effects on unsensitized preparations, showing that PKG reverses both PKC and Rho-mediated Ca 2؉ sensitization. CPI-17 was present in permeabilized intestinal tissue. In PKC-sensitized preparations, CPI-17 phosphorylation decreased in response to 8-Br-cGMP. The rate of PKC-mediated phosphorylation in the presence of the MLCP inhibitor microcystin-LR was not influenced by 8-Br-cGMP. PKC-induced Ca 2؉ sensitization also was reversed in vascular smooth muscle tissues (portal vein and femoral artery). We conclude that actions downstream of cGMP/PKG can reverse PKC-mediated phosphorylation of CPI-17 and Ca 2؉ sensitization in smooth muscle.Smooth muscle contraction involves a rise in intracellular [Ca 2ϩ ]. The Ca 2ϩ -calmodulin complex activates myosin light chain kinase, which phosphorylates the 20-kDa myosin regulatory light chains (MLC 20 MLCP (PP1M) belongs to the PP1 group of phosphatases and is a heteromeric enzyme consisting of three subunits: a catalytic subunit (PP1M C ); a myosin-targeting subunit (MYPT1); and the M20 subunit (5). MLCP has been reported to be inhibited directly by phosphorylation of its myosin-targeting subunit, MYPT1, on residue Thr-695 performed by Rho kinase (6), MYPT1-associated kinase (sometimes referred to as ZIP-like kinase) (7), and integrin-linked kinase (8). Phosphorylation of MYPT1 at Thr-34 by protein kinase C (PKC) has been shown in vitro (9); the relevance of this result and the effect on the holoenzyme are unknown. MLCP is blocked and inhibited by the binding of the phosphorylated form of protein kinase Cpotentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kDa (CPI-17). CPI-17 is a major target for phosphorylation by PKC, which phosphorylates the Thr-38 residue. This process has been shown to be crucial for PKCmediated increase in Ca 2ϩ sensitivity of force (Ca 2ϩ sensitization) in smooth muscle (10,36,43). In vitro, Thr-38 on CPI-17 is also phosphorylated by Rho kinase (11), MYPT1-associated kinase (12), protein kinase N (13), integrin-linked kinase (14), p21-activated protein kinase (15), cAMP-activated protein kinase A (16), and cGMP-activated pro...

Increased Rho activation and PKC-mediated smooth muscle contractility in the absence of caveolin-1

“…To examine whether Ca 2ϩ sensitization might be affected in the absence of caveolae, we used ␣-toxin permeabilized smooth muscle. In the ileum, Rho/Rho-kinase mediated modulation of Ca 2ϩ sensitivity is prominent and well characterized (8,18,33). The pCa-force relationships were identical in ␣-toxin permeabilized strips from WT and KO (Fig.…”

“…In the present study, using KO mice, we investigate whether PKC and RhoA/Rho-kinase-mediated contractile mechanisms depend on caveolae. We have used longitudinal smooth muscle from the small intestine, which shows robust RhoA/Rhokinase-mediated Ca 2ϩ sensitization, but weak sensitization in response to PKC activation with phorbol ester (8,21,33), and femoral artery, which displays prominent Ca 2ϩ sensitization in response to phorbol ester (8, 38). …”

“…Femoral artery segments (2 mm long, cut as spiral strips from the media) were mounted on two 30 m steel wires (31). All preparations were mounted horizontally between two tungsten wires, one of which was attached to a force transducer (model AE 801, SensoNor, Horten, Norway) and the other to a micrometer screw (8). Experiments were performed on "bubble plates" (140 l solution) with stirring.…”

“…The intestine and femoral artery were removed and placed in cold HEPES-buffered physiological saline solution (for composition, see below). Strips from the outer longitudinal small intestinal muscle layer were obtained by tearing the longitudinal layer off from the underlying circular layer (8).…”

Beyond the dogma: novel 2-adrenoceptor signalling in the airways

“…These events correlate with elevated levels of cGMP, increased MLCP activity and a concomitant loss of CPI-17 phosphorylation [72]; the cGMP analogue, 8-bromo-cGMP, is also reported to reduce CPI-17 phosphorylation ( fig. 4) [73]. Taken together, these data are potentially significant as cAMP can cross-activate PKG in smooth muscle ( fig.…”