New Roads Leading to Ca2+ Sensitization
Gprotein–coupled receptors (GPCRs) activated by a wide variety of agonists can switch on myosin light chain phosphorylation and force in smooth muscle. The extent of myosin light chain phosphorylation reflects activity of both the Ca2+/calmodulin–dependent myosin light chain kinase (MLCK) and the myosin light chain phosphatase. Thus, at constant Ca2+ and MLCK activity, processes that inhibit myosin phosphatase activity cause a leftward shift of the Ca2+ force-response curve, a physiological relevant phenomenon known as Ca2+ sensitization.1,2⇓ There are two well-described myosin phosphatase inhibitory pathways. The first is through the small GTPase RhoA, in which GTP-bound RhoA translocates to the membrane and activates Rho-kinase that either directly or indirectly acts on the regulatory phosphatase subunit (MYPT-1) to inhibit phosphatase activity. The second signaling pathway, which is present in only some smooth muscles, is through phosphorylation of a phosphatase inhibitor, CPI-17 that, when phosphorylated, potently inhibits the catalytic subunit of myosin phosphatase.3 Crosstalk between the two pathways has been implicated from experiments using the Rho-kinase inhibitor Y-27632.4 A new signaling messenger of Ca2+ sensitization, which converges on the RhoA pathway at the level of Rho-kinase, is presented by Kobayashi and colleagues5 in this issue of Circulation Research: they demonstrate that sphingosylphosphorylcholine (SPC), a product of sphingomyelin deacylation, leads to an increase in force in the absence of an increase in the fura 2 Ca2+ signal. This force is inhibited by a Rho-kinase inhibitor, but not by inhibition of conventional or novel PKCs. Interestingly, the Ca2+-independent contraction was maintained for ≈2 hours, even after removal of SPC from the bathing medium, and was completely and reversibly relaxed by Y-27632: subsequent stimulation with 118 mmol/L K+ induced a normal increase in [Ca2+] and contraction. The redevelopment of force after a quick-release step during the maintained contraction indicated that SPC did not interfere with the ability of myosin crossbridges to hydrolyze MgATP and cycle normally. Provided that SPC is indeed released into the subarachnoid space, this study, carried out on cerebral arteries, has important implications for the therapy of cerebral vasospasm, a major health risk in some populations. Further studies on SPC production, regulation, and misregulation should establish its significance in normal and/or pathological states.
Apart from their metabolic roles, lysophospholipids such as lysophosphatidic acid (LPA), sphingosine 1-phosphate (S1P), and sphingosylphosphorylcholine have long been known to be associated with cell growth and differentiation but are now also recognized as important signaling molecules functioning in a variety of intracellular pathways that target cell migration, cell growth, and cell survival (antiapoptosis), through effects on such processes as calcium mobilization, inhibition of adenylate cyclase, and activation of mitogen-activated protein kinases.6–9⇓⇓⇓ S1P has also been implicated in cerebral vasospasm.10 It has been suggested6 that lysophospholipids are likely to be as important a class of intracellular signaling molecules as the glycerophospholipid metabolites, which give rise to inositol 1,4,5-trisphosphate (InsP3), diacylglycerol, phosphatidic acid, and arachidonic acid.
A major advance in this still young field has been the identification and cloning of GPCRs for some of the lysophospholipids. Five of the eight GPCRs, commonly called Edg receptors for endothelial differentiation gene, bind S1P with nanomolar affinities and SPC ≈10- to 100-fold less efficiently. The remaining three receptors preferentially bind LPA. (For a review of current nomenclature and structure activity relationships of lysophospholipid messengers, see References 9 and 11.) SPC is reported to be a high-affinity ligand (KD=33 nmol/L) for the ovarian cancer G protein–coupled receptor 1 (OGR1); it is expressed in several tissues and upon activation leads to Ca2+ mobilization and activation of p42/44 mitogen-activated protein kinases.12 A second closely related GPCR, also claimed to be activated by SPC, GPR4, has also been identified.11 Surprisingly, SPC-mediated Ca2+ sensitization in the cerebral artery described in the present study5 does not appear to signal through a GPCR. This conclusion was largely based on the finding that, unlike agonist-induced Ca2+ sensitization, SPC does not require the addition of GTP to the permeabilized smooth muscles, nor was SPC-induced Ca2+ sensitization affected by the nonhydrolyzable GDPβS, which inhibits signaling by GPCRs. Some of these same investigators have also reported similar effects of SPC (1 to 50 μmol/L) on intact, denuded of endothelium, and permeabilized pig coronary artery smooth muscle.13 On the other hand, in intact endothelial cells, SPC and S1P increase cytoplasmic [Ca2+] through release of intracellular calcium as well as Ca2+ influx.14,15⇓ SPC also increases production of NO, leading to endothelium-dependent vasorelaxation with an EC50=5 μmol/L.14 The different concentrations used by both groups make it probable that Ca2+ mobilization is through lysophospholipid GPCRs, whereas the activation of NO may be through direct action of SPC on the eNOS/NO pathway, because SPC readily crosses cell membranes. Evaluation of the contributions of SPC as a signaling molecule in vascular biology will require determination of the intracellular and extracellular concentrations of SPC in normal and pathological states.
If SPC, as suggested, directly activates Rho-kinase, it may be that it interacts with the putative negative regulatory region of Rho-kinase, possibly the pleckstrin homology domain (PH), based on consideration of the effects of arachidonic acid in vitro.16–18⇓⇓ In this model, the Rho binding domain (RB) and PH domains interact with the catalytic domain to inactivate the kinase (Figure). The enzyme is thought to be activated by the binding of RhoA · GTP to RB, thereby releasing the inhibition of the catalytic site. Perhaps SPC and other lipid messengers activate Rho-kinase through a similar mechanism, releasing the inhibitory from the catalytic domain through a conformational change and exposing the catalytic site. Direct activation of Rho-kinase by lipid messengers should not be inhibited by upstream inactivation of RhoA by the bacterial exoenzyme C3, whereas an inhibitor of SPC production (eg, sphingomyelin deacylation) would be expected to inhibit vasospasm due to subarachnoid hemorrhage.19 These experiments remain to be performed.
In summary, these and other studies revealing the activities of intracellular and extracellular phospholipid mediators point to new mechanisms of signaling and potential new therapeutic targets for pathophysiological states of the cardiovascular system.
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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- ↵Eto M, Ohmori T, Suzuki M, Furuya K, Morita F. A novel protein phosphatase-1 inhibitory protein potentiated by protein kinase C: isolation from porcine aorta media and characterization. J Biochem. 1995; 118: 1104–1107.
- ↵Kitazawa T, Eto M, Woodsome TP, Brautigan DL. Agonists trigger G protein-mediated activation of the CPI-17 inhibitor phosphoprotein of myosin light chain phosphatase to enhance vascular smooth muscle contractility. J Biol Chem. 2000; 275: 9897–9900.
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- ↵Sato M, Tani E, Fujikawa H, Kaibuchi K. Involvement of Rho-kinase mediated phosphorylation of myosin light chain in enhancement of cerebral vasospasm. Circ Res. 2000; 87: 195–200.
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