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Review
. 2018 Jul;153:91-122.
doi: 10.1016/j.bcp.2018.02.012. Epub 2018 Feb 13.

Evolving Mechanisms of Vascular Smooth Muscle Contraction Highlight Key Targets in Vascular Disease

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Free PMC article
Review

Evolving Mechanisms of Vascular Smooth Muscle Contraction Highlight Key Targets in Vascular Disease

Zhongwei Liu et al. Biochem Pharmacol. .
Free PMC article

Abstract

Vascular smooth muscle (VSM) plays an important role in the regulation of vascular function. Identifying the mechanisms of VSM contraction has been a major research goal in order to determine the causes of vascular dysfunction and exaggerated vasoconstriction in vascular disease. Major discoveries over several decades have helped to better understand the mechanisms of VSM contraction. Ca2+ has been established as a major regulator of VSM contraction, and its sources, cytosolic levels, homeostatic mechanisms and subcellular distribution have been defined. Biochemical studies have also suggested that stimulation of Gq protein-coupled membrane receptors activates phospholipase C and promotes the hydrolysis of membrane phospholipids into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 stimulates initial Ca2+ release from the sarcoplasmic reticulum, and is buttressed by Ca2+ influx through voltage-dependent, receptor-operated, transient receptor potential and store-operated channels. In order to prevent large increases in cytosolic Ca2+ concentration ([Ca2+]c), Ca2+ removal mechanisms promote Ca2+ extrusion via the plasmalemmal Ca2+ pump and Na+/Ca2+ exchanger, and Ca2+ uptake by the sarcoplasmic reticulum and mitochondria, and the coordinated activities of these Ca2+ handling mechanisms help to create subplasmalemmal Ca2+ domains. Threshold increases in [Ca2+]c form a Ca2+-calmodulin complex, which activates myosin light chain (MLC) kinase, and causes MLC phosphorylation, actin-myosin interaction, and VSM contraction. Dissociations in the relationships between [Ca2+]c, MLC phosphorylation, and force have suggested additional Ca2+ sensitization mechanisms. DAG activates protein kinase C (PKC) isoforms, which directly or indirectly via mitogen-activated protein kinase phosphorylate the actin-binding proteins calponin and caldesmon and thereby enhance the myofilaments force sensitivity to Ca2+. PKC-mediated phosphorylation of PKC-potentiated phosphatase inhibitor protein-17 (CPI-17), and RhoA-mediated activation of Rho-kinase (ROCK) inhibit MLC phosphatase and in turn increase MLC phosphorylation and VSM contraction. Abnormalities in the Ca2+ handling mechanisms and PKC and ROCK activity have been associated with vascular dysfunction in multiple vascular disorders. Modulators of [Ca2+]c, PKC and ROCK activity could be useful in mitigating the increased vasoconstriction associated with vascular disease.

Keywords: Blood vessels; Calcium; Channels; Protein kinase; Sarcoplasmic reticulum; Signaling.

Conflict of interest statement

CONFLICT OF INTEREST

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Figures

Fig. 1
Fig. 1
Mechanisms of VSM contraction. A vasoconstrictor agonist (A) binding to its receptor (R) is coupled to heterotrimeric GTP-binding protein (Gq) and activates phospholipase C (PLCβ) which stimulates the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). The agonist also activates phospholipase D (PLD) which hydrolyzes phosphatidylcholine (PC) into choline and DAG. IP3 stimulates Ca2+ release from sarcoplasmic reticulum (SR). The agonist also stimulates Ca2+ influx through Ca2+ channels. Ca2+ binds calmodulin (CaM), activates MLC kinase (MLCK), causes MLC phosphorylation, and initiates VSM contraction. DAG, phosphatidylserine (PS), and Ca2+ (for cPKCs) cause activation and translocation of PKC. PKC inhibits K+ channels leading to membrane depolarization and activation of voltage-dependent Ca2+ channels. PKC phosphorylates CPI-17, which in turn inhibits MLC phosphatase and enhances the myofilament force sensitivity to Ca2+. PKC phosphorylates calponin (CaP), allowing more actin to bind myosin. PKC may activate a protein kinase cascade involving Raf, MAPK kinase (MEK), and MAPK (ERK1/2), leading to phosphorylation of the actin-binding protein caldesmon (CaD). DAG is transformed by DAG lipase into arachidonic acid (AA), and activation of phospholipase A2 (PLA2) increases the hydrolysis of phosphatidylethanolamine (PE) into AA, which in turn inhibits MLC phosphatase. Agonist-induced activation of RhoA/ROCK also inhibits MLC phosphatase and further enhances Ca2+ sensitivity of the contractile proteins. Dashed line indicates inhibition.
Fig. 2
Fig. 2
Ca2+ mobilization and Ca2+ removal mechanisms in VSM. Agonist (A)-receptor (R) interaction causes Ca2+ release from sarcoplasmic reticulum (SR) in response to 1,4,5-inositol trisphosphate (IP3) and to Ca2+ via Ca2+-induced Ca2+ release (CICR). VSMC activation also stimulates Ca2+ influx through nonspecific Ca2+ leak, voltage-dependent Ca2+ channels (VDCC), receptor-operated channels (ROC), transient receptor potential (TRP) channels, and stretch-activated channels. Depletion of intracellular Ca2+ stores in SR causes the release of stromal interaction molecule (STIM1) which in turn stimulates Orai1 store-operated Ca2+ channels. The increased intracellular Ca2+ is taken up by SR Ca2+-ATPase (SERCA) or extruded by the plasmalemmal Ca2+-ATPase (PMCA) or Na+-Ca2+ exchanger (NCX), and the resulting excess Na+ is extruded via Na+/K+ pump and Na+/H+ exchanger. At very high and pathological increases in intracellular Ca2+, the mitochondria play a role in Ca2+ uptake and homeostasis. When Ca2+ is taken up by mitochondria, HPO42− is also taken up via HPO42−:2OH exchange and calcium phosphate is formed. Under favorable conditions and when Ca2+ can be handled by SERCA, PMCA and NCX, mitochondrial Ca2+ is slowly released via a Ca2+ efflux pathway involving a Ca2+:2H+ or Ca2+:2Na+ antiporter. PIP2, phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C; DAG, diacyglycerol
Fig. 3
Fig. 3
PKC structure and isoforms. PKC comprises a N-terminal regulatory domain and a C-terminal catalytic domain, connected by a V3 hinge region. The regulatory domain contains two conserved C1 and C2 regions, and the pseudosubstrate region. The catalytic or kinase activity domain contains a C3 ATP-binding site and a C4 binding site for the substrate. The catalytic domain also contains phosphorylation sites in the activation loop, turn-motif and hydrophobic-motif (The figure illustrates PKCδ phosphorylation sites, which vary in different PKCs). The PKC family is classified into conventional cPKCs α, βI, βII, and γ; novel nPKCs δ, ε, η and θ; and atypical aPKCs ζ and ι/λ isoforms. cPKCs consist of 4 conserved (C1–C4) and 5 variable regions (V1–V5) and are activated by DAG, PS and Ca2+. The C1 region binds phosphatidylserine (PS), DAG, and phorbol esters, and the C2 region contains the binding site for Ca2+. PS can also bind to the C2 region. Both cPKCs and nPKCs have twin C1 regions (C1A and C1B) and a C2 region, but the order of C1 and C2 regions is switched in nPKCs compared to cPKCs. The nPKCs have a variant form of C2 region that is insensitive to Ca2+, but still binds lipids. The aPKCs do not have a C2 region and hence not activated by Ca2+, and have a variant form of C1 that is not duplicated, but retains lipid-binding activity and sensitivity to PS. The aPKCs also have a protein–protein-interacting region Phox and Bem 1 (PB1) that controls their cellular localization. Other related kinases include PKCμ (PKD). PKC inhibitors compete with DAG at the C1 region (calphostin C), ATP at the ATP-binding site (H-7, staurosporine) or the PKC true substrate (pseudosubstrate inhibitor peptide).
Fig. 4
Fig. 4
Activation, translocation, substrate interaction and deactivation of cPKCs. In the PKC cytosolic and inactive state, the pseudosubstrate binds the catalytic site in the C4 region, and the regulatory and catalytic domains are folded. Before it becomes catalytically competent, nascent PKC undergoes phosphorylation at three phosphorylation sites. Phosphorylation of the activation loop by phosphoinositide-dependent kinase (PDK) introduces a negative charge that properly aligns residues to form a competent catalytic domain, facilitate subsequent autophosphorylation at the turn motif and hydrophobic motif, and keep PKC in a catalytically competent and protease resistant conformation. Phosphate groups are indicated as green ovals labeled “P”. PKC activators such as PS, DAG, phorbol esters, and Ca2+ promote allosteric activation, translocation of PKC to the plasma membrane, and subsequent interaction with the substrate. Allosteric activation also induces an open conformation state, making PKC susceptible to phosphatases and proteases and allows PKC to either enter an autophosphorylation/dephosphorylation cycle, or undergo proteolytic degradation. PKC dephosphorylation terminates its kinase activity and is carried out by the PP2C member pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP) at the hydrophobic motif, which starts the process that consequently drives further dephosphorylation of PKC by PP1/PP2A protein phosphatases at the turn motif. Dephosphorylation also predisposes “naked” PKC to ubiquitination and degradation, leading to de novo synthesis and regeneration of the enzyme.
Fig. 5
Fig. 5
Structure and activation of ROCKs. (A) ROCK amino acid sequence comprises a kinase domain located at the N-terminus, a coiled-coil region containing the Rho-binding domain, and a pleckstrin-homology domain (PHD) with a cysteine-rich domain (CRD). ROCK-1 and ROCK-2 are highly homologous with an overall amino acid sequence identity of 65%. (B) In the inactive form, the C-terminus region of ROCK is folded over the N-terminus region, allowing the autoinhibitory region to block the kinase site. Binding of activated GTP-bound RhoA causes unfolding and activation of ROCK, and thereby exposes the kinase domain and allows phosphorylation of the true substrate.

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