Role of Ca ²⁺ -Independent Phospholipase A ₂ and Store-Operated Pathway in Urocortin-Induced Vasodilatation of Rat Coronary Artery

Abstract

Abstract-Urocortin has been shown to produce vasodilatation in several arteries, but the precise mechanism of its action is still poorly understood. Here we demonstrate the role of store operated Ca 2ϩ entry (SOCE) regulated by Ca 2ϩ -independent phospholipase A 2 (iPLA 2 ) in phenylephrine hydrochloride (PE)-induced vasoconstriction, and we present the first evidence that urocortin induces relaxation by the modulation of SOCE and iPLA 2 in rat coronary artery. Urocortin produces an endothelium independent relaxation, and its effect is concentrationdependent (IC 50 Ϸ4.5 nmol/L). We show in coronary smooth muscle cells (SMCs) that urocortin inhibits iPLA 2 activation, a crucial step for SOC channel activation, and prevents Ca 2ϩ influx evoked by the emptying of the stores via a cAMP and protein kinase A (PKA)-dependent mechanism. Lysophophatidylcholine and lysophosphatidylinositol, products of iPLA 2 , exactly mimic the effect of the depletion of the stores in presence of urocortin. Furthermore, we report that long treatment with urocortin downregulates iPLA 2 mRNA and proteins expression in rat coronary smooth muscle cells. In summary, we propose a new mechanism of vasodilatation by urocortin which involves the regulation of iPLA 2 and SOCE via the stimulation of a cAMP/PKA-dependent signal transduction cascade in rat coronary artery. Key Words: urocortin Ⅲ iPLA 2 Ⅲ vasoconstriction Ⅲ store operated Ca 2ϩ entry Ⅲ cAMP-PKA C oronary artery smooth muscle cells (SMCs) regulate vascular tone influencing perfusion of the heart, peripheral resistance, and as a consequence heart function. Agonist induces a contraction of vascular SMCs by a rise in cytosolic free Ca 2ϩ concentration 1,2 because of a rapid Ca 2ϩ release by InsP 3 from intracellular stores and a transmembrane Ca 2ϩ influx through L-type voltage-dependent Ca 2ϩ channels or nonvoltage-gated channels such as store-operated Ca 2ϩ (SOC) channels. The relative contribution of each channel depends on the smooth muscle type. [2][3][4] The use of selective inhibitors of sarcoplasmic reticulum Ca 2ϩ -ATPase pump, as thapsigargin (TG), to activate SOC channels not only increases Ca 2ϩ influx but also enhances tone in a variety of SMCs. 3,5 Recently we showed Ca 2ϩ -independent phospholipase A 2 (iPLA 2 ) to be a crucial determinant of storeoperated Ca 2ϩ entry (SOCE). We demonstrated that the emptying of the stores activated iPLA 2 and its lysophospholipid products opened the SOC channels in aortic SMCs and nonexcitable cells. 6 -8 Thus iPLA 2 became a potential physiological target for regulation and fine-tuning of SOCE by other signaling cascades in SMCs.A few years ago a new 40-aa peptide, urocortin, 9,10 related to corticotropin-releasing factor (CRF) was described as a new player in cardiac control, 11,12 and was proposed to protect cardiac myocytes during ischemia/reperfusion by downregulating iPLA 2 expression. 13,14 Urocortin also emerged as a potent vasodilator peptide, and its mechanism of action appears to be complex, eg, vasodilatation has been reported...

Urocortin increased endothelial ICAM1 by cPLA2-dependent NF-κB and PKA pathways in HUVECs

“…A full colour version of this figure is available via http:dx.doi.org/10.1530/ JME-13-0182. rat coronary artery (Smani et al 2007). Consistent with the results of Lawrence et al, Takatani-Nakase & Takahashi (2010) found that Ucn1 could significantly suppress the expression and activity of iPLA2, which results in prevention of the ischemia-induced cell death.…”

G protein-coupled estrogen receptor 1 mediates relaxation of coronary arteries via cAMP/PKA-dependent activation of MLCP

“…In rat mesenteric artery, vasoactive intestinal polypeptide caused relaxation through PKA-stimulated K ATP channels (73). Storeoperated Ca 2ϩ entry was also demonstrated to be involved in coronary artery relaxation mediated by a PKA-dependent mechanism under urocortin treatment (62). Thus, it seems that ion channels, especially BK Ca channels, play an important role in PKA-mediated vascular smooth muscle relaxation by lowering intracellular Ca .…”

Urocortin is a novel regulator of osteoclast differentiation and function through inhibition of a canonical transient receptor potential 1-like cation channel

“…However, other pathways have been reported to be activated by stimulation of CRFRs such as (ERK)-MAP kinase, AKT/protein kinase B and nitric oxide synthase-guanylyl cyclase (Grammatopoulos et al 2000, Cantarella et al 2001, Bayatti et al 2003, Karteris et al 2005. The intracellular signalling pathways activated by UCN in osteoclasts remain unknown, but intracellular Ca 2C signalling has been shown to be crucial for maintaining osteoclastic differentiation (Boyce & Xing 2007) and UCN has been shown to inhibit store-operated Ca 2C entry in vascular smooth muscle cells (Smani et al 2007, Negishi-Koga & Takayanagi 2009). Therefore, channels that regulate Ca 2C entry are potential candidates as a target for UCN in osteoclasts.…”

“…In other cell types, UCN has been shown to regulate cellular functions by inhibiting ion channels involved in promoting Ca 2C influx, such as voltage-dependent and -independent Ca 2C -permeable channels (Tao et al 2004, Smani et al 2007. Extracellular Ca…”

“…UCN has been shown to act predominantly through both G as and G aq G-protein subunits to stimulate diverse signalling cascades, through respectively, adenylate cyclase (AC) and phospholipase C (PLC) pathways, depending on the tissue type (Grammatopoulos et al 2000, Cantarella et al 2001, Graziani et al 2002, McEvoy et al 2002, Bayatti et al 2003, Karteris et al 2005. UCN-mediated activation of these signal transduction pathways modulates a variety of end effectors including ion channels involved in calcium signalling and energy metabolism, such as L-type voltage-gated calcium channels, store-operated channels and K ATP channels (Lawrence et al 2002, Tao et al 2004, Smani et al 2007.…”