Cholesterol Depletion Impairs Vascular Reactivity to Endothelin-1 by Reducing Store-Operated Ca ²⁺ Entry Dependent on TRPC1

Abstract

Abstract-The reactivity of the vascular wall to endothelin-1 (ET-1) is influenced by cholesterol, which is of possible importance for the progression of atherosclerosis. To elucidate signaling steps affected, the cholesterol acceptor methyl-␤-cyclodextrin (m␤cd, 10 mmol/L) was used to manipulate membrane cholesterol and disrupt caveolae in intact rat arteries. In endothelium-denuded caudal artery, contractile responsiveness to 10 nmol/L ET-1 (mediated by the ET A receptor) was reduced by m␤cd and increased by cholesterol. Neither ligand binding nor colocalization of ET A and caveolin-1 was affected by m␤cd. Ca 2ϩ inflow via store-operated channels after depletion of intracellular Ca 2ϩ stores was reduced in m␤cd-treated caudal arteries, as shown by Mn 2ϩ quench rate and intracellular [Ca 2ϩ ] response. Expression of TRPC1, 3, and 6 was detected by reverse transcriptase-polymerase chain reaction, and colocalization of TRPC1 with caveolin-1 was reduced by m␤cd, as seen by immunofluorescence. Part of the contractile response to ET-1 was inhibited by Ni 2ϩ (0.5 mmol/L) and by a TRPC1 blocking antibody. In the basilar artery, exhibiting less store-operated channel activity than the caudal artery, ET-1-induced contractions were insensitive to the TRPC1 blocking antibody and to m␤cd. Increased store-operated channel activity in basilar arteries after organ culture correlated with increased sensitivity of ET-1 contraction to m␤cd. These results suggest that cholesterol influences vascular reactivity to ET-1 by affecting the caveolar localization of TRPC1. Key Words: arterial smooth muscle Ⅲ methyl-␤-cyclodextrin Ⅲ caveolae Ⅲ endothelin Ⅲ store-operated Ca 2ϩ channels H ypercholesterolemia increases reactivity to endothelin-1 (ET-1) in experimental animals and humans. [1][2][3][4] This has been pointed out as one possible factor in the progression of atherosclerosis. [5][6][7] The mechanism of action has not been elucidated, although both endothelial dysfunction and altered smooth muscle reactivity have been proposed. 5 Lipoprotein particles may directly influence endothelial membrane-associated endothelial NO synthase activity by interfering with cholesterol-rich domains referred to as caveolae. 8 Although these effects modulate the endothelial influence on vascular tone, less is known regarding direct effects of cholesterol on vascular smooth muscle functions.Caveolae are 50-to 100-nm membrane invaginations that integrate many cellular receptor functions. 9 For instance, ET A receptors expressed in COS cells colocalize with the caveolae-associated protein caveolin. 10,11 The caveolar structure is disrupted after depletion of cholesterol with cyclodextrins, 12 and this correlates with a decreased contractility to ET-1, but not to depolarization or ␣ 1 -receptor stimulation, in endothelium-denuded rat caudal arteries. 13 Cholesterol might thus modulate the strength of caveolae-associated signaling, providing a basis for altered contractility in response to ET-1.Activation of the ET A receptor stimulates Ca 2ϩ inflow ov...

Effects of caveolin-1 and P-ERK1/2 on Ang II-induced glomerular mesangial cell proliferation

“…It was reported that PD98059 (5 Â 10 À5 mol/L) and MbCD (10 À2 mol/L) did not produce toxic effects on cells but inhibited them. 14,15 Furthermore, both agents inhibited Ang II-induced GMC proliferation, suggesting that p-ERK1/2 and caveolin-1 were involved in regulating Ang II-induced GMC proliferation.…”

Enhanced expression of transient receptor potential channels in idiopathic pulmonary arterial hypertension

“…In rat aorta, antisense inhibition of TRPC6 also markedly inhibited the ␣ 1 -adrenoreceptor-mediated increase in [Ca 2ϩ ] cyt (53). Therefore, which TRPC isoform(s) form ROC or SOC may depend on (i) cell type (e.g., TRPC6 may be involved in forming SOC in PASMC, but ROC in aortic smooth muscle cells), (ii) composition (or ratio) of different TRP subunits in the heterotetram (e.g., a channel made up of one TRPC3 and three TRPC6 may be a ROC and a channel made of one TRPC1, two TRPC3, and one TRPC6 may be a SOC), (iii) heteromultimeric assembly of functional channels (e.g., a ROC-TRPC6 may form heterologous SOC with SOC-TRPCs like TRPC3 and TRPC7) (44,45,47,48,51,52), and (iv) localization or compartmentalization (e.g., ligand-receptor interaction and store depletion can activate a TRPC1-dominant channel in caveolae) (30). In addition, human TRP genes often show tissue-specific expression patterns and likely encode both ROC and SOC (29).…”

“…A central aspect of pulmonary vascular remodeling is medial hypertrophy caused by sustained pulmonary vasoconstriction (2-4), excessive pulmonary artery smooth muscle cell (PASMC) proliferation (5), and inhibited PASMC apoptosis (6, 7), resulting in a narrowed vascular lumen and increased PVR. Although its etiology remains unclear, elevated levels of circulating mitogens, dysfunction or down-regulation of receptors and ion channels, upregulation of transporters, and heightened activity of elastases and glycoproteins have been implicated in IPAH (5,6,(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) Transient receptor potential (TRP) channel genes may encode subunits that form receptor-(ROC) and store-(SOC) operated Ca 2ϩ channels in many cell types, including PASMC and pulmonary artery endothelial cells (PAEC) (28,(30)(31)(32)(33)(34). Ca 2ϩ entry through ROC and SOC increases [Ca 2ϩ ] cyt , allowing for phosphorylation of signal transduction proteins and transcription factors (23,24,(35)(36)(37)(38), that are essential for the progression of the cell cycle (21).…”

“…Transient receptor potential (TRP) channel genes may encode subunits that form receptor-(ROC) and store-(SOC) operated Ca 2ϩ channels in many cell types, including PASMC and pulmonary artery endothelial cells (PAEC) (28,(30)(31)(32)(33)(34). Ca 2ϩ entry through ROC and SOC increases [Ca 2ϩ ] cyt , allowing for phosphorylation of signal transduction proteins and transcription factors (23,24,(35)(36)(37)(38), that are essential for the progression of the cell cycle (21).…”

Regulation of Pulmonary Vasoconstriction by Agonists and Caveolae

“…In smooth muscle and endothelial cells, caveolae are important sites for membrane receptors and channels to associate closely with the endo-plasmic/ sarcoplasmic reticulum [12] and for ligand-mediated Ca 2+ entry through ROC and SOC [13][14][15]. Intracellular messenger pathways regulated by cAMP and Ca 2+ are tightly integrated and functionally colocal-ized in caveolae [16,17].…”