Modification of the cytoplasmic tails of the integrinIntegrin ␣ IIb ␤ 3 (GPIIb-IIIa) is one of the receptors on the cellular surface of platelets and megakaryocytes. It binds to various adhesive proteins including fibrinogen, von Willebrand factor, vitronectin, and fibronectin that contain a core amino acid sequence of arginine-glycine-aspartic acids (RGD). Binding of fibrinogen to ␣ IIb ␤ 3 leads to platelet aggregation and finally to thrombus formation at the injured vascular sites. A pivotal role of ␣ IIb ␤ 3 in hemostasis is supported by the clinical observation that the congenital deficiency of ␣ IIb ␤ 3 , Glanzmann's thrombasthenia, results in lifelong bleeding tendency (1). Whereas ␣ IIb ␤ 3 on resting platelets does not bind soluble fibrinogen, once platelets are activated, conformation of the extracellular domains of the ␣ IIb ␤ 3 is altered and its ligandbinding affinity is increased (affinity modulation) (2). This process of the inside-out signaling is considered to be mediated by modification of the short cytoplasmic tails of ␣ IIb and ␤ 3 subunits; however, the mechanism remains to be elucidated.The nuclear magnetic resonance structural analysis of the ␣ IIb cytoplasmic tail revealed a closed conformation where the highly conserved N-terminal membrane-proximal region forms an ␣-helix followed by a turn, and the acidic C-terminal loop interacts with the N-terminal helix (3). Deletion of almost the entire ␣ IIb -cytoplasmic tail and mutations in its N-terminal sequence (GFFKR) conserved among the integrin ␣ subunits enhance the affinity of ␣ IIb ␤ 3 for ligands (4 -6). The cytoplasmic tail of the ␤ 3 subunit also has an amino acid sequence that is conserved among integrin ␤ subunits: a stretch of 8 amino acids (KLLITIHD) adjacent to the transmembrane domain. In a similar fashion to the ␣ IIb subunit, deletion or mutation in this conserved region induces activation of ␣ IIb ␤ 3 (6, 7). These observations suggest that membrane-proximal regions of the cytoplasmic domains of both subunits exert a negative regulatory function and lock ␣ IIb ␤ 3 in a low affinity state. Negative regulation may be mediated by the interaction between ␣ IIb and ␤ 3 cytoplasmic tails, possibly through a salt bridge between Arg-995 in ␣ IIb and Asp-723 in ␤ 3 (6), or binding of intracellular proteins to ␣ IIb and/or ␤ 3 subunits. Two candidates for the modulator proteins have been reported: calcium-and integrinbinding protein (CIB) 1 (8) and ␤ 3 -endonexin (9, 10), which bind to ␣ IIb and ␤ 3 cytoplasmic tails, respectively. Although CIB is unlikely to have a regulatory effect on ␣ IIb ␤ 3 ligand binding function (11), ␤ 3 -endonexin fused to GST protein induces the conformational change of ␣ IIb ␤ 3 and activates it when co-transfected with ␣ IIb and ␤ 3 subunits in Chinese hamster ovary cells. Another mechanism of modification has been recently suggested: an interaction between cytoplasmic tails of ␣ IIb ␤ 3 and the actin cytoskeleton. ␣ IIb ␤ 3 and the actin cytoskeleton are physically linked by binding of talin to the ␤ 3 cy...

Abstract. We evaluated the effect of alacepril, CV-11974, and spironolactone on the production of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-alpha (TNF-α) in cultured human peripheral blood mononuclear cells stimulated with angiotensin (Ang) II. Alacepril, CV-11974, and spironolactone significantly reduced the enhanced production of MCP-1 and TNF-α induced by exogenous Ang II. Specifically, 10 µM of spironolactone significantly reduced cytokine production, compared to the same dose of alacepril or CV-11974. These findings indicate that spironolactone may contribute to ameliorate the prognosis of patients with cardiovascular diseases by reducing Ang II-induced inflammation, although further exploration including determining the mechanisms would be required.

Abstract. Previously, we reported that spironolactone reduced cytokine production in cultured human peripheral blood mononuclear cells (PBMCs) with angiotensin (Ang) II stimulation. To address the mechanisms underlying this effect, we examined the contribution of aldosterone to cytokine production in cultured human PBMCs with Ang II stimulation. PBMCs expressed the messenger RNA (mRNA) of Ang II type 1 receptor (AT1R) and mineralocorticoid receptor (MR) both spontaneously and after Ang II stimulation, but expressed Ang II type 2 receptor (AT2R) under neither condition. After 24 h of incubation, exogenous Ang II induced the expression of CYP11B2 (a key enzyme of aldosterone synthesis) mRNA and caused aldosterone synthesis. CV-11974 (an AT1R antagonist) reduced Ang II-induced aldosterone synthesis, whereas PD-123319 (an AT2R antagonist) had no effect. The concentration of aldosterone peaked earlier than those of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α). After 48 h of incubation (under the influence of synthesized aldosterone), CV-11974 and spironolactone significantly reduced the Ang II-enhanced production of MCP-1 and TNF-α, whereas PD-123319 also had no effect. In conclusion, Ang II induces aldosterone synthesis through AT1R and enhances cytokine production through an AT1R-dependent mechanism and, at least partly, through a MR-dependent mechanism in human PBMCs.