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    • AT signaling is distinct to that

    2022-11-18

    AT2 signaling is distinct to that of AT1 (Kaschina and Unger, 2003). As shown here, opposite to the stimulating effect of AT1 signaling, it inhibits apelin secretion. Activation of AT2 receptor, which is also G-protein coupled, is known to decrease cAMP and cGMP levels by inhibiting adenylyl cyclase (Zhang and Pratt, 1996, Hansen et al., 2000) and guanylate cyclase (Sumners et al., 1991, Takekoshi et al., 2002), respectively. Decrease of cAMP or cGMP level leads to suppression of protein kinase A (PKA) activities or protein kinase G (PKG) activities. As a consequence, apelin expression and secretion are reduced. Such scenario of signal transduction is supported by our observations when AT2 receptors were selectively activated by blocking AT1 receptors. Specifically, both adenylyl cyclase activator (forskolin) and membrane permeable cGMP analogue (8-Bromo-cGMP) were able to increase basal apelin secretion and expression, and block the inhibitory effect of AT2 activation (Fig. 3). In addition to the cAMP and cGMP pathways, AT2 activation can stimulate various phosphatases, resulting in reduction of ERK 1/2 phosphorylation (Fig. 5C) (Dinh et al., 2001). This may also partly account for the inhibitory effect of AT2 receptors. The differential regulations by AT1 and AT2 receptors underlie the observed biphasic dose-dependent response of apelin secretion to AngII. This phenomenon has also been observed in other protein phosphatase inhibitor (Zheng et al., 2003, Utsugisawa et al., 2005). For example, high-dose AngII inhibits fluid transport in rat jejunum through actions of AT1 receptors, while low-dose AngII stimulates it via AT2 receptors (Jin et al., 1998). Similarly, we show that low-dose AngII inhibits apelin secretion through AT2 receptors whereas high-dose AngII stimulates it through AT1 receptors. The low-dose responsiveness (sensitivity) of AT2 receptor may be ascribed to its higher binding affinity to AngII (Cox and Rosenfeld, 1999, McMullen et al., 2002) and its dominant abundance in 3T3-L1 adipocytes (Jones et al., 1997, Hung et al., 2010). The concentration of AngII and the expression level of AT1 or AT2 receptors change in different physiological and pathological conditions. For instances, AngII concentration is elevated in obesity and hypertension (Aneja et al., 2004, Kotsis et al., 2010); AT1 receptor expression is up-regulated in obesity (Gorzelniak et al., 2002); and AT2 receptor is up-regulated during adipo-differentiation of 3T3-L1 as well as human preadipose cells (Schling, 2002, Hung et al., 2010). The co-existence of the antagonizing AT1 and AT2 signaling pathways allows the cells to respond differently to different situations. In contrast to 3T3-L1 adipocytes, AT1 receptors dominate in human adipocytes and other cells in adult tissues (Engeli et al., 1999, de Gasparo et al., 2000). Due to its low expression, the physiological and clinical significance of AT2 receptor is largely overlooked. However, mounting evidence indicates that expression of AT2 receptors increases in various pathological conditions and in response to stimulation of several agents including AngII and insulin (Dinh et al., 2001, Lemarie and Schiffrin, 2010). The AngII regulation on apelin secretion cannot be solely attributed to the alteration on protein expression which is a slow process. We therefore speculate that apelin molecules may be packed into abundant readily releasable vesicles to support acute secretion and regulation. Several adipokines have been shown to segregate in vesicles (Xie et al., 2008, Ye et al., 2010). Using confocal imaging and total internal reflection fluorescence microscopy (TIRFM), we indeed found that apelin molecules are also packed into numerous intracellular vesicles (Supplementary Fig. S2A and B). In addition, we found that apelin secretion was significantly reduced by Brefeldin A which blocks vesicular traffic between the endoplasmic reticulum and Golgi (Supplementary Fig. S3). These suggest that apelin is secreted through the classic vesicular secretory pathway. In contrast to the biochemical assays or electrophysiological assays (Chen and Gillis, 2000, Chen et al., 2001), TIRFM is able to visualize the individual secretory vesicle in the thin subplasmalemmal region (<200nm) and resolve the events of vesicular fusion (release) (Zhang et al., 2009, Soo et al., 2010, Than et al., 2011b). As shown in Supplementary Fig. S2 (C and D), activation of AT1 receptors significantly increased the frequency of the fusion events of apelin vesicles, whereas activation of AT2 decreased it.