The multiple actions of Ang II are mediated via specific, highly complex intracellular signaling pathways, which are stimulated following initial binding of the peptide to its specific receptors. In mammalian cells, Ang II mediates effects via at least two high-affinity plasma membrane receptors, ATi and AT2. Both receptor subtypes have been cloned and pharmacologically characterized (37,38). Two other Ang II receptors have been described, namely AT3 and AT4. The AT3 receptor is peptide-specific, recognizing mainly Ang II. This receptor does not bind nonpeptide ligands such as losartan (selective ATi receptor antagonist) or PDi233i9 (selective AT2 receptor antagonist), and has only been observed in cell lines. The AT4 receptor, which is present in heart, lung, kidney, brain, and liver, binds Ang IV (39), but not losartan or PDi233i9. We will focus on the signaling pathways mediated by ATi and AT2.
The gene for the ATi was first cloned in i99i (37), and consists of 359 amino acids with a molecular mass of 4i kDa. Two ATi receptor subtypes have been described in rodents, ATia and ATib, with greater than 94% amino acid sequence homology (40), and which have similar pharmacological properties and tissue distribution patterns. ATia and ATib genes in rats are mapped to chromosomes i7 and 2 (41), respectively; whereas, the human ATi gene is mapped to chromosome 3 (42). ATi receptors are primarily found in the brain, adrenals, heart, vasculature, and kidney, and serve to regulate blood pressure and fluid and electrolyte balance. In the heart, the highest density of AT1 is found in the conducting system (43). Punctate AT1 binding is found in the epicardium surrounding the atria, with low binding seen throughout the atrial and ventricular myocardium (44). Moreover, AT1 in the vasculature, including the aorta, pulmonary and mesenteric arteries, are present in high levels on VSMC, with low levels in the adventitia (45). Virtually all of the known biological actions of Ang II are mediated by ATp including the elevation of blood pressure, vasoconstriction, increase in cardiac contractility, release of aldo-sterone and vasopressin, renal tubular sodium reabsorption, stimulation of sympathetic transmission, and cellular growth (46). In addition, a recent in vitro and in vivo evidence supports the notion that Ang II, mediated by ATp may participate directly in the pathogenesis of various cardiovascular diseases (47-49). Thus, the molecular and cellular actions of Ang II in cardiovascular diseases are almost exclusively mediated by ATr
AT1 belongs to the seven transmembrane class of GPCRs. Four cysteine residues are located in the extracellular domain, which represent the sites of disulfide bridge formation and are critical tertiary structure determinants. The transmembrane domain and the extracellular loop play an important role in Ang II binding (50). The Ang II binding site with AT1 is different from the binding site for AT1 antagonists, which interact only with the transmembrane domain of the receptor (51). Like most G protein-coupled receptors, AT1 is also subject to internalization when stimulated by Ang II, a process dependent on specific residues on the cytoplasmic tail (52). AT1 receptors interact with various hetero-trimeric G proteins, including Gq/11, Gi/o, Ga12, and Ga13. The different G protein isoforms couple to distinct signaling cascades.
The second major isoform of the Ang II receptor, AT2, has been cloned in a variety of species, including human (53), rat (54), and mouse (38). AT2 is also a seven-transmembrane glycoprotein, encoded by a 363-amino-acid protein with a molecular mass of 41 kDa, and shares only 34% sequence identity with ATj (55). The AT2 receptor gene is localized as a single copy on the X chromosome. Unlike ATp there is no evidence for subtypes of AT2. AT2 is normally expressed at high levels in developing fetal tissues, and decreases rapidly after birth (56). In the adult, AT2 expression is detectable in the pancreas, heart, kidney, adrenals, myometrium, ovary, brain and vasculature. AT2 is re-expressed in adults after vascular and cardiac injury and during wound healing and renal obstruction (57-60). Thus, AT2 receptors appear to be involved in the control of cell proliferation, cell differentiation and development, angiogenesis, wound healing, tissue regeneration, and even apoptosis, namely, biological processes that counteract the trophic responses mediated through ATj (61,62).
In the heart, AT2 inhibits growth and remodeling, induces vasodilation, and is up-regulated in pathological states (63). Conflicting data on antigrowth effects have emerged from the studies of mice lacking AT2 (64). However, recent studies have helped to clarify the role of AT2 in cardiac remodeling following myocardial infarction (65) and in cardiac hypertrophy and fibrosis because of Ang II infusion in mice overexpressing AT2 selectively in the myocardium. After myocardial infarction, AT2 overexpression resulted in preservation of left ventricular global and regional function, indicating a beneficial role of AT2 in volume-overload states, including post-myocardial infarction remodeling
(65,66). In blood vessels, in addition to vasodilatory actions, AT2 exerts antiproliferative and apoptotic effects in VSMC and decreases neointima formation in response to an injury by counteracting Ang II actions at the ATj receptor (67). Part of the actions of AT2 in blood vessels may be to downregulate the expression of ATj and TGF-P receptors via the bradykinin-nitric oxide (NO) pathway.
Signaling pathways through which AT2 mediates cardiovascular actions have recently been explained. Four major cascades are involved that include: (1) activation of protein phosphatases and protein dephosphorylation, (2) regulation of the NO-cGMP system,
(3) stimulation of phospholipase A2 (PLA2) and release of arachidonic acid (AA), and
(4) sphingolipid-derived ceramide (61,62,68,69). In contrast to the extensive data on the molecular and cellular functions and pathophysiological significance of ATp the role of AT2 in cardiovascular diseases remains to be defined.
There are five classical signal transduction mechanisms for ATp as follows: activation of PLA2, phospholipase C (PLC), phospholipase D (PLD) and L-type Ca2+ channels and inhibition of adenylyl cyclase (Fig. 2). AT1 couples to Gq/n protein, and induces the activation of PLC-^, which results in generation of two secondary messengers, inositol (1,4,5) trisphosphate (IP3) and diacylglycerol (DAG). IP3 stimulates the release of Ca2+ from intracellular stores, and DAG activates protein kinase C (PKC), both of which are involved in cardiac hypertrophy, heart failure, and vasoconstriction (70-73). Activation of PLA2 and PLD stimulates the release of AA, the precursor molecule for the generation of prostaglandins, and is involved in the Ang II-induced growth of VSMC and cardiac hypertrophy (74). Ang II-mediated stimulation of AT1 coupled with Gi/o protein can also inhibit adenylyl cyclase in several target tissues, thereby attenuating the production of the second messenger cAMP (75). cAMP is a vasodilator and when production is decreased by AT1 activation, vasoconstriction ensues. Moreover, AT1 is also involved in the opening of Ca2+ channels and influx of extracellular Ca2+ into cells (76,77), and the activation of L-channels is mediated by AT1 coupled with G^/^ proteins (78).
A recent development in the field of Ang II signaling is the demonstration that AT1 activation is associated with increased protein tyrosine phosphorylation. These processes are characteristically associated with growth factors and cytokines. Accordingly, it is becoming increasingly evident that in addition to its potent vasoconstrictor properties, Ang II has mitogenic- and inflammatory-like characteristics. Ang II stimulates phos-phorylation of many non-receptor tyrosine kinases including PLCy, Src family kinases, Janus kinase (JAK), focal adhesion kinase (FAK), Ca2+-dependent tyrosine kinases (e.g., proline-rich tyrosine kinase, Pyk2), p130Cas, and phosphatidylinositol 3-kinase (PI3K). In addition, Ang II influences the activity of receptor tyrosine kinases (RTK), such as epidermal growth factor receptor (EGFR), PDGF receptor (PDGFR), and IGF receptor (IGFR) (Fig. 3). Ang II mediated stimulation of cellular proliferation and growth has been demonstrated in cardiac myocytes and VSMC. These growth-like effects are associated with increased tyrosine phosphorylation and activation of MAP kinase and related pathways, which result in increased expression of early response genes, such as c-fos,
c-jun and c-myc, which control cellular proliferation and growth (79). Such actions have been linked to cardiovascular diseases, including hypertension, cardiac hypertrophy, heart failure, and atherosclerosis. The role of tyrosine kinases in Ang II-mediated signaling has been extensively reviewed (74,79-81) and only recent developments are discussed here.
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