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Am J Physiol Heart Circ Physiol 281: H2337-H2365, 2001;
0363-6135/01 $5.00
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Vol. 281, Issue 6, H2337-H2365, December 2001

REVIEW
Angiotensin receptors: signaling, vascular pathophysiology, and interactions with ceramide

C. Berry1, R. Touyz2, A. F. Dominiczak1, R. C. Webb3, and D. G. Johns4

1 Department of Medicine and Therapeutics, Western Infirmary, University of Glasgow, G11 6NT Glasgow, United Kingdom; 2 Laboratory of Experimental Hypertension, Clinical Research Institute of Montreal, University of Montreal, Montreal, Quebec, Canada H2W 1R7; 3 Department of Physiology and Endocrinology, Medical College of Georgia, Augusta, Georgia 30912; and 4 Department of Medicine, Boston University, Boston, Massachusetts 02118


    ABSTRACT
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ABSTRACT
INTRODUCTION
PHYSIOLOGICAL ROLE OF ANG...
ANGIOTENSIN RECEPTORS
PHYSIOLOGICAL ACTIONS OF...
INTRACELLULAR SIGNALING THROUGH...
PHYSIOLOGICAL AND...
CERAMIDE SIGNALING
CERAMIDE AND ANG II
AREAS FOR FUTURE INVESTIGATION
REFERENCES

Angiotensin II (ANG II) is a pleiotropic vasoactive peptide that binds to two distinct receptors: the ANG II type 1 (AT1) and type 2 (AT2) receptors. Activation of the renin-angiotensin system (RAS) results in vascular hypertrophy, vasoconstriction, salt and water retention, and hypertension. These effects are mediated predominantly by AT1 receptors. Paradoxically, other ANG II-mediated effects, including cell death, vasodilation, and natriuresis, are mediated by AT2 receptor activation. Our understanding of ANG II signaling mechanisms remains incomplete. AT1 receptor activation triggers a variety of intracellular systems, including tyrosine kinase-induced protein phosphorylation, production of arachidonic acid metabolites, alteration of reactive oxidant species activities, and fluxes in intracellular Ca2+ concentrations. AT2 receptor activation leads to stimulation of bradykinin, nitric oxide production, and prostaglandin metabolism, which are, in large part, opposite to the effects of the AT1 receptor. The signaling pathways of ANG II receptor activation are a focus of intense investigative effort. We critically appraise the literature on the signaling mechanisms whereby AT1 and AT2 receptors elicit their respective actions. We also consider the recently reported interaction between ANG II and ceramide, a lipid second messenger that mediates cytokine receptor activation. Finally, we discuss the potential physiological cross talk that may be operative between the angiotensin receptor subtypes in relation to health and cardiovascular disease. This may be clinically relevant, inasmuch as inhibitors of the RAS are increasingly used in treatment of hypertension and coronary heart disease, where activation of the RAS is recognized.

renin-angiotensin system; angiotensin receptor antagonist; second messenger


    INTRODUCTION
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ABSTRACT
INTRODUCTION
PHYSIOLOGICAL ROLE OF ANG...
ANGIOTENSIN RECEPTORS
PHYSIOLOGICAL ACTIONS OF...
INTRACELLULAR SIGNALING THROUGH...
PHYSIOLOGICAL AND...
CERAMIDE SIGNALING
CERAMIDE AND ANG II
AREAS FOR FUTURE INVESTIGATION
REFERENCES

IN THIS REVIEW, we compare and contrast the mechanisms of action and vascular effects of the angiotensin type 1 (AT1) and type 2 (AT2) receptors. The physiology of angiotensin II (ANG II) continues to be a major field of investigation. Recently reported mechanisms of AT1 receptor activation, such as receptor transactivation of tyrosine kinase receptors and stimulation of reactive oxygen species (ROS) production, suggest that ANG II has growth factor and cytokine-like properties in addition to its vasoconstrictor actions. The AT2 receptor has only recently been identified, and its mechanisms of action continue to be elaborated. Therefore, we consider the emergent physiological systems that are activated by the AT2 receptor.

One such pathway is the interaction between ANG II and ceramide. Ceramide is a lipid second messenger that is involved in a variety of physiological pathways, including cytokine-induced apoptosis and vasodilation. We summarize the recently described novel interaction of ANG II and ceramide and consider the potential importance of ceramide as an intracellular second messenger of the AT2 receptor. Finally, we discuss the physiological antagonism and cross talk that may exist between the two angiotensin receptor subtypes in relation to health and cardiovascular disease.


    PHYSIOLOGICAL ROLE OF ANG II
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ABSTRACT
INTRODUCTION
PHYSIOLOGICAL ROLE OF ANG...
ANGIOTENSIN RECEPTORS
PHYSIOLOGICAL ACTIONS OF...
INTRACELLULAR SIGNALING THROUGH...
PHYSIOLOGICAL AND...
CERAMIDE SIGNALING
CERAMIDE AND ANG II
AREAS FOR FUTURE INVESTIGATION
REFERENCES

ANG II, an octapeptide hormone, is the active component of the renin-angiotensin system (RAS). It plays an important physiological role in the regulation of blood pressure, plasma volume, sympathetic nervous activity, and thirst responses. ANG II also has a pathophysiological role in cardiac hypertrophy, myocardial infarction, hypertension, and atherosclerosis. It is produced systemically via the classical RAS and locally via tissue RAS. In the classical RAS, circulating renal-derived renin cleaves hepatic-derived angiotensinogen to form the decapeptide angiotensin I (ANG I), which is converted by angiotensin-converting enzyme (ACE) in the lungs to the active ANG II (59, 228, 278). ANG I can also be processed into the heptapeptide ANG-(1-7) by tissue endopeptidases (75).

Although the RAS was originally regarded as a circulating system, many of its components are localized in tissues, indicating the existence of a local tissue RAS as well (48, 66). ACE exists in plasma, in the interstitium, and intracellularly. Tissue ACE is present in all major organs, including the heart, brain, blood vessels, adrenals, kidney, liver, and reproductive organs (115), and is already functional in utero (73). All components of the RAS, except renin, have been demonstrated to be produced in the vasculature (66, 198). Because vascular renin is absent, local generation of ANG II in the interstitium is regulated by tissue ACE that is probably dependent on circulating renin. In addition to ACE-dependent pathways of ANG II formation, non-ACE pathways have been demonstrated. Chymotrypsin-like serine protease (chymase) may represent an important mechanism for conversion of ANG I to ANG II in the human heart (307), kidney (115), and vasculature (115, 289) and may be particularly important in pathological conditions, such as coronary heart disease (227).


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INTRODUCTION
PHYSIOLOGICAL ROLE OF ANG...
ANGIOTENSIN RECEPTORS
PHYSIOLOGICAL ACTIONS OF...
INTRACELLULAR SIGNALING THROUGH...
PHYSIOLOGICAL AND...
CERAMIDE SIGNALING
CERAMIDE AND ANG II
AREAS FOR FUTURE INVESTIGATION
REFERENCES

In mammalian cells, ANG II mediates its effects via at least two plasma membrane receptors: AT1 and AT2 receptors. Both receptor subtypes have been cloned and pharmacologically characterized (149, 201, 205, 251). The ANG II receptors can be distinguished according to inhibition by specific antagonists. AT1 receptors are selectively antagonized by biphenylimidazoles, such as losartan, whereas tetrahydroimidazopyridines specifically inhibit AT2 receptors (8). The AT2 receptor may also be selectively activated by CGP-42112A. This is a hexapeptide analog of ANG II, which may also inhibit the AT2 receptor, depending on concentration (46). Two other angiotensin receptors have been described: AT3 and AT4 subtypes (20, 39, 285). However, the pharmacology of AT3 and AT4 receptors has not been fully characterized, and therefore these receptors are not included in a definitive classification of mammalian angiotensin receptors as defined by the International Union of Pharmacology Nomenclature Subcommittee for Angiotensin Receptors (50, 51).

AT1 Receptor

The AT1 receptor belongs to the seven-membrane-spanning G protein-coupled receptor family and typically activates phospholipase C (PLC) through the heterotrimeric Gq protein, although it may also signal through Gi, G11/13, and Gs (19, 51, 201, 205, 251). The human AT1 receptor gene is mapped to chromosome 3 (47). AT1 receptors are widely distributed throughout the cardiovascular, renal, endocrine, and nervous system in humans (6). Allen et al. (7) and Zhuo and colleagues (344) demonstrated that, in the human vasculature, AT1 receptors are present at high levels in smooth muscle cells and at relatively low levels in the adventitia. Freeman et al. (82) and Pueyo et al. (231) reported that AT1 receptors are also expressed in cultured rat aortic endothelial cells. In studies undertaken in human myocardial biopsies, Regitz-Zagrosek et al. (238) demonstrated that, in the heart, AT1 receptors are present in atrial and ventricular myocytes and in fibroblasts. Using radioligand binding techniques, they also demonstrated that the abundance of the AT1 receptor protein is reduced in patients with heart failure, which may be due to a reduction in the abundance of myocardial AT1 receptor mRNA (237).

In rodents, the AT1 receptor has two functionally distinct subtypes, AT1A and AT1B, with >95% amino acid sequence homology (126, 134). On the basis of the cDNA sequence, the AT1 receptor is composed of 359 amino acids (250). It is a glycoprotein and contains extracellular glycosylation sites at the amino terminus (Asn4) and the second extracellular loop (Asp176 and Asn188) (54). The transmembrane domain at the amino-terminal extension and segments in the first and third extracellular loops are responsible for G protein interactions with the receptor (113). Internalization of G protein-coupled receptors involves receptor phosphorylation, which may be mediated, in part, via caveola (123).

Although G protein-coupled receptors do not contain intrinsic kinase activity, they are phosphorylated on serine and threonine residues by members of the G protein receptor kinase (GRK) family (293, 294). AT1 receptors may be phosphorylated in the basal state and in response to ANG II stimulation (147). Threonine and serine residues between Thr332 and Ser338 of the cytoplasmic tail are essential for receptor internalization (for review see Ref. 124). The AT1 receptor may also be phosphorylated at tyrosine residues. Potential tyrosine phosphorylation sites within the AT1 receptor include amino acids 302, 312, 319, and 339 within the carboxy terminal (19, 127). Tyr319 is important, inasmuch as it is part of the motif Tyr-Ile-Pro-Pro, which is analogous to an Src homology 2 (SH2) binding motif in the platelet-derived growth factor (PDGF) receptor (PDGFR), Tyr-Ile-Pro, and in the epidermal growth factor (EGF) receptor (EGFR), Tyr-Leu-Pro-Pro (74). In EGFR and PDGFR, these motifs are target sequences for tyrosine phosphorylation. The question of agonist-induced tyrosine phosphorylation of the AT1 receptor and the possible effects that may be consequent on this remain controversial (293, 294). Initial studies in vascular smooth muscle cells (VSMC) reported that agonist-induced phosphorylation of the AT1 receptor was mediated by tyrosine and serine kinase-mediated phosphorylation (147). However, studies by Thomas et al. (294) demonstrated that a serine/threonine-rich segment of the carboxy terminus was essential for phosphorylation and internalization of this receptor. Other studies in cells transfected with the AT1 receptor (218) and VSMC (133) determined that GRKs, such as GRK2 and GRK5, and second-messenger-activated kinases, such as protein kinase C (PKC), mediate predominantly serine phosphorylation, which results in desensitization of the agonist-occupied receptor.

AT2 Receptor

The second major angiotensin receptor isoform is the AT2 receptor. The gene of this receptor is localized as a single copy on the X chromosome (161). The AT2 receptor is a seven-transmembrane-type, G protein-coupled receptor comprising 363 amino acids. It has low amino acid sequence homology (~34%) with AT1A or AT1B receptors (128, 201). Although the exact signaling pathways and the functional roles of AT2 receptors are unclear, these receptors may antagonize, under physiological conditions, AT1-mediated actions (42, 334) by inhibiting cell growth and by inducing apoptosis and vasodilation (84, 107, 118, 119, 273, 306). The exact role of AT2 receptors in cardiovascular disease remains to be defined.

The AT2 receptor is ubiquitously expressed in human fetal mesenchymal tissues. However, the expression of this receptor rapidly declines after birth (208). In adults, AT2 receptor expression is detectable in the pancreas, heart, kidney, adrenals, brain, and vasculature (162, 222, 295, 299, 318, 324, 325). In blood vessels, the AT2 receptor has been detected in the mesenteric blood vessels of adult rats, but this receptor is not detectable in all vascular beds (6).

The distribution of the AT2 receptor in the human cardiovascular system is poorly characterized. AT2 receptor mRNA has been demonstrated in cultured coronary artery endothelial cells and in the medial layer of interlobular renal arteries (170). Most recently, Malendowicz et al. (181), using RT-PCR techniques, investigated whether AT2 receptor mRNA could be detected in vastus lateralis muscle biopsies obtained from healthy individuals or patients with severe chronic heart failure treated with an ACE inhibitor or an AT1 receptor antagonist. Control studies identified the presence of von Willebrand factor mRNA in muscle biopsy homogenates. This was important to confirm the presence of endothelial cells and, therefore, vascular tissue in these biopsies. Although AT1 receptor transcripts were detected in these homogenates, the AT2 receptor was undetectable in any biopsy other than that obtained from a human fetus. Although these data suggest that the AT2 receptor is absent in human skeletal muscle vasculature, further investigations to detect AT2 receptor protein are required to confirm or refute this thesis.

Studies in ex vivo human cardiac tissue have demonstrated the AT2 receptor to be present in tissue from healthy (327) and failing human hearts (10, 108). The AT2 receptor is present in human cardiac myocytes (10) and fibroblasts (304). In healthy human myocardium, the AT2 receptor predominates (327). In heart failure, although expression of the AT2 receptor gene in cardiomyocytes, as measured by competitive RT-PCR, may be unchanged, the overall abundance of the receptor protein (and also that of the AT1 receptor) falls (108).

The expression of both angiotensin receptor types is tightly regulated. The AT1 receptor may be subject to "negative feedback" by ANG II (1), whereas expression of the AT2 receptor is upregulated by sodium depletion (222) and is inhibited by ANG II and growth factors such as PDGF and EGF (125).


    PHYSIOLOGICAL ACTIONS OF ANGIOTENSIN RECEPTORS
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ABSTRACT
INTRODUCTION
PHYSIOLOGICAL ROLE OF ANG...
ANGIOTENSIN RECEPTORS
PHYSIOLOGICAL ACTIONS OF...
INTRACELLULAR SIGNALING THROUGH...
PHYSIOLOGICAL AND...
CERAMIDE SIGNALING
CERAMIDE AND ANG II
AREAS FOR FUTURE INVESTIGATION
REFERENCES

Physiological Effects Mediated by the AT1 Receptor

ANG II mediates its effects by acting directly through ANG II receptors, indirectly through the release of other factors, and via cross talk with intracellular signaling cascades of other vasoactive agents, growth factors, and cytokines. Integrated responses to ANG II are the result of combined AT1- and AT2-mediated actions. The established cardiovascular effects of AT1 and AT2 receptor activation in humans are shown in Table 1 (8, 129). AT1 receptor activation stimulates vasoconstriction, vascular cell hypertrophy and hyperplasia, and sodium retention. Other more recently described physiological effects of this receptor include stimulation of ROS production (23, 94, 234, 234) and induction of inflammatory (202), thrombotic (315), and fibrotic processes (26, 211, 280).

                              
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  		Table 1.   Physiological effects of AT1 and AT2 receptors

Some of the pathophysiological effects of ANG II may be mediated through activation of the transcription factor nuclear factor-kappa B (NF-kappa B) (247), which participates in a variety of inflammatory responses (13). For example, studies in experimental rats overexpressing the human renin and angiotensinogen genes (double-transgenic rats) suggest that AT1 receptor-coupled NF-kappa B activation may be of pathological importance (202). Chronic treatment of these animals with the antioxidant pyrrolidine dithiocarbamate was associated with reductions in blood pressure, cardiac hypertrophy, macrophage tissue infiltration, and albuminuria. Furthermore, electrophoretic mobility shift assay demonstrated that pyrrolidine dithiocarbamate inhibited NF-kappa B binding activity in heart and kidney.

Increasingly, ANG II is recognized to trigger diverse effects, some of which may be opposite to what might be anticipated (84, 246, 247). For example, under some circumstances, AT1 receptor activation is reported to lead to apoptosis in some cell types (57, 169).

Physiological Effects Mediated by the AT2 Receptor

Most of the biological actions of ANG II are thought to be mediated by the AT1 receptor. However, recent evidence suggests that the AT2 receptor may have a physiological role in the regulation of blood pressure and renal function, counterbalancing the vasoconstrictor and antinatriuretic actions of ANG II. The significance of AT2 receptors in blood pressure regulation was recently demonstrated by Tsutsumi et al. (303), who selectively overexpressed the AT2 receptors in VSMC of transgenic mice. In animals overexpressing the AT2 receptor, ANG II infusion did not cause a pressor response, which was present in wild-type mice. Furthermore, in the presence of AT1 receptor blockade, ANG II infusion decreased blood pressure not only in transgenic, but also in wild-type, mice. These findings suggest that AT2 receptors do regulate blood pressure, probably by modulating vasoconstrictor responses. In general, cardiovascular effects of the AT2 receptor appear to be opposite to those of the AT1 receptor (189) (Table 1). The vasodilator, antigrowth, and apoptotic actions of the AT2 receptor are in contradistinction to those of the AT1 receptor. Other novel, physiological effects attributed to the AT2 receptor include modulation of thirst, behavior, and locomotor activity (110, 126).

Are all the effects of the AT2 receptor beneficial? Paradoxically, AT2 receptor activation may have proinflammatory effects, such as in induction of NF-kappa B (244), and trophic effects, leading to vascular (36, 220) and cardiac (30, 177, 265, 304) hypertrophy. These observations underline the complexity and our limited understanding of the angiotensin receptor systems.


    INTRACELLULAR SIGNALING THROUGH ANGIOTENSIN RECEPTORS
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ANGIOTENSIN RECEPTORS
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CERAMIDE AND ANG II
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AT1 Receptor-Mediated Signaling Events

AT1 receptors are coupled to multiple, specific signaling cascades, leading to diverse biological actions. The signaling processes are multiphasic with distinct temporal characteristics. The trophic effects of ANG II are mediated by activation of pathways that involve tyrosine phosphorylation and enhanced gene expression (19, 65, 67, 98, 127) (Fig. 1). Processes involved in these and other ANG II-stimulated pathways have only recently been elucidated. In this section, mechanisms of AT1 receptor-induced activation of tyrosine kinase and phospholipase pathways, prostaglandin (PG) metabolism, and transcription factor and ROS activities are discussed.


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  		Fig. 1.   Signaling mechanisms of the angiotensin type 1 (AT1) and type 2 (AT2) receptors, interactions with ceramide, and physiological effects. NOS, nitric oxide synthase; PLA2, PLB, and PLD, phospholipase A2, B, and D, respectively; PGF2alpha , prostaglandin F2alpha ; DAG, diacylglycerol; PA, phosphatidic acid.

Ligand-receptor binding results in activation of heterotrimeric G proteins through exchange of GTP for GDP, resulting in the release of Galpha -GTP and beta gamma complexes, which induce downstream actions (102). These intracellular responses are dependent on the identity of the G protein subunits. Activation of Galpha i and related subunits results in cGMP, which is sensitive to pertussis toxin, whereas Galpha s and Galpha olf activate adenylate cyclases and Galpha q leads to activation of PLC (102). AT1 receptor-Gi activation inhibits adenylate cyclase, leading to a reduction in cAMP (8).

AT1 receptor-induced phosphorylation by tyrosine kinase. Vascular cell growth involves non-receptor- and receptor-associated tyrosine kinase-mediated intracellular protein phosphorylation (19). Activation of these pathways is important for the physiological growth and contractile responses consequent on AT1 receptor activation (19). This is supported by studies demonstrating that inhibition of tyrosine kinases attenuates ANG II-induced hypertrophic (163), proliferative (299), and contractile (114) responses in cultured VSMC.

In VSMC, the nonreceptor tyrosine kinases (non-RTKs) include PLC-gamma 1, Src family kinases, Janus kinases (Jak and Tyk), focal adhesion kinase (FAK), Ca2+-dependent tyrosine kinases (e.g., PYK2), p130Cas (a Crk-associated substrate), and phosphatidylinositol 3-kinase (PI3K) (19, 67, 246). The receptor tyrosine kinases (RTKs) involved in vascular ANG II signaling include EGFR, PDGFR, and insulin-like growth factor I. In this section, we discuss recent developments relating to ANG II signaling and tyrosine kinases and consider the emerging evidence of ANG II transactivation of RTKs.

ACTIVATION OF NONRECEPTOR TYROSINE KINASES. Src family kinases. ANG II induces rapid phosphorylation of c-Src, measured by autophosphorylation or kinase activity toward enolase (132, 225, 226, 302). This appears to be a redox-sensitive process, as recently demonstrated by Ushio-Fukai et al. (310). Src plays an important role in ANG II-induced phosphorylation of PLC-gamma and inositol trisphosphate formation. Src, intracellular Ca2+, and PKC regulate ANG II-induced phosphorylation of p130Cas, a signaling molecule involved in integrin-mediated cell adhesion (246, 254) (Fig. 2). Src has also been associated with ANG II-induced activation of PYK2 (56, 245) and with phosphorylation of extracellular signal-regulated kinases (ERKs) (132) as well as activation of other downstream proteins including pp120, p125Fak, paxillin, Jak2, signal transducer and activator of transcription 1 (STAT1), Galpha , caveolin, and the adapter protein Shc (49, 172). From a physiological viewpoint, phosphorylation of these proteins is important. For example, Shc is a linker protein involved in mediating intracellular signaling of G protein-coupled receptors (GPCRs) (19). Studies in VSMC isolated from human resistance arteries suggest that c-Src may also be important in the regulation of ANG II-stimulated Ca2+ mobilization (132). Furthermore, c-Src mediates ANG II regulation of plasminogen activity in bovine aortic endothelial cells (16).


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  		Fig. 2.   AT1 receptor (AT1R)-induced tyrosine phosphorylation of signal transducer and activator of transcription 1 (STAT1) is mediated by binding of Janus kinase 2 (Jak2) by this receptor at a YIPP motif in the COOH-terminal domain of this receptor. Activation of Jak2 may occur independent of any physical association with the AT1 receptor. p59fyn serves as a docking protein for Jak2 and STAT1. Phosphorylation of STAT1 results in homo/heterodimerization and translocation to the nucleus, where binding with specific promoter elements leads to enhanced gene transcription. STAT1 phosphorylation is negatively regulated by MKP-1, a nuclear mitogen-activated protein kinase phosphatase. VSMC, vascular smooth muscle cell.

Although there is increasing evidence that Src family kinases are functionally linked to the AT1 receptor (132, 183, 302, 310), the exact mechanisms whereby AT1 receptors associate with Src are unclear. The interaction between the Gbeta gamma -subunits, their associated kinases, and kinase substrates could provide the signaling complex that activates and binds c-Src (345). It has also been suggested that Src interacts indirectly with the receptor, via other proteins such as Jak2 (255) or possibly via beta -arrestin (179).

JANUS FAMILY KINASES, TYROSINE KINASE, AND STAT ACTIVATION. Similar to classical cytokine receptors, the AT1 receptor stimulates Jak2 and Tyk2, members of the Janus family kinases (185). Using immunoprecipitation and immunoblotting techniques with an antiphosphotyrosine antibody, Marrero et al. (185) demonstrated that AT1 receptor activation leads to rapid phosphorylation (within minutes) of the intracellular kinases Jak2 and Tyr2 (Fig. 3). This pathway involves an association between Jak2 and the AT1 receptor, which is dependent on a YIPP sequence in the carboxy-terminal intracellular domain of this receptor (5). In addition, it has recently been shown that Jak2 autophosphorylates on tyrosine in response to ANG II, which is important for the interaction between Jak2 and STAT1, a process that seems to be independent of physical binding to the AT1 receptor (4).


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  		Fig. 3.   Non-receptor kinase AT1 receptor-dependent tyrosine phosphorylation. The AT1 receptor-dependent interaction of Src and PYK2 (also known as CADTK or CAKbeta ) involves a PYK2 kinase-mediated autophosphorylation, which is sensitive to Ca2+ and protein kinase C. This may in turn result in Src-mediated Shc (adapter protein) phosphorylation, resulting in the association of this complex with Grb2 and Sos and with Ras-Raf-MEK-ERK1/2 activation. This then stimulates JNK activation, leading to activator protein 1 (AP-1) DNA binding, gene expression, and protein synthesis. AT1 receptor activation may also lead to mitogen-activated protein kinase (MAPK) activation by Ras-independent pathways. MEK, MAPK kinase; ERK, extracellular signal-regulated kinase.

Venema et al. (316) demonstrated in rat VSMC that ANG II induced tyrosine phosphorylation of STAT1, which could be prevented by cotreatment with a Jak2 inhibitor or p59Fyn kinase (a member of the Src family of kinases) inhibitor. These authors proposed that neither of these kinases were "upstream" but, rather, that p59Fyn acted as a docking protein for Jak2 and STAT1 (316), which facilitates Jak2-mediated phosphorylation of STAT1, resulting in nuclear translocation of this transcription factor (185). By contrast, treatment of VSMC with sense or antisense MKP-1, a nuclear mitogen-activated protein kinase (MAPK) phosphatase, demonstrated that this enzyme induced dephosphorylation of STAT1 (316). Gene expression of this phosphatase is stimulated by treatment with ANG II, suggesting a negative-feedback mechanism in Jak-STAT signaling. Electroporation of antibodies against STAT1 and STAT3 abolished VSMC proliferative responses to ANG II, but not to other growth factors, implicating an essential role of STAT proteins in ANG II-induced cell proliferation (184). The Jak-STAT signaling pathway activates early growth response genes and may be a mechanism whereby ANG II influences vascular and cardiac growth, remodeling, and repair (19, 109).

FAK. ANG II promotes cell migration and induces changes in cell shape and volume by activating FAK-dependent signaling cascades (120, 164, 337). Focal adhesion complexes, specialized sites of cell adhesion, act as supramolecular structures for the assembly of signal transduction mediators. The best-characterized tyrosine kinase localized to focal adhesion complexes is a 125-kDa protein, FAK (99). p125Fak and the related cytoplasmic tyrosine kinase PYK2 are nonreceptor kinases associated with the cytoskeleton (34, 259, 342). Ligand induction of FAK autophosphorylation, such as by ANG II, requires the enzyme to associate with cell surface integrins. By contrast, inhibition of FAK induces cytoskeletal disassembly (93). FAK exhibits extracellular matrix-dependent tyrosine autophosphorylation and physically associates with two non-RTKs, c-Src and Fyn (pp59), via their SH2 domains (256). FAK autophosphorylation may also result in physical associations with PI3K, which is a "downstream" tyrosine kinase involved in trophic cellular responses (41). As a consequence of its association with c-Src, FAK undergoes further tyrosine phosphorylation, which results in FAK binding to Grb2, an association with the GDP-GTP exchange protein Sos and Ras. This in turn leads to ERK1/2 activation (259) (Figs. 2 and 4).


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  		Fig. 4.   AT1 receptor-receptor kinase transactivation. AT1 receptor activation may stimulate intracellular protein tyrosine phosphorylation, although the AT1 receptor has no intrinsic tyrosine kinase activity. Reactive oxygen species and Ca2+-dependent activation of PYK2 and Src lead to activation of, for example, epidermal growth factor receptor (EGFR) kinase and phosphorylation of Shc. This in turn triggers downstream activation of Ras-Raf-mediated MAPK pathways, c-fos/c-jun expression, and increased protein synthesis. PKB, protein kinase B; FAK, focal adhesion kinase; PLC-gamma 1, phospholipase C-gamma 1; PI3K, phosphatidylinositol 3-kinase.

FAK is abundant in developing blood vessels, and elevation of its phosphotyrosine content in VSMC is a rapid response to ANG II (215, 230). ANG II-induced activation of FAK causes its translocation to sites of focal adhesion with the extracellular matrix and phosphorylation of paxillin and talin, which may be involved in the regulation of cell morphology and movement. The functional importance of ANG II-induced FAK activation in VSMC has recently been investigated by Govindarajan et al. (93). In these studies, FAK activation was inhibited by treatment with an actin depolymerizing agent, cytochalasin D, and by transfection of these cells with an adenovirus encoding FAK-related nonkinase (FNKNK) (93). Both of these treatments attenuated ANG II-induced ERK1/2 activation, c-fos mRNA expression, and new protein synthesis in these cells. These observations implicate FAK as an upstream promoter of ANG II-induced hypertrophic responses in VSMC. The link between the AT1 receptor and FAK is unknown, but the Rho family of GTPases may be important (11, 242).

P130CAS. p130Cas is an ANG II-activated tyrosine kinase that plays a role in cytoskeletal rearrangement (242). This protein serves as an adapter molecule, because it contains proline-rich domains, an SH3 domain, and binding motifs for the SH2 domains of Crk and Src (Fig. 2). In cultured VSMC, ANG II induces a transient increase in p130Cas tyrosine phosphorylation (254). Sayeski et al. (254) found that this phosphorylation is dependent on Ca2+, c-Src, and PKC and that it requires an intact cytoskeletal network. Other investigators reported that ANG II-induced activation of p130Cas is Ca2+ and PKC independent (287). Although the exact functional significance of ANG II-induced activation of p130Cas is unclear, it might regulate alpha -actin expression, cellular proliferation, migration, and cell adhesion (210, 214, 246, 254).

PI3K. PI3Ks, a large family of intracellular signal transducers that phosphorylate inositol lipids to generate the 3-phosphoinositides phosphatidylinositol 3-phosphate, phosphatidylinositol 3,4-diphosphate, and phosphatidylinositol 3,4,5-trisphosphate, are heterodimeric proteins composed of 85- and 110-kDa subunits (166). These kinases influence cell survival, metabolism, cytoskeletal reorganization, and membrane trafficking and have recently been identified to play an important role in the regulation of VSMC growth (166, 253). PI3K, characteristically associated with tyrosine kinase receptors, is also activated by AT1 receptors (253). PI3K inhibition by pharmacological agents completely blocks ANG II-stimulated hyperplasia in cultured rat VSMC, suggesting the important regulatory role of this non-RTK in cell growth (253). Several molecular targets for PI3K have been identified, including the protein serine/threonine kinase Akt/protein kinase B (PKB) (330). Akt/PKB regulates protein synthesis by activating p70 S6 kinase (p70S6K) (43, 69), and it modulates ANG II-mediated Ca2+ responses in aortic VSMC by stimulating Ca2+ channel currents (263). Akt/PKB has also been implicated to protect VSMC from apoptosis and to promote cell survival by influencing Bcl-2 and c-Myc expression and by inhibiting caspases (43). Mechanisms whereby the AT1 receptor mediates activation of PI3K-dependent Akt/PKB are unclear, but redox-sensitive pathways and c-Src may be important (292, 309).

SMALL GTP-BINDING PROTEINS AND MAPK ACTIVATION BY TYROSINE KINASES. Small GTP-binding proteins include Ras, a cell membrane protein, and Cdc42, Rho, and Rac, which are cytosolic proteins (100). On activation by GPCR, tyrosine kinase receptors, or cytokine receptors, these pleiotropic GTP proteins participate in signaling pathways that result in a variety of cell functions, such as differentiation, proliferation, and contraction (100) (Figs. 2 and 4).

MAPKs, a family of serine/threonine protein kinases, mediate nuclear transduction of extracellular signals by intracellular protein phosphorylation, leading to a cascade of transcription factor activation, enhanced gene expression, and trophic cellular and vascular responses (19, 19, 186, 192). Furthermore, these AT1 receptor systems are causally implicated in the pathophysiology of vascular disease (112, 159).

Mammalian MAPKs are grouped into six major subfamilies: 1) ERK1/2 (also known as p42MAPK and p44MAPK, respectively), 2) c-Jun NH2-terminal protein kinases/stress-activated protein kinases (JNK/SAPK), 3) p38, 4) ERK6 (p38-like MAPK), 5) ERK3, and 6) ERK5 (also called Big MAPK1) (78, 240, 296, 300). ERK1/2 is activated in response to growth and differentiation factors, whereas JNKs and p38MAPK are usually activated in response to inflammatory cytokines and cellular stress (78, 130, 157, 199, 213, 240). ANG II differentially activates the three major members of the MAPK family: ERK1/2, JNKs, and p38MAPK (156, 164, 297, 298). Induction of MAPK activation typically involves phosphorylation by an MAPK kinase, also known as MEK (45). MEK is, in turn, regulated by other MEK kinases, including Raf-1 (157). Although activated by similar stimuli, the signaling processes leading to JNK and p38MAPK activation are quite different. The best-characterized MAPK cascade is the Raf-Ras-MEK-ERK1/2 pathway (Fig. 4).

ERK1/2 is a proximal kinase that phosphorylates and activates numerous transcription factors, such as Elk-1, leading to c-fos protooncogene expression and formation of the activator protein-1 (AP-1) complex. This is a heterodimeric transcriptional factor, formed by binding of the Fos and the Jun family gene products, which mediates cell growth (132, 186). ANG II-induced activation of ERK1/2 is an important step in the induction of VSMC trophic responses by this hormone in rat (331) and human VSMC (296). Studies by Xi et al. (331) in rat VSMC demonstrated that inhibition of ERK1/2, by treatment with an MEK inhibitor or by transfection of these cells with ERK1 and ERK2 antisense oligodeoxynucleotides, was associated with reductions in AT1 receptor-dependent ERK1/2 activation, c-fos induction, DNA synthesis, and VSMC migration.

Touyz et al. (296) examined the importance of AT1 receptor-induced MEK/ERK1/ERK2 activation for Ca2+ handling and contraction in cultured human VSMC that had been obtained from isolated small resistance arteries. They demonstrated the involvement of ERK1/2 in AT1 receptor-mediated stimulation of Ca2+ currents and VSMC contraction.

Boffa et al. (26) recently explored the role of ERK1/2 in ANG II-induced tissue fibrosis. In initial studies in transgenic mice that overexpressed the alpha 2-chain of the collagen I gene, induction of hypertension by inhibition of nitric oxide (NO) synthesis was associated with renal and vascular fibrosis, which could be prevented by cotreatment of these animals with an AT1 receptor. Treatment of ex vivo aortic and renal cortical sections with ANG II was associated with increased expression of c-fos and increased abundance of collagen I-alpha 2 gene mRNA (291). These effects were inhibited by treatment with an AT1 receptor antagonist, by blockade of the MAPK-ERK cascade, and by an inhibitor of the transcriptional factor AP-1 (291). Furthermore, inhibition of transforming growth factor-beta (TGF-beta ) abolished the ANG II-induced effect on collagen I gene expression, implicating TGF-beta and ERK activation in this pathway.

There is considerable interest in dissecting the role of GTP-binding proteins in ANG II-stimulated MAPK activation. GTP-binding proteins are intermediary factors in the activation of ERK1/2 and JNK/stress-activated protein kinases (SAPK) (70, 249). In studies undertaken in rat VSMC, Eguchi et al. (70) demonstrated that AT1 receptor activation stimulates a rapid, Ca2+-calmodulin, tyrosine kinase-dependent increase in the binding of GTP to p21Ras. Activation of Ras by binding GTP is one important event in AT1 receptor-Gq-induced activation of MAPK in cultured VSMC (70). Furthermore, the activation of Ras appears to involve a signaling cascade via c-Src (258). AT1 receptor activation of Ras involves the phosphorylation of Shc linker protein, which then binds the adapter protein, Grb2, via an SH2 domain (249). The guanine nucleotide exchange protein Sos then stimulates GTP binding by Ras, leading to formation of the activated Shc-Grb2-Sos-Ras adapter protein complex. Raf may then be recruited into the plasma membrane, inducing MEK phosphorylation, which in turn triggers phosphorylation and activation of ERK1/2 (19).

Interestingly, in studies using VSMC treated with an adenovirus dominant-negative mutant of Ras, Takahashi et al. (286) observed that AT1 receptor-stimulated MAPK activation and stimulation of protein synthesis were preserved. This suggests that AT1 receptor activation may stimulate MAPK and VSMC hypertrophy by Ras-independent pathways. By contrast, other studies by Eguchi et al. (69) demonstrated that Ras was required for AT1 receptor-induced ERK activation in these cells.

ANG II-stimulated ERK1/2, JNK, and p38MAPK activation involves cell-specific signaling pathways. For example, Kudoh et al. (156) reported that, in neonatal rat cardiomyocytes, ANG II-induced activation of the transcription factor AP-1 is mediated by ERK1/2- and JNK-dependent pathways, which is PKC and AT1 receptor dependent. Molloy et al. (196) demonstrated that ANG II induced rapid tyrosine phosphorylation of ERK1/2 and hyperphosphorylation of Raf in VSMC. Furthermore, Rac and Cdc42 GTP proteins mediated JNK activation in these cells (193). By contrast, in VSMC, AT1 receptor-induced activation of p38MAPK appears to be tyrosine kinase and PKC independent (260, 317). Whereas ERK1/2 and p38MAPK are rapidly phosphorylated in response to ANG II, JNK activation is delayed, indicating differential regulation of MAPK in VSMC by ANG II (260, 297, 317).

Schmitz et al. (260) demonstrated that p21-activated kinase (PAK) is an upstream mediator for ANG II-induced activation of JNK in cultured rat VSMC. Rac and Cdc42 GTP-bound proteins associate with PAK, suggesting that ANG II induces activation of the Rac and Cdc42. Importantly, ANG II activation of PAK involved a Ca2+-dependent kinase other than Src. This suggests that multiple tyrosine kinase pathways may exist for the AT1 receptor/GPCR-induced activation of small GTP-binding proteins.

Inactivation of ANG II-stimulated MAPKs occurs via MKP-1-induced dephosphorylation of tyrosine and threonine on MAPKs. Inhibition of MKP-1 results in sustained activation of MAPK in response to ANG II, suggesting that this enzyme is primarily responsible for the termination of the MAPK signal (63, 64). In VSMC, ANG II modulates MKP-1 activity. MKP-1 expression is stimulated by ANG II, and activities of MKP-1 as well as tyrosine phosphatase (PTP-1C) and serine/threonine phosphatase 2A (PP2A) are increased by ANG II (15, 118, 149). Various studies have shown that these effects may be mediated, at least in part, via the AT2 receptor subtype, which has been associated with inhibition of cell growth and apoptosis (15, 77, 118). It is thus possible that AT1 receptors induce growth via stimulation of ERK-dependent signaling pathways, whereas AT2 receptors oppose these effects by stimulating MKP-1 activity to inhibit ERK activity and to arrest the cell growth signal. Termination of ANG II-stimulated MAPK activity may also involve activation of protein kinase A (PKA), which inhibits the phosphorylation of Raf-1 (44).

RECEPTOR TYROSINE KINASES. RTKs mediate a variety of cellular growth responses. Ligand binding to the RTK results in activation of an intrinsic kinase, which in turn results in autophosphorylation and formation of new binding sites (e.g., for SH2 or phosphotyrosine binding domains). This results in RTK binding of adapter proteins, such as SHC, or tyrosine phosphorylation of other proteins, such as Src, PLC-gamma 1, and PI3K. RTK may also be activated by a variety of nonligand stimuli, such as ROS (235), and ultraviolet radiation (178).

Recent evidence suggests that mitogenic responses to GPCR, such as the AT1 receptor, may also be mediated by activation of RTKs, such as the EGFR (71, 204). Receptor transactivation may be defined as that process whereby ligand stimulation of one receptor leads to activation of another, distinct receptor. Three mechanisms have been proposed for ANG II-induced RTK transactivation in VSMC (67): tyrosine kinase phosphorylation (71, 204), ROS activation, or cleavage of the EGFR (67).

MECHANISMS OF AT1 RECEPTOR-INDUCED RECEPTOR KINASE TRANSACTIVATION. Studies by Murusawa et al. (204) in cardiac fibroblasts demonstrated that inhibition of EGFR activity by a dominant-negative EGFR mutant or by treatment with a specific EGFR antagonist abrogated ANG II-induced ERK1/2 activation, induction of c-fos gene expression, and DNA synthesis. This mechanism does not involve production of autocrine factors (71) but appears to be mediated by a Ca2+-dependent kinase, such as PYK2, in VSMC (71) and cardiac fibroblasts (204).

Recent studies in cultured rat aortic VSMC by Bokemeyer et al. (27) demonstrated that ANG II-induced EGFR transactivation depends on c-Src. ANG II also resulted in EGFR kinase-induced phosphorylation of p52 and p66 isoforms of Shc adapter protein, leading to formation of an EGFR-Shc complex. These findings suggest an obligatory role for EGFR kinase in ANG II-induced signaling through the Shc adapter protein (Fig. 4).

Ushio-Fukai et al. (310) recently explored the possibility that ROS may mediate ANG II-induced EGFR transactivation. In these studies, pretreatment of VSMC with antioxidants prevented ANG II-induced tyrosine phosphorylation of the EGFR, but not EGF-induced phosphorylation of its own receptor. Alternatively, direct treatment of these cells with H2O2 and the superoxide-generating compound LY-83583, in the absence of any other ligand, was associated with a concentration-dependent increase in EGFR phosphorylation. These observations suggest that ROS may induce EGFR phosphorylation through activation of an upstream intermediary, rather than activation of EGFR kinase. In this case, redox-sensitive candidates include Ca2+ (284), PYK2 (80), and c-Src (67, 96). Further studies by Ushio-Fukai et al. in VSMC demonstrated that EGFR transactivation could be prevented by inhibition of tyrosine kinases, c-Src kinases, or Ca2+ chelation, but not by Jak2 kinase or PI3K inhibition. In addition, transfection of these cells with an adenovirus containing DNA for a kinase-inactive form of c-Src led to inhibition of the activity of c-Src compared with inactive (Ad.LacZ) control transfected cells. These data suggest that c-Src is an upstream effector for ANG II-induced EGFR transactivation by tyrosine phosphorylation.

Similarly, in VSMC, ANG II may transactivate the PDGF beta -receptor independent of autocrine PDGF production (173). Treatment of VSMC with ANG II leads to tyrosine phosphorylation of Shc proteins, resulting in subsequent complex formation between Shc proteins and the PDGFR. This in turn is associated with Src activation. Moreover, these events could be inhibited by treatment with an AT1 receptor antagonist. Other studies have also demonstrated that ANG II induces rapid transactivation of the mitogenic insulin-like growth factor I receptor in VSMC (61). This effect involves autophosphorylation of the beta -subunit of the tyrosine kinase receptor and phosphorylation of insulin receptor substrate-1.

EFFECTS OF AT1 RECEPTOR KINASE TRANSACTIVATION. AT1 receptor-elicited tyrosine phosphorylation and activation of EGFR result in downstream activation of ERK1/2 and VSMC hyperplasia (Fig. 4) (27). In rat VSMC, ANG II-induced nuclear protooncogene expression and increase in c-Fos protein were prevented by treatment with an MEK or an EGFR kinase inhibitor (68). By contrast, this mechanism is not involved in ANG II-stimulated c-Jun expression in these cells (68). Alternatively, ANG II-induced expression of this growth-response gene may be mediated by PYK2 (260).

ANG II-mediated EGFR transactivation is also reported to be operative in other growth-promoting signaling pathways (69, 288). The p70 ribosomal protein S6 kinase (p70S6K), which is activated on phosphorylation (233), is a major component of the cellular machinery involved in protein synthesis (319). ANG II may stimulate protein synthesis in rat VSMC through p70S6K activation (87). Studies by Eguchi et al. (69) demonstrated that transfection of a dominant-negative mutant of Ras inhibited AT1 receptor-induced p70S6K Ser411 phosphorylation, implicating an Ras-PI3K-PKB cascade, rather than the Raf-MEK-ERK system, in the activation of p70S6K.

Could AT1 receptor transactivation participate in vascular pathobiology? In studies of cultured VSMC isolated from hypertensive rats, ANG II-induced increase in TGF-beta mRNA abundance was mediated through ERK1/2 activation, enhanced c-Fos/c-Jun expression, and increased activation of NF-kappa B and AP-1 transcription factors (101). These observations suggest that AT1 RTK transactivation may be involved in cellular processes associated with vascular remodeling.

Phospholipid second-messenger signaling. AT1 receptor-Gq protein stimulation leads to activation of phospholipase A2 (PLA2), PLC-beta , and PLC-gamma (8). The AT1 receptor also activates phospholipase D (PLD) by a mechanism that involves the G protein subunit Gbeta gamma and smaller G proteins (194, 309). PLA2 activation results in catabolism of arachidonic acid (AA) and the production of PG metabolites (248).

PLC ACTIVATION BY AT1 RECEPTORS. One of the earliest detectable events resulting from ANG II stimulation of VSMC is a rapid, PLC-dependent hydrolysis of phosphatidylinositol 4,5-bisphosphate (3, 18, 95, 97). The PLC family includes three related enzymes: PLC-beta , PLC-gamma , and PLC-delta , which are regulated by G proteins alpha  and beta gamma in the case of PLC-beta (290), by tyrosine phosphorylation in the case of PLC-gamma (152), or by Ca2+ in the case of PLC-delta (19, 239). PLC-beta 1, PLC-gamma 1, and PLC-delta 1 have been identified in VSMC (182). AT1 receptor activation results in a rapid production of inositol 1,4,5-trisphosphate and a more sustained release of diacylglycerol (DAG; Fig. 5A) (127), which are involved in Ca2+ mobilization from the sarcoplasmic reticulum (20) and stimulation of PKC (320), respectively. ANG II-stimulated inositol trisphosphate generation may also be mediated, in part, via tyrosine kinase-dependent pathways (92). Increased intracellular Ca2+ results in VSMC contraction (60), whereas PKC activation regulates intracellular pH through the Na+/H+ exchanger (313). PLC activation correlates temporally with initiation of contraction in isolated VSMC, as well as in intact small resistance arteries and, most likely, constitutes the early signaling pathway for initiation of the Ca2+-dependent, calmodulin-activated phosphorylation of the myosin light chain that leads to cellular contraction (158, 252, 296, 299, 321).


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  		Fig. 5.   A: AT1 receptor-induced activation of PLC. IP3, inositol trisphosphate. B: AT1 receptor-induced activation of PLD.

ACTIVATION OF PLA2 AND AA METABOLISM BY ANG II. ANG II stimulates PLA2 activity, which is responsible for release of AA from cell membrane phospholipids (29, 53, 236). PLA2-derived eicosanoids influence vascular and renal mechanisms important in blood pressure regulation (212). In VSMC and endothelial cells, these effects are mediated via AT1 receptors (82, 231), whereas in neonatal rat cardiac myocytes, neuronal cells, and renal proximal tubule epithelial cells, ANG II-induced activation of PLA2 occurs via AT2 receptors (65, 176, 241, 343). ANG II-elicited activation of vascular PLA2 is dependent on intracellular Ca2+ concentration, Ca2+-calmodulin-dependent protein kinase II, and MAPKs (206, 207). Activated PLA2 and its metabolites in turn activate Ras/MAPK-dependent signaling pathways, amplifying PLA2 activity and releasing additional AA by a positive-feedback mechanism (206). In renal epithelial cells, ANG II activates PLA2 via an AT2-mediated Ca2+-independent mechanism (14, 135). Renal-derived arachidonate phosphorylates the adapter protein Shc and stimulates its association with Grb2 and Sos1 (65). ANG II-generated eicosanoids regulate vascular contraction and growth, possibly by activating MAPKs and redox-sensitive pathways (65, 212). Thromboxanes are involved in ANG II-induced contraction, whereas vasorelaxant PGs such as PGE2 and PGI2 attenuate ANG II-mediated vasoconstriction in some vascular beds (328). Lipoxygenase-derived eicosanoids also influence ANG II-mediated actions in VSMC. 12-Hydroxyeicosatetraenoic acid (HETE) facilitates the stimulatory actions of ANG II on Ca2+ transients in cultured cells. Lipoxygenase inhibitors attenuate the vasoconstrictor action of ANG II and decrease blood pressure in spontaneously hypertensive rats (221, 281). Some of these effects may be mediated via modulation of the oxidative state of the cell (339).

PLD ACTIVATION BY ANG II. Hydrolysis of phosphatidylcholine by PLD leads to the production of phosphatidic acid and subsequent generation of DAG by phosphatidic acid phosphohydrolase (24, 55, 89, 158). DAG contributes to prolonged activation of PKC. This pathway probably represents the major cascade by which ANG II-induced activation of PKC remains sustained in VSMC. The downstream pathways associated with ANG II-induced activation of PLD in VSMC are PKC independent (81) but involve intracellular Ca2+ mobilization (81) and Ca2+ influx that is tyrosine kinase dependent (283). ANG II-induced PLD signaling has been implicated in cardiac hypertrophy as well as in proliferation of VSMC (55, 200). PLD-dependent signaling cascades also influence cardiac and vascular contraction (332). These effects are mediated via phosphatidic acid and other PLD metabolites (25, 55, 329) that influence vascular generation of superoxide anions by stimulating NADH/NADPH oxidase (89, 94, 309), activate tyrosine kinases and Raf, and modulate intracellular Ca2+ signaling (25, 72, 89). The long-term signaling pathways associated with ANG II-stimulated growth and remodeling in the cardiovascular system are dependent, in part, on PLD-mediated responses.

Molecular mechanisms coupling AT1 receptors to PLD have recently been identified. AT1 receptor-induced PLD activation involves a Gq/11- and Gi/O-independent mechanism (Fig. 5B). Using immunoprecipitation techniques in astrocytoma cells, Mitchell et al. (194) demonstrated that AT1 receptor activation resulted in translocation of ARF/RhoA to the plasma membrane, thereby forming an AT1 receptor-GTP-binding protein functional complex. This in turn resulted in PLD activation. This pathway appears dependent on a specific Asn-Pro-XX-Tyr amino acid sequence within the AT1 receptor. In summary, these observations confirmed that the AT1 receptor and other GPCR can physically associate with intracellular proteins other than Gq/11, creating membrane-delimited signal transduction complexes similar to those observed for classic growth factor receptors (194, 246). These observations are supported by studies by Ushio-Fukai et al. (309), which demonstrated that ANG II-induced stimulation of PLD was inhibited by electroporation of anti-Gbeta , anti-Galpha 12, anti-c-Src, and anti-Rho antibodies, whereas anti-Galpha i and -Gaalpha q/11 antibodies had no effect. These results implicate Gbeta gamma and Galpha 12 subunits in AT1 receptor-induced PLD activation through a c-Src-RhoA-mediated pathway.

AT1 receptor-mediated transcription factor activation. Transcription factors, such as NF-kappa B and AP-1, are important upstream mediators of ANG II trophic effects (156, 202, 312). In its inactive form, NF-kappa B is a heterotrimeric, cytoplasmic protein that is bound to inhibitory-kappa B (Ikappa B) (13). Activation of NF-kappa B involves release from Ikappa B when the latter undergoes phosphorylation, which occurs because of the activity of cytokine-inducible oxidant-sensitive kinases (138, 151, 244). On activation, NF-kappa B enters the nucleus and binds to the promoter region of target, inflammatory genes such as intercellular adhesion molecule and vascular cell adhesion molecule (13).

ANG II-induced activation of NF-kappa B has been causally implicated in the inflammatory vasculopathy in rats made hypertensive by chronic blockade of NO synthesis (312) or by chronic infusion of ANG II (202). Treatment with an AT1 receptor antagonist or an antioxidant inhibited these effects. In other studies in rat VSMC, Ruiz-Ortega et al. (244) demonstrated that ANG II treatment stimulated degradation of cytosolic Ikappa B-alpha binding protein, which was paralleled by translocation of the activated heterotrimeric protein form of NF-kappa B, p50/p65, to the nucleus. These effects were attenuated by AT1 receptor and phosphotyrosine kinase inhibition. Furthermore, AT1 receptor inhibition abrogated NF-kappa B-induced gene transcription.

AT1 receptor-mediated generation of ROS. ANG II increases VSMC superoxide (O<UP><SUB>2</SUB><SUP>−</SUP></UP>·) production by activation of a membrane-bound NAD(P)H oxidase, suggesting that enhanced ROS activity may be involved in the vasoactive effect of this hormone (94, 160, 234). Zafari et al. (340) demonstrated that the ANG II-stimulated activation of NAD(P)H oxidase occurred through release of AA metabolites, triggering PKC activation. This in turn led to phosphorylation of the phox subunits of NAD(P)H oxidase and activation of this enzyme. Ushio-Fukai et al. (311) demonstrated that transfection of antisense p22phox cDNA into cultured rat VSMC abrogated any ANG II-stimulated increase in O<UP><SUB>2</SUB><SUP>−</SUP></UP>· concentrations or hypertrophy of these cells. These observations implicated the p22phox subunit as a key element in NAD(P)H oxidase-dependent O<UP><SUB>2</SUB><SUP>−</SUP></UP>· production. Further work in rat VSMC treated with diethyldithiocarbamate, a superoxide dismutase inhibitor, demonstrated that O<UP><SUB>2</SUB><SUP>−</SUP></UP>· conversion to H2O2 was important for ANG II-stimulated VSMC hypertrophy (339). In studies undertaken by Pagano's group, cotreatment of ANG II-treated aortae with actinomycin D, an inhibitor of transcription, and cycloheximide, an inhibitor of protein synthesis, attenuated ANG II-stimulated O<UP><SUB>2</SUB><SUP>−</SUP></UP>· production in these arteries (323). These data suggest that ANG II augments NAD(P)H oxidase-mediated O<UP><SUB>2</SUB><SUP>−</SUP></UP>· production by enhancing the abundance of mRNA via transcriptional and nontranscriptional pathways (223, 322). Interestingly, the AT1 receptor antagonist losartan inhibited ANG II-stimulated O<UP><SUB>2</SUB><SUP>−</SUP></UP>· production in rat thoracic aortae; in similar studies in rabbits, losartan had no such action (323). This suggests that important species differences may exist in this pathway, inasmuch as ANG II-stimulated O<UP><SUB>2</SUB><SUP>−</SUP></UP>· production in rabbits could be inhibited by the nonspecific receptor antagonist [Sar1,Thr8]ANG II (224). Berry et al. (23) demonstrated that ANG II stimulates O<UP><SUB>2</SUB><SUP>−</SUP></UP>· production in human internal mammary arteries by an AT1 receptor-dependent, NAD(P)H oxidase-mediated pathway. They also found that the AT2 receptor activation does not contribute to ANG II-stimulated O<UP><SUB>2</SUB><SUP>−</SUP></UP>· production in these arteries (22).

ROS are involved in modulating a variety of intracellular signaling pathways for vascular cell growth regulation (80, 131). ROS are second messengers for AT1 receptor activation, such as ANG II-induced EGFR transactivation (310). Schieffer et al. (257) recently investigated the possibility that ROS may act as signaling messengers for AT1 receptor activation of Jak and STAT factors in rat aortic VSMC. Treatment of these cells with ANG II (10 µmol/l) stimulated an increase in the concentrations of O<UP><SUB>2</SUB><SUP>−</SUP></UP>· and the cytokine interleukin (IL)-6. Both of these effects were abolished by cotreatment with the AT1 receptor antagonist losartan or the flavoprotein inhibitor diphenyleneiodonium or by inhibition of p47phox by electroporation of p47phox antibodies into these cells. Similarly, treatment of these cells with ANG II led to Jak2, STAT1alpha /beta , and STAT3 tyrosine phosphorylation, which could also be inhibited by treatment with losartan, diphenyleneiodonium, or electroporation of p47phox antibodies. In other studies, these investigators demonstrated that treatment of rat VSMC with AG-940 (10 µmol/l), a selective antagonist of Jak2, or STAT1alpha /beta antisera prevented ANG II-induced synthesis of IL-6. These studies demonstrated that, in rat VSMC, ANG II-induced NAD(P)H oxidase-dependent O<UP><SUB>2</SUB><SUP>−</SUP></UP>· production may be important for activation of the Jak-STAT cascade, which in turn stimulates an increase in the synthesis of IL-6.

In other studies in rat VSMC, Viedt et al. (317) reported that AT1 receptor-induced ROS production stimulated JNK and p38MAPK, but not ERK1/2, leading to an increase in AP-1 DNA binding. Inhibition of p22phox activity by treatment with a specific antibody or antisense DNA abolished AT1 receptor-induced JNK and p38MAPK activation and reduced AP-1 DNA binding. In this study, ANG II induced ERK1/2 activation by a tyrosine kinase-, PKC-, and MEK-dependent pathway. Ushio-Fukai et al.(308) also demonstrated that ANG II-induced ERK1/2 activation may be ROS independent, whereas other studies by Frank et al. (79) demonstrated that NAD(P)H oxidase inhibition inhibits ERK1/2 activation. Taken together, these observations suggest that ANG II may activate tyrosine kinase pathways, such as ERK1/2, by ROS-dependent and ROS-independent pathways.

The activity of NF-kappa B is also regulated by ROS activity (13, 202, 312, 312), suggesting the possibility that AT1 receptor-induced ROS production (234) leads to activation of NF-kappa B (23). This thesis has been recently investigated by Pueyo et al. (232), who demonstrated that AT1 receptor-induced activation of NF-kappa B, which was associated with enhanced vascular cell adhesion molecule-1 expression, is a redox-sensitive pathway.

AT2 Receptor

The signaling pathways involved in AT2 receptor activation are not fully understood but appear to involve G protein-dependent (106, 341) and -independent (31) pathways (32, 84, 116). Immunoselection studies in the rat fetus demonstrated that AT2 receptors are associated with Galpha i2 and Galpha i3, rather than AT1 receptor-Gq/11 (341). This raises the possibility that this receptor may be G protein coupled (Fig. 1).

Hansen et al. (106) recently investigated the pharmacology of the AT2 receptor in relation to interactions of this receptor with G proteins. Studies with cells transfected with plasmids encoding the AT2 receptor, Galpha i2, Gbeta 2, and Ggamma 1 sequences, facilitated the selective investigation of AT2 receptor ligands and G protein activation. In other studies, NIH/3T3 cells, which express native AT2 receptor, were also used. They found that the AT2 receptor may catalyze the exchange of GDP for guanosine 5'-O-(3-thiotriphosphate) on Galpha i or Galpha 0, but not Galpha q or Galpha s. Their other findings included evidence that the pseudopeptide CGP-41112A is a partial agonist of the AT2 receptor.

The AT2 receptor-G protein hypothesis is supported by studies in other cell types. For example, in cultured hypothalamic neurons, the AT2 receptor-mediated delayed rectifier K+ current is abolished by treatment with a selective anti-Galpha i binding protein or pertussis toxin, a selective Galpha i protein inhibitor (150). Furthermore, in biochemical studies in cultured neurons, AT2 receptor-stimulated activation of serine/threonine PP2A has also been shown to be selectively inhibited by pertussis toxin (121, 122). PP2A activation results in dephosphorylation and inactivation of growth factor-activated MAPK and, in particular, inactivation of ERK1/2. In studies in transgenic mice, cardiac overexpression of AT2 receptor was associated with an inhibition of AT1 receptor-mediated MAPK activation, which may have been involved in the AT2 receptor-mediated negative chronotropic effect in these animals (187).

Using cultured fibroblasts that selectively express the AT2 receptor but not the AT1 receptor, Tsuzuki et al. (305) demonstrated that EGF-induced cell proliferation was attenuated by cotreatment of these cells with ANG II. This growth-retardant response was enhanced by the specific AT2 receptor agonist CGP-42112A and inhibited by the AT2 receptor antagonist PD-123319. Again, the mechanism for this appeared to involve activation of PP2A, supporting the thesis that AT2 receptor-induced PP2A activation inhibits cell proliferation by counterregulation of MAPK phosphorylation.

Yamada et al. (333) demonstrated that AT2 receptor activation may also lead to programmed cell death. In PC12W cells, a rat pheochromocytoma cell line that selectively expresses AT2 receptors rather than AT1 receptors, ANG II activation antagonized the growth-promoting effects of nerve growth factor and resulted in apoptosis of these cells. This mechanism is G protein coupled, inasmuch as pertussis toxin treatment inhibited ERK1/2 activation and apoptosis. Bcl-2 is an intracellular membrane protein that, when phosphorylated, inhibits apoptosis (262). In further studies in PC12W cells, Horiuchi et al. (118) demonstrated that the activity of Bcl-2 could be modulated by MAPK-induced phosphorylation and M