I. Introduction

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The vascular wall is an active, pliable and integrated organ made up of cellular (endothelial cells, vascular smooth muscle cells, and fibroblasts) and noncellular (extracellular matrix) components. It is not a static organ; the components dynamically change shape, increase, decrease, or reorganize, in response to physiological and pathological stimuli (Dubey, 1997). In the intact arterial media, smooth muscle cells and matrix are responsible for structural and functional characteristics of the vessel wall, including contraction-relaxation, growth, development, remodeling, and repair, and for the pathogenesis of cardiovascular disease, such as atherosclerosis, restenosis and hypertension (Mulvany and Aalkjaer, 1990; Schiffrin, 1992; Katoh and Periasamy, 1996; Bornfeldt, 1996). Many local and systemic factors regulate vascular smooth muscle cell function, including vasoactive peptides, such as Ang² II and endothelin-1 (ET-1), that stimulate vasoconstriction and growth and vasorelaxing factors, such as nitric oxide, prostacyclin, and C-type natriuretic peptide that induce vasodilation by increasing levels of cyclic nucleotides (Rubanyi, 1991; Lüscher, 1993; Lüscher and Barton, 1997; Stein and Levin, 1998).

Ang II is a multifunctional peptide that has numerous actions on vascular smooth muscleit modulates vasomotor tone, it regulates cell growth and apoptosis, it influences cell migration and extracellular matrix deposition, it is proinflammatory, and it stimulates production of other growth factors [e.g., platelet-derived growth factor (PDGF)] and vasoconstrictors (e.g., ET-1). Accordingly, Ang II plays a fundamental role in controlling the functional and structural integrity of the arterial wall and may be important in physiological processes regulating blood pressure and in pathological mechanisms underlying vascular diseases. The multiple actions of Ang II are mediated via specific, highly complex intracellular signaling pathways that are stimulated following an initial binding of the peptide to its cell-surface receptors (Matsusaka and Ichikawa, 1997). The term "intracellular signaling pathway" includes the interconnected molecular cascades that transmit information from the cell membrane receptor to the intracellular proteins that regulate cell activities such as contraction, cell growth, mitogenesis, apoptosis, differentiation, migration, and other specialized functions. Identification of such signal transduction processes is essential for understanding mechanisms that regulate vascular smooth muscle cell function, both physiologically and pathophysiologically. This review focuses on Ang II-mediated signaling in vascular smooth muscle cells and implications of altered Ang II-induced signal transduction in vascular pathological processes, concentrating specifically on hypertension. The molecular and cellular mechanisms of Ang II in cardiac and renal diseases have recently been reviewed and will not be discussed in detail here (Kim and Iwao, 2000).

	II. Physiological Actions of Angiotensin II in Vascular Smooth Muscle Cells

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Ang II, an octapeptide hormone, is the active component of the renin-angiotensin system (RAS). It regulates blood pressure, plasma volume via aldosterone-regulated sodium excretion, sympathetic nervous activity, and thirst responses. It also plays a fundamental role in pathological adaptation, as manifested in myocardial remodeling after myocardial infarction and in vascular remodeling in hypertension. Ang II is produced systemically via the classical or renal RAS, and locally via tissue RAS. In the classical RAS, circulating renal-derived renin cleaves hepatic-derived angiotensinogen at the N terminus to form the decapeptide, angiotensin I, which is converted by the dipeptidyl carboxypeptidase, angiotensin-converting enzyme (ACE), in the lungs, to the active Ang II (Skeggs et al., 1967; Dorer et al., 1972; Phillips et al., 1993; Inagami, 1998) (Fig. 1). Ang I can also be processed into the heptapeptide Ang-(1-7) by three tissue endopeptidases, neutral endopeptidase (NEP) 24.11, NEP 24.15, and NEP 24.26 (Ferrario et al., 1997). Ang II is degraded by aminopeptidases to Ang III and Ang IV (Fig. 1).

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Scheme of the classical renin-angiotensin system. Circulating renal-derived renin cleaves hepatic-derived angiotensinogen to form the decapeptide angiotensin I (Ang I). Ang I is converted by ACE in the lungs and tissue to active angiotensin II (Ang II), which is further metabolized to angiotensin III, angiotensin IV, and Ang II (1-7). Several non-ACEs, such as chymase, carboxypeptidase and cathepsin G, may also cleave Ang I to Ang II.

The RAS was originally regarded as a circulating system. However, many of its components are localized in tissues indicating the existence of a local tissue RAS as well (Dzau, 1989; Danser, 1996). ACE exists in plasma (as the circulating hormone), in the interstitium and intracellularly. Tissue ACE is present in all major organs, heart, brain, blood vessels, adrenals, kidney, liver, and reproductive organs (Hollenberg, 1998), and is already functional in utero (Schutz et al., 1996; Esther et al., 1997). Tissue ACE activity seems to peak during the phase of major organ development and declines thereafter (Esther et al., 1997). All components of the RAS, except renin, have been demonstrated to be produced in the vasculature. ACE is found in high concentrations in the adventitia, as well as in cultured vascular smooth muscle and endothelial cells (Dzau, 1989; Ekker et al., 1989; Naftilan, 1994). Angiotensinogen mRNA and protein have been detected in vascular smooth muscle, endothelium, and perivascular fat (Naftilan et al., 1991; Naftilan, 1994; Morgan et al., 1996). Since vascular renin is absent, local generation of Ang II in the interstitium is regulated by tissue ACE that is probably dependent on circulating renin (Fig. 2). Although the function of tissue ACE is currently unclear, it may contribute to regulation of regional blood flow as recently demonstrated in the human forearm where in situ generated Ang II is more important for vasoconstriction than circulating Ang II (Saris et al., 2000).

View larger version (51K): [in this window] [in a new window]   Fig. 2.   Scheme of the tissue renin-angiotensin system. Angiotensinogen, ACE, and angiotensin receptors have been demonstrated in endothelial and vascular smooth muscle cells, as well as in perivascular fat. Tissue-derived angiotensinogen is converted to Ang I by renal-derived renin that is adsorbed from the circulation. Ang I is cleaved to Ang II by tissue ACE. endoth, endothelium; other abbreviations as in Fig. 1.

In addition to ACE-dependent pathways of Ang II formation, non-ACE pathways, which could be particularly important in pathological states, have been demonstrated. Chymotrypsin-like serine protease (chymase) may represent an important pathway for conversion of Ang I to Ang II in the human heart (Urata et al., 1990, 1996) and kidney (Hollenberg, 1998). Functional chymase and a non-ACE pathway have also been demonstrated in human vascular tissue (Hollenberg et al., 1998; Takai et al., 1998) and in dog carotid artery (Shiota et al., 1999).

In mammalian cells, Ang II mediates its effects via at least two high-affinity plasma membrane receptors, AT₁ and AT₂. Both receptor subtypes have been cloned and pharmacologically characterized (Murphy et al., 1991; Sasaki et al., 1991; Kambayashi et al., 1993; Mukoyama et al., 1993). Two other Ang receptors have been described, AT₃ and AT₄ subtypes. The AT₃ receptor subtype, initially described in the neuro 2A neuroblastoma cell line (Chaki and Inagami, 1992) is peptide-specific recognizing mainly Ang II. This subtype does not bind nonpeptide ligands such as losartan (selective AT₁ receptor antagonist) or PD123319 (selective AT₂ receptor antagonist), and has only been observed in cell lines. The AT₄ receptor, which is distributed in heart, lung, kidney, brain, and liver, binds Ang IV (Swanson et al., 1992) but not losartan or PD123319. Since the pharmacology of AT₃ and AT₄ receptors has not been fully characterized, these receptors are not yet included in a definitive classification of mammalian AT receptors as defined by the International Union of Pharmacology Nomenclature Subcommittee for Angiotensin Receptors (de Gasparo, 1995).

The AT₁ receptor belongs to the seven membrane-spanning G protein-coupled receptor family and typically activates phospholipase C through the heterotrimeric Gq protein (de Gasparo et al., 1995; Inagami, 1995) (Table 1). Human AT₁ receptor gene is mapped to chromosome 3. To date, AT₁ receptors have been shown to mediate most of the physiological actions of Ang II, and this subtype is predominant in the control of Ang II-induced vascular functions (Sadoshima, 1998). In the vasculature, AT₁ receptors are present at high levels in smooth muscle cells and relatively low levels in the adventitia and are undetectable in the endothelium (Zhuo et al., 1998; Allen et al., 2000). Two AT₁ receptor subtypes have been described in rodents, AT_1A and AT_1B, with greater than 95% amino acid sequence identity (Iwai and Inagami, 1992). AT_1A and AT_1B receptor genes in rats are mapped to chromosome 17 and 2, respectively. Based on the cDNA sequence, the AT₁ receptor is composed of 359 amino acids (Sandberg, 1994). It is a glycoprotein and contains extracellular glycosylation sites at the amino terminus (Asn⁴) and the second extracellular loop (Asp¹⁷⁶ and Asn¹⁸⁸) (Desarnaud et al., 1993). 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 (Hjorth et al., 1994). Internalization of G protein-coupled receptors involves receptor phosphorylation, which may be mediated, in part via caveola (Berk and Corson, 1997; Ishizaka et al., 1998). 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. AT₁ receptors are phosphorylated both in the basal state and in response to Ang II stimulation (Kai et al., 1994). Threonine and serine residues between Thr³³² and Ser³³⁸ of the cytoplasmic tail are essential for receptor internalization (Hunyady et al., 1994). The AT₁ receptor is also phosphorylated at tyrosine residues. Potential tyrosine phosphorylation sites within the AT₁ receptor include amino acids 302, 312, 319, and 339 within the carboxyl terminus (Berk and Corson, 1997). Tyrosine at position 319 is important as it is part of the motif Tyr-Ile-Pro-Pro, which is analogous to a Src homology 2 (SH2) binding motif in the PDGF receptor (Tyr-Ile-Pro) and in the epidermal growth factor (EGF) receptor (Tyr-Leu-Pro-Pro) (Fantl et al., 1993). In EGF and PDGF receptors, these motifs are target sequences for tyrosine phosphorylation. Various tyrosine kinases, including Janus kinases (JAK and TYK), Src family kinases, and focal adhesion kinase (FAK) can tyrosine phosphorylate AT₁ receptors.

The second major isoform of the Ang receptor, AT₂, is normally expressed at high levels in fetal tissues and decreases rapidly after birth (Nahmias and Strosberg, 1995). The AT₂ receptor gene is localized as a single copy on the X chromosome. In adults, AT₂ receptor expression is detectable in the pancreas, heart, kidney, adrenals, brain, and vasculature (Viswanathan and Saavedra, 1994; Touyz et al., 1999a). In the vasculature, AT₂ receptors predominate in the adventitia and are detectable in the media (Zhuo et al., 1998). AT₂ receptors are also expressed in several cell lines, including PC12W, R3T3, and N1E115 (Inagami, 1995). The AT₂ receptor is a seven transmembrane-type, G protein-coupled receptor, comprising 363 amino acids. It has low amino acid sequence homology (~32%) with AT_1A or AT_1B receptors (Mukoyama et al., 1993). Although the exact signaling pathways and the functional roles of AT₂ receptors are unclear, these receptors, which appear to be regulated by intracellular cations, particularly Na⁺ (Tamura et al., 1999), may antagonize, under physiological conditions, AT₁-mediated effects (Ciuffo et al., 1998; Yamada et al., 1998) by inhibiting cell growth, and by inducing apoptosis and vasodilation (Hayashida et al., 1996; Horiuchi, 1997a,b; Gallinat et al., 2000; Unger, 1999; Siragy, 2000). The exact role of AT₂ receptors in cardiovascular disease remains to be defined.

Ang II promotes its effects by acting directly through Ang II receptors present on vascular cells, indirectly through the release of other factors, and possibly via cross-talk with intracellular signaling pathways of other vasoactive agents and growth factors. Although the principal function of smooth muscle cells is vasoconstriction, it has become evident that vascular smooth muscle cells have important synthetic properties during development and vascular remodeling (Table 2) and are the major source of extracellular matrix components of the vascular media (Katoh and Periasamy, 1996). During blood vessel development, immature smooth muscle cells are in a dynamic state of growth and differentiation characterized by proliferation and migration (Glukhova et al., 1991). In the adult vessel, they become quiescent and assume a fibroblast-like appearance, and become filled with contractile fibers (Gordon et al., 1990). Although mature smooth muscle cells remain quiescent until injury or insult occurs, they undergo physiological hypertrophy in response to increased load (Bucher et al., 1982; Katoh and Periasamy, 1996). Ang II plays a role in these developmental processes, acting via AT₁ and AT₂ receptors, which are differentially expressed in vascular smooth muscle cells during normal development and during pathological processes. In vascular disease, smooth muscle cells undergo hyperplasia and/or hypertrophy as an adaptive or reactive response (Table 2). (Geisterfer et al., 1988; Berk et al., 1989; Paquet et al., 1990; Stouffer and Owens, 1992; Dubey, 1997; Touyz and Schiffrin, 1997a; Touyz et al., 1999b) and may be critical in vascular remodeling associated with hypertension, atherosclerosis, or neointimal formation. Both Ang II receptor subtypes appear to be necessary for a complete vascular smooth muscle cell response to injury (Zahradka et al., 1998).

Integrated vascular responses to Ang II are the result of combined AT₁- and AT₂-mediated actions, as well as effects of bioactive end products of the RAS, such as Ang-(1-7). Whereas Ang II induces vasoconstriction, growth, migration, production of extracellular matrix components, and inflammation via AT₁ receptors, it promotes apoptosis, and inhibits proliferation and hypertrophy via AT₂ receptors (Allen et al., 2000; Siragy, 2000). Ang-(1-7) has been described as a naturally occurring competitive inhibitor of Ang II, as it has potent vasodepressor and antihypertensive effects. It can stimulate release of vasopressin, act as an excitatory neurotransmitter, augment synthesis and release of vasodilator prostaglandins, potentiate the actions of bradykinin and release nitric oxide (Ferrario et al., 1997). In addition, ACE inhibitors augment circulating levels of the vasodilator peptide, which may contribute to the antihypertensive effects associated with ACE inhibitors (Chappel et al., 1998; Iyer et al., 1998). The receptor mediating the vascular actions of Ang-(1-7) has been tentatively characterized as a non-AT₁/AT₂ subtype (Ferrario et al., 1997). Although the exact role of this peptide in the physiological and pathophysiological regulation of vascular function awaits clarification, its potential to antagonize AT₁-mediated actions suggests that Ang-(1-7) could modulate vascular tone by promoting vasodilation.

Ang II elicits complex highly regulated cascades of intracellular signal transduction that lead to short-term vascular effects, such as contraction, and to long-term biological effects, such as cell growth, migration, extracellular matrix deposition, and inflammation. Ligand-receptor binding on the external cell membrane surface induces the interaction between the receptor and effector protein on the internal cell membrane surface via G proteins (heterotrimeric proteins comprised of ,  and  subunits). Intracellular signaling via the AT₂ receptor subtype will not be discussed in detail here, as progress in Ang II type 2 receptor research in the cardiovascular system has recently been reviewed (Csikos et al., 1998; Horiuchi et al., 1999; Unger, 1999). Unless otherwise indicated, signaling events described in the present review are mediated via AT₁ receptors. AT₁ receptors are coupled to multiple, distinct signal transduction processes, leading to diverse biological actions. The signaling processes are multiphasic with distinct temporal characteristics (Fig. 3). Immediate, early, and late signaling events occur within seconds, minutes, and hours, respectively (Fig. 3). Ang II-induced phospholipase C (PLC) phosphorylation and Src activation occur within seconds and constitute immediate signaling events, activation of phospholipase A₂ (PLA₂), phospholipase D (PLD), tyrosine kinases and mitogen-activated protein kinases (MAPKs) occurs within minutes and are early signaling processes, whereas generation of oxidative stress, proto-oncogene expression, and protein synthesis, which occur within hours, make up late signaling events.

View larger version (44K): [in this window] [in a new window]   Fig. 3.   Diagram demonstrating the multiphasic nature of Ang II-mediated signaling events in vascular smooth muscle cells. Binding of Ang II to the AT1 receptor (AT1 rec) stimulates activation of PLC and Src within seconds and constitutes the immediate signaling events. Activation of PLA2, PLD, tyrosine kinases, and MAP kinases occurs within minutes and are the early signaling processes. Generation of reactive oxygen species, proto-oncogene expression, and protein synthesis occurs within hours and makes up the late signaling events.

Ang II-elicited vascular contraction is rapid and utilizes various signaling mechanisms that occur within seconds of Ang II binding to its receptor. These immediate signal transduction processes include: a) G protein-mediated activation of PLC, leading to phosphatidylinositol hydrolysis and formation of inositol trisphosphate (IP₃) and diacylglycerol accumulation (DAG); b) increase in cytosolic free calcium concentration ([Ca²⁺]_i) by increasing Ca²⁺ influx and mobilizing intracellular Ca²⁺; c) activation of protein kinase C (PKC); d) changes in intracellular pH (alkalinization) via stimulation of the Na⁺/H⁺ exchanger; e) changes in intracellular free concentrations of Na⁺ ([Na⁺]_i) and Mg²⁺ ([Mg²⁺]_i); and f) activation of the Src family of kinases (Fig. 4).

View larger version (46K): [in this window] [in a new window]   Fig. 4.   Immediate signaling events induced by Ang II stimulation in vascular smooth muscle cells. Angiotensin receptor (AT1) binding leads to G protein-coupled activation of PLC, resulting in phosphatidylinositol hydrolysis and formation of IP3 and DAG accumulation. IP3 mobilizes Ca2+ from sarcoplasmic reticular stores, and DAG activates PKC, which in turn activates the Na+/H+ exchanger. These events result in increased intracellular free Ca2+ concentration ([Ca2+]i) and intracellular alkalinization. Ang II also activates the Na+-dependent Mg2+ exchanger that induces Mg2+ efflux and Na+ influx leading to increased intracellular free Na+ concentration ([Na+]i) and decreased intracellular free Mg2+ concentration ([Mg2+]i). These signaling events stimulate actin-myosin interaction resulting in vascular smooth muscle cell contraction. Src family kinases, which are also activated by Ang II within seconds, are major upstream regulators of signaling pathways associated with cell growth. Src-dependent pathways may also modulate [Ca2+]i a possible pathway that has not yet been fully elucidated (dashed line).

1. Stimulation of Phospholipase C and Phosphatidylinositol Hydrolysis. One of the earliest detectable events resulting from Ang II stimulation of vascular smooth muscle cells is a rapid, PLC-dependent hydrolysis of phosphatidylinositol-4,5-bisphosphate (PtdInsP₂) to yield water soluble IP₃ and membrane bound DAG (Alexander, 1985; Griendling et al., 1985; Berk et al., 1987a; Griendling et al., 1989). PLC is a family of at least three related genes: PLC-, PLC-, and PLC- (Rhee and Choi, 1992). PLC- isoforms are regulated by  and subunits of G proteins (Smrcka et al., 1991), whereas PLC- isoforms are regulated by tyrosine phosphorylation (Rhee, 1991; Homma et al., 1993; Marrero et al., 1995b). PLC- regulation is unclear, but may involve intracellular Ca²⁺. PLC-1, PLC-1, and PLC-1 have been identified in vascular smooth muscle cells (Marrero et al., 1994; Ushio-Fukai et al., 1998b). The AT₁ receptor sequentially couples to PLC-1 via a heterotrimeric G protein and to PLC-1 via a tyrosine kinase (Ushio-Fukai et al., 1998b; Venema et al., 1998). The initial AT₁ receptor-PLC-1 coupling is mediated by G_q/11 and G₁₂. The dimer acts as a signal transducer for activation of PLC (Touhara et al., 1995; Ushio-Fukai et al., 1998b). Both PLC-1 and PLC- isoforms play a role in IP₃ formation. PLC-1 appears to be important in the rapid generation of IP₃ (within 15 s), whereas PLC- seems to play a role in the later phase of IP₃ formation (Ushio-Fukai et al., 1998b). Ang II-stimulated IP₃ generation may also be mediated, in part, via tyrosine kinase-dependent pathways (Goutsouliak and Rabkin, 1997). Ang II induces a dose-dependent increase in phosphatidylinositol turnover resulting in rapid transient IP₃ formation (Griendling et al., 1989) and biphasic and sustained DAG generation (Alexander et al., 1985; Griendling et al., 1985). Losartan, the selective AT₁ receptor blocker, inhibits Ang II-induced hydrolysis of PtdInsP₂, indicating that Ang II stimulation of the PLC pathway is mediated exclusively via AT₁ receptors. IP₃ stimulates release of Ca²⁺ from sarcoplasmic/endoplasmic reticular stores and DAG, with cofactors phosphatidylserine and Ca²⁺, activates PKC. Ang II-elicited IP₃ signal slightly precedes a rapid increase in cytoplasmic free calcium concentration ([Ca²⁺]_i), which is in large part independent of calcium influx. These events correlate temporally with initiation of contraction in isolated vascular smooth muscle cells, as well as in intact small resistance arteries, and most likely constitute the early signaling pathway for initiation of the calcium-dependent, calmodulin-activated phosphorylation of the myosin light chain, which leads to cellular contraction (Lassegue et al., 1993; Walsh et al., 1995; Savineau and Marthan, 1997; Touyz and Schiffrin, 1997a; Touyz et al., 1999c). DAG can also be formed by the PLD-mediated hydrolysis of other phospholipids such as phosphatidylcholine and phosphatidylethanolamine.

2. Increased Intracellular Free Calcium Concentration. Ang II-stimulated Ca²⁺ signaling is complex and occurs via multiple pathways to elicit an integrated Ca²⁺ signal. Ang II typically mediates a biphasic [Ca²⁺]_i response comprising a rapid initial transient phase and a sustained plateau phase (Dostal, 1990; Touyz et al., 1994; Assender et al., 1997; Touyz and Schiffrin, 1997b). Both AT1_A and AT1_B receptors have been shown to mediate calcium signaling in rodent vascular smooth muscle cells (Zhu et al., 1998b). The first [Ca²⁺]_i transient is generated primarily by IP₃-induced mobilization of intracellular Ca²⁺ and to a lesser extent by Ca²⁺-induced Ca²⁺ release (Touyz and Schiffrin, 1997b). The second [Ca²⁺]_i phase, which appears to contribute to the sustained Ang II-induced vasoconstriction, is dependent on external Ca²⁺ and is the result of transmembrane Ca²⁺ influx (Rembold, 1992; Ruan and Arendshorst, 1996a; Inscho et al., 1997; Iverson and Arendshorst, 1998; Touyz et al., 1999c). Exact mechanisms whereby Ang II stimulates Ca²⁺ influx are unclear but may involve voltage-dependent calcium channels, which are directly or indirectly activated by Ang II, Ca²⁺-permeable, nonspecific dihydropyridine-insensitive cation channels, receptor-gated Ca²⁺ channels, Ca²⁺-activated Ca²⁺ release channels, and activation of the Na⁺/Ca²⁺ exchanger (Arnaudeau et al., 1996; Lu et al., 1996). In addition to IP₃-mediated mobilization of intracellular Ca²⁺ and influx of extracellular Ca²⁺, tyrosine kinase-dependent increases in [Ca²⁺]_i have been demonstrated in vascular smooth muscle cells (Hughes and Bolton, 1995; Touyz and Schiffrin, 1996a; Di Salvo et al., 1998).

3. Activation of Protein Kinase C. Ang II-induced DAG production, together with Ca²⁺ and phosphatidylserine, activate PKC, a serine/threonine kinase that is a member of a multigene family consisting of at least 11 isoenzymes (Hug and Sarre, 1993; Newton, 1997). Ang II stimulates the translocation of cytosolic PKC to the plasma membrane where the activated enzyme phosphorylates specific proteins associated with vascular function (Walsh et al., 1996; Damron et al., 1998). PKC is implicated in Ang II-induced vascular contraction as well as in vascular smooth muscle cell growth (Rasmussen et al., 1987; Ruan and Arendshorst, 1996b; Orjii and Keiser, 1997; Kiron and Loutzenhiser, 1998; Bauer, 1999). These effects are mediated via activation of the Na⁺/H⁺ exchanger leading to intracellular alkalinization, an important modulator of actin-myosin interaction, and of contraction (Aalkjaer and Peng, 1997). In addition, Ang II-stimulated PKC induces its actions through phosphorylation of tyrosine kinases, such as proline-rich tyrosine kinase (PYK2) (Sabri et al., 1998), p130^Cas (Sayeski et al., 1998), and Src family tyrosine kinases (Zou et al., 1998), and by stimulating MAP kinase signaling pathways (Zou et al., 1996; Wilkie et al., 1997; Kudoh, 1997; Li et al., 1998a). The PKC isoform that activates ERK-1 and ERK-2 (extracellular signal-regulated kinases) in vascular smooth muscle cells has been identified as PKC- (Liao et al., 1997). Some studies failed to demonstrate that Ang II effects are PKC-dependent and others reported only a partial dependence on PKC (Berk et al., 1987b, 1989; Assender et al., 1997). Thus both PKC-dependent and -independent mechanisms are involved in Ang II-stimulated vascular contraction and growth. In addition to its second messenger function, PKC has been implicated in the rapid-agonist-induced desensitization of AT₁ receptors (Balmforth et al., 1997).

4. Stimulation of Na⁺/H⁺ Exchange. Ang II elicits a biphasic change in intracellular pH (pH_i), comprising an initial acidification followed by a sustained alkalinization (Griendling et al., 1989; Touyz and Schiffrin, 1997a; Touyz et al., 1999d). The rapid acidification is associated with Ca²⁺-ATPase-regulated Ca²⁺ mobilization (Berk et al., 1987b). Ang II-stimulated alkalinization is entirely dependent on activation of the Na⁺/H⁺ exchanger (Berk et al., 1987b; Touyz and Schiffrin, 1997a; Touyz et al., 1999d), which is modulated by PKC-dependent and PKC-independent mechanisms (Berk et al., 1987b). MAPKs also play a role in Ang II-stimulated activation of the Na⁺/H⁺ exchanger. ERK-1/ERK-2 and p38 activate the Na⁺/H⁺ exchanger in vascular smooth muscle cells (Kusuhara, 1998; Touyz et al., 1999d) and p90^rsk has been identified as a putative potent Na⁺/H⁺ kinase (Takahashi et al., 1997a). Activation of the Na⁺/H⁺ exchanger and akalinization induce vasoconstriction in various vascular beds by increasing [Na⁺]_i and [Ca²⁺]_i and by sensitizing the contractile machinery to Ca²⁺ (Grinstein et al., 1989; Carr et al., 1995; Ye, 1996; Tepel et al., 1998b; Touyz et al., 1999). In addition, increased intracellular pH_i is a potent stimulus for DNA synthesis (Sachinidis et al., 1996). Thus alkalinization is an important mechanism whereby Ang II modulates vascular smooth muscle function by stimulating both contraction and growth.

5. Angiotensin II Increases Intracellular Free Concentrations of Na⁺ and Decreases Intracellular Free Concentrations of Mg²⁺. In addition to increasing [Ca²⁺]_i and pH_i, Ang II raises [Na⁺]_i and reduces [Mg²⁺]_i in a concentration-dependent fashion in vascular smooth muscle cells (Johnson et al., 1991; Ye et al., 1996; Touyz and Schiffrin, 1999). These effects are rapid and maximal responses occur within 40 to 60 s (Touyz and Schiffrin, 1998). [Na⁺]_i is regulated by the Na⁺/H⁺ exchanger, the Na⁺/Ca²⁺ exchanger, the Na⁺/K⁺ ATPase pump, and Na⁺ channels (Shigekawa et al., 1996; Juhaszova and Blaustein, 1997; Cox et al., 1998). The cellular mechanisms regulating [Mg²⁺]_i are unknown, but we and others have shown that a putative Na⁺/Mg²⁺ exchanger regulates [Mg²⁺]_i by inducing Mg²⁺ efflux and by stimulating Na⁺ influx (Touyz and Schiffrin, 1996b; Touyz and Schiffrin, 1999a; Murphy, 2000). Ang II-stimulated increase in [Na⁺]_i and reduction in [Mg²⁺]_i influence vascular smooth muscle contraction directly or indirectly by modulating [Ca²⁺]_i.

6. Activation of Src Family Kinases. The Src family of protein tyrosine kinases that characteristically interact with transmembrane tyrosine kinase receptors, also interact functionally with G protein-coupled receptors, such as AT₁ (Paxton et al., 1994; Marrero et al., 1995b; Parsons and Parsons, 1997; Thomas and Brugge, 1997; Ishida et al., 1998). To date, at least 14 Src-related kinases have been identified, of which the 60-kDa c-Src is the best characterized (Thomas and Brugge, 1997). The Src family kinases are subdivided into three groups based on their pattern of expression. Src, Fyn, and Yes are expressed ubiquitously, Blk, Fgr, Hck, Lck, and Lyn are found primarily in hematopoietic cells and Frk-related kinases (Frk/Rak and Iyk/Bsk) are expressed predominantly in epithelial-derived cells (Thomas and Brugge, 1997). Src family kinases share a high degree of structural similarity, with common domain architecture and regulatory mechanisms. They consist of one or more amino-terminal acylation sites (required for membrane localization), a unique domain (which defines the individual members), an SH3 domain, an SH2 domain, a catalytic domain, and a carboxyl-terminal noncatalytic domain. Regulation of Src activity is complex. Phosphorylation of Tyr⁵²⁷ by Csk inhibits Src activity, whereas dephosphorylation of this residue activates Src. Activation may also occur by autophosphorylation of Tyr⁴¹⁹ in the catalytic domain, by displacement of the intramolecular interactions of the SH2 or SH3 domains by high-affinity ligands or modification of certain residues (Erpel and Courtneidge, 1995). Src family kinases are activated in response to various stimuli in many cell types and have been suggested to play an important role in signal transduction pathways that control growth and cellular architecture.

In addition to rapid signaling events associated with contraction, the AT₁ receptor couples to multiple intracellular transduction pathways that are linked to long-term regulation of vascular smooth muscle cell function, such as growth, migration, deposition of extracellular matrix, and production of growth factors. These processes are initiated by signaling pathways that are stimulated by Ang II within minutes and include: a) phosphorylation of tyrosine kinases; b) activation of MAPKs; c) activation of PLA₂ and arachidonic acid metabolism; d) activation of PLD; and e) modulation of cyclic nucleotides (Fig. 5).

View larger version (35K): [in this window] [in a new window]   Fig. 5.   Early signaling events mediated by Ang II in vascular smooth muscle cells. Ang II phosphorylates multiple tyrosine kinases (TK) such as Janus family kinases (JAK/TYK), focal adhesion kinases (FAK and Pyk2), p130Cas and phosphatidylinositol 3-kinase (PI3K), within minutes of stimulation. Activated tyrosine kinases phosphorylate many downstream targets including the mitogen-activated protein kinase cascade (detailed in Figs. 7 and 8). Src associates with the adapter protein complex, Shc-GRB2-Sos that induces guanine nucleotide exchange on the small G protein Ras-GDP/GTP. Activated Ras-GTP interacts with Raf (MAPK kinase kinase) resulting in phosphorylation of two serine residues present in MEK (MAPK/ERK kinase) which, in turn, phosphorylates MAPKs, including ERK1/2, JNK/SAPK, and p38. Ang II also activates PLD, a major source of DAG, and phospholipase A2, which induces arachidonic acid production. PLD- and PLA2-dependent signaling pathways may also activate MAPKs. Ptd, phosphatidylcholine; PtdOH, phosphatidic acid.

1. Activation of Tyrosine Kinases. Ang II stimulates phosphorylation of a tyrosine residue of many vascular smooth muscle cell proteins. These include the AT₁ receptor itself, PLC-1 and Src family kinases (activated within seconds), as well as JAK and TYK, FAK, Pyk2, p130^Cas (a Crk-associated substrate), and phosphatidylinositol 3-kinase (PI3K), all of which are activated within minutes (Fig. 6). The role of tyrosine kinases in Ang II-mediated signal transduction pathways in cardiovascular cells was extensively reviewed in 1997 (Marrero et al., 1995a; Berk et al., 1997; Berk and Corson, 1997; Dostal et al., 1997; Griendling et al., 1997). Only recent developments relating to Ang II signaling and tyrosine kinases will be discussed in detail here.

View larger version (39K): [in this window] [in a new window]   Fig. 6.   Tyrosine kinase pathways stimulated by Ang II in vascular smooth muscle cells. Ang II rapidly activates Src, which regulates PLC-- and ERK-dependent signaling pathways. Ang II binding to the AT1 receptor induces the physical association and activation of JAK2/TYK2 (Janus kinases) as indicated by dashed line. JAK2/TYK2 phosphorylates STAT proteins that are translocated to the nucleus where they activate gene transcription. Ang II also activates FAK, which possesses sites favored for phosphorylation by Src. FAK associates with paxillin and talin that associate with actin. The link between AT1 receptor and FAK is unknown, but the Rho family of GTPases are potential candidates. Pyk2 and CADTK are activated by Ang II through Ca2+-dependent pathways. Activated Pyk2 regulates Src and ERK-dependent signaling cascades. p130cas is transiently activated by Ang II, possibly via a Ca2+-dependent pathway. Phosphorylated p130cas may be important in the regulation of -actin expression. PI3K activation by Ang II leads to Akt/PKB activation, which in turn stimulates cell survival pathways and activation of p70S6K. p70S6K, p70 S6-kinase.

2. Mitogen-Activated Protein Kinase Pathways. MAP kinases constitute a superfamily of serine/threonine protein kinases involved in the regulation of a number of intracellular pathways. Mammalian MAPKs are grouped into six major subfamilies: a) ERK-1/ERK-2; b) JNK/stress-activated protein kinases (SAPK); c) p38; d) ERK-6, p38-like MAPK; e) ERK-3; and f) ERK-5 (also called Big MAP kinase 1) (Robinson and Cobb, 1997) (Fig. 7). MAP kinase-dependent signaling pathways have been associated with cellular growth and apoptosis, with cellular differentiation and transformation and with vascular contraction (Mii et al., 1996; Force and Bonventre, 1998; Touyz et al., 1999b,c). The ERKs are activated in response to growth and differentiation factors, whereas JNKs and p38 are usually activated in response to inflammatory cytokines and cellular stress (Robinson and Cobb, 1997; Force and Bonventre, 1998; Morinville et al., 1998; New and Han, 1998; Ip and Davis, 1998). Ang II activates the three major members of the MAP kinase family, ERKs, JNKs, and p38 (Leduc and Meloche, 1995; Kudoh et al., 1997; Touyz et al., 1999d). MAP kinase pathways comprise a three-component protein kinase cascade consisting of a serine/threonine protein kinase (MAPKKK), which phosphorylates and activates a dual-specificity protein kinase (MAPKK), which in turn phosphorylates and activates another protein kinase (MAPK) (Cobb and Goldsmith, 1995; Robinson and Cobb, 1997). In the Ras/Raf/MEK/ERK pathway, Raf corresponds to MAPKKK, MEK corresponds to MAPKK, and ERK corresponds to MAPK (Fig. 7).

View larger version (25K): [in this window] [in a new window]   Fig. 7.   Schematic diagram of the currently known mammalian MAP kinase signaling pathways. Individual components are discussed in the text. The question marks denote the signaling components that remain to be elucidated. PAK, p21-activated protein kinase; TAK, TGF--activated kinase; TAO, thousand and one amino acid kinase; BMK, big MAP kinase

View larger version (26K): [in this window] [in a new window]   Fig. 8.   Upstream regulators of Ang II-stimulated extracellular signal-regulated kinase (ERK)-dependent signaling pathways in vascular smooth muscle cells. The ERK phosphorylation cascade is initiated by Ang II-binding to AT1 receptors that induces Shc-Grb2-Sos formation (tyrosine phosphorylation of Shc), which in turn facilitates guanine nucleotide exchange on the small G protein Ras-GDP/GTP. Activated Ras-GTP interacts with the Ser/Thr kinase Raf (MAPK kinase kinase (MAPKKK)) which translocates to the cell membrane. Activation of Raf leads to phosphorylation of two serine residues present in MEK (MAPK/ERK kinase), which in turn phosphorylates Thr/Tyr and activates MAPK, present as a 44- (ERK-1) and a 42-kDa (ERK-2) isoform. Phosphorylated ERK has diverse intracellular protein targets, which it phosphorylates and activates (Fig. 9). Dephosphorylation of ERK is accomplished by activation of MAP kinase phosphatase-1 (MKP-1).

View larger version (38K): [in this window] [in a new window]   Fig. 9.   Downstream effectors of activated ERK. Once phosphorylated, ERK activates various intracellular proteins. These substrates include: 1) proteins involved in transcriptional activation such as Elk-1, TAL 1, RNA polymerase II; 2) proteins involved in protein translation such as PHAS I; 3) structural proteins such as myelin basic protein (MBP), microtubule-associated protein (MAP) and caldesmon; and 4) secondary enzymes such as PLA2, S6 kinase, Ca2+ channels, Na+/H+ exchanger, and MAP kinase-activated protein kinase (MAPKAPK2). Activation of these downstream proteins regulates cellular functions associated with cell growth and contraction.