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(Hypertension. 1997;29:366.)
© 1997 American Heart Association, Inc.

State-of-the-Art-Lecture

Angiotensin II Signaling in Vascular Smooth Muscle

New Concepts

Kathy K. Griendling; Masuko Ushio-Fukai; Bernard Lassègue; R. Wayne Alexander

From the Emory University School of Medicine, Division of Cardiology, Atlanta, Ga.

Correspondence to Kathy K. Griendling, PhD, Emory University, Division of Cardiology, 319 WMB, 1639 Pierce Dr, Atlanta, GA 30322. E-mail kgriend{at}emory.edu

	Abstract

Top Abstract Introduction Early Signaling Events:... Tyrosine Phosphorylation MAP Kinase Pathway Receptor Desensitization and... Long-term Signaling Events:... Downregulation of Ang II... Conclusions References

 
Angiotensin II is a multifunctional hormone that affects both contraction and growth of vascular smooth muscle cells through a complex series of intracellular signaling events initiated by the interaction of angiotensin II with the AT₁ receptor. The cellular response to angiotensin II is multiphasic, involving stimulation within seconds of phospholipase C and Ca²⁺ mobilization; activation within minutes of phospholipase D, A₂, protein kinase C, and MAP kinase; and stimulation after a period of hours of gene transcription and NADH/NADPH oxidase activity. Angiotensin II also activates numerous intracellular tyrosine kinases. In this respect, it shares some aspects of signaling with growth factor and cytokine receptors, including activation of phospholipase C-, src, and ras; association of shc with grb2; and stimulation of the Jak/STAT pathway. The cellular events responsible for this unique series of events may involve receptor movement and the creation of a signaling domain. Elucidation of these pathways is important to our understanding of AT₁ receptor function as a final effector of the renin-angiotensin system.

Key Words: angiotensin II • vascular smooth muscle • receptor sequestration • phospholipase • tyrosine • phosphorylation • oxidant stress

Abbreviations: Ang II = angiotensin II • AT₁ = angiotensin type 1 receptor • GRK(s) = G protein-coupled receptor kinase(s) • MAP = mitogen-activated protein • PLA₂, PLC, PLD = phospholipases A₂, C, D • VSMC(s) = vascular smooth muscle cell(s)

	Introduction

Top Abstract Introduction Early Signaling Events:... Tyrosine Phosphorylation MAP Kinase Pathway Receptor Desensitization and... Long-term Signaling Events:... Downregulation of Ang II... Conclusions References

 
Ang II is a multifunctional hormone that has pleiotropic effects on vascular smooth muscle. It was originally identified as an acute regulator of vasomotor tone¹ and has since been shown to promote vascular hypertrophy^2,3 and in some cases, hyperplasia.⁴ It also affects vascular cell migration⁵ and extracellular matrix production.⁶ As such, it plays an important role in the normal and pathological physiology of the vessel wall. Virtually all of the hemodynamic effects of ang II are mediated by the AT₁ receptor, which was cloned simultaneously by two groups.^7,8 The biochemical pathways that are activated upon stimulation of the AT₁ receptor are complex, but recent work has provided new insights into the signaling mechanisms utilized by this system.

	Early Signaling Events: Phospholipase Activation

Top Abstract Introduction Early Signaling Events:... Tyrosine Phosphorylation MAP Kinase Pathway Receptor Desensitization and... Long-term Signaling Events:... Downregulation of Ang II... Conclusions References

 
Ang II rapidly activates multiple phospholipases to hydrolyze membrane phospholipids and generate signaling molecules. In cultured VSMCs, PLC is activated within 5 seconds, hydrolyzing phosphatidylinositol 4,5-bisphosphate to inositol trisphosphate and diacylglycerol.^9,10 This response is transient, returning toward baseline by 2 minutes. These second messengers in turn release calcium from internal stores and activate protein kinase C, leading to a cascade of protein phosphorylations that direct the subsequent response of the cell. Protein kinase C also serves as a negative-feedback regulator of PLC, since it is apparently responsible, at least in part, for terminating the PLC response.¹¹

Recently the identity of the PLC that is coupled to AT₁ receptors has come into question. Marrero et al¹² showed that in VSMCs, ang II phosphorylates PLC- and that incubation of cells with the tyrosine kinase inhibitor genistein nearly abolishes inositol trisphosphate formation. These results suggest that the AT₁ receptor is coupled to PLC- via a tyrosine phosphorylation mechanism. However, other data support the concept that ang II receptors are coupled to a G protein-linked PLC. Thus, in permeabilized VSMCs, GTPS synergistically enhances ang IIinduced inositol trisphosphate generation.¹³ Furthermore, downregulation of G_q/11 by prolonged agonist incubation markedly abrogates the ability of ang II to stimulate PLC,¹⁴ implying that a G protein of the Gq family is involved in coupling the AT₁ receptor to PLC. It is possible that a G protein-coupled tyrosine kinase is upstream of PLC-, but it is also possible that another PLC isotype is involved. The identity of this putative G protein-coupled PLC is unclear, since the prototypical subtype, PLC-ß, is not found in VSMCs.¹² A leading candidate is PLC-, which is robustly expressed in vascular tissue.¹⁵

Ang II also activates PLD, hydrolyzing phosphatidylcholine to choline and phosphatidic acid. In VSMCs, phosphatidic acid is rapidly converted to diacylglycerol by phosphatidic acid phosphohydrolase. PLD activation is subsequent to that of PLC: it is detectable at 1 to 2 minutes and remains elevated for as long as 1 hour.¹⁶ In contrast to the PLC response, PLD activation does not appear to desensitize significantly during this time period. This pathway is the most important source of phosphatidic acid and diacylglycerol and probably represents the major pathway by which protein kinase C remains activated. There are two major unresolved questions concerning the AT₁ receptor and the PLD pathway. First, the mechanism by which the receptor couples to PLD is unknown. Smallmolecular-weight G proteins (arf, rho), pertussis toxinsensitive and -insensitive heterotrimeric G proteins, protein kinase C activation, and tyrosine phosphorylation have been variously suggested as coupling mechanisms.¹⁷ Recent data indicate that there is a family of PLD isoforms,¹⁸ which may explain the plethora of proposed coupling mechanisms. Our own data in VSMCs strongly suggest that a heterotrimeric G protein is involved (K.K.G. et al, unpublished data, 1997), although the identity of the G protein and even the responsible subunit remains to be determined. The second major issue concerning PLD is its function in VSMCs. It is without doubt the major source of diacylglycerol for activation of protein kinase C,¹⁶ but it is likely to have other functions as well. For growth factors such as platelet-derived growth factor, it has been suggested that PLD is integral to the growth response.¹⁹ In neutrophils, the phosphatidic acid that results from PLD serves to activate an NADPH oxidase.²⁰ Although a similar oxidase is activated by ang II in VSMCs,²¹ a role for PLD in this response remains to be established.

In addition to PLC and PLD, ang II rapidly activates PLA₂ to release free fatty acids such as arachidonate.²² PLA₂ activity in response to ang II is evident within minutes and is sustained for at least 30 minutes. The arachidonic acid that is produced is then converted via cyclooxygenase to prostaglandins, via lipoxygenase to unstable hydroperoxy intermediates that go on to form the leukotrienes, hydroxyeicosatetraenoic acids and lipoxins, or via cytochrome P450 monooxygenase to epoxides, midchain cis/trans conjugated dienols, and C-19/C-20 alcohols. Natarajan et al²³ have shown that the lipoxygenase pathway has a role in VSMC hypertrophy. In addition, our recent data suggest that arachidonic acid metabolites may be involved in activation of NADH/NADPH oxidase (see below), thus regulating the oxidative state of the cell.²⁴

	Tyrosine Phosphorylation

Top Abstract Introduction Early Signaling Events:... Tyrosine Phosphorylation MAP Kinase Pathway Receptor Desensitization and... Long-term Signaling Events:... Downregulation of Ang II... Conclusions References

 
Recently it has become apparent that ang II, like many growth factors, stimulates the phosphorylation of numerous proteins on tyrosine residues (Table). Several of these proteins have been identified, and it is clear that phosphorylation and dephosphorylation of specific tyrosinecontaining proteins are important signaling events. In one of the earliest reports of ang II-induced tyrosine phosphorylation in VSMCs, Tsuda et al²⁵ demonstrated that at least nine proteins are phosphorylated by ang II on tyrosine. This phosphorylation was mimicked either by phorbol 12-myristate 13-acetate activation of protein kinase C or by addition of the calcium ionophore ionomycin, suggesting that tyrosine kinase activation is downstream of PLC and PLD. Subsequent reports have identified numerous additional proteins phosphorylated on tyrosine by ang II in both VSMCs and cardiac fibroblasts.^12,25–32

View this table: [in this window] [in a new window]   Ang II-Stimulated Protein Phosphorylation in the Cardiovascular System

Several of these tyrosine-phosphorylated proteins deserve specific comment. Ang II has been shown to phosphorylate and activate pp60src.^27,28,30 This is potentially quite important, because src activates several other molecules involved in intracellular signaling, including PLC-, pp120, p125^FAK, paxillin, JAK2, STAT1, G, and caveolin.^33–36 In addition, src-family kinases have been implicated in the G protein receptor-mediated phosphorylation of the adapter protein shc.³⁷ shc proteins (p46, p56, and p66) are tyrosine phosphorylated by ang II, and this leads to association with Grb2 via its SH2 domain.²⁷ In growth factor receptor systems, Grb2 associates with the SH3 domain of SOS, which serves as a guanine nucleotide exchange factor to activate p21^ras.³⁸ It is unclear whether a similar system is utilized by the AT₁ receptor. Ang II also phosphorylates and activates Jak2 and Tyk2 (Fig 1), tyrosine kinases usually associated with cytokine receptors.³⁹ In VSMCs, Jak apparently phosphorylates STATs 1, 2, and 3, albeit with different time courses. STAT1 and STAT2 phosphorylation in response to ang II is maximal by 15 minutes, while STAT3 phosphorylation is not detectable until 60 minutes.³⁹ Upon phosphorylation, STATs form dimers that associate with p48 and are translocated to the nucleus where they activate gene transcription, suggesting a possible role for this pathway in the activation of early growth response genes by ang II. Finally, ang II phosphorylates the cytosolic focal adhesion kinase (FAK), causing its translocation to sites of focal adhesion with the extracellular matrix and phosphorylation of talin and paxillin,^40,41 which may be involved in the regulation of cell morphology and movement. Thus, it seems clear that ang II activates multiple tyrosine kinase pathways in VSMCs that, by analogy with growth factors, are likely to be important in mediating the growth effects of ang II.

View larger version (13K): [in this window] [in a new window]   FIG 1. Stimulation of Jak/STAT pathway by ang II. Ang II has been shown to activate both Jak2 and Tyk2 by phosphorylation. These activated kinases then phosphorylate STAT/ß and STAT2, which associate with p48 to form an active transcriptional complex.

	MAP Kinase Pathway

Top Abstract Introduction Early Signaling Events:... Tyrosine Phosphorylation MAP Kinase Pathway Receptor Desensitization and... Long-term Signaling Events:... Downregulation of Ang II... Conclusions References

 
MAP kinases comprise a superfamily of serine/threonine protein kinases involved in cell growth and differentiation, as well as in cell transformation.⁴² ERK1 (p44^mapk) and ERK2 (p42^mapk), the most well studied of the MAP kinases, are activated by dual phosphorylation on the threonine and tyrosine occurring in the motif TEY⁴³ and in turn phosphorylate numerous cellular proteins, including MBP, pp90^rsk, p62^TCF, 4E-BP-1, cPLA2, c-jun, and PHAS-1.⁴⁴ Since the initial identification of ERK1 and ERK2, several other members of the MAP kinase superfamily have been identified, including ERK3 isoforms, Jun N-terminal kinases/stress-activated protein kinases (JNK/SAPK), p38^mapk, p57^mapk, and BMK-1 (Big MAP kinase-1 or ERK5).

It is now well documented that ang II activates the MAP kinase pathway in VSMCs. ERK1 and ERK2 were among the first tyrosine-phosphorylated proteins to be identified in ang II-stimulated cells.^25,29,45,46 MAP kinase activation by ang II in VSMCs appears to be transient with a peak around 2 to 5 minutes, although activity remains elevated above baseline for at least 60 minutes.^45–47 Stimulation of MAP kinase is at least partially dependent on protein kinase C⁴⁵ and apparently requires prior activation of a Ca²⁺dependent tyrosine kinase.⁴⁷

Recent work has focused on the biochemical pathways leading to MAP kinase activation (Fig 2). The immediate activator of MAP kinase is MEK (MAP or ERK kinase), which is a dual-specificity kinase that phosphorylates ERK1 and ERK2 on both threonine and tyrosine.⁴⁸ MEK in turn is activated when it is phosphorylated on serine by a MAP kinase kinase. At least three MAP kinase kinases have been identified, raf-1, c-mos, and MEK kinase,⁴⁹ two of which (raf-1 and MEK kinase) are activated by ang II.^29,50 raf-1 is stimulated by phosphorylation on threonine and serine and is recruited to the plasma membrane by the small-molecular-weight G-binding protein ras.⁵¹ It is at this point that our knowledge of the MAP kinase activation pathway becomes sketchy. The stimulation of ras by growth factor receptors is complex, involving receptor autophosphorylation and binding to the SH2 domain of the adaptor protein Grb2, which then binds to the guanine nucleotide exchange factor SOS via its SH3 domain.⁵² A similar pathway may be used by G protein-coupled receptors, except that the initial tyrosine kinase is a nonreceptor tyrosine kinase. This has been clearly shown for Gi-coupled receptors,⁵³ and evidence is accumulating that some Gqcoupled receptors may utilize this pathway as well.⁵⁴ The most proximal kinase activated by these receptors is unknown, but current data suggest that a member of the src kinase family, src itself, lyn, or syk, may be involved.⁵⁴ It has also been postulated that PI3 kinase activation may be upstream of ras activation.⁵⁵ The primary signal initiating this cascade has been proposed to involve a Ca²⁺-dependent protein kinase⁴⁷ or the ß subunits of a coupling G protein.³⁷ With regard to this pathway, ang II has been shown to activate pp60^src,²⁸ to promote association of Grb and SOS,²⁷ to activate ras,⁴⁷ and to phosphorylate raf-1²⁹ and MEK,⁵⁰ although the links between each of these events remain to be established.

View larger version (16K): [in this window] [in a new window]   FIG 2. Ang II-induced MAP kinase activation in vascular smooth muscle. This schematic shows biochemical events that have been proposed to be involved in MAP kinase activation in VSMCs. Dotted lines indicate links that have not yet been demonstrated. Ang II binds to its receptor and activates an src family kinase, either directly or via activation of PI3K. src kinase phosphorylates shc, which then associates with Grb2 and SOS to promote guanine nucleotide exchange on ras. There is some evidence that ras is also activated by an upstream Ca2+-dependent tyrosine kinase. ras activates raf-1, perhaps by recruiting it to the membrane. raf or some other unknown MAP kinase kinase phosphorylates MEK, which then phosphorylates MAP kinase. Known substrates for MAP kinase are indicated. Dephosphorylation of MAP kinase is accomplished by activation of MKP-1. indicates addition of a phosphate group to the amino acid indicated by its one-letter abbreviation; AT1R, angiotensin type1 receptor; PI3K, phosphatidylinositol 3-kinase; SH2, src homology 2 domain; SH3, src homology 3 domain; MEK, MAP kinase kinase; MEKK, MAP kinase kinase kinase; TK, tyrosine kinase; MBP, myelin basic protein; and MKP-1, MAP kinase phosphatase-1.

As noted above, MAP kinase activity in response to ang II is transient, raising the issue of the mechanism(s) responsible for signal termination. Inactivation appears to be achieved by dephosphorylation of MAP kinase. The phosphatase responsible for this event, MKP-1 (mitogen-activated protein kinase phosphatase-1), was originally isolated as a nonspecific tyrosine phosphatase (3CH134) and was subsequently shown to have high specificity for MAP kinase.⁵⁶ Like MEK, MKP-1 has dual specificity: It dephosphorylates MAP kinase on both threonine and tyrosine.⁵⁶ In VSMCs, inhibition of MKP-1 leads to sustained activation of MAP kinase in response to ang II, suggesting that it is this enzyme that is primarily responsible for the termination of the MAP kinase signal.⁵⁷ MKP-1 expression in turn is stimulated by ang II, so that MKP-1 serves as a long-term negative-feedback regulator of this pathway.⁵⁸ Termination of MAP kinase activity may also involve activation of protein kinase A.⁵⁹ This mechanism, however, most likely results from inhibition of the events leading to MAP kinase activation. Specifically, it appears that activators of protein kinase A inhibit the activity of raf-1.⁶⁰

	Receptor Desensitization and Creation of Signaling Domains

Top Abstract Introduction Early Signaling Events:... Tyrosine Phosphorylation MAP Kinase Pathway Receptor Desensitization and... Long-term Signaling Events:... Downregulation of Ang II... Conclusions References

 
It is somewhat paradoxical that the most intense focus on AT_1A receptor coupling mechanisms in VSMCs has been on PLC activation, a response that, regardless of the isozyme involved, is markedly desensitized and attenuated in 5 minutes. In contrast, ang II-stimulated PLD is robustly activated as long as it has been followed to date— 1 hour. The mechanisms responsible for this selective desensitization to PLC are not entirely understood. Desensitization to PLC prominently and perhaps predominantly involves protein kinase C, since downregulation of protein kinase C results in prolonged stimulation of PLC.⁶¹ In contrast, PLD activity cannot be sustained in the absence of protein kinase C activation (K.K.G. et al, unpublished observations, 1997). Termination of the PLC response may also involve phosphorylation of the receptor by GRKs, which in the ß-adrenergic receptor system phosphorylate ligand-bound receptors, promoting the binding of arrestin and the consequent interruption of G protein-mediated signal transduction.⁶² The AT_1A receptor has been shown to be phosphorylated on serine and tyrosine in response to ang II, with a peak at 20 minutes of ang II stimulation.⁶³ Although the kinases responsible for this phosphorylation in vivo are not known, in HEK293 cells cotransfected with the AT_1A receptor and GRK-2, -3, or -5, receptor phosphorylation is augmented.⁶⁴ Overexpression of a dominant negative K220R mutant GRK2 and inhibition of protein kinase C both attenuated, in an additive fashion, ang IIinduced AT_1A receptor phosphorylation,⁶⁴ suggesting that not only are GRKs involved in receptor phosphorylation but that receptor phosphorylation may also be an additional mechanism by which protein kinase C desensitizes PLC. In contrast, PLD is stimulated by exogenous activators of protein kinase C, and signaling through the PLD pathway in response to ang II continues even when the receptor is phosphorylated.^16,63 These observations suggest that the two responses, PLC and PLD, are independently regulated and that PLD activation is resistant to conventional mechanisms of desensitization.

A recent hypothesis developed to explain these disparate signaling events involves the creation of signaling domains by ang II. It has long been known that the ang II receptor internalizes rapidly upon binding of the ligand.^65,66 In fact, it was originally proposed as a mechanism of desensitization. However, the AT₁ receptor internalizes with a half-time of 1.5 minutes,⁶⁵ whereas PLD signaling commences at about this time and continues for 1 hour.¹⁶ This simple observation has led to the proposal that the receptor moves within the plane of the membrane (Fig 3) to a specialized domain from which prolonged signals are generated. Anderson et al⁶⁷ showed that AT₁ receptors rapidly move to coated pits, which internalize as vesicles and fuse with lysosomes. Zhang et al⁶⁸ found that AT₁ receptors also move to noncoated pits, suggesting that there may be two internalization mechanisms. The recent identification of caveolae as membrane signaling domains associated with growth factor receptors⁶⁹ raises the possibility that the noncoated pit regions in which AT₁ receptors aggregate may represent similar signaling domains. This is a particularly attractive notion, in view of the apparent association of src with caveolae⁷⁰ and the recent observation that AT₁ receptors activate src.²⁸

View larger version (103K): [in this window] [in a new window]   FIG 3. Sequestration of the AT1 receptor. VSMCs were exposed to vehicle or 100 nmol/L ang II for 2 minutes and then stained with AT1 receptor antibody. After counterstaining with Texas red, immunofluorescence was visualized with a confocal microscope at x60. In control cells, fluorescence is distributed across the cytoplasm and is intensified in the area of the nucleus where the cell is thickest. In ang II-stimulated cells, fluorescence has moved to the perinuclear region and has become concentrated in punctate regions on the edges of the cells.

	Long-term Signaling Events: Modulation of the Oxidative State

Top Abstract Introduction Early Signaling Events:... Tyrosine Phosphorylation MAP Kinase Pathway Receptor Desensitization and... Long-term Signaling Events:... Downregulation of Ang II... Conclusions References

 
Although much emphasis has been placed on the rapid, transient biochemical pathways stimulated by ang II, it is clear that ang II has prolonged effects on VSMCs. As mentioned previously, one of these is PLD, which remains activated for as long as the agonist is present.¹⁶ Another important tonic signaling pathway is activation of NADH/NADPH oxidase (Fig 4). This enzyme transfers electrons from NADH or NADPH to molecular oxygen, producing superoxide anion (·O₂^-). Ang II activation of the NADH/NADPH oxidase is delayed: It is first detectable after about 1 hour and is sustained for at least 24 hours.²¹ Importantly, when NADH/NADPH oxidase activity is inhibited, ang II-stimulated protein synthesis is also inhibited, implying that activation of this pathway is integral to the growth response.

View larger version (10K): [in this window] [in a new window]   FIG 4. The NADPH/NADH oxidase pathway stimulated by ang II. Ang II activates a membrane NADPH/NADH oxidase to produce ·O2-, which is converted by superoxide dismutase (SOD) to H2O2. Both of these species have been implicated in gene induction and cell growth.

The structure and activation mechanisms of the VSMC NADH/NADPH oxidase are unknown. Recent work suggests that ang II-stimulated production of arachidonic acid metabolites may be required for oxidase activation, although this pathway has not been fully elucidated.²⁴ In terms of structure, there is some evidence that this enzyme is similar to the bactericidal neutrophil enzyme, which consists of five subunits: a 22-kD -subunit (p22^phox) and a glycosylated 91-kD ß-subunit (gp91^phox), which together compose cytochrome b₅₅₈, the electron transfer element; cytosolic components p47^phox and p67^phox; and a low-molecular-weight G protein, rac 1 or rac 2.⁷¹ We have recently shown that smooth muscle oxidase is a p22phox-based enzyme,⁷² but the presence of other subunits has not been demonstrated in VSMCs. Although VSMC oxidase shares some similarity to the neutrophil enzyme, it differs in several important aspects. First, vascular oxidase preferentially utilizes NADH rather than NADPH as a substrate for its activity, and its output is much lower than that of phagocytic oxidase.^21,73,74 Second, the peak of the absorbance spectrum of the cytochrome component of vascular oxidase is shifted slightly to the left (553 nm).⁷² Finally, knockout of p22phox in vascular cells does not affect baseline NADH-mediated ·O₂^- production but only attenuates ang II-stimulated oxidase activity,⁷² suggesting that the p22phox-containing oxidase is an inducible protein that has little constitutive activity in untreated cells.

The ·O₂^- that is produced by NADH/NADPH oxidase is presumably rapidly converted by superoxide dismutase to H₂O₂. We have recently shown that 4 hours of ang II treatment leads to H₂O₂ accumulation in VSMCs and that this H₂O₂ is a consequence of activation of the p22phoxbased enzyme.⁷² This is important, because the altered redox state of the cell promotes lipid peroxidation, and both lipid hydroperoxides and H₂O₂ increase expression of several growth-related and inflammatory genes and activate intracellular signaling pathways. Of potential importance to the VSMC response to ang II is the activation of PLA₂,⁷⁵ MAP kinases,⁷⁶ and ras,⁷⁷ as well as the induction of c-fos⁷⁸ and c-jun.⁷⁹ There is some evidence that this pathway is not involved in ang II stimulation of the MAP kinase pathway,⁷⁶ but in general, the role of reactive oxygen species in growth-related signal transduction is an unexplored area. It is possible that the redox state of the cell acts as a rheostat to modulate the activation of cellular signaling enzymes, such as protein kinase C and tyrosine kinases, and may also directly alter the activity of transcription factors. That reactive oxygen species have a role in VSMC growth is fairly clear. H₂O₂ stimulates VSMC proliferation,⁸⁰ inhibition of NADH/NADPH oxidase attenuates ang II-induced hypertrophy,^21,72 and treatment of VSMCs with antioxidants induces apoptosis.⁸¹ The precise mechanisms await further definition.

	Downregulation of Ang II Responsiveness

Top Abstract Introduction Early Signaling Events:... Tyrosine Phosphorylation MAP Kinase Pathway Receptor Desensitization and... Long-term Signaling Events:... Downregulation of Ang II... Conclusions References

 
Many investigators have observed a diminution of responsiveness (termed either tachyphylaxis, desensitization, or downregulation) to repeated applications of ang II.⁸² There is no doubt that prolonged exposure to ang II alters the signaling pathways involved in subsequent signal generation. In VSMCs, ang II downregulates its own receptor,⁸³ decreases the amount of and coupling to G_q,¹⁴ and increases GRK5 mRNA and protein expression.⁸⁴ The overall effect is an attenuation of responsiveness to ang II. Thus, there are fewer surface receptors to be stimulated, and those that are present are unable to couple to G_q, unable to activate PLC, are phosphorylated, and therefore become inactivated, by increased GRKs. It is clear that not only does ang II acutely affect intracellular signal generation, but also that it regulates expression of key components of the signaling pathways to modulate long-term responsiveness.

	Conclusions

Top Abstract Introduction Early Signaling Events:... Tyrosine Phosphorylation MAP Kinase Pathway Receptor Desensitization and... Long-term Signaling Events:... Downregulation of Ang II... Conclusions References

 
Ang II is a unique hormone that generates complex signaling events in VSMCs. Not only does it activate classic G protein-coupled phospholipases, but it also activates some of the same tyrosine kinase pathways that are coupled to growth factor receptors. VSMCs respond to ang II multiphasically: PLC activation and Ca²⁺ mobilization occurs within seconds, PLD and protein kinase C activation occur within minutes, and modulation of NADH/NADH oxidase activity occur within hours. The end result, contraction, hypertrophy, or proliferation, is influenced by the phenotype of the cell but also almost certainly results from selective activation of these multiple pathways. The cellular events responsible for this unique series of events may involve receptor movement and creation of a signaling domain. Elucidation of these pathways is important to our understanding of AT₁ receptor function as a final effector of the renin-angiotensin system.

	Acknowledgments

 
This work was supported by NIH grants HL47557 and HL38206 (to R.W.A. and K.K.G.) from the National Heart, Lung, and Blood Institute, Bethesda, Md. We thank Barbara Merchant for editorial assistance and for preparing the figures.

	References

Top Abstract Introduction Early Signaling Events:... Tyrosine Phosphorylation MAP Kinase Pathway Receptor Desensitization and... Long-term Signaling Events:... Downregulation of Ang II... Conclusions References

 

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