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Novel aspects of angiotensin II signalling
in vascular smooth muscle



The AT1 subtype of angiotensin II (AngII) receptors mediates most of the effects of this agonist in the healthy adult cardiovascular system [1, 2], thus we will review here new advances in AT1-mediated signalling.

 

Phospholipases


Phospholipase C (PLC)

Protein sequence homologies indicate that AT1 belongs to the vast family of receptors with seven transmembrane domains, a notion consistent with experiments indicating that AngII signals via heterotrimeric G-proteins [3]. Phospholipase C (PLC) is activated by AngII within 5 s in vascular smooth muscle cells (vsmc), thus generating diacylglycerol (DG) and inositol trisphosphate (IP3), an activator of intracellular calcium mobilization [1, 2]. G-proteins usually activate the ß isoform of PLC [4]. Thus the discovery that AngII activates PLC via tyrosine phoshorylation, just like a typical growth factor, appeared paradoxical [5]. AngII increased PLC1 phosphorylation 4.5 fold at 30 s in rat vsmc. Electroporation of c-Src antibodies or preincubation with the tyrosine kinase inhibitors genistein or tyrphostin A inhibited AngII-induced PLC phosphorylation, IP3 production and calcium mobilization [5-8]. AngII induced phosphorylation of tyrosine 319 in the cytoplasmic domain of AT1, thus permitting binding of PLC via its C-terminal SH2 (Src homology 2) domain [9]. Opposite results were obtained in human vsmc, in which anti PLCß1 antibodies inhibited AngII-induced IP3 generation. In this system, neither anti PLC1 antibodies, nor incubation with genistein or herbimycin A had any effect on IP3 formation or the calcium signal [10]. These discrepancies were reconciled in a recent study in rat vsmc which showed that both mechanisms sequentially contributed to AngII-induced IP3 production. Before 30 s, IP3 generation was insensitive to genistein and was inhibited by anti PLCß1 antibodies. In contrast, after 30 s both genistein and anti PLC1 antibodies reduced IP3 accumulation [11]. The presence of PLCß1 in rat vsmc was confirmed by RT-PCR and western blotting [10, 11]. In additional experiments, early coupling of AT1 to G-proteins was demonstrated by inhibition of AngII-induced IP3 generation at 15 s with anti Gq/11, anti G12 and Gß antibodies, as well as by overexpression of the ß binding domain of an adrenergic receptor kinase [11]. These studies demonstrate that AngII couples sequentially, first to PLCß1, through Gß as well as two different G subunits (Fig. 1, left panel); and second to PLC1 (Fig. 1, middle panel).

 

Figure 1. Sequential activation of different phospholipase subtypes by AngII in vsmc

 

Phospholipase D (PLD)

Following transient generation of IP3 and diacylglycerol (DG) by PLC (terminated within minutes), AngII induced a prolonged activation of phospholipase D (PLD) in vsmc [12]. This resulted in a long-lasting accumulation of phosphatidic acid and its dephosphorylation product DG, that accounted for the sustained stimulation of protein kinase C observed in vsmc [2, 13]. Coupling of AT1 to PLD activation was dependent on several pathways in vsmc. The heterotrimeric G protein activator sodium fluoride almost doubled PLD activity in vsmc. Furthermore, AngII-induced PLD activation was inhibited by 80% in cells overexpressing a ß binding protein and more than 50% by electroporation of anti G12 or anti Gß antibodies and 75% by anti-Src antibodies [14]. PLD activation by AngII in vsmc also required extracellular calcium, protein kinase C [13, 15] and sequestration of AT1 in a compartment such as caveolae [16]. Thus, initial activation of PLD presumably results from AT1-induced dissociation of G12 and ß subunits which may activate PLD through a tyrosine kinase pathway. Sustained activation of PLD may result from a positive feedback loop in which protein kinase C, stimulated by calcium influx and PLD-generated DG, phosphorylates G12 [17], thus keeping ß subunits dissociated (Fig. 1, right panel). These recent studies have clarified the earliest coupling events important for AngII responses, leading to a greater understanding of AT1 coupling to G proteins at the molecular level.

 

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ERK1/2 (p42/44) MAP kinases

 

Several important AngII-induced signalling cascades start with activation of the Src family of tyrosine kinases [18-21]. Src does not bind to AT1, but may be activated by ß subunits of G-proteins [14, 19]. In addition to activating PLC and PLD, Src is probably responsible for phosphorylation of Shc in vsmc [18] as seen in similar systems [22]. A complex of Shc, GRB2 and SOS may stimulate the small G-protein Ras [23], leading to activation of the serine/threonine kinase Raf-1 [24-26]. Raf-1 is one of the activators of the threonine/tyrosine kinase MEK1 [24, 25, 27] which activates the serine/threonine MAP kinases ERK1/2 [28]. In vsmc, AngII activates ERK1/2 from about 2 to 30 min [23, 25, 29-33]. Activation of ERK1/2 is terminated by the MKP-1 phosphatase [34, 35].

ERK1/2 can phosphorylate a number of proteins, at least in vitro, such as cytosolic phospholipase A2, myelin basic protein, transcription factors such as c-jun and p62TCF and may increase protein synthesis by activation of p90 ribosomal S6 kinase and the mRNA cap-binding protein eIF-4A [1, 2, 36, 37]. Tyrosine kinase inhibitors such as genistein or herbimycin A reduced AngII-induced activation of the ERK1/2 and JNK MAP kinases, vessel contraction and protein synthesis [1, 20, 38].

AngII-induced ERK1/2 activity in vsmc is dependent on calcium signalling since it was blocked by inhibition of PLC, calmodulin or intracellular calcium mobilization [23]. AngII-induced ERK1/2 activity was not affected by PKC inhibition or down-regulation with phorbol esters [23, 24]. However, antisense inhibition of the atypical PKC zeta subtype, which is not down-regulated by phorbol esters, decreased AngII-induced ERK1/2 activity [39].

AngII may also activate the signalling pathways of other typical tyrosine kinase-coupled receptors. Thus, AngII induced transactivation of the EGF receptor, without EGF secretion [40], and stimulated secretion of autocrine mediators such as IGF-I [41]. These pathways probably contribute to the sustained activation of tyrosine kinases by AngII.

 

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Reactive Oxygen Species

 

Reactive oxygen species, such as superoxide and hydrogen peroxide (H2O2), can chemically alter many cellular components. However, oxidants once only known for their toxicity are now being recognized as possible signalling molecules at moderate concentration [42]. Inside the cell the superoxide free radical can be quickly metabolized by superoxide dismutase (SOD) to hydrogen peroxide which is then more slowly converted to water by catalase.

Oxidants may have both rapid and long-term signalling effects. Exogenous superoxide generated for example with xanthine/xanthine oxidase doubled IP3-induced calcium release from the vsmc sarcoplasm. This effect appeared to be mediated by superoxide since it was sensitive to SOD, but not to catalase [42, 43]. H2O2 was also capable of inducing rapid and sustained mobilization of calcium from agonist-sensitive stores in another vsm model [44]. Exogenous superoxide increased DNA synthesis [45-48] and cell number [45, 48] in quiescent vsmc. Similarly, incubation with H2O2 increased DNA synthesis [46-50] in the same model. In some instances the effect of exogenous oxidants may be indirect, since it may be dependent upon IGF-I, bFGF or the EGF receptor [47, 51, 52].



NADH/NADPH oxidase

The physiological relevance of treatments with exogenous oxidants was recently established by the discovery of an AngII-activated endogenous superoxide-generating pathway in vsmc [53]. The source of superoxide is a membrane-bound NADH or NADPH-dependent oxidase with features similar to the phagocyte NADPH oxidase [53-58]. This latter enzyme complex is composed of a catalytic membrane-bound cytochrome b558 heterodimer (gp91- and p22-phox) that is activated by binding at least three cytosolic components (p47-, p67-phox and Rac2). p47phox may be activated by protein kinase C [59, 60] and Rac2 by exchange of GTP for GDP (Fig. 2). AngII induced almost a 3-fold increase in superoxide production and a 5-fold accumulation of H2O2 in vsmc [57, 61]. Superoxide was the major source of H2O2 because inhibition of oxidase activity with diphenylene iodonium (DPI) or p22-phox antisense mRNA inhibited the formation of both compounds [33, 53, 57, 61, 62]. This pathway was critical to growth in vsmc since inhibition of the oxidase or SOD, or overexpression of catalase, greatly reduced AngII-induced protein synthesis [53, 57, 61].

The function of oxidants was confirmed in-vivo. Hypertension induced by infusion of AngII (but not norepinephrine) in the rat was accompanied by up-regulation of p22phox mRNA, cytochrome b558 protein, and NAD(P)H oxidase-dependent activity, as well as an impairment of endothelium-dependent relaxation. Importantly, AngII-induced hypertension was corrected by infusion of heparin-binding SOD. Moreover, the biochemical effects of AngII infusion were inhibited in vitro by DPI or liposomal SOD [58, 63, 64].

 

Figure 2. The NAD(P)H signalling cascade in vsmc

 

Oxidants and MAP kinases

Exogenous superoxide increased the activity of ERK1/2, an effect that was inhibited by the superoxide scavenger tiron and protein kinase C down-regulation [48]. The effect of exogenous H2O2 on ERK1/2 activation has been controversial. ERK1, ERK2 or both were phosphorylated by exposure to H2O2 in some studies [51, 65, 66], but not in others [33, 48, 67]. Stimulation of vsmc with AngII increases endogenous H2O2 accumulation and is accompanied by ERK1/ERK2 activation [33, 67]. However, the fact that AngII-induced ERK1/2 phosphorylation was not inhibited by DPI, tiron or by overexpression of catalase suggests that these kinases are not redox-sensitive within cells [33, 48]. Exogenous H2O2 increased phosphorylation and/or activity of other MAP kinases present in vsmc, namely JNK, p38MAPK and BMK1 (ERK5) [33, 66, 67]. AngII induced p38MAPK activation appeared to result from endogenous H2O2 generation via the NAD(P)H oxidase pathway, since it was blocked by DPI or catalase overexpression [33]. It is worth noting that ERK1/2 and p38MAPK control together the major portion of AngII-mediated hypertrophy. While specific inhibitors of either kinase were partially effective, simultaneous incubation with both compounds blocked most of AngII-induced protein synthesis [33]. This may be explained by the fact that these two kinases regulate complementary sets of proteins and transcription factors. For example, p70 and p90 ribosomal S6 kinases are phosphorylated by p38MAPK and ERK1/2, respectively. Similarly, ATF-2 and CHOP-1 are activated by p38MAPK, while Elk-1 and AP-1 are stimulated by ERK1/2 (Fig. 2).

 

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Conclusion

Following an immediate stimulation of vascular smooth muscle contraction, AngII chronically induces the cellular hypertrophy and proliferation observed in vascular diseases. These effects are mediated by a vast array of signals, notably heterotrimeric and small G-proteins, phospholipases, and kinase cascades which control gene transcription. It is interesting to note that the most recently recognized of these mediators, superoxide and H2O2, now fulfill the conditions required for classification as signalling molecules. Both agents are rapidly synthesized in response to agonist stimulation, have specific molecular targets and are rapidly degraded enzymatically. Although formerly known for their toxicity, these oxidants are now gaining acceptance as redox signal mediators. Their complex roles in AngII responsiveness are only beginning to be understood.

 

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References

1. Berk, B. C. and Corson, M. A. (1997) Circ Res 80: 607-16
2. Griendling, K. K., Ushio-Fukai, M., Lassègue, B. et al. (1997) Hypertension 29: 366-73
3. Kai, H., Fukui, T., Lassegue, B. et al. (1996) Mol Pharmacol 49: 96-104
4. Rhee, S. G. and Bae, Y. S. (1997) J Biol Chem 272: 15045-8
5. Marrero, M. B., Paxton, W. G., Duff, J. L. et al. (1994) J Biol Chem 269: 10935-9
6. Marrero, M. B., Schieffer, B., Paxton, W. G. et al. (1995) J Biol Chem 270: 15734-8
7. Touyz, R. M. and Schiffrin, E. L. (1997) Hypertension 30: 222-9
8. Di Salvo, J. and Nelson, S. R. (1998) FEBS Lett 422: 85-8
9. Venema, R. C., Ju, H., Venema, V. J. et al. (1998) J Biol Chem 273: 7703-8
10. Schelling, J. R., Nkemere, N., Konieczkowski, M. et al. (1997) Am J Physiol 272: C1558-C66
11. Ushio-Fukai, M., Griendling, K. K., Akers, M. et al. (1998) J Biol Chem 273: 19772-7
12. Lassègue, B., Alexander, R. W., Clark, M. et al. (1991) Biochem. J. 276: 19-25
13. Lassègue, B., Alexander, R. W., Clark, M. et al. (1993) Biochem. J. 292: 509-17
14. Ushio-Fukai, M., Alexander, R. W., Akers, M. et al. (in press) Mol Pharmacol
15. Exton, J. H. (1997) J Biol Chem 272: 15579-82
16. Ishizaka, N., Griendling, K. K., Lassegue, B. et al. (1998) Hypertension 32: 459-66
17. Kosaka, T. and Gilman, A. G. (1996) J Biol Chem 271: 12562-7
18. Linseman, D. A., Benjamin, C. W. and Jones, D. A. (1995) J Biol Chem 270: 12563-8
19. Ishida, M., Marrero, M. B., Schieffer, B. et al. (1995) Circ Res 77: 1053-9
20. Ishida, M., Ishida, T., Thomas, S. M. et al. (1998) Circ Res 82: 7-12
21. Schieffer, B., Paxton, W. G., Chai, Q. et al. (1996) J Biol Chem 271: 10329-33
22. Wan, Y., Kurosaki, T. and Huang, X. Y. (1996) Nature 380: 541-4
23. Eguchi, S., Matsumoto, T., Motley, E. D. et al. (1996) J Biol Chem 271: 14169-75
24. Liao, D. F., Duff, J. L., Daum, G. et al. (1996) Circ Res 79: 1007-14
25. Molloy, C. J., Taylor, D. S. and Weber, H. (1993) J Biol Chem 268: 7338-45
26. Schieffer, B., Drexler, H., Ling, B. N. et al. (1997) Am J Physiol 272: C2019-30
27. Ishida, Y., Kawahara, Y., Tsuda, T. et al. (1992) FEBS Lett 310: 41-5
28. Marrero, M. B., Schieffer, B., Li, B. et al. (1997) J Biol Chem 272: 24684-90
29. Malarkey, K., McLees, A., Paul, A. et al. (1996) Cell Signal 8: 123-9
30. Tsuda, T., Kawahara, Y., Shii, K. et al. (1991) FEBS Lett. 285: 44-8
31. Tsuda, T., Kawahara, Y., Ishida, Y. et al. (1992) Circ Res 71: 620-30
32. Duff, J. L., Berk, B. C. and Corson, M. A. (1992) Biochem Biophys Res Commun 188: 257-64
33. Ushio-Fukai, M., Alexander, R. W., Akers, M. et al. (1998) J Biol Chem 273: 15022-9
34. Duff, J. L., Marrero, M. B., Paxton, W. G. et al. (1993) J Biol Chem 268: 26037-40
35. Duff, J. L., Monia, B. P. and Berk, B. C. (1995) J Biol Chem 270: 7161-6
36. Duff, J. L., Marrero, M. B., Paxton, W. G. et al. (1995) Cardiovasc Res 30: 511-7
37. Giasson, E. and Meloche, S. (1995) J Biol Chem 270: 5225-31
38. Leduc, I., Haddad, P., Giasson, E. et al. (1995) Mol Pharmacol 48: 582-92
39. Liao, D. F., Monia, B., Dean, N. et al. (1997) J Biol Chem 272: 6146-50
40. Eguchi, S., Numaguchi, K., Iwasaki, H. et al. (1998) J Biol Chem 273: 8890-6
41. Delafontaine, P. and Lou, H. (1993) J Biol Chem 268: 16866-70
42. Suzuki, Y. J., Forman, H. J. and Sevanian, A. (1997) Free Radic Biol Med 22: 269-85
43. Suzuki, Y. J. and Ford, G. D. (1992) Am J Physiol 262: H114-6
44. Roveri, A., Coassin, M., Maiorino, M. et al. (1992) Arch Biochem Biophys 297: 265-70
45. Li, P. F., Dietz, R. and von Harsdorf, R. (1997) Circulation 96: 3602-9
46. Rao, G. N. and Berk, B. C. (1992) Circ Res 70: 593-9
47. Delafontaine, P. and Ku, L. (1997) Cardiovasc Res 33: 216-22
48. Baas, A. S. and Berk, B. C. (1995) Circ Res 77: 29-36
49. Stauble, B., Boscoboinik, D., Tasinato, A. et al. (1994) Eur J Biochem 226: 393-402
50. Fiorani, M., Cantoni, O., Tasinato, A. et al. (1995) Biochim Biophys Acta 1269: 98-104
51. Rao, G. N. (1996) Oncogene 13: 713-9
52. Herbert, J. M., Bono, F. and Savi, P. (1996) FEBS Lett 395: 43-7
53. Griendling, K. K., Minieri, C. A., Ollerenshaw, J. D. et al. (1994) Circ. Res. 74: 1141-8
54. Mohazzab, K. M. and Wolin, M. S. (1994) Am J Physiol 267: L823-31
55. Mohazzab, K. M. and Wolin, M. S. (1994) Am J Physiol 267: L815-22
56. Pagano, P. J., Ito, Y., Tornheim, K. et al. (1995) Am J Physiol 268: H2274-80
57. Ushio-Fukai, M., Zafari, A. M., Fukui, T. et al. (1996) J Biol Chem 271: 23317-21
58. Rajagopalan, S., Kurz, S., Munzel, T. et al. (1996) J Clin Invest 97: 1916-23
59. Rotrosen, D., Yeung, C. L., Leto, T. L. et al. (1992) Science 256: 1459-62
60. Jones, O. T. (1994) Bioessays 16: 919-23
61. Zafari, A. M., Ushio-Fukai, M., Akers, M. et al. (1998) Hypertension 32: 488-95
62. Fukui, T., Lassegue, B., Kai, H. et al. (1995) Biochim Biophys Acta 1231: 215-9
63. Fukui, T., Ishizaka, N., Rajagopalan, S. et al. (1997) Circ Res 80: 45-51
64. Bech Laursen, J., Rajagopalan, S., Galis, Z. et al. (1997) Circulation 95: 588-93
65. Guyton, K. Z., Liu, Y., Gorospe, M. et al. (1996) J Biol Chem 271: 4138-42
66. Zhang, J., Jin, N., Liu, Y. et al. (1998) Am J Respir Cell Mol Biol 19: 324-32
67. Abe, J., Kusuhara, M., Ulevitch, R. J. et al. (1996) J Biol Chem 271: 16586-90


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