Involvement of insulin-like growth factor 1 receptor transactivation in endothelin-1-induced signaling in vascular smooth muscle cells.

Endothelin-1 (ET-1) is a potent vasoactive peptide that exerts hypertrophic, migratory, and mitogenic effects in vascular smooth muscle cells. ET-1-induced activation of several signaling events has been shown to mediate the cellular effects of ET-1. In the past several years, transactivation of growth factor receptor has gained much recognition in transducing the signaling responses of ET-1. Among various growth factor receptors studied, the involvement of epidermal growth factor receptor transactivation in triggering ET-1-induced responses has been studied in some detail. However, recent studies have implicated insulin-like growth factor 1 receptor transactivation in this process. There are also some suggestions for a role of the Src family of nonreceptor protein tyrosine kinases, such as c-Src, in transducing the signaling responses of vasoactive peptides. In this review, we will examine the contribution of both insulin-like growth factor 1 receptor and c-Src in mediating ET-1-induced signaling responses in vascular smooth muscle cells.

Role of insulin-like growth factor 1 receptor and c-Src in endothelin-1- and angiotensin II-induced PKB phosphorylation, and hypertrophic and proliferative responses in vascular smooth muscle cells.

Endothelin-1 (ET-1) and angiotensin II (Ang II) are vasoactive peptides believed to contribute to the pathogenesis of vascular abnormalities such as hypertension, atherosclerosis, hypertrophy, and restenosis. The concept of transactivation of growth factor receptors, such as epidermal growth factor receptor (EGFR), in triggering vasoactive peptide-induced signaling events has gained much recognition during the past several years. We have demonstrated that insulin-like growth factor type 1 receptor (IGF-1R) plays a role in transducing the effect of H2O2, leading to protein kinase B (PKB) phosphorylation. Since vasoactive peptides elicit their responses through generation of reactive oxygen species, including H2O2, we investigated whether IGF-1R transactivation plays a similar role in ET-1- and Ang II-induced PKB phosphorylation and hypertrophic responses in vascular smooth muscle cells (VSMC). AG1024, a specific inhibitor of IGF-1R protein tyrosine kinase (PTK), attenuated both ET-1- and Ang II-induced PKB phosphorylation in a dose-dependent manner. ET-1 and Ang II treatment also induced the phosphorylation of tyrosine residues in the autophosphorylation sites of IGF-1R, which were blocked by AG1024. In addition, both ET-1 and Ang II evoked tyrosine phosphorylation of c-Src, a nonreceptor PTK, whereas pharmacological inhibition of c-Src PTK activity by PP2, a specific inhibitor of Src-family tyrosine kinase, significantly reduced PKB phosphorylation as well as tyrosine phosphorylation of IGF-1R induced by the 2 vasoactive peptides. Furthermore, protein and DNA synthesis enhanced by ET-1 and Ang II were attenuated by AG1024 and PP2. In conclusion, these data suggest that IGF-1R PTK and c-Src PTK play a critical role in mediating PKB phosphorylation as well as hypertrophic and proliferative responses induced by ET-1 and Ang II in A10 VSMC.

Endothelin-1-induced signaling pathways in vascular smooth muscle cells.

Endothelin-1 (ET-1), a vasoactive peptide, is believed to contribute to the pathogenesis of vascular abnormalities such as hypertension, atherosclerosis, hypertrophy and restenosis. ET-1 elicits its biological effects through the activation of two receptor subtypes, ET-A and ET-B that belong to a large family of transmembrane guanine nucleotide-binding protein-coupled receptors (GPCRs). ET-1 receptor activation results in the stimulation of several signaling pathways including mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3-kinase (PI3-K) and protein kinase B (PKB). An intermediary role of Ca(2+)/calmodulin-dependent protein kinases (CaMK), protein kinase C (PKC) as well as receptor and non-receptor protein tyrosine kinases in triggering the activation of MAPK and PI3-K/PKB signaling in response to ET-1 has been suggested. Activation of these pathways by ET-1 is intimately linked with the regulation of cellular hypertrophy, growth, proliferation and cell survival. Here we provide an overview of these signaling pathways in vascular smooth muscle cells (VSMCs) with an emphasis on their potential role in vascular pathophysiology.

Ca2+/calmodulin-dependent protein kinase- II in vasoactive peptide- induced responses and vascular biology.

Vasoactive peptides such as angiotensin II and endothelin-1 as well as growth factors regulate vascular homeostasis through signaling pathways that are triggered in both normal and disease states. These vasoactive peptides and growth factors also increase the cellular levels of calcium which, through calcium binding effector systems initiating the downstream signaling and physiological responses in target cells. A multifunctional calcium-calmodulin-dependent protein kinase II (CaMKII) has emerged as an important transducer of vasoactive peptide-induced responses in vascular smooth muscle cells (VSMC). The catalytic activity of CaMKII can be stimulated by autophosphorylation and oxidation leading to the activation of signaling events that mediate growth, proliferation, migration, and gene transcription in VSMC. Pharmacological and gene deletion approaches have demonstrated a requirement of CaMKII in mediating the mitogen- activated protein kinase and phosphatidyl-inositol 3-kinase/protein kinase B signalling, as well as the proliferative, migratory and transcriptional responses of vasoactive peptides. In addition, a potential involvement of hyperactive CaMKII in animal models of vascular disease has also been reported. Therefore, this review aims to highlight the role of CaMKII in mediating signaling and physiological responses in VSMC and discuss its potential role in vascular pathophysiology.

ET-1-induced growth promoting responses involving ERK1/2 and PKB signaling and Egr-1 expression are mediated by Ca2+/CaM-dependent protein kinase-II in vascular smooth muscle cells.

Endothelin-1 (ET-1), a potent vasoactive peptide with a pathogenic role in vascular diseases, has been shown to induce the activation of ERK1/2, PKB and the expression of a transcriptional regulator, the early growth response 1 (Egr-1), key mediators of hypertrophic and proliferative responses in vascular smooth muscle cells (VSMC). We have demonstrated earlier that ET-1 requires H2O2 generation to activate these signaling pathways and Ca2+, calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CaMKII), play a critical role to trigger H2O2-induced effects in VSMC. However, an involvement of CaMKII in mediating ET-1-induced responses in VSMC remains unknown. Therefore, by utilizing pharmacological inhibitors of CaM, CaMKII, a CaMKII inhibitor peptide and CaMKII knockdown techniques, we have investigated the contribution of CaM and CaMKII in ET-1-induced ERK1/2 and PKB signaling, Egr-1 expression and hypertrophic and proliferative responses in VSMC. W-7 and calmidazolium, antagonists of CaM, as well as KN-93, an inhibitor of CaMKII activity, attenuated ET-1-induced ERK1/2 and PKB phosphorylation. In addition, transfection of VSMC with a CaMKII inhibitory peptide suppressed ET-1-evoked ERK1/2 and PKB phosphorylation. Similarly, siRNA-mediated CaMKII silencing reduced ET-1-produced ERK1/2 and PKB phosphorylation. CaM and CaMKII blockade also significantly lowered the ET-1-induced protein and DNA synthesis as well as Egr-1 expression. These findings demonstrate that CaMKII plays a critical role in ET-1-induced growth promoting signaling pathways as well as hypertrophic and proliferative responses in VSMC.

Modulatory Role of Nitric Oxide/cGMP System in Endothelin-1-Induced Signaling Responses in Vascular Smooth Muscle Cells.

Nitric oxide (NO) is an important vasoprotective molecule that serves not only as a vasodilator but also exerts antihypertrophic and antiproliferative effects in vascular smooth muscle cells (VSMC). The precise mechanism by which the antihypertrophic and antiproliferative responses of NO are mediated remains obscure. However, recent studies have suggested that one of the mechanisms by which this may be achieved includes the attenuation of signal transduction pathways responsible for inducing the hypertrophic and proliferative program in VSMC. Endothelin-1 is a powerful vasoconstrictor peptide with mitogenic and growth stimulatory properties and exerts its effects by activating multiple signaling pathways which include ERK 1/2, PKB and Rho-ROCK. Both cGMP-dependent and independent events have been reported to mediate the effect of NO on these pathways leading to its vasoprotective response. This review briefly summarizes some key studies on the modulatory effect of NO on these signaling pathways and discusses the possible role of cGMP system in this process.

CaMKII knockdown attenuates H2O2-induced phosphorylation of ERK1/2, PKB/Akt, and IGF-1R in vascular smooth muscle cells.

We have shown earlier a requirement for Ca(2+) and calmodulin (CaM) in the H(2)O(2)-induced activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and protein kinase B (PKB), key mediators of growth-promoting, proliferative, and hypertrophic responses in vascular smooth muscle cells (VSMC). Because the effect of CaM is mediated through CaM-dependent protein kinase II (CaMKII), we have investigated here the potential role of CaMKII in H(2)O(2)-induced ERK1/2 and PKB phosphorylation by using pharmacological inhibitors of CaM and CaMKII, a CaMKII inhibitor peptide, and siRNA knockdown strategies for CaMKII alpha. Calmidazolium and W-7, antagonists of CaM, as well as KN-93, a specific inhibitor of CaMKII, attenuated H(2)O(2)-induced responses of ERK1/2 and PKB phosphorylation in a dose-dependent fashion. Similar to H(2)O(2), calmidazolium and KN-93 also exhibited an inhibitory effect on glucose/glucose oxidase-induced phosphorylation of ERK1/2 and PKB in these cells. Transfection of VSMC with CaMKII autoinhibitory peptide corresponding to the autoinhibitory domain (aa 281-309) of CaMKII and with siRNA of CaMKII alpha attenuated the H(2)O(2)-induced phosphorylation of ERK1/2 and PKB. In addition, calmidazolium and KN-93 blocked H(2)O(2)-induced Pyk2 and insulin-like growth factor-1 receptor (IGF-1R) phosphorylation. Moreover, treatment of VSMC with CaMKII alpha siRNA abolished the H(2)O(2)-induced IGF-1R phosphorylation. H(2)O(2) treatment also induced Thr(286) phosphorylation of CaMKII, which was inhibited by both calmidazolium and KN-93. These results demonstrate that CaMKII plays a critical upstream role in mediating the effects of H(2)O(2) on ERK1/2, PKB, and IGF-1R phosphorylation.

Nitric oxide attenuates endothelin-1-induced activation of ERK1/2, PKB, and Pyk2 in vascular smooth muscle cells by a cGMP-dependent pathway.

Nitric oxide (NO), in addition to its vasodilator action, has also been shown to antagonize the mitogenic and hypertrophic responses of growth factors and vasoactive peptides such as endothelin-1 (ET-1) in vascular smooth muscle cells (VSMCs). However, the mechanism by which NO exerts its antimitogenic and antihypertrophic effect remains unknown. Therefore, the aim of this study was to determine whether NO generation would modify ET-1-induced signaling pathways involved in cellular growth, proliferation, and hypertrophy in A-10 VSMCs. Treatment of A-10 VSMCs with S-nitroso-N-acetylpenicillamine (SNAP) or sodium nitroprusside (SNP), two NO donors, attenuated the ET-1-enhanced phosphorylation of several key components of growth-promoting and hypertrophic signaling pathways such as ERK1/2, PKB, and Pyk2. On the other hand, inhibition of the endogenous NO generation with N(G)-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor, increased the ET-1-induced phosphorylation of these signaling components. Since NO mediates its effect principally through a cGMP-soluble guanylyl cyclase (sGC) pathway, we investigated the role of these molecules in NO action. 8-Bromoguanosine 3',5'-cyclic monophosphate, a nonmetabolizable and cell-permeant analog of cGMP, exhibited a effect similar to that of SNAP and SNP. Furthermore, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of sGC, reversed the inhibitory effect of NO on ET-1-induced responses. SNAP treatment also decreased the protein synthesis induced by ET-1. Together, these data demonstrate that NO, in a cGMP-dependent manner, attenuated ET-1-induced phosphorylation of ERK1/2, PKB, and Pyk2 and also antagonized the hypertrophic effects of ET-1. It may be suggested that NO-induced generation of cGMP contributes to the inhibition of ET-1-induced mitogenic and hypertrophic responses in VSMCs.

Nitric oxide inhibits the bradykinin B2 receptor-mediated adrenomedullary catecholamine release but has no effect on adrenal blood flow response in vivo.

The role of nitric oxide (NO) in bradykinin (BK)-induced adrenal catecholamine secretion still remains obscure. The present study was to investigate whether an inhibition of NO synthase with N(omega)-nitro-L-arginine methyl ester (L-NAME) would modulate BK-induced adrenal catecholamine secretion (ACS) and adrenal vasodilating response (AVR) in anesthetized dogs. Plasma catecholamine concentrations were determined with an HPLC coupled with an electrochemical detector. All drugs were locally administered to the left adrenal gland via intra-arterial infusion. BK dose-dependently increased both ACS and AVR. Hoe-140, a selective B(2) antagonist, significantly blocked the BK-induced increases in both ACS and AVR. In the presence of L-NAME, the BK-induced ACS was significantly enhanced, while the simultaneous AVR remained unaffected. These results suggest that the both BK-induced ACS and AVR are primarily mediated by B(2) receptors in the canine adrenal gland. Our results also suggest that the enhanced ACS in response to BK in the presence of L-NAME may have resulted from a specific inhibition of NO formation in the adrenal gland. It is concluded that the BK-induced NO may play an inhibitory role in the B(2)-receptor-mediated mechanisms regulating ACS, while it may not be implicated in the B(2)-receptor-mediated AVR under in vivo conditions.