Increased vulnerability of nigral dopamine neurons after expansion of their axonal arborization size through D2 dopamine receptor conditional knockout.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNc). Rare genetic mutations in genes such as Parkin, Pink1, DJ-1, α-synuclein, LRRK2 and GBA are found to be responsible for the disease in about 15% of the cases. A key unanswered question in PD pathophysiology is why would these mutations, impacting basic cellular processes such as mitochondrial function and neurotransmission, lead to selective degeneration of SNc DA neurons? We previously showed in vitro that SNc DA neurons have an extremely high rate of mitochondrial oxidative phosphorylation and ATP production, characteristics that appear to be the result of their highly complex axonal arborization. To test the hypothesis in vivo that axon arborization size is a key determinant of vulnerability, we selectively labeled SNc or VTA DA neurons using floxed YFP viral injections in DAT-cre mice and showed that SNc DA neurons have a much more arborized axon than those of the VTA. To further enhance this difference, which may represent a limiting factor in the basal vulnerability of these neurons, we selectively deleted in mice the DA D2 receptor (D2-cKO), a key negative regulator of the axonal arbour of DA neurons. In these mice, SNc DA neurons have a 2-fold larger axonal arborization, release less DA and are more vulnerable to a 6-OHDA lesion, but not to α-synuclein overexpression when compared to control SNc DA neurons. This work adds to the accumulating evidence that the axonal arborization size of SNc DA neurons plays a key role in their vulnerability in the context of PD.

Angiotensin II-induced overexpression of sirtuin 1 contributes to enhanced expression of Giα proteins and hyperproliferation of vascular smooth muscle cells.

Angiotensin II (ANG II) plays an important role in the regulation of various physiological functions including proliferation, hypertrophy of vascular smooth muscle cells (VSMCs) through the overexpression of Giα proteins. Sirtuin 1 (Sirt1), a class III histone deacetylase and epigenetic regulator is implicated in a wide range of cellular functions, including migration and growth of VSMCs and in ANG II-induced hypertension. The present study was undertaken to examine the role of Sirt1 in ANG II-induced overexpression of Giα proteins and hyperproliferation of aortic VSMCs. We show that ANG II treatment of VSMCs increased the expression of Sirt1, which was attenuated by AT1 and AT2 receptor antagonists, losartan, and PD123319, respectively. In addition, the knockdown of Sirt1 by siRNA attenuated ANG II-induced overexpression of Giα-2 and Giα-3 proteins, hyperproliferation of VSMCs and the overexpression of cell cycle proteins, cyclin D1, Cdk4, and phosphorylated retinoblastoma proteins. Furthermore, ANG II-induced increased levels of superoxide anion (O2-) and NADPH oxidase activity and increased phosphorylation of ERK1/2 and Akt that are implicated in enhanced expression of Giα proteins and hyperproliferation of VSMCs were also attenuated to control levels by silencing of Sirt1. In addition, depletion of Sirt1 by siRNA also attenuated ANG II-induced enhanced phosphorylation of platelet-derived growth factor receptor (PDGFR), epidermal growth factor receptor (EGFR), and insulin-like growth factor receptor (IGFR) in VSMCs. In summary, our results demonstrate that ANG II increased the expression of Sirt1, which through oxidative stress, growth factor receptor-mediated mitogen-activated protein (MAP) kinase/Akt signaling pathway enhances the expression of Giα proteins and cell cycle proteins and results in the hyperproliferation of VSMCs.NEW & NOTEWORTHY ANG II regulates various physiological functions including proliferation of VSMCs through the overexpression of Giα proteins. Sirt1, a class III histone deacetylase, is implicated in several cellular functions, including VSMC growth and ANG II-induced hypertension. We showed for the first time that ANG II increased the expression of Sirt1, which through oxidative stress, growth factor receptor-mediated MAP kinase/Akt signaling pathway enhances the levels of Giα and cell cycle proteins resulting in the hyperproliferation of VSMCs.

Angiotensin II-induced histone deacetylase 5 phosphorylation, nuclear export, and Egr-1 expression are mediated by Akt pathway in A10 vascular smooth muscle cells.

Angiotensin II (ANG II) regulates an array of physiological and pathological responses in vascular smooth muscle cells (VSMCs) by activating ERK1/2 and phosphoinositide 3-kinase (PI3K)/Akt signaling pathways. We have demonstrated that ANG II and insulin-like growth factor-1 (IGF-1) induce the expression of early growth response protein-1 (Egr-1), a zinc finger transcription factor, which regulates the transcription of cell cycle regulatory genes network in VSMCs. We have reported that IGF-1 induces the phosphorylation of histone deacetylase 5 (HDAC5), which has been implicated in the expression of genes linked to VSMC growth and hypertrophy, via a PI3K/Akt-dependent pathway in VSMCs. However, the involvement of PI3K/Akt pathways in ANG II-induced HDAC5 phosphorylation and the contribution of HDAC5 in Egr-1 expression and hypertrophy in VSMCs remain unexplored. Here, we show that pharmacological blockade of the PI3K/Akt pathway either by wortmannin/SC66 or siRNA-induced silencing of Akt attenuated ANG II-induced HDAC5 phosphorylation and its nuclear export. Moreover, SC66 or Akt knockdown also suppressed ANG II-induced Egr-1 expression. Furthermore, pharmacological inhibition of HDAC5 by MC1568 or TMP-195 or knockdown of HDAC5 and the blockade of the nuclear export of HDAC5 by leptomycin B or KPT-330 significantly reduced ANG II-induced Egr-1 expression. In addition, depletion of either HDAC5 or Egr-1 by siRNA attenuated VSMC hypertrophy in response to ANG II. In summary, our results demonstrate that ANG II-induced HDAC5 phosphorylation and its nuclear exclusion are mediated by PI3K/Akt pathway and HDAC5 is an upstream regulator of Egr-1 expression and hypertrophy in VSMCs.NEW & NOTEWORTHY ANG II-induced histone deacetylase 5 (HDAC5) phosphorylation and nuclear export occurs via the phosphoinositide 3-kinase/Akt pathway. Akt, through HDAC5, regulates ANG II-induced expression of early growth response protein-1 (Egr-1), which is a transcription factor linked with vascular dysfunction. Inhibition of HDAC5 exclusion by nuclear export inhibitors suppresses ANG II-induced Egr-1 expression. HDAC5 is an upstream mediator of Egr-1 expression and cell hypertrophy in response to ANG II in vascular smooth muscle cells.

Role of the JAK2/STAT3 pathway in angiotensin II-induced enhanced expression of Giα proteins and hyperproliferation of aortic vascular smooth muscle cells.

We earlier showed that angiotensin (Ang) II-induced overexpression of Giα proteins contributes to the hyperproliferation of vascular smooth muscle cells (VSMC). In addition, the implication of the JAK2/STAT3 pathway in Ang II-induced hyperproliferation of VSMC has also been reported. However, the role of the JAK2/STAT3 pathway in Ang II-induced overexpression of Giα proteins and hyperproliferation of VSMC remains unexplored. In the present study, we show that inhibition or knockdown of the JAK2/STAT3 pathway by a specific inhibitor "cucurbitacin I" (CuI) or siRNAs attenuated Ang II-induced overexpression of Giα proteins and hyperproliferation of VSMC. In addition, the enhanced expression of cell cycle proteins induced by Ang II was also attenuated by CuI. Furthermore, Ang II-induced enhanced production of the superoxide anion (O2 -), H2O2, and NADPH oxidase activity, as well as the enhanced expression of NADPH oxidase subunits implicated in enhanced expression of Giα proteins and hyperproliferation, were also attenuated by inhibition of the JAK2/STAT3 pathway. On the other hand, Ang II-induced inhibition and augmentation of the levels of nitric oxide and peroxynitrite, respectively, in VSMC were restored to control levels by CuI. In summary, our results demonstrate that Ang II through the JAK2/STAT3 pathway increases nitroxidative stress, which contributes to the overexpression of Giα proteins and cell cycle proteins and the hyperproliferation of VSMC.

Role of cyclic AMP response element binding protein (CREB) in angiotensin II-induced responses in vascular smooth muscle cells.

Cyclic AMP response element (CRE) binding protein (CREB) is a nuclear transcription factor that regulates the transcription of several genes containing the CRE sites on their promoters. CREB is activated by phosphorylation on a key serine residue, Ser311, in response to a wide variety of extracellular stimuli including angiotensin II (Ang II). Ang II is an important vasoactive peptide and mitogen for vascular smooth muscle cells (VSMC) that in addition to regulating the contractile response in VSMC also plays an important role in phenotypic switch of VSMC from contractile to a synthetic state. The synthetic VSMC are known to exhibit proliferative and migratory properties due to hyperactivation of Ang II-induced signaling events. Ang II has been shown to induce CREB phosphorylation/activation and transcription of genes implicated in proliferation, growth, and migration. Here, we have highlighted some key studies that have demonstrated an important role of CREB in Ang II-mediated gene transcription, proliferation, hypertrophy, and migration of VSMC.

Sodium nitroprusside attenuates hyperproliferation of vascular smooth muscle cells from spontaneously hypertensive rats through the inhibition of overexpression of AT1 receptor, cell cycle proteins, and c-Src/growth factor receptor signaling pathways.

We recently showed that sodium nitroprusside (SNP), a NO donor, attenuated hypertension in spontaneously hypertensive rats (SHR). Since hypertension is associated with enhanced proliferation and hypertrophy of vascular smooth muscle cells (VSMC), the present study examines whether in vivo treatment of SHR with SNP could also inhibit the augmented proliferation of VSMC and explore the signaling mechanisms. Treatment of 8 week old SHR and Wistar Kyoto rats with SNP twice a week for 2 weeks inhibited the enhanced proliferation of VSMC from SHR, the enhanced expression of angiotensin II type 1 (AT1) receptor, and enhanced activation of c-Src and growth factor receptors and ERK1/2 signaling pathways. In addition, SNP also inhibited the overexpression of cell cycle proteins including cyclins D1, Cdk4, and phosphorylated pRB and restored the downregulated Cdk inhibitors p21Cip1 and p27Kip1 expression towards control levels. Furthermore, SNP-induced inhibition of enhanced levels of the AT1 receptor and enhanced proliferation was reversed by L-NAME, an inhibitor of nitric oxide synthase. These results suggest that the SNP-induced antiproliferative effect may be mediated through the inhibition of enhanced expression of the AT1 receptor, cell cycle proteins and activation of c-Src, growth factor receptors, and MAP kinase signaling.

Protein kinase B/AKT mediates insulin-like growth factor 1-induced phosphorylation and nuclear export of histone deacetylase 5 via NADPH oxidase 4 activation in vascular smooth muscle cells.

Insulin-like growth factor 1 (IGF-1) mediates the generation of reactive oxygen species (ROS) and the activation of growth promoting signaling pathways. Histone deacetylases (HDACs) regulate gene transcription by deacetylating lysine residues in histone and nonhistone proteins and a heightened HDAC activation, notably of HDAC5, is associated with vascular disorders, such as atherosclerosis. Although the contribution of IGF-1 in these pathologies is well documented, its role in HDAC phosphorylation and activation remains unexplored. Here, we examined the effect of IGF-1 on HDAC5 phosphorylation in vascular smooth muscle cells (VSMCs) and identified the signaling pathways involved in controlling HDAC5 phosphorylation and nuclear export. Treatment of A10 VSMCs with IGF-1 enhanced HDAC5 phosphorylation. Blockade of the IGF-1 receptor tyrosine kinase (TK) activity with the specific pharmacological inhibitor, AG1024, significantly inhibited IGF-1-induced HDAC5 phosphorylation, whereas the epidermal growth factor receptor (EGFR) TK antagonist, AG1478, had no effect. Inhibition of the mitogen-activated protein kinase pathway with U0126, SP600125, or SB203580, did not affect HDAC5 phosphorylation, whereas two inhibitors of the phosphoinositide 3-kinase (PI3K)/AKT pathways, wortmannin and SC66, almost completely attenuated IGF-1-induced responses as confirmed by immunoblotting of phospho-HDAC5 and by small interfering RNA (siRNA)-induced AKT silencing. Moreover, the NAD(P)H oxidase (Nox) inhibitor, diphenyleneiodonium (DPI), and Nox4 siRNA, attenuated IGF-1-induced phosphorylation of HDAC5 and AKT. The HDAC5 phosphorylation resulted in its nuclear export, which was reversed by SC66 and DPI. Our results indicate that IGF-1-induced phosphorylation and nuclear export of HDAC5 involve Nox4-dependent ROS generation and PI3K/AKT signaling pathways.

Resveratrol prevents the development of high blood pressure in spontaneously hypertensive rats through the inhibition of enhanced expression of Giα proteins 1.

Resveratrol (RV), a polyphenolic component of red wine, has been shown to attenuate high blood pressure (BP) in spontaneously hypertensive rats (SHRs). We previously found that the enhanced expression of Giα proteins plays a role in the pathogenesis of hypertension in SHRs. In the present study, we investigated whether this RV-induced decrease in BP in SHRs can be attributed to the ability of RV to inhibit the enhanced expression of Giα proteins and the upstream signaling molecules implicated in the overexpression of Giα proteins. Administration of RV (50 mg/kg per day) to prehypertensive 2-week-old SHRs for 6 weeks prevented the development of high BP and inhibited the enhanced expression of Giα proteins, the enhanced levels of superoxide anion (O2-) and NADPH oxidase activity, the enhanced activation (phosphorylation) of c-Src and growth factor receptors, as well as the enhanced levels of extracellular signal-regulated kinase 1/2 (ERK1/2) and protein kinase B (Akt) exhibited by vascular smooth muscle cells isolated from SHRs. In conclusion, these results indicate that RV attenuates the development of high BP in SHRs through the inhibition of enhanced levels of Giα proteins, oxidative stress, and the upstream signaling molecules that contribute to the overexpression of Giα proteins. These findings suggest that RV could potentially be used as a therapeutic agent in the treatment of cardiovascular complications including hypertension.

Natriuretic peptide receptor-C activation attenuates angiotensin II-induced enhanced oxidative stress and hyperproliferation of aortic vascular smooth muscle cells.

We showed previously that natriuretic peptide receptor-C (NPR-C) agonist, C-ANP4-23, attenuated the enhanced expression of Giα proteins in vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) through the inhibition of enhanced oxidative stress. Since the enhanced levels of endogenous angiotensin II (Ang II) contribute to the overexpression of Giα proteins and augmented oxidative stress in VSMC from SHR, the present study was undertaken to investigate if C-ANP4-23 could also attenuate angiotensin II (Ang II)-induced oxidative stress and associated signaling. Ang II treatment of aortic VSMC augmented the levels of superoxide anion (O2-), NADPH oxidase activity, and the expression of NADPH oxidase subunits and C-ANP4-23 treatment attenuated all these to control levels. In addition, Ang II-induced enhanced levels of thiobarbituric acid-reactive substances (TBARS) and protein carbonyl content were also attenuated toward control levels by C-ANP4-23 treatment. On the other hand, Ang II inhibited the levels of nitric oxide (NO) and augmented the levels of peroxynitrite (OONO-) in VSMC which were restored to control levels by C-ANP4-23 treatment. Furthermore, C-ANP4-23 treatment attenuated Ang II-induced enhanced expression of Giα proteins, phosphorylation of p38, JNK, and ERK 1,2 as well as hyperproliferation of VSMC as determined by DNA synthesis, and metabolic activity. These results indicate that C-ANP4-23, via the activation of NPR-C, attenuates Ang II-induced enhanced nitroxidative stress, overexpression of Giα proteins, increased activation of the p38/JNK/ERK 1,2 signaling pathways, and hyperproliferation of VSMC. It may be suggested that C-ANP4-23 could be used as a therapeutic agent in the treatment of vascular remodeling associated with hypertension and atherosclerosis.

Resveratrol prevents angiotensin II-induced hypertrophy of vascular smooth muscle cells through the transactivation of growth factor receptors.

We previously showed that augmented levels of endogenous angiotensin II (AngII) contribute to vascular smooth muscle cell (VSMC) hypertrophy through the transactivation of growth factor receptors in spontaneously hypertensive rats. Resveratrol (RV), a polyphenolic component of red wine, has also been shown to attenuate AngII-evoked VSMC hypertrophy; however, the molecular mechanism mediating this response is obscure. The present study was therefore undertaken to examine whether RV could prevent AngII-induced VSMC hypertrophy through the transactivation of growth factor receptor and associated signaling pathways. AngII treatment of VSMC enhanced the protein synthesis that was attenuated towards control levels by RV pretreatment as well as by the inhibitors of NADPH oxidase, c-Src, and growth factor receptors. Furthermore, RV pretreatment also inhibited enhanced levels of superoxide anion, NADPH oxidase activity, increased expression of NADPH oxidase subunits, and phosphorylation of c-Src, EGF-R, PDGE-R, ERK1/2, and AKT1/2. In conclusion, these results indicate that RV attenuates AngII-induced VSMC hypertrophy through the inhibition of enhanced oxidative stress and activation of c-Src, growth factor receptors, and MAPK/AKT signaling. We suggest that RV could be used as a therapeutic agent in the treatment of vascular complications associated with hypertension and hypertrophy.

Oxidative stress contributes to the enhanced expression of Gqα/PLCß1 proteins and hypertrophy of VSMC from SHR: role of growth factor receptor transactivation.

We showed previously that vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHRs) exhibit overexpression of Gqα/PLCß1 proteins, which contribute to increased protein synthesis through the activation of MAP kinase signaling. Because oxidative stress has been shown to be increased in hypertension, the present study was undertaken to examine the role of oxidative stress and underlying mechanisms in enhanced expression of Gqα/PLCß1 proteins and VSMC hypertrophy. Protein expression was determined by Western blotting, whereas protein synthesis and cell volume, markers for VSMC hypertrophy, were determined by [(3)H]-leucine incorporation and three-dimensional confocal imaging, respectively. The increased expression of Gqα/PLCß1 proteins, increased protein synthesis, and augmented cell volume exhibited by VSMCs from SHRs were significantly attenuated by antioxidants N-acetyl-cysteine (NAC), a scavenger of superoxide anion, DPI, an inhibitor of NAD(P)H oxidase. In addition, PP2, AG1024, AG1478, and AG1295, inhibitors of c-Src, insulin-like growth factor receptor (IGFR), epidermal growth factor receptor (EGFR), and platelet-derived growth factor receptor (PDGFR), respectively, also attenuated the enhanced expression of Gqα/PLCß1 proteins and enhanced protein synthesis in VSMCs from SHRs toward control levels. Furthermore, the levels of IGF-1R and EGFR proteins and not of PDGFR were also enhanced in VSMCs from SHRs, which were attenuated significantly by NAC, DPI, and PP2. In addition, NAC, DPI, and PP2 also attenuated the enhanced phosphorylation of IGF-1R, PDGFR, EGFR, c-Src, and EKR1/2 in VSMCs from SHRs. These data suggest that enhanced oxidative stress in VSMCs from SHRs activates c-Src, which through the transactivation of growth factor receptors and MAPK signaling contributes to enhanced expression of Gqα/PLCß1 proteins and resultant VSMC hypertrophy.

Enhanced expression of Giα proteins contributes to the hyperproliferation of vascular smooth muscle cells from spontaneously hypertensive rats via MAP kinase- and PI3 kinase-independent pathways.

Vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) exhibit hyperproliferation, enhanced MAP kinase (MAPK) activity, and overexpression of Giα proteins. This study was undertaken to examine whether the overexpression of Giα proteins contributes to the hyperproliferation of VSMC of SHR through MAPK signaling. The hyperproliferation of VSMC from SHR in the absence and presence of angiotensin II was restored towards those in Wistar-Kyoto (WKY) rats levels by pertussis toxin (PT) treatment. In addition, siRNA knockdown of Giα proteins also resulted in the attenuation of hyperproliferation towards control levels. The overexpression of Giα proteins was also inhibited by MAPK and PI3 kinase (PI3K) inhibitors. In addition, the hyperproliferation and enhanced phosphorylation of ERK1/2 and Akt in VSMC from SHR were attenuated towards WKY levels by the inhibitors of MAPK, PI3K, c-Src, and antioxidants, whereas PT was unable to attenuate the enhanced phosphorylation of ERK1/2 and Akt. Furthermore, 8Br-cAMP and forskolin also attenuated the hyperproliferation of VSMC from SHR. These results suggest that the hyperproliferation of VSMC from SHR may be attributed to the enhanced expression of Giα proteins and increased activation of MAPK and PI3 kinase. However, Giα-mediated hyperproliferation may not be mediated through MAPK- and PI3 kinase-dependent pathways and may involve decreased levels of intracellular cAMP.

Enhanced expression of Gqα and PLC-ß1 proteins contributes to vascular smooth muscle cell hypertrophy in SHR: role of endogenous angiotensin II and endothelin-1.

Vascular Gqα signaling has been shown to contribute to cardiac hypertrophy. In addition, angiotensin II (ANG II) was shown to induce vascular smooth muscle cell (VSMC) hypertrophy through Gqα signaling; however, the studies on the role of Gqα and PLC-ß1 proteins in VSMC hypertrophy in animal model are lacking. The present study was therefore undertaken to examine the role of Gqα/PLC-ß1 proteins and the signaling pathways in VSMC hypertrophy using spontaneously hypertensive rats (SHR). VSMC from 16-wk-old SHR and not from 12-wk-old SHR exhibited enhanced levels of Gqα/PLC-ß1 proteins compared with age-matched Wistar-Kyoto (WKY) rats as determined by Western blotting. However, protein synthesis as determined by [(3)H]leucine incorporation was significantly enhanced in VSMC from both 12- and 16-wk-old SHR compared with VSMC from age-matched WKY rats. Furthermore, the knockdown of Gqα/PLC-ß1 in VSMC from 16-wk-old SHR by antisense and small interfering RNA resulted in attenuation of protein synthesis. In addition, the enhanced expression of Gqα/PLC-ß1 proteins, enhanced phosphorylation of ERK1/2, and enhanced protein synthesis in VSMC from SHR were attenuated by the ANG II AT1 and endothelin-1 (ET-1) ETA receptor antagonists losartan and BQ123, respectively, but not by the ETB receptor antagonist BQ788. In addition, PD98059 decreased the enhanced expression of Gqα/PLC-ß1 and protein synthesis in VSMC from SHR. These results suggest that the enhanced levels of endogenous ANG II and ET-1 through the activation of AT1 and ETA receptors, respectively, and MAP kinase signaling, enhanced the expression of Gqα/PLC-ß1 proteins in VSMC from 16-wk-old SHR and result in VSMC hypertrophy.

Regulation of Giα protein expression by vasoactive peptides in hypertension: molecular mechanisms.

Guanine nucleotide regulatory proteins (G proteins) play a key role in the regulation of various signal transduction systems, including adenylyl cyclase/cAMP and phospholipase C (PLC)/phosphatidyl inositol (PI) turnover, which are implicated in the modulation of a variety of physiological functions, such as platelet functions, including platelet aggregation, secretion, and clot formation and cardiovascular functions, including arterial tone and reactivity. Several abnormalities in adenylyl cyclase activity, cAMP levels and G proteins have been shown to be responsible for the altered cardiac performance and vascular functions observed in cardiovascular disease states. The enhanced or unaltered levels of inhibitory G proteins (Giα) and mRNA have been reported in different models of hypertension, whereas Gsα levels are shown to be unaltered. The enhanced levels of Giα proteins precede the development of blood pressure and suggest that overexpression of Gi proteins may be one of the contributing factors for the pathogenesis of hypertension. The levels of vasoactive peptides including ET-1 and Ang II and growth factors are augmented in hypertension and contribute to the enhanced expression of Giα proteins in hypertension. In addition, oxidative stress due to enhanced levels of Ang II and ET-1 is enhanced in hypertension and may also be responsible for the enhanced expression of Giα proteins observed in hypertension. Furthermore, Ang II- and ET-1-induced transactivation of growth factor receptor through the activation of MAP kinase signaling is also shown to contribute to the augmented levels of Giα in hypertension. Thus, it appears that the enhanced levels of vasoactive peptides by increasing oxidative stress and transactivation growth factor receptors enhance MAP kinase activity that contribute to the enhanced expression of Giα proteins responsible for the pathogenesis of hypertension. In this review, we describe the role of vasoactive peptides and the signaling mechanisms responsible for the enhanced expression of Giα proteins in hypertension.

cAMP attenuates the enhanced expression of Gi proteins and hyperproliferation of vascular smooth muscle cells from SHR: role of ROS and ROS-mediated signaling.

We previously showed that angiotensin II (ANG II)-induced overexpression of inhibitory G proteins (Gi) was attenuated by dibutyryl-cAMP (db-cAMP) in A10 vascular smooth muscle cells (VSMC). Since enhanced levels of endogenous ANG II contributed to the overexpression of Gi protein and hyperproliferation of VSMC from spontaneously hypertensive rats (SHR), the present study was therefore undertaken to examine if cAMP could also attenuate the overexpression of Gi proteins and hyperproliferation of VSMC from SHR and to explore the underlying molecular mechanisms responsible for this response. The enhanced expression of Giα proteins in VSMC from SHR and Nω-nitro-L-arginine methyl ester hypertensive rats was decreased by db-cAMP. In addition, enhanced inhibition of adenylyl cyclase by inhibitory hormones and forskolin-stimulated adenylyl cyclase activity by low concentration of GTPγS in VSMC from SHR was also restored to Wistar-Kyoto (WKY) levels by db-cAMP. Furthermore, db-cAMP also attenuated the hyperproliferation and the increased production of superoxide anion, NAD(P)H oxidase activity, overexpression of Nox1/Nox2/Nox4 and p47phox proteins, increased phosphorylation of PDGF-receptor (R), EGF-R, c-Src, and ERK1/2 to control levels. In addition, the protein kinase A (PKA) inhibitor reversed the effects of db-cAMP on the expression of Nox4 and Giα proteins and hyperproliferation of VSMC from SHR to WKY levels, while stimulation of the exchange protein directly activated by cAMP did not have any effect on these parameters. These results suggest that cAMP via PKA pathway attenuates the overexpression of Gi proteins and hyperproliferation of VSMC from SHR through the inhibition of ROS and ROS-mediated transactivation of EGF-R/PDGF-R and MAPK signaling pathways.

Role of epidermal growth factor receptor transactivation in endothelin-1-induced enhanced expression of Gi protein and proliferation in A10 vascular smooth muscle cells.

We have recently shown that vasoactive peptides such as angiotensin II (Ang II) and endothelin-1 (ET-1) increase the expression of Gi proteins and the proliferation of A10 vascular smooth muscle cells (VSMC) through mitogen-activated protein (MAP) kinase-phosphoinositide (PI) 3-kinase pathways. This study was intended to examine the implication of epidermal growth factor receptor (EGFR) activation in ET-1-induced enhanced expression of Gi proteins and proliferation of A10 VSMC, and to further investigate the underlying mechanisms responsible for these increases. Cell proliferation was determined by [(3)H]thymidine incorporation and the expression of Gi proteins; extracellular signal-regulated kinases 1 and 2 (ERK1/2) and EGFR phosphorylation was determined by Western blotting. Treatment of A10 VSMC with ET-1 enhanced the expression of Gi proteins, which was attenuated by BQ123 and BQ788, antagonists of ET(A) and ET(B) receptor respectively. In addition, ET-1 enhanced the phosphorylation of EGFR in A10 VSMC, which was restored to the control levels by EGFR inhibitor and ETA and ETB receptor antagonists. Furthermore, ET-1 also augmented the proliferation and ERK1/2 phosphorylation of A10 VSMC, which were restored to the control levels by inhibition of EGFR. These data suggest that ET-1 transactivates EGFR, which, through MAP kinase signaling, may contribute to the enhanced expression of Gi proteins and thus increased proliferation of A10 VSMC.

Role of Gi proteins in the regulation of blood pressure and vascular remodeling.

Heterotrimeric guanine nucleotide regulatory proteins (G-proteins) through the activation of several signaling mechanisms including adenylyl cyclase/cAMP and phospholipase C (PLC)/phosphatidyl inositol (PI) turnover. regulate a variety of cellular functions, including vascular reactivity, proliferation and hypertrophy of VSMC. Activity of adenylyl cyclase is regulated by two G proteins, stimulatory (Gsα) and inhibitory (Giα). Gsα stimulates adenylyl cyclase activity and increases the levels of cAMP, whereas Giα inhibits the activity of adenylyl cyclase and results in the reduction of cAMP levels. Abnormalities in Giα protein expression and associated adenylyl cyclase\cAMP levels result in the impaired cellular functions and contribute to various pathological states including hypertension. The expression of Giα proteins is enhanced in various tissues including heart, kidney, aorta and vascular smooth muscle cells (VSMC) from genetic (spontaneously hypertensive rats (SHR)) and experimentally - induced hypertensive rats and contribute to the pathogenesis of hypertension. In addition, the enhanced expression of Giα proteins exhibited by VSMC from SHR is also implicated in the hyperproliferation and hypertrophy, the two key players contributing to vascular remodelling in hypertension. The enhanced levels of endogenous vasoactive peptides including angiotensin II (Ang II), endothelin-1 (ET-1) and growth factors contribute to the overexpression of Giα proteins in VSMC from SHR. In addition, enhanced oxidative stress, activation of c-Src, growth factor receptor transactivation and MAP kinase/PI3kinase signaling also contribute to the augmented expression of Giα proteins in VSMC from SHR. This review summarizes the role of Giα proteins, and the underlying molecular mechanisms implicated in the regulation of high blood pressure and vascular remodelling.

Hydrogen peroxide enhances the expression of Giα proteins in aortic vascular smooth cells: role of growth factor receptor transactivation.

Oxidative stress has been shown to increase the expression of G(i)α proteins in vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats. The present study was undertaken to examine if H(2)O(2), which induces oxidative stress, could also enhance the expression of G(i)α proteins in VSMC and to further explore the underlying signaling pathways responsible for this response. Treatment of VSMC with H(2)O(2) increased the expression of G(i)α proteins and not of G(s)α protein in a concentration- and time-dependent manner. A maximal increase of ∼40-50% was observed at 100 µM and 1 h and was restored to control levels by AG1295 and AG1478, inhibitors of epidermal growth factor receptor (EGF-R) and platelet-derived growth factor receptor (PDGF-R), respectively, and PD98059 and U126, inhibitors of extracellular signal-regulated kinase (ERK1/2), and wortmannin and AKT inhibitor VIII, inhibitors of PKB/AKT, respectively. In addition, H(2)O(2) also increased the phosphorylation of EGF-R, PDGF-R, ERK1/2, and AKT, which was attenuated by the respective inhibitors, whereas the inhibitors of EGF-R and PDGE-R also inhibited the enhanced phosphorylation of ERK1/2 and AKT. Furthermore, transfection of cells with short interfering RNA of EGF-R and PDGF-R restored the H(2)O(2)-induced enhanced expression of G(i)α proteins to control levels. The increased expression of G(i)α proteins was reflected in enhanced G(i) functions as demonstrated by enhanced inhibition of adenylyl cyclase by inhibitory hormones and forskolin-stimulated adenylyl cyclase activity by a low concentration of GTPγS, whereas G(s)α-mediated stimulations of AC were significantly decreased. Furthermore, H(2)O(2)-induced enhanced proliferation of VSMC was attenuated by dibutyryl-cAMP. These results suggest that H(2)O(2) increases the expression of G(i)α proteins in VSMC through the transactivation of EGF-R/PDGF-R and ERK1/2 and phosphatidylinositol-3 kinase signaling pathways.

Implication of multiple signaling pathways in the regulation of angiotensin II induced enhanced expression of Giα proteins in vascular smooth muscle cells.

We have previously shown that A10 vascular smooth muscle cells (VSMC) exposed to angiotensin II (Ang II) exhibited overexpression of Giα proteins. In the present study, we examined the involvement of different signaling pathways in regulating Ang II induced enhanced expression of Giα proteins in VSMC by using pharmacological inhibitors. Ang II induced increased expression of Giα proteins in A10 VSMC was markedly attenuated by actinomycin D, losartan (an AT(1) receptor antagonist), dibutyryl cAMP, phospholipase C (PLC) inhibitor U73122, protein kinase C (PKC) inhibitors staurosporine and GP109203X, but not by PD123319 (an AT(2) receptor antagonist). In addition, BAPTA-AM and TMB-8 (chelators of intracellular Ca(2+)); and nifedipine (a blocker of L-type Ca(2+) channels) significantly inhibited Ang II induced enhanced expression of Giα proteins. On the other hand, extracellular Ca(2+) chelation using EGTA did not affect the Ang II evoked enhanced levels of Giα proteins. Furthermore, pretreatment of A10 VSMC with calmidazolium (an inhibitor of calmodulin), or KN93 (an inhibitor of CaM kinase), or genistein (an inhibitor of protein tyrosine kinase, PTK), also attenuated the increased levels of Giα proteins induced by Ang II. These results suggest that Ang II induced enhanced expression of Giα proteins may be regulated by different signaling pathways through AT(1) receptors in A10 VSMC.