Induction of the proto-oncogene c-jun by angiotensin II.

Angiotensin (Ang) II causes hypertrophy of rat aortic smooth muscle cells in culture and results in the rapid activation of c-fos. This study demonstrated that Ang II also activated c-jun and, in addition, could activate the AP-1 enhancer element. These data add support for a role of Ang II as an important mediator of vascular smooth muscle cell growth.     

心房钠尿肽对血管紧张素II促血管平滑肌细胞增殖...

By means of technique of cell culture, 3H-thymidine incorporation and dot blot, it was demonstrated that angiotensin II (AGT II) stimulated proliferation and c-fos oncogene expression in cultured SHR vascular smooth muscle cells (VSMC) in a dose-dependent manner. This effect of AGT II was significantly inhibited by co-incubation with ANP. The results suggest that proliferation of VSMC is regulated by some interaction between AGT II and ANP.     

Angiotensin II can regulate gene expression by the AP-1 binding sequence via a protein kinase C-dependent pathway

An expression vector containing three copies of the AP-1 binding element (TRE) upstream of a thymidine kinase promotor which controlled the expression of the chloramphenicol acetyl transferase (CAT) gene was transiently transfected into vascular smooth muscle (VSM) cells and a human hepatocarcinoma cell line, Hep G2. Twelve hours of angiotensin (Ang) II exposure stimulated significantly CAT expression by 3.4 fold and 2.7 fold in Hep G2 and VSM cells, respectively. AngII had no effect on CAT expression of a control vector. This AngII-induced stimulation was attenuated significantly by an AngII receptor antagonist, Sar1 Ile8 AngII, and abolished completely by a PKC inhibitor, staurosporine. Our data suggest that the TRE plays a crucial role in AngII-induced gene expression that is mediated by PKC. We concluded that TRE is one of the AngII-responsive elements.     

Role of nitric oxide on atrial natriuretic peptide release induced by angiotensin II in superfused rat atrial tissue

The present study investigated the role of nitric oxide (NO) on atrial natriuretic peptide (ANP) release stimulated by angiotensin II (Ang II) (10(-7) M) in superfused sliced rat atrial tissue. The use of N(G)-nitro-L-arginine methyl ester (L-NAME) at 10(-4) M, an inhibitor of nitric oxide synthase did not modify basal ANP release. In presence of Ang II (10(-7) M), we observed that L-NAME enhanced ANP secretion induced by Ang II. Furthermore, cGMP levels increased significantly in the presence of Ang II and was attenuated by L-NAME. On the other hand, the perfusion of 8 bromo-cGMP (10(-5) M) with Ang II reduced the effect of this octapeptide on ANP secretion. Secondly, we evaluated the effect of authentic NO on ANP release and observed that perfusion of NO reduced significantly the effect of Ang II on ANP release. We propose that the effect of Ang II on ANP secretion was modulated by NO likely via cGMP pathway.     

Rapid effects of stretched myocardial and vascular cells on gene expression of neonatal rat cardiomyocytes with emphasis on autocrine and paracrine mechanisms

Passive stretch of the heart has a direct effect on cardiomyocytes and other cell types including cardiac fibroblasts, endothelial cells, and vascular smooth muscle cells (VSMCs). Cardiomyocytes are targets for the action of peptide growth factors found in myocardium, suggesting an autocrine or paracrine model of the hypertrophic process. In this study we examined stretch-dependent cellular communication between cardiomyocytes, cardiac fibroblasts, endothelial cells, and VSMCs. Stationary cardiomyocytes were incubated with stretch-conditioned medium (CM0-CM60) derived from stretched (for 0-60 min) cardiomyocytes, cardiac fibroblasts, endothelial cells, and VSMCs. The expression levels of protooncogenes (as c-fos, c-jun, and fra-1) were measured, and as an indication of a hypertrophic response the expression of atrial natriuretic peptide (ANP) was measured. Stationary cardiomyocytes that have been incubated for 30 min with CM from stretched (for 0-60 min) cardiomyocytes, cardiac fibroblasts, endothelial cells, and VSMCs showed distinct gene expression patterns that were time-dependent and cell-type specific. In stationary cardiomyocytes, CM derived from stretched cardiomyocytes caused decreased c-fos and fra-1 expression by 37 and 20%, respectively (CM30), elevated c-jun expression by 20% (CM45-CM60), and increased ANP expression by 106% (CM45). CM derived from stretched cardiac fibroblasts caused increased c-fos expression by 41% (CM60), no significant changes in c-jun expression, and increased fra-1 and ANP expression by 39 and 20%, respectively (CM45). CM derived from stretched VSMCs induced an initial decrease in c-fos expression followed by an increase of 13% (CM45) and induced increased c-jun, fra-1, and ANP expression by 39, 24, and 22%, respectively. CM15-CM60 derived from stretched endothelial cells caused decreased c-fos, c-jun and fra-1 expression by 20, 25, and 25%, respectively, and increased ANP expression by 18%. Our data indicate that gene expression of cardiomyocytes in stretched myocardium is regulated by mediators released by cardiomyocytes, cardiac fibroblasts, endothelial cells, and VSMCs. This observation emphasizes the involvement of nonmyocyte cells in the early stages of cardiomyocyte hypertrophy caused by cardiac stretch.     

Comparison of angiotensin II (Ang II) effects in the internal anal sphincter (IAS) and lower esophageal sphincter smooth muscles

Studies were performed to compare the actions of Ang II in the internal anal sphincter (IAS) vs. lower esophageal sphincter (LES) smooth muscles in vitro, in opossum and rabbit. Studies also were carried out in isolated smooth muscle cells. In opossum, Ang II produced no discernible effects in the IAS, but did produce a concentration-dependent contraction in the LES. Conversely, in the rabbit, while Ang II caused a modest response in the LES, it caused a significant contraction in the IAS. The contractile responses of Ang II in the opossum LES were mostly resistant to different neurohumoral antagonists but were antagonized by AT1 antagonist losartan. AT2 antagonist PD 123,319, rather than inhibiting, prolonged the contractile action of Ang II. The contractile actions of Ang II in the opossum LES were not modified by the tyrosine kinase inhibitors (genistein and tyrphostin 1 x 10(-6) M) but were partially attenuated by the PKC inhibitor H-7 (1 x 10(-6) M), Ca2+ channel blocker nicardipine (1 x 10(-5) M), Rho kinase inhibitor HA-1077 (1 x 10(-7) M) or p(44/42) MAP kinase inhibitor PD 98059 (5 x 10(-5) M). The combination of HA-1077 and H-7 did not cause an additive attenuation of Ang II responses. Western blot analyses revealed the presence of both AT1 and AT2 receptors. We conclude that Ang lI-induced contraction of sphincteric smooth muscle occurs primarily by the activation of AT1 receptors at the smooth muscle cells and involves multiple pathways, influx of Ca2+, and PKC, Rho kinase and p(44/42) MAP kinase.     

Angiotensin II and atrial natriuretic peptide in the cow oviductal contraction in vitro: direct effect and local secretion of prostaglandins, endothelin-1, and angiotensin II

Angiotensin II (Ang II) and atrial natriuretic peptide (ANP) may be involved in local regulation of the oviductal contraction during the estrous cycle. Thus, the in vitro effects of Ang II and ANP on the secretion and contraction of bovine oviduct during the follicular, postovulatory, and luteal phases were investigated. An in vitro microdialysis system (MDS) was utilized to determine the intraluminal release of prostaglandins (PGs), Ang II, and endothelin-1 (ET-1) from the bovine oviducts as well as to observe the effect of Ang II and ANP on the local secretion of these substances. The basal release of PGs, ET-1, and Ang II was higher (P < 0.05) during the follicular and postovulatory phases than during the luteal phase. Stimulation by infusion of Ang II (10(-6) M) or ANP (10(-7) M) into the MDS was carried out for 4 h between 4 and 8 h of incubation. In the oviducts from the follicular and postovulatory phases, the infusion of ANP increased the release of Ang II, but not of ET-1. Infusion of Ang II stimulated the release of ET-1. Both Ang II and ANP increased PGE(2) and PGF(2alpha) release. In the contraction study, direct administration of Ang II (10(-7) M) or ANP (10(-8) M) into the medium during the follicular and postovulatory phases increased the amplitude of oviductal contraction. In contrast, these substances did not show any effect in the contraction and secretion of oviducts from cows during the midluteal phase. These results indicate that during the periovulatory period, Ang II and ANP stimulate the contractile amplitude of the oviduct in vitro. In addition to their direct action on oviductal contraction, Ang II may activate oviductal secretion of ET-1 and PGs. Likewise, ANP stimulates oviductal secretion of PGs and Ang II. Hence, the overall results suggest the existence of a functional endothelin-angiotensin-ANP system in the bovine oviduct during the periovulatory period, which may regulate the oviductal contraction to ensure maximum efficiency of gamete/embryo transport through the oviduct.     

The effects of thiazolidinediones on vascular smooth muscle cell activation by angiotensin II

Angiotensin II (Ang II) stimulates the activation of extracellular signal-regulated kinase (ERK), a subgroup of the mitogen-activated protein kinase (MAPK) family, in cultured vascular smooth muscle cells (VSMC). This ERK activation was recently shown to be a critical regulatory factor for Ang II-mediated migration and growth. It has been demonstrated that the thiazolidinedione troglitazone (TRO) blocked Ang II-induced DNA synthesis and migration in VSMC. Here we provide evidence for TRO to inhibit Ang II-induced ERK activation which was suggested to constitute the mechanism by which this agent blocks Ang II-induced VSMC growth and migration. We have found that pretreatment with PD98059, which selectively blocks the activity of ERK pathway at the level of MAPK kinase, decreased Ang II-induced AP-1 activation and that TRO is capable of inhibiting Ang II-induced AP-1 activation. On the other hand, the other thiazolidinediones pioglitazone (PIO) and rosiglitazone (ROSI) had little effect on Ang II-induced activation of ERK or AP-1, suggesting the inhibitory effects of TRO on VSMC activation by Ang II be independent of the peroxisome proliferator-activated receptor-gamma (PPARgamma) for which thiazolidinediones are ligands. Ang II-induced ERK activation was inhibited by protein kinase C (PKC)-specific inhibitor GF109203X, while TRO was also able to block PKC activator phorbol 12 myristate 13-acetate (PMA)-induced ERK activation. Accordingly, TRO may inhibit Ang II-induced MAPK activation at least partly by an inhibition of PKC. These results support the assumption that by targeting MAPK activation, TRO may inhibits the critical signaling steps leading to restenosis and atherosclerosis that may result in part from dysregulated VSMC growth and migration induced by Ang II.     

Dimethylarginine dimethylaminohydrolase-1 mediates inhibitory effect of interleukin-10 on angiotensin II-induced hypertensive effects in vascular smooth muscle cells of spontaneously hypertensive rats

In hypertension studies, anti-inflammatory cytokine interleukin-10 (IL-10) has been shown to prevent angiotensin II (Ang II)-induced vasoconstriction and regulate vascular function by down-regulating pro-inflammatory cytokine and superoxide production in vascular cells. However, little is known about the mechanism behind the down-regulatory effect of IL-10 on Ang II-induced hypertensive mediators. In this study, we demonstrated the effects of IL-10 on expression of dimethylarginine dimethylaminohydrolase (DDAH)-1, a regulator of NO bioavailability, as well as the down-regulatory mechanism of action of IL-10 in relation to Ang II-induced hypertensive mediator expression and cell proliferation in vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR). IL-10 increased DDAH-1 but not DDAH-2 expression and increased DDAH activity. Additionally, IL-10 attenuated Ang II-induced DDAH-1 inhibition in SHR VSMCs. Increased DDAH activity due to IL-10 was mediated mainly through Ang II subtype II receptor (AT₂ R) and AMP-activated protein kinase (AMPK) activation. DDAH-1 induced by IL-10 partially mediated the inhibitory action of IL-10 on Ang II-induced 12-lipoxygenase (LO) and endothelin (ET)-1 expression in SHR VSMCs. In addition, the inhibitory effect of IL-10 on proliferation of Ang II-induced VSMCs was mediated partially via DDAH-1 activity. These results suggest that DDAH-1 plays a potentially important role in the anti-hypertensive activity of IL-10 during Ang II-induced hypertension.     

Angiotensin II induces c-fos mRNA in aortic smooth muscle. Role of Ca2+ mobilization and protein kinase C activation

Vasoconstrictors such as angiotensin II (Ang II) play an important role in the pathogenesis of hypertension. These agonists may be responsible for the abnormal vascular smooth muscle cell (VSMC) growth seen in hypertension, either indirectly as a consequence of elevating blood pressure or directly as a result of receptor-mediated effects on VSMC growth. To investigate whether Ang II might directly initiate or modulate some of the "early" genetic programs associated with growth in VSMC, the expression of the proto-oncogene c-fos was studied in cultured rat aortic VSMC. Ang II rapidly induced the accumulation of c-fos mRNA, with maximal levels occurring at approximately 30 min. Induction of c-fos mRNA by Ang II was concentration-dependent, with a maximal response at 100 nM. Ang II induction of c-fos mRNA was blocked by its competitive inhibitor, [sarcosine 1,isoleucine 8]angiotensin II. Induction of c-fos mRNA was not dependent upon Ang II-stimulated intracellular alkalinization or activation of Na+/H+ exchange, but was dependent upon mobilization of intracellular Ca2+ and protein kinase C activation. Epidermal growth factor, a VSMC mitogen, also induced c-fos mRNA in VSMC, but by a mechanism different from that of Ang II. These results demonstrate that the vasoconstrictor hormone Ang II induces in VSMC one of the earliest genes, c-fos, associated with the proliferative response.     

Hydrogen peroxide-induced c-fos expression is mediated by arachidonic acid release: role of protein kinase C.

We found previously that stimulation of c-fos and c-myc mRNA expression are early events in hydrogen peroxide-induced growth in rat aortic smooth muscle (RASM) cells. In the present study, we investigated the role of phospholipase A2 (PLA2) and protein kinase C (PKC) in mediating hydrogen peroxide-induced c-fos mRNA expression in RASM cells. Mepacrine and p-bromophenacylbromide, potent inhibitors of PLA2 activity, blocked hydrogen peroxide-induced c-fos mRNA expression. Arachidonic acid, a product of PLA2 activity, stimulated the expression of c-fos mRNA with a time course similar to that of hydrogen peroxide. PKC down-regulation attenuated both hydrogen peroxide and arachidonic acid-induced c-fos mRNA expression by 50%. Nordihydroguaiaretic acid (a lipoxygenase-cytochrome P450 monooxygenase inhibitor) significantly inhibited both hydrogen peroxide and arachidonic acid-induced c-fos mRNA expression, whereas indomethacin (a cyclooxygenase inhibitor) had no effect. Together, these findings indicate that 1) hydrogen peroxide-induced c-fos mRNA expression is mediated by PLA2-dependent arachidonic acid release, 2) both PKC-dependent and independent mechanisms are involved in hydrogen peroxide-induced expression of c-fos mRNA and 3) arachidonic acid metabolism via the lipoxygenase-cytochrome P450 monooxygenase pathway appears to be required for hydrogen peroxide-induced expression of c-fos mRNA.     

Angiotensin-II-induced enhanced expression of Gi proteins is attenuated by losartan in A10 vascular smooth muscle cells: role of AT1 receptors

We have previously shown that treatment of A10 vascular smooth muscle cells (VSMCs) with angiotensin II (Ang II) enhanced the expression of inhibitory guanine nucleotide regulatory proteins (Gi alpha2 and Gi alpha3). In the present studies, we have investigated the role of type 1 angiotensin receptors (AT1) in the Ang-II-induced enhanced expression of Gi alpha proteins and their functions in A10 SMCs. Ang II enhanced the levels of Gi alpha2 and Gi alpha3 proteins and their mRNA, as determined by Western and Northern blot analysis, respectively; losartan treatment attenuated the enhanced expression of Gi alpha2 and Gi alpha3 proteins and their mRNA in a concentration-dependent manner. In addition, the inhibition of adenylyl cyclase induced by Ang II and des(Glu18,Ser19,Glu20,Leu21,Gly22)ANP(4-23)-NH2 (C-ANP(4-23)), which was attenuated by Ang-II treatment, was partially restored by losartan treatment. Similarly, losartan was also able to restore the Ang-II-induced stimulatory responses of isoproterenol and N-ethylcarboxamide adenosine (NECA) on adenylyl cyclase activity. These results suggest a role for AT1 receptors in Ang-II-evoked increases in Gi alpha protein expression and Gs-mediated stimulation in VSMCs.     

ERK is regulated by sodium-proton exchanger in rat aortic vascular smooth muscle cells

The purposes of this study were to test 1) the relationship between two widely studied mitogenic effector pathways, and 2) the hypothesis that sodium-proton exchanger type 1 (NHE-1) is a regulator of extracellular signal-regulated protein kinase (ERK) activation in rat aortic smooth muscle (RASM) cells. Angiotensin II (Ang II) and 5-hydroxytryptamine (5-HT) stimulated both ERK and NHE-1 activities, with activation of NHE-1 preceding that of ERK. The concentration-response curves for 5-HT and Ang II were superimposable for both processes. Inhibition of NHE-1 with pharmacological agents or by isotonic replacement of sodium in the perfusate with choline or tetramethylammonium greatly attenuated ERK activation by 5-HT or Ang II. Similar maneuvers significantly attenuated 5-HT- or Ang II-mediated activation of MEK and Ras but not transphosphorylation of the epidermal growth factor (EGF) receptor. EGF receptor blockade attenuated ERK activation, but not NHE-1 activation by 5-HT and Ang II, suggesting that the EGF receptor and NHE-1 work in parallel to stimulate ERK activity in RASM cells, converging distal to the EGF receptor but at or above the level of Ras in the Ras-MEK-ERK pathway. Receptor-independent activation of NHE-1 by acute acid loading of RASM cells resulted in the rapid phosphorylation of ERK, which could be blocked by pharmacological inhibitors of NHE-1 or by isotonic replacement of sodium, closely linking the proton transport function of NHE-1 to ERK activation. These studies identify NHE as a new regulator of ERK activity in RASM cells.     

Differential stimulation of growth related metabolism in cultured smooth muscle cells from SHR and WKY rats by combinations of EGF and LDL

We have reported previously that vascular smooth muscle cells from spontaneously hypertensive rats (SHR) were more responsive to epidermal growth factor (EGF) than their normotensive derived Wistar Kyoto (WKY) controls. This differential responsiveness is evident for several cellular processes including activation of S6-kinase, elevation of intracellular pH and stimulation of both phosphoinositide metabolism and DNA synthesis. Quiescent smooth muscle cells exposed to low density lipoprotein (LDL) exhibited a similar differential responsiveness (SHR greater than WKY) in terms of S6-kinase activation, which was time- and dose-dependent (10(-10)-10(7) M), but neither cell type responded appreciably to LDL in terms of a stimulation in [3H]-thymidine incorporation. Exposure of the same cells to EGF and LDL in combination elicited a marked synergistic stimulation in DNA synthesis, the extent of which was greater for SHR than WKY. The sensitivity of both cell types to EGF was increased in the presence of LDL, although cells from hypertensive animals still exhibited their greater (vs. WKY) sensitivity. In both cell types, activation of nuclear protooncogenes c-fos and c-myc by LDL was minimal, whereas oncogene induction by EGF was approximately five-fold greater for SHR-derived cells compared to those from WKY animals. No marked synergistic effect on the time-dependent induction of either entity was observed for cells exposed to EGF and LDL simultaneously, and the response of SHR-cells remained greater than WKY-cells.     

Crosstalk between the signaling pathways triggered by angiotensin II and adenosine in the renal proximal tubules: implications for modulation of Na(+)-ATPase activity

We have previously demonstrated that adenosine (Ado) reverses the stimulatory effect of angiotensin II (Ang II) on Na(+)-ATPase activity via the A(2A) receptor. In this work, the molecular mechanism involved in Ado-induced shutdown in the signaling pathway triggered by 10(-8)M Ang II was investigated. It was observed that: (1) both 10(-12)M PMA (a PKC activator) and 5x10(-8)M U73122 (an inhibitor of PI-PLCbeta) prevent the reversion effect induced by 10(-6)M Ado (only observed in the presence of 10(-6)M DPCPX (an A(1) receptor antagonist)) on Ang II-stimulated Na(+)-ATPase and PKC activities; (2) Ang II-stimulated PKC activity was reversed by 10(-6)M forskolin (an adenylyl cyclase activator) or 10(-8)M PKA inhibitory peptide and 10(-8)M DMPX (an A(2) receptor-selective antagonist). Considering that PMA prevents the inhibitory effect of Ado on Ang II-stimulated Na(+)-ATPase and PKC activities, it is likely that the PMA-induced effect, i.e. PKC activation, is downstream of the target for Ado-induced reversion of Ang II stimulation of Na(+)-ATPase activity. We investigated the hypothesis that PI-PLCbeta could be the target for Ado-induced PKA activation. Our data demonstrate that Ang II-stimulated PI-PLCbeta activity was reversed by Ado or 10(-7)M cAMP; the reversibility of the Ado-induced effect was prevented by either DMPX or PKA inhibitory peptide. These data demonstrate that Ado-induced PKA activation reduces Ang II-induced stimulation of PI-PLCbeta.