心房钠尿肽对血管紧张素Ⅱ促血管平滑肌细胞增殖和c-fos原癌基因表达的影响

本工作应用细胞培养、~3氢·胸腺嘧啶核苷参入和斑点杂交的方法,观察到血管紧张素Ⅱ(AGTⅡ)明显促进培养的自发性高血压大鼠的主动脉平滑肌细胞(VSMC)的增殖和c-fos原癌基因的表达;该效应可显著为心房钠尿肽(ANP)所抑制。     

心房钠尿肽以血管紧张素及禁水引起大鼠饮水行为的影响

Atrial natriuretic peptide (ANP) present in the brain has been reported to have profound effects on water and salt metabolism. This study was designed to observe the effect of intracerebroventricular (ICV) injection of ANP on drinking behavior of rats, induced by centrally administered angiotensin II (Ang II) and 24-hours water deprivation, by using a T-maze to measure the speed they ran in a runway for water rewards. In 24-hours water deprived rats ICV injection of ANP resulted in a significant decrease of either running speed or water intake. Drinking behavior induced by ICV injection of Ang II in normally hydrated rats was also significantly inhibited by a prior injection of ANP. These findings suggest that ANP in the brain plays an important role in the central control of drinking behavior.     

心房钠尿肽对血管紧张素及禁水引起大鼠饮水行为的影响

利用T-迷宫,以S-D大鼠在直跑道内的钦水跑速及大鼠在饲养笼内的饮水量作为指标,观察ANP对AugⅡ及禁水引起大鼠饮水行为的影响。结果显示,通过脑室导管将100ng AngⅡ注入第三脑室后,立即引起已饱水大鼠快速奔跑的饮水行为。iCV.注射2μgANP3min后,再注射AngⅡ,大鼠即失去快速奔跑饮水行为。禁水24h后,大鼠放入迷宫内亦出现快速奔跑饮水行为,icv.注射ANP 3min及20min时,跑速明显减慢。观察同组实验动物的饮水量变化,与跑速比较,两者变化方向和程度基本一致。本实验证明,ANP可以使AngⅡ或禁水致渴的大鼠饮水动机减低,跑速减慢,饮水量减少。由此可见,ANP不仅具有利钠、利尿、降低血压作用,还可以影响神经性致渴机制,抑制饮水行为,是机体维持体液平衡的重要调节物。     

血管紧张素Ⅱ、心房钠尿肽和血管升压素对大鼠脑片穹窿下器神经元活动的影响

在31个脑片观察了血管紧张素Ⅱ(AGⅡ)、心房钠尿肽(ANP)和血管升压素(AVP)三种多肽对87个穹窿下器(SFO)神经元单位电活动的影响。脑片灌流AGⅡ(10~(-7)mol/L,3min)后,40/55个单应(72.73％)放电频率明显增加,3/55个单位(5.45％)放电频率降低,12/55个单位(21.82％)无明显反应。AGⅡ对SFO放电单位的兴奋作用可被AGⅡ受体阻断剂saralasin(10~(-6)mol/L)完全阻断。脑片灌流心房肽Ⅲ(APⅢ)(10~(-7)mol/L,3min)后,7/17个单位(41.18％)放电频率明显降低,2/17个单位(11.76％)放电频率增加,8/17个单位(47.06％)无明显反应。脑片灌流AVP(10~(-7)mol/L,3min)后,8/15个单位(53.33％)放电频率明显增加,3/15个单位(20.00％)放电频率降低,4/15个单位(26.67％)无明显反应。在观察这三种多肽对同一SFO神经元的作用时,1个单位对AGⅡ和AVP均产生兴奋反应;3个单位对AGⅡ呈兴奋和被APⅢ所抑制;1个单位对AVP呈兴奋,而对APⅢ为抑制,未见到既对AGⅡ和AVP呈兴奋,又为APⅢ所抑制的单位。结果提示:AGⅡ,ANP和AVP三种多肽都能影响SFO神经元的自发电活动,SFO可能也是三者调节机体水盐平衡和血压的中枢部位之一。     

血管紧张素Ⅱ、心房钠尿肽Ⅲ和血管升压素对大鼠脑片室旁核神经元放电活动的影响

在28个脑片观察了血管紧张素Ⅱ(AGⅡ)、心房钠尿肽Ⅲ(ANPⅢ)和血管升压素(AVP)三种多肽对101个下丘脑室旁核(PVN)神经元单位电活动的影响。脑片灌流AGⅡ(10~(-7)mol/L,3 min)后,28/50个单位(56.0％)放电频率明显增加,5/50个单位(10.0％)放电频率降低,17/50个单位(34.0％)无明显反应。AGⅡ对PVN放电单位的兴奋和抑制作用均可为AGⅡ受体阻断剂saralasin(10~(-6)mol/L)所阻断。脑片灌流ANPⅢ(10~(-7)mol/L,3 min)后,16/26个单位(61.5％)放电频率明显降低,1/26个单位(3.9％)放电频率增加,9/26个单位(34.6％)无明显反应。脑片灌流AVP,(10~(-7)mol/L,3min)后,19/25个单位(76.0％)放电频率明显增加,1/25个单位(4.0％)放电频率降低,5/25个单位(20,0％)无明显反应。在观察这三种多肽对同一PVN神经元的作用时,4个单位对AGⅡ和AVP均产生兴奋反应;2个单位对AGⅡ呈兴奋和被ANPⅢ所抑制;7个单位对AVP呈兴奋,而对ANPⅢ为抑制,未见到既对AGⅡ和AVP呈兴奋,又为ANPⅢ所抑制的单位。结果提示:AGⅡ,ANP和AVP三种多肽都能影响PVN神经元的自发电活动,PVN可能是神经内分泌和植物性功能调节的中枢整合部位之一。     

粉防己碱对血管紧张素II促进心肌成纤维细胞增殖的抑制作用

血管紧张素（ＡＮＧ）Ⅱ对新生大鼠无血清培养第二代心肌成纤维细胞（ＦＢ）具有剂量依赖性的促进增殖,ＤＮＡ和ＲＮＡ以及胶原合成的作用。ＡＮＧⅡ１０＾－７ｍｏｌ／Ｌ时浓度时,ＦＢ的（＾３Ｈ）胸腺嘧啶核苷（Ｔｈｙｍｉｄｉｎｅ）,（＾３Ｈ）尿嘧啶核苷（Ｕｒｉｄｉｍｅ）（＾３Ｈ）脯氨酸（Ｐｒｏｌｉｎｅ）掺入较对照组分别增加了１６７％,５８％,１７４％,此刺激作用可被特异性ＡＮＧⅡ受体拮抗剂阻断,粉防己碱预处理     

吗啡和血管紧张素II对大鼠脑突触小体Ca^2＋摄取的拮抗效应

Behavioral observations have repeatedly shown that the analgesic effect of morphine can be antagonized by intracerebroventricular injection of angiotensin I (A I), although mechanisms underlying the action were obscure. Since a prevention of Ca2+ uptake into the nerve terminals was considered as one of the mechanisms for morphine analgesia, we examined the effect of A I and morphine on the 45Ca uptake by rat brain synaptosomal preparations. Morphine of 10(-8)-10(-6) mol/L produced a dose-related suppression on synaptosomal 45Ca uptake, which was completely reversed by the opioid antagonist naloxone of 10(-6) mol/L. A I of 10(-8)-10(-6) mol/L, on the contrary, enhanced 45Ca uptake. This effect was totally abolished by saralasin, a A I antagonist, at 10(-6) mol/L. When synaptosomal preparations were incubated in a mixture of morphine (10(-6) mol/L) and A I (10(-8)-10(-6) mol/L), the effect of morphine was almost completely reversed. The results suggest that the distinct effect of A I may account for, at least in part, the antagonistic effect of A I on morphine analgesia.     

Endothelin stimulates c-fos and c-myc expression and proliferation of vascular smooth muscle cells

Recently, a potent vasoconstrictor peptide, endothelin (EDT), was isolated from vascular endothelial cells. We examined its effect on rat vascular smooth muscle cells (VSMCs). EDT induced the elevation of intracellular calcium, which was dependent on extracellular calcium and inhibited by a calcium-channel antagonist in a competitive manner. EDT caused a rapid and transient increase in the c-fos and c-myc mRNA levels and stimulated the DNA synthesis of VSMCs in a dose-dependent manner. This effect of EDT on the proliferation of VSMCs might be related to the development of atherosclerosis.     

Endothelium-independent conversion of angiotensin I by vascular smooth muscle cells

The conversion of angiotensin I (AT-I) to angiotensin II (AT-II) by angiotensin I-converting enzyme (ACE) is a key step in the action of angiotensins. ACE is constitutively expressed in endothelial cells, but can also be detected at low levels in smooth muscle cells (SMC). Furthermore, in rats the ACE activity can be induced in SMC in vivo by experimental hypertension or vascular injury and in vivo by corticoid treatment. This study was therefore undertaken to evaluate the conversion of AT-I and its subsequent effects in SMC in basal conditions and after stimulation by dexamethasone. Using rat and human SMC, showed that dexamethasone induced ACE expression and that this enzyme was functional, leading to AT-II-dependent intracellular signaling. A fourfold increase in phospholipase C activity in response to AT-I was observed in dexamethasone-activated SMC compared with quiescent SMC. This effect of dexamethasone on signal transduction is dependent on ACE activity, whereas AT-II receptor parameters remain unchanged. The action of AT-I was blocked by an AT1 receptor antagonist, suggesting that it was mediated by AT-II. Similarly, dexamethasone-induced ACE expression was present in human SMC, and calcium signaling was mobilized in response to AT-I in activated human cells. Experiments performed with cocultures of endothelial cells and SMC in a Transwell system showed that the response to AT-I was limited to the compartment where AT-I was localized, suggesting that AT-I does not pass through the endothelial cell barrier to interact with underlying SMC. Our data suggest that in rat, as in human SMC, the conversion of AT-I into AT-II and the signal transduction in response to AT-I are ACE expression-dependent. In addition, the present findings show that this SMC response to AT-I is endothelium-independent, supporting the idea of a local generation of AT-II in the vascular wall.     

Resveratrol attenuates angiotensin II-induced senescence of vascular smooth muscle cells

Resveratrol (3,5,4'-trihydroxystilbene), a polyphenol abundant in red wine, is known to extend the life span of diverse species. On the contrary, it was reported that angiotensin (Ang) II enhances senescence of vascular smooth muscle cells (VSMCs). We, therefore, examined whether resveratrol attenuates Ang II-induced senescence of VSMC. Senescence-associated β-galactosidase (SA β-gal) assay showed that Ang II induced senescence of VSMC. The Ang II-induced senescence was inhibited by losartan, an Ang II type 1 receptor (AT1R) antagonist but not by PD123319, Ang II type 2 receptor antagonist, indicating that AT1R is responsible for the induction of senescence. Resveratrol suppressed Ang II-induced senescence of VSMC in a dose-dependent manner. In addition, resveratrol suppressed Ang II-induced induction of p53 and its downstream target gene p21, both of which play an important role in the induction of senescence. Resveratrol suppressed senescence of VSMC possibly through inhibition of AT1R-dependent induction of p53/p21. Suppression of p53 induction may be involved in the longevity by resveratrol.     

Functional angiotensin II receptors in cultured vascular smooth muscle cells

To study cellular mechanisms influencing vascular reactivity, vascular smooth muscle cells (VSMC) were obtained by enzymatic dissociation of the rat mesenteric artery, a highly reactive, resistance-type blood vessel, and established in primary culture. Cellular binding sites for the vasoconstrictor hormone angiotensin II (AII) were identified and characterized using the radioligand 125I-angiotensin II. Freshly isolated VSMC, and VSMC maintained in primary culture for up to 3 wk, exhibited rapid, saturable, and specific 125I-AII binding similar to that seen with homogenates of the intact rat mesenteric artery. In 7-d primary cultures, Scatchard analysis indicated a single class of high-affinity binding sites with an equilibrium dissociation constant (Kd) of 2.8 +/- 0.2 nM and a total binding capacity of 81.5 +/- 5.0 fmol/mg protein (equivalent to 4.5 x 10(4) sites per cell). Angiotensin analogues and antagonists inhibited 125I-AII binding to cultured VSMC in a potency series similar to that observed for the vascular AII receptor in vivo. Nanomolar concentrations of native AII elicited a rapid, reversible, contractile response, in a variable proportion of cells, that was inhibited by pretreatment with the competitive antagonist Sar1,Ile8-AII. Transmission electron microscopy showed an apparent loss of thick (12-18 nm Diam) myofilaments and increased synthetic activity, but these manifestations of phenotypic modulation were not correlated with loss of 125I-AII binding sites or hormonal responsiveness. Primary cultures of enzymatically dissociated rat mesenteric artery VSMC thus may provide a useful in vitro system to study cellular mechanisms involved in receptor activation-response coupling, receptor regulation, and the maintenance of differentiation in vascular smooth muscle.     

Cross-talk between aldosterone and angiotensin signaling in vascular smooth muscle cells

In hypertension or other forms of cardiovascular disease, the chronic activation of the renin-angiotensin-aldosterone system (RAAS) leads to dysfunction of the vasculature, including, increased vascular tone, inflammation, fibrosis and thrombosis. Cross-talk between the main mediators of the RAAS, aldosterone and angiotensin (Ang) II, participates in the development of this vascular dysfunction. Recent studies have highlighted the molecular mechanisms supporting this cross-talk in vascular smooth muscle cells (VSMCs). Some of the signaling pathways activated by the Ang II type 1 receptor (AT₁R) are dependent on the mineralocorticoid receptor (MR) and vice versa. VSMC signaling pathways involved in migration and growth are under the control of cross-talk between aldosterone and Ang II. A synergistic mechanism leads to potentiation of signaling pathways activated by each agent. The genomic and non-genomic mechanisms activated by aldosterone cooperate with Ang II to regulate vascular tone and gene expression of pro-inflammatory and pro-fibrotic molecules. This cross-talk is dependent on the non-receptor tyrosine kinase c-Src, and on receptor tyrosine kinases, EGFR and PDGFR, and leads to activation of MAP kinases and growth, migration and inflammatory effects. These new findings will contribute to development of better treatments for conditions in which the RAAS is excessively activated.     

Rho expression and activation in vascular smooth muscle cells

Phenotypic modulation of smooth muscle cells (SMC) involves dramatic changes in expression and organization of contractile and cytoskeletal proteins, but little is known of how this process is regulated. The present study used a cell culture model to investigate the possible involvement of RhoA, a known regulator of the actin cytoskeleton. In rabbit aortic SMC seeded into primary culture at moderate density, Rho activation was high at two functionally distinct time-points, first as cells modulated to the "synthetic" phenotype, and again upon confluence and return to the "contractile" phenotype. Rho expression increased with time, such that maximal expression occurred upon return to the contractile state. Transient transfection of synthetic state cells with constitutively active RhoA (Val14RhoA) caused a reduction in cell size and reorganization of cytoskeletal proteins to resemble that of the contractile phenotype. Actin and myosin filaments were tightly packed and highly organised while vimentin localised to the perinuclear region; focal adhesions were enlarged and concentrated at the cell periphery. Conversely, inhibition of endogenous Rho by C3 exoenzyme resulted in complete loss of contractile filaments without affecting vimentin distribution; focal adhesions were reduced in size and number. Treatment of synthetic state SMC with known regulators of SMC phenotype, heparin and thrombin, caused a modest increase in Rho activation. Long-term confluence and serum deprivation induced cells to return to a more contractile phenotype and this was augmented by heparin and thrombin. The results implicate RhoA for a role in regulating SMC phenotype and further show that activation of Rho by heparin and thrombin correlates with the ability of these factors to promote the contractile phenotype.     

Role of integrins in angiotensin II-induced proliferation of vascular smooth muscle cells

Angiotensin II (AII) binds to G protein-coupled receptor AT(1) and stimulates extracellular signal-regulated kinase (ERK), leading to vascular smooth muscle cells (VSMC) proliferation. Proliferation of mammalian cells is tightly regulated by adhesion to the extracellular matrix, which occurs via integrins. To study cross-talk between G protein-coupled receptor- and integrin-induced signaling, we hypothesized that integrins are involved in AII-induced proliferation of VSMC. Using Oligo GEArray and quantitative RT-PCR, we established that messages for α(1)-, α(5)-, α(V)-, and β(1)-integrins are predominant in VSMC. VSMC were cultured on plastic dishes or on plates coated with either extracellular matrix or poly-d-lysine (which promotes electrostatic cell attachment independent of integrins). AII significantly induced proliferation in VSMC grown on collagen I or fibronectin, and this effect was blocked by the ERK inhibitor PD-98059, suggesting that AII-induced proliferation requires ERK activity. VSMC grown on collagen I or on fibronectin demonstrated approximately three- and approximately sixfold increases in ERK phosphorylation after stimulation with 100 nM AII, respectively, whereas VSMC grown on poly-d-lysine demonstrated no significant ERK activation, supporting the importance of integrin-mediated adhesion. AII-induced ERK activation was reduced by >65% by synthetic peptides containing an RGD (arginine-glycine-aspartic acid) sequence that inhibit α(5)β(1)-integrin, and by ～60% by the KTS (lysine-threonine-serine)-containing peptides specific for integrin-α(1)β(1). Furthermore, neutralizing antibody against β(1)-integrin and silencing of α(1), α(5), and β(1) expression by transfecting VSMC with short interfering RNAs resulted in decreased AII-induced ERK activation. This work demonstrates roles for specific integrins (most likely α(5)β(1) and α(1)β(1)) in AII-induced proliferation of VSMC.