Angiotensin II-induced ET and PGI2 release in rat aortic endothelial cells is mediated by PKC.

Angiotensin II (Ang II) has been shown to stimulate the release of immunoreactive endothelin (ET) from cultured bovine ECs. Also, Ang II activates phospholipase A2 (PLA2) in various tissues, resulting in the release of arachidonic acid and formation of prostaglandins. We used rat aortic endothelial cells to investigate the role of protein kinase C (PKC) in Ang II-induced release of both ET and prostacyclin (PGI2). The amount of ET and PGI2 produced were determined by radioimmunoassay. Ang II-induced the release of both ET and PGI2. Pretreatment with 10(-6) M of any one of the PKC inhibitors: 1-(5-isoquinolinesulfonyl) piperazine(CL), staurosporine, 1-(5-isoquinolinesulfonylmethyl)piperazine(H7), and calphostin C blocked AII-induced release of both ET and PGI2. In rat aortic endothelial cells that were treated with either AII or PDBu, PKC enzyme assay showed PKC was translocated from the cytosol to the membrane which indicates activation. This suggests that PKC mediates AII-induced ET and PGI2 release. In summary, AII activates PKC which inhibits rat aortic endothelial cells ET and PGI2 formation, and this inhibition can be overcome by pretreatment with PKC inhibitors.     

Angiotensin II-induced fluid phase endocytosis in human cerebromicrovascular endothelial cells is regulated by the inositol-phosphate signaling pathway

The involvement of the early signaling messengers, inositol tris-phosphate (IP₃), intracellular calcium, [Ca²⁺]_i, and protein kinase C (PKC), in angiotensin II (AII)-induced fluid phase endocytosis was investigated in human brain capillary and microvascular endothelial cells (HCEC). AII (0.01–10 μM) stimulated the uptake of Lucifer yellow CH, an inert dye used as a marker for fluid phase endocytosis, in HCEC by 50–230%. AII also triggered a fast accumulation of IP₃ and a rapid increase in [Ca²⁺]_i in cells loaded with the Ca²⁺-responsive fluorescent dye fura-2. The prompt AII-induced [Ca²⁺]_i spike was not affected by incubating HCEC in Ca²⁺-free medium containing 2 mM EGTA or by pretreating the cultures with the Ca²⁺ channel blockers, methoxyverapamil (D600; 50 μM), nickel (1 mM), or lanthanum (1 mM), suggesting that the activation of AII receptors on HCEC triggers the release of Ca²⁺ from intracellular stores. The AII-triggered increases in IP₃, [Ca²⁺]_i, and Lucifer yellow uptake were inhibited by the nonselective AII receptor antagonist, Sar¹, Val⁵, Ala⁸-AII (SVA-AII), and by the phospholipase C (PLC) inhibitors, neomycin and U-73122. By contrast, the protein kinase C (PKC) inhibitors, staurosporine and calphostin C, failed to affect any of these AII-induced events. This study demonstrates that increased fluid phase endocytotosis induced by AII in human brain capillary endothelium, an event thought to be linked to the observed increases in blood-brain barrier permeability in acute hypertension, is likely dependent on PLC-mediated changes in [Ca²⁺]_i and independent of PKC. © 1996 Wiley-Liss, Inc.     

Angiotensin II-induced Akt activation is mediated by metabolites of arachidonic acid generated by CaMKII-stimulated Ca2(+)-dependent phospholipase A2

Angiotensin II (ANG II) promotes vascular smooth muscle cell (VSMC) growth, stimulates Ca(2+)-calmodulin (CaM)-dependent kinase II (CaMKII), and activates cytosolic Ca(2+)-dependent phospholipase A2 (cPLA2), which releases arachidonic acid (AA). ANG II also generates H2O2 and activates Akt, which have been implicated in ANG II actions in VSMC. This study was conducted to investigate the relationship of these signaling molecules to Akt activation in rat aortic VSMC. ANG II increased Akt activity, as measured by its phosphorylation at serine-473. ANG II (200 nM)-induced Akt phosphorylation was decreased by extracellular Ca2+ depletion and calcium chelator EGTA and inhibitors of CaM [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide] and CaMKII [(2-[N-(2-hydroxyethyl)]-N-(4-me-thoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzyl-amine)]. cPLA2 inhibitor pyrrolidine-1, antisense oligonucleotide, and retroviral small interfering RNA also attenuated ANG II-induced Akt phosphorylation. AA increased Akt phosphorylation, and AA metabolism inhibitor 5,8,11,14-eicosatetraynoic acid (ETYA) blocked ANG II- and AA-induced Akt phosphorylation (199.03 +/- 27.91% with ANG II and 110.18 +/- 22.40% with ETYA + ANG II; 405.00 +/- 86.22% with AA and 153.97 +/- 63.26% with ETYA + AA). Inhibitors of lipoxygenase (cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate) and cytochrome P-450 (ketoconazole and 17-octadecynoic acid), but not cyclooxygenase (indomethacin), attenuated ANG II- and AA-induced Akt phosphorylation. Furthermore, 5(S)-, 12(S)-, 15(S)-, and 20-hydroxyeicosatetraenoic acids and 5,6-, 11,12-, and 14,15-epoxyeicosatrienoic acids increased Akt phosphorylation. Catalase inhibited ANG II-increased H2O2 production but not Akt phosphorylation. Oleic acid, which also increased H2O2 production, did not cause Akt phosphorylation. These data suggest that ANG II-induced Akt activation in VSMC is mediated by AA metabolites, most likely generated via lipoxygenase and cytochrome P-450 consequent to AA released by CaMKII-activated cPLA2 and independent of H2O2 production.     

Angiotensin II-induced process of angiogenesis is mediated by spleen tyrosine kinase via VEGF receptor-1 phosphorylation

Spleen tyrosine kinase (Syk), expressed in endothelial cells, has been implicated in migration and proliferation and in vasculogenesis. This study was conducted to determine the contribution of Syk and the underlying mechanism to the angiogenic effect of ANG II and VEGF. Angiogenesis was determined by tube formation from the endothelial cell line EA.hy926 (EA) and human umbilical vein endothelial cells (HUVECs) and microvessel sprouting in rat aortic rings. ANG II (10 nM), EGF (30 ng/ml), and VEGF (50 ng/ml) stimulated EA cells and HUVECs to form tubular networks and increased aortic sprouting; these effects were blocked by VEGF receptor-1 and Flt-1 antibody (Flt-1/Fc) but not by the VEGF receptor-2 (Flk-1) antagonist SU-1498. ANG II increased the phosphorylation of Flt-1 but not Flk-1, whereas VEGF increased the phosphorylation of both receptors in EA cells and HUVECs. VEGF expression elicited by ANG II was not altered by Flt-1/Fc or SU-1498. EGF stimulated tube formation from EA cells and HUVECs and Flt-1 phosphorylation and aortic sprouting, which were blocked by the EGF receptor antagonist AG-1478 and Flt-1/Fc but not by SU-1498. ANG II-, EGF-, and VEGF-induced tube formation and aortic sprouting were attenuated by the Syk inhibitor piceatannol and by Syk short hairpin interfering (sh)RNA and small interfering RNA, respectively. ANG II, EGF, and VEGF increased Syk phosphorylation, which was inhibited by piceatannol and Syk shRNA in EA cells and HUVECs. Neither piceatannol nor Syk shRNA altered ANG II-, EGF-, or VEGF-induced phosphorylation of Flt-1. These data suggest that ANG II stimulates angiogenesis via transactivation of the EGF receptor, which promotes the phosphorylation of Flt-1 and activation of Syk independent of VEGF expression.     

Angiotensin II-induced smooth muscle cell migration is mediated by LDL receptor-related protein 1 via regulation of matrix metalloproteinase 2 expression

Angiotensin II (Ang II), one of the main vasoactive hormones of the renin-angiotensin system, contributes to the development and progression of atherosclerosis by inducing vascular smooth muscle cells (VSMCs) migration. Although previous studies have shown that Ang II upregulates low density lipoprotein receptor-related protein 1 (LRP1) expression in VSMCs and increases VSMCs migration, the role of LRP1 in Ang II-induced VSMCs migration remains unclear. Here, we reveal a novel mechanism by which LRP1 induces the expression of matrix metalloproteinase 2 (MMP2) and thereby promotes the migration of rat aortic SMCs (RAoSMCs). Knockdown of LRP1 expression greatly decreased RAoSMCs migration, which was rescued by forced expression of a functional LRP1 minireceptor, suggesting that LRP1 is a key regulator of Ang II-induced RAoSMCs migration. Inhibition of ligand binding to LRP1 by the specific antagonist receptor-associated protein (RAP) also led to reduced RAoSMCs migration. Because MMPs play critical roles in RAoSMCs migration, we examined the expression of several MMPs and found that the expression of functional MMP2 was selectively increased by Ang II treatment and decreased in LRP1-knockdown RAoSMCs. More interestingly, reduced MMP2 expression in LRP1-knockdown cells was completely rescued by exogenous expression of mLRP4, suggesting that MMP2 is a downstream regulator of LRP1 in Ang II-induced RAoSMCs migration. Together, our data strongly suggest that LRP1 promotes the migration of RAoSMCs by regulating the expression and function of MMP2.     

Bartonella-induced endothelial cell proliferation is mediated by release of calcium from intracellular stores

The facultative intracellular bacterium Bartonella henselae induces unique angiogenic lesions in immunocompromised hosts. To determine the role of intracellular calcium pools in B. henselae-induced endothelial cell proliferation, we generated B. henselae-conditioned medium (BCM) and tested the ability of these cell-free proteins to induce human umbilical vein endothelial cell (HUVEC) proliferation, CXCL8 production, and intracellular Ca2+ signals. HUVECs incubated with BCM for 3 days had higher cell numbers than controls. In addition, HUVECs produced increased amounts of CXCL8 in response to BCM when compared to medium controls. When BCM was added to HUVECs and the intracellular Ca2+ response measured with the calcium-sensitive dye fura-2/AM, a Ca2+ rise was demonstrated. It was determined that this Ca2+ rise originated from intracellular Ca2+ stores through the use of the Ca2+ ATPase inhibitor thapsigargin. Further, it was demonstrated that BCM enhanced CXCL8 production and HUVEC proliferation in a Ca2+-dependent manner. Conditioned medium from B. henselae causes an intracellular Ca2+ rise in HUVECs, which is involved in B. henselae-induced HUVEC proliferation and CXCL8 production. These results implicate intracellular Ca2+ pools in B. henselae-induced angiogenesis and may lead to increased understanding of the mechanisms of pathogen-induced angiogenesis.     

Angiotensin II-induced mononuclear leukocyte interactions with arteriolar and venular endothelium are mediated by the release of different CC chemokines

Angiotensin II (Ang-II) is associated with atherogenesis and arterial subendothelial mononuclear leukocyte infiltration. We have demonstrated that Ang-II causes the initial attachment of mononuclear cells to the arteriolar endothelium. We now report on the contribution of CC chemokines to this response. Intraperitoneal administration of 1 nM Ang-II induced MCP-1, RANTES, and MIP-1alpha generation, maximal at 4 h, followed by mononuclear leukocyte recruitment at 8 and 24 h. Using intravital microscopy within the rat mesenteric microcirculation 4 h after exposure to 1 nM Ang-II, arteriolar mononuclear cell adhesion was 80-90% inhibited by pretreatment with Met-RANTES, a CCR1 and CCR5 antagonist, or an anti-MCP-1 antiserum, without affecting the increased endothelial expression of P-selectin and VCAM-1. Conversely, leukocyte interactions with the venular endothelium, although inhibited by Met-RANTES, were little affected by the anti-MCP-1. Using rat whole blood in vitro, Ang-II (100 nM) induced the expression of monocyte CD11b that was inhibited by Met-RANTES but not by anti-MCP-1. Stimulation of human endothelial cells (human umbilical arterial endothelial cells and HUVECs) with 1-1000 nM Ang-II, predominantly acting at its AT(1) receptor, induced the release of MCP-1 within 1 h, RANTES within 4 h, and MCP-3 within 24 h. Eotaxin-3, a natural CCR2 antagonist, was released within 1 h and may delay mononuclear cell responses to MCP-1. Therefore, Ang-II-induced mononuclear leukocyte recruitment at arterioles and venules is mediated by the production of different CC chemokines. Thus, Ang-II may be a key molecule in the initial attachment of mononuclear cells to the arterial endothelium in cardiovascular disease states where this event is a characteristic feature.     

Angiotensin II-induced stimulation of p21-activated kinase and c-Jun NH2-terminal kinase is mediated by Rac1 and Nck

p21-activated kinase (PAK) has been shown to be an upstream mediator of JNK in angiotensin II (AngII) signaling. Little is known regarding other signaling molecules involved in activation of PAK and JNK by AngII. Rho family GTPases Rac and Cdc42 have been shown to enhance PAK activity by binding to p21-binding domain of PAK (PAK-PBD). In vascular smooth muscle cells (VSMC) AngII stimulated Rac1 binding to GST-PAK-PBD fusion protein. Pretreatment of VSMC by genistein inhibited AngII-induced Rac1 activation, whereas Src inhibitor PP1 had no effect. Inhibition of protein kinase C by phorbol 12,13-dibutyrate pretreatment also decreased AngII-mediated activation of Rac1. The adaptor molecule Nck has been shown previously to mediate PAK activation by facilitating translocation of PAK to the plasma membrane. In VSMC AngII stimulated translocation of Nck and PAK to the membrane fraction. Overexpression of dominant-negative Nck in Chinese hamster ovary (CHO) cells, stably expressing the AngII type I receptor (CHO-AT1), inhibited both PAK and JNK activation by AngII, whereas it did not affect ERK1/2. Finally, dominant-negative Nck inhibited AngII-induced DNA synthesis in CHO-AT1 cells. Our data provide evidence for Rac1 and Nck as upstream mediators of PAK and JNK in AngII signaling and implicate JNK in AngII-induced growth responses.     

Angiotensin II-induced transcriptional activation of the cyclin D1 gene is mediated by Egr-1 in CHO-AT(1A) cells

Cyclin D1 protein expression is regulated by mitogenic stimuli and is a critical component in the regulation of G(1) to S phase progression of the cell cycle. Angiotensin II (Ang II) binds to specific G protein-coupled receptors and is mitogenic in Chinese hamster ovary cells stably expressing the rat vascular Ang II type 1A receptor (CHO-AT(1A)). We recently reported that in these cells, Ang II induced cyclin D1 promoter activation and protein expression in a phosphatidylinositol 3-kinase (PI3K)-, SHP-2-, and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK)-dependent manner (Guillemot, L., Levy, A., Zhao, Z. J., Béréziat, G., and Rothhut, B. (2000) J. Biol. Chem. 275, 26349-26358). In this report, transfection studies using a series of deleted cyclin D1 promoters revealed that two regions between base pairs (bp) -136 and -96 and between bp -29 and +139 of the human cyclin D1 promoter contained regulatory elements required for Ang II-mediated induction. Mutational analysis in the -136 to -96 bp region provided evidence that a Sp1/early growth response protein (Egr) motif was responsible for cyclin D1 promoter activation by Ang II. Gel shift and supershift studies showed that Ang II-induced Egr-1 binding involved de novo protein synthesis and correlated well with Egr-1 promoter activation. Both U0126 (an inhibitor of the MAPK/ERK kinase MEK) and wortmannin (an inhibitor of PI3K) abrogated Egr-1 endogenous expression and Egr-1 promoter activity induced by Ang II. Moreover, using a co-transfection approach, we found that Ang II induction of Egr-1 promoter activity was blocked by dominant-negative p21(ras), Raf-1, and tyrosine phosphatase SHP-2 mutants. Identical effects were obtained when inhibitors and dominant negative mutants were tested on the -29 to +139 bp region of the cyclin D1 promoter. Taken together, these findings demonstrate that Ang II-induced cyclin D1 up-regulation is mediated by the activation and specific interaction of Egr-1 with the -136 to -96 bp region of the cyclin D1 promoter and by activation of the -29 to +139 bp region, both in a p21(ras)/Raf-1/MEK/ERK-dependent manner, and also involves PI3K and SHP-2.     

Cytotoxicity of IFN-gamma and TNF-alpha for vascular endothelial cell is mediated by nitric oxide

Endothelial cell injury is a critical event in tissue damage accompanying inflammation, in which both inflammatory cytokines and reactive oxygen species may play pivotal roles, although the exact mechanism has not yet been clarified. We found that combined stimulation with interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) induced both cytotoxicity to murine vascular endothelial cell line F-2 and an increase in nitric oxide (NO). Therefore, in the present study, the implication of NO in cytotoxicity was examined. A potent iNOS-specific inhibitor ONO-1714 completely blocked both cytokine-induced cytotoxicity and NO production. NO scavengers such as carboxy-PTIO and hemoglobin blocked cytotoxicity. Moreover, exogenous NO from NOC 18 also caused cytotoxicity. These results together demonstrated that cytotoxicity of IFN-gamma and TNF-alpha for endothelial cell F-2 was mediated by NO, suggesting a pathogenic role of cytokine-induced NO production in endothelial damage under inflammatory conditions.     

Angiotensin II-induced JNK activation is mediated by NAD(P)H oxidase in isolated rat pancreatic islets

Angiotensin II (AII), the active component of the renin angiotensin system (RAS), plays a vital role in the regulation of physiological processes of the cardiovascular system, but also has autocrine and paracrine actions in various tissues and organs. Many studies have shown the existence of RAS in the pancreas of humans and rodents. The aim of this study was to evaluate potential signaling pathways mediated by AII in isolated pancreatic islets of rats. Phosphorylation of MAPKs (ERK1/2, JNK and p38MAPK), and the interaction between proteins JAK/STAT were evaluated. AII increased JAK2/STAT1 (42%) and JAK2/STAT3 (100%) interaction without altering the total content of JAK2. Analyzing the activation of MAPKs (ERK1/2, JNK and p38MAPK) in isolated pancreatic islets from rats we observed that AII rapidly (3 min) promoted a significant increase in the phosphorylation degree of these proteins after incubation with the hormone. Curiously JNK protein phosphorylation was inhibited by DPI, suggesting the involvement of NAD(P)H oxidase in the activation of protein.     

Angiotensin II-induced skeletal muscle insulin resistance mediated by NF-kappaB activation via NADPH oxidase

Reduced insulin sensitivity is a key factor in the pathogenesis of type 2 diabetes and hypertension. Skeletal muscle insulin resistance is particularly important for its major role in insulin-mediated glucose disposal. Angiotensin II (ANG II) is integral in regulating blood pressure and plays a role in the pathogenesis of hypertension. In addition, we have documented that ANG II-induced skeletal muscle insulin resistance is associated with generation of reactive oxygen species (ROS). However, the linkage between ROS and insulin resistance in skeletal muscle remains unclear. To explore potential mechanisms, we employed the transgenic TG(mRen2)27 (Ren-2) hypertensive rat, which harbors the mouse renin transgene and exhibits elevated tissue ANG II levels, and skeletal muscle cell culture. Compared with Sprague-Dawley normotensive control rats, Ren-2 skeletal muscle exhibited significantly increased oxidative stress, NF-kappaB activation, and TNF-alpha expression, which were attenuated by in vivo treatment with an angiotensin type 1 receptor blocker (valsartan) or SOD/catalase mimetic (tempol). Moreover, ANG II treatment of L6 myotubes induced NF-kappaB activation and TNF-alpha production and decreased insulin-stimulated Akt activation and GLUT-4 glucose transporter translocation to plasma membranes. These effects were markedly diminished by treatment of myotubes with valsartan, the antioxidant N-acetylcysteine, NADPH oxidase-inhibiting peptide (gp91 ds-tat), or NF-kappaB inhibitor (MG-132). Similarly, NF-kappaB p65 small interfering RNA reduced NF-kappaB p65 subunit expression and nuclear translocation and TNF-alpha production but improved insulin-stimulated phosphorylation (Ser(473)) of Akt and translocation of GLUT-4. These findings suggest that NF-kappaB plays an important role in ANG II/ROS-induced skeletal muscle insulin resistance.     

TNF-α在冻融嗜中性粒细胞介导血管内皮细胞损伤中的作用

目的:探讨肿瘤坏死因子-α(TNF-α)在组织冻融造成的血管内皮细胞(VEC)损伤过程中的作用.方法:以右旋糖酐沉降法分离的大鼠嗜中性粒细胞(PMN)和体外培养的大鼠VEC为实验材料,建立细胞冻融模型.通过测定培养液中LDH活性确定VEC损伤程度,采用活性染料吞噬法检测与VEC粘附的PMN数,以流式细胞技术分析淋巴细胞功能相关抗原-1(LFA-1)表达.结果:TNF-α上调冻融PMN表面LFA-1表达,促进冻融PMN与正常VEC粘附,增强VEC损伤.抗LFA-1Mab部分阻断冻融PMN与正常VEC粘附,减轻VEC损伤.结论:在冻融损伤过程中TNF-α促进PMN表面LFA-1表达,增强PMN-VEC粘附,加重VEC损伤.     

Thrombin-induced release of von Willebrand factor from endothelial cells is mediated by phospholipid methylation. Prostacyclin synthesis is independent of phospholipid methylation

The biochemical events that lead to thrombin-stimulated release of von Willebrand factor and prostacyclin synthesis in cultured endothelial cells are examined. Treatment of human umbilical vein endothelial cells with thrombin results in an instantaneous increase in phospholipid methylation which can be blocked by 3-deazaadenosine, a methyltransferase inhibitor. 3-Deazaadenosine also blocks the thrombin-induced Ca2+ influx into endothelial cells and the release of von Willebrand factor, indicating that these processes are coupled. The phorbol ester 4 beta-phorbol 12-myristate 13-acetate (PMA) and the Ca2+ ionophore A23187 both bypass the phospholipid methylation and directly stimulate Ca2+ influx and von Willebrand factor release. In contrast to the stimulus-induced von Willebrand factor release, the thrombin-induced prostacyclin synthesis cannot be blocked by 3-deazaadenosine. Similarly, incubation of endothelial cells with EDTA has no influence on the thrombin-induced prostacyclin synthesis, and PMA has no stimulatory effect on prostacyclin synthesis. These observations indicate that thrombin induces different metabolic responses in endothelial cells: phospholipid methylation followed by a Ca2+ influx, which subsequently leads to release of von Willebrand factor, and liberation of arachidonic acid from phospholipids for prostacyclin formation, which is independent of phospholipid methylation and Ca2+ influx.     

Halothane potentiates the alcohol-adduct induced TNF-alpha release in heart endothelial cells

BACKGROUND: The possibility exists for major complications to occur when individuals are intoxicated with alcohol prior to anesthetization. Halothane is an anesthetic that can be metabolized by the liver into a highly reactive product, trifluoroacetyl chloride, which reacts with endogenous proteins to form a trifluoroacetyl-adduct (TFA-adduct). The MAA-adduct which is formed by acetaldehyde (AA) and malondialdehyde reacting with endogenous proteins, has been found in both patients and animals chronically consuming alcohol. These TFA and MAA-adducts have been shown to cause the release of inflammatory products by various cell types. If both adducts share a similar mechanism of cell activation, receiving halothane anesthesia while intoxicated with alcohol could exacerbate the inflammatory response and lead to cardiovascular injury. METHODS: We have recently demonstrated that the MAA-adduct induces tumor necrosis factor-alpha (TNF-alpha) release by heart endothelial cells (HECs). In this study, pair and alcohol-fed rats were randomized to receive halothane pretreatments intra peritoneal. Following the pretreatments, the intact heart was removed, HECs were isolated and stimulated with unmodified bovine serum albumin (Alb), MAA-modified Alb (MAA-Alb), Hexyl-MAA, or lipopolysaccharide (LPS), and supernatant concentrations of TNF-alpha were measured by ELISA. RESULTS: Halothane pre-treated rat HECs released significantly greater TNF-alpha concentration following MAA-adduct and LPS stimulation than the non-halothane pre-treated in both pair and alcohol-fed rats, but was significantly greater in the alcohol-fed rats. CONCLUSION: These results demonstrate that halothane and MAA-adduct pre-treatment increases the inflammatory response (TNF-alpha release). Also, these results suggest that halothane exposure may increase the risk of alcohol-induced heart injury, since halothane pre-treatment potentiates the HEC TNF-alpha release measured following both MAA-Alb and LPS stimulation.     

Angiotensin IV-mediated pulmonary artery vasorelaxation is due to endothelial intracellular calcium release

Angiotensin (ANG) IV stimulation of pulmonary artery (PA) endothelial cells (PAECs) but not of PA smooth muscle cells (PASMCs) resulted in significant increased production of cGMP in PASMCs. ANG IV receptors are not present in PASMCs, and PASMC nitric oxide synthase activity was not altered by ANG IV. ANG IV caused a dose-dependent vasodilation of U-46619-precontracted endothelium-intact but not endothelium-denuded PAs, and this response was blocked by the ANG IV receptor antagonist divalinal ANG IV but not by ANG II type 1 and 2 receptor blockers. ANG IV receptor-mediated increased intracellular Ca(2+) concentration ([Ca(2+)](i)) release from intracellular stores in PAECs was blocked by divalinal ANG IV as well as by the G protein, phospholipase C, and phosphoinositide (PI) 3-kinase inhibitors guanosine 5'-O-(2-thiodiphosphate), U-73122, and LY-294002, respectively, and was regulated by both PI 3-kinase- and ryanodine-sensitive Ca(2+) stores. Basal and ANG IV-mediated vasorelaxation of endothelium-denuded PAs was restored by exogenous PAECs but not by exogenous PAECs pretreated with the intracellular Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM. These results demonstrate that ANG IV-mediated vasodilation of PAs is endothelium dependent and regulated by [Ca(2+)](i) release through receptor-coupled G protein-phospholipase C-PI 3-kinase signaling mechanisms.     

Alpha-galactosylceramide-induced liver injury in mice is mediated by TNF-alpha but independent of Kupffer cells

NKT cells expressing phenotypic markers of both T and NK cells seem to be pivotal in murine models of immune-mediated liver injury, e.g., in Con A-induced hepatitis. Also alpha-galactosylceramide (alpha-GalCer), a specific ligand for invariant Valpha14 NKT cells, induces hepatic injury. To improve the comprehension of NKT-cell mediated liver injury, we investigated concomitants and prerequisites of alpha-GalCer-induced hepatitis in mice. Liver injury induced by alpha-GalCer injection into C57BL/6 mice was accompanied by intrahepatic caspase-3 activity but appeared independent thereof. alpha-GalCer injection also induces pronounced cytokine responses, including TNF-alpha, IFN-gamma, IL-2, IL-4, and IL-6. We provide a detailed time course for the expression of these cytokines, both in liver and plasma. Cytokine neutralization revealed that, unlike Con A-induced hepatitis, IFN-gamma is not only dispensable for alpha-GalCer-induced hepatotoxicity but even appears to exert protective effects. In contrast, TNF-alpha was clearly identified as an important mediator for hepatic injury in this model that increased Fas ligand expression on NKT cells. Whereas intrahepatic Kupffer cells are known as a pivotal source for TNF-alpha in Con A-induced hepatitis, they were nonessential for alpha-GalCer-mediated hepatotoxicity. In alpha-GalCer-treated mice, TNF-alpha was produced by intrahepatic lymphocytes, in particular NKT cells. BALB/c mice were significantly less susceptible to alpha-GalCer-induced liver injury than C57BL/6 mice, in particular upon pretreatment with d-galactosamine, a hepatocyte-specific sensitizer to TNF-alpha-mediated injury. Finally, we demonstrate resemblance of murine alpha-GalCer-induced hepatitis to human autoimmune-like liver disorders. The particular features of this model compared with other immune-mediated hepatitis models may enhance comprehension of basic mechanisms in the etiopathogenesis of NKT cell-comprising liver disorders.