Peptide synthesis through evolution.

Ribosome-catalyzed peptide bond formation is a crucial function of all organisms. The ribosome is a ribonucleoprotein particle, with both RNA and protein components necessary for the various steps leading to protein biosynthesis. Evolutionary theory predicts an early environment devoid of complex biomolecules, and prebiotic peptide synthesis would have started in a simple way. A fundamental question regarding peptide synthesis is how the current ribosome-catalyzed reaction evolved from a primitive system. Here we look at both prebiotic and modern mechanisms of peptide bond formation and discuss recent experiments that aim to connect these activities. In particular, RNA can facilitate peptide bond formation by providing a template for activated amino acids to react and can catalyze a variety of functions that would have been necessary in a pre-protein world. Therefore, RNA may have facilitated the emergence of the current protein world from an RNA or even prebiotic world.

Cholesterol depletion inhibits epidermal growth factor receptor transactivation by angiotensin II in vascular smooth muscle cells: role of cholesterol-rich microdomains and focal adhesions in angiotensin II signaling.

Angiotensin II (Ang II) induces transactivation of the epidermal growth factor (EGF) receptor (EGF-R), which serves as a scaffold for various signaling molecules in vascular smooth muscle cells (VSMCs). Cholesterol and sphingomyelin-enriched lipid rafts are plasma membrane microdomains that concentrate various signaling molecules. Caveolae are specialized lipid rafts that are organized by the cholesterol-binding protein, caveolin, and have been shown to be associated with EGF-Rs. Angiotensin II stimulation promotes a rapid movement of AT(1) receptors to caveolae; however, their functional role in angiotensin II signaling has not been elucidated. Here we show that cholesterol depletion by beta-cyclodextrin disrupts caveolae structure and concomitantly inhibits tyrosine phosphorylation of the EGF-R and subsequent activation of protein kinase B (PKB)/Akt induced by angiotensin II. Similar inhibitory effects were obtained with other cholesterol-binding agents, filipin and nystatin. In contrast, EGF-R autophosphorylation and activation of Akt/PKB in response to EGF are not affected by cholesterol depletion. The early Ang II-induced upstream signaling events responsible for transactivation of the EGF-R, such as the intracellular Ca(2+) increase and c-Src activation, also remain intact. The EGF-R initially binds caveolin, but these two proteins rapidly dissociate following angiotensin II stimulation during the time when EGF-R transactivation is observed. The activated EGF-R is localized in focal adhesions together with tyrosine-phosphorylated caveolin. These findings suggest that 1) a scaffolding role of caveolin is essential for EGF-R transactivation by angiotensin II and 2) cholesterol-rich microdomains as well as focal adhesions are important signal-organizing compartments required for the spatial and temporal organization of angiotensin II signaling in VSMCs.

Epidermal growth factor receptor transactivation by angiotensin II requires reactive oxygen species in vascular smooth muscle cells.

Angiotensin II (Ang II) is a vasoactive hormone with critical roles in vascular smooth muscle cell growth, an important feature of hypertension and atherosclerosis. Many of these effects are dependent on the production of reactive oxygen species (ROS). Ang II induces phosphorylation of the epidermal growth factor (EGF) receptor (EGF-R), which serves as a scaffold for various signaling molecules. Here, we provide novel evidence that ROS are critical mediators of EGF-R transactivation by Ang II. Pretreatment of vascular smooth muscle cells with the antioxidants diphenylene iodonium, Tiron, N-acetylcysteine, and ebselen significantly inhibited ( approximately 80% to 90%) tyrosine phosphorylation of the EGF-R by Ang II but not by EGF. Of the 5 autophosphorylation sites on the EGF-R, Ang II mainly phosphorylated Tyr1068 and Tyr1173 in a redox-sensitive manner. The Src family kinase inhibitor PP1, overexpression of kinase-inactive c-Src, or chelation of intracellular Ca(2+) attenuated EGF-R transactivation. Although antioxidants had no effects on the Ca(2+) mobilization or phosphorylation of Ca(2+)-dependent tyrosine kinase Pyk2, they inhibited c-Src activation by Ang II, suggesting that c-Src is 1 signaling molecule that links ROS and EGF-R phosphorylation. Furthermore, Ang II-induced tyrosine phosphorylation of the autophosphorylation site and the SH2 domain of c-Src was redox sensitive. These findings emphasize the importance of ROS in specific Ang II-stimulated growth-related signaling pathways and suggest that redox-sensitive EGF-R transactivation may be a potential target for antioxidant therapy in vascular disease.

Convergence of redox-sensitive and mitogen-activated protein kinase signaling pathways in tumor necrosis factor-alpha-mediated monocyte chemoattractant protein-1 induction in vascular smooth muscle cells.

Monocyte chemoattractant protein-1 (MCP-1) is an important component of the inflammatory response of the vessel wall and has been shown to be regulated by cytokines, such as tumor necrosis factor-alpha (TNF-alpha). However, the precise signaling pathways leading to MCP-1 induction have not been fully elucidated in vascular smooth muscle cells (VSMCs). Cytokine signal transduction involves protein kinases as well as reactive oxygen species (ROS). The relation between these 2 factors is not clear. In this study, we show that TNF-alpha induces a parallel phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (p38MAPK) and increases MCP-1 mRNA expression in cultured VSMCs. Inhibition of ERK1/2 but not p38MAPK caused a partial attenuation of MCP-1 induction (43+/-10% inhibition). Incubation of VSMCs with multiple antioxidants (diphenylene iodonium, liposomal superoxide dismutase, catalase, N-acetylcysteine, dimethylthiourea, and pyrrolidine dithiocarbamate) had no effect on TNF-alpha-mediated MCP-1 upregulation. However, simultaneous blockade of the ERK1/2 and ROS pathways by using PD098059 combined with diphenylene iodonium or N-acetylcysteine potently enhanced the ability of MAPK kinase inhibitors to abrogate MCP-1 mRNA expression (100+/-2% inhibition). Thus, parallel ROS-dependent and ERK1/2-dependent pathways converge to regulate TNF-alpha-induced MCP-1 gene expression in VSMCs. These data unmask a complex but organized integration of ROS and protein kinases that mediates cytokine-induced vascular inflammatory gene expression.

Reactive oxygen species mediate the activation of Akt/protein kinase B by angiotensin II in vascular smooth muscle cells.

Angiotensin II, a hypertrophic/anti-apoptotic hormone, utilizes reactive oxygen species (ROS) as growth-related signaling molecules in vascular smooth muscle cells (VSMCs). Recently, the cell survival protein kinase Akt/protein kinase B (PKB) was proposed to be involved in protein synthesis. Here we show that angiotensin II causes rapid phosphorylation of Akt/PKB (6- +/- 0.4-fold increase). Exogenous H(2)O(2) (50-200 microM) also stimulates Akt/PKB phosphorylation (maximal 8- +/- 0.2-fold increase), suggesting that Akt/PKB activation is redox-sensitive. Both angiotensin II and H(2)O(2) stimulation of Akt/PKB are abrogated by the phosphatidylinositol 3-kinase (PI3-K) inhibitors wortmannin and LY294002 (2(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one), suggesting that PI3-K is an upstream mediator of Akt/PKB activation in VSMCs. Furthermore, diphenylene iodonium, an inhibitor of flavin-containing oxidases, or overexpression of catalase to block angiotensin II-induced intracellular H(2)O(2) production significantly inhibits angiotensin II-induced Akt/PKB phosphorylation, indicating a role for ROS in agonist-induced Akt/PKB activation. In VSMCs infected with dominant-negative Akt/PKB, angiotensin II-stimulated [(3)H]leucine incorporation is attenuated. Thus, our studies indicate that Akt/PKB is part of the remarkable spectrum of angiotensin II signaling pathways and provide insight into the highly organized signaling mechanisms coordinated by ROS, which mediate the hypertrophic response to angiotensin II in VSMCs.

Angiotensin II receptor coupling to phospholipase D is mediated by the betagamma subunits of heterotrimeric G proteins in vascular smooth muscle cells.

In cultured vascular smooth muscle cells (VSMCs), activation of phospholipase D (PLD) by angiotensin II (Ang II) represents a major source of sustained generation of second messengers. Understanding the molecular mechanisms controlling activation of this pathway is essential to clarify the complexities of Ang II signaling, but the most proximal mechanisms coupling AT1 receptors to PLD have not been defined. Here we examine the role of heterotrimeric G proteins in AT1 receptor-PLD coupling. In alpha-toxin permeabilized VSMCs, GTPgammaS enhanced Ang II-stimulated PLD activation. In intact cells, Ang II activation of PLD was pertussis toxin-insensitive and was not additive with sodium fluoride, a cell-permeant activator of heterotrimeric G proteins, indicating that AT1 receptor-PLD coupling requires pertussis toxin-insensitive heterotrimeric G proteins. Ang II-stimulated PLD activity was significantly inhibited in VSMCs electroporated with anti-Gbeta antibody (56 +/- 5%) and in cells overexpressing the Gbetagamma-binding region of the carboxyl terminus of beta-adrenergic receptor kinase1 (79 +/- 8%), suggesting a critical role for Gbetagamma in PLD activation by Ang II. This effect may be mediated by pp60(c-src), because in beta-adrenergic receptor kinase1 overexpressing cells, pp60(c-src) activation was inhibited, and in normal cells anti-pp60(c-src) antibody inhibited Ang II-stimulated PLD activity. Galpha12 may also contribute to AT1 receptor-PLD coupling because electroporation of anti-Galpha12 antibody significantly inhibited PLD activity, whereas anti-Galphai and Galphaq/11 antibodies had no effect. Furthermore, electroporation of anti-RhoA antibody also attenuated Ang II-induced PLD activation, suggesting a role for small molecular weight G protein RhoA in this response. Thus, we provide evidence here that Gbetagamma as well as Galpha12 subunits mediate AT1 receptor coupling to tonic PLD activation via pp60(c-src)-dependent mechanisms, and that RhoA is involved in these signaling pathways in rat VSMCs. These results may provide insight into the molecular mechanisms underlying the highly organized, complex, chronic signaling programs associated with vascular smooth muscle growth and remodeling in response to Ang II.

Sexual and physical abuse in women with fibromyalgia: association with outpatient health care utilization and pain medication usage.

OBJECTIVE: To evaluate the relationship between sexual and/or physical abuse and health care usage in patients with fibromyalgia (FM) and identify variables that may influence this relationship. METHODS: We assessed history of sexual/physical abuse, health care utilization, and medication usage, as well as related variables in 75 women with FM using standardized questionnaires, structured interviews, and laboratory pain perception tasks. RESULTS: Fifty-seven percent of FM patients reported a history of sexual/physical abuse. Compared to non-abused patients, abused patients reported significantly greater utilization of outpatient health care services for problems other than FM and greater use of medications for pain (P < or = 0.025). Consistent with our expectations, abused patients also were characterized by significantly greater pain, fatigue, functional disability, and stress, as well as by a tendency to label dolorimeter stimuli as painful regardless of their intensities (P < or = 0.05). Additional analyses suggested that the high frequency of sexual/physical abuse in our patients was associated primarily with seeking health care for chronic pain rather than the FM syndrome itself or genetic factors. CONCLUSION: There is an association in FM patients between sexual/physical abuse and increased use of outpatient health care services and medications for pain. This association may be influenced by clinical symptoms, functional disability, psychiatric disorders, stress, and abnormal pain perception. The relationships among these variables should be further tested in prospective, population-based studies.

Temporal dispersion of activation of phospholipase C-beta1 and -gamma isoforms by angiotensin II in vascular smooth muscle cells. Role of alphaq/11, alpha12, and beta gamma G protein subunits.

Activation of phospholipase C (PLC) is one of the earliest events in angiotensin II (Ang II) type 1 (AT1) receptor (R)-mediated signal transduction in vascular smooth muscle cells (VSMCs). The coupling mechanisms of AT1 Rs to PLC, however, are controversial, because both tyrosine phosphorylation of PLC-gamma and G protein-dependent PLC-beta activation pathways have been reported. The expression of PLC-beta1, furthermore, has not been consistently demonstrated in VSMCs. Here we identified the PLC subtypes and subunits of heterotrimeric G proteins involved in AT1 R-PLC coupling using cultured rat VSMCs. Western analysis revealed the expression of PLC-beta1, -gamma1, and -delta1 in VSMCs. Ang II-stimulated inositol trisphosphate (IP3) formation measured at 15 s, which corresponds to the peak response, was significantly inhibited by electroporation of antibodies against PLC-beta1, but not by anti-PLC-gamma and -delta antibodies. Electroporation of anti-Galphaq/11 and -Galpha12 antibodies also showed significant inhibition of the Ang II-induced IP3 generation at 15 s, while anti-Galphai and Galpha13 antibodies were ineffective. Furthermore, in VSMCs electroporated with anti-Gbeta antibody and cells stably transfected with the plasmid encoding the Gbetagamma-binding region of the carboxyl terminus of beta-adrenergic receptor kinase1, the peak Ang II-stimulated PLC activity (at 15 s) was significantly inhibited. The tyrosine kinase inhibitor, genistein, had no effect on the peak response to Ang II stimulation, but significantly inhibited IP3 production after 30 s, a time period which temporally correlated with PLC-gamma tyrosine phosphorylation in response to Ang II. Moreover, electropor-ation of anti-PLC-gamma antibody markedly inhibited the IP3 production measured at 30 s, indicating that tyrosine phosphorylation of PLC-gamma contributes mainly to the later phase of PLC activation. Thus, these results suggest that: 1) AT1 receptors sequentially couple to PLC-beta1 via a heterotrimeric G protein and to PLC-gamma via a downstream tyrosine kinase; 2) the initial AT1 receptor-PLC-beta1 coupling is mediated by Galphaq/11beta gamma and Galpha12 beta gamma; 3) Gbeta gamma acts as a signal transducer for activation of PLC in VSMCs. The sequential coupling of AT1 receptors to PLC-beta1 and PLC-gamma, as well as dual coupling of AT1 receptors to distinct Galpha proteins, suggests a novel mechanism for a temporally controlled, highly organized and convergent Ang II-signaling network in VSMCs.

G-Protein binding domains of the angiotensin II AT1A receptors mapped with synthetic peptides selected from the receptor sequence.

The vascular angiotensin II type 1 receptor (AT1AR) is a member of the G-protein-coupled receptor superfamily. We mapped the G-protein binding domains of the AT1AR using synthetic peptides selected from the receptor sequence, which interfere with AT1AR-G-protein coupling. Membrane GTPase activity was used as a measure of the functional coupling in rat vascular smooth muscle cells. Peptides corresponding to the N-terminal region of the second intracellular loop (residues 125-137), the N-terminal region of the third intracellular loop (217-227) and the juxtamembranous region of the C-terminal tail (304-316) inhibited angiotensin II-induced GTPase activation by 30%, 30%, and 70%, respectively. The latter two domains (217-227 and 304-316) are predicted to form amphiphilic alpha-helices. Only the peptide representing residues 217-227 stimulated basal activity (45%). No synthetic peptide had a significant effect on either the number or the affinity of the AT1AR binding. These observations indicate that domains of the second and third regions and the cytoplasmic tail of the AT1AR interact with G-proteins, and that multiple contacts with these receptor domains may be important for binding and activation of the G-proteins.

Oscillatory shear stress stimulates adhesion molecule expression in cultured human endothelium.

Low and oscillatory shear stresses are major features of the hemodynamic environment of sites opposite arterial flow dividers that are predisposed to atherosclerosis. Atherosclerosis is a focal inflammatory disease characterized initially by the recruitment of mononuclear cells into the arterial wall. The specific characteristics of the hemodynamic environment that facilitate the generation of arterial inflammatory responses in the presence of, for example, hyperlipidemia are unknown. We show here that prolonged oscillatory shear stress induces expression of endothelial cell leukocyte adhesion molecules, which are centrally important in mediating leukocyte localization into the arterial wall. Vascular cell adhesion molecule-1 was upregulated an average 9-fold relative to endothelial monolayers in static culture. Intercellular adhesion molecule-1 and E-selectin exhibited 11-fold and 7.5-fold increases, respectively. Upregulation of these adhesion molecules was associated with enhanced monocyte adherence. Cytokine stimulation of surface vascular cell adhesion molecule-1 was maximally induced after 6 and 8 hours of cytokine incubation. Oscillatory shear stress for these time periods elicited respective vascular cell adhesion molecule-1 levels of 16% and 30% relative to those observed for cytokine stimulation. Surface intercellular adhesion molecule-1 induction by cytokine stimulation for 24 hours was found to be approximately five times the level detected after 24 hours of oscillatory shear stress. Experiments performed in the presence of the antioxidant N-acetylcysteine demonstrated that the expression of vascular cell adhesion molecule-1 could be almost totally abolished, whereas that of intercellular adhesion molecule-1 was typically reduced by approximately 70%. These results imply that oscillatory shear stress per se is sufficient to stimulate mononuclear leukocyte adhesion and, presumptively, migration into the arterial wall. These results further indicate that atherosclerotic lesion initiation is likely related, at least in part, to unique signals generated by oscillatory shear stress and that the mechanism of upregulation is, to some extent, redox sensitive.

Tumour necrosis factor alpha activates a p22phox-based NADH oxidase in vascular smooth muscle.

Increasing experimental evidence suggests that non-phagocytic cells express a potent superoxide (O2-.)-producing NADH oxidase that might be related to the phagocytic NADPH oxidase. Here we show that the cytokine tumour necrosis factor alpha (TNF-alpha) activates, in a time- and dose-dependent manner, a O2-.-producing NADH oxidase in cultured rat aortic smooth-muscle cells. Dose-response experiments for NADH showed an upward shift of the curve for TNF-alpha-treated cells, suggesting that TNF-alpha increased the amount of available enzyme. Using the anti-sense transfection technique, we further demonstrate that the molecular identity of this oxidase includes p22(phox) (the alpha subunit of cytochrome b558 and part of the electron transfer component of the phagocytic NADPH oxidase), which we recently cloned from a rat vascular smooth-muscle cell cDNA library. In addition, prolonged treatment with TNF-alpha increased p22phox mRNA expression without affecting p22phox mRNA stability, and only when transcriptional activity was intact. These findings identify a p22phox-containing NADH oxidase as a source for cytokine-induced free radical production in vascular smooth-muscle cells and clarify some of the mechanisms involved in the regulation of vascular oxidase activity.

Monocyte chemoattractant protein-1 expression in aortic tissues of hypertensive rats.

Monocyte chemoattractant protein-1 (MCP-1), a potent monocyte chemoattractant synthesized by vascular cells and monocytes, has been proposed to be an important mediator of inflammatory responses in the arterial vasculature. It was recently demonstrated that hypertension is associated with an inflammatory response in the arterial wall. To determine the effect of hypertension on arterial MCP-1 expression, we induced hypertension in Sprague-Dawley rats by infusing angiotensin II (0.75 mg x kg[-1] x d[-1] SC) for 7 days. Using Northern blot analysis, we detected a 3.6-fold increase in MCP-1 mRNA in the aortas of hypertensive rats. When we normalized blood pressure in angiotensin II-treated rats through oral administration of the nonspecific vasodilator hydralazine (15 mg x kg[-1] x d[-1]), aortic MCP-1 mRNA expression was significantly reduced. Similar results were obtained with a norepinephrine model of hypertension. Taken together, these data suggest that mechanical factors may be responsible in part for the upregulation of expression. Consistent with this interpretation, we found that cultured rat aortic vascular smooth muscle cells exposed to mechanical strain (20% peak deformation at 1 Hz) exhibited a marked increase in MCP-1 expression, suggesting the hemodynamic strain imparted onto arterial cells in hypertension is an important stimulus underlying this phenomenon. These results provide important insights into the in vivo regulation of MCP-1 and have potential implications for understanding the influence of hypertension on atherosclerosis.

Perceived physical and emotional trauma as precipitating events in fibromyalgia. Associations with health care seeking and disability status but not pain severity.

OBJECTIVE: We examined relationships between perceived physical and emotional trauma that occur prior to, or that initiate, pain onset and health care seeking for fibromyalgia syndrome (FMS). We also assessed associations between perceived trauma and levels of health care usage, symptom severity, functional disability, and receipt of disability compensation among patients with FMS. METHODS: We evaluated these variables using interviews and standardized instruments in a consecutive series of FMS patients and community residents who met the American College of Rheumatology criteria for FMS but had not sought medical care ("nonpatients"). RESULTS: Emotional trauma was associated with status as an FMS patient independently of demographics, physical trauma, and sexual/physical abuse (P = 0.007). Among patients, emotional trauma was related to a high number of physician visits (P = 0.013), functional disability ratings (P = 0.012), and fatigue (P = 0.029), but physical trauma was associated with receipt of disability compensation (P = 0.019). Trauma history was not related to pain severity or pain thresholds. CONCLUSION: Perception of physical trauma is a greater determinant of disability compensation for FMS than is perceived emotional trauma, symptom severity, or functional disability. Effort should be devoted to understanding the social and legal factors underlying this observation, as well as to reducing high health care usage among FMS patients with emotional trauma.

Nitric oxide regulates vascular cell adhesion molecule 1 gene expression and redox-sensitive transcriptional events in human vascular endothelial cells.

Decreased nitric oxide (NO) activity, the formation of reactive oxygen species, and increased endothelial expression of the redox-sensitive vascular cell adhesion molecule 1 (VCAM-1) gene in the vessel wall are early and characteristic features of atherosclerosis. To explore whether these phenomena are functionally interrelated, we tested the hypothesis that redox-sensitive VCAM-1 gene expression is regulated by a NO-sensitive mechanism. In early passaged human umbilical vein endothelial cells and human dermal microvascular endothelial cells, the NO donor diethylamine-NO (DETA-NO, 100 microM) reduced VCAM-1 gene expression induced by the cytokine tumor necrosis factor alpha (TNF-alpha, 100 units/ml) at the cell surface level by 65% and intracellular adhesion molecule 1 (ICAM-1) gene expression by 35%. E-selectin gene expression was not affected. No effect on expression of cell adhesion molecules was observed with DETA alone. Moreover, DETA-NO suppressed TNF-alpha-induced mRNA accumulation of VCAM-1 and TNF-alpha-mediated transcriptional activation of the human VCAM-1 promoter. Conversely, treatment with NG-monomethyl-L-arginine (L-NMMA, 1 mM), an inhibitor of NO synthesis, augmented cytokine induction of VCAM-1 and ICAM-1 mRNA accumulation. By gel mobility shift analysis, DETA-NO inhibited TNF-alpha activation of DNA binding protein activity to the VCAM-1 NF-kappa B like binding sites. Peroxy-fatty acids such as 13-hydroperoxydodecanoeic acid (linoleyl hydroperoxide) may serve as an intracellular signal for NF-kappa B activation. Using thin layer chromatography, DETA-NO (100 microM) suppressed formation of this metabolite, suggesting that DETA-NO modifies the reactivity of oxygen intermediates in the vascular endothelium. Through this mechanism, NO may function as an immunomodulator of the vessel wall and thus mediate inflammatory events involved in the pathogenesis of atherosclerosis.

Psychiatric diagnoses in patients with fibromyalgia are related to health care-seeking behavior rather than to illness.

OBJECTIVE: To compare the frequency of lifetime psychiatric disorders among 3 groups of subjects: patients with fibromyalgia syndrome (FMS) from a tertiary care setting, community residents with FMS who had not sought medical care for their FMS symptoms ("FMS nonpatients"), and healthy controls. METHODS: We used the Computerized Diagnostic Interview Schedule to assess lifetime psychiatric diagnoses, as well as the Center for Epidemiological Studies Depression scale and the Trait Anxiety Inventory to assess current psychological distress, among 64 patients with FMS, 28 FMS nonpatients, and 23 healthy individuals. RESULTS: Patients with FMS, relative to FMS nonpatients and healthy controls, were characterized by a significantly greater number of lifetime psychiatric diagnoses (P = 0.002). Nonpatients did not differ from controls in psychiatric diagnoses. Patients also exhibited higher psychological distress levels than nonpatients, and nonpatients showed greater distress than controls. Differences in psychological distress between patients and nonpatients were eliminated after controlling for pain threshold and fatigue ratings. CONCLUSION: Psychiatric disorders are not intrinsically related to the FMS syndrome. Instead, multiple lifetime psychiatric diagnoses may contribute to the decision to seek medical care for FMS in tertiary care settings.

Angiotensin II down-regulates the vascular smooth muscle AT1 receptor by transcriptional and post-transcriptional mechanisms: evidence for homologous and heterologous regulation.

The vascular angiotensin II (ANG II) receptor (AT1) is a central component of the renin-angiotensin system; thus, regulation of its expression is likely to be important in cardiovascular responsiveness. We demonstrate that ANG II down-regulates its receptor in rat aortic vascular smooth muscle cells. Incubation for 4 hr with 100 nM ANG II decreased AT1 mRNA and protein by 70% and 35%, respectively. This homologous down-regulation was concentration and time dependent and was blocked by the AT1 antagonist losartan. It did not appear to be mediated by protein kinase C or other protein kinases but was dependent on the sustained signaling pathway sensitive to phenylarsine oxide. Heterologous down-regulation was observed with the agonists alpha-thrombin and ATP and the cAMP-increasing agent forskolin. ANG II inhibited transcription by 50% and destabilized the AT1 mRNA. Down-regulation of AT1 mRNA was blocked by transcription and translation inhibitors, suggesting that it required expression of a protein factor or factors. These results indicate that ANG II down-regulates its vascular receptor by both transcriptional and post-transcriptional mechanisms. Homologous and heterologous down-regulation of the AT1 receptor may participate in the coordinated physiological adaptation of vascular tone to vasoactive hormones.