Delicate conformational balance of the redox enzyme cytochrome P450cam

The energy landscapes of proteins are highly complex and can be influenced by changes in physical and chemical conditions under which the protein is studied. The redox enzyme cytochrome P450cam undergoes a multistep catalytic cycle wherein two electrons are transferred to the heme group and the enzyme visits several conformational states. Using paramagnetic NMR spectroscopy with a lanthanoid tag, we show that the enzyme bound to its redox partner, putidaredoxin, is in a closed state at ambient temperature in solution. This result contrasts with recent crystal structures of the complex, which suggest that the enzyme opens up when bound to its partner. The closed state supports a model of catalysis in which the substrate is locked in the active site pocket and the enzyme acts as an insulator for the reactive intermediates of the reaction.During catalysis, an enzyme will traverse a conformational energy landscape that can be highly complex and easily changed by external factors, such as temperature, ionic strength, and crystallization. Cytochromes P450 (CYPs) are b-type heme-containing monooxygenases found throughout the three domains of life. These enzymes catalyze the regiospecific and stereospecific hydroxylation of various aliphatic and aromatic compounds and are involved in a considerable number of metabolic processes, such as steroid biosynthesis and metabolism of xenobiotics in mammals. The CYP superfamily has been extensively studied over the past five decades, and an understanding of its mechanism is crucial for the development of pharmaceutical compounds that do not inhibit these enzymes. Under other circumstances, it is desirable to inhibit a CYP for therapeutic reasons. For example, compounds that inhibit CYP1B1 and CYP1A2 in humans have been proposed as promising anticancer agents (1). The archetypal member of this superfamily is CYP101A1 (more commonly known as P450cam) from Pseudomonas putida, which was the first CYP for which the 3D structure was determined by X-ray crystallography (2). P450cam catalyzes the hydroxylation of d-camphor to 5-exohydroxycamphor using two electrons, two protons, and molecular oxygen. Putidaredoxin (Pdx) reductase oxidizes NADH and transfers the electrons to Pdx, which, in turn, shuttles electrons to P450cam in two consecutive steps (3–5). This complex reaction is controlled by the enzyme to ensure an efficient coupling between dioxygen reduction and substrate hydroxylation and to avoid side reactions. P450cam must go through a cycle of several conformational and electronic states to perform its catalytic task (6).The first X-ray crystal structures of P450cam showed that the enzyme occupied a closed conformation, both in the presence and absence of substrate; therefore, it was unclear how the substrate was able to bind into the active site (2). Moreover, structures of the intermediates of the catalytic cycle were also in a closed conformation (7), leading to the idea that during the catalytic cycle, P450cam opens to bind camphor, closes to perform hydroxylation, and then reopens to release the product. The first structures of a more open state of P450cam were obtained by using ligands that forced a channel open (8, 9), and, subsequently, an open structure of P450cam in the absence of substrate was published (10). These structures showed that the F- and G-helices and the F/G loop of P450cam moved to reveal an opening through which the active site could be accessed by the substrate.The conformational landscape of P450cam is, however, more complex, owing to the fact that the catalytic cycle also comprises two electron transfer steps involving binding of its redox partner Pdx, several spin state changes of the heme iron, and a conformational change upon the binding of substrate. A resonance Raman study published by Sjodin et al. (11) demonstrated that additional conformational substates of the oxy-heme were present when Pdx is bound to P450cam, and they argued that these states may be linked to the electron donation properties of the cysteinate ligated to the heme. NMR spectroscopy studies have revealed changes in the chemical shifts of P450cam when Pdx binds (12, 13).Recently, a structure of the complex of oxidized P450cam tethered to oxidized Pdx obtained using X-ray crystallography has been reported, and it shows P450cam in the open state in this complex [Protein Data Bank (PDB) ID code 4JWS (14)]. It was proposed that oxidized P450cam favors the open conformation when Pdx binds. A crystal structure of the native complex, without cross-links, was simultaneously but independently obtained using X-ray crystallography, and it suggests that P450cam occupies an intermediate state when Pdx binds [PDB ID code 3W9C (15)]. It is somewhat counterintuitive that P450cam would be in the open state when Pdx binds. Changing to the open state during the catalytic cycle could cause the release of the substrate and reactive oxygen species from the enzyme. However, a recent EPR study supported the finding that oxidized Pdx induces oxidized P450cam to adopt an open conformation, and the authors argue that this observation eliminates the possibility that crystal contacts are the reason why P450cam adopts the open state in the complex with Pdx (16). However, all of the EPR measurements were performed at 50 K or less. Kinetic experiments at ambient temperatures reported in the same article (16) show that the rate constant for camphor dissociation is low and hardly affected by Pdx binding, at least at the concentrations of potassium ions present in the EPR solution and the crystallization buffers. Because it is generally assumed that the dissociation rate of camphor is much lower in the closed state than in the open state of the enzyme, these results suggest that the enzyme remains closed at ambient temperatures upon binding to Pdx, implying the presence of a subtle balance between open and closed states that can be affected by crystallography and low temperature.In this work, we studied the state of P450cam in complex with Pdx in solution at ambient temperature (25 °C) using paramagnetic NMR spectroscopy. A pseudocontact shift (PCS) is the difference in chemical shift observed for a nucleus in paramagnetically and diamagnetically tagged protein (17, 18). PCSs can provide long-range distance information and are highly sensitive to structural changes within a protein. The PCS depends on the distance of the nucleus to the paramagnetic center, as well as on the position of the nucleus in the frame defined by the anisotropic component of the magnetic susceptibility of the paramagnetic center, described by the Δχ tensor. The PCS can be used to aid in protein structure determination (19–21), for example, as recently reported for the structure of the seven-transmembrane phototactic receptor sensory rhodopsin II (22). This approach complements the use of paramagnetic relaxation enhancements in structure determination using both liquid and solid-state NMR spectroscopy, as has been demonstrated by various research groups (23–26). Furthermore, PCSs are very useful to obtain long-range distance information for protein docking (17, 27). Recently, the use of PCSs in the refinement of X-ray crystal structures was described, as shown for the catalytic domain of matrix metalloproteinase 1, the third IgG-binding domain of protein G, ubiquitin, free calmodulin, and calmodulin–peptide complexes (28). Similar to residual dipolar couplings (RDCs) (29–32), which are also caused by anisotropic interactions, PCSs can also be used to study conformation changes in proteins, for example, as demonstrated for calmodulin–peptide complexes (33). When a paramagnetic probe is attached to part of a protein that moves between two conformations, the vector that connects the nucleus under observation and the probe will change (Fig. 1). As a consequence, a nucleus experiences distinct distances to the paramagnetic center and positions in the Δχ tensor frame in the two states, and the ensuing differences in PCSs provide a means of differentiating between the two states. We used this approach by mutating residues E195 and A199 to Cys for attachment of the two-armed caged lanthanoid NMR probe-7 (CLaNP-7) (15, 34). This mutation site is at the top of the G-helix, which undergoes a significant conformational change when the protein transitions from the closed state to the open state. Our data demonstrate that Pdx binding does not induce opening of P450cam in solution, in contrast to what is observed in the crystalline state. A closed state of the enzyme is better in line with a model of catalysis in which the substrate and the reactive intermediates remain enclosed within the enzyme during the reaction.Open in a separate windowFig. 1.Use of PCSs to distinguish conformational states. The paramagnetic center (small black circle) is attached to a part of a protein that exists in two conformations. The nucleus (large gray circle) experiences a distinct PCS in each state because the distance to the center (double-pointed arrows) and the orientation relative to the direction of the anisotropic component of the magnetic susceptibility tensor (transparent shapes) differ.     

花生四烯酸细胞色素p450表氧化酶基因对野百合碱诱导大鼠肺动脉高压的治疗作用

目的研究过表达花生四烯酸细胞色素p450表氧化酶基因CYP2J2对野百合碱(MCT)诱导的大鼠原发性肺动脉高压的治疗作用,并对其机制进行初步探讨。方法模型组18只雄性大鼠一次性注射MCT(60 mg/kg·w),对照组18只。三周后分六组:MCT 生理盐水(n=6),MCT pCB6(n=6),MCT pCB6-2J2(n=6),生理盐水(n=6),pCB6(n=6),pCB6-2J2(n=6)。两周后,测右室收缩压RVSP和右室肥厚指数RV/(LV s),western blot检测CYP2J2、Smad4蛋白的表达。结果模型组注射生理盐水、pCB6组RVSP分别为(41.48±0.45)和(38.13±3.68)mm Hg,显著高于正常对照组动物(20.89±3.09)mm Hg,(P<0.05)和模型组注射pCB6-2J2组(29.48±5.67)mm Hg,(P<0.05)。模型组注射生理盐水、pCB6组RV/(LV S)分别为(0.425±0.08)和(0.358±0.07)显著高于正常对照组动物(0.24±0.03)(P<0.01)和模型组注射pCB6-2J2组(0.28±0.02) (P<0.05)。Western blots显示模型组注射生理盐水、pCB6组Smad4蛋白表达水平均明显低于注射pCB6-2J2组。结论过表达CYP2J2基因显著缓解野百合碱诱导的大鼠肺动脉高压,降低右室收缩压和减轻右心室肥厚程度,上调了TGFβ信号通道中的Smad4蛋白。     

Structural basis for the wobbler mouse neurodegenerative disorder caused by mutation in the Vps54 subunit of the GARP complex

The multisubunit Golgi-associated retrograde protein (GARP) complex is required for tethering and fusion of endosome-derived transport vesicles to the trans-Golgi network. Mutation of leucine-967 to glutamine in the Vps54 subunit of GARP is responsible for spinal muscular atrophy in the wobbler mouse, an animal model of amyotrophic lateral sclerosis. The crystal structure at 1.7 Å resolution of the mouse Vps54 C-terminal fragment harboring leucine-967, in conjunction with comparative sequence analysis, reveals that Vps54 has a continuous α-helical bundle organization similar to that of other multisubunit tethering complexes. The structure shows that leucine-967 is buried within the α-helical bundle through predominantly hydrophobic interactions that are critical for domain stability and folding in vitro. Mutation of this residue to glutamine does not prevent integration of Vps54 into the GARP complex but greatly reduces the half-life and levels of the protein in vivo. Severely reduced levels of mutant Vps54 and, consequently, of the whole GARP complex underlie the phenotype of the wobbler mouse.     

Uncoupling of the cytochrome P-450cam monooxygenase reaction by a single mutation, threonine-252 to alanine or valine: possible role of the hydroxy amino acid in oxygen activation.

Site-directed mutants of cytochrome P-450cam (the cytochrome P-450 that acts as the terminal monooxygenase in the d-camphor monooxygenase system), in which threonine-252 had been changed to alanine, valine, or serine, were employed to study the role of the hydroxy amino acid in the monooxygenase reaction. The mutant enzymes were expressed in Escherichia coli and were purified by a conventional method. All the mutant enzymes in the presence of d-camphor exhibited optical absorption spectra almost indistinguishable from those of the wild-type enzyme in their ferric, ferrous, oxygenated, and carbon monoxide ferrous forms. In a reconstituted system with putidaredoxin and its reductase, the alanine enzyme consumed O2 at a rate (1100 per min per heme) comparable to that of the wild-type enzyme (1330 per min per heme), whereas the amount of exo-5-hydroxycamphor formed was less than 10% of that formed by the wild-type enzyme. About 85% of the O2 consumed was recovered as H2O2. The valine enzyme also exhibited an oxidase activity to yield H2O2 accompanied by a relative decrease in the monooxygenase activity. On the other hand, the serine enzyme exhibited essentially the same monooxygenase activity as that of the wild-type enzyme. Thus, uncoupling of O2 consumption from the monooxygenase function was produced by the substitution of an amino acid without a hydroxyl group. When binding of O2 to the ferrous forms was examined, the alanine and valine enzymes formed instantaneously an oxygenated form, which slowly decomposed to the ferric form with rates of 5.5 and 3.2 x 10(-3) sec-1 for the former and latter enzymes, respectively. Since these rates were too slow to account for the overall rates of O2 consumption, the formation of H2O2 was considered to proceed not by way of this route but through the decomposition of a peroxide complex formed by reduction of the oxygenated form by reduced putidaredoxin. Based on these findings, a possible mechanism for oxygen activation in this monooxygenase reaction has been discussed.     

Essential role of the cytochrome P450 CYP4F22 in the production of acylceramide,the key lipid for skin permeability barrier formation

A skin permeability barrier is essential for terrestrial animals, and its impairment causes several cutaneous disorders such as ichthyosis and atopic dermatitis. Although acylceramide is an important lipid for the skin permeability barrier, details of its production have yet to be determined, leaving the molecular mechanism of skin permeability barrier formation unclear. Here we identified the cytochrome P450 gene CYP4F22 (cytochrome P450, family 4, subfamily F, polypeptide 22) as the long-sought fatty acid ω-hydroxylase gene required for acylceramide production. CYP4F22 has been identified as one of the autosomal recessive congenital ichthyosis-causative genes. Ichthyosis-mutant proteins exhibited reduced enzyme activity, indicating correlation between activity and pathology. Furthermore, lipid analysis of a patient with ichthyosis showed a drastic decrease in acylceramide production. We determined that CYP4F22 was a type I membrane protein that locates in the endoplasmic reticulum (ER), suggesting that the ω-hydroxylation occurs on the cytoplasmic side of the ER. The preferred substrate of the CYP4F22 was fatty acids with a carbon chain length of 28 or more (≥C28). In conclusion, our findings demonstrate that CYP4F22 is an ultra-long-chain fatty acid ω-hydroxylase responsible for acylceramide production and provide important insights into the molecular mechanisms of skin permeability barrier formation. Furthermore, based on the results obtained here, we proposed a detailed reaction series for acylceramide production.A skin permeability barrier protects terrestrial animals from water loss from inside the body, penetration of external soluble materials, and infection by pathogenetic organisms. In the stratum corneum, the outermost cell layer of the epidermis, multiple lipid layers (lipid lamellae) play a pivotal function in barrier formation (Fig. S1) (1–3). Impairment of the skin permeability barrier leads to several cutaneous disorders, such as ichthyosis, atopic dermatitis, and infectious diseases.Open in a separate windowFig. S1.Structures of the epidermis, the stratum corneum, acylceramide, and protein-bound ceramide. Acylceramides are produced mainly in the stratum granulosum and partly in the stratum spinosum and are stored in lamellar bodies as glucosylated forms (acyl glucosylceramides). At the interface of the stratum granulosum and stratum corneum, the lamellar bodies fuse with the plasma membrane and release their contents into the extracellular space, where acyl glucosylceramides are converted to acylceramides. Thus, released acylceramides, FAs, and cholesterol form lipid lamellae in the stratum corneum. Some acylceramide is hydrolyzed to ω-hydroxyceramide, followed by covalent binding to corneocyte surface proteins to create corneocyte lipid envelopes. Acylceramide contains ULCFAs with carbon chain lengths of C28–C36. The FA elongase ELOVL1 produces VLCFAs, which are further elongated to ULCFAs by ELOVL4 (29). The ceramide synthase CERS3 creates an amide bond between ULCFA and LCB (17). ω-Hydroxylation of ULCFA is required for acylceramide production. However, the responsible ω-hydroxylase had not been identified previously; its identification is the subject of this research.The major components of the lipid lamellae are ceramide (the sphingolipid backbone), cholesterol, and free fatty acid (FA). In most tissues, ceramide consists of a long-chain base (LCB; usually sphingosine) and an amide-linked FA with a chain length of 16–24 (C16–C24) (4, 5). On the other hand, ceramide species in the epidermis are strikingly unique (Fig. S2A). For example, epidermal ceramides contain specialized LCBs (phytosphingosine and 6-hydroxysphingosine) and/or FAs with α- or ω-hydroxylation (1–3). In addition, substantial amounts of epidermal ceramides have ultra-long-chain FAs (ULCFAs) with chain lengths of 26 or more (≥C26) (4, 5). Unique epidermal ceramides are acylceramides having C28–C36 ULCFAs, which are ω-hydroxylated and esterified with linoleic acid [EOS in Fig. S1; EODS, EOS, EOP, and EOH in Fig. S2A; EOS stands for a combination of an esterified ω-hydroxy FA (EO) and sphingosine (S); DS, dihydrosphingosine; P, phytosphingosine; H, 6-hydroxysphingosine] (1–3, 6, 7). These characteristic molecules may be important to increase the hydrophobicity of lipid lamellae and/or to stabilize the multiple lipid layers. Linoleic acid is one of the essential FAs, and its deficiency causes ichthyosis symptoms resulting from a failure to form normal acylceramide (8). Ichthyosis is a cutaneous disorder accompanied by dry, thickened, and scaly skin; it is caused by a barrier abnormality. In patients who have atopic dermatitis, both total ceramide levels and the chain length of ceramides are decreased, and ceramide composition is altered also (9–11).Open in a separate windowFig. S2.Structure and synthetic pathways of ceramides in mammals. (A) Structure and nomenclature of epidermal ceramides. Epidermal ceramides are classified into 12 classes depending on their differences in the LCB and FA moieties. N-type and A-type ceramides contain C16–C30 FAs (n = 1–15), whereas EO-type ceramides contain C28–C36 FAs (n = 13–21) (6, 7). (B) FA elongation and ceramide synthesis in mammals. The FA elongation pathways of saturated and monounsaturated FAs and the ceramide-synthetic pathways are illustrated. E1–E7 and C1–C6 indicate the ELOVL (ELOVL1–7) and CERS (CERS1–6) isozymes involved in each step, respectively. The differences in the letter size of E1–E7 reflect their enzyme activities in each FA elongation reaction. Cer, ceramide; MUFA, monounsaturated FA; SFA, saturated FA.In addition to its essential function in the formation of lipid lamellae, acylceramide also is important as a precursor of protein-bound ceramide, which functions to connect lipid lamellae and corneocytes (Fig. S1) (12, 13). After the removal of linoleic acid, the exposed ω-hydroxyl group of acylceramide is covalently bound to corneocyte proteins, forming a corneocyte lipid envelope. Acylceramides and protein-bound ceramides are important in epidermal barrier formation, and mutations in the genes involved in their synthesis, including the ceramide synthase CERS3, the 12(R)-lipoxygenase ALOX12B, and the epidermal lipoxygenase-3 ALOXE3, can cause nonsyndromic, autosomal recessive congenital ichthyosis (ARCI) (3, 14–16). CERS3 catalyzes the amide bond formation between an LCB and ULCFA, producing ULC-ceramide, which is the precursor of acylceramide (Fig. S1 and Fig. S2B) (17). ALOX12B and ALOXE3 are required for the formation of protein-bound ceramides (13, 18). Other ARCI genes include the ATP-binding cassette (ABC) transporter ABCA12, the transglutaminase TGM1, NIPAL4 (NIPA-like domain containing 4)/ICHTHYIN, CYP4F22/FLJ39501, LIPN (lipase, family member N), and PNPLA1 (patatin-like phospholipase domain containing 1) (16, 19). The exact functions of NIPAL4, LIPN, and PNPLA1 are currently unclear. Causative genes of syndromic forms of ichthyosis also include a gene required for acylceramide synthesis: the FA elongase ELOVL4, which produces ULCFA-CoAs, the substrate of CERS3 (20).Although acylceramide is essential for the epidermal barrier function, the mechanism behind acylceramide production is still poorly understood, leaving the molecular mechanisms behind epidermal barrier formation unclear. For example, acylceramide production requires ω-hydroxylation of the FA moiety of ceramide. However, the ω-hydroxylase responsible for this reaction was unidentified heretofore (Fig. S1). Here, we identified the cytochrome P450, family 4, subfamily F, polypeptide 22 (CYP4F22), also known as “FLJ39501,” as this missing FA ω-hydroxylase required for acylceramide production. CYP4F22 had been identified as one of the ARCI genes (21), although its function in epidermal barrier formation remained unsolved. Our findings clearly demonstrate a relationship between ARCI pathology, acylceramide levels, and ω-hydroxylase activity.     

Hepatic cytochrome P450 is directly inactivated by nitric oxide, not by inflammatory cytokines, in the early phase of endotoxemia

BACKGROUND/AIMS: Although the activity of the liver in metabolizing and eliminating various drugs decreases in endotoxemia, the mechanism remains to be elucidated. The generation of nitric oxide by the inducible type of nitric oxide synthase increases in endotoxemia. Nitric oxide readily reacts with heme proteins such as cytochrome P450 that metabolize various compounds, including steroids and eicosanoids. The purpose of this study was to determine the effect of nitric oxide on the function of hepatic cytochrome P450 in endotoxemic rats. METHODS: To determine the dynamic aspects of nitric oxide metabolism, hepatic levels of the inducible type of nitric oxide synthase and heme-iron nitrosyl complexes, and plasma levels of nitrite and nitrate were determined in rats before and after intravenous administration of lipopolysaccharide. Changes in the levels of P450 isoforms and testosterone hydroxylation activity in hepatic microsomes were also determined. To evaluate in vivo CYP3A2 activity, midazolam sleep time was measured. RESULTS: When lipopolysaccharide increased the hepatic inducible type of nitric oxide synthase and plasma levels of nitric oxide metabolites, the intensity of low-spin signal of electron spin resonance responsible for the ferric form of P450 decreased with a concomitant increase in heme-iron nitrosyl complexes in the liver. Lipopolysaccharide-related nitric oxide generation is followed by an early decrease in the levels of cytochrome P450 and of testosterone hydroxylation activity in liver microsomes. Midazolam sleep time was prolonged by lipopolysaccharide. All these early changes were prevented by the inhibitor of nitric oxide synthase, N(G)-iminoethyl-L-ornithine. Moreover, lipopolysaccharide suppressed the gene expression of CYP2C11 and CYP3A2. Decreases in levels of cytochrome P450 and their mRNAs were more pronounced at 24 h after LPS administration, but apparently they are NO-independent. CONCLUSIONS: These results suggest that lipopolysaccharide-induced modulation of cytochrome P450 may occur via the interplay of two different mechanisms and that, especially in the early phase, nitric oxide-dependent inhibition is more important.     

Identification and structural basis of the reaction catalyzed by CYP121, an essential cytochrome P450 in Mycobacterium tuberculosis

The gene encoding the cytochrome P450 CYP121 is essential for Mycobacterium tuberculosis. However, the CYP121 catalytic activity remains unknown. Here, we show that the cyclodipeptide cyclo(l-Tyr-l-Tyr) (cYY) binds to CYP121, and is efficiently converted into a single major product in a CYP121 activity assay containing spinach ferredoxin and ferredoxin reductase. NMR spectroscopy analysis of the reaction product shows that CYP121 catalyzes the formation of an intramolecular C-C bond between 2 tyrosyl carbon atoms of cYY resulting in a novel chemical entity. The X-ray structure of cYY-bound CYP121, solved at high resolution (1.4 Å), reveals one cYY molecule with full occupancy in the large active site cavity. One cYY tyrosyl approaches the heme and establishes a specific H-bonding network with Ser-237, Gln-385, Arg-386, and 3 water molecules, including the sixth iron ligand. These observations are consistent with low temperature EPR spectra of cYY-bound CYP121 showing a change in the heme environment with the persistence of the sixth heme iron ligand. As the carbon atoms involved in the final C-C coupling are located 5.4 Å apart according to the CYP121-cYY complex crystal structure, we propose that C-C coupling is concomitant with substrate tyrosyl movements. This study provides insight into the catalytic activity, mechanism, and biological function of CYP121. Also, it provides clues for rational design of putative CYP121 substrate-based antimycobacterial agents.     

Using U0126 to dissect the role of the extracellular signal-regulated kinase 1/2 (ERK1/2) cascade in the regulation of gene expression by endothelin-1 in cardiac myocytes

The hypertrophic agonist endothelin-1 rapidly but transiently activates the extracellular signal-regulated kinase 1/2 (ERK1/2) cascade (and other signalling pathways) in cardiac myocytes, but the events linking this to hypertrophy are not understood. Using Affymetrix rat U34A microarrays, we identified the short-term (2-4 h) changes in gene expression induced in neonatal myocytes by endothelin-1 alone or in combination with the ERK1/2 cascade inhibitor, U0126. Expression of 15 genes was significantly changed by U0126 alone, and expression of an additional 78 genes was significantly changed by endothelin-1. Of the genes upregulated by U0126, four are classically induced through the aryl hydrocarbon receptor (AhR) by dioxins suggesting that U0126 activates the xenobiotic response element in cardiac myocytes potentially independently of effects on ERK1/2 signalling. The 78 genes showing altered expression with endothelin-1 formed five clusters: (i) three clusters showing upregulation by endothelin-1 according to time course (4 h > 2 h; 2 h > 4 h; 2 h approximately 4 h) with at least partial inhibition by U0126; (ii) a cluster of 11 genes upregulated by endothelin-1 but unaffected by U0126 suggesting regulation through signalling pathways other than ERK1/2; (iii) a cluster of six genes downregulated by endothelin-1 with attenuation by U0126. Thus, U0126 apparently activates the AhR in cardiac myocytes (which must be taken into account in protracted studies), but careful analysis allows identification of genes potentially regulated acutely via the ERK1/2 cascade. Our data suggest that the majority of changes in gene expression induced by endothelin-1 are mediated by the ERK1/2 cascade.