Role of the Membrane-Proximal Domain in the Initial Stages of Human Immunodeficiency Virus Type 1 Envelope Glycoprotein-Mediated Membrane Fusion

We have examined mutations in the ectodomain of the human immunodeficiency virus type 1 transmembrane glycoprotein gp41 within a region immediately adjacent to the membrane-spanning domain for their effect on the outcome of the fusion cascade. Using the recently developed three-color assay (I. Muñoz-Barroso, S. Durell, K. Sakaguchi, E. Appella, and R. Blumenthal, J. Cell Biol. 140:315–323, 1998), we have assessed the ability of the mutant gp41s to transfer lipid and small solutes from susceptible target cells to the gp120-gp41-expressing cells. The results were compared with the syncytium-inducing capabilities of these gp41 mutants. Two mutant proteins were incapable of mediating both dye transfer and syncytium formation. Two mutant proteins mediated dye transfer but were less effective at inducing syncytium formation than was wild-type gp41. The most interesting mutant proteins were those that were not capable of inducing syncytium formation but still mediated dye transfer, indicating that the fusion cascade was blocked beyond the stage of small fusion pore formation. Fusion mediated by the mutant gp41s was inhibited by the peptides DP178 and C34.

The human immunodeficiency virus type 1 (HIV-1) gp120-gp41 fusion machine consists of an assembly of viral envelope glycoprotein oligomers which forms a molecular scaffold responsible for bringing the viral membrane close to the target cell membrane and creating the architecture that enables lipid bilayers to merge (7). The fusion reaction undergoes multiple steps before the final event occurs which allows delivery of the nucleocapsid into the cell. In the case of influenza virus hemagglutinin (HA), we have dissected these steps kinetically and analyzed the molecular features of the kinetic intermediates (1). In order to examine the modus operandi of the fusion machine, mutations in various domains of viral envelope glycoproteins have been examined for their effect on the outcome of the fusion cascade. For instance, replacement of the membrane-spanning domain of influenza virus HA with a glycosylphosphatidylinositol anchor results in a very stable hemifusion intermediate (6). Moreover, single-site mutations in the fusion peptide of HA significantly affect fusion pore dilation (9). Recently, cytoplasmic tail acylation mutants of influenza virus HA were identified which induce transfer of lipids and small aqueous molecules but do not induce syncytium formation (4a).High-resolution crystallographic determinations (4, 10, 11) of gp41 fragments from HIV-1 have revealed a bent-in-half, antiparallel, heterotrimeric coiled-coil structure. This is made up of a triple-stranded coiled coil of α-helices from the leucine zipper-like 4-3 repeat domain in gp41 close to the N-terminal fusion peptide termed HR1 (8) flanked by α-helices from the domain in gp41 close to the C-terminal membrane anchor termed HR2 (8). Comparison with the crystal structure of the influenza virus HA2 subunits in a low-pH-induced conformation (2) reveals common structural motifs which provide growing support for the “spring-loaded” type of mechanistic models (3). In this scenario, activation of the fusion protein results in release of the fusion peptide and extension of the central coiled-coil structure. The new positioning of the fusion peptides at the tip of the stalk provides for easy contact with the target cell membrane. A small group of proximal fusion proteins which are simultaneously inserted into both the viral and target membranes would constitute a potential fusion site. A concerted collapse of this protein complex, actuated by the bending in half of the stalks at a central hinge region, would presumably position the C-terminal transmembrane anchors and N-terminal fusion peptides on top of each other in the center, bring the two membranes into contact, and thus allow formation of the fusion pore (7). In this study, we examined the effects on the various stages of the fusion reaction of mutations in the region between HR2 and the transmembrane (TM) anchor (Fig. ​(Fig.1)1) described in detail by Salzwedel et al. (8a). Open in a separate windowFIG. 1Amino acid sequence and mutations in gp41. FP is the predicted fusion peptide region, and HR1 and HR2 (8) represent, respectively, the N-terminal and C-terminal α-helices of the triple-stranded coiled coil (4, 11). Mutations in the region between HR2 and the TM anchor include deletions of amino acids 665 to 682 and 678 to 682, insertion of a FLAG sequence (YKDDDD), insertion of a DAF sequence (PNKGSGTTS), scrambling of the underlined sequence to SC7 (INNWNFT), and replacement of the five tryptophans with alanines [W(1-5)A]. Peptide C34 represents HR2 amino acids 628 to 663, and peptide DP178 represents amino acids 638 to 673.Mutagenesis of HIV-1 env, construction of plasmids, cell surface expression, CD4 binding, and cell fusion were performed as previously described (8a). The simian virus 40-based env expression plasmids (1 μg of DNA) were transfected into COS-1 cells in 35-mm-diameter plates by using DEAE-dextran (1 mg/ml). At 14 h posttransfection, the cells were replated, and starting at 36 to 48 h posttransfection, they were incubated with 20 μM CMAC (7-amino-4-chloromethylcoumarin) in Dulbecco modified Eagle medium overnight at 37°C. All constructs expressed similar amounts of envelope glycoprotein on the cell surface (8a). The transfected cells were then washed and incubated in fresh medium for 2 h at 37°C before addition of HeLa-CD4 cells which were labeled in the membrane with octadecyl indocarbocyanine (DiI) and in the cytosol with calcein as previously described (7). The method used to detect cell-cell fusion was a three-color assay (7) based on the redistribution of fluorescent probes between effector and target cells upon fusion. The application of three different probes was used to monitor lipid versus cytosolic mixing in the same cell population. Fluorescently labeled gp120-gp41-expressing cells and CD4⁺ cells were cocultured at a 1:10 ratio for 2 h at 37°C in uncoated microwells (MatTek Corp., Ashland, Mass.). Bright-field and fluorescent images were acquired with an Olympus IX70 microscope coupled to a charge-coupled device camera (Princeton Instruments, Trenton, N.J.) with a 40× UplanApo oil immersion objective. Fluorescein isothiocyanate (exciter, BP470-490; beam splitter, DM505; emitter, BA515-550), rhodamine (exciter, BP530-550; beam splitter, DM570; emitter, BA590), and 4′,6-diamidino-2-phenylindole (DAPI) (exciter, D360/40; beam splitter, 400DCLP; emitter, D450/60) optical filter cubes were carefully chosen to avoid spillover when observing the fluorescence of the three dyes. For each sample, three or four different fields were collected, and data were analyzed by overlaying the images using Metamorph software (Universal Imaging Corporation, West Chester, Pa.). The percentage of lipid mixing and cytoplasmic mixing was calculated as 100 times the number of COS-1 cells stained with DiI and calcein divided by the total number of COS-1–HeLa-CD4 conjugates. Although not all COS-1 cells express env since the transfection efficiency is not 100%, env-expressing COS-1 cells are more likely to adhere to HeLa-CD4 cells.Figure ​Figure22 shows a montage of video images taken 2 h following incubation of COS-1 cells expressing wild-type (WT), W(1-5)A, and +DAF env with HeLa-CD4 cells at 37°C. As described in detail in the legend to Fig. ​Fig.2,2, we clearly observed COS-1 cells attached to HeLa-CD4 cells, which showed continuity of all three dyes (CMAC, calcein, and DiI). We know that for +DAF and W(1-5)A env-expressing COS-1 cells, these images do not represent syncytia since even small heterokaryons will show up in the MAGI cell assay (6a), which is based on the transfer of HIV-1 Tat coexpressed with env in COS-1 cells to HeLa-CD4 cells as a result of cell fusion. This transfer induces the expression of a β-galactosidase reporter gene engineered in HeLa-CD4 (MAGI) cells under the control of the viral long terminal repeat promoter (8a). Because the β-galactosidase has been modified to contain a nuclear targeting signal, the nuclei of the resulting heterokaryons stain dark blue with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) in situ. The MAGI assay is extremely sensitive and clearly identifies syncytia as small as two nuclei. Such nuclei were common for the Δ678-682 mutant, which produced syncytia with an average size of ∼5 nuclei (Fig. ​(Fig.3).3). The assay can detect even a few of these fusion events per 100,000 cells. In the MAGI assay, we did not observe any blue nuclei with the W(1-5)A and +DAF constructs, an experiment repeated several times. The three-color assay therefore reveals a distinct phenotype exhibited by the +DAF and W(1-5)A mutant envelope glycoproteins, which form small fusion pores allowing movement of lipids and small molecules (<1,000 Da) but not of large molecules (HIV-1 Tat is about 14 kDa [4b]). Open in a separate windowFIG. 2Three-color assay for WT and mutant HIV-1 gp41s. Simian virus 40-based env expression plasmids (1 μg) containing WT (A to D), W(1-5)A (E to H), and +DAF (I to L) env genes were transfected into COS-1 cells in 35-mm plates using DEAE-dextran (1 μg/ml). At 14 h posttransfection, the cells were replated, and starting at 36 to 48 h posttransfection they were incubated with 20 μM CMAC in Dulbecco modified Eagle medium overnight at 37°C. All constructs expressed similar amounts of envelope glycoprotein on the cell surface (8a). The transfected cells were then washed and incubated in fresh medium for 2 h at 37°C before addition of HeLa-CD4 cells which were labeled in the membrane with DiI and in the cytosol with calcein as previously described (7). The COS-1 cells, labeled with CMAC, were cocultured 1:10 at 37°C for 2 h with HeLa-CD4 cells labeled with DiI and calcein, and images were examined by bright-field microscopy (A, E, and I) and fluorescence microscopy for CMAC staining (B, F, and J), for DiI staining (C, G, and K), and for calcein staining (D, H, and L). CMAC is a fluorescent chloromethyl derivative that freely diffuses through the membranes of live cells. Once inside the cell, this mildly thiol-reactive probe undergoes what is believed to be a glutathione S-transferase-mediated reaction to produce membrane-impermeant fluorescent dye adducts with glutathione, as well as with other intracellular components. Staining of COS-1 cells with CMAC gives rise to bright fluorescence due to reaction with proteins in the perinuclear, endoplasmic reticulum, and Golgi regions, which are immobile, as well as to weaker fluorescence due to the fluorescent glutathione adduct (molecular mass, ∼600 Da) in the cytosol, which is able to diffuse through small fusion pores. The COS-1 cells identified by CMAC staining (B, F, and J) are large and often appear multinuclear, although we do not know whether the round granular structures seen by bright-field microscopy of the COS-1 cells are nuclei or large granules. Panels A to D show one large cell triple stained with CMAC, DiI, and calcein (indicated by a star). DiI is internalized after 2 h at 37°C and appears punctate with nuclear sparing due to its localization in membranes of intracellular organelles. Calcein (465 Da) is evenly distributed throughout the cell (D). One large, granular COS-1 cell (A, left) is only stained with CMAC (B); its lack of staining with DiI (C) and calcein (D) indicates that it has not fused with HeLa-CD4 cells. In panel F, a large structure is seen which seems in continuity with CMAC. However, since the bottom left part of this structure is not in continuity with DiI (G) and calcein (H), it represent two cells. The top right cell (indicated by a star) is in continuity with CMAC, DiI, and calcein. Since COS-1 cells expressing W(1-5)A env do not produce blue nuclei when incubated with MAGI cells (see Fig. ​Fig.3),3), which requires transfer of the 14-kDa HIV-1 Tat protein (see text), we conclude that this COS-1–HeLa-CD4 conjugate represents a phenotype in which small fusion pores form, allowing movement of lipids and small molecules (<1,000 Da) but not of large molecules. The same phenotype is seen with COS-1 cells expressing DAF env: the COS-1–HeLa-CD4 conjugate indicated by a star in panels J, K, and L is in continuity with CMAC, DiI, and calcein but does not allow transfer of HIV-1 Tat (see Fig. ​Fig.33).Open in a separate windowFIG. 3Fusogenic activity of WT and mutant HIV-1 gp41s. The three-color assay was performed as described in the legend to Fig. ​Fig.2.2. Since multiple rounds of fusion may interfere with quantitation in the case of WT and mutant env genes which produce a large number of blue nuclei after 24 h at 37°C (grey bars), incubations were done for 2 h at 37°C. Black bars represent 100 times the number of COS-1 cells stained with DiI and calcein over the total number of COS-1–HeLa-CD4 conjugates measured in the three-color assay. The data are representative of five separate experiments. In each experiment, a total of 30 to 50 COS-1–HeLa-CD4 conjugates were counted. The number of nuclei per syncytium (grey bars) was obtained from the MAGI assay (8a) and represents the ability of HIV-1 Tat to transfer from COS-1 cells to HeLa-CD4 cells.We tallied data from many cell pairs similar to those shown in Fig. ​Fig.22 and plotted the average percentage of COS-1 cells stained with DiI and calcein. Figure ​Figure33 shows the data for the WT and a number of mutants described by Salzwedel et al. (8a). The data fall into three groups, in which the envelope glycoproteins mediate (i) both dye and HIV-1 Tat redistribution (WT, Δ678-682, and SC7), (ii) neither dye nor HIV-1 Tat redistribution (Δ665-682 and +FLAG), or (iii) dye but not HIV-1 Tat redistribution [W(1-5)A and +DAF]. The latter represents a nonexpanding fusion pore phenotype.Dye redistribution induced by WT and mutant gp41s was inhibited by the peptide inhibitors DP178 and C34 (Fig. ​(Fig.4).4). The latter peptide is from the HR2 sequence (residues 628 to 663) which forms the flanking peptide of the heterotrimeric coiled coil in the crystal structure. DP178 is frameshifted 10 amino acids toward the C terminus (residues 638 to 673). The inhibition data indicate that dye redistribution mediated by WT and mutant gp41 molecules is specific for the gp120-gp41-induced fusion reaction and not due to nonspecific transfer. Interestingly, W(1-5)A and SC7 exhibited greater sensitivity than the WT to DP178 inhibition. In the case of C34, inhibition was about the same for the WT and the two mutants. We observed no inhibition by DP178 or C34 of HIV-2 env-mediated fusion at up to 100 nM peptide (data not shown). Open in a separate windowFIG. 4Inhibition of cell-cell fusion by DP178 and C34 peptides. Cell fusion was calculated as a percentage of the control by using the three-color assay method shown in Fig. ​Fig.22 and ​and33 and described in the text for the WT, W(1-5)A, and SC7.Although the crystal structure of the gp41 core (4, 11) is based on the HR1-HR2 coiled coil, it is possible that in intact gp41 the bundle is extended to include amino acids downstream from HR2 and upstream from HR1. Extension of the coiled coil might lead to tilting of the TM anchor, which is presumably important for producing sufficient lipid curvature to form a fusion junction (1). Removal of amino acids 665 to 682 may leave no possibility to form this extended coiled coil. Similarly, insertion of the FLAG sequence, which contains four aspartic acid residues, would presumably insert charged residues into a hydrophobic domain, which could also prevent extension of the coiled coil. The other mutations presumably allow extended coiled-coil formation but reduce its efficiency because of weaker interactions between the amino acids in the extended region. The coiled-coil structure might be so frail in mutant gp41s W(1-5)A and +DAF that it is not present for a sufficient amount of time to create the fusion pore dilation necessary to allow transfer of HIV-1 Tat. Since the Δ678-682 and SC7 proteins are, to a limited extent, capable of inducing syncytium formation and dye transfer, we surmise that they possess intermediate extended coiled-coil-forming propensities.Based on the structural information about the gp41 core (4, 10, 11), it has been proposed that the binding site for the peptide inhibitors is in the HR1 bundle. The C34 and DP178 peptides presumably bind in the same way as the corresponding amino acid sequence regions of the three HR2 helices in the crystal structures. At this position, the peptides would sterically block the regular binding of the HR2 helices to the inner core of HR1 helices and thus prevent formation of the bent-in-half, antiparallel, heterotrimeric coiled-coil structure presumably required to bring the viral and target cell membranes into contact for fusion. Since C34 corresponds to HR2 with no amino acids in the extended region, we do not expect any enhanced inhibitory effect on fusion mediated by the mutant gp41s. Figure ​Figure4b4b shows that this is the case. Since DP178 does contain 10 amino acids downstream from HR2 whose interaction with amino acids upstream from HR1 is weaker in the mutants, we expect greater sensitivity to DP178 inhibition in the mutant proteins. This does seem to be the case, as shown in Fig. ​Fig.44a.The recent high-resolution X-ray crystallographic determination of the structure of the gp41 core from HIV-1 provides well-defined landmarks in the terrain the viral envelope glycoproteins navigate following CD4 and coreceptor-induced conformational changes (5). The structures include neither fusion peptides and TM anchors nor regions between those domains and HR1 and HR2, respectively, which are crucial for fusion activity. Therefore, mutagenesis of those undetermined domains combined with sensitive assays for the activity of the modified proteins will lead to refinement of our thinking about the HIV-1 gp120-gp41 fusion machine.     

Differential impairment of lytic and cytokine functions in senescent human immunodeficiency virus type 1-specific cytotoxic T lymphocytes

Telomere length is abnormally short in the CD8(+) T-cell compartment of human immunodeficiency virus type 1 (HIV-1)-infected persons, likely because of chronic cell turnover. Although clonal exhaustion of CD8(+) cytotoxic T lymphocytes (CTL) has been proposed as a mechanism for loss of antigen-specific responses, the functional consequences of exhaustion are poorly understood. Here we used telomerase transduction to evaluate the impact of senescence on CTL effector functions. Constitutive expression of telomerase in an HIV-1-specific CTL clone results in enhanced proliferative capacity, in agreement with prior studies of other human cell types. Whereas the CTL remain phenotypically normal in terms of antigenic specificity and requirements for proliferation, their cytolytic and antiviral capabilities are superior to those of control CTL. In contrast, their ability to produce gamma interferon and RANTES is essentially unchanged. The selective enhancement of cytolytic function in memory CTL by ectopic telomerase expression implies that loss of this function (but not cytokine production) is a specific consequence of replicative senescence. These data suggest a unifying mechanism for the in vivo observations that telomere lengths are shortened in the CD8(+) cells of HIV-1-infected persons and that HIV-1-specific CTL are deficient in perforin. Telomerase transduction could therefore be a tool with which to explore a potential therapeutic approach to an important pathophysiologic process of immune dysfunction in chronic viral infection.     

Butterfly–host plant synchrony determines patterns of host use across years and regions

Variation in the degree of synchrony among host plants and herbivores can disrupt or intensify species interactions, alter the strength of natural selection on traits associated with phenological timing, and drive novel host plant associations. We used field observations from three regions during four seasons to examine how timing of the butterfly herbivore Anthocharis cardamines relative to six host plant species (Arabis hirsuta, Cardamine pratensis, Arabis glabra, Arabidopsis thaliana, Thlaspi caerulescens and Capsella bursa‐pastoris) influenced host species use and the choice of host plant individuals within populations. Butterflies laid a larger fraction of their eggs on species that were closer to the butterfly's preferred stage of development than on other host species. Within host plant populations, butterflies showed a stronger preference for individuals with a late phenology when plants within the population were on average more developed at the time of butterfly flight. Our results suggest that changes in synchrony between herbivores and their host plants are associated with changes in both host species use and the choice of host plant individuals differing in phenology within populations. This is likely to be an important mechanism generating variation in interaction intensities and trait selection in the wild, and therefore also relevant for understanding how anthropogenic induced changes, such as global warming, will influence natural communities.     

Silencing the expression of the salivary sheath protein causes transgenerational feeding suppression in the aphid Sitobion avenae

Aphids produce gel saliva during feeding which forms a sheath around the stylet as it penetrates through the apoplast. The sheath is required for the sustained ingestion of phloem sap from sieve elements and is thought to form when the structural sheath protein (SHP) is cross‐linked by intermolecular disulphide bridges. We investigated the possibility of controlling aphid infestation by host‐induced gene silencing (HIGS) targeting shp expression in the grain aphid Sitobion avenae. When aphids were fed on transgenic barley expressing shp double‐stranded RNA (shp‐dsRNA), they produced significantly lower levels of shp mRNA compared to aphids feeding on wild‐type plants, suggesting that the transfer of inhibitory RNA from the plant to the insect was successful. shp expression remained low when aphids were transferred from transgenic plants and fed for 1 or 2 weeks, respectively, on wild‐type plants, confirming that silencing had a prolonged impact. Reduced shp expression correlated with a decline in growth, reproduction and survival rates. Remarkably, morphological and physiological aberrations such as winged adults and delayed maturation were maintained over seven aphid generations feeding on wild‐type plants. Targeting shp expression therefore appears to cause strong transgenerational effects on feeding, development and survival in S. avenae, suggesting that the HIGS technology has a realistic potential for the control of aphid pests in agriculture.     

Male aggression,limited female choice and the ontogeny of mating behaviour in the flesh fly Sarcophaga crassipalpis

Previous work has shown that male flesh flies (Sarcophaga crassipalpis Macquart) exhibit an ontogeny of behaviour from eclosion through sexual maturity that includes extensive changes in the expression of aggressive, non‐aggressive interactive and non‐interactive behaviours. To determine how the presence of a female flesh fly influences the manifestation of these behaviours, male flesh flies of different ages post‐eclosion are paired with same‐age females and their behaviours are monitored in a simple arena during a 50‐min observation period. All flies are socially isolated until pairing. Although the levels of expression of aggressive and non‐aggressive interactive behaviours are depressed relative to previous findings in male‐opponent pairs, the ontogeny of aggression still occurs as indicated by a significant increase, with age, in the agonistic behaviour ‘hold’. Similar to male‐opponent pairs and individual males, the performance by males of the non‐interactive behaviours ‘walking’ and ‘standing’ diminishes, whereas ‘upside‐down’ increases with age. By contrast, ‘grooming’ shows a significant age‐related decline. No courtship behaviours are observed in the males, although the aggressive behaviour ‘hold’ is a significant transition to mating. Females show no obvious courtship or rejection behaviours, although the significant increase in ‘upside‐down’ with age could possibly be a behavioural gateway to mating. The results of this study indicate that extensive age‐related changes encompassing the entire behavioural repertoire are intrinsic to male flesh flies and persist under a variety of different social contexts.     

Disease-associated polyglutamine stretches in monomeric huntingtin adopt a compact structure

Abnormal polyglutamine (polyQ) tracts are the only common feature in nine proteins that each cause a dominant neurodegenerative disorder. In Huntington's disease, tracts longer than 36 glutamines in the protein huntingtin (htt) cause degeneration. In situ, monoclonal antibody 3B5H10 binds to different htt fragments in neurons in proportion to their toxicity. Here, we determined the structure of 3B5H10 Fab to 1.9?? resolution by X-ray crystallography. Modeling demonstrates that the paratope forms a groove suitable for binding two β-rich polyQ strands. Using small-angle X-ray scattering, we confirmed that the polyQ epitope recognized by 3B5H10 is a compact two-stranded hairpin within monomeric htt and is abundant in htt fragments unbound to antibody. Thus, disease-associated polyQ stretches preferentially adopt compact conformations. Since 3B5H10 binding predicts degeneration, this compact polyQ structure may be neurotoxic.     

Structural basis of transfer between lipoproteins by cholesteryl ester transfer protein

Human cholesteryl ester transfer protein (CETP) mediates the net transfer of cholesteryl ester mass from atheroprotective high-density lipoproteins to atherogenic low-density lipoproteins by an unknown mechanism. Delineating this mechanism would be an important step toward the rational design of new CETP inhibitors for treating cardiovascular diseases. Using EM, single-particle image processing and molecular dynamics simulation, we discovered that CETP bridges a ternary complex with its N-terminal β-barrel domain penetrating into high-density lipoproteins and its C-terminal domain interacting with low-density lipoprotein or very-low-density lipoprotein. In our mechanistic model, the CETP lipoprotein-interacting regions, which are highly mobile, form pores that connect to a hydrophobic central cavity, thereby forming a tunnel for transfer of neutral lipids from donor to acceptor lipoproteins. These new insights into CETP transfer provide a molecular basis for analyzing mechanisms for CETP inhibition.     

Both estrogen receptor subtypes, ERα and ERβ, prevent aldosterone-induced oxidative stress in VSMC via increased NADPH bioavailability

Activation of vascular mineralocorticoid (MR) or estrogen receptors (ER) exerts opposing effects on vascular remodeling. As we have previously shown, activation of either estrogen receptor subtype, ERα or ERβ, is fully sufficient to attenuate vascular remodeling in aldosterone salt-treated rats. To further elucidate the underlying mechanism(s) we tested the hypothesis that ER and MR activation might differentially modulate vascular reactive oxygen species (ROS) generation. In support of this concept, aldosterone increased ROS generation in vascular smooth muscle cells as determined by quantitative dihydroethidium fluorescence microscopy. Co-treatment with the selective ERα agonist 16α-LE2, the selective ERβ agonist 8β-VE2 or the non-selective ER agonist 17β-estradiol (E2) significantly reduced aldosterone-induced ROS generation. The pure ER antagonist ICI 182,780 completely blocked these salutary effects of E2, 16α-LE2 and 8β-VE2. Activation of ERα or ERβ fully blocked the reduction of intracellular nicotinamide adenine dinucleotide phosphate (NADPH) levels observed in aldosterone treated vascular smooth muscle cells. Intracellular NADPH levels were closely associated with expression and activity of the NADPH generating enzyme glucose-6-phosphate dehydrogenase. In conclusion, estrogens attenuate the detrimental vascular effects of excessive MR activation at least in part by preventing the depletion of intracellular NADPH levels.