Compromised Aortic Vasoreactivity in Male Estrogen Receptor-α-Deficient Mice during Acute Lipopolysaccharide-Induced Inflammation

0013-7227/07/$15.00/0 Printed in U.S.A.

Endocrinology 148(3):1403–1411 Copyright © 2007 by The Endocrine Society doi: 10.1210/en.2006-0399

Compromised Aortic Vasoreactivity in Male Estrogen Receptor-␣-Deficient Mice during Acute Lipopolysaccharide-Induced Inflammation Ana M. Corbacho, Jason P. Eiserich, Luis A. Zuniga, Giuseppe Valacchi, and Amparo C. Villablanca Department of Internal Medicine, Divisions of Pulmonary/Critical Care Medicine (A.M.C., G.V.), Nephrology (J.P.E.), and Cardiovascular Medicine (L.A.Z., A.C.V.), and Department of Physiology and Membrane Biology (J.P.E.), School of Medicine, University of California, Davis, California 95616 Activation of the estrogen receptor-␣ (ER␣) mediates the vasculoprotective effects of estrogen, in part, through modulating nitric oxide (NO) production and vasodilation. Whereas inflammation is accompanied by altered vascular reactivity and underlies the pathogenesis of vascular disease, the role of the ER␣ in the vascular responses associated with acute systemic inflammation remains poorly characterized. Contractile and relaxation responses of isolated aortic segments were investigated 12 h after ip injection of saline or lipopolysaccharide (LPS, 10 mg/kg) in male wild-type (ER␣ⴙ/ⴙ) and ER␣deficient (ER␣ⴚ/ⴚ) mice. As previously observed, LPS-injected ER␣ⴙ/ⴙ mice displayed reduced contractile responses to phenylephrine and enhanced vasodilation in response to acetylcholine. In contrast, aortic tissues from LPS-injected ER␣ⴚ/ⴚ mice displayed enhanced contractile responses and reduced sensitivity to acetylcholine- and sodium nitroprusside-in-

A

ROLE FOR ESTROGEN in normal cardiovascular development and function in both males and females has been established (1). Two estrogen receptors (ER), ER␣ and ER␤, have been characterized (2– 6), and they are both present in endothelial (7) and vascular smooth muscle cells (8, 9). The direct actions of estrogen on the cardiovascular system include rapid nongenomic effects and long-term effects involving changes in gene expression (10, 11). Estrogen rapidly induces arterial vasodilation by activating the endothelial nitric oxide synthase (eNOS) through a nongenomic mechanism, presumably through the activation of a tyrosine kinase pathway (11–14). Estrogen’s genomic effects include the stimulation of eNOS expression (10, 15–17). Thus, both nongenomic and genomic effects of estrogen can increase endothelium-derived nitric oxide, inducing vasodilation and increased blood flow. Although eNOS activity is crucial in the regulation of basal vascular tone, NO can also be generated by two other NOS isoforms: namely, the neuronal NO synthase and the inducible NO synthase (iNOS) (18). Particularly, iNOS is of imFirst Published Online December 7, 2006 Abbreviations: ACh, Acetylcholine; eNOS, endothelial nitric oxide synthase; ER, estrogen receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; PBST, PBS with 0.5% Tween 20; PE, phenylephrine; sGC, soluble guanylate cyclase; SNP, sodium nitroprusside. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

duced vasodilation. LPS treatment in ER␣ⴙ/ⴙ and ER␣ⴚ/ⴚ mice resulted in similar increased levels of systemic NO production and inducible NO synthase expression in the vascular wall. However, expression of mRNA and protein for endothelial NOS and soluble guanylate cyclase (␣- and ␤-subunits) were significantly reduced in aortic tissues from LPS-treated ER␣ⴚ/ⴚ animals, possibly accounting for reduced endothelial NO production and reduced smooth muscle responses to NO. These findings represent new evidence of the functional role of ER␣ in the male vasculature and suggest that during acute LPS-induced inflammatory responses, the ER␣ mediates the sustained expression of the molecular machinery essential for endothelial NO synthesis (i.e. endothelial NOS) and the vascular responses to NO (i.e. soluble guanylate cyclase). (Endocrinology 148: 1403–1411, 2007)

portance because its expression and function are enhanced in inflammatory immune processes, which have been associated with the development of cardiovascular disease (19 –21). Lipopolysaccharide (LPS), a cell wall component of Gramnegative bacteria, alone or in combination with inflammatory cytokines, induces the expression of iNOS in the vascular wall (22–25). Once expressed, iNOS produces relatively large and sustained fluxes of NO, which mediates vasodilatation, hypotension, and shock (22–25). Although it has also been demonstrated that estrogen can induce the expression of iNOS in vascular smooth muscle cells (26, 27), the role of the ER␣ in vascular contractile and relaxant responses under conditions of acute inflammation remains poorly characterized. Epidemiological studies have revealed an increased mortality risk in men during sepsis (28 –30), and several lines of evidence indicate that estrogen may play a protective role during injury and sepsis (21, 31). Although circulating estrogen levels are low in males, peripheral production of estrogen from testosterone occurs in several tissues via the enzymatic activity of P450 aromatase (1), and several pieces of evidence of the effect of estrogen in the vasculature of males exist (1, 32). A recent epidemiological study has shown an association between the presence of ER␣ polymorphism and cardiovascular disease in males (33). Furthermore, endothelial dysfunction and premature coronary arteriosclerosis have been described in a male patient with genetic mutation in the ER␣ gene (34 –36), and a role for ER␣ in early

1403

1404

Endocrinology, March 2007, 148(3):1403–1411

susceptibility to atherosclerosis has been described in male mice (37). Contraction-relaxation vascular studies on male mice have shown that aromatase deficiency (resulting in estrogen production deficiency) results in an enhanced response to noradrenaline and diminished sensitivity to acetylcholine (ACh) (1, 38). Despite the fact that several studies have demonstrated 1) the contribution of the ER␣ on normal vascular responses in males (1, 32, 38), 2) that estrogen modulate the expression of NO synthases in vascular cells (26, 27, 39), and 3) that estrogen may play a protective role during sepsis (21, 31), the extent to which ER␣ plays a role in vascular reactivity in males during sepsis and inflammation remains unknown. Based upon these premises, we have hypothesized that in the absence of the full-length ER␣, vascular responses typically observed during sepsis will be altered due to changes in the expression and activity of NO synthases localized in the vascular wall. To understand the functional role of ER␣ during acute inflammation, we determined vascular reactivity changes after LPS-induced inflammation in aortas of male mice deficient in ER␣ (ER␣⫺/⫺) compared with wild-type mice (ER␣⫹/⫹). Materials and Methods Animals ␣ERKO mice (40), also named ER␣ Neo knockout mice, were used in this study. Heterozygous animals were obtained from Dr. Dennis Lubahn (University of Missouri) and mated to yield progeny deficient in the full-length ER␣ (ER␣⫺/⫺) and wild-type littermate controls having intact ER␣ (ER␣⫹/⫹). ER␣⫺/⫺ animals are deficient in the full-length wild-type ER␣ protein, and their reproductive function is abolished; however, the disrupting neo sequence used to generate the knockout results in an alternative variant of ER␣ protein, which retains estradiolbinding activity and can mediate some of estradiol’s effects (41). Mice were housed in standard conditions of temperature, 12-h light, 12-h dark cycle, and humidity-controlled environment in a dedicated pathogen-free barrier facility at the University of California, Davis. Tail DNA from progeny of heterozygous matings were genotyped as previously described (40) using DNeasy spin columns (QIAGEN, Valencia, CA) per the manufacturer’s instructions. PCR amplification was used to distinguish homozygous mutants from heterozygotes and from normal animals. Primers used to amplify products specific for the wild-type ER␣ gene and for the disrupted ER␣ gene were synthesized as custom primers (Life Technologies, Inc., Rockville, MD). Primer sequences for presence of the targeted ER␣ gene in homozygous mutants (a 649-bp fragment only) and animals with the normal wild-type gene (a 239-bp fragment only) were as follows: ER␣⫺/⫺ (KO) forward 5⬘-TGAATGAACTGCAGGACGAG3-⬘ and reverse 5⬘-AATATCACGGGTAGCCAACG-3⬘; ER␣⫹/⫹ (WT) forward 5⬘-CTACGGCCAGTCGGGCAT-3⬘; and reverse 5⬘-AGACCTGTAGAAGGCGGGAG-3⬘ (40).

Experimental design Six- to seven-week-old ER␣⫹/⫹ and ER␣⫺/⫺ littermate male mice were used. Animals were ip injected with either saline or a sublethal dose of LPS (10 mg/kg) and killed 12 h later by pentobarbital overdose (200 mg/kg, ip) (42). At the LPS dose and time point selected, cytokine levels were previously shown to be significantly increased (42). Blood was taken by cardiac puncture, and plasma was collected and stored at ⫺70 C. The descending thoracic aorta was excised, cleansed of adhering tissue, and cut in 3- to 4-mm-long segments. For each mouse, a segment of the aorta was harvested for vascular contraction-relaxation studies, and two segments were stored at ⫺70 C for subsequent RNA and protein extraction.

Corbacho et al. • Vasoreactivity of ER␣⫺/⫺ Male Mice in Endotoxemia

Vascular contraction-relaxation studies Individual aortic rings were suspended from a Radnoti isometric transducer in oxygenated tissue baths containing bicarbonate-buffered Krebs-Henseleit (K-H) solution (118 mm NaCl, 4.6 mm KCl, 27.2 mm NaHCO3, 1.2 mm KH2PO4, 1.2 mm MgSO4, 1.75 mm CaCl2, 0.03 mm Na2-EDTA, and 11.1 mm glucose, pH 7.4). According to length-tension curves previously established in our laboratory, a passive load of 1.8 g was applied and the aortic segments were allowed to equilibrate for approximately 1 h with frequent readjustment of tension until reaching a stable baseline. Contractions induced with KCl (70 mm) were elicited in the presence of indomethacin (5 ␮m) to determine the maximal contractile capacity. Rings were washed, allowed to equilibrate for 40 min, and contracted with phenylephrine (PE). To determine the maximum contractile response, a dose-response curve to PE (1 nm to 3 ␮m) was obtained. To evaluate the vasodilatory response, a single dose of PE (50 –150 nm) was used to develop similar tension values in all groups and subsequently cumulative doses of either ACh (0.1 nm to 3 ␮m) or sodium nitroprusside (SNP, 1–300 nm) were added to the tissue bath to invoke endothelial-cell-dependent or endothelial-cell-independent relaxation, respectively. Doses were added with an interval of approximately 1 min between them. All aortas were studied with intact endothelium. Data were collected and analyzed using PowerLab software (ADI Instruments, Colorado Springs, CO).

Quantitation of nitric oxide metabolites in plasma Quantitative measurements of plasma nitrite (NO2⫺) and nitrate (NO3⫺) were performed as an index of global nitric oxide production 12 h after LPS injection, as described previously (43). Briefly, the analytical procedure is based on acidic reduction of NO2⫺ and NO3⫺ to NO by vanadium (III) at 90 C, and purging of NO with helium into an Antek 7020 chemiluminescence NO detector (Antek Instruments, Houston, TX).

Real-time PCR analysis Total RNA from tissues was purified using TRIzol reagent, and firststrand cDNA was generated using 1 ␮g total RNA and the Superscript II system, according to the manufacturer’s instructions (Invitrogen Life Technologies, Carlsbad, CA). Primers were designed using Primer Express software (Applied Biosystems, Foster City, CA) and synthesized as custom primers (Life Technologies). Sequences were as follows: iNOS forward 5⬘-CAGCTGGGCTGTACAAACCTT-3⬘ and reverse 5⬘-CATTGGAAGTGAAGCGTTTCG-3⬘; eNOS forward 5⬘-CCTTCCGCTACCAGCCAGA-3⬘ and reverse 5⬘-CAGAGATCTTCACTGCATTGGCTA3⬘; sGC3␣ forward 5⬘-TTTGTCATCCGGGTGAGGAG-3⬘ and reverse 5⬘-CCTTGACGATTTCTTCACCGAG-3⬘; sGC␤ forward 5⬘-TTGCGTGTCCTGGGATCTAAT-3⬘ and reverse 5⬘-GGCATCGAGGTTCTGCAAAA-3⬘; and GAPDH forward 5⬘-TGCACCACCAACTGCTTAG-3⬘ and reverse 5⬘-GGATGCAGGGATGATGTTC-3⬘. PCR were set up in 25-␮l volumes, consisting of 2.5 ␮l cDNA, 0.5 ␮l forward and 0.5 ␮l reverse 10 ␮m primers, 12.5 ␮l SYBR Green PCR Master Mix (Applied Biosystems), and 9 ␮l RNase-/DNase-free water (Invitrogen). For all primer sets, a denaturing step at 94 C for 10 min was followed by 40 cycles of denaturing at 94 C for 30 sec, annealing at 60 C for 45 sec, and extension at 72 C for 30 sec. Real-time PCR was performed using an AbiPrism 7900HT sequence detector, and data were analyzed using SDS 2.1 software (Applied Biosystems). The relative concentration of the corresponding mRNA was measured as the number of cycles of PCR required to reach threshold fluorescence and normalized against that of an internal standard gene (GAPDH).

Western blot analysis Aortic tissues were homogenized in RIPA buffer [50 mm Tris (pH 7.4), 0.5% Nonidet P-40, 0.2% sodium deoxycholate, 100 mm NaCl, 1 mm EGTA, 1 mm phenylmethylsulfonyl fluoride, 1 ␮g/ml aprotinin, 1 mm sodium orthovanadate, and 1 mm NaF). Protein quantitation was performed using Bio-Rad Dc Protein Assay (Bio-Rad Laboratories, Hercules, CA). Whole lysate proteins were denatured in 4⫻ electrophoresis buffer and boiled for 5 min. Samples (40 ␮g total protein per lane) were

Corbacho et al. • Vasoreactivity of ER␣⫺/⫺ Male Mice in Endotoxemia

resolved in 4 –20% Tris-HCl Ready Gel Precast Gel (Bio-Rad) and electroblotted onto polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA). Membranes were blocked in 3% milk in PBS with 0.5% Tween 20 (PBST) overnight and probed with the primary antibody in 1% milk/PBST for 2 h at room temperature. Primary antibodies were all rabbit polyclonal antibodies, and dilutions were as follows: 1:500 anti-iNOS (sc-651; Santa Cruz Biotechnology, Santa Cruz, CA); 1:1000 anti-eNOS (no. 9572; Cell Signaling Technology, Inc., Beverly, MA); 1:100 anti-GC␣ and 1:500 anti-sGC␤1 (nos. 160895 and 160897; Cayman Chemical, Ann Arbor, MI); and 1:1000 anti-GAPDH (Abcam Inc., Cambridge, MA). Membranes were washed with PBST and incubated for 1 h at room temperature with horseradish-peroxidase-conjugated secondary antibody (1:10,000; Pierce, Rockford, IL) in 1% milk/ PBST. Membranes were washed, incubated with West Pico chemiluminescent substrate (Pierce), and visualized using Super RX film (Fuji Photo Film, Du¨sseldorf, Germany). Densitometry was performed using a FujiFilm LAS-3000 imaging system and Image Gauge 4.22 software. Relative intensities for iNOS, eNOS, soluble guanylate cyclase (sGC) ␣-subunit (sGC␣) and ␤-subunit (sGC␤) were normalized against that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH, used as internal loading control).

Statistical analysis Results from vascular contraction-relaxation studies were subjected to statistical analysis using the two-way ANOVA test followed by Bonferroni’s post hoc test to compare individual means (Prism, version 4.0b; GraphPad Software, Inc., San Diego, CA). Real-time and Western blot data were analyzed by two-way ANOVA followed by one-way ANOVA and the Bonferroni’s multiple comparison test. Data are presented as mean ⫾ sem. P ⬍ 0.05 denoted statistical significance.

Results Survival after ip LPS injection

Twelve hours after saline or LPS injection, all ER␣⫹/⫹ and ER␣⫺/⫺ mice had survived.

Endocrinology, March 2007, 148(3):1403–1411

1405

Vasomotor responses after LPS-induced inflammation are impaired in male ER␣⫺/⫺ mice

To evaluate the role of the ER␣ in the vascular contractile responses during acute inflammation, isolated aortic segments from saline- or LPS-injected ER␣⫹/⫹ and ER␣⫺/⫺ were subjected to ex vivo PE-induced contraction. Representative traces of PE dose responses are shown in Fig. 1A, and the graphical representation of four independent experiments (total n ⫽ 8 nice per group) is shown in Fig. 1B. No statistical differences were observed between saline-injected ER␣⫹/⫹ and saline-injected ER␣⫺/⫺ mice, between salineinjected ER␣⫹/⫹ and LPS-injected ER␣⫹/⫹ groups, and between saline-injected ER␣⫺/⫺ and LPS-injected ER␣⫺/⫺ groups. However, maximum tension values for LPS-injected ER␣⫺/⫺ mice were statistically higher than those for LPSinjected ER␣⫹/⫹ mice. Data were analyzed by two wayANOVA, an interaction (P ⫽ 0.45) was observed between LPS-injected ER␣⫹/⫹ and LPS-injected ER␣⫺/⫺ mice, the posttest analysis indicated significant differences at 1–3 ␮m PE (P ⬍ 0.05). To evaluate the role of the ER␣ in vasodilatory responses during acute inflammation, aortic rings were precontracted with PE (50 –150 nm) to reach equivalent tension values in all groups, and endothelium-dependent relaxation responses were determined by adding increasing doses of ACh (Fig. 2A). No statistical differences were observed between salineinjected ER␣⫹/⫹ and saline-injected ER␣⫺/⫺ mice. However, aortic rings from LPS-injected ER␣⫹/⫹ mice were more sensitive to ACh-dependent relaxation (EC50 ⫽ 30 nm; 100% relaxation at 300 nm ACh) than saline-injected ER␣⫹/⫹ mice (EC50 ⫽ 110 nm; 80% relaxation at maximum 1 ␮m ACh dose). In contrast, whereas saline-injected ER␣⫺/⫺ mice showed a normal vasodilatory response (EC50 ⫽ 110 nm; 80% relax-

FIG. 1. Isometric tension developed by aortic rings contracted with PE. A, Representative traces of isometric tension developed by aortic segments with the addition of increasing concentrations of PE (1 nM to 3 ␮M). The addition of consecutive PE doses was separated by 1-min intervals. B, Dose-response effect of PE on isometric tension values developed by aortic segments. Data were analyzed by two-way ANOVA; an interaction (P ⫽ 0.45) was observed between LPS-injected ER␣⫹/⫹ and LPS-injected ER␣⫺/⫺ mice, and the posttest analysis indicated a significant difference at 1–3 ␮M PE (*, P ⬍ 0.05). Data summarize the results of four independent experiments. Data are mean ⫾ SEM; n ⫽ 8 mice per group.

1406

Endocrinology, March 2007, 148(3):1403–1411

Corbacho et al. • Vasoreactivity of ER␣⫺/⫺ Male Mice in Endotoxemia

FIG. 2. Vasodilatory effects of ACh and SNP. A, Endothelium-dependent relaxation induced by ACh. Dose responses to ACh (0.1 nM to 3 ␮M) were obtained in PE (50 –150 nM) precontracted aortic rings of saline- or LPS-injected ER␣⫹/⫹ and ER␣⫺/⫺ male mice. Analysis by two-way ANOVA revealed an interaction between LPS-injected ER␣⫹/⫹ and LPS-injected ER␣⫺/⫺ mice (P ⬍ 0.0001), and the posttest analysis indicated significant differences at 30 nM to 3 ␮M ACh (*, P ⬍ 0.01 or P ⬍ 0.001; see text for details). An interaction was observed between saline-injected ER␣⫺/⫺ and LPS-injected ER␣⫺/⫺ mice (P ⬍ 0.0001), and significant differences were observed at 100 nM to 3 ␮M ACh (⫹, P ⬍ 0.001). No statistical differences were observed between saline-injected ER␣⫹/⫹ and saline-injected ER␣⫺/⫺ mice. Data summarize the results of four independent experiments. Data are mean ⫾ SEM; n ⫽ 8 mice per group. B, Endothelium-independent relaxation induced by SNP. Dose-response profiles to SNP (1–300 nM) were obtained in aortic rings contracted with PE (50 –150 nM). Analysis by two-way ANOVA revealed an interaction between saline-injected ER␣⫺/⫺ and LPS-injected ER␣⫺/⫺ mice (P ⬍ 0.46), and the posttest analysis indicated significant differences at 100 –300 nM SNP (*, P ⬍ 0.05). Data summarize the results of three independent experiments. Data are mean ⫾ SEM; n ⫽ 5 mice per group.

ation at 1 ␮m ACh), aortic rings from LPS-treated ER␣⫺/⫺ mice remained highly contracted despite the addition of ACh, reaching only 40% of relaxation values at maximum doses of 1–3 ␮m ACh. Statistical analysis by the two-way ANOVA test revealed an interaction between LPS-injected ER␣⫹/⫹ and LPS-injected ER␣⫺/⫺ mice (P ⬍ 0.0001) and the posttest analysis indicated significant differences at ACh doses starting at 30 nm (P ⬍ 0.01) with higher significance at 100 nm to 3 ␮m ACh (P ⬍ 0.001). Also, an interaction was observed between saline-injected ER␣⫺/⫺ and LPS-injected ER␣⫺/⫺ mice (P ⬍ 0.0001), and significant differences were observed at 100 nm to 3 ␮m ACh (P ⬍ 0.001). Endothelium-independent relaxation responses were determined by adding increasing doses of SNP to PE precontracted aortic segments (Fig. 2B). Aortic rings from LPSinjected ER␣⫺/⫺ mice had slightly reduced sensitivity to SNP than other groups, suggesting that the smooth muscle cell response to NO (mediated by the activation of sGC in smooth muscle cells) may be impaired in these animals. Statistical analysis by the two-way ANOVA test revealed an interaction between saline-injected ER␣⫺/⫺ and LPS-injected ER␣⫺/⫺ mice (P ⬍ 0.46), and the posttest analysis indicated significant differences at 100 –300 nm SNP (P ⬍ 0.05). Systemic NO production and iNOS levels are not altered in ER␣⫺/⫺ mice compared with ER␣⫹/⫹ mice

A possible explanation for the altered vasoactivity observed in LPS-injected ER␣⫺/⫺ mice is that LPS-induced NO production is impaired in these animals. To assess this possibility, we determined the plasma concentration of the NO metabolites, nitrite and nitrate, 12 h after LPS injection. Compared with saline injection, LPS induced the elevation of

nitrite and nitrate levels in plasma to similar values in ER␣⫹/⫹ and ER␣⫺/⫺ mice (Fig. 3A), and no statistical differences were observed between these groups. To determine whether the expression of iNOS was locally modulated in aortic tissues of ER␣⫺/⫺ mice, iNOS protein expression was determined by Western blotting (Fig. 3B). Compared with saline-injected controls, iNOS protein expression was elevated to similar levels in LPS-injected ER␣⫹/⫹ and LPS-injected ER␣⫺/⫺ mice, and no statistical differences were observed between these groups. LPS injection reduces the expression of eNOS and sGC in aortic tissue of ER␣⫺/⫺ mice

To determine whether the expression of eNOS was locally modulated in aortic tissues of ER␣⫺/⫺ mice, mRNA levels were assayed by real-time PCR and correlated to protein levels by Western blotting. Significant reductions in eNOS mRNA and protein levels were observed in LPS-injected ER␣⫺/⫺ mice compared with all other groups (Fig. 4, A and B). For eNOS mRNA and protein, statistical analysis by twoway ANOVA showed an interaction (P ⬍ 0.05), and significant differences related to the genotype were detected (P ⬍ 0.01). Further analysis by one-way ANOVA and the Bonferroni’s multiple comparison test showed significant differences between LPS-injected ER␣⫹/⫹ and LPS-injected ER␣⫺/⫺ mice (P ⬍ 0.01 for mRNA and P ⬍ 0.05 for protein). Because we observed a reduced sensitivity to SNP (endothelium-independent relaxation), we hypothesized that LPSinjected ER␣⫺/⫺ mice had altered expression of sGC␣ and -␤ subunits. Indeed, mRNA and protein levels for sGC␣ and -␤ subunits were significantly reduced in LPS-injected ER␣⫺/⫺ mouse aortas when compared with all other groups (Figs. 5

Corbacho et al. • Vasoreactivity of ER␣⫺/⫺ Male Mice in Endotoxemia

Endocrinology, March 2007, 148(3):1403–1411

1407

FIG. 3. LPS injection induced the elevation of NO plasma levels and iNOS protein expression in aortic tissues to similar values in ER␣⫹/⫹ and ER␣⫺/⫺ mice. A, Plasma concentration of the NO metabolites, nitrite and nitrate, 12 h after saline or LPS injection. Data summarize the results of four independent experiments. Data are mean ⫾ SEM of eight mice per group. B, Western blot detection of iNOS in lysates of total aortic tissue. GAPDH detection is shown as loading control. Lanes correspond to individual mice. Densitometry of three independent experiments is shown; values for iNOS were normalized to those for GAPDH. Data are expressed as mean ⫾ SEM.

and 6). For sGC␣ mRNA, statistical analysis by two-way ANOVA showed an interaction (P ⬍ 0.05), and significant differences related to the genotype were detected (P ⬍ 0.01). Further analysis by one-way ANOVA and the Bonferroni’s multiple comparison test showed significant differences between LPS-injected ER␣⫹/⫹ and LPS-injected ER␣⫺/⫺ mice (P ⬍ 0.01). For sGC␣ protein, statistical analysis by two-way ANOVA showed an interaction (P ⬍ 0.005), and significant differences related to treatment (LPS vs. saline, P ⬍ 0.0001) and genotype (P ⬍ 0.0001) were detected. Analysis by oneway ANOVA and Bonferroni’s multiple comparison test showed significant differences between LPS-injected ER␣⫹/⫹ and LPS-injected ER␣⫺/⫺ mice (P ⬍ 0.001). For sGC␤ mRNA and protein, statistical analysis by two-way ANOVA showed an interaction (P ⬍ 0.05), and significant differences related to the genotype were detected (P ⬍ 0.01). Further analysis by one-way ANOVA and the Bonferroni’s multiple comparison test showed significant differences be-

tween LPS-injected ER␣⫹/⫹ and LPS-injected ER␣⫺/⫺ mice (P ⬍ 0.01 for mRNA and P ⬍ 0.05 for protein). Discussion

We investigated the contractile-relaxant responses of isolated thoracic aortic segments from ER␣⫹/⫹ and ER␣⫺/⫺ male mice using a well-characterized LPS model of inflammation. This model mimics several aspects of the immune reaction otherwise elicited by tissue injury, infection, or inflammation. It is characterized by profound immune, neuroendocrine, and metabolic changes that can lead to sepsis and multiple organ failure (44). The direct effect of LPS, and the previously characterized associated early systemic release of inflammatory cytokines, induce the expression of iNOS and the release of high levels of NO, causing excessive vasodilation and severe hypotension (24, 25). Herein we demonstrate that during experimental inflam-

FIG. 4. LPS injection reduces expression of eNOS in aortic tissue of ER␣⫺/⫺ mice. A, eNOS mRNA was analyzed by RT-PCR performed on total RNA extracted from aortic tissues of saline- or LPS-treated ER␣⫹/⫹ and ER␣⫺/⫺ male mice. Values for eNOS mRNA were normalized to the expression of GAPDH. Data summarize the results of three independent experiments. Data are the mean ⫾ SEM of five mice per group. B, Western blot detection of eNOS in lysates of total aortic tissue. GAPDH detection is shown as loading control. Lanes correspond to individual mice. Densitometry of three independent experiments is shown; values for eNOS were normalized to those for GAPDH. Data are expressed as mean ⫾ SEM. For A and B, data analysis was performed by two-way ANOVA followed by one-way ANOVA and Bonferroni’s multiple comparison test. Significant differences between LPS-injected ER␣⫹/⫹ and ER␣⫺/⫺ mice are shown: *, P ⬍ 0.01 (A) and P ⬍ 0.05 (B).

1408

Endocrinology, March 2007, 148(3):1403–1411

Corbacho et al. • Vasoreactivity of ER␣⫺/⫺ Male Mice in Endotoxemia

FIG. 5. LPS injection reduces expression of sGC␣ in aortic tissue of ER␣⫺/⫺ mice. A, sGC␣ mRNA was analyzed by RT-PCR performed on total RNA extracted from aortic tissues of saline- or LPS-treated ER␣⫹/⫹ and ER␣⫺/⫺ male mice. Values for sGC␣ mRNA were normalized to the expression of GAPDH. Data summarize the results of three independent experiments. Data are the mean ⫾ SEM of five mice per group. B, Western blot detection of sGC␣ in lysates of total aortic tissue. GAPDH detection is shown as loading control. Lanes correspond to individual mice. Densitometry of three independent experiments is shown; values for sGC␣ were normalized to those for GAPDH. Data are expressed as mean ⫾ SEM. For A and B, data analysis was performed by two-way ANOVA followed by one-way ANOVA and Bonferroni’s multiple comparison test. Significant differences between LPS-injected ER␣⫹/⫹ and ER␣⫺/⫺ mice are shown: *, P ⬍ 0.05 (A and B).

mation and endotoxemia induced by ip injection of Escherichia coli LPS, disruption of the full-length ER␣ gene leads to significant alterations in aortic vasoreactivity in male mice, including hypersensitive responses to PE-induced contraction, and failure of effective vasodilatory responses to increasing concentrations of ACh. Furthermore, reduced sensitivity to SNP-induced vasodilation was observed in ER␣deficient mice after LPS injection. These alterations are functionally linked to reductions in gene and protein expression of eNOS and sGC subunits (␣ and ␤) in aortic tissue. However, the absence of the ER␣ did not alter LPS-induced systemic NO production or iNOS protein expression in the

aortic wall. These findings suggest that during endotoxemia, the ER␣ mediates the sustained expression of the molecular machinery essential for endothelial NO synthesis (i.e. eNOS) and the vascular responses to NO (i.e. sGC). In the vascular wall, ACh effects are mediated by the stimulation of eNOS activity in endothelial cells and the subsequent production of NO (45, 46). Endothelial NO diffuses to the vascular smooth muscle cells to induce relaxation through the activation of sGC. On the other hand, LPS induces the expression of iNOS, which releases high levels of NO. NO produced by iNOS also induces vascular smooth muscle relaxation (22, 23). When iNOS is highly expressed,

FIG. 6. LPS injection reduces expression of sGC␤ in aortic tissue of ER␣⫺/⫺ mice. A, sGC␤ mRNA was analyzed by RT-PCR performed on total RNA extracted from aortic tissues of saline- or LPS-treated ER␣⫹/⫹ and ER␣⫺/⫺ male mice. Values for sGC␤ mRNA were normalized to the expression of GAPDH. Data summarize the results of three independent experiments. Data are the mean ⫾ SEM of five mice per group. B, Western blot detection of sGC␤ in lysates of total aortic tissue. GAPDH detection is shown as loading control. Lanes correspond to individual mice. Densitometry of three independent experiments is shown; values for sGC␤ were normalized to those for GAPDH. Data are expressed as mean ⫾ SEM. For A and B, data analysis was performed by two-way ANOVA followed by one-way ANOVA and Bonferroni’s multiple comparison test. Significant differences between LPS-injected ER␣⫹/⫹ and ER␣⫺/⫺ mice are shown: *, P ⬍ 0.01 (A) and P ⬍ 0.05 (B).

Corbacho et al. • Vasoreactivity of ER␣⫺/⫺ Male Mice in Endotoxemia

the vascular wall is continuously exposed to NO and tends to relax further than the control vessels, tending to counteract the PE contractile effect (22). Furthermore, in aortic rings from LPS-injected ER␣⫹/⫹, an additive effect of NO produced by iNOS and of NO produced by eNOS after ACh stimulation is anticipated (23). Consistent with high levels of NO being produced in the vascular wall, LPS-injected ER␣⫹/⫹ showed lower response to PE-stimulated contraction and earlier response to ACh, a cumulative effect resulting in profound vasodilation. In contrast, aortic rings from LPS-injected ER␣⫺/⫺ mice developed higher tension levels after PE stimulation and failed to relax to the same extent as the corresponding control (saline-injected ER␣⫺/⫺) in response to ACh. These results suggested that vascular NO production and/or smooth muscle cell responsiveness to NO are impaired in LPS-injected ER␣⫺/⫺ mice during acute inflammatory responses. Furthermore, aortic rings from LPStreated ER␣⫺/⫺ mice displayed reduced sensitivity to SNP, further suggesting a dysfunctional smooth muscle cell response to NO, because SNP acts directly to activate sGC and it represents an indirect measurement of smooth muscle response to NO. In our experiments, no differences were observed in the PE-dependent contraction and the ACh-dependent vasodilatory responses of aortas from control (saline-injected) ER␣⫹/⫹ and ER␣⫺/⫺ mice. These results are in agreement with previous studies where no significant differences were observed in contractile responses [KCl-induced (32) or U-46619-induced (32, 47)] of aortic segments of ER␣⫹/⫹ and ER␣⫺/⫺ male mice. Also, no differences were observed in endothelium-dependent (ACh-induced) (32, 47) or endothelium-independent (nitroglycerin-induced) (32) relaxation of aortic segments from ER␣⫹/⫹ and ER␣⫺/⫺ male mice. Nonetheless, Rubanyi et al. (32) reported that basal aortic NO production (estimated by l-nitro-arginine inhibition of NO production that results in contraction) was decreased in ER␣⫺/⫺ compared with ER␣⫹/⫹ male mice. Having established that the ER␣ appeared to play a critical role in vascular reactivity after acute inflammation, we determined whether or not NO production was altered in ER␣⫺/⫺ mice after LPS injection. Because no differences were detected in NO metabolite levels in plasma from LPS-injected ER␣⫹/⫹ or ER␣⫺/⫺ mice, we investigated whether NO production was locally modulated in aortic tissue via alterations in the expression of iNOS and eNOS. A similar level of iNOS protein induction was observed in aortic tissues of LPSinjected ER␣⫹/⫹ and ER␣⫺/⫺ mice. Taken together, our results suggest that the impaired contraction-relaxation response observed in ER␣⫺/⫺ 12 h after LPS injection is not explained by impaired systemic NO production or by a significant reduction in the expression of aortic iNOS protein. However, our studies revealed significant reductions in aortic eNOS mRNA and protein levels in LPS-injected ER␣⫺/⫺ mice, suggesting an impaired endothelium-derived NO production. Reduction in endothelial-derived NO may account, in part, for the development of exacerbated contractile responses and decreased relaxation responses (endotheliumdependent and -independent) observed in the functional experiments (Figs. 1 and 2). Previously, basal expression of eNOS in ER␣⫺/⫺ female mice was reported normal in the

Endocrinology, March 2007, 148(3):1403–1411

1409

aorta (47), but reduced in the coronary artery (48). In our study, impaired expression of eNOS in the aorta of ER␣⫺/⫺ male mice was manifested only after systemic acute inflammation induced by LPS. These findings suggest that in male mice, the ER␣ helps maintain normal aortic eNOS expression during acute inflammation. Nonetheless, a local reduction in eNOS expression may not have a significant physiological effect on the vascular tone considering that during sepsis, high amounts of NO are being produced by iNOS activity, which leads to vasodilation. Because LPS-injected ER␣⫺/⫺ mice showed reduced sensitivity to SNP, a compound that releases NO inside of smooth muscle cells (i.e. endothelium-independent vasodilation), we further analyzed the expression level of sGC, the presence of which is a requisite for NO-dependent vasodilatory effects in the smooth muscle. Reduced levels of mRNA and protein for sGC ␣- and ␤-subunits were detected in aortic tissues from LPS-injected ER␣⫺/⫺ mice. A reduced expression of sGC in the aortic wall suggests that the smooth muscle response to NO is impaired in ER␣⫺/⫺ male mice during sepsis. The reduction in the expression of sGC isoforms in LPS-injected ER␣⫺/⫺ mice provide a strong explanation for the impaired response to ACh and SNP that these mice displayed after LPS treatment. Previous studies have shown that LPS can induce a reduction in sGC expression or activity (49, 50), and this may represent a mechanism to counteract the massive levels of NO being produced by iNOS activity. Our studies reveal that after LPS injection, the expression of sGC isoforms was further reduced in ER␣⫺/⫺ than in ER␣⫹/⫹ mice, suggesting that ER␣ contributes to support aortic sGC expression during sepsis. The ER␣-deficient mouse used in the present study (ER␣ Neo knockout) is not fully null (41, 51, 52). In this mouse, the ER␣ gene was disrupted by the insertion of the neomycin gene into the first coding exon, resulting in the deficiency of the full-length wild-type ER␣ protein (40). However, this approach also resulted in the expression of two alternatively spliced transcripts of the disrupted ER␣ gene, called E1 and E2 (41), and the expression of two variants of ER␣ proteins (only one of them found in the aorta) (41, 51). These variants have estrogen-dependent transcriptional activity (41, 51, 52), which explains the observation of similar vascular responses to estradiol in ER␣⫹/⫹ and ER␣⫺/⫺ mice (51). More recently, Dupont et al. (53) generated mice completely deficient in ER␣ gene (called ER␣-⌬2KO) that resulted in the complete abrogation of estradiol effect in vascular responses (51). Although in our study we used the ER␣ Neo knockout model, the results presented here clearly indicate that the activity of the variant of ER␣ present in the aorta of this mouse is not sufficient to maintain the expression of eNOS and sGC under acute inflammatory conditions in male mice, indicating that the expression of the full-length ER␣ is required. Recently, contraction-relaxation vascular studies on male aromatase-deficient mice have demonstrated that aromatization of testosterone to estrogen is required for the maintenance of normal endothelial function and vascular tone in males (1, 38). Estrogen deficiency, as observed in aromatasedeficient male mice, results in an enhanced response to noradrenaline and diminished sensitivity to ACh (endothelialdependent relaxation). In our study, similar vascular

1410

Endocrinology, March 2007, 148(3):1403–1411

responses were observed only after LPS injection to ER␣⫺/⫺ mice but not under basal conditions. These differences may be explained by the fact that this ER␣⫺/⫺ mouse model represents a condition of partial estrogen-activity deficiency, in which only the activity of the full-length ER␣ is abrogated, whereas the ER␤ and variants of ER␣ are present. Under these circumstances, a phenotype may become apparent only during a challenge (i.e. acute endotoxemia) and not under basal conditions (i.e. saline injection) as it was observed in aromatase-deficient male mice. Previously, Rubanyi et al. (32) have shown that the levels of circulating estrogen (plasma 17␤-estradiol) are similar in ER␣⫹/⫹ and ER␣⫺/⫺ male mice, and the role of endogenous estradiol in males has been previously demonstrated in male mice (38). Although our results suggest that endogenous levels of estradiol are sufficient to distinguish between the presence or absence of the full-length ER␣, the study of exogenous estradiol effects on this experimental model remains to be investigated. This is of particular importance in relationship to the role of estrogen in male vascular reactivity during sepsis and in regard to the proposed protective role of estradiol during sepsis. Finally, to further understand the physiological significance of the results presented herein, additional research is needed, including comparisons with female mice, the use of denuded aortic rings, and the evaluation of changes in blood pressure during sepsis in ER␣⫹/⫹ and ER␣⫺/⫺ mice. In summary, our results provide evidence of the functional and modulatory role of the ER␣ in the vascular contractile and relaxation responses of male mice during acute inflammation. In our model, and under conditions of acute inflammation, the ER␣ appears to mediate the sustained expression of eNOS and sGC and thus facilitates vasodilatory responses by preserving endothelium-derived NO production and perhaps more importantly smooth muscle responsiveness to NO. The observations made herein have implications to further define the role of ER␣ in the vascular responses associated with acute inflammation as a condition related to the development of cardiovascular disease. Acknowledgments Received March 31, 2006. Accepted November 29, 2006. Address all correspondence and requests for reprints to: Ana M. Corbacho, Ph.D., Center for Biophotonics Science and Technology (CBST), University of California Davis, One Shields Avenue, Hunt Hall 225, Davis, California 95616. E-mail: [email protected]. This work was supported by the National Heart, Lung, and Blood Institute Training Grant in Comparative Lung Biology and Medicine (T32 HL07013-27 support to A.M.C.), the National Institutes of Health (K01-HL04142 to A.V.), the Paul F. Gulyassy Endowed Professorship (to J.P.E.), a UC Davis Health Systems Research Grant (to J.P.E.), and a Nora Eccles Treadwell Innovative Research Grant (to A.V. and J.P.E.). Disclosure Statement: The authors have nothing to declare.

References 1. Mendelsohn ME, Rosano GM 2003 Hormonal regulation of normal vascular tone in males. Circ Res 93:1142–1145 2. Green S, Walter P, Greene G, Krust A, Goffin C, Jensen E, Scrace G, Waterfield M, Chambon P 1986 Cloning of the human oestrogen receptor cDNA. J Steroid Biochem 24:77– 83 3. Green S, Walter P, Kumar V, Krust A, Bornert JM, Argos P, Chambon P 1986 Human oestrogen receptor cDNA: sequence, expression and homology to v-erb-A. Nature 320:134 –139

Corbacho et al. • Vasoreactivity of ER␣⫺/⫺ Male Mice in Endotoxemia 4. Greene GL, Gilna P, Waterfield M, Baker A, Hort Y, Shine J 1986 Sequence and expression of human estrogen receptor complementary DNA. Science 231:1150 –1154 5. Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA 1996 Cloning of a novel receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 93:5925–5930 6. Kuiper GG, Carlsson B, Grandien K, Enmark E, Haggbland J, Nilsson S, Gustafsson JA 1997 Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors ␣ and ␤. Endocrinology 138: 863– 870 7. Mendelsohn ME, Karas RH 1999 The protective effects of estrogen on the cardiovascular system. N Engl J Med 340:1801–1811 8. Campisi D, Bivona A, Paterna S, Valenza M, Albiero R 1987 Oestrogen binding sites in fresh human aortic tissue. Int J Tissue React 9:393–398 9. Karas RH, Patterson BL, Mendelsohn ME 1994 Human vascular smooth muscle cells contain functional estrogen receptor. Circulation 89:1943–1950 10. Mendelsohn ME 2002 Genomic and nongenomic effects of estrogen in the vasculature. Am J Cardiol 90:3F– 6F 11. Chen Z, Yuhanna IS, Galcheva-Gargova Z, Karas RH, Mendelsohn ME, Shaul PW 1999 Estrogen receptor ␣ mediates the nongenomic activation of endothelial nitric oxide synthase by estrogen. J Clin Invest 103:401– 406 12. Edwards DP 2005 Regulation of signal transduction pathways by estrogen and progesterone. Annu Rev Physiol 67:335–376 13. Chambliss KL, Yuhanna IS, Anderson RG, Mendelsohn ME, Shaul PW 2002 ER␤ has nongenomic action in caveolae. Mol Endocrinol 16:938 –946 14. Mendelsohn ME, Karas RH 2005 Molecular and cellular basis of cardiovascular gender differences. Science 308:1583–1587 15. MacRitchie AN, Jun SS, Chen Z, German Z, Yuhanna IS, Sherman TS, Shaul PW 1997 Estrogen upregulates endothelial nitric oxide synthase gene expression in fetal pulmonary artery endothelium. Circ Res 81:355–362 16. Kleinert H, Wallerath T, Euchenhofer C, Ihrig-Biedert I, Li H, Forstermann U 1998 Estrogens increase transcription of the human endothelial NO synthase gene: analysis of the transcription factors involved. Hypertension 31:582–588 17. Hishikawa K, Nakaki T, Marumo T, Suzuki H, Kato R, Saruta T 1995 Up-regulation of nitric oxide synthase by estradiol in human aortic endothelial cells. FEBS Lett 360:291–293 18. Knowles RG, Moncada S 1994 Nitric oxide synthases in mammals. Biochem J 298:249 –258 19. Roivainen M, Viik-Kajander M, Palosuo T, Toivanen P, Leinonen M, Saikku P, Tenkanen L, Manninen V, Hovi T, Manttari M 2000 Infections, inflammation, and the risk of coronary heart disease. Circulation 101:252–257 20. Danesh J, Appleby P 1998 Persistent infection and vascular disease: a systematic review. Expert Opin Investig Drugs 7:691–713 21. Kher A, Wang M, Tsai BM, Pitcher JM, Greenbaum ES, Nagy RD, Patel KM, Wairiuko GM, Markel TA, Meldrum DR 2005 Sex differences in the myocardial inflammatory response to acute injury. Shock 23:1–10 22. Szabo C, Mitchell JA, Thiemermann C, Vane JR 1993 Nitric oxide-mediated hyporeactivity to noradrenaline precedes the induction of nitric oxide synthase in endotoxin shock. Br J Pharmacol 108:786 –792 23. Thiemermann C, Szabo C, Mitchell JA, Vane JR 1993 Vascular hyporeactivity to vasoconstrictor agents and hemodynamic decompensation in hemorrhagic shock is mediated by nitric oxide. Proc Natl Acad Sci USA 90:267–271 24. Kilbourn RG, Traber DL, Szabo C 1997 Nitric oxide and shock. Dis Mon 43:277–348 25. Li H, Forstermann U 2000 Nitric oxide in the pathogenesis of vascular disease. J Pathol 190:244 –254 26. Binko J, Majewski H 1998 17␤-Estradiol reduces vasoconstriction in endothelium-denuded rat aortas through inducible NOS. Am J Physiol 274:H853– H859 27. Mershon JL, Baker RS, Clark KE 2002 Estrogen increases iNOS expression in the ovine coronary artery. Am J Physiol Heart Circ Physiol 283:H1169 –H1180 28. Mostafa G, Huynh T, Sing RF, Miles WS, Norton HJ, Thomason MH 2002 Gender-related outcomes in trauma. J Trauma 53:430 – 434; discussion 434 – 435 29. Offner PJ, Moore EE, Biffl WL 1999 Male gender is a risk factor for major infections after surgery. Arch Surg 134:935–938; discussion 938 –940 30. Gannon CJ, Pasquale M, Tracy JK, McCarter RJ, Napolitano LM 2004 Male gender is associated with increased risk for postinjury pneumonia. Shock 21:410 – 414 31. Chaudry IH, Samy TS, Schwacha MG, Wang P, Rue 3rd LW, Bland KI 2003 Endocrine targets in experimental shock. J Trauma 54:S118 –S125 32. Rubanyi GM, Freay AD, Kauser K, Sukovich D, Burton G, Lubahn DB, Couse JF, Curtis SW, Korach KS 1997 Vascular estrogen receptors and endothelium-derived nitric oxide production in the mouse aorta. Gender difference and effect of estrogen receptor gene disruption. J Clin Invest 99:2429 – 2437 33. Shearman AM, Cupples LA, Demissie S, Peter I, Schmid CH, Karas RH, Mendelsohn ME, Housman DE, Levy D 2003 Association between estrogen receptor ␣ gene variation and cardiovascular disease. JAMA 290:2263–2270 34. Sudhir K, Chou TM, Chatterjee K, Smith EP, Williams TC, Kane JP, Malloy MJ, Korach KS, Rubanyi GM 1997 Premature coronary artery disease associated with a disruptive mutation in the estrogen receptor gene in a man. Circulation 96:3774 –3777

Corbacho et al. • Vasoreactivity of ER␣⫺/⫺ Male Mice in Endotoxemia 35. Sudhir K, Chou TM, Messina LM, Hutchinson SJ, Korach KS, Chatterjee K, Rubanyi GM 1997 Endothelial dysfunction in a man with disruptive mutation in oestrogen-receptor gene. Lancet 349:1146 –1147 36. Rubanyi GM, Kauser K, Johns A 2002 Role of estrogen receptors in the vascular system. Vascul Pharmacol 38:81– 88 37. Villablanca A, Lubahn D, Shelby L, Lloyd K, Barthold S 2004 Susceptibility to early atherosclerosis in male mice is mediated by estrogen receptor ␣. Arterioscler Thromb Vasc Biol 24:1055–1061 38. Kimura M, Sudhir K, Jones M, Simpson E, Jefferis AM, Chin-Dusting JP 2003 Impaired acetylcholine-induced release of nitric oxide in the aorta of male aromatase-knockout mice: regulation of nitric oxide production by endogenous sex hormones in males. Circ Res 93:1267–1271 39. Zhu Y, Bian Z, Lu P, Karas RH, Bao L, Cox D, Hodgin J, Shaul PW, Thoren P, Smithies O, Gustafsson JA, Mendelsohn ME 2002 Abnormal vascular function and hypertension in mice deficient in estrogen receptor ␤. Science 295:505–508 40. Lubahn DB, Moyer JS, Golding TS, Couse JF, Korach KS, Smithies O 1993 Alteration of reproductive function but not prenatal sexual development after insertional disruption of the mouse estrogen receptor gene. Proc Natl Acad Sci USA 90:11162–11166 41. Couse JF, Curtis SW, Washburn TF, Lindzey J, Golding TS, Lubahn DB, Smithies O, Korach KS 1995 Analysis of transcription and estrogen insensitivity in the female mouse after targeted disruption of the estrogen receptor gene. Mol Endocrinol 9:1441–1454 42. Corbacho AM, Valacchi G, Kubala L, Olano-Martı´n E, Schock BC, Kenny TP, Cross CE 2004 Tissue-specific gene expression of prolactin receptor in the acute-phase response induced by lipopolysaccharides. Am J Physiol Endocrinol Metab 287:E750 –E757 43. Van Der Vliet A, Nguyen MN, Shigenaga MK, Eiserich JP, Marelich GP, Cross CE 2000 Myeloperoxidase and protein oxidation in cystic fibrosis. Am J Physiol Lung Cell Mol Physiol 279:L537–L546

Endocrinology, March 2007, 148(3):1403–1411

1411

44. Berczi I, Bertok L, Chow DA 2000 Natural immunity and neuroimmune host defense. Ann NY Acad Sci 917:248 –257 45. Moncada S, Palmer RM, Higgs EA 1991 Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 43:109 –142 46. Chu A, Chambers DE, Lin CC, Kuehl WD, Palmer RM, Moncada S, Cobb FR 1991 Effects of inhibition of nitric oxide formation on basal vasomotion and endothelium-dependent responses of the coronary arteries in awake dogs. J Clin Invest 87:1964 –1968 47. Darblade B, Pendaries C, Krust A, Dupont S, Fouque MJ, Rami J, Chambon P, Bayard F, Arnal JF 2002 Estradiol alters nitric oxide production in the mouse aorta through the ␣-, but not ␤-, estrogen receptor. Circ Res 90:413– 419 48. Muller-Delp JM, Lubahn DB, Nichol KE, Philips BJ, Price EM, Curran EM, Laughlin MH 2003 Regulation of nitric oxide-dependent vasodilation in coronary arteries of estrogen receptor-␣-deficient mice. Am J Physiol Heart Circ Physiol 285:H2150 –H2157 49. da Silva-Santos JE, Terluk MR, Assreuy J 2002 Differential involvement of guanylate cyclase and potassium channels in nitric oxide-induced hyporesponsiveness to phenylephrine in endotoxemic rats. Shock 17:70 –76 50. Scott WS, Nakayama DK 1998 Escherichia coli lipopolysaccharide downregulates soluble guanylate cyclase in pulmonary artery smooth muscle. J Surg Res 80:309 –314 51. Pendaries C, Darblade B, Rochaix P, Krust A, Chambon P, Korach KS, Bayard F, Arnal JF 2002 The AF-1 activation-function of ER␣ may be dispensable to mediate the effect of estradiol on endothelial NO production in mice. Proc Natl Acad Sci USA 99:2205–2210 52. Kos M, Denger S, Reid G, Korach KS, Gannon F 2002 Down but not out? A novel protein isoform of the estrogen receptor ␣ is expressed in the estrogen receptor ␣ knockout mouse. J Mol Endocrinol 29:281–286 53. Dupont S, Krust A, Gansmuller A, Dierich A, Chambon P, Mark M 2000 Effect of single and compound knockouts of estrogen receptors ␣ (ER␣) and ␤ (ER␤) on mouse reproductive phenotypes. Development 127:4277– 4291

Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community.