AJP Regulatory Integrative and Comparative Physiology

Published by American Physiological Society
Online ISSN: 1522-1490
Publications
Transfer function relationships between HR and SBP. Each point represents the mean SE of 5 rabbits. Solid horizontal line represents 0 phase. Broken horizontal line represents the coherence value of 0.5. *Data points for which the coherence was significantly 0.5.
Power spectra of the DP (F) and RevHRSBP signal (E). Each point represents the mean SE of 5 rabbits. Data are presented in normalized units in A and in original units in B. *Points at which the DP and RevHRSBP signals are significantly different. Log-log plots of the DP and RevHRSBP spectra are shown in inset in B. AU, arbitrary units.
Contribution of HR and SBP to the variability of the DP. A : power spectra of the DP, HR ϫ MSBP, MHR ϫ SBP, and the sum of HR ϫ MSBP and MHR ϫ SBP. B : replotting of the spectra in A with spectral power expressed as a fraction of that of the DP. The line at y ϭ 1 highlights the position at which the spectral power is at the level of that of the DP. Each point represents the mean of 5 rabbits. Error bars are omitted for clarity. *Signi fi cant difference between the DP and the sum of the HR ϫ MSBP and MHR ϫ SBP spectra. 
Article
The product of heart rate (HR) and systolic blood pressure (SBP), the double product (DP), is an indirect index of cardiac oxygen consumption. We used spectral analysis to test the hypothesis that baroreflex adjustments of HR stabilize the DP during spontaneous variations in SBP. SBP and HR were recorded by telemetry in five male conscious rabbits. HR and SBP power spectra each exhibited a low frequency peak at approximately 0.05 Hz that was associated with high (>0.5) spectral coherence and a positive phase relationship between SBP and HR (SBP leading). A prominent peak was absent in the spectra of their product, suggesting that SBP and HR interacted to reduce DP variability in this frequency region. In contrast, a prominent 0.05-Hz peak was present in the power spectrum of calculated surrogates of the DP in which reflex interactions between HR and SBP had been removed. Our results suggest that baroreflex adjustments of HR stabilize the DP during spontaneous low-frequency variations in SBP in conscious rabbits.
 
Article
This study quantified the effect of interrupting the descending input to the sympathetic preganglionic neurons on the dynamic behavior of arterial blood pressure (BP) in the unanesthetized rat. BP was recorded for approximately 4-h intervals in six rats in the neurally intact state and in the same animals after complete spinal cord transection (SCT) between T(4) and T(5). In the intact state, power within the frequency range of 0.35-0.45 Hz was 1.53 +/- 0.38 mmHg(2)/Hz (mean +/- SD by fast Fourier transform). One week after SCT, power within this range decreased significantly (P < 0.05) to 0.43 +/- 0.62 mmHg(2)/Hz. To test for self-similarity before and after SCT, we analyzed data using a wavelet (i.e., functionally, a digital bandpass filter) tuned to be maximally sensitive to fluctuations with periods of approximately 2, 4, 8, 16, 32, or 64 s. In the control state, all fluctuations with periods of >/=4 s conformed to a "self-similar" (i.e., fractal) distribution. In marked contrast, the oscillations with a period of approximately 2 s (i.e., approximately 0.4 Hz) were significantly set apart from those at lower frequencies. One day and seven days after the complete SCT, however, the BP fluctuations at approximately 0.4 Hz now also conformed to the same self-similar behavior characteristic of the lower frequencies. We conclude that 1) an intact sympathetic nervous system endows that portion of the power spectrum centered around approximately 0.4 Hz with properties (e.g., a periodicity) that differ significantly from the self-similar behavior that characterizes the lower frequencies and 2) even within the relatively high frequency range at 0.4 Hz self-similarity is the "default" condition after sympathetic influences have been eliminated.
 
Article
Dexamethasone (DM) was administered to pregnant ewes as three weekly courses of four injections of 2 mg at 12-h intervals. DM (n = 7) or saline (n = 7) was given starting at 103 days of gestation (dGA; term approximately 149 days). Fetal femoral arteries (approximately 300-microm internal diameter) were evaluated using wire myography at 119 dGA. DM-exposed fetuses were significantly smaller than saline-exposed fetuses. DM exposure increased maximal contraction to 125 mM KCl, and maximum tension developed along with sensitivity to endothelin-1 and relaxation to bradykinin. Preincubation with the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester shifted the dose-response curves to endothelin-1 and acetylcholine to the right in controls but not in the DM-exposed group. Relaxation to acetylcholine and to the nitric oxide donor sodium nitroprusside was similar in both groups. The combination of enhanced endothelin-induced vasoconstriction, abnormal endothelium-dependent relaxation, and normal endothelium-independent relaxation indicates microvessel dysfunction following antenatal DM administration. Because such dysfunction is associated with several forms of adult hypertension, our results indicate the potential for consequences of antenatal glucocorticoid exposure on adult cardiovascular health.
 
Article
The processes that trigger severe muscle atrophy and loss of myosin in critical illness myopathy (CIM) are poorly understood. It has been reported that muscle disuse alters Ca(2+) handling by the sarcoplasmic reticulum. Since inactivity is an important contributor to CIM, this finding raises the possibility that elevated levels of the proteins involved in Ca(2+) handling might contribute to development of CIM. CIM was induced in 3- to 5-mo-old rats by sciatic nerve lesion and infusion of dexamethasone for 1 wk. Western blot analysis revealed increased levels of ryanodine receptor (RYR) isoforms-1 and -2 as well as the dihydropyridine receptor/voltage-gated calcium channel type 1.1 (DHPR/Ca(V) 1.1). Immunostaining revealed a subset of fibers with elevation of RYR1 and Ca(V) 1.1 that had severe atrophy and disorganization of sarcomeres. These findings suggest increased Ca(2+) release from the sarcoplasmic reticulum may be an important contributor to development of CIM. To assess the endogenous functional effects of increased intracellular Ca(2+) in CIM, proteolysis of α-fodrin, a well-known target substrate of Ca(2+)-activated proteases, was measured and found to be 50% greater in CIM. There was also selective degradation of myosin heavy chain relative to actin in CIM muscle. Taken together, our findings suggest that increased Ca(2+) release from the sarcoplasmic reticulum may contribute to pathology in CIM.
 
Article
Adeno-associated virus delivery systems and shRNA were used to selectively silence the voltage gated sodium channel NaV 1.7 in the nodose ganglia of guinea pigs. The cough reflex in these animals was subsequently assessed. NaV 1.7 shRNA was delivered to the majority of nodose ganglia neurons (50-60% transfection rate determined by green fluorescent protein (GFP) gene co-transfection) and action potential conduction in the nodose vagal nerve fibers, as evaluated using an extracellular recording technique, was markedly and significantly reduced. By contrast, <5% of neurons in the jugular vagal ganglia neurons were transfected, and action potential conduction in the jugular vagal nerve fibers was unchanged. The control virus (with GFP expression) was without effect on action potential discharge and conduction in either ganglia. In vivo, NaV 1.7 silencing in the nodose ganglia nearly abolished cough evoked by mechanically probing the tracheal mucosa in anesthetized guinea pigs. Stimuli such as capsaicin and bradykinin that are known to stimulate both nodose and jugular C-fibers evoked coughing in conscious animals was unaffected by NaV 1.7 silencing in the nodose ganglia. Nodose C-fiber selective stimuli including adenosine, 2-methyl-5-HT and ATP all failed to evoke coughing upon aerosol challenge. These results indicate that cough is independently regulated by two vagal afferent nerve subtypes in guinea pigs, with nodose Aδ fibers regulating cough evoked mechanically from the trachea and bradykinin- and capsaicin-evoked cough regulated by C-fibers arising from the jugular ganglia.
 
Article
We reported impaired endothelium-derived relaxation factor/nitric oxide (EDRF/NO) responses and constitutive nitric oxide synthase (cNOS) activity in subcutaneous vessels dissected from patients with essential hypertension (n = 9) compared with normal controls (n = 10). We now test the hypothesis that the patients in this study have increased circulating levels of the cNOS inhibitor, asymmetric dimethylarginine (ADMA), or the lipid peroxidation product of linoleic acid, 13-hydroxyoctadecadienoic acid (HODE), which is a marker of reactive oxygen species. Patients had significantly (P < 0.001) elevated (means +/- SD) plasma levels of ADMA (P(ADMA), 766 +/- 217 vs. 393 +/- 57 nmol/l) and symmetric dimethylarginine (P(SDMA): 644 +/- 140 vs. 399 +/- 70 nmol/l) but similar levels of L-arginine accompanied by significantly (P < 0.015) increased rates of renal ADMA excretion (21 +/- 9 vs. 14 +/- 5 nmol/mumol creatinine) and decreased rates of renal ADMA clearance (18 +/- 3 vs. 28 +/- 5 ml/min). They had significantly increased plasma levels of HODE (P(HODE): 309 +/- 30 vs. 226 +/- 24 nmol/l) and renal HODE excretion (433 +/- 93 vs. 299 +/- 67 nmol/micromol creatinine). For the combined group of normal and hypertensive subjects, the individual values for plasma levels of ADMA and HODE were both significantly (P < 0.001) and inversely correlated with microvascular EDRF/NO and positively correlated with mean blood pressure. In conclusion, elevated levels of ADMA and oxidative stress in a group of hypertensive patients could contribute to the associated microvascular endothelial dysfunction and elevated blood pressure.
 
Expression of mRNA for myotrophin, endothelial nitric oxide synthase (eNOS, NOS-3), and VEGF in left ventricles from fetuses of the control (white bars), high (black bars), and low maternal cortisol (gray bars) groups. Data are expressed as in Fig. 1. *P 0.05 vs. control. 
Expression of mRNA for IGF-I, IGF-II, IGF receptors 1 (IGF-1R), and IGF-2R in left ventricles from fetuses of the control (white bars), high (black bars), and low maternal cortisol (gray bars) groups. Data are expressed as in Fig. 1. *P 0.05 vs. control. 
Article
Moderately elevated maternal cortisol levels late in gestation cause enlargement of the fetal sheep heart. We have used quantitative real-time PCR to examine expression of candidate genes in fetal hearts from mothers in whom cortisol levels were increased (by infusion of 1 mg cortisol.kg(-1).day(-1)) or decreased (by adrenalectomy and replacement to 0.5 mg cortisol.kg(-1).day(-1)) from 115 to 130 days gestation. Control ewes were not treated with steroid. Expression of mineralocorticoid receptor (MR), glucocorticoid receptor (GR), 11beta-hydroxysteroid dehydrogenases 1 and 2 (11beta-HSD1 and -2), IGF I and II, IGF receptors 1 and 2 (IGF-1R and IGF-2R), endothelial nitric oxide synthase, VEGF, myotrophin, angiotensinogen, the angiotensin receptors 1 and 2 (AT1R and AT2R), and the angiotensin converting enzymes 1 and 2 were measured. MR mRNA abundance in fetal hearts was found to be similar to that in adult kidney and hippocampus. Although there were no significant changes in most genes, 11beta-HSD2 and IGF-1R expression were significantly decreased in the high cortisol group and 11beta-HSD2 expression negatively correlated to left ventricular wall thickness. There was also a significant change in the ratio of AT receptor expression, with increased AT2R and decreased AT1R in the high cortisol group. MR, GR, and 11beta-HSD1 immunoreactivity was found in cardiomyocytes and cardiac blood vessels in 126-128 day fetal sheep; in contrast 11beta-HSD2 staining was predominantly in blood vessels. These results indicate that cortisol could indeed act in the fetal heart to induce enlargement and suggest that the renin-angiotensin system may play a role.
 
Article
Conjugated linoleic acid (CLA) is reported to have health benefits, including reduction of body fat. Previous studies have shown that brown adipose tissue (BAT) is particularly sensitive to CLA-supplemented diet feeding. Most of them use mixtures containing several CLA isomers, mainly cis-9, trans-11 and trans-10, cis-12 in equal concentration. Our aim was to characterize the separate effects of both CLA isomers on thermogenic capacity in cultured brown adipocytes. The CLA isomers showed opposite effects. Hence, on the one hand, trans-10, cis-12 inhibited uncoupling protein (UCP) 1 induction by norepinephrine (NE) and produced a decrease in leptin mRNA levels. These effects were associated with a blockage of CCAAT-enhancer-binding protein-alpha and peroxisome proliferator-activated receptor-gamma(2) mRNA expression. On the other hand, cis-9, trans-11 enhanced the UCP1 elicited by NE, an effect reported earlier for polyunsaturated fatty acids and also observed here for linoleic acid. These findings could explain, at least in part, the effects observed in vivo when feeding a CLA mixture supplemented diet as a result of the combined action of CLA isomers (reduction of adipogenesis and defective BAT thermogenesis that could be through trans-10, cis-12 and enhanced UCP1 thermogenic capacity through cis-9, trans-11).
 
Article
Intrauterine growth restriction (IUGR) increases the risk of serious adult morbidities such as hypertension. In an IUGR rat model of hypertension, we reported a persistent decrease in kidney 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) mRNA and protein levels from birth through postnatal (P) day 21. This enzyme deficiency can lead to hypertension by limiting renal glucocorticoid deactivation. In the present study, we hypothesized that IUGR affects renal 11beta-HSD2 epigenetic determinants of chromatin structure and alters key transcription factor binding to the 11beta-HSD2 promoter in association with persistent downregulation of its mRNA expression. To test this hypothesis, we performed bilateral uterine artery ligation on embryonic day 19.5 pregnant rats and harvested kidneys at day 0 (P0) and P21. Key transcription factors that can affect 11beta-HSD2 expression include transcriptional enhancers specificity protein 1 (SP1) and NF-kappaB p65 and transcriptional repressors early growth response factor (Egr-1) and NF-kappaB p50. Our most important findings were as follows: 1) IUGR significantly decreased SP1 and NF-kappaB (p65) binding to the 11beta-HSD2 promoter in males, while it increased Egr-1 binding in females and NF-kappaB (p50) binding in males; 2) IUGR increased CpG methylation status, as well as modified the pattern of methylation in several CpG sites of 11beta-HSD2 promoter at P0 also in a sex-specific manner; and 3) IUGR decreased trimethylation of H3K36 in exon 5 of 11beta-HSD2 at P0 and P21 in both genders. We conclude that IUGR is associated with altered transcriptional repressor/activator binding in connection with increased methylation in the 11beta-HSD2 promoter region in a sex-specific manner, possibly leading to decreased transcriptional activity. Furthermore, IUGR decreased trimethylation of H3K36 of the 11beta-HSD2 gene in both genders, which is associated with decreased transcriptional elongation. We speculate that alterations in transcription factor binding and chromatin structure play a role in in utero reprogramming.
 
Article
The Na(+)-K(+)-ATPase is the primary electrogenic component driving transepithelial ion transport in the teleost gill; thus regulation of its level of activity is of critical importance for osmotic homeostasis. In the present study, we examined the dynamics of the gill-specific FXYD-11 protein, a putative regulatory subunit of the pump, in Atlantic salmon during seawater (SW) acclimation, smoltification, and treatment with cortisol, growth hormone, and prolactin. Dual-labeling immunohistochemistry showed that branchial FXYD-11 is localized in Na(+)-K(+)-ATPase immunoreactive cells, and coimmunoprecipitation experiments confirmed a direct association between FXYD-11 and the Na(+)-K(+)-ATPase α-subunit. Transfer of freshwater (FW)-acclimated salmon to SW induced a parallel increase in total α-subunit and FXYD-11 protein expression. A similar concurrent increase was seen during smoltification in FW. In FW fish, cortisol induced an increase in both α-subunit and FXYD-11 abundance, and growth hormone further stimulated FXYD-11 levels. In SW fish, prolactin induced a decrease in FXYD-11 and α-subunit protein levels. In vitro cortisol (18 h, 10 μg/ml) stimulated FXYD-11, but not FXYD-9, mRNA levels in gills from FW and SW salmon. The data show that Na(+)-K(+)-ATPase expressed in branchial mitochondrion-rich cells is accompanied by FXYD-11, and that regulation of the two proteins is highly coordinated. The demonstrated association of FXYD-11 and α-subunit strengthens our hypothesis that FXYD-11 has a role in modulating the pump's kinetic properties. The presence of putative phosphorylation sites on the intracellular domain of FXYD-11 suggests the possibility that this protein also may transmit external signals that regulate Na(+)-K(+)-ATPase activity.
 
Patient demographics Male Female 
Article
Placental 11beta-hydroxysteroid dehydrogenase-2 (11betaHSD2) limits fetal glucocorticoid exposure and is associated with physiological stability in the premature newborn infant. Antenatal betamethasone alters 11betaHSD2 activity and confers sex-specific advantages in neonatal outcome. We investigated the influence of betamethasone and sex on 11betaHSD2 activity, neonatal adrenal function and clinical course in 24- to 36-wk gestation neonates from birth to day 5 of life. Univariate analyses demonstrated an interaction between timing of betamethasone exposure and sex for 11betaHSD2 activity rate (P = 0.02) and umbilical arterial cortisol (P = 0.01). For infants born < 72 h following antenatal betamethasone, females had higher 11betaHSD2 activity (P < 0.01) and umbilical arterial cortisol (P = 0.01) than males. Females born < 72 h of betamethasone exposure had higher day 1 urinary cortisol, if exposed to perinatal stress, than males (P < 0.01). For infants born < 72 h after betamethasone exposure, 11betaHSD2 activity was negatively correlated with Clinical Illness Severity Score score (r = -0.79 P = 0.01) and positively correlated with mean arterial blood pressure (r = 0.8 P = 0.01) only in females. Sex-specific placental 11BHSD2 autoregulation following antenatal betamethasone exposure may limit adrenal suppression in females influencing physiological stability following preterm birth. A lack of adjustment in 11betaHSD2 and adrenal response may contribute to the increased incidence of poor outcome observed in preterm males.
 
Article
Insulin covalently and allosterically regulates glycogen synthase (GS) and may also cause the translocation of GS from glycogen-poor to glycogen-rich locations. We examined the possible role of subcellular localization of GS and glycogen in insulin activation of GS in skeletal muscle of six obese monkeys and determined whether 1) insulin stimulation during a hyperinsulinemic euglycemic clamp and/or peroxisome proliferator-activated receptor (PPAR)-alpha agonist treatment (K-111, 3 mg.kg(-1).day(-1); Kowa) induced translocation of GS and 2) translocation of GS was associated with insulin activation of GS. GS and glycogen were present in all fractions obtained by differential centrifugation, except for the cytosolic fraction, under both basal and insulin-stimulated conditions. We found no evidence for translocation of GS by insulin. GS total (GST) activity was strongly associated with glycogen content (r = 0.70, P < 0.001). Six weeks of treatment with K-111 increased GST activity in all fractions, except the cytosolic fraction, and mean GST activity, GS independent activity, and glycogen content were significantly higher in the insulin-stimulated samples compared with basal samples, effects not seen with vehicle. The increase in GST activity was strongly related to the increase in glycogen content during the hyperinsulinemic euglycemic clamp after K-111 administration (r = 0.74, P < 0.001). Neither GS protein expression nor GS gene expression was affected by insulin or by K-111 treatment. We conclude that 1) in vivo insulin does not cause translocation of GS from a glycogen-poor to a glycogen-rich location in primate skeletal muscle and 2) the mechanism of action of K-111 to improve insulin sensitivity includes an increase in GST activity without an increase in GS gene or protein expression.
 
Article
Rat offspring prenatally exposed to alcohol display features of metabolic syndrome characterized by a low birth weight, catch-up growth, dyslipidemia, and insulin-resistant diabetes with increased gluconeogenesis, during adult life. Gluconeogenesis is partly regulated by cyclic AMP- and glucocorticoid-dependent mechanisms. Glucocorticoid action at the receptor level depends on its circulating concentrations and is amplified at the prereceptor level by 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), which regenerates active glucocorticoids from inactive forms. To determine whether 11beta-HSD1 is dysregulated in this rat model, we examined the expression and enzyme activity of 11beta-HSD1 and its regulator enzyme hexose-6-phosphate dehydrogenase (H6PD) in the liver of postnatal day 7 (neonatal) and 3-mo-old (adult) rat offspring prenatally exposed to alcohol. Measurements of 11beta-HSD1 and H6PD were also performed in the omental fat of adult rat offspring. In both neonatal and adult rats, prenatal alcohol exposure resulted in increased tissue corticosterone concentrations, increased expression, and oxoreductase activity of 11beta-HSD1, and a parallel increase of H6PD expression. The data suggest that due to both transcriptional and posttranscriptional dysregulations, rats exposed to alcohol early in life have increased 11beta-HSD1 activity, which may explain insulin-resistant diabetes in these animals later in life.
 
Effect of bile duct ligation (BDL) on the renin-angiotensin aldosterone system. A: plasma renin concentrations in rats followed for 5 and 7 wk (W). Values are means SE *P 0.05. B: plasma concentrations of corticosterone and aldosterone after 7 wk compared with sham-operated controls (sham). Values are means SE. **P 0.05 vs. control.  
Effect of 7 wk of BDL on 11-hydroxysteroid dehydrogenase type 2 (11-HSD-2) and epithelial sodium channel (ENaC) in kidney cortex. A–C: renocortical expression of 11-HSD-2 mRNA (A) and protein (B) and 11-HSD-2 activity (C) compared with sham rats. Bars represent means SE. *P 0.05 vs. sham; n 8 –12 for RNA measurements, n 4 for protein determination, and n 6 –7 for activity assay. D: distribution of immunoreactive 11-HSD-2 protein in kidney sections. Labeling was associated with connecting tubules and cortical and outer medullary collecting ducts in sham rats (left), and staining was less intense in decompensated BDL rats (right). Bar, 50 m. E: results of a ribonuclease protection assay for ENaC -, -, and -subunit mRNAs in kidney cortex from sham and BDL rats. F: quantitative evaluation of the protection assay in which ENaC subunit mRNAs in BDL rats (filled bars, n 8) were compared with those in sham rats (open bars, n 12). Bars represent mean SE counts per minute. *P 0.05. G: distribution of immunoreactive ENaC -subunit protein in BDL kidney (right) compared with kidney from sham rat (left). Top, immunoreactive protein visualized with diaminobenzidine (DAB ) as chromogen substrate; bottom, micrographs with fluorescence detection.  
Effect of dexamethasone and K-canrenoate on urinary and fecal Na /K ratio in BDL rats. A: effect of Dex and Can on urinary Na /K ratio in BDL compared with sham rats. B: effect of Dex and Can on fecal Na /K ratio in BDL rats compared with sham rats. Values are shown as means SE. Numbers of rats in each group are as reported in Fig. 4. *P 0.05; **P 0.01.  
Article
Downregulation of the renal glucocorticoid-metabolizing enzyme 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD-2) during liver cirrhosis may allow activation of the mineralocorticoid receptor (MR) by glucocorticoids and contribute to sodium retention. We tested this hypothesis in male Wistar rats with decompensated liver cirrhosis and ascites 7 wk after bile duct ligation (BDL). Renal 11beta-HSD-2 mRNA, protein, and activity were significantly decreased in decompensated rats. The urinary Na(+)/K(+) ratio was reduced by 40%. Renal epithelial sodium channel (ENaC) mRNA and immunostaining were only slightly affected. Complete metabolic studies, including fecal excretion, showed that the BDL rats had avid renal sodium retention. Treatment of the BDL rats with dexamethasone suppressed endogenous glucocorticoid production, normalized total sodium balance and renal sodium excretion, and reduced ascites formation to the same degree as direct inhibition of MR with K-canrenoate. Total potassium balance was negative in the BDL rats, whereas renal potassium excretion was unchanged. In the distal colon, expression of ENaC was increased in BDL rats. Fecal potassium excretion was increased in cirrhotic rats, and this was corrected by treatment with K-canrenoate but not dexamethasone. We conclude that development of sodium retention and decompensation in cirrhotic rats is associated with downregulation of renal 11beta-HSD-2 activity and inappropriate activation of renal sodium reabsorption by endogenous glucocorticoids. In addition, the overall potassium loss in the BDL model is due to increased fecal potassium excretion, which is associated with upregulation of ENaC in distal colon.
 
Article
11beta-hydroxysteroid dehydrogenases (HSDs) interconvert active 11-hydroxy glucocorticoids (cortisol, corticosterone) and their inert 11-keto derivatives (cortisone, 11-dehydrocorticosterone). 11beta-HSD type 1 is a predominant reductase that regenerates active glucocorticoids in expressing cells, thus amplifying local glucocorticoid action, whereas 11beta-HSD type 2 catalyzes rapid dehydrogenation, potently inactivating intracellular glucocorticoids. Both isozymes thus regulate receptor activation by substrate availability. Spatial and temporal regulation of expression are important determinants of the physiological roles of 11beta-HSDs, with each isozyme exhibiting a distinct, tissue-restricted pattern together with dynamic regulation during development and in response to environmental challenges, including diet and stress. Transgenic approaches in the mouse have contributed significantly toward an understanding of the importance of these prereceptor regulatory mechanisms on corticosteroid receptor activity and have highlighted its potential relevance to human health and disease. Here we discuss current ideas of the physiological roles of 11beta-HSDs, with emphasis on the key contributions made by studies of 11beta-HSD gene manipulation in vivo.
 
Article
The syndrome of apparent mineralocorticoid excess (SAME) is an autosomal recessive form of salt-sensitive hypertension caused by deficiency of the kidney type 2 11beta-hydroxysteroid dehydrogenase (11betaHSD2). In this disorder, cortisol is not inactivated by 11betaHSD2, occupies mineralocorticoid receptors (MRs), and causes excessive sodium retention and hypertension. In renal medulla, prostaglandins derived from cyclooxygenase-2 (COX-2) stimulate sodium and water excretion, and renal medullary COX-2 expression increases after mineralocorticoid administration. We investigated whether medullary COX-2 also increases in rats with 11betaHSD2 inhibition and examined its possible role in the development of hypertension. 11betaHSD2 inhibition increased medullary and decreased cortical COX-2 expression in adult rats and induced high blood pressure in high-salt-treated rats. COX-2 inhibition had no effect on blood pressure in control animals but further increased blood pressure in high-salt-treated rats with 11betaHSD2 inhibition. COX-1 inhibition had no effect on blood pressure in either control or experimental animals. 11betaHSD2 inhibition also led to medullary COX-2 increase and cortical COX-2 decrease in weaning rats, primarily through activation of MRs. In the suckling rats, medullary COX-2 expression was very low, consistent with a urinary concentrating defect. 11betaHSD2 inhibition had no effect on either cortical or medullary COX-2 expression in the suckling rats, consistent with low levels of circulating corticosterone in these animals. These data indicate that COX-2 plays a modulating role in the development of hypertension due to 11betaHSD2 deficiency and that 11betaHSD2 regulates renal COX-2 expression by preventing glucocorticoid access to MRs during postnatal development.
 
Article
The placenta expresses high levels of 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2) that converts cortisol into inactive 11-keto metabolites and effectively protects the developing fetus from maternal cortisol during pregnancy. Impairment of this glucocorticoid barrier has adverse effects on fetal outcomes. A similar spectrum of adverse fetal effects is induced by antenatal stress during pregnancy. To examine the hypothesis that physiological stress may regulate placental 11betaHSD2 gene expression, we examined the effects of the catecholamines norepinephrine (NE) and epinephrine (E) on 11betaHSD2 expression in human trophoblastic cells. With the use of Northern blotting and semiquantitative RT-PCR, we determined that NE and E rapidly downregulate 11betaHSD2 steady-state mRNA levels in early- and late-gestation human trophoblasts and BeWo trophoblastic cells. Experiments using different adrenoceptor subtype-selective agonists and antagonists demonstrated that this catecholamine suppression of 11betaHSD2 mRNA expression is mediated via both alpha(1)- and alpha(2)-adrenoceptors and is independent of beta-adrenergic stimulation. To examine transcriptional regulation, BeWo cells were transiently transfected with a reporter construct in which an 11betaHSD2 human promoter sequence was inserted upstream of the luciferase gene. Treatment with 10(-7) M NE decreased luciferase activity by ~60% (n = 3, P < 0.01). These results suggest the NE/E-mediated decrease in placental 11betaHSD2 gene expression is an instance of alpha-adrenoceptor-specific rapid transcriptional inhibition of an adrenergic target gene. This molecular mechanism may be involved in the deleterious effects of antenatal physiological stress on fetoplacental growth and development.
 
Article
Transient receptor potential vanilloid 4 (TRPV4) is one member of the TRP superfamily of nonselective cation channels. Recently, the possibility has been raised that TRPV4 is an osmoreceptor, because it is found in the circumventricular organs where osmoreceptors are supposed to be distributed and because it is sensitive to osmotic pressure in in vitro experiments. In addition, TRPV4 knockout mice have abnormal osmosensitivity. In this study, effects of 4alpha-phorbol 12,13-didecanoate (4alpha-PDD), a TRPV4 agonist, on drinking behavior were examined to investigate roles for TRPV4 as an osmoreceptor in vivo in wild-type animals. Intracerebroventricular injections of 4alpha-PDD inhibited water intake under normal conditions in both light and dark periods of the day, after food deprivation, and after administration of angiotensin II. However, this drug did not influence increased water intake after administration of a hypertonic solution or after water deprivation that significantly increased plasma osmolality. Locomotor activity of the 4alpha-PDD-injected group decreased slightly compared with that of the vehicle-injected group; however, sweet taste, food intake, and body temperature were not different between the two groups. The antidipsogenic effects of 4alpha-PDD were blocked by preinjection into the ventricle of TRPV4 antagonists such as ruthenium red or gadolinium. These findings suggest that TRPV4 regulates drinking behavior under certain conditions, and the regulation interacts with the angiotensin II pathway.
 
MSNA in HUT tests
Baroreflex slopes for MSNA ( A ) and R-R interval ( B ) in Valsalva maneuver before (Pre-HDBR), after 60 (HDBR 60 ) and 120 days (HDBR 120 ) of HDBR, and during recovery (Rec- 
Heart rate and mean blood pressure in HUT tests Supine (0°) 30° HUT 60° HUT
Article
To examine how long-lasting microgravity simulated by 6 degrees head-down bed rest (HDBR) induces changes in the baroreflex control of muscle sympathetic nerve activity (MSNA) at rest and changes in responses of MSNA to orthostasis, six healthy male volunteers (range 26-42 yr) participated in Valsalva maneuver and head-up tilt (HUT) tests before and after 120 days of HDBR. MSNA was measured directly using a microneurographic technique. After long-term HDBR, resting supine MSNA and heart rate were augmented. The baroreflex slopes for MSNA during Valsalva maneuver (in supine position) and during 60 degrees HUT test, determined by least-squares linear regression analysis, were significantly steeper after than before HDBR, whereas the baroreflex slopes for R-R interval were significantly flatter after HDBR. The increase in MSNA from supine to 60 degrees HUT was not different between before and after HDBR, but mean blood pressure decreased in 60 degrees HUT after HDBR. In conclusion, the baroreflex control of MSNA was augmented, whereas the same reflex control of R-R interval was attenuated after 120 days of HDBR.
 
Article
C57BL/6J (B6) mice consume more sugar and fat solutions than do 129 mice in 24-h preference tests. Previous studies have attributed this observation to strain differences in taste responsiveness to these nutrients. We tested the hypothesis that differences in postingestive responsiveness contribute to the strain differences. In experiment 1, B6 and 129 mice were trained to associate consumption of a flavored solution (CS+) with intragastric (IG) infusions of 16% sucrose and a different flavored solution (CS-) with IG water infusions (22 h/day). They were then retrained with new flavors paired with IG infusions of 5.6% soybean oil and water. Although both strains developed preferences for the nutrient-paired CS+ solutions, the B6 mice displayed significantly stronger preferences. The B6 mice consumed more CS+ during training, which may have contributed to their enhanced preference. In a second experiment, training intakes were equated by giving B6 and 129 mice "isosweet" CS solutions prepared with different amounts of sucrose and saccharin. The B6 and 129 mice consumed more of the sugar- or fat-paired CS+ than the water-paired CS- during training. The two strains also displayed equally strong preferences for the CS+ over CS- in choice tests, indicating that they had similar postingestive responsiveness to the sucrose and soybean oil. We propose that B6 mice consume more sugar and fat than 129 mice because their stronger orosensory response stimulates greater intake, which leads to greater stimulation of postingestive nutrient detectors and further enhancement of consumption.
 
Selective inhibitors of NADPH oxidase or short interfering RNA (siRNA)-p47 phox inhibits glucose-stimulated reactive oxygen species (ROS) generation in insulin-secreting cells. INS 832/13 cells were incubated with either diluent or apocynin (100 M, 12 h; A) or diphenyleneiodonium chloride (DPI; 5 M, 2 h; A) or transfected with p47 phox siRNA (B and C), following which they were stimulated with either low (2.5 mM; LG) or high glucose (20 mM; HG) for 1 h. ROS generated was quantified as dichlorofluorescein (DCF) fluorescence and expressed as arbitrary units (AU). B: transfection efficiency of p47 phox siRNA was determined by immunoblotting. Values are means SE from three independent experiments done in triplicates in each case. *P 0.05 vs. LG alone or mock transfected LG. **P 0.05 vs. HG alone or mock transfected HG.  
Depletion of endogenous GTP markedly attenuates glucose-induced ROS generation in INS 832/13 cells Condition Degree of ROS Generation, fold over basal glucose
Pertussis toxin (Ptx) pretreatment attenuates glucose-induced ROS generation in INS 832/13 cells or normal rat islets. Untreated or Ptx-treated (100 ng/ml) INS 832/13 cells (A) or normal rat islets (B) were stimulated with either LG (2.5 mM) or HG (20 mM) for 1 h. ROS generated was quantified as DCF fluorescence and expressed as AU. Values are means SE from three independent experiments done in triplicates (in INS 832/13 cell) and in duplicates (in islets) in each case. *P 0.05 vs. LG alone. **P 0.05 vs. HG alone.  
Article
Recent evidence suggests that an acute increase in the generation of phagocyte-like NADPH-oxidase (Nox)-mediated reactive oxygen species (ROS) may be necessary for glucose-stimulated insulin secretion. Using rat islets and INS 832/13 cells, we tested the hypothesis that activation of specific G proteins is necessary for nutrient-mediated intracellular generation of ROS. Stimulation of β-cells with glucose or a mixture of mitochondrial fuels (mono-methylsuccinate plus α-ketoisocaproic acid) markedly elevated intracellular accumulation of ROS, which was attenuated by selective inhibitors of Nox (e.g., apocynin or diphenyleneiodonium chloride) or short interfering RNA-mediated knockdown of p47(phox), one of the subunits of Nox. Selective inhibitors of protein prenylation (FTI-277 or GGTI-2147) markedly inhibited nutrient-induced ROS generation, suggesting that activation of one (or more) prenylated small G proteins and/or γ-subunits of trimeric G proteins is involved in this signaling axis. Depletion of endogenous GTP levels with mycophenolic acid significantly reduced glucose-induced activation of Rac1 and ROS generation in these cells. Other immunosuppressants, like cyclosporine A or rapamycin, which do not deplete endogenous GTP levels, failed to affect glucose-induced ROS generation, suggesting that endogenous GTP is necessary for glucose-induced Nox activation and ROS generation. Treatment of INS 832/13 cells or rat islets with pertussis toxin (Ptx), which ADP ribosylates and inhibits inhibitory class of trimeric G proteins (i.e., G(i) or G(o)), significantly attenuated glucose-induced ROS generation in these cells, implicating activation of a Ptx-sensitive G protein in these signaling cascade. Together, our findings suggest a prenylated Ptx-sensitive signaling step couples Rac1 activation in the signaling steps necessary for glucose-mediated generation of ROS in the pancreatic β-cells.
 
Subject characteristics Variable Means SE 
Average 75-km metabolic and performance data Variable Means SE 
Changes in 13-and 9-hydroxy-octadecadienoic acid (13-HODE 9-HODE) across four time points: preexercise, immediately post-75-km cycling time trial, and 1.5-h and 21-h postexercise. *P 0.01 compared with preexercise. Time main effect, P 0.001. 
Changes in (Z)-9,10-dihydroxyoctadec-12-enoic acid (9,10-DiHOME) and (Z)-12,13-dihydroxyoctadec-9-enoic acid (12,13-DiHOME) and across four time points: preexercise, immediately post-75-km cycling time trial, and 1.5-h and 21-h postexercise. *P 0.01 compared with preexercise. Time main effect, P 0.001. 
Article
Bioactive oxidized linoleic acid metabolites (OXLAMs) include 13- and 9-hydroxy-octadecadienoic acid (13-HODE + 9-HODE), and have been linked to oxidative stress, inflammation, and pathological and physiological states. The purpose of this study was to measure changes in plasma 13-HODE + 9-HODE following a 75-km cycling bout and identify potential linkages to linoleate metabolism and established biomarkers of oxidative stress (F2-isoprostanes) and inflammation (cytokines) using a metabolomics approach. Trained male cyclists (N=19, age 38.0±1.6 y, wattsmax 304±10.5) engaged in a 75-km cycling time trial on their own bicycles using electromagnetically-braked cycling ergometers (2.71±0.07 h). Blood samples were collected pre-exercise, and immediately post-, 1.5-h post-, and 21-h post-exercise, and analyzed for plasma cytokines (IL-6, IL-8, IL-10, TNFα, MCP-1, GCSF), F2-isoprostanes, and shifts in metabolites using global metabolomics procedures with GC-MS and LC-MS. 13-HODE + 9-HODE increased 3.1-fold and 1.7-fold immediately post- and 1.5-h post-exercise (both p<0.001), and returned to pre-exercise levels by 21-h post-exercise. Post-75-km cycling plasma levels of 13-HODE + 9-HODE were not significantly correlated with increases in plasma cytokines, but were positively correlated with post-exercise F2-isoprostanes (r=0.75, p<0.001), linoleate (r=0.54, P=0.016), arachidate (r=0.77, p<0.001), 12,13-dihydroxy-9Z-octadecenoate (12,13-DiHOME) (r=0.60, p=0.006), dihomo-linolenate (r=0.57, p=0.011), and adrenate (r=0.56, p=0.013). These findings indicate that prolonged and intensive exercise caused a transient, 3.1-fold increase in the stable linoleic acid oxidation product 13-HODE + 9-HODE, and was related to increases in F2-isoprostanes, linoleate, and fatty acids in the linoleate conversion pathway. These data support the use of 13-HODE + 9-HODE as an oxidative stress biomarker in acute exercise investigations.
 
Effects of intracerebroventricular injection of interleukin-13 (IL-13) on the time spent in non-rapid eye movement sleep (NREMS) and rapid eye movement sleep (REMS), electroencephalograph (EEG) slow-wave activity (SWA) and brain temperature (Tbr). E, Vehicle treatment group; s, IL-13 treatment group. Horizontal shaded bars denote dark phase. IL-13 (200 ng) was injected either during the light phase (A) or at dark onset (B). IL-13 significantly inhibited NREMS. All data shown are averages obtained from 2-h intervals and expressed as means SE.
Effects of intracerebroventricular injection of transforming growth factor-1 (TGF-1) on time spent in NREMS, REMS, EEG SWA, and Tbr. E, vehicle treatment group; s, TGF-1 treatment group. TGF-1 (200 ng) was injected either during light phase (A) or at dark onset (B). TGF-1 significantly inhibited NREMS when given during light phase; however, it failed to affect NREMS if injected at dark onset. All data shown are averages obtained from 2-h time blocks SE. 
Article
Proinflammatory cytokines, including interleukin-1beta and tumor necrosis factor-alpha are involved in physiological sleep regulation. Interleukin (IL)-13 and transforming growth factor (TGF)-beta1 are anti-inflammatory cytokines that inhibit proinflammatory cytokines by several mechanisms. Therefore, we hypothesized that IL-13 and TGF-beta1 could attenuate sleep in rabbits. Three doses of IL-13 (8, 40, and 200 ng) and TGF-beta1 (40, 100, and 200 ng) were injected intracerebroventricularly 3 h after the beginning of the light period. In addition, one dose of IL-13 (200 ng) and one dose of TGF-beta1 (200 ng) were injected at dark onset. The two higher doses of IL-13 and the highest dose of TGF-beta1 significantly inhibited spontanenous non-rapid eye movement sleep (NREMS) when they were given in the light period. IL-13 also inhibited NREMS after dark onset administration; however, the inhibitory effect was less potent than that observed after light period administration. The 40-ng dose of IL-13 inhibited REMS duration during the dark period. TGF-beta1 administered at dark onset had no effect on sleep. These data provide additional evidence for the hypothesis that a brain cytokine network is involved in regulation of physiological sleep.
 
Article
Hibernating mammals have developed many physiological adaptations to extreme environments. During hibernation, 13-lined ground squirrels (Ictidomys tridecemlineatus) must suppress hemostasis to survive prolonged body temperatures of 4-8°C and 3-5 heartbeats per minute without forming lethal clots. Upon arousal in the spring, these ground squirrels must be able to quickly restore normal clotting activity to avoid bleeding. Here we show that ground squirrel platelets stored in vivo at 4-8°C were released back into the blood within 2 h of arousal in the spring with a body temperature of 37°C but were not rapidly cleared from circulation. These released platelets were capable of forming stable clots and remained in circulation for at least 2 days before newly synthesized platelets were detected. Transfusion of autologous platelets stored at 4°C or 37°C showed the same clearance rates in ground squirrels, whereas rat platelets stored in the cold had a 140-fold increase in clearance rate. Our results demonstrate that ground squirrel platelets appear to be resistant to the platelet cold storage lesions observed in other mammals, allowing prolonged storage in cold stasis and preventing rapid clearance upon spring arousal. Elucidating these adaptations could lead to the development of methods to store human platelets in the cold, extending their shelf life.
 
Confocal microscopic images of sections of Xenopus oocytes (stage IV or V) injected with 50 ng zglut1a-green fluorescent protein (GFP)-capped mRNA (cRNA) (A), 50 ng zglut6-GFP cRNA (B), 50 ng zglut13.1GFP cRNA (C), RNase-free modified Barth's saline (MBS) (D), and pCS2GFPXLT cRNA (E). Green florescence indicates the GFP signal. F: the zebrafish glucose transporter (zGLUT)-GFP chimeric protein. The zGLUT protein was tagged with GFP at its carboxyl terminus; the length, in amino acids (aa) or base pairs (bp), of each region and of the junction between zGLUT (white box) and GFP (dark gray box) is indicated. The amino (N) and carboxyl (C) termini of the fusion protein are indicated. The SP6 promoter region is shown as a light-gray arrow box. F: Western blots of GFP and-tubulin in oocytes injected with cRNAs of zGLUT1a-,-6-, and-13.1-pCS2GFPXLT, the vector (pCS2GFPXLT only), and MBS.
Effects of the translational knockdown of zglut1a , -6, 
Article
The hexose supply and subsequent metabolism are crucial for the operations of the iono- and osmoregulatory mechanisms in fish, but how hexose is transported and supplied to cells of the ionoregulatory epithelia is unknown. Three zebrafish glucose transporters (zGLUTs), zGLUT1a, -13.1, and -6, were previously found to respectively be expressed by ionocytes (Na(+)-K(+)-ATPase-rich, Na(+)-Cl(-) cotransporter-expressing, and H(+)-ATPase-rich cells) and adjacent energy-depositing cells [glycogen-rich (GR) cells] in zebrafish skin and gills (32). The present study aimed to test if the transport kinetics of these three zGLUTs differ, and if the transport functional differences are of physiological relevance to the respective functions of epithelial cells. The three zGLUTs expressed by Xenopus laevis oocytes revealed different d-glucose transport kinetics; zGLUT13.1 showed the lowest Michaelis constant (K(m)), whereas zGLUT6 had the highest K(m) and maximal velocity. In morpholino injection experiments, translational knockdown of zGLUT1a and -13.1, respectively, impaired Cl(-)/Ca(2+) and Na(+)/Ca(2+) uptake, but loss-of-function of zGLUT6 did not cause a significant effect on ion uptake functions in zebrafish. Based on these results, zGLUT1a and -13.1 appear to be superior to zGLUT6 in competing for glucose under a situation of low blood glucose due to extensive energy consumption, whereas, in a high blood glucose situation, zGLUT6 is able to absorb the excess glucose for energy deposition. The timely and sufficient supply of energy to ionocytes so that they can carry out ion regulation is definitely a more important event than storing energy in GR cells, particularly when acute environmental change disturbs the ion balance in zebrafish.
 
Effect of a single third cerebral ventricle (i3vt) injection of 1 nmol agouti-related peptide [AgRP-(83O132)] or saline at the onset of dark on food intake at 2 h (A), 24 h (B), and the ensuing effect on body weight as the percent change from preinjection weight (C). *** P 0.001, ** P 0.01, and * P 0.05 vs. saline.  
Long-lasting effect of a single i3vt injection of 0.01 and 0.1 nmol AgRP-(83O132) or saline (A) and 1 and 5 nmol AgRP-(83O132) or saline on noncumulative, 24-h food intakes (B) for 2 to 7 days after injection. Because of missing time points (due to severe weather), days 3 and 4 and 5 and 6 in A, and 4 and 5 in B represent the average daily intake over a 48-h period. *** P 0.001, ** P 0.01, and * P 0.05.  
Article
Overexpression of agouti-related peptide (AgRP), an endogenous melanocortin (MC) 3 and 4 receptor antagonist (MC3/4-R), causes obesity. Exogenous AgRP-(83---132) increases food intake, but its duration and mode of action are unknown. We report herein that doses as low as 10 pmol can have a potent effect on food intake of rats over a 24-h period after intracerebroventricular injection. Additionally, a single third ventricular dose as low as 100 pmol in rats produces a robust increase in food intake that persists for an entire week. AgRP-(83---132) completely blocks the anorectic effect of MTII (MC3/4-R agonist), given simultaneously, consistent with a competitive antagonist action. However, when given 24 h prior to MTII, AgRP-(83---132) is ineffective at reversing the anorectic effects of the agonist. These results support a critical role of MC tone in limiting food intake and indicate that the orexigenic effects of AgRP-(83---132) are initially mediated by competitive antagonism at MC receptors but are sustained by alternate mechanisms.
 
Article
Agouti-related peptide (AgRP) is a receptor antagonist of central nervous system (CNS) melanocortin receptors and appears to have an important role in the control of food intake since exogenous CNS administration in rats and overexpression in mice result in profound hyperphagia and weight gain. Given that AgRP is heavily colocalized with neuropeptide Y (NPY) and that orexigenic effects of NPY depend on activity at opioid receptors, we hypothesized that AgRP's food-intake effects are also mediated by opioid receptors. Subthreshold doses of the opioid receptor antagonist naloxone blocked AgRP-induced intake when given simultaneously but not 24 h after AgRP injection. Opioids not only influence food intake but food selection as well. Hence, we tested AgRP's effect to alter food choice between matched diets with differing dietary fat content. AgRP selectively enhanced intake of the high-fat but not the low-fat diet. Additionally, AgRP selectively increased chow intake in rats given ad libitum access to a 20% sucrose solution and standard rat chow. The current results indicate that AgRP influences not only caloric intake but food selection as well and that the early effects of AgRP depend critically on an interaction with opioid receptors.
 
Oxidative stress elicits muscle fiber atrophy with mechanical unloading. Fiftyfour hours of hindlimb unloading reduced muscle fiber size as displayed by fiber crosssectional area (CSA) distribution (A) for CON, hindlimb-unloaded (HU), and HU- EUK rats (n 8/group). EUK-134 protected against the left shift in muscle fiber CSA distribution, indicating that oxidative stress was causal in fiber atrophy in the early phase of unloading. Muscle fiber atrophy was apparent in slow-twitch of Type I fibers of the soleus (B) (P 0.01), but not in Type IIa fibers (C) or Type IIb/d fibers (D) of the soleus. a,b Letters that differ are significantly different from each other (P 0.05).  
EUK-134 has no effect on soleus mass or fiber cross-sectional area in ambulatory rats. Seventy-eight hours EUK-134 or saline injection had no differential effect on soleus muscle mass (A), soleus mass/body mass ratio (B), or soleus muscle cross-sectional area (C).  
EUK-134 reduces 4-hydroxynonenal (4-HNE) in the HU soleus. We demonstrated that oxidative stress (A) was increased by hindlimb unloading using 4-hydroxynonenal as a marker. Highest positive staining occurred near the sarcolemma as localized via immunofluorescence with laminin (B) used as a control for identifying muscle cells. Quantification of 4-HNE fluorescence intensity can be found in Fig. 6C. a Significantly different than the CON group (P 0.001). b Significantly different than the HU group (P 0.01).
Immunofluorescence of gp91phox in soleus muscles from control (CON) and hindlimb unloaded (HU) mice. -sarcoglycan is used as a sarcolemmal marker. Merged images are also displayed. Rabbit IgG is used as a negative control for the primary antibody.  
Article
Reduced mechanical loading during bedrest, spaceflight, casting, etc. causes rapid morphological changes in skeletal muscle: fiber atrophy and reduction of slow-twitch fibers. An emerging signaling event in response to unloading is the translocation of neuronal nitric oxide synthase (nNOSµ) from the sarcolemma to the cytosol. We used EUK-134, a cell-permeable mimetic of superoxide dismutase and catalase, to test the role of redox signaling in nNOSµ translocation and muscle fiber atrophy as a result of short-term (54 hours) hindlimb unloading. Fischer-344 rats were divided into ambulatory controls (CON), hindlimb unloaded (HU), and hindlimb unloaded + EUK-134 (HU-EUK) groups. EUK-134 mitigated the unloading-induced phenotype, including muscle fiber atrophy and muscle fiber-type shift from slow to fast. nNOSµ immunolocalization at the sarcolemma of the soleus was reduced with HU, while nNOSµ protein content in the cytosol increased with unloading. Translocation of nNOS from the sarcolemma to cytosol was virtually abolished by EUK-134. EUK-134 also mitigated dephosphorylation at Thr32 of FoxO3a during HU. Hindlimb unloading elevated oxidative stress (4-hydroxynonenal) and increased sarcolemmal localization of Nox2 subunits gp91phox (Nox2) and p47phox, effects normalized by EUK-134. Thus, our findings are consistent with the hypothesis that oxidative stress triggers nNOSµ translocation from the sarcolemma and FoxO3a dephosphorylation as an early event during mechanical unloading. Thus redox signaling may serve as a biological switch for nNOS to initiate morphological changes in skeletal muscle fibers.
 
Article
Endogenous endothelin (ET)-1 modulates hypoxic pulmonary vasoconstriction (HPV). Accordingly, intravenously applied ET(A) receptor antagonists reduce HPV, but this is accompanied by systemic vasodilation. We hypothesized that inhalation of an ET(A) receptor antagonist might act selectively on the pulmonary vasculature and investigated the effects of aerosolized LU-135252 in an experimental model of HPV. Sixteen piglets (weight: 25 +/- 1 kg) were anesthetized and mechanically ventilated at an inspiratory oxygen fraction (Fi(O(2))) of 0.3. After 1 h of hypoxia at Fi(O(2)) 0.15, animals were randomly assigned either to receive aerosolized LU-135252 as bolus (0.3 mg/kg for 20 min; n = 8, LU group), or to receive aerosolized saline (n = 8, controls). In all animals, hypoxia significantly increased mean pulmonary arterial pressure (32 +/- 1 vs. 23 +/- 1 mmHg; P < 0.01; means +/- SE) and increased arterial plasma ET-1 (0.52 +/- 0.04 vs. 0.37 +/- 0.05 fmol/ml; P < 0.01) compared with mild hyperoxia at Fi(O(2)) 0.3. Inhalation of LU-135252 induced a significant and sustained decrease in mean pulmonary arterial pressure compared with controls (LU group: 27 +/- 1 mmHg; controls: 32 +/- 1 mmHg; values at 4 h of hypoxia; P < 0.01). In parallel, mean systemic arterial pressure and cardiac output remained stable and were not significantly different from control values. Consequently, in our experimental model of HPV, the inhaled ET(A) receptor antagonist LU-135252 induced selective pulmonary vasodilation without adverse systemic hemodynamic effects.
 
Article
The aim of this study was to determine the effect of 14 days of 5-aminoimidazole-4-carboxamide-1β-4-ribofuranoside (AICAR) treatment on mammalian target of rapamycin (mTOR) signaling and mTOR-regulated processes (i.e., translation initiation) in obese mouse skeletal muscle. Our hypothesis was that daily treatment (14 days) with AICAR would normalize obesity-induced alterations in skeletal muscle mTOR signaling and mTOR-regulated processes to lean levels and positively affect muscle mass. Fourteen-week-old male, lean (L; 31.3 g body wt) wild-type and ob/ob (O; 59.6 g body wt) mice were injected with the AMP-activated kinase (AMPK) activator AICAR (A) at 0.5 mg·g body wt(-1)·day(-1) or saline control (C) for 14 days. At 24 h after the last injection (including a 12-h fast), all mice were killed, and the plantar flexor complex muscle (gastrocnemius, soleus, and plantaris) was excised for analysis. Muscle mass was lower in OC (159 ± 12 mg) than LC, LA, and OA (176 ± 10, 178 ± 9, and 166 ± 16 mg, respectively) mice, independent of a body weight change. A decrease in obese muscle mass corresponded with higher muscle cross section staining intensity for lipid and glycogen, higher blood glucose and insulin levels, and lower nuclear-enriched fractions for peroxisome proliferator-activated receptor-γ coactivator-1α protein expression in OC skeletal muscle, which was normalized with AICAR treatment. AMPK and acetyl-cocarboxylase phosphorylation was reduced in OC mice and augmented by AICAR treatment in OA mice. Conversely, OC mice displayed higher activation of downstream targets (S6 kinase-1 and ribosomal protein S6) of mTOR and lower raptor-associated mTOR than LC mice, which were reciprocally altered after 14 days of AICAR treatment. Dysregulation of translational capacity was improved in OA mice, as assessed by sucrose density gradient fractionation of ribosomes, total and ribosome-associated RNA content, eukaryotic initiation factor 4F complex formation, and eukaryotic initiation factor 4G phosphorylation. These data show that short-term (14 days) AMPK agonist treatment augments regulatory processes in atrophic obese mouse skeletal muscle through the normalization of mTOR signaling and mRNA translation closer to lean levels.
 
Article
Clinical therapies for both obesity and obese non-insulin-dependent diabetes mellitus require maintenance of reduced body weight after the initial successful reduction resulting from calorie control, exercise, or medication. Although beta(3)-adrenergic receptor (beta(3)-AR) agonists have been shown to stimulate whole body energy expenditure and lipid mobilization, whether stimulatory effects on oxygen consumption and lipolysis are influenced by chronic exposure to agonists has not been fully characterized. We therefore examined the acute and chronic effects of FR-149175, a selective beta(3)-AR agonist, on whole body oxygen consumption in genetically obese Zucker fatty rats. Chronic treatment with FR-149175 caused a decrease in both body weight gain and white fat pad weight at doses that induced lipolysis in acute treatment (1 and 3.2 mg/kg p.o.). Single administration of FR-149175 (0.1, 1, and 3.2 mg/kg p.o.) dose dependently increased whole body oxygen consumption. Repetitive administration did not cause attenuation of the thermogenic response at lower doses (0.1 and 1 mg/kg 2 times daily), whereas the highest dose (3.2 mg/kg 2 times daily) induced a progressive increase in oxygen consumption. PCR analyses of retroperitoneal white adipose tissue indicated little or no change in beta(3)-AR mRNA levels. Uncoupling protein 1 gene expression increased at 1 mg/kg, and drastic upregulation was detected at 3.2 mg/kg. FR-149175 also increased HSL mRNA levels in a dose-related manner, whereas there was no effect on genes involved in beta-oxidation. These results support that the thermogenic effect of beta(3)-AR agonists is not attenuated by chronic exposure to agonists.
 
Article
[(3)H]SSR-149415 is the first tritiated nonpeptide vasopressin V(1b) receptor (V(1b)R) antagonist ligand. It was used for studying rodent (mouse, rat, hamster) and human V(1b)R from native or recombinant origin. Moreover, a close comparison between the human and the mouse V(1b)R was performed using SSR-149415/[(3)H]SSR-149415 in binding and functional studies in vitro. [(3)H]SSR-149415 binding was time-dependent, reversible, and saturable. Scatchard plot analysis gave a single class of high-affinity binding sites with apparent equilibrium dissociation constant (K(d)) approximately 1 nM and maximum binding density (B(max)) values from 7,000 to 300,000 sites/cell according to the cell line. In competition experiments, [(3)H]SSR-149415 binding was stereospecific and dose-dependently displaced by reference peptide and nonpeptide arginine vasopressin (AVP)/OT ligands following a V(1b) rank order of affinity: SSR-149415 = AVP > dCha > dPen > dPal > dDavp > SSR-126768A > SR-49059 > SSR-149424 > OT > SR-121463B. Species differences between human, rat, mouse, and hamster V(1b)R were observed. Autoradiography studies with [(3)H]SSR-149415 on rat and human pituitary showed intense specific labeling confined to corticotroph cells and absence of labeling in the other tissues examined. SSR-149415 potently and stereospecifically antagonized the AVP-induced inositol phosphate production and intracellular Ca(2+) increase (EC(50) from 1.83 to 3.05 nM) in recombinant cell lines expressing either the mouse or the human V(1b)R. AVP (10(-7) M) exposure of AtT20 cells expressing mouse or human EGFP-tagged V(1b)R induced their rapid internalization. Preincubation with 10(-6) M SSR-149415 counteracted the internalization process. Moreover, recycling of internalized receptors was observed upon 10(-6) M SSR-149415 treatment. Thus SSR-149415/[(3)H]SSR-149415 are unique tools for studying animal and human V(1b)R.
 
Article
In this study, we have investigated the hypothesis that an exercise protocol designed to repeatedly induce a large dependence on carbohydrate and large increases in glycolytic flux rate would result in rapid increases in the principal glucose and lactate transporters in working muscle, glucose transporter (GLUT)-4 and monocarboxylate transporter (MCT)4, respectively, and in activity of hexokinase (Hex), the enzyme used to phosphorylate glucose. Transporter abundance and Hex activity were assessed in homogenates by Western blotting and quantitative chemiluminescence and fluorometric techniques, respectively, in samples of tissue obtained from the vastus lateralis in 12 untrained volunteers [peak aerobic power (.VO(2peak)) = 44.3 +/- 2.3 ml.kg(-1).min(-1)] before cycle exercise at repetitions 1 (R1), 2 (R2), 9 (R9), and 16 (R16). The 16 repetitions of the exercise were performed for 6 min at approximately 90% .VO(2peak), once per hour. Compared with R1, GLUT-4 increased (P < 0.05) by 28% at R2 and remained elevated (P < 0.05) at R9 and R16. For MCT-4, increases (P < 0.05) of 24% were first observed at R9 and persisted at R16. No changes were observed in GLUT-1 and MCT-1 or in Hex activity. The approximately 17- to 24-fold increase (P < 0.05) in muscle lactate observed at R1 and R2 was reduced (P < 0.05) to an 11-fold increase at R9 and R16. It is concluded that an exercise protocol designed to strain muscle carbohydrate reserves and to result in large increases in lactic acid results in a rapid upregulation of both GLUT-4 and MCT-4.
 
Article
The X-linked ANG II type 2 receptor (AT2) is supposed to be involved in cardiovascular disorders. Two studies associated the A allele of the AT2 gene polymorphism (PM) 1675 G/A with left ventricular hypertrophy in men and coronary ischemia in women. Because the PM is located in the short intron 1 of the AT2 gene within a sequence motif similar to the splice branch site consensus, we tested whether it might affect pre-mRNA splicing and/or modulate AT2 receptor expression. We first analyzed the AT2 mRNA splice pattern by RT-PCR in myocardial samples from 12 explanted human hearts and compared it with the respective genotypes. All 12 patients, 10 hemizygous males (7 A, 3 G allele carriers) and 2 homozygous females (2 G/G allele carriers), exhibited the same myocardial AT2 splice pattern with a relative abundance of transcript exon 1/2/3 compared with exon 1/3. Next, AT2 minigene constructs were cloned from both alleles, comprising the core promoter and the complete transcribed region up to the translation start codon, upstream of a luciferase reporter gene. These constructs were transfected into human (HT1080) and rat (PC12W) cell lines and rat vascular smooth muscle cells, and luciferase activities were assessed, as well as the splice patterns of the chimeric AT2/luciferase mRNAs. In all transfected cell types, the mRNA expressed from the AT2 constructs was spliced like the endogenous myocardial AT2 mRNA. However, luciferase activities driven by the G allele construct were significantly higher than those expressed from the A allele. Taken together, these data indicate that individuals carrying the G allele may express higher levels of AT2 receptor protein, which may be protective during the development of ventricular hypertrophy and coronary ischemia.
 
Article
In the present study, the effects of 17beta-estradiol (E(2)) treatment on the expression of preprosomatostatin (PPSS) I, PPSS II', and PPSS II" mRNA in the hypothalamus and endocrine pancreas (Brockmann body), as well as the effects of E(2) treatment on plasma somatostatin (SS)-14 and -25 concentrations in sexually immature rainbow trout (Oncorhynchus mykiss), were investigated. E(2) treatment significantly (P < 0.001) depressed both plasma SS-14 and SS-25. In the hypothalamus, E(2) treatment significantly (P < 0.001) decreased the levels of PPSS I and PPSS II" mRNA. However, there was no effect of E(2) treatment on PPSS II' mRNA levels. In the pancreas, E(2) treatment had no significant effect on the levels of either PPSS II' mRNA or PPSS II" mRNA. However, E(2) treatment significantly (P < 0.005) decreased levels of PPSS I mRNA. These data suggest that E(2) acts, in part, to increase plasma growth hormone levels in rainbow trout by decreasing the endogenous inhibitory somatostatinergic tone by inhibiting plasma levels of both SS-14 and SS-25 and hypothalamic levels of mRNA encoding these proteins.
 
Chromosome 17 and the position of the B6.CAST-17 congenic segment responsible for increased carbohydrate and total energy consumption. This region contains an estimated 1138 genes. Map distance is illustrated in both cM and Mb. The gray shaded areas represent yet undefined regions of CAST- or B6-derived genomic DNA. 
Daily calorie intake per 20 g body wt of carbohydrate/protein (C/P) ( A ), fat/protein (F/P) ( B ) and total kcal ( C ) in mice self- selecting from a choice between the F/P and C/P diets. A hyperphagic response, specific for the C/P diet, was indicated by main effects of day on both C/P kcal [ F (9,591) ϭ 15.34, P Ͻ 0.0001] and total kcal [ F (9,571) ϭ 19.32, P Ͻ 0.0001]. Over the 10-day study, homozygous B6.CAST-17 mice (HOMO) consumed 27% more total daily calories than the heterozygous congenic (HET) or wild-type (WT) strains [ F (2,102) ϭ 12.72, P Ͻ 0.0001], due to a genotype effect on intake of the C/P [ F (2,75.8) ϭ 3.39, P Ͻ 0.05] but not F/P diet [ F (2,78.1) ϭ 0.87, P ϭ 0.42]. * P Ͻ 0.05, genotype effect by day. 
Expression pattern of Glp1r in parental strain tissues implicated in the regulation of food intake. A : qRT-PCR expression of Glp1r mRNA in pancreas, hypothalamus, fundal, and antral stomach of C57BL/6J (B6) and CAST/Ei (C) mice. Data are means Ϯ SE from 12 mice per strain analyzed in triplicate. B : Western blot analyses of pancreas and stomach mucosa yielded a single ϳ 50-kDa band in both B6 and CAST corresponding to the known molecular weight of the human GLP-1R. Mouse ␤ -actin antibody was applied as a loading control. Significance in t -test is indicated by * P Ͻ 0.01; † P Ͻ 0.001; # P Ͻ 10 Ϫ 5 . GLP1R, glucagon-like peptide-1 receptor. 
Expression pattern of Glp1r in congenic strain tissues. A : qRT-PCR expression of Glp1r mRNA in pancreas, stomach, and hypothalamus of wild-type B6 and homozygous B6.CAST-17 congenic mice. Data are means Ϯ SE from 7 to 11 mice per strain analyzed in duplicate. B : Western blot analyses of pancreas and gastric mucosa yielded a single ϳ 50-kDa band in wild-type B6 and homozygous B6.CAST-17 corresponding to the known molecular weight of the human GLP-1R. Mouse ␤ -actin antibody was applied as a loading control. Significance in t -test is indicated by * P Ͻ 0.01; # P Ͻ 0.005. 
Article
Quantitative trait loci (QTL) for carbohydrate (Mnic1) and total energy (Kcal2) intake on proximal mouse chromosome 17 were identified previously from a C57BL/6J (B6) X CAST/Ei (CAST) intercross. Here we report that a new congenic strain developed in our laboratory has confirmed this complex locus by recapitulating the original linked phenotypes: B6.CAST-17 homozygous congenic mice consumed more carbohydrate (27%) and total energy (17%) compared with littermate wild-type mice. Positional gene candidates with relevance to carbohydrate metabolism, glyoxalase I (Glo1) and glucagon-like peptide-1 receptor (Glp1r), were evaluated. Glo1 expression was upregulated in liver and hypothalamus of congenic mice when compared with B6 mice. Analyses of Glp1r mRNA and protein expression revealed tissue-specific strain differences in pancreas (congenic>B6) and stomach (B6>congenic). These results suggest the possibility of separate mechanisms for enhanced insulin synthesis and gastric accommodation in the presence of high carbohydrate intake and larger food volume, respectively. Sequence analysis of Glp1r found a G insert at nt position 1349, which results in earlier termination of the open reading frame, thus revealing an error in the public sequence. Consequently, the predicted length of GLP-1R is 463 aa compared with 489 aa, as previously reported. Also, we found a polymorphism in Glp1r between parental strains that alters the amino acid sequence. Variation in Glp1r could influence nutrient intake in this model through changes in the regulatory or protein coding regions of the gene. These congenic mice offer a powerful tool for investigating gene interactions in the control of food intake.
 
IL-17-induced hypertension is attenuated with a superoxide dismutase (SOD) mimetic (tempol) or AT1 receptor blockade. Chronic infusion of IL-17 into normal pregnant (NP) rats causes hypertension during pregnancy. This blood pressure (MAP) response is attenuated by administration of a SOD mimetic or losartan, an At1 receptor blocker. Data are expressed as means Ϯ SE. *** P Ͻ 0.05 vs. NP controls. 
Infusion of IL-17 into NP rats increased circulating CD4/RORT cells compared with NP control rats. Data are expressed as means SE. **P 0.05 vs. NP controls.
IL-17 infusion increases urinary isoprostane excretion. Urinary isoprostane excretion is increased in response to chronic IL-17 infusion, indicating that IL-17 increases oxidative stress. Data are expressed as means Ϯ SE. * P Ͻ 0.05 vs. NP controls. 
IL-17-infused placental oxidative stress is blunted by a SOD mimetic (tempol) or losartan. A : chronic infusion of IL-17 into NP rats significantly produces greater NADPH-stimulated placental reactive oxygen species (ROS). B : chronic administration of tempol to IL-17-infused rats decreases NADPH-stimulated placental oxidative stress to levels no longer significantly different from that of tempol-treated controls. C : chronic AT1 receptor blockade with losartan significantly decreased NADPH-stimulated placental oxidative stress IL-17-treated rats. Data are expressed as means Ϯ SE. RLU, relative light units. *** P Ͻ 0.05 vs. NP controls. 
Tempol administration decreases AT1-AA in response to chronic infusion of IL-17. AT1-AA isolated from serum of rats chronically infused with IL-17 is significantly increased compared with NP rats but is blunted in response to tempol administration. Data are expressed as means Ϯ SE. *** P Ͻ 0.001 vs. IL-17-infused rats. 
Article
Preeclampsia is associated with autoimmune cells T(H)17, secreting interleukin-17, autoantibodies activating the angiotensin II type I receptor (AT1-AA), and placental oxidative stress (ROS). The objective of our study was to determine whether chronic IL-17 increases blood pressure by stimulating ROS and AT1-AAs during pregnancy. To answer this question four groups of rats were examined: normal pregnant (NP, n = 20), NP+IL-17 (n = 12), NP+tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) (n = 7) (a superoxide dismutase mimetic that scavenges ROS), and NP+IL-17+tempol (n = 11). IL-17 (150 pg/day) was infused into NP rats while tempol was administered via the drinking water ad libitum. On day 19 blood pressure (MAP) was recorded, and plasma, urine, and tissue were collected for isolation of ROS detected by chemilluminescent technique. Urinary isoprostane was measured by ELISA. AT1-AAs were determined via cardiomyocyte assay and expressed as beats per minute. MAP increased from 98 ± 3 mmHg in NP to 123 ± 3 mmHg in IL-17-infused NP rats. Urinary isoprostane increased from 1,029 ± 1 in NP to 3,526 ± 2 pg·mg(-1)·day(-1) in IL-17-infused rats (P < 0.05). Placental ROS was 436 ± 4 RLU·ml(-1)·min(-1) (n = 4) in NP and 702 ± 5 (n = 5) RLU·ml(-1)·min(-1) in IL-17-treated rats. Importantly, AT1-AA increased from 0.41 ± 0.05 beats/min in NP rats (n = 8) to 18.4 ± 1 beats/min in IL-17 rats (n = 12). Administration of tempol attenuated the hypertension (101 ± 3 mmHg) ROS (459 ± 5 RLU·ml(-1)·min(-1)) and blunted AT1-AAs (7.3 ± 0.6 beats/min) in NP+IL-17+tempol-treated rats. Additionally, AT1 receptor blockade inhibited IL-17-induced hypertension and placental oxidative stress. MAP was 105 ± 5 mmHg and ROS was 418 ± 5 RLU·ml(-1)·min(-1) in NP+IL 17-treated with losartan. These data indicate that IL-17 causes placental oxidative stress, which serves as stimulus modulating AT1-AAs that may play an important role in mediating IL-17-induced hypertension during pregnancy.
 
Article
17β-estradiol (E2) attenuates exercise-induced muscle damage and inflammation in some models. Eighteen men completed 150 eccentric contractions after random assignment to placebo (Control group) or E2 supplementation (Experimental group). Muscle biopsies and blood samples were collected at baseline, following 8-day supplementation and 3 h and 48 h after exercise. Blood samples were analyzed for sex hormone concentration, creatine kinase (CK) activity and total antioxidant capacity. The mRNA content of genes involved in lipid and cholesterol homeostasis [forkhead box O1 (FOXO1), caveolin 1, and sterol regulatory element binding protein-2 (SREBP2)] and antioxidant defense (SOD1 and -2) were measured by RT-PCR. Immunohistochemistry was used to quantify muscle neutrophil (myeloperoxidase) and macrophage (CD68) content. Serum E2 concentration increased 2.5-fold with supplementation (P < 0.001), attenuating neutrophil infiltration at 3 h (P < 0.05) and 48 h (P < 0.001), and the induction of SOD1 at 48 h (P = 0.02). Macrophage density at 48 h (P < 0.05) and SOD2 mRNA at 3 h (P = 0.01) increased but were not affected by E2. Serum CK activity was higher at 48 h for both groups (P < 0.05). FOXO1, caveolin 1 and SREBP2 expression were 2.8-fold (P < 0.05), 1.4-fold (P < 0.05), and 1.5-fold (P < 0.001) and higher at 3 h after exercise with no effect of E2. This suggests that E2 attenuates neutrophil infiltration; however, the mechanism does not appear to be lesser oxidative stress or membrane damage and may indicate lesser neutrophil/endothelial interaction.
 
Angiotensin-converting enzyme (ACE) activity at 2 and 5 wk ( A ), ACE binding densities at 5 wk ( B ) and ANG II type 1 receptor (AT 1 R) binding 
Representative autoradiographs of ACE and AT 1 R binding densities within adrenal and paraventricular nucleus (PVN) of 
Changes in ACE activity and ACE and AT 1 R binding densities at 2 and 
ACE and AT 1 R binding densities in brain nuclei of female rats 5 wk after sham or OVX surgery and E2 pellet implantation
Article
Estrogens have been implicated in both worsening and protecting from cardiovascular disease. The effects of 17beta-estradiol (E2) on the cardiovascular system may be mediated, at least in part, by its modulation of local tissue renin-angiotensin systems (RAS). We assessed two critical components, angiotensin-converting enzyme (ACE) and ANG II type 1 receptor (AT(1)R), in the heart, lung, abdominal aorta, adrenal, kidney, and brain in four groups of female Wistar rats (n = 5-6/group): 1) sham ovariectomized, 2) ovariectomized (OVX) treated with subcutaneous vehicle, 3) OVX treated with 25 mug/day (regular) E2 subcutaneously, and 4) OVX treated with 250 mug/day (high) subcutaneous E2 for 2 or 5 wk. After 2 wk, plasma ACE activity was not altered by OVX, but it was 34-38% lower in OVX + regular E2 and OVX + high E2 rats compared with sham OVX rats, and these decreases were no longer present after 5 wk. After 5 wk, OVX alone increased ACE activity and binding densities, and AT(1)R binding densities by 15-100% in right ventricle, left ventricle (LV), kidney, lung, abdominal aorta, adrenal and several cardiovascular regulatory nuclei in the brain. These effects were, for the most part, prevented by regular E2 replacement and were reversed to decreases by high E2 treatment. This regulation of tissue ACE and AT(1)R is significant as the activity of these tissue RAS contributes to the pathogenesis and/or progression of hypertension, atherosclerosis, and LV remodeling after myocardial infarction.
 
Article
To study the mechanisms by which missense mutations in alpha-tropomyosin cause familial hypertrophic cardiomyopathy, we generated transgenic rats overexpressing alpha-tropomyosin with one of two disease-causing mutations, Asp(175)Asn or Glu(180)Gly, and analyzed phenotypic changes at molecular, morphological, and physiological levels. The transgenic proteins were stably integrated into the sarcomere, as shown by immunohistochemistry using a human-specific anti-alpha-tropomyosin antibody, ARG1. In transgenic rats with either alpha-tropomyosin mutation, molecular markers of cardiac hypertrophy were induced. Ca(2+) sensitivity of cardiac skinned-fiber preparations from animals with mutation Asp(175)Asn, but not Glu(180)Gly, was decreased. Furthermore, elevated frequency and amplitude of spontaneous Ca(2+) waves were detected only in cardiomyocytes from animals with mutation Asp(175)Asn, suggesting an increase in intracellular Ca(2+) concentration compensating for the reduced Ca(2+) sensitivity of isometric force generation. Accordingly, in Langendorff-perfused heart preparations, myocardial contraction and relaxation were accelerated in animals with mutation Asp(175)Asn. The results allow us to propose a hypothesis of the pathogenetic changes caused by alpha-tropomyosin mutation Asp(175)Asn in familial hypertrophic cardiomyopathy on the basis of changes in Ca(2+) handling as a sensitive mechanism to compensate for alterations in sarcomeric structure.
 
Effect of E2 on oxyradical and nitrite (NO 2 ) production by mussel hemocytes. A and B: extracellular oxyradical production, evaluated as cytochrome c reduction. Effect of PMA (10 g/ml, 60 min) is shown as positive control. OD, optical density. A: time course of cytochrome c reduction in control hemocytes and hemocytes treated with 5 and 25 nM E2 (E) and effect of tamoxifen (Tam/ E). B: effects of cell pretreatment with different inhibitors on cytochrome c reduction in control and 25 nM E2-treated hemocytes at 60 min. SB, SB-203580; GF, GF-109203X. SOD (300 mU/ml) and N G -monomethyl-Larginine (L-NMMA, 1 mM) were utilized as described in MATERIALS AND METHODS. C: intracellular oxyradical production, evaluated as nitro blue tetrazolium (NBT) reduction, in hemocytes incubated with 25 nM E2 for 60 min in the absence and presence of inhibitors. D: nitric oxide (NO) production, evaluated as NO 2 concentration by the Griess reaction, in control hemocytes and hemocytes incubated with 25 nM E2 for 4 h. Effect of E2 on NO production was also evaluated in the presence of different inhibitors. NO production induced by PMA (50 g/ml, 4 h) is shown as positive control. Values are means SD of 3 experiments in triplicate. *P 0.05, E2 or PMA vs. C. °P 0.05, inhibitors/E2 vs. E2 alone.  
Effect of hemocyte pretreatment with the PKC inhibitor GF-109203X (20 min, 2.5 M) on lysosomal membrane destabilization induced by 25 nM E2. Lysosomal membrane destabilization induced by hemocyte incubation with E. coli is shown as positive control. Values are means SD of 4 experiments in triplicate. *P 0.05.  
Effect of incubation of mussel hemocytes with E2 (25 nM) for different periods of time (5– 60 min) on CREB phosphorylation. Protein extracts from control and E2-treated hemocytes were subjected to 12% SDS-PAGE followed by Western blot analysis using polyclonal phosphospecific antibodies to CREB (p-CREB; A), stripped and reprobed with antibodies directed towards the unphosphorylated (total) form (CREB; B). Bands were detected using enhanced chemiluminescence reagents (see MATERIALS AND METHODS). Results are representative of 3 independent experiments. C, control. Inset: densitometric analysis of blots from three independent experiments (means SD). P/Tot, ratio between the phosphorylated and total protein band. *P 0.05. Relative increases in band optical densities (arbitrary units) were normalized for control band in each series.  
Effects of 17-estradiol (E2) on phagocytosis of neutral red (NR)conjugated zymosan particles. A and B: effects of 5–50 nM and 25 nM E2, respectively, on hemocytes preincubated with the antiestrogen tamoxifen (100 nM, TAM/E2) or the specific kinase inhibitors SB-203580 (20 M, SB/E2) and GF-109203X (2.5 M, GF/E2). C, control. Effect of 200 nM TNF-is shown as positive control. Values are means SD of 4 experiments in triplicate. *P 0.05.  
Article
In mammals, estrogens have dose- and cell-type-specific effects on immune cells and may act as pro- and anti-inflammatory stimuli, depending on the setting. In the bivalve mollusc Mytilus, the natural estrogen 17beta-estradiol (E(2)) has been shown to affect neuroimmune functions. We have investigated the immunomodulatory role of E(2) in Mytilus hemocytes, the cells responsible for the innate immune response. E(2) at 5-25 nM rapidly stimulated phagocytosis and oxyradical production in vitro; higher concentrations of E(2) inhibited phagocytosis. E(2)-induced oxidative burst was prevented by the nitric oxide (NO) synthase inhibitor N(G)-monomethyl-L-arginine and superoxide dismutase, indicating involvement of NO and O(2)(-); NO production was confirmed by nitrite accumulation. The effects of E(2) were prevented by the antiestrogen tamoxifen and by specific kinase inhibitors, indicating a receptor-mediated mechanism and involvement of p38 MAPK and PKC. E(2) induced rapid and transient increases in the phosphorylation state of PKC, as well as of a aCREB-like (cAMP responsive element binding protein) transcription factor, as indicated by Western blot analysis with specific anti-phospho-antibodies. Localization of estrogen receptor-alpha- and -beta-like proteins in hemocytes was investigated by immunofluorescence confocal microscopy. The effects of E(2) on immune function were also investigated in vivo at 6 and 24 h in hemocytes of E(2)-injected mussels. E(2) significantly affected hemocyte lysosomal membrane stability, phagocytosis, and extracellular release of hydrolytic enzymes: lower concentrations of E(2) resulted in immunostimulation, and higher concentrations were inhibitory. Our data indicate that the physiological role of E(2) in immunomodulation is conserved from invertebrates to mammals.
 
Article
The present study tested the hypothesis that 17beta-estradiol (E2) inhibits increases in angiotensin-converting enzyme (ACE) and ANG II type 1 receptor (AT1R) in the brain and heart after myocardial infarction (MI) and, thereby, inhibits development of left ventricular (LV) dysfunction after MI. Age-matched female Wistar rats were treated as follows: 1) no surgery (ovary intact), 2) ovariectomy + subcutaneous vehicle treatment (OVX + Veh), or 3) OVX + subcutaneous administration of a high dose of E2 (OVX + high-E2). After 2 wk, rats were randomly assigned to coronary artery ligation (MI) and sham operation groups and studied after 3 wk. E2 status did not affect LV function in sham rats. At 2-3 wk after MI, impairment of LV function was similar across MI groups, as measured by echocardiography and direct LV catheterization. LV ACE mRNA abundance and activity were increased severalfold in all MI groups compared with respective sham animals and to similar levels across MI groups. In most brain nuclei, ACE and AT1R densities increased after MI. Unexpectedly, compared with the respective sham groups the relative increase was clearest (20-40%) in OVX + high-E2 MI rats, somewhat less (10-15%) in ovary-intact MI rats, and least (< 10-15%) in OVX + Veh MI rats. However, because in the sham group brain ACE and AT1R densities increased in the OVX + Veh rats and decreased in the OVX + high-E2 rats compared with the ovary-intact rats, actual ACE and AT1R densities in most brain nuclei were modestly higher (< 20%) in OVX + Veh MI rats than in the other two MI groups. Thus E2 does not inhibit upregulation of ACE in the LV after MI and amplifies the percent increases in ACE and AT1R densities in brain nuclei after MI, despite E2-induced downregulation in sham rats. Consistent with these minor variations in the tissue renin-angiotensin system, during the initial post-MI phase, E2 appears not to enhance or hinder the development of LV dysfunction.
 
Article
Estrogen has diverse effects on inflammation and immune responses. That pregnancy is associated with remission of some autoimmune diseases and exacerbation of others suggests that physiological fluctuation in estrogen levels could affect the immune responses in humans. However, the molecular basis for these phenomena is poorly understood. We hypothesized that fluctuations of estrogen levels modulate intracellular signaling for immune responses via estrogen receptors (ERs). In reporter assays, 17beta-estradiol (E2) at a physiologically high concentration increased the activity of NF-kappaB in Jurkat cells stimulated by PMA/ionomycin or TNF-alpha. Overexpression and RNA interference experiments suggested that the effects were mediated through ERbeta. Immunoprecipitation assay showed that both ERalpha and ERbeta are directly associated with NF-kappaB in the cell nucleus. Using chromatin immunoprecipitation assay, we confirmed that ERalpha and ERbeta associated with NF-kappaB and steroid hormone coactivators at the promoter region of NF-kappaB regulated gene. Considering that NF-kappaB regulates the expression of various genes essential for cell growth and death, estrogen could regulate the fate of T cells by affecting the activity of NF-kappaB. To determine whether E2 alters the fate of T cells, we investigated E2 actions on T cell apoptosis, a well-known NF-kappaB-mediated phenomenon. E2 increased apoptosis of Jurkat cells and decreased that of human peripheral blood T cells. Our results indicate that E2 at a physiologically high concentration modulates NF-kappaB signaling in human T cells via ERbeta and affects T cell survival, suggesting that these actions may underlie the gender differences in autoimmune diseases.
 
Effect of single intraperitoneal injection of saline (control), 0.2, 1, and 5 g/g PTHrP(1-34) (A) and PTHrP(7-34) (B) on blood plasma levels of free and total calcium in sea bream juveniles sampled 24 h after injection. PTHrP, parathyroid hormone-related protein. Results are shown as means SE (n 8). *Significant difference from controls (P 0.05). 
The effect of estradiol-17 (E2) implants (10 g/g) in sea bream juveniles (30 g) at different times (1, 4, and 8 days) in total calcium (A), phosphate (B), and E2 (C). Results are shown as means SE (n 7). *Significant differences from controls at a given time (P 0.05). 
The effect of E2 implants (10 g/g) alone or in combination with PTHrP(7-34) (PTHrP antagonist) in sea bream juveniles (70 g) at different times (1, 4, and 8 days) in plasma PTHrP (A); plasma phosphate (B), and plasma total calcium (C). Each point represents means SE of 6-9 fish. a,b,c Different letters indicate significant differences (P 0.05, Bonferroni test) between treatments at a given time. 
Article
Estradiol (E(2)) increases circulating calcium and phosphate levels in fish, thus acting as a hypercalcemic and hyperphosphatemic factor during periods of high calcium requirements, such as during vitellogenesis. Since parathyroid hormone (PTH)-related protein (PTHrP) has been shown to be calciotropic in fish, we hypothesized that the two hormones could be mediating the same process. Sea bream (Sparus auratus) juveniles receiving a single intraperitoneal injection of piscine PTHrP(1-34) showed an elevation in calcium plasma levels within 24 h. In contrast, injections of the PTH/PTHrP receptor antagonist PTHrP(7-34) decreased circulating levels of calcium in the same period. Intraperitoneal implants of estradiol-17beta (E(2); 10 microg/g) evoked significant increases of circulating plasma levels of calcium and phosphorus and a sustained increases of circulating plasma levels of PTHrP. However, a combined treatment of E(2) and PTHrP(7-34) evoked a markedly lower calcium response compared with E(2) alone. We conclude that PTHrP or a related peptide that binds the PTH/PTHrP receptor mediates, at least in part, the hypercalcemic effect of E(2) in calcium and phosphate balance in fish.
 
Means Ϯ SE values for hemodynamic parameters in basal condition from Sham-operated (Sham, n ϭ 6), ovariectomized (Ovx, n ϭ 8), and ovariectomized with estrogen replacement (Ovx ϩ est, n ϭ 6) rats. MAP, mean arterial pressure. * P Ͻ 0.05 vs. Sham; † P Ͻ 0.05 vs. Ovx. 
Hemodynamic response to administration of N G -nitro- L -arginine methyl ester (3 mg/kg ϩ 50 ␮ g ⅐ kg Ϫ 1 ⅐ min Ϫ 1 during 30 min) in Sham ( n ϭ 6), Ovx ( n ϭ 8), and Ovx ϩ est ( n ϭ 6). * P Ͻ 0.05 vs. Sham; † P Ͻ 0.05 vs. Ovx. 
Means Ϯ SE values of plasma reduced thiol groups ( Ϫ SH groups), plasma nitrites/nitrates concentration, total antioxidant status (TAS), and plasma lipoperoxides from Sham ( n ϭ 7), Ovx ( n ϭ 7), and Ovx ϩ est ( n ϭ 7). MDA ϩ HNE, malonaldehyde plus (E)-4-hydroxy-2-nonenal. * P Ͻ 0.05 vs. Sham; † P Ͻ 0.05 vs. Ovx. 
Article
In this study, we tested whether estrogen deficiency is associated with oxidative stress and decreased nitric oxide (NO) production, which could be responsible for an increased blood pressure in ovariectomized rats. Hemodynamic studies were performed on conscious, chronically instrumented rats. Chronic estrogen replacement on ovariectomized rats lowered blood pressure approximately 13 mmHg, from 119 +/- 3 mmHg in ovariectomized rats to 106 +/- 3 mmHg in ovariectomized-treated rats; it was also accompanied by an increase in cardiac index and vascular conductance, achieving hemodynamic values similar to those shown by sham-operated rats. N(G)-nitro-L-arginine methyl ester administration lowered significantly less the vascular conductance (0.14 +/- 0.01 vs. 0.22 +/- 0.03 and 0.26 +/- 0.01 ml. min(-1). mmHg(-1)/100 g; P < 0.05) in ovariectomized rats than in the sham-operated and estrogen-treated ovariectomized rats, respectively. Estrogen replacement prevented the lower plasma levels of nitrites/nitrates observed in ovariectomized rats. The lower plasma total antioxidant status and reduced thiol groups and the increase in plasma lipoperoxides presented in ovariectomized animals were reestablished with the estrogen treatment. These results show that estrogen administration decreases blood pressure and increases vascular conductance in ovariectomized rats. This effect may be related to an increase in NO synthesis and/or preventing oxidative stress, then improving endothelial function.
 
Sex hormone concentrations with and without estrogen supplementation in men. PL ES 
Article
Sex-based differences in inflammatory responses to exercise may be mediated by estrogen through increased muscle membrane stability and/or inhibited cytokine production. In this study, in vivo effects of estrogen on systemic inflammation-related responses to exercise were assessed in healthy men. In a double-blind, placebo-controlled, crossover design, 11 men cycled for 90 min at 65% Vo2 max after 8 days of 17beta-estradiol supplementation (ES; 2 mg/day) or placebo (PL; glucose polymer). After a 2-wk washout, exercise was repeated after 8 days on the alternate treatment. Blood was collected pre- and postexercise to determine IL-6, soluble intercellular adhesion molecule-1 (sICAM-1), neutrophil counts, and cortisol. Preexercise serum was assayed for sex hormones. ES increased estradiol (133+/-71 to 840+/-633 pmol/l, P=0.005) and reduced testosterone (19.9+/-3.7 to 16.1+/-3.9 nmol/l, P=0.007). Exercise increased cortisol (P=0.02), IL-6 (P<0.001) and neutrophil counts (P<0.001) with no influence on sICAM-1 (P=0.34) and no effect of ES on these changes. Postexercise IL-6 and neutrophil counts were correlated (r=0.58, P=0.005); postexercise IL-6 and cortisol (r=0.18, P=0.43) and postexercise cortisol and neutrophil counts (r=0.06, P=0.78) were not. Postexercise sICAM-1 was not correlated with the above variables (P>or=0.79). In conclusion, 8 days of ES in healthy men did not influence systemic inflammation-related responses to acute exercise. Future studies should investigate 17beta-estradiol effects on IL-6 production and neutrophil infiltration within skeletal muscle during and after exercise.
 
Deduced amino acid sequence of squirrelfish MT (sq) compared with the sequences of viviparous eelpout (zoa), winter flounder (wf), rainbow trout (rbt), Atlantic cod (cod), zebra fish (zeb), human (hu), mouse (mou), and domestic pigeon (pi). Differences in amino acid residues relative to squirrelfish MT are indicated by substitution of the appropriate one-letter code. 1 Data from Kille and Olsson (41); 2 Chan et al. (6); 3 Bonham et al. (3); 4 Stennard et al. (39); 5 Durnam et al. (9); 6 Lin et al. (24). 
Article
Females of the squirrelfish family (Holocentridae) accumulate higher levels of zinc in the liver than any other known animal. This zinc accumulation is made possible by high expression of the zinc-binding protein, metallothionein (MT). In the present study, the squirrelfish (Holocentrus ascensionis) MT cDNA was cloned and sequenced. The deduced amino acid sequence was very similar to other teleost MT. The role of estrogens on zinc metabolism was investigated by injecting male and immature female squirrelfish with 17 beta-estradiol (E(2)). E(2) treatment triggered transient increases in plasma zinc and vitellogenin (VTG) levels, and both of these variables showed very similar time courses. These results suggest that E(2) is responsible for the large hepatoovarian translocation of zinc observed in female squirrelfish and that VTG might be a vehicle for zinc. E(2) did not directly alter the levels of zinc or MT mRNA in the liver. However, the hepatic MT protein concentration increased differentially in the nuclear fraction. Thus E(2) is probably responsible for the association of MT with the nuclear fraction previously observed in untreated mature female squirrelfish.
 
A : Western analysis of endothelial nitric oxide synthase (eNOS) in kidneys from animals treated with E2- ␤ or vehicle and exposed to hypoxia (12% O 2 ) or normoxia for 8 h. Quantitative normalized densitometric analysis. E2- ␤ treatment did not increase 
Article
Exposure to chronic hypoxia induces erythropoietin (EPO) production to facilitate oxygen delivery to hypoxic tissues. Previous studies from our laboratory found that ovariectomy (OVX) exacerbates the polycythemic response to hypoxia and treatment with 17beta-estradiol (E2-beta) inhibits this effect. We hypothesized that E2-beta decreases EPO gene expression during hypoxia. Because E2-beta can induce nitric oxide (NO) production and NO can attenuate EPO synthesis, we further hypothesized that E2-beta inhibition of EPO gene expression is mediated by NO. These hypotheses were tested in OVX catheterized rats treated with E2-beta (20 microg/day) or vehicle for 14 days and exposed to 8 or 12 h of hypoxia (12% O(2)) or normoxia. We found that E2-beta treatment significantly decreased EPO synthesis and gene expression during hypoxia. E2-beta treatment did not induce endothelial NO synthase (eNOS) expression in the kidney but potentiated hypoxia-induced increases in plasma nitrates. We conclude that E2-beta decreases hypoxic induction of EPO. However, this effect does not appear to be related to changes in renal eNOS expression.
 
Article
Women are exposed to estrogen in several forms, such as oral contraceptive pills and hormone replacement therapy. Although estrogen was believed to be cardioprotective, lately, its beneficial effects are being questioned. Recent studies indicate that oxidative stress in the rostral ventrolateral medulla (RVLM) may play a role in the development of hypertension. Therefore, we hypothesized that chronic exposure to low levels of estradiol-17β (E(2)) leads to hypertension in adult-cycling female Sprague Dawley (SD) rats potentially through generation of superoxide in the RVLM. To test this hypothesis, young adult (3 or 4 mo old) female SD rats were either sham-implanted or implanted (subcutaneously) with slow-release E(2) pellets (20 ng/day) for 90 days. A group of control and E(2)-treated animals were fed lab chow or chow containing resveratrol (0.84 g/kg of chow), an antioxidant. Rats were implanted with telemeters to continuously monitor blood pressure (BP) and heart rate (HR). At the end of treatment, the RVLM was isolated for measurements of superoxide. E(2) treatment significantly increased mean arterial pressure (mmHg) and HR (beats/min) compared with sham rats (119.6 ± 0.8 vs. 105.1 ± 0.7 mmHg and 371.7 ± 1.5 vs. 354.4 ± 1.3 beats/min, respectively; P < 0.0001). Diastolic and systolic BP were significantly increased in E(2)-treated rats compared with control animals. Superoxide levels in the RVLM increased significantly in the E(2)-treated group (0.833 ± 0.11 nmol/min·mg) compared with control (0.532 ± 0.04 nmol/min·mg; P < 0.05). Treatment with resveratrol reversed the E(2)-induced increases in BP and superoxide levels in the RVLM. In conclusion, these findings support the hypothesis that chronic exposure to low levels of E(2) induces hypertension and increases superoxide levels in the RVLM and that this effect can be reversed by resveratrol treatment.
 
Article
We previously reported that supplementation with 17beta-estradiol (E2) attenuates albuminuria, glomerulosclerosis, and tubulointerstitial fibrosis in diabetic nephropathy. The present study examined the mechanisms by which E2 regulates extracellular matrix (ECM) metabolism, a process that contributes to the development of glomerulosclerosis and tubulointerstitial fibrosis. The study was performed in female nondiabetic (ND), streptozotocin-induced diabetic (D), and diabetic with E2 supplementation (D+E2) Sprague-Dawley rats for 12 wk. Diabetes was associated with an increase in the renal expression of collagen alpha type IV [ND, 0.22 +/- 0.02; D, 0.99 +/- 0.09 relative optical density (ROD); P < 0.05] and fibronectin protein (ND, 0.36 +/- 0.08; D, 1.47 +/- 0.08 ROD; P < 0.05), as measured by Western blot analysis. E2 supplementation partially attenuated this increase in collagen alpha type IV (D+E2, 0.47 +/- 0.10 ROD) and fibronectin (D+E2, 0.71 +/- 0.16 ROD) protein expression associated with D. Diabetes was also associated with a decrease in the expression of matrix metalloproteinase (MMP) isoform MMP-2 (ND, 0.79 +/- 0.01; D, 0.62 +/- 0.06 ROD; P < 0.05) and MMP-9 protein (ND, 0.49 +/- 0.02; D, 0.33 +/- 0.03 ROD; P < 0.05). E2 supplementation restored MMP-2 and MMP-9 protein to levels similar or even greater than in the ND kidneys (MMP-2, 0.75 +/- 0.06; MMP-9, 0.73 +/- 0.01 ROD). The activities of MMP-2 (ND, 7.88 +/- 0.44; D, 5.60 +/- 0.54 ROD; P < 0.05) and MMP-9 (ND, 29.9 +/- 1.8; D, 12.9 +/- 2.3 ROD; P < 0.05), as measured by zymography, were also decreased with D. E2 supplementation restored MMP-2 and MMP-9 activity to levels similar to that in ND kidneys (MMP-2, 7.66 +/- 0.35; MMP-9, 21.4 +/- 2.9 ROD). We conclude that E2 supplementation is renoprotective by attenuating glomerulosclerosis and tubulointerstitial fibrosis by reducing ECM synthesis and increasing ECM degradation.
 
Top-cited authors
Andrew M Jones
  • University of Exeter
Stephen J Bailey
  • Loughborough University
James Tidball
  • University of California, Los Angeles
Armando Villalta
  • University of California, San Francisco
Jamie R Blackwell
  • University of Exeter