The application of epiphenotyping approaches to DNA methylation array studies of the human placenta.

BACKGROUND: Genome-wide DNA methylation (DNAme) profiling of the placenta with Illumina Infinium Methylation bead arrays is often used to explore the connections between in utero exposures, placental pathology, and fetal development. However, many technical and biological factors can lead to signals of DNAme variation between samples and between cohorts, and understanding and accounting for these factors is essential to ensure meaningful and replicable data analysis. Recently, "epiphenotyping" approaches have been developed whereby DNAme data can be used to impute information about phenotypic variables such as gestational age, sex, cell composition, and ancestry. These epiphenotypes offer avenues to compare phenotypic data across cohorts, and to understand how phenotypic variables relate to DNAme variability. However, the relationships between placental epiphenotyping variables and other technical and biological variables, and their application to downstream epigenome analyses, have not been well studied. RESULTS: Using DNAme data from 204 placentas across three cohorts, we applied the PlaNET R package to estimate epiphenotypes gestational age, ancestry, and cell composition in these samples. PlaNET ancestry estimates were highly correlated with independent polymorphic ancestry-informative markers, and epigenetic gestational age, on average, was estimated within 4 days of reported gestational age, underscoring the accuracy of these tools. Cell composition estimates varied both within and between cohorts, as well as over very long placental processing times. Interestingly, the ratio of cytotrophoblast to syncytiotrophoblast proportion decreased with increasing gestational age, and differed slightly by both maternal ethnicity (lower in white vs. non-white) and genetic ancestry (lower in higher probability European ancestry). The cohort of origin and cytotrophoblast proportion were the largest drivers of DNAme variation in this dataset, based on their associations with the first principal component. CONCLUSIONS: This work confirms that cohort, array (technical) batch, cell type proportion, self-reported ethnicity, genetic ancestry, and biological sex are important variables to consider in any analyses of Illumina DNAme data. We further demonstrate the specific utility of epiphenotyping tools developed for use with placental DNAme data, and show that these variables (i) provide an independent check of clinically obtained data and (ii) provide a robust approach to compare variables across different datasets. Finally, we present a general framework for the processing and analysis of placental DNAme data, integrating the epiphenotype variables discussed here.

Adjusting for Batch Effects in DNA Methylation Microarray Data, a Lesson Learned.

It is well-known, but frequently overlooked, that low- and high-throughput molecular data may contain batch effects, i.e., systematic technical variation. Confounding of experimental batches with the variable(s) of interest is especially concerning, as a batch effect may then be interpreted as a biologically significant finding. An integral step toward reducing false discovery in molecular data analysis includes inspection for batch effects and accounting for this signal if present. In a 30-sample pilot Illumina Infinium HumanMethylation450 (450k array) experiment, we identified two sources of batch effects: row and chip. Here, we demonstrate two approaches taken to process the 450k data in which an R function, ComBat, was applied to adjust for the non-biological signal. In the "initial analysis," the application of ComBat to an unbalanced study design resulted in 9,612 and 19,214 significant (FDR < 0.05) DNA methylation differences, despite none present prior to correction. Suspicious of this dramatic change, a "revised processing" included changes to our analysis as well as a greater number of samples, and successfully reduced batch effects without introducing false signal. Our work supports conclusions made by an article previously published in this journal: though the ultimate antidote to batch effects is thoughtful study design, every DNA methylation microarray analysis should inspect, assess and, if necessary, account for batch effects. The analysis experience presented here can serve as a reminder to the broader community to establish research questions a priori, ensure that they match with study design and encourage communication between technicians and analysts.

A de novo mosaic mutation in SPAST with two novel alternative alleles and chromosomal copy number variant in a boy with spastic paraplegia and autism spectrum disorder.

Here we report a 12 year old male with an extreme presentation of spastic paraplegia along with autism and dysmorphisms. Whole exome sequencing identified a predicted pathogenic pair of missense variants in SPAST at the same chromosomal location, each with a different alternative allele, while a chromosome microarray identified a 1.73 Mb paternally inherited copy gain of 1q21.1q21.2 resulting in a blended phenotype of both Spastic paraplegia 4 and 1q21.1 microduplication syndrome. We believe that the extreme phenotype observed is likely caused by the presence of cells which contain only mutant SPAST, but that the viability of the patient is possible due mosaicism of mutant alleles observed in different proportions across tissues.

Prenatal and perinatal environmental influences on the human fetal and placental epigenome.

Drug, hormonal, and nutritional exposures in pregnancy can cause birth defects, or have more subtle influences that may affect adult health through epigenetic developmental programming. Characterizing these epigenetic changes may provide valuable insight into the mechanism by which our health is influenced by the environment and provide biomarkers of exposure; however, currently there are few data from human studies directly addressing this. We review data on three types of exposures-nutritional, glucocorticoid, and endocrine-disrupting chemicals-and discuss how these may be associated with epigenetic changes in the fetus and the placenta, a key mediator of in utero environment. In addition, we outline some considerations for investigation of epigenetic effects in the fetus and placenta that are important to consider in the design of studies for the future.

Production of endothelial nitric oxide synthase (eNOS) over-expressing piglets.

Vascular function, vascular structure, and homeostasis are thought to be regulated in part by nitric oxide (NO) released by endothelial cell nitric oxide synthase (eNOS), and NO released by eNOS plays an important role in modulating metabolism of skeletal and cardiac muscle in health and disease. The pig is an optimal model for human diseases because of the large number of important similarities between the genomic, metabolic and cardiovascular systems of pigs and humans. To gain a better understanding of cardiovascular regulation by eNOS we produced pigs carrying an endogenous eNOS gene driven by a Tie-2 promoter and tagged with a V5 His tag. Nuclear transfer was conducted to create these animals and the effects of two different oocyte activation treatments and two different culture systems were examined. Donor cells were electrically fused to the recipient oocytes. Electrical fusion/activation (1 mM calcium in mannitol: Treatment 1) and electrical fusion (0.1 mM calcium in mannitol)/chemical activation (200 microM Thimerosal for 10 min followed by 8 mM DTT for 30 min: Treatment 2) were used. Embryos were surgically transferred to the oviducts of gilts that exhibited estrus on the day of fusion or the day of transfer. Two cloned transgenic piglets were born from Treatment 1 and low oxygen, and another two from Treatment 2 and normal oxygen. PCR, RT-PCR, Western blotting and immunohistochemistry confirmed that the pigs were transgenic, made message, made the fusion protein and that the fusion protein localized to the endothelial cells of placental vasculature from the conceptuses as did the endogenous eNOS. Thus both activation conditions and culture systems are compatible with development to term. These pigs will serve as the founders for a colony of miniature pigs that will help to elucidate the function of eNOS in regulating muscle metabolism and the cardiorespiratory system.

Thyroid status and nitric oxide in rat arterial vessels.

Thyroid disease has profound effects on cardiovascular function. Hypo- and hyperthyroidism, for example, are associated with reduced and increased maximal endothelium-dependent vasodilation respectively. We therefore hypothesized that the capacity for vascular nitric oxide (NO) formation is decreased in hypothyroidism and increased in hyperthyroidism. To test this hypothesis, rats were made hypothyroid (HYPO) with propylthiouracil or hyperthyroid (HYPER) with triiodothyronine over 3-4 months. Compared with euthyroid control rats (EUT), HYPO exhibited blunted growth and lower citrate synthase activity in the soleus muscle; HYPER exhibited left ventricular hypertrophy and higher citrate synthase activity in the soleus muscle (P<0.05 for all effects). The capacity for NO formation was determined in aortic extracts by formation of [3H]L-citrulline from [3H]L-arginine, i.e. NO synthase (NOS) activity. Thyroid status modulated NOS activity (EUT, 36.8 +/- 5.5 fmol/h per mg protein; HYPO, 26.0 +/- 7.9; HYPER, 64.6 +/- 12.7; P<0.05, HYPER vs HYPO). Expression of endothelial and neural isoforms of NOS was modulated by thyroid status in a parallel fashion. Capacity for responding to NO was also determined via measuring cGMP concentration in aortae incubated with sodium nitroprusside. Stimulated cGMP formation was also modulated by thyroid status (EUT, 73.0 +/- 20.2 pmol/mg protein; HYPO, 152.4 +/- 48.7; HYPER, 10.4 +/- 2.6; P<0.05, HYPER vs HYPO). These data indicate that thyroid status alters capacities for both formation of and responding to NO. The former finding may contribute to previous findings concerning vascular function in thyroid disease states.

Thyroid status and endothelium-dependent vasodilation in skeletal muscle.

Cardiovascular dysfunction is characteristic of both hypo- and hyperthyroidism. Endothelium-dependent dilation of conductance vessels is impaired in hypothyroidism but augmented in hyperthyroidism. We hypothesized that these alterations in dilation extend into the resistance vasculature of skeletal muscle. To test this hypothesis, rats were made hypothyroid with propylthiouracil (Hypo; n = 13) or hyperthyroid with triiodothyronine (Hyper; n = 9) over 3-4 mo. Compared with euthyroid controls (Eut; n = 14), Hypo rats were characterized by reduced skeletal muscle oxidative capacity and blunted growth; Hyper rats exhibited increased muscle oxidative capacity and left ventricular hypertrophy (P < 0.05 for all effects). Vasodilation to the endothelium-dependent agent acetylcholine ( approximately 2 x 10(-4) M) in skeletal muscle was determined in situ. Conductance in certain muscles increased from control [e.g., soleus: 0.98 +/- 0.15 (Eut), 0.79 +/- 0.14 (Hypo), and 1.06 +/- 0.24 ml.min(-1).100 g(-1).mmHg(-1) (Hyper); not significant among groups] to acetylcholine [1.91 +/- 0.21 (Eut), 2.28 +/- 0.26 (Hypo), and 2.15 +/- 0.33 ml.min(-1).100 g(-1).mmHg(-1) (Hyper); P < 0.05 vs. control values for all groups] but did not differ among groups. Expression of mRNA for the endothelial isoform of nitric oxide synthase in resistance vessels isolated from various muscles was similarly unchanged with alterations in thyroid status [e.g., soleus 1A arterioles: 33.15 +/- 0.58 (Eut), 32.73 +/- 0.27 (Hypo), and 32.80 +/- 0.54 (Hyper) cycles at threshold; not significant]. These data suggest that endothelium-dependent dilation of resistance vasculature in skeletal muscle is unchanged in both hypo- and hyperthyroidism. These data also emphasize the importance of examining resistance vasculature to improve understanding of effects of chronic disease on integrated cardiovascular function.

Hypercholesterolemia inhibits L-type calcium current in coronary macro-, not microcirculation.

Hypercholesterolemia (HC) is a mary risk factor for the development of coronary heart disease. Coronary ion regulation, especially calcium, is thought to be important in coronary heart disease development; however, the influence of high dietary fat and cholesterol on coronary arterial smooth muscle (CASM) ion channels is unknown. The purpose of this study was to determine the effect of diet-induced HC on CASM voltage-gated calcium current (I(Ca)). Male miniature swine were fed a high-fat, high-cholesterol diet (40% kcal fat, 2% wt cholesterol) for 20-24 wk, resulting in elevated serum total and low-density lipoprotein cholesterol. Histochemistry indicated early atherosclerosis in large coronary arteries. CASM were isolated from the right coronary artery (>1.0 mm ID), small arteries ( approximately 200 microm), and large arterioles ( approximately 100 microm). I(Ca) was determined by whole cell voltage clamp. L-type I(Ca) was reduced approximately 30% by HC compared with controls in the right coronary artery (-5.29 +/- 0.42 vs. -7.59 +/- 0.41 pA/pF) but not the microcirculation (small artery, -8.39 +/- 0.80 vs. -10.13 +/- 0.60; arterioles, -10.78 +/- 0.93 vs. -11.31 +/- 0.95 pA/pF). Voltage-dependent activation was unaffected by HC in both the macro- and microcirculation. L-type voltage-gated calcium channel (Ca(v)1.2) mRNA and membrane protein levels were unaffected by HC. Inhibition of I(Ca) by HC was reversed in vitro by the cholesterol scavenger methyl-beta-cyclodextrin and mimicked in control CASM by incubation with the cholesterol donor cholesterol:methyl-beta-cyclodextrin. These data indicate that CASM L-type I(Ca) is decreased in large coronary arteries in early stages of atherosclerosis, whereas I(Ca) in the microcirculation is unaffected. The inhibition of calcium channel activity in CASM of large coronary arteries is likely due to increases in membrane free cholesterol.

Interval sprint training enhances endothelial function and eNOS content in some arteries that perfuse white gastrocnemius muscle.

The purpose of this study was to test the hypothesis that interval sprint training (IST) selectively increases endothelium-dependent dilation (EDD) and endothelial nitric oxide synthase and/or superoxide dismutase-1 protein content in arteries and/or arterioles that perfuse the white portion of rat gastrocnemius muscle (WG). Male Sprague-Dawley rats completed 10 wk of IST (n = 62) or remained sedentary (Sed) (n = 63). IST rats performed six 2.5-min exercise bouts, with 4.5 min of rest between bouts (60 m/min, 15% incline), 5 days/wk. EDD was assessed from acetylcholine (ACh)-induced increases in muscle blood flow measured in situ and by ACh-induced dilation of arteries and arterioles [first to third order (1A-3A)] that perfuse red gastrocnemius muscle (RG) and WG. Artery protein content was determined with immunoblot analysis. ACh-induced increases in blood flow were enhanced in WG of IST rats. eNOS content was increased in conduit arteries, gastrocnemius feed artery, and fourth-order arterioles from WG and fifth-order arterioles of RG but not in 2As from RG. EDD was examined in 2As and 3As from a subset of IST and Sed rats. Arterioles were canulated with micropipettes, and intraluminal pressure was set at 60 cmH2O. Results indicate that passive diameter (measured in 0 calcium PSS) of WG 2As was similar in IST and Sed, whereas diameter of WG 3As was greater in IST (96 +/- 8 microm) than Sed (73 +/- 4 microm). WG 2As and 3As of IST rats exhibited greater spontaneous tone, but sensitivity to stretch, phenylephrine, and sodium nitroprusside was similar to Sed arterioles. ACh-induced dilation was enhanced by IST in WG 2As but not in RG 2As or WG 3As. We conclude that IST induces vascular adaptations nonuniformly among arteries that perfuse WG muscle.

Influence of coronary artery diameter on eNOS protein content.

The purpose of this study was to test the hypothesis that the content of endothelial nitric oxide synthase (eNOS) protein (eNOS protein/g total artery protein) increases with decreasing artery diameter in the coronary arterial tree. Content of eNOS protein was determined in porcine coronary arteries with immunoblot analysis. Arteries were isolated in six size categories from each heart: large arteries [301- to 2,500-microm internal diameter (ID)], small arteries (201- to 300-microm ID), resistance arteries (151- to 200-microm ID), large arterioles (101- to 150-microm ID), intermediate arterioles (51- to 100-microm ID), and small arterioles(<50-microm ID). To obtain sufficient protein for analysis from small- and intermediate-sized arterioles, five to seven arterioles 1-2 mm in length were pooled into one sample for each animal. Results establish that the number of smooth muscle cells per endothelial cell decreases from a number of 10 to 15 in large coronary arteries to 1 in the smallest arterioles. Immunohistochemistry revealed that eNOS is located only in endothelial cells in all sizes of coronary artery and in coronary capillaries. Contrary to our hypothesis, eNOS protein content did not increase with decreasing size of coronary artery. Indeed, the smallest coronary arterioles had less eNOS protein per gram of total protein than the large coronary arteries. These results indicate that eNOS protein content is greater in the endothelial cells of conduit arteries, resistance arteries, and large arterioles than in small coronary arterioles.

Short-term training enhances endothelium-dependent dilation of coronary arteries, not arterioles.

Our objective was to test the hypothesis that short-term exercise training (STR) of pigs increases endothelium-dependent dilation (EDD) of coronary arteries but not coronary arterioles. Female Yucatan miniature swine ran on a treadmill for 1 h, at 3.5 mph, twice daily for 7 days (STR; n = 28). Skeletal muscle citrate synthase activity was increased in STR compared with sedentary controls (Sed; n = 26). Vasoreactivity was evaluated in isolated segments of conduit arteries (1-2 mm ID, 3-4 mm length) mounted on myographs and in arterioles (50-100 microm ID) isolated and cannulated with micropipettes with intraluminal pressure set at 60 cmH(2)O. EDD was assessed by examining responses to increasing concentrations of bradykinin (BK) (conduit arteries 10(-12)-10(-6) M and arterioles 10(-13)-10(-6) M). There were no differences in maximal EDD or BK sensitivity of coronary arterioles from Sed and STR hearts. In contrast, sensitivity of conduit arteries (precontracted with PGF(2alpha)) to BK was increased significantly (P < 0.05) in STR (EC(50), 2.33 +/- 0.62 nM, n = 12) compared with Sed animals (EC(50), 3.88 +/- 0.62 nM, n = 13). Immunoblot analysis revealed that coronary arteries from STR and Sed animals had similar levels of endothelial nitric oxide synthase (eNOS). In contrast, eNOS protein was increased in STR aortic endothelial cells. Neither protein nor mRNA levels of eNOS were different in coronary arterioles from STR compared with Sed animals. STR did not alter expression of superoxide dismutase (SOD-1) protein in any artery examined. We conclude that pigs exhibit increases in EDD of conduit arteries, but not in coronary arterioles, at the onset of exercise training. These adaptations in pigs do not appear to be mediated by alterations in eNOS or SOD-1 expression.

Endothelium-mediated relaxation of porcine collateral-dependent arterioles is improved by exercise training.

BACKGROUND: Endothelium-dependent modulation of coronary tone is impaired in the collateral-dependent coronary microcirculation. We used a porcine model of chronic coronary occlusion and collateral development to evaluate the hypothesis that exercise training enhances endothelium-mediated relaxation and increases endothelial nitric oxide synthase (ecNOS) mRNA levels of collateral-dependent microvasculature. METHODS AND RESULTS: Adult female miniature swine were subjected to chronic, progressive ameroid occlusion of the proximal left circumflex coronary artery (LCx); after 2 months, animals were randomly exposed to 16-week exercise-training (EX group; treadmill running) or sedentary (SED group; cage confinement) protocols. After completion of EX or SED programs, coronary arterioles ( approximately 100 microm in diameter) were isolated from collateral-dependent LCx (distal to occlusion) and nonoccluded left anterior descending coronary artery (LAD) regions of each heart. Arterioles were studied by in vitro videomicroscopy or frozen for ecNOS mRNA analysis (RT-PCR techniques). Relaxation to the endothelium-dependent vasodilator bradykinin was decreased (P<0.05) in arterioles isolated from collateral-dependent LCx versus nonoccluded LAD regions of SED animals. Bradykinin-mediated relaxation, however, was not different in LCx versus LAD arterioles isolated from EX animals. Nitroprusside-induced relaxation was unaffected by either chronic occlusion or exercise. Importantly, ecNOS mRNA expression was significantly decreased in arterioles isolated from LCx versus LAD regions of SED animals. After training, ecNOS mRNA expression was not different between LAD and LCx arterioles. CONCLUSIONS: These data indicate that exercise training enhances bradykinin-mediated relaxation of collateral-dependent LCx arterioles isolated after chronic coronary occlusion, most likely because of effects on ecNOS mRNA expression and increased production of NO.

Hindlimb unweighting decreases endothelium-dependent dilation and eNOS expression in soleus not gastrocnemius.

We tested the hypothesis that hindlimb unweighting (HLU) decreases endothelium-dependent vasodilation and expression of endothelial nitric oxide synthase (eNOS) and superoxide dismutase-1 (SOD-1) in arteries of skeletal muscle with reduced blood flow during HLU. Sprague-Dawley rats (300-350 g) were exposed to HLU (n = 15) or control (n = 15) conditions for 14 days. ACh-induced dilation was assessed in muscle with reduced [soleus (Sol)] or unchanged [gastrocnemius (Gast)] blood flow during HLU. eNOS and SOD-1 expression were measured in feed arteries (FA) and in first-order (1A), second-order (2A), and third-order (3A) arterioles. Dilation to infusion of ACh in vivo was blunted in Sol but not Gast. In arteries of Sol muscle, HLU decreased eNOS mRNA and protein content. eNOS mRNA content was significantly less in Sol FA (35%), 1A arterioles (25%) and 2A arterioles (18%). eNOS protein content was less in Sol FA (64%) and 1A arterioles (65%) from HLU rats. In arteries of Gast, HLU did not decrease eNOS mRNA or protein. SOD-1 mRNA expression was less in Sol 2A arterioles (31%) and 3A arterioles (29%) of HLU rats. SOD-1 protein content was less in Sol FA (67%) but not arterioles. SOD-1 mRNA and protein content were not decreased in arteries from Gast. These data indicate that HLU decreases endothelium-dependent vasodilation, eNOS expression, and SOD-1 expression primarily in arteries of Sol muscle where blood flow is reduced during HLU.

Sarcoplasmic reticulum Ca(2+) uptake is impaired in coronary smooth muscle distal to coronary occlusion.

After chronic occlusion, collateral-dependent coronary arteries exhibit alterations in both vasomotor reactivity and associated myoplasmic free Ca(2+) levels that are prevented by chronic exercise training. We tested the hypotheses that coronary occlusion diminishes Ca(2+) uptake by the sarcoplasmic reticulum (SR) and that exercise training would prevent impaired SR Ca(2+) uptake. Ameroid constrictors were surgically placed around the proximal left circumflex (LCx) artery of female swine 8 wk before initiating 16-wk sedentary (pen confined) or exercise-training (treadmill run) protocols. Twenty-four weeks after Ameroid placement, smooth muscles cells were enzymatically dissociated from both the LCx and nonoccluded left anterior descending (LAD) arteries of sedentary and exercise-trained pigs, and myoplasmic free Ca(2+) was studied using fura 2 microfluorometry. After the SR Ca(2+) store was partially depleted with caffeine (5 mM), KCl-induced membrane depolarization produced a significant decrease in the time to half-maximal (t(1/2)) myoplasmic free Ca(2+) accumulation in LCx versus LAD cells of sedentary pigs. Furthermore, inhibition of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA; 10 microM cyclopiazonic acid) significantly reduced t(1/2) in cells isolated from the LAD but not from the LCx. Exercise training did not prevent the differences in t(1/2) myoplasmic free Ca(2+) accumulation observed between LCx and LAD cells. Occlusion or exercise training did not alter SERCA protein levels. These results support our hypothesis of impaired SR Ca(2+) uptake in coronary smooth muscle cells isolated distal to chronic occlusion. Impaired SR Ca(2+) uptake was independent of SERCA protein levels and was not prevented by exercise training.

Short-term exercise training increases ACh-induced relaxation and eNOS protein in porcine pulmonary arteries.

We tested the hypothesis that short-term exercise (STEx) training and the associated increase in pulmonary blood flow during bouts of exercise cause enhanced endothelium-dependent vasorelaxation in porcine pulmonary arteries and increased expression of endothelial cell nitric oxide synthase (eNOS) and superoxide dismutase-1 (SOD-1) protein. Mature, female Yucatan miniature swine exercised 1 h twice daily on a motorized treadmill for 1 wk (STEx group, n = 7); control pigs (Sed, n = 6) were kept in pens. Pulmonary arteries were isolated from the left caudal lung lobe, and vasomotor responses were determined in vitro. Arterial tissue from the distal portion of this pulmonary artery was processed for immunoblot analysis. Maximal endothelium-dependent (ACh-stimulated) relaxation was greater in STEx (71 +/- 5%) than in Sed (44 +/- 6%) arteries (P < 0.05), and endothelium-independent (sodium nitroprusside-mediated) responses did not differ. Sensitivity to ACh was not altered by STEx training. Immunoblot analysis indicated a 3.9-fold increase in eNOS protein in pulmonary artery tissue from STEx pigs (P < 0.05) with no change in SOD-1 or glyceraldehyde-3-phosphate dehydrogenase protein levels. We conclude that STEx training enhances ACh-stimulated vasorelaxation in pulmonary arterial tissue and that this adaptation is associated with increased expression of eNOS protein.

Training induces nonuniform increases in eNOS content along the coronary arterial tree.

Exercise training produces enhanced nitric oxide (NO)-dependent, endothelium-mediated vasodilator responses of porcine coronary arterioles but not conduit coronary arteries. The purpose of this study was to test the hypothesis that exercise training increases the amount of endothelial NO synthase (eNOS) in the coronary arterial microcirculation but not in the conduit coronary arteries. Miniature swine were either exercise trained or remained sedentary for 16--20 wk. Exercise-trained pigs exhibited increased skeletal muscle oxidative capacity, exercise tolerance, and heart weight-to-body weight ratios. Content of eNOS protein was determined with immunoblot analysis in conduit coronary arteries (2- to 3-mm ID), small arteries (301- to 1,000-microm ID), resistance arteries (151- to 300-microm ID), and three sizes of coronary arterioles [large (101- to 150-microm ID), intermediate (51- to 100-microm ID), and small (<50-microm ID)]. Immunoblots revealed increased eNOS protein in some sizes of coronary arteries and arterioles but not in others. Content of eNOS was increased by 60--80% in small and large arterioles, resistance arteries, and small arteries; was increased by 10--20% in intermediate-sized arterioles; and was not changed or decreased in conduit arteries. Immunohistochemistry revealed that eNOS was located in the endothelial cells in all sizes of coronary artery. We conclude that exercise training increases eNOS protein expression in a nonuniform manner throughout the coronary arterial tree. Regional differences in shear stress and intraluminal pressures during exercise training bouts may be responsible for the distribution of increased eNOS protein content in the coronary arterial tree.

SOD-1 expression in pig coronary arterioles is increased by exercise training.

Coronary arterioles of exercise-trained (EX) pigs have enhanced nitric oxide (NO.)-dependent dilation. Evidence suggests that the biological half-life of NO. depends in part on the management of the superoxide anion. The purpose of this study was to test the hypothesis that expression of cytosolic copper/zinc-dependent superoxide dismutase (SOD)-1 is increased in coronary arterioles as a result of exercise training. Male Yucatan pigs either remained sedentary (SED, n = 4) or were EX (n = 4) on a motorized treadmill for 16-20 wk. Individual coronary arterioles ( approximately 100-microm unpressurized internal diameter) were dissected and frozen. Coronary arteriole SOD-1 protein (via immunoblots) increased as a result of exercise training (2.16 +/- 0.35 times SED levels) as did SOD-1 enzyme activity (measured via inhibition of pyrogallol autooxidation; approximately 75% increase vs. SED). In addition, SOD-1 mRNA levels (measured via RT-PCR) were higher in EX arterioles (1.68 +/- 0.16 times the SED levels). There were no effects of exercise training on the levels of SOD-2 (mitochondrial), catalase, or p67(phox) proteins. Thus chronic aerobic exercise training selectively increases the levels of SOD-1 mRNA, protein, and enzymatic activity in porcine coronary arterioles. Increased SOD-1 could contribute to the enhanced NO.-dependent dilation previously observed in EX porcine coronary arterioles by improving management of superoxide in the vascular cell environment, thus prolonging the biological half-life of NO.

Estrogen inhibition of cystic fibrosis transmembrane conductance regulator-mediated chloride secretion.

Cystic fibrosis (CF) is an autosomal genetic disease associated with impaired epithelial ion transport. Mutations in the CF gene alter the primary sequence of the CF transmembrane conductance regulator (CFTR). Several therapeutic modalities have been proposed for CF patients, including the phytoestrogen genistein. Experiments were completed in cellular and subcellular systems to evaluate the impact of naturally occurring and synthetic estrogens on epithelial ion transport, and specifically on the CF protein CFTR. 17beta-Estradiol, a naturally occurring estrogen, caused a rapid and reversible inhibition of forskolin-stimulated chloride secretion across T84 epithelial cell monolayers with a K(i) of 8 microM. In addition, 17alpha-estradiol, a stereoisomer that fails to bind and activate nuclear estrogen receptors was equipotent with 17beta-estradiol, arguing against a genomic-mediated mechanism of action. Synthetic estrogens, including diethylstilbesterol and the antiestrogen tamoxifen likewise inhibited forskolin-stimulated ion transport. Aldosterone, dexamethasone, and cholesterol were without effect at the highest concentrations tested (>/=1 mM). Studies indicated that diethylstilbesterol and other synthetic estrogens that inhibited anion secretion in intact monolayers likewise inhibited CFTR chloride channel activity with similar concentration dependencies in excised membrane patches. Experiments with radioactive photoactivatable estrogen derivatives demonstrated that these compounds bind directly to CFTR expressed in insect cells. Taken together, the data suggest that estrogens can interact directly with CFTR to alter anion transport.

Evidence that reversed glutamate uptake contributes significantly to glutamate release following experimental injury to the rat spinal cord.

Released excitatory amino acids contribute significantly to secondary damage following spinal cord injury. Reversal of normal transport due to cell membrane depolarization may contribute to this release. We tested this by administering dihydrokainic acid (DHK), a non-transported glutamate uptake blocker, into the rat spinal cord by microdialysis in association with contusion spinal cord injury. Glutamate release in response to injury was reduced by 34% (P<0.05) when 3 mM DHK was administered within the microdialysis fiber, suggesting that reversed transport is an important contributor to glutamate release upon spinal cord injury.

Activation of metabotropic glutamate receptors inhibits synapsin I phosphorylation in visceral sensory neurons.

Activation of glutamate metabotropic receptors (mGluRs) in nodose ganglia neurons has previously been shown to inhibit voltage-gated Ca++ currents and synaptic vesicle exocytosis. The present study describes the effects of mGluRs on depolarization-induced phosphorylation of the synaptic-vesicle-associated protein synapsin I. Depolarization of cultured nodose ganglia neurons with 60 mM KCl resulted in an increase in synapsin I phosphorylation. Application of mGluR agonists 1-aminocyclopentane-1s-3r-dicarboxylic acid (t-ACPD) and L(+)-2-Amino-4-phosphonobutyric acid (L-AP4) either in combination or independently inhibited the depolarization induced phosphorylation of synapsin I. Application of the mGluR antagonist (RS)-alpha-Methyl-4-carboxyphenylglycine (MCPG) blocked t-ACPD-induced inhibition of synapsin phosphorylation but not the effects of L-AP4. In addition, application of either t-ACPD or L-AP4 in the absence of KCl induced depolarization had no effect on resting synapsin I phosphorylation. RT-PCR analysis of mGluR subtypes in these nodose ganglia neurons revealed that these cells only express group III mGluR subtypes 7 and 8. These results suggest that activation of mGluRs modulates depolarization-induced synapsin I phosphorylation via activation of mGluR7 and/or mGluR8 and that this process may be involved in mGluR inhibition of synaptic vesicle exocytosis in visceral sensory neurons of the nodose ganglia.