Muscle growth requires a constant supply of amino acids (AAs) from the blood. Therefore, plasma AA profile is a critical factor for maximizing the growth performance of animals, including pigs. This research was conducted to study how dietary lysine intake affects plasma AA profile in pigs at the late production stage. Eighteen crossbred (Large White × Landrace) finishing pigs (nine barrows and nine gilts; initial BW 92.3 ± 6.9 kg) were individually penned in an environment controlled barn. Pigs were assigned randomly to one of the three dietary treatments according to a randomized complete block design with sex as block and pig as experiment unit (6 pigs/treatment). Three corn- and soybean meal-based diets contained 0.43 % (lysine-deficient, Diet I), 0.71 % (lysine-adequate, Diet II), and 0.98 % (lysine-excess, Diet III) l-lysine, respectively. After a 4-week period of feeding, jugular vein blood samples were collected from the pigs and plasma was obtained for AA analysis using established HPLC methods. The change of plasma lysine concentration followed the same pattern as that of dietary lysine supply. The plasma concentrations of threonine, histidine, phenylalanine, isoleucine, valine, arginine, and citrulline of pigs fed Diet II or III were lower (P < 0.05) than that of pigs fed Diet I. The plasma concentrations of alanine, glutamate, and glycine of pigs fed Diet II or III were higher (P < 0.05) than that of pigs fed Diet I. The change of plasma leucine and asparagine concentrations followed the patterns similar to that of plasma lysine. Among those affected AAs, arginine was decreased (P < 0.05) in the greatest proportion with the lysine-excess diet. We suggest that the skeletal muscle growth of finishing pigs may be further increased with a lysine-excess diet if the plasma concentration of arginine can be increased through dietary supplementation or other practical nutritional management strategies.

The mycotoxin deoxynivalenol (DON), one of the most common food contaminants, primarily targets the gastrointestinal tract to affect animal and human health. This study was conducted to examine the protective function of glutamic acid on intestinal injury and oxidative stress caused by DON in piglets. Twenty-eight piglets were assigned randomly into 4 dietary treatments (7 pigs/treatment): 1) uncontaminated control diet (NC), 2) NC+DON at 4 mg/kg (DON), 3) NC+2% glutamic acid (GLU), and 4) NC+2% glutamic acid + DON at 4 mg/kg (DG). At day 15, 30 and 37, blood samples were collected to determine serum concentrations of CAT (catalase), T-AOC (total antioxidant capacity), H2O2 (hydrogen peroxide), NO (nitric oxide), MDA (maleic dialdehyde), DAO (diamine oxidase) and D-lactate. Intestinal morphology, and the activation of Akt/mTOR/4EBP1 signal pathway, as well as the concentrations of H2O2, MDA, and DAO in kidney, liver and small intestine, were analyzed at day 37. Results showed that DON significantly (P<0.05) induced oxidative stress in piglets, while this stress was remarkably reduced with glutamic acid supplementation according to the change of oxidative parameters in blood and tissues. Meanwhile, DON caused obvious intestinal injury from microscopic observations and permeability indicators, which was alleviated by glutamic acid supplementation. Moreover, the inhibition of DON on Akt/mTOR/4EBP1 signal pathway was reduced by glutamic acid supplementation. Collectively, these data suggest that glutamic acid may be a useful nutritional regulator for DON-induced damage manifested as oxidative stress, intestinal injury and signaling inhibition.

Uterine glands are essential for pregnancy in mice and likely humans, because they secrete or transport bioactive substances that regulate uterine receptivity for blastocyst implantation. In mice, the uterus becomes receptive to blastocyst implantation on day 4, but is refractory by day 5. Here, blastocysts could be recovered from progesterone-induced uterine gland (PUGKO) but not wildtype (WT) mice on day 5 post-mating. Anti-adhesive Muc1 protein and microvilli were present on the luminal epithelium of PUGKO but not WT uteri. A number of known uterine receptivity genes and gland-specific genes were altered in the PUGKO uterus. Next, the uterus and uterine luminal fluid (ULF) were obtained from WT and PUGKO mice on day 3, 4 and 5. Transcriptome analysis revealed that 580 genes were decreased in the PUGKO uterus, however ULF secrotome analysis revealed that many proteins and several amino acids were increased in the PUGKO ULF. Of note, many proteins encoded by many gland-specific genes were not identified in the ULF of WT mice. These results support the ideas that uterine glands secrete factors that regulate ULF homeostasis and interact with other cell types in the uterus to influence uterine receptivity and blastocyst implantation for the establishment of pregnancy.

Childhood obesity has become a prevalent risk to health of children and teenagers. To develop biomarkers in serum for altered lipid metabolism, genetically obese (Ningxiang strain) and lean (Duroc×Landrace×Large Yorkshire strain) growing pigs were used as models to identify potential differences in the serum metabonome between the two strains of pigs after consuming the same diet for 46 days. At the end of the study, pigs were euthanized for analysis of the serum metabonome and determination of body composition. Obese pigs had higher fat mass (42.3±8.8% vs. 21.9±4.5%) and lower muscle mass (35.4±4.5% vs. 58.9±2.5%) than lean pigs (P<.01). Serum concentrations of insulin and glucagon were higher (P<.02) in obese than in lean pigs. With the use of an NMR-based metabonomic technology, orthogonal projection to latent structure with discriminant analysis showed that serum HDL, VLDL, lipids, unsaturated lipids, glycoprotein, myo-inositol, pyruvate, threonine, tyrosine and creatine were higher in obese than in lean pigs (P<.05), while serum glucose and urea were lower in obese pigs (P<.05). In addition, changes in gut microbiota-related metabolites, including trimethylamine-N-oxide and choline, were observed in sera of obese pigs relatively to lean pigs (P<.05). These novel findings indicate that obese pigs have distinct metabolism, including lipogenesis, lipid oxidation, energy utilization and partition, protein and amino acid metabolism, and fermentation of gastrointestinal microbes, compared with lean pigs. The obese Ningxiang pig may be a useful model for childhood obesity research.

Porcine epidemic diarrhea virus (PEDV) has caused enormous economic losses to the swine industry worldwide in recent years. Puerarin (PR), a major isoflavonoid isolated from the Chinese herb Gegen, possesses many pharmacological activities, including anti-inflammatory, and anti-viral activities. This study was conducted with both PEDV-infected African green monkey kidney cells (Vero) and neonatal pigs to determine the effect of PR on PEDV infection and to elucidate the underlying mechanisms by using proteomic analyses. Twenty-four piglets fed a milk replacer were randomly allocated into one of three groups (Control, PEDV, and PEDV + PR). After a 5-day period of adaption, piglets (n = 8/group) in the PEDV + PR were orally administered with PR (0.5 mg/kg body weight) between days 5 and 9, whereas piglets in the other two groups received the same volume of liquid milk replacer. On day 9, piglets were orally administered with either sterile saline or PEDV (Yunnan province strain) at 104.5 TCID50 (50% tissue culture infectious dose) per pig. On day 12 of the trial, jugular vein blood and intestinal samples were collected. In addition, Vero cells were assigned randomly into three groups (Control, PEDV, PEDV + PR). Cells in the PEDV and PEDV + PR groups were infected with PEDV at a multiplicity of infection of 0.01, while cells in the control group were treated with the same volume of sterile saline. One hour later, cells in the Control and PEDV groups were cultured in serum-free DMEM, while cells in the PEDV + PR group were supplemented with PR. After 36 h of culture, cells were harvested. PR attenuated the reductions in cell proliferation in vitro and growth performance in PEDV-infected piglets, and inhibited PEDV replication and the expression of several cytokines (including IL-8) both in vitro and in vivo. Proteomic analyses identified that the abundances of 29 proteins in the ileum were altered by PEDV infection and restored to the control level by PR. Pathway analyses revealed that PR restored the expression of several interferon-stimulated genes and selectively upregulated the expression of guanylate-binding proteins. Western blot analyses showed that PR supplementation inhibited the PEDV-induced NF-κB activation. Collectively, these results indicate that PR could exert antiviral and anti-inflammatory effects in piglets infected with PEDV and have the potential to be an effective antiviral feed additive.

Intrauterine growth restriction (IUGR) is associated with fetal mortality and morbidity. One of the most common causes of IUGR is placental insufficiency, including placental vascular defects, and mitochondrial dysfunction. In addition, a high level of oxidative stress induces placental vascular lesions. Here, we evaluated the oxidative stress status, mitochondrial function, angiogenesis, and nutrient transporters in placentae of piglets with different birth weights: <500 g (L), 500-600 g (LM), 600-700 g (M), and >700 g (H). Results showed that placentae from the L group had higher oxidative damage, lower adenosine triphosphate and citrate synthase levels, and lower vascular density, compared to those from the other groups. Protein expression of angiogenic markers, including vascular endothelial cadherin, vascular endothelial growth factor A, and platelet endothelial cell adhesion molecule-1, was the lowest in the L group placentae compared to the other groups. In addition, the protein levels of glucose transporters GLUT1 and GLUT3 were downregulated in the L group, compared to the other groups. Furthermore, oxidative stress induced by H2O2 inhibited tube formation and migration in porcine vascular endothelial cells. Collectively, placentae for lower birth weight neonates are vulnerable to oxidative damage, mitochondrial dysfunction, and impaired angiogenesis.

l-proline (Pro) is a precursor of ornithine, which is converted into polyamines via ornithine decarboxylase (ODC). Polyamines plays a key role in the proliferation of intestinal epithelial cells. The study investigated the effect of Pro on polyamine metabolism and cell proliferation on porcine enterocytes in vivo and in vitro. Twenty-four Huanjiang mini-pigs were randomly assigned into 1 of 3 groups and fed a basal diet that contained 0.77% alanine (Ala, iso-nitrogenous control), 1% Pro or 1% Pro + 0.0167% α-difluoromethylornithine (DFMO) from d 15 to 70 of gestation. The fetal body weight and number of fetuses per litter were determined, and the small and large intestines were obtained on d 70 ± 1.78 of gestation. The in vitro study was performed in intestinal porcine epithelial (IPEC-J2) cells cultured in Dulbecco's modified Eagle medium-high glucose (DMEM-H) containing 0 μmol/L Pro, 400 μmol/L Pro, or 400 μmol/L Pro + 10 mmol/L DFMO for 4 d. The results showed that maternal dietary supplementation with 1% Pro increased fetal weight; the protein and DNA concentrations of the fetal small intestine; and mRNA levels for potassium voltage-gated channel, shaker-related subfamily, member 1 (Kv1.1) in the fetal small and large intestines (P < 0.05). Supplementing Pro to either gilts or IPEC-J2 cells increased ODC protein abundances and polyamine concentrations in the fetal intestines and IPEC-J2 cells (P < 0.05). In comparison with the Pro group, the combined administration of Pro and DFMO reduced the expression of ODC protein and spermine concentration in the fetal intestine, as well as the concentrations of putrescine, spermidine and spermine in IPEC-J2 cells (P < 0.05). Meanwhile, the percentage of cells in the S-phase and the mRNA levels of proto-oncogenes c-fos and c-myc were increased in response to Pro supplementation, whereas depletion of cellular polyamines with DFMO increased tumor protein p53 (p53) mRNA levels (P < 0.05). Taken together, dietary supplementation with Pro improved fetal pig growth and intestinal epithelial cell proliferation via enhancing polyamine synthesis.

Puerarin has been reported to be an excellent antioxidant, anti-inflammatory and antimicrobial agent, but the potential effect of puerarin on porcine epidemic diarrhea virus (PEDV) is unclear. This study aimed to determine whether puerarin could alleviate intestinal injury in piglets infected with PEDV. A PEDV (Yunnan province strain) infection model was applied to 7-day-old piglets at 104.5 TCID50 (50% tissue culture infectious dose). Piglets were orally administered with puerarin at the dosage of 0.5 mg/kg body weight from day 5 to day 9. On day 9 of the trial, piglets were inoculated orally with PEDV. Three days later, jugular vein blood and intestinal samples were collected. Results showed puerarin reduced morbidity of piglets infected with PEDV. In addition, puerarin reduced the activities of aspartate aminotransferase and alkaline phosphatase, the ratio of serum aspartate aminotransferase to serum alanine aminotransferase, the number of white blood cells and neutrophils, and the plasma concentrations of interleukin-6, interleukin-8 and tumor necrosis factor-α, as well as protein abundances of heat shock protein-70 in PEDV-infected piglets. Moreover, puerarin increased D-xylose concentration but decreased intestinal fatty acid-binding protein concentration and diamine oxidase activity in the plasma of piglets infected with PEDV. Puerarin increased the activities of total superoxide dismutase, glutathione peroxidase and catalase, while decreasing the activities of myeloperoxidase and concentration of hydrogen peroxide in both the intestine and plasma of PEDV-infected piglets. Puerarin decreased mRNA levels of glutathione S-transferase omega 2 but increased the levels of nuclear factor erythroid 2-related factor 2. Furthermore, puerarin increased the abundance of total eubacteria (16S rRNA), Enterococcus genus, Lactobacillus genus and Enterobacteriaceae family in the intestine, but reduced the abundance of Clostridium coccoides in the caecum. These data indicate puerarin improved intestinal function in piglets infected by PEDV and may be a promising supplement for the prevention of PEDV infection.

Polyamines are essential for cell growth and beneficial for intestinal maturation. To evaluate the effects of putrescine on alleviating intestinal atrophy and underlying molecular mechanisms, both in vivo feeding trial and in vitro cell culture were conducted. Weanling pigs were fed a diet supplemented with 0, 0.1%, 0.2% or 0.3% putrescine dihydrochloride, whereas porcine intestinal epithelial cells (IPEC-J2) were challenged with lipopolysaccharide (LPS) in the presence of 200 μmol/L putrescine.

Intrauterine growth restriction (IUGR) has negative impacts on the postnatal survival, growth and development of humans and animals, with not only on newborns but also adulthood. However, the characteristics for nutrient digestion and absorption in IUGR offspring are still largely unknown. Therefore, the normal birth weight (NBW) and IUGR growing pigs were used in this study to investigate their differences in nutrient utilization, with an expectition for further nutritional optimization of the IUGR offspring during their later life.

This study was designed to determine the presence of Eph B4 or ephrin B2 in human retinal endothelial cells (REC) and their signal transduction. Human retinal endothelial cells were stimulated with an Eph B4/Fc chimera and probed for phosphorylation of phosphatidylinositol-3-kinase (PI3K), Src, and mitogen-activated protein kinase (MAPK) pathways. Proliferation and migration were investigated after Eph B4/Fc stimulation in the presence of various pathway inhibitors. Human retinal endothelial cells express ephrin B2, with little expression of Eph B4. Treatment with EphB4/Fc chimera resulted in activation of PI3K, Src, and MAPK pathways. Eph B4-stimulated endothelial cell proliferation was mediated via PI3K, nitric oxide synthase, and extracellular signal-regulated kinase 1/2 (ERK1/2). Blockade of Src-PI3K pathways produced significant attenuation of Eph B4/Fc-stimulated migration. These results demonstrate for the first time that ephrin B2 is present in human retinal endothelial cells. Additionally, it appears that vascular growth may be modulated in the retina through activation of the PI3K pathway and its downstream components.

Over the past 2 decades, there have been revolutionary developments in life science technologies characterized by high throughput, high efficiency, and rapid computation. Nutritionists now have the advanced methodologies for the analysis of DNA, RNA, protein, low-molecular-weight metabolites, as well as access to bioinformatics databases. Statistics, which can be defined as the process of making scientific inferences from data that contain variability, has historically played an integral role in advancing nutritional sciences. Currently, in the era of systems biology, statistics has become an increasingly important tool to quantitatively analyze information about biological macromolecules. This article describes general terms used in statistical analysis of large, complex experimental data. These terms include experimental design, power analysis, sample size calculation, and experimental errors (Type I and II errors) for nutritional studies at population, tissue, cellular, and molecular levels. In addition, we highlighted various sources of experimental variations in studies involving microarray gene expression, real-time polymerase chain reaction, proteomics, and other bioinformatics technologies. Moreover, we provided guidelines for nutritionists and other biomedical scientists to plan and conduct studies and to analyze the complex data. Appropriate statistical analyses are expected to make an important contribution to solving major nutrition-associated problems in humans and animals (including obesity, diabetes, cardiovascular disease, cancer, ageing, and intrauterine growth retardation).

Endometria and placentae undergo developmental changes that affect the stability of genes used as references for normalization of qPCR data. We identified genes that are stable within the porcine endometrium and placenta throughout pregnancy, and elucidated the temporal/spatial mRNA localization of the glucose and arginine transporters, solute carrier family (SLC) 5A1 and SLC7A3, respectively.

Wnt/β-catenin is a crucial repressor of adipogenesis. We have shown that E2 promoter binding factor 1 (E2F1) suppresses Wnt/β-catenin activity through transactivation of β-catenin interacting protein 1 (CTNNBIP1), also known as inhibitor of β-catenin and TCF4 (ICAT) in human colorectal cancers. However, it remains unknown whether ICAT is required for E2F1 to promote differentiation by inhibiting β-catenin activity in pre-adipocytes. In the present study, we found that 1-methyl-3-isobutylxanthine, dexamethasone, and insulin (MDI)-induced differentiation and lipid accumulation in 3T3-L1 pre-adipocytes was reversed by activation of β-catenin triggered by CHIR99021, a GSK3β inhibitor. Intriguingly, we observed a reduced protein level of E2F1 and ICAT at a later stage of pre-adipocytes differentiation. Importantly, overexpression of ICAT in 3T3-L1 pre-adipocytes markedly promote the adipogenesis and partially reversed the inhibitory effect of CHIR99021 on MDI-induced adipogenesis and lipid accumulation by regulating adipogenic regulators and Wnt/β-catenin targets. Moreover, pre-adipocytes differentiation induced by MDI were markedly inhibited in siE2F1 or siICAT transfected 3T3-L1 cells. Gene silencing of ICAT in the E2F1 overexpressed adipocytes also inhibited the adipogenesis. These data indicated that E2F1 is a metabolic regulator with an ability to promote pre-adipocyte differentiation by activating ICAT, therefore represses Wnt/β-catenin activity in 3T3-L1 cells. We also demonstrated that ICAT overexpression did not affect oleic acid-induced lipid accumulation at the surface of Hela and HepG2 cells. In conclusion, we show that E2F1 is a critical regulator with an ability to promote differentiation and adipogenesis by activating ICAT in pre-adipocytes.

Apoptosis of intestinal epithelial cells following oxidative stress is a major cause of mucosal barrier dysfunction and is associated with the pathogenesis of various gastrointestinal diseases. Although L-tryptophan (Trp) is known to improve intestinal integrity and function, a beneficial effect of N-acetyl serotonin (NAS), a metabolite of Trp, on the apoptosis of enterocytes and the underlying mechanisms remain largely unknown. In the present study, we showed that porcine enterocytes treated with 4-hydroxy-2-nonenal (4-HNE), a metabolite of lipid peroxidation, led to upregulation of apoptotic proteins, including Bax and cleaved caspase-3, and reduction of tight junction proteins. These effects of 4-HNE were significantly abrogated by NAS. In addition, NAS reduced ROS accumulation while increasing the intracellular concentration of glutathione (GSH), and the abundance of the Nrf2 protein in the nucleus and its downstream target proteins. Importantly, these protective effects of NAS were abrogated by Atra, an inhibitor of Nrf2, indicating a dependence on Nrf2 signaling. Taken together, we demonstrated that NAS attenuated oxidative stress-induced cellular injury in porcine enterocytes by regulating Nrf2 signaling. These findings provide new insights into a functional role of NAS in maintaining intestinal homeostasis.

The conceptuses (embryo/fetus and placental membranes) of pigs require energy to support elongation and implantation, and amounts of glucose and fructose increase in the uterine lumen during the peri-implantation period. Conceptuses from day 16 of pregnancy were incubated with either 14C-glucose or 14C-fructose and amounts of radiolabeled CO2 released from the conceptuses measured to determine rates of oxidation of glucose and fructose. Glucose and fructose both transport into conceptuses, and glucose is preferentially metabolized in the presence of fructose, whereas fructose is actively metabolized in the absence of glucose and to a lesser extent in the presence of glucose. Endometrial and placental expression of glucose transporters SLC2A1, SLC2A2, SCL2A3, and SLC2A4 were determined. SLC2A1 messenger RNA (mRNA) and protein, and SLC2A4 mRNA were abundant in the uterine luminal epithelium of pregnant compared to cycling gilts, and increased in response to progesterone and conceptus-secreted estrogen. SLC2A2 mRNA was expressed weakly by conceptus trophectoderm on day 15 of pregnancy, whereas SLC2A3 mRNA was abundant in trophectoderm/chorion throughout pregnancy. Therefore, glucose can be transported into the uterine lumen by SLC2A1, and then into conceptuses by SLC2A3. On day 60 of gestation, the cell-specific expression of these transporters was more complex, suggesting that glucose and fructose transporters are precisely regulated in a spatial-temporal pattern along the uterine-placental interface of pigs to maximize hexose sugar transport to the pig conceptus/placenta.

We hypothesized that supplementation of nursery and grower pig diets with coconut oil in the absence of antibiotics would yield maintenance of glucose homeostasis, growth performance, and immune function similar to what is achieved with nursery and grower pig diets containing antibiotics. Pigs received the same base treatment diets from d24 (weaning) to d71 of age and had blood and fecal samples collected on d24, d31, d45 and d71 for measurement of whole blood glucose, serum insulin, cortisol and cytokines, and fecal microbiome. Pigs had weekly weights and daily feed consumption measured throughout the study. Animals were euthanized at d71 and subcutaneous fat and ileal contents were collected for assessment for fatty acids and microbiome, respectively. Diet treatments consisted of 2% soybean oil plus antibiotics (ABX; n = 22), 2% soybean oil without antibiotics (NABX; n = 22), and 2% coconut oil without antibiotics (COC; n = 22). Statistical analysis examined the effect of diet within each timepoint using a repeated measures ANOVA.

Morphogenesis and differentiation of organs is required for subsequent functional maturation. The morphological features of Peyer's patches vary among species. In pigs, they develop extensively in the ileum as ileal Peyer's patches (IPPs). However, the role of IPPs in the porcine immune system remains to be elucidated because of a lack of complete understanding of IPP organogenesis. Results of the present study revealed that development of porcine IPPs is initiated prenatally between embryonic days 76 and 91. The process of IPP organogenesis is concomitant with increased transcriptional patterns of CXCL13 and CCL19. IPPs undergo further development postnatally by forming central, marginal, and subepithelial zones. Importantly, a large number of proliferating B cells and apoptotic cells are found in porcine IPPs postnatally, but not prenatally. The expression level of IgM in proliferating B cells depends on the zone in which distinct B cells are separately localized after birth. Specifically, IgM+ cells are predominantly found in the central zone, whereas IgM-/low cells are abundant in the marginal zone. Importantly, the cellular feature of IPPs differs from that of mesenteric lymph nodes (MLNs) where such distinct zones are not formed both prenatally and postnatally. Our findings suggest that IPPs (not MLNs) in postnatal pigs are involved in complementing functions of the primary lymphoid tissue that promotes the differentiation and maturation of B cells.

Alpha-ketoglutarate, a key intermediate in the Krebs cycle, has been reported to benefit intestinal health. We tested whether alpha-ketoglutarate can protect intestinal cells against hydrogen peroxide induced damage and aimed to reveal the underlying mechanism. Intestinal porcine epithelial cell line J2 were cultured in Dulbecco's Modified Eagle Medium-High glucose with or without alpha-ketoglutarate and hydrogen peroxide. Cell viability, proliferation, mitochondrial respiration, mitochondrial membrane potential, antioxidant function, apoptosis and mitochondrial-dependent apoptotic pathways were determined. Our experiments demonstrated that, first, exposure to 100μM hydrogen peroxide decreased cell viability, DNA synthesis, mitochondrial respiration and antioxidant function, and increased apoptosis. Second, 2mM alpha-ketoglutarate addition attenuated hydrogen peroxide-induced cell cycle arrest, and improved cell viability, DNA synthesis, mitochondrial respiration and antioxidant function. Third, alpha-ketoglutarate enhanced tricarboxylic acid cycle activity, mitochondrial respiration, and decrease the intracellular content of reactive oxygen species. Finally, alpha-ketoglutarate stabilized the mitochondrial membrane potential, increased the ratio of Bcl-2/Bax, decreased the release of cytochrome c and activation of caspase-3, thereby prevented cell apoptosis. Altogether, we proposed that alpha-ketoglutarate protects intestinal cells against hydrogen peroxide-induced damage partly via mitochondria dependent pathway.

The objective of this study was to elucidate the mechanisms by which nebivolol, a cardio-selective beta-adrenergic receptor antagonist, inhibits rat aortic smooth muscle cell (RASMC) proliferation. Nebivolol was compared with DETA-NO and S-nitroso-N-acetylpenicillamine (SNAP), two nitric oxide (NO) donor agents, and alpha-difluoromethylornithine (DFMO), a known inhibitor of ornithine decarboxylase (ODC). All four test agents inhibited RASMC proliferation in a concentration-dependent manner, with nebivolol being the most potent (IC(50) = 4.5 microM), whereas atenolol, another relatively selective beta(1)-blocker, was inactive. DFMO, nebivolol, and DETA-NO interfered with cell proliferation in a cell-density-dependent manner, the lower the cell density the greater the inhibition of cell proliferation. The cytostatic effects of nebivolol and DETA-NO were completely independent of cyclic GMP, as neither ODQ (cytosolic guanylyl cyclase inhibitor) nor zaprinast (cyclic GMP phosphodiesterase inhibitor) affected the antiproliferative action of nebivolol or DETA-NO. The cytostatic effects of nebivolol, SNAP, and DFMO were largely prevented by the addition of excess putrescine, but not ornithine, to cell cultures. Moreover, nebivolol caused a marked reduction in the intracellular levels of putrescine, spermidine, and spermine. Like DFMO, nebivolol and DETA-NO interfered with the G(1)-phase to S-phase cell cycle transition in RASMC. These observations confirm previous findings that DFMO and NO interfere with RASMC proliferation by inhibiting ODC and polyamine production and provide evidence that nebivolol works by the same mechanism.