A macrocyclic quinol-containing ligand enables high catalase activity even with a redox-inactive metal at the expense of the ability to mimic superoxide dismutase.

Previously, we found that linear quinol-containing ligands could allow manganese complexes to act as functional mimics of superoxide dismutase (SOD). The redox activity of the quinol enables even Zn(ii) complexes with these ligands to catalyze superoxide degradation. As we were investigating the abilities of manganese and iron complexes with 1,8-bis(2,5-dihydroxybenzyl)-1,4,8,11-tetraazacyclotetradecane (H4qp4) to act as redox-responsive contrast agents for magnetic resonance imaging (MRI), we found evidence that they could also catalyze the dismutation of H2O2. Here, we investigate the antioxidant behavior of Mn(ii), Fe(ii), and Zn(ii) complexes with H4qp4. Although the H4qp4 complexes are relatively poor mimetics of SOD, with only the manganese complex displaying above-baseline catalysis, all three display extremely potent catalase activity. The ability of the Zn(ii) complex to catalyze the degradation of H2O2 demonstrates that the use of a redox-active ligand can enable redox-inactive metals to catalyze the decomposition of reactive oxygen species (ROS) besides superoxide. The results also demonstrate that the ligand framework can tune antioxidant activity towards specific ROS.

Diquinol Functionality Boosts the Superoxide Dismutase Mimicry of a Zn(II) Complex with a Redox-Active Ligand while Maintaining Catalyst Stability and Enhanced Activity in Phosphate Solution.

In the current work, we demonstrate ligand design concepts that significantly improve the superoxide dismutase (SOD) activity of a zinc complex; the catalysis is enhanced when two quinol groups are present in the polydentate ligand. We investigate the mechanism through which the quinols influence the catalysis and determine the impact of entirely removing a chelating group from the original hexadentate ligand. Our results suggest that SOD mimicry with these compounds requires a ligand that coordinates Zn(II) strongly in both its oxidized and reduced forms and that the activity proceeds through Zn(II)-semiquinone complexes. The complex with two quinols displays greatly enhanced catalytic ability, with the activity improving by as much as 450% over a related complex with a single quinol. In the reduced form of the diquinol complex, one quinol appears to coordinate to the zinc much more weakly than the other. We believe that superoxide can more readily displace this portion of the ligand, facilitating its coordination to the metal center and thereby hastening the SOD reactivity. Despite the presence of two redox-active groups that may communicate through intramolecular hydrogen bonding and redox tautomerism, only one quinol undergoes two-electron oxidation to a para-quinone during the catalysis. After the formation of the para-quinone, the remaining quinol deprotonates and binds tightly to the metal, ensuring that the complex remains intact in its oxidized state, thereby maintaining its catalytic ability. The Zn(II) complex with the diquinol ligand is highly unusual for a SOD mimic in that it performs more efficiently in phosphate solution.

The Herbicide Atrazine Potentiates Angiotensin II-Induced Aldosterone Synthesis and Release From Adrenal Cells.

Atrazine is one of the most commonly used pre-emergence and early post-emergence herbicides in the world. We have shown previously that atrazine does not directly stimulate the pituitary or adrenal to trigger hormone release but acts centrally to activate a stress-like activation of the hypothalamic-pituitary-adrenal axis. In doing so, atrazine treatment has been shown to cause adrenal morphology changes characteristic of repeated stress. In this study, adrenals from atrazine treated and stressed animals were directly compared after 4 days of atrazine treatment or restraint stress. Both atrazine and stressed animals displayed reduced adrenocortical zona glomerulosa thickness and aldosterone synthase (CYP11B2) expression, indicative of repeated adrenal stimulation by adrenocorticotropic hormone. To determine if reduced CYP11B2 expression resulted in attenuated aldosterone synthesis, stressed and atrazine treated animals were challenged with angiotensin II (Ang II). As predicted, stressed animals produced less aldosterone compared to control animals when stimulated. However, atrazine treated animals had higher circulating aldosterone concentrations compared to both stressed and control groups. Ang II-induced aldosterone release was also potentiated in atrazine pretreated human adrenocortical carcinoma cells (H295R). Atrazine pretreated did not alter the expression of the rate limiting steroidogenic StAR protein or angiotensin II receptor 1. Atrazine treated animals also presented with higher basal blood pressure than vehicle treated control animals suggesting sustained elevations in circulating aldosterone levels. Our results demonstrate that treatment with the widely used herbicide, atrazine, directly increases stimulated production of aldosterone in adrenocortical cells independent of expression changes to rate limiting steroidogenic enzymes.

Superoxide dismutase activity enabled by a redox-active ligand rather than metal.

Reactive oxygen species are integral to many physiological processes. Although their roles are still being elucidated, they seem to be linked to a variety of disorders and may represent promising drug targets. Mimics of superoxide dismutases, which catalyse the decomposition of O2•- to H2O2 and O2, have traditionally used redox-active metals, which are toxic outside of a tightly coordinating ligand. Purely organic antioxidants have also been investigated but generally require stoichiometric, rather than catalytic, doses. Here, we show that a complex of the redox-inactive metal zinc(II) with a hexadentate ligand containing a redox-active quinol can catalytically degrade superoxide, as demonstrated by both reactivity assays and stopped-flow kinetics studies of direct reactions with O2•- and the zinc(II) complex. The observed superoxide dismutase catalysis has an important advantage over previously reported work in that it is hastened, rather than impeded, by the presence of phosphate, the concentration of which is high under physiological conditions.

Adding a Second Quinol to a Redox-Responsive MRI Contrast Agent Improves Its Relaxivity Response to H2O2.

The overproduction of reactive oxygen species has been linked to a wide array of health disorders. The ability to noninvasively monitor oxidative stress in vivo could provide substantial insight into the progression of these conditions and, in turn, could facilitate the development of better diagnosis and treatment options. A mononuclear Mn(II) complex with the redox-active ligand N,N'-bis(2,5-dihydroxybenzyl)-N,N'-bis(2-pyridinylmethyl)-1,2-ethanediamine (H4qtp2) was made and characterized. A previously prepared Mn(II) complex with a ligand containing a single quinol subunit was found to display a modest T1-derived relaxivity response to H2O2. The introduction of a second redox-active quinol both substantially improves the relaxivity response of the complex to H2O2 and reduces the cytotoxicity of the sensor but renders the complex more susceptible to transmetalation. The addition of H2O2 partially oxidizes the quinol subunits to para-quinones, concomitantly increasing the r1 from 5.46 mM-1 s-1 to 7.17 mM-1 s-1. The oxidation of the ligand enables more water molecules to coordinate to the metal ion, providing an explanation for the enhanced relaxivity. That the diquinol complex is only partially oxidized by H2O2 is attributed to its activity as an antioxidant; the complex can both catalytically degrade superoxide and serve as a hydrogen atom donor.

Abnormal coagulation factor VIII transcript in a Tennessee Walking Horse colt with hemophilia A.

Hemophilia A is an X-chromosome-linked disorder caused by a deficiency in factor VIII (FVIII). Although foals have been diagnosed with hemophilia A based on deficiency in FVIII activity, causative gene mutations have not been identified. The genomic DNA and cDNA encoding FVIII of a Tennesee Walking Horse colt affected with hemophilia A and the genomic DNA of his dam and a normal unrelated horse were analyzed with no splice site or coding sequence abnormalities identified in any of the horses. Polymerase chain reactions (PCR) were then performed on hepatic cDNA from the affected colt and an unrelated normal horse, and no product was obtained for the sequence between and including exon 1 and exon 2 in the affected colt. Based on these results, suspected mutations were identified in the noncoding region of FVIII (intron 1), and genomic sequencing of intron 1 in the dam and the affected colt suggested maternal inheritance.

Characterization of endothelial colony-forming cells from peripheral blood samples of adult horses.

OBJECTIVE: To isolate and characterize endothelial colony-forming cells (ECFCs; a subtype of endothelial progenitor cells) from peripheral blood samples of horses. SAMPLE: Jugular venous blood samples from 24 adult horses. PROCEDURES: Blood samples were cultured in endothelial cell growth medium. Isolated ECFCs were characterized by use of functional assays of fluorescence-labeled acetylated low-density lipoprotein (DiI-Ac-LDL) uptake and vascular tubule formation in vitro. Expression of endothelial (CD34, CD105, vascular endothelial growth factor receptor 2, and von Willebrand factor) and hematopoietic (CD14) cell markers was assessed through indirect immunofluorescence assay and flow cytometry. The number of passages before senescence was determined through serial evaluation of DiI-Ac-LDL uptake, vascular tubule formation, and cell doubling rates. RESULTS: Samples from 3 horses produced colonies at 12 ± 2.5 days with characteristic endothelial single layer cobblestone morphology and substantial outgrowth on expansion. Equine ECFCs formed vascular tubules in vitro and had uptake of DiI-Ac-LDL (74.9 ± 14.7% positive cells). Tubule formation and DiI-Ac-LDL uptake diminished by passage 5. Equine ECFCs tested positive for von Willebrand factor, vascular endothelial growth factor receptor 2, CD34, and CD105 with an immunofluorescence assay and for CD14 and CD105 via flow cytometry. CONCLUSIONS AND CLINICAL RELEVANCE: ECFCs can be isolated from peripheral blood of horses and have characteristics similar to those described for other species. These cells may have potential therapeutic use in equine diseases associated with ischemia or delayed vascularization.

A mononuclear manganese(II) complex demonstrates a strategy to simultaneously image and treat oxidative stress.

A manganese(II) complex with a ligand containing an oxidizable quinol group serves as a turn-on sensor for H2O2. Upon oxidation, the relaxivity of the complex in buffered water increases by 0.8 mM(-1) s(-1), providing a signal that can be detected and quantified by magnetic resonance imaging. The complex also serves as a potent antioxidant, suggesting that this and related complexes have the potential to concurrently visualize and alleviate oxidative stress.

Expression of melanocortin receptors in human prostate cancer cell lines: MC2R activation by ACTH increases prostate cancer cell proliferation.

The melanocortin receptors (MCRs 1-5) are G protein coupled-receptors (GPCRs) that regulate food intake, inflammation, skin pigmentation, sexual function and steroidogenesis. Their peptide ligands, the melanocortins, are α-, ß- and γ-melanocyte-stimulating hormone and adrenocorticotropic hormone (ACTH) all of which are secreted from the anterior pituitary gland under hypothalamic control. MC2R binds ACTH but has no affinity for the other melanocortins and is, thereby, pharmacologically different from MCRs that bind those ligands. Evidence suggests that elevated GPCRs transactivate the androgen receptor (AR), the critical mediator of prostate cell growth, and consequently promote prostate cancer cell proliferation. It may be that reduced central melanocortin signaling is coincidental with reversal of prostate cancer cachexia, but no data are available on the expression of, or the role for, MCRs in prostate cancer. Here, we show that MCR (1-5) mRNAs are expressed in androgen-dependent LNCaP and androgen-independent PC3 and DU-145 human prostate cancer cell lines. Further, MC2R, the specific target of ACTH, is expressed in LNCaP, PC3 and DU-145 cells. Among the several synthetic MCR peptide ligands that we used, only ACTH promoted concentration-dependent cell proliferation in the three cell lines as shown by MTT cell proliferation assay. In LNCaP cells, the effect was additive with testosterone stimulation and was partially blunted with SHU9119, a non-selective MCR antagonist. In the same cells, ACTH induced cAMP production and increased AR nuclear labeling in immunocytochemical assays. Our observations suggest that MC2R is involved in prostate carcinogenesis and that targeting MC2R signaling may provide a novel avenue in prostate carcinoma treatment.

Thiazolidinediones/PPARγ agonists and fatty acid synthase inhibitors as an experimental combination therapy for prostate cancer.

The prostate cancer (PCa) cell lines LNCaP, PC-3, and DU-145 express peroxisome proliferator-activated receptor γ (PPARγ) but its role in PCa is unclear. Thiazolidinediones (TZDs), a family of PPARγ activators and type 2 anti-diabetic drugs, exhibit anti-tumor apoptotic effects in human PCa cell lines. Likewise, pharmacological inhibitors of fatty acid synthase (FASN), a metabolic enzyme highly expressed in PCa, induce apoptosis in prostate and other cancer cells. Here, we show positive correlation between PPARγ and FASN protein in PCa cell lines and synergism between TZDs and FASN blockers in PCa cell viability reduction and apoptosis induction. Combined TZDs/FASN has enhanced anti-tumor properties in both androgen-dependent LNCaP and androgen-independent PC-3 and DU-145 cells when compared with single drug exposure. Low concentrations (5-10 µM) of the TZD drug rosiglitazone failed to alter cell viability but, paradoxically, upregulated lipogenic genes [PPARγ, FASN, sterol regulatory element binding protein-1c (SREBP-1c) and acetyl-Co A carboxylase-1 (ACC1)], which diminish the apoptotic effects of rosiglitazone. The mean IC50 in all cell lines was 45 ± 2 µM for rosiglitazone compared with significantly lower 5 ± 1 µM for rosiglitazone plus the FASN blocker cerulenin, and 10.2 ± 2 µM for rosiglitazone plus the cerulenin synthetic analog C75. The IC50 for the combined rosiglitazone and FASN blockers contrasts with the relatively higher IC50 for rosiglitazone (45 ± 2 µM), the TZD drug troglitazone (13 ± 2 µM), cerulenin (32 ± 1 µM), or C75 (26 ± 3 µM) when these drugs were used alone. In summary, this study shows proof-of-principle for combining FASN blockers and TZDs for PCa treatment.

Activation of PPARγ by Rosiglitazone does not negatively impact male sex steroid hormones in diabetic rats.

Peroxisome proliferator-activated receptor gamma (PPARγ) activation decreased serum testosterone (T) in women with hyperthecosis and/or polycystic ovary syndrome and reduced the conversion of androgens to estradiol (E2) in female rats. This implies modulation of female sex steroid hormones by PPARγ. It is not clear if PPARγ modulates sex steroid hormones in diabetic males. Because PPARγ activation by thiazolidinedione increased insulin sensitivity in type 2 diabetes, understanding the long term impact of PPARγ activation on steroid sex hormones in males is critical. Our objective was to determine the effect of PPARγ activation on serum and intratesticular T, luteinizing hormone (LH), follicle stimulating hormone (FSH) and E2 concentrations in male Zucker diabetic fatty (ZDF) rats treated with the PPARγ agonist rosiglitazone (a thiazolidinedione). Treatment for eight weeks increased PPARγ mRNA and protein in the testis and elevated serum adiponectin, an adipokine marker for PPARγ activation. PPARγ activation did not alter serum or intratesticular T concentrations. In contrast, serum T level but not intratesticular T was reduced by diabetes. Neither diabetes nor PPARγ activation altered serum E2 or gonadotropins FSH and LH concentrations. The results suggest that activation of PPARγ by rosiglitazone has no negative impact on sex hormones in male ZDF rats.

Activation of Penile Proadipogenic Peroxisome Proliferator-Activated Receptor gamma with an Estrogen: Interaction with Estrogen Receptor Alpha during Postnatal Development.

Exposure to the estrogen receptor alpha (ERalpha) ligand diethylstilbesterol (DES) between neonatal days 2 to 12 induces penile adipogenesis and adult infertility in rats. The objective of this study was to investigate the in vivo interaction between DES-activated ERalpha and the proadipogenic transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma). Transcripts for PPARs alpha, beta, and gamma and gamma1a splice variant were detected in Sprague-Dawley normal rat penis with PPARgamma predominating. In addition, PPARgamma1b and PPARgamma2 were newly induced by DES. The PPARgamma transcripts were significantly upregulated with DES and reduced by antiestrogen ICI 182, 780. At the cellular level, PPARgamma protein was detected in urethral transitional epithelium and stromal, endothelial, neuronal, and smooth muscular cells. Treatment with DES activated ERalpha and induced adipocyte differentiation in corpus cavernosum penis. Those adipocytes exhibited strong nuclear PPARgamma expression. These results suggest a biological overlap between PPARgamma and ERalpha and highlight a mechanism for endocrine disruption.

Molecular and functional characterization of bovine beta-defensin-1.

We report the biochemical and functional properties of a novel bovine beta-defensin (bBD-1). Cloned from bovine mammary papillary duct epithelia, the bBD-1 cDNA predicts a 69 amino acid propeptide that is much more similar to human beta-defensin-1 (hBD-1) than to other bovine defensins. The bBD-1 gene contains two exons and one 8.5 kb intron. Using RT-PCR, we detected the bBD-1 transcript in the teat mucosa, kidney, vagina, ovary, oviduct, and colon. A synthetic bBD-1 peptide demonstrates potent antibacterial activity against Escherichia coli. The widespread expression of bBD-1 mRNA indicates that bBD-1 may play an important role in the bovine host defense against infections.

Hypotonic swelling stimulates L-type Ca2+ channel activity in vascular smooth muscle cells through PKC.

It has been suggested that L-type Ca(2+) channels play an important role in cell swelling-induced vasoconstriction. However, there is no direct evidence that Ca(2+) channels in vascular smooth muscle are modulated by cell swelling. We tested the hypothesis that L-type Ca(2+) channels in rabbit portal vein myocytes are modulated by hypotonic cell swelling via protein kinase activation. Ba(2+) currents (I(Ba)) through L-type Ca(2+) channels were recorded in smooth muscle cells freshly isolated from rabbit portal vein with the conventional whole cell patch-clamp technique. Superfusion of cells with hypotonic solution reversibly enhanced Ca(2+) channel activity but did not alter the voltage-dependent characteristics of Ca(2+) channels. Bath application of selective inhibitors of protein kinase C (PKC), Ro-31-8425 or Go-6983, prevented I(Ba) enhancement by hypotonic swelling, whereas the specific protein kinase A (PKA) inhibitor KT-5720 had no effect. Bath application of phorbol 12,13-dibutyrate (PDBu) significantly increased I(Ba) under isotonic conditions and prevented current stimulation by hypotonic swelling. However, PDBu did not have any effect on I(Ba) when cells were first exposed to hypotonic solution. Furthermore, downregulation of endogenous PKC by overnight treatment of cells with PDBu prevented current enhancement by hypotonic swelling. These data suggest that hypotonic cell swelling can enhance Ca(2+) channel activity in rabbit portal vein smooth muscle cells through activation of PKC.

Regulation of adiponectin secretion by endothelin-1.

Adiponectin is an adipocyte-derived hormone best known for its insulin-sensitizing ability. The expression and circulating concentration of adiponectin are decreased in type 2 diabetics and increase following treatment with thiazolidinediones. Endothelin-1 (ET-1) is a potent vasoconstrictor peptide whose levels are elevated in numerous disease states, including obesity and diabetes. ET-1 has profound effects on adipose tissue metabolism and alters the release of adipose-derived factors such as leptin and resistin, therefore we investigated the role of ET-1 in adiponectin secretion. 3T3-L1 adipocytes were treated with insulin (100 nM), ET-1 (100 nM), or the appropriate vehicle and adiponectin secretion into the media was determined by immunoblotting and densitometric analysis. Adiponectin secretion significantly increased 1h following insulin or ET-1 treatment, respectively. Pretreatment with ET-1 for 24h significantly inhibited the ability of insulin or ET-1 to acutely stimulate adiponectin secretion. The specific ET(A) receptor antagonist, BQ-610 (1 microM), significantly inhibited ET-1-stimulated adiponectin secretion. In summary, ET-1 acutely stimulates adiponectin secretion through the ET(A) receptor. Chronic exposure to ET-1 dramatically decreases the stimulatory effect of insulin and ET-1 on adiponectin secretion. Our findings suggest vascular factors such as ET-1 may play a role in the regulation of adiponectin secretion and whole body energy metabolism.

Effect of myocardial volume overload and heart failure on lactate transport into isolated cardiac myocytes.

The purpose of this study was to determine lactate transport kinetics in single isolated rat ventricular cardiac myocytes after 1) 8 wk of myocardial volume overload (MVO) and 2) congestive heart failure (CHF). Twenty male Sprague-Dawley rats were assigned to one of four groups: myocardial hypertrophy (MH), MH sham (MHS), CHF, or CHF sham (CHFS). A chronic MVO was induced in the MH and CHF groups by an infrarenal arteriovenous fistula. Postdeath heart and lung weights were significantly greater (P < 0.05) for the MH and CHF groups compared with controls. Isolated cardiac myocytes were loaded with BCECF to determine intracellular pH (pH(i)) changes after the addition of lactate to the extracellular superfusate. Alterations in pH(i) with the addition of varied lactate concentrations were attenuated 72-89% by 5.0 mM alpha-cyano-4-hydroxycinnamate. Significant differences (P < 0.05) were found in estimated maximal lactate transport rates between the experimental and sham groups (MH = 19.4 +/- 1.1 nmol x microl(-1) x min(-1) vs. MHS = 15.1 +/- 1.1 nmol x microl(-1) x min(-1); CHF = 20.2 +/- 2.0 nmol x microl(-1) x min(-1) vs. CHFS = 14.0 +/- 0.9 nmol x microl(-1) x min(-1)). Western blot analysis confirmed a 270% increase in monocarboxylate symport protein 1 (MCT1) protein content in CHF compared with CHFS rats. The results of this study suggest that MH and CHF induced by MVO engender a greater maximal lactate transport capacity across the cardiac myocyte sarcolemma along with an increase in MCT1 protein content. These alterations would likely benefit the cell by attenuating intracellular acidification during a period of increased myocardial load.

Endothelin-1 inhibits resistin secretion in 3T3-L1 adipocytes.

Resistin is an adipocyte-derived hormone whose role in the development of insulin resistance is controversial. Endothelin-1 (ET-1) is a 21 amino acid peptide demonstrated to possess vasoconstrictor, positive inotropic, mitogenic, and metabolic properties. In numerous disease states, including congestive heart failure, obesity, and diabetes, elevated levels of ET-1 have been reported and are thought to contribute to the pathology of the disease. A recent study demonstrated that ET-1 induces the expression and stimulates the secretion of the adipose tissue-derived hormone leptin. However, the effect of ET-1 on resistin secretion has not been determined. To characterize the effect of ET-1 on resistin secretion, 3T3-L1 fibroblasts were differentiated into adipocytes and allowed to mature for 14 days. Cells were incubated for 24h with ET-1 (1-100 nM), insulin (1-100 nM), insulin+ET-1 (100 nM I+E) or the appropriate vehicle or antagonist. At the end of the incubation period, resistin secretion was determined in the media by immunoblotting and densitometric analysis. ET-1 (1-100 nM) significantly decreased basal resistin secretion by 49% (1 nM), 43% (10nM), and 59% (100 nM). Insulin (1-100 nM) produced a concentration-dependent increase in resistin secretion from 3T3-L1 adipocytes (1 nM-42%, 10nM-55%, and 100 nM-86% vs. control). Insulin-stimulated resistin secretion (100 nM) was almost completely inhibited (94%) by ET-1 (100 nM). The effects of ET-1 on resistin protein secretion were inhibited by co-incubation with the ET(A) receptor antagonist BQ-610. In conclusion, our studies demonstrate that basal and hormonal stimulation of resistin secretion by insulin are inhibited by ET-1. Such findings demonstrate that resistin secretion is regulated in a similar manner to other adipose tissue factors, including leptin, in 3T3-L1 adipocytes. In addition, our findings suggest that vascular factors such as ET-1 may regulate whole body energy metabolism through adipocyte-derived hormones, including leptin and resistin.