The effect of allopurinol administration on mitochondrial respiration and gene expression of xanthine oxidoreductase, inducible nitric oxide synthase, and inflammatory cytokines in selected tissues of broiler chickens.

Birds have a remarkable longevity for their body size despite an increased body temperature, higher metabolic rate, and increased blood glucose concentrations compared to most mammals. As the end-product of purine degradation, uric acid (UA) is generated in the xanthine/hypoxanthine reactions catalyzed by xanthine oxidoreductase (XOR). In the first study, Cobb × Cobb broilers (n = 12; 4 weeks old) were separated into 2 treatments (n = 6); control (CON) and allopurinol (AL) 35 mg/kg BW (ALLO). The purpose of this study was to assess mitochondrial function in broiler chickens in response to potential oxidative stress generated from the administration of AL for 1 wk. There was a significant reduction in state 3 respiration (P = 0.01) and state 4 respiration (P = 0.007) in AL-treated birds compared to the controls. The purpose of the second study was to assess the effect of AL on gene expression of inflammatory cytokines interferon-γ (IFN)-γ, IL-1ß, IL-6, and IL-12p35, as well as inducible nitric oxide synthase and XOR in liver tissue. Cobb × Cobb broilers were separated into two groups at 4 wk age (n = 10); CON and ALLO. After 1 wk AL treatment, half of the birds in each group (CON 1 and ALLO 1) were euthanized while the remaining birds continued on AL treatment for an additional week (CON 2 and ALLO 2). A significant increase in gene expression of XOR, IFN-γ, IL-1ß, and IL-12p35 in ALLO 2 birds as compared to birds in CON 2 was detected. Liver UA content was significantly decreased in both ALLO 1(P = 0.003) and ALLO 2 (P = 0.012) birds when compared to CON 1 and CON 2, respectively. The AL reduced liver UA concentrations and increased expression of inflammatory cytokines. Additional studies are needed to determine if AL causes a direct effect on mitochondria or if mitochondrial dysfunction observed in liver mitochondria was due indirectly through increased oxidative stress or increased inflammation.

Effects of a Phytogenic Feed Additive Versus an Antibiotic Feed Additive on Oxidative Stress in Broiler Chicks and a Possible Mechanism Determined by Electron Spin Resonance.

Phytogenic feed additives are plant-derived products used in poultry feeding to improve overall performance of broilers. In this study, 588 one day-old Cobb 500 chicks were fed one of four diets and housed on either dirty or clean litter for 3wks. Treatments included: Group I: commercial diet with no additive and housed on clean litter; Group II: commercial diet with no additive and housed on dirty litter; Group III: commercial diet with a 0.05% inclusion of the anitobiotic, BMD (bacitracin methylene disalicylate); Group IV: commercial diet with a 0.05% inclusion of a phytogenic feed additive (PFA). The study was designed around a random block assignment of treatments allocated to groups of twenty-one birds per pen. Blood samples were obtained from chicks at 18 days of age for measurement of leukocyte oxidative activity by a bioluminescence technique. Results of the study showed that chicks in the treatment groups fed the PFA had significantly lower oxidative stress (p<0.02) when compared to the BMD treatment group. Once this was determined, electron spin resonance (ESR) spin trapping was used to detect and measure hydroxyl or superoxide radicals in. Fenton chemistry was utilized for production of hydroxyl radicals and a xanthine/xanthine oxidase reaction for the production of superoxide radicals in the diet and in RAW 264.7 mouse peritoneal monocytes exposed to the diet. Results from the reactions showed that the antibiotic scavenges hydroxyl and superoxide radicals more efficiently than the phytogenic. The results were comparable to those measured in the RAW 264.7 cells.

The effects of allopurinol, uric acid, and inosine administration on xanthine oxidoreductase activity and uric acid concentrations in broilers.

The purpose of these studies was to determine the effects of uric acid (UA) and inosine administration on xanthine oxidoreductase activity in broilers. In experiment one, 25 broilers were assigned to 5 treatment groups: control, AL (25 mg of allopurinol/kg of body mass), AR (AL for 2 wk followed by allopurinol withdrawal over wk 3), UAF (AL plus 6.25 g of UA sodium salt/kg of feed), and UAI (AL plus 120 mg of UA sodium salt injected daily). The UA administration had no effect on plasma concentration of UA (P > 0.05), and all allopurinol-treated birds had lower (P < 0.05) UA levels than controls. The UA concentrations were restored in both plasma and kidney of AR birds at wk 3, but liver UA concentrations remained lower. Whereas xanthine oxidoreductase (XOR) activity in the liver (LXOR) was reduced (P < 0.05) by allopurinol treatment, XOR activity in the kidney (KXOR) was not affected (P = 0.05). In experiment two, 3 groups of 5 birds each were fed 0 (control), 0.6 M inosine/kg of feed (INO), or INO plus 50 mg of allopurinol/kg of body mass (INOAL). The INOAL birds showed lower total LXOR activity, but KXOR activity was not affected. Both INO and INOAL birds had higher plasma and kidney UA concentrations than controls. The results suggest that regulation of UA production is tissue dependent.

Effects of allopurinol on uric acid concentrations, xanthine oxidoreductase activity and oxidative stress in broiler chickens.

The purpose of this study was to determine the effects of allopurinol (AL) on xanthine oxidoreductase (XOR) activity and uric acid (UA) levels in chickens. Thirty 5-week-old broilers were divided into three groups and fed 0 (control), 25 (AL25) or 50 (AL50) mg AL per kg of body mass for 5 weeks. Chicks were weighed twice weekly and leukocyte oxidative activity (LOA) and plasma purine levels were determined weekly in five birds per group. Chicks were sacrificed after 2 or 5 weeks, and samples from tissues were taken for analysis of XOR activity. Plasma UA concentrations were lower (P<0.001) and xanthine and hypoxanthine concentrations were greater (P<0.001) in AL25 and AL50 birds compared to controls, whereas no differences (P=0.904) were detected in allantoin concentrations. By week 5, body mass was reduced (P<0.001) to 84.0 and 65.1% of that in controls for AL25 and AL50 broilers, respectively, and LOA was 4.1 times greater (P<0.05) in AL25 compared to control birds. Liver XOR activity was increased by 1.1 and 1.2 times in AL25 and AL50 birds, but there was no change (P>0.05) in XOR activity in the pancreas and intestine. These results suggest that AL effect on XOR activity is tissue dependent.

Determination of xanthine oxidoreductase activity in broilers: effect of pH and temperature of the assay and distribution in tissues.

Xanthine oxidoreductase (XOR) is the enzyme responsible for the synthesis of uric acid, which exists primarily in the dehydrogenase form in birds. Uric acid is the major end product of the metabolism of nitrogen-containing compounds in birds and it functions as an antioxidant to reduce oxidative stress. Despite the importance of this enzyme, the tissue distribution of XOR in physiologically normal chickens is not well known. In this study, we analyzed XOR activity in extracts of 8 tissues from broilers at 7 and 10 wk of age. No differences in XOR activity due to the age were found in any tissue. Liver and kidney showed the greatest activity, that in the kidney being about 89% of the activity in the liver. Enzyme activity in intestine and pancreas was about 60 and 37% of that in the liver. All breast muscle, heart, and lung samples showed enzyme activity, but values were only 3.0, 1.2, and 0.6% of those found in the liver. Traces of enzyme activity were also detected in 3 out of 10 brain samples, and no activity was found in the plasma. Our results show that XOR distribution in chickens differs from that in mammals, in which the highest levels have been found in liver and intestine. An additional objective was the evaluation of the effect of pH (7.2, 7.7, 8.2, and 8.7) and temperature (25 and 41 degrees C) on the enzyme activity in liver and kidney samples. Temperature had a similar effect on both tissues, with the activity at 25 degrees C being about 30% of that measured at 41 degrees C. At 41 degrees C, the enzyme activity in liver and kidney decreased quadratically as pH decreased from 8.7 to 7.2. The highest activity in kidney was measured at pH 8.2, although there were no differences between enzyme activities at pH 8.7 or 8.2 in the liver. Our results indicate that the optimum pH of the enzyme in chicken liver and kidney is around 8.2.

Classic and non-classic progesterone receptors are both expressed in human spermatozoa.

Progesterone is one of the physiological inducers of the acrosome reaction in mammalian spermatozoa. The receptor that responds to progesterone is not yet identified, and its properties differ in many aspects from the properties of the classic nuclear progesterone receptor, suggesting the participation of a novel or non-classic receptor. In this study, we investigated the expression of a novel progesterone-binding protein (hmPR1/PGMRC1) and its ortholog (hmPR2/PGMRC2), which have previously been identified in liver microsomes and are considered receptor candidates, along with the nuclear progesterone receptor. The purification procedure was optimized with special emphasis on the control of leukocyte contamination in single donor samples. The results indicate that all three proteins are expressed in human sperm, as transcripts have been detected in 46 %, 42 % and 37.5 % of individual samples, respectively (n = 24).

The human membrane progesterone receptor gene: genomic structure and promoter analysis.

Rapid, nongenomic effects of steroids are likely to be mediated by membrane receptors not by intracellular steroid receptors. We recently identified a progesterone membrane binding protein (mPR) from human liver. The corresponding hmpr gene is comprised of 3 exons and 2 introns. The promoter sequence of hmpr lacks a typical TATA box but contains instead a high homology to a transcription Initiatior consensus sequence, which overlaps the experimentally determined transcriptional start site. The major proximal promoter is GC-rich and sequence analysis revealed a CpG island spanning the transcriptional start site. Several putative cis-regulatory DNA-motifs, which represent possible binding sites for transcription factors like AP2, NF-AT, Ahr/Arnt and C/EBP were identified in the genomic upstream region by sequence homology. Functional analysis of differently deleted fragments of the hmpr upstream region in a GFP-reportergene assay in transiently transfected cultured cells indicates the general testability of the hmpr promoter in vivo.

Chemical modification and structural analysis of the progesterone membrane binding protein from porcine liver membranes.

In addition to the classical genomic steroid actions on modulation of transcription and protein synthesis, rapid, nongenomic effects have been described for various steroids. These effects on cellular signaling and function are supposed to be transmitted by membrane binding sites unrelated to the classical intracellular receptors. Recently, a high affinity progesterone membrane binding protein (mPR) has been characterized in porcine liver membranes. In the present study, amino acid residues that are essential for progesterone binding to porcine liver microsomal mPR have been identified by the use of protein modifying reagents. Among all reagents tested, agents with specificity for carboxyl groups, methionine and tryptophan such as N,N'-dicyclohexylcarbodiimide, chloramine T and N-bromosuccinimide induced a reduction in [3H]progesterone binding. To evaluate the presence of essential disulfide bridges, porcine liver microsomes were incubated with the disulfide reducing agent dithiothreitol (DTT) and [3H]progesterone binding was measured. This treatment also resulted in a reduction of binding activity with an IC50 of 20 mM for DTT. Western-blotting analysis in the presence or absence of the reducing agent suggested that mPR--in its binding state--consists of at least two identical subunits with an apparent molecular mass of 28 kDa which are linked by a disulfide bridge. In conclusion, in the present study evidence for an involvement of carboxyl-, tryptophan- and methionine residues in [3H]progesterone binding to porcine liver microsomes is given. In addition, it is shown that mPR can form disulfide-linked homodimers.

Multiple actions of steroid hormones--a focus on rapid, nongenomic effects.

According to the traditional model, steroid hormones bind to intracellular receptors and subsequently modulate transcription and protein synthesis, thus triggering genomic events finally responsible for delayed effects. Based upon similarities in molecular structure, specific receptors for steroids, vitamin D(3) derivatives, thyroid hormone, retinoids, and a variety of orphan receptors are considered to represent a superfamily of steroid receptors. In addition, very rapid effects of steroids mainly affecting intracellular signaling have been widely recognized that are clearly incompatible with the genomic model. These rapid, nongenomic steroid actions are likely to be transmitted via specific membrane receptors. Evidence for nongenomic steroid effects and distinct receptors involved is presented for all steroid groups including related compounds like vitamin D(3) and thyroid hormones. The physiological and clinical relevance of these rapid effects is still largely unclear, but their existence in vivo has been clearly shown in various settings including human studies. Drugs that specifically affect nongenomic steroid action may find applications in various clinical areas such as cardiovascular and central nervous disorders, electrolyte homeostasis, and infertility. In addition to a short description of genomic steroid action, this review pays particular attention to the current knowledge and important results on the mechanisms of nongenomic steroid action. The modes of action are discussed in relation to their potential physiological or pathophysiological relevance and with regard to a cross-talk between genomic and nongenomic responses.

Nongenomic actions of steroids--from the laboratory to clinical implications.

In the classical concept of steroid action, steroids bind to cytoplasmic receptors and modulate nuclear transcription after translocation of steroid-receptor complexes into the nucleus. Due to similarities of molecular structure, receptors for steroids, retinoids, vitamin D3 and thyroid hormone are considered to represent a superfamily of receptors. While genomic steroid effects been evident for several decades, rapid effects of steroids have been characterized only recently. These rapid actions are likely to be transmitted by specific membrane receptors. Binding sites in membranes have been characterized which display binding features compatible with an involvement in rapid steroid signaling. Characteristics of putative membrane receptors are completely distinct from those of intracellular steroid receptors, a fact which is further supported by the inability of classic steroid receptor antagonists to suppress nongenomic steroid actions. The cloning and functional expression of a putative progesterone membrane receptor has been achieved. Drugs that specifically modulate nongenomic action alone or even both genomic and nongenomic actions may be applied in various areas such as the cardiovascular and central nervous systems, and treatment of infertility and electrolyte homeostasis.

Mannheim classification of nongenomically initiated (rapid) steroid action(s).

There is increasing evidence for rapid effects of steroids that are incompatible with the classical model of genomic steroid action. To address the diversity of mechanisms for rapid steroid signaling described over the past years, a classification of rapid steroid effects has been proposed to promote the discussion and understanding of nongenomic steroid action.

Specific nongenomic actions of aldosterone.

BACKGROUND: According to the traditional model, steroid hormones modulate gene transcription and protein synthesis. The considerable latency of these genomic steroid effects is the consequence of these time-consuming steps of action. Over the years, it has become increasingly clear that rapid actions of steroids exist that are incompatible with this "classic" genomic model of action. These rapid, nongenomic effects, which recently have been shown for virtually all groups of steroids, are likely to be transmitted by specific membrane receptors. METHODS: A review of data mainly focusing on the nongenomic in vitro and in vivo effects of aldosterone is presented. RESULTS: For rapid aldosterone effects, a prominent example of a receptor/effector cascade for nongenomic steroid effects has been described in various cell types. Nonclassic membrane receptors with a high affinity for aldosterone, but not for cortisol, seem to be involved. As an important second messenger, [Ca2+]i is consistently increased within minutes after the addition of aldosterone. The effects are half maximal at physiological concentrations of free aldosterone (approximately 0.1 nmol/L), while the classic mineralocorticoid antagonist canrenone is ineffective in blocking the action of aldosterone. In addition, cortisol is active only at supramicromolar concentrations. Aldosterone rapidly acts on further cell signaling systems, for example, phosphoinositide hydrolysis and cAMP generation. CONCLUSIONS: For a better understanding of nongenomic aldosterone action even in a clinical context, future research will have to target the cloning of the first membrane receptor for aldosterone and the evaluation of the clinical relevance of rapid steroid effects in general.

Rapid, nongenomic steroid actions: A new age?

In the traditional theory of steroid action, steroids bind to intracellular receptors and modulate nuclear transcription after translocation of steroid-receptor complexes into the nucleus. Due to similarities of molecular structure, specific receptors for steroids, vitamin D(3) derivatives, and thyroid hormone are considered to represent a superfamily of steroid receptors. While genomic steroid effects characterized by their delayed onset of action and their sensitivity to blockers of transcription and protein synthesis have been known for several decades, rapid actions of steroids have been more widely recognized and characterized in detail only recently. Rapid effects of steroids, thyroid hormones, and the steroid hormone metabolite of vitamin D(3), 1alpha, 25-dihydroxyvitamin D(3), on cellular signaling and function may be transmitted by specific membrane receptors. Binding sites in membranes have been characterized, exposing binding features compatible with an involvement in rapid steroid signaling. Characteristics of putative membrane receptors are completely distinct from intracellular steroid receptors, a fact which is further supported by the inability of classic steroid receptor antagonists to block nongenomic steroid actions. A putative progesterone membrane receptor has been cloned and functionally expressed with regard to progesterone binding. Development of drugs that specifically affect nongenomic action alone or even both modes of action may find applications in various, areas such as in the cardiovascular and central nervous systems and treatment of preterm labor, infertility, and electrolyte abnormalities.

Growth/differentiation factor-15/macrophage inhibitory cytokine-1 is a novel trophic factor for midbrain dopaminergic neurons in vivo.

Transforming growth factor-betas (TGF-betas) constitute an expanding family of multifunctional cytokines with prominent roles in development, cell proliferation, differentiation, and repair. We have cloned, expressed, and raised antibodies against a distant member of the TGF-betas, growth/differentiation factor-15 (GDF-15). GDF-15 is identical to macrophage inhibitory cytokine-1 (MIC-1). GDF-15/MIC-1 mRNA and protein are widely distributed in the developing and adult CNS and peripheral nervous systems, including choroid plexus and CSF. GDF-15/MIC-1 is a potent survival promoting and protective factor for cultured and iron-intoxicated dopaminergic (DAergic) neurons cultured from the embryonic rat midbrain floor. The trophic effect of GDF-15/MIC-1 was not accompanied by an increase in cell proliferation and astroglial maturation, suggesting that GDF-15/MIC-1 probably acts directly on neurons. GDF-15/MIC-1 also protects 6-hydroxydopamine (6-OHDA)-lesioned nigrostriatal DAergic neurons in vivo. Unilateral injections of GDF-15/MIC-1 into the medial forebrain bundle just above the substantia nigra (SN) and into the left ventricle (20 microgram each) immediately before a 6-OHDA injection (8 microgram) prevented 6-OHDA-induced rotational behavior and significantly reduced losses of DAergic neurons in the SN. This protection was evident for at least 1 month. Administration of 5 microgram of GDF-15/MIC-1 in the same paradigm also provided significant neuroprotection. GDF-15/MIC-1 also promoted the serotonergic phenotype of cultured raphe neurons but did not support survival of rat motoneurons. Thus, GDF-15/MIC-1 is a novel neurotrophic factor with prominent effects on DAergic and serotonergic neurons. GDF-15/MIC-1 may therefore have a potential for the treatment of Parkinson's disease and disorders of the serotonergic system.

Nongenomically initiated steroid actions.

The classical theory of steroid hormone action comprises binding to an intracellular receptor followed by modulation of transcriptional and translational events. These cumbersome model explains the characteristic latency of these genomic steroid effects. Over the past two decades, increasing evidence for rapid nongenomic effects of steroids, incompatible with the traditional model, has accumulated. These alternative steroid effects have been described for all classes of steroids and a multitude of species and tissues with different mechanisms of action.

Aldosterone- and progesterone-membrane-binding proteins: new concepts of nongenomic steroid action.

n the classical theory of steroid action steroids penetrate into cells and bind to intracellular receptors resulting in modulation of nuclear transcription and protein synthesis within hours. In addition, rapid actions of steroids have been identified, which are incompatible with the classic model of steroid action. Specific binding sites for aldosterone and progesterone have been reported in membrane preparations of liver, vascular smooth muscle cells and kidney. These sites are discussed to be involved in rapid nongenomic steroid actions, such as the rapid activation of the Na(+)/H(+) exchanger and elevation of [Ca(2+)]i in vascular smooth muscle cells by aldosterone. In addition, rapid progesterone-induced increases of [Ca(2+)]i have been reported in spermatozoa. A high affinity progesterone-membrane binding protein from porcine liver has been identified and cloned. The derived amino acid sequence showed no significant identity with any functional protein suggesting a binding site completely different to classic progesterone receptors. These binding sites are possibly involved in rapidly induced meiotic maturation of amphibian oocytes and the spermatozoan acrosome reactions as evidenced by recent studies, where the progesterone induced acrosome reactions and calcium signaling was blocked by a specific antibody raised against the membrane binding site for progesterone. In addition to data on specific steroid binding and rapid steroid signaling in vitro, results of nongenomic steroid effects in vivo are presented and their physiological relevance are discussed in the review.

Inhibition of platelet aggregation after intake of acetylsalicylic acid detected by a platelet function analyzer (PFA-100).

OBJECTIVE: The aim of the investigation was to determine the inhibition of platelet aggregation detected by the platelet function analyzer PFA-100 in patients with coronary artery disease who routinely take 100 mg acetylsalicylic acid once daily. METHOD: The PFA-100 (Dade International Inc., Miami, USA) is a new device for in vitro measurement of platelet function in citrated whole blood. The time needed to form a platelet plug occluding the aperture cut into a collagen/epinephrine- or collagen/ADP-coated membrane is determined under high shear conditions. Typically, acetylsalicylic acid-induced inhibition of platelet aggregation is detected by prolonged closure time in collagen/epinephrine or and normal values for collagen/ADP. Blood samples of 48 patients were investigated, the control group consisted of 10 healthy volunteers without intake of acetylsalicylic acid. The upper limits of normal values of the control group were 137 sec closure time for collagen/epinephrine and 150 sec for collagen/ADP (mean + 2 SD). RESULTS: Statistical analysis (Wilcoxon test) did not show a significant difference (p = 0.46) between the patient group (129 +/- 11 sec) and the control group (92 +/- 7 sec) for collagen/epinephrine (mean +/- SEM). Only 31% of patients had closure time values above those upper limits defined above. CONCLUSION: The effect of 100 mg acetylsalicylic acid daily appears to be too small and too variable to detect a sufficient inhibition of platelet aggregation by the PFA-100 in all patients with coronary artery disease.

Rapid aldosterone effects on tyrosine phosphorylation in vascular smooth muscle cells.

Non-genomic aldosterone effects are characterized by their rapid onset, their specificity for mineralocorticoids and their insensitivity both to the mineralocorticoid type 1 receptor antagonist spironolactone and to the inhibitors of transcription and translation, cycloheximide and actinomycin D. The aim of the present study was to further characterize the second messenger system involved in the non-genomic pathway of aldosterone with particular emphasis on protein phosphorylation. The rapid increase of free intracellular calcium by aldosterone in VSMC is sensitive to genistein, so that tyrosine kinase activity appears likely to be involved in the signaling pathway. Here, the effect of 100 nmol/l aldosterone (10 min.) on tyrosine protein-phosphorylation was determined in VSMC. Our findings show that aldosterone (100 nmol/l) in combination with shear stress as additional stimulus induces a rapid (within 10 min.) small but consistent increase in tyrosine-phosphorylation compared with aldosterone or shear stress alone. Immunoprecipitation of the MAPK-isoforms ERK 1 and ERK 2 showed an increased phosphorylation after 3 and 5 min.

Specific progesterone binding to a membrane protein and related nongenomic effects on Ca2+-fluxes in sperm.

Rapid, nongenomic effects of steroids are supposed to be transmitted by membrane receptors unrelated to the classic intracellular steroid receptors. In this context, a putative progesterone membrane binding protein (mPR) has been identified, recently. Here we show that expression of mPR-cDNA in CHO cells leads to increased microsomal progesterone binding. This result is mirrored by effects of an antibody raised against the recombinant E. coli mPR which suppressed the rapid progesterone-initiated Ca2+ increase in sperm. Our results support the assumption that mPR represents the first steroid membrane receptor or a part of it involved in rapid, nongenomic steroid signalling.