Antisteroidogenic actions of hydrogen peroxide on rat Leydig cells

It has been well known that reactive oxygen species (ROS) are produced in the steroidogenic pathway and spermatozoa. H2O2, one of ROS produced by spermatozoa, appears to be a primary toxic agent. In the present study, we examined the effects of H2O2 on the basal and evoked-testosterone release from primary Leydig cells, the protein expressions of cytochrome P450 side chain cleavage enzyme (P450scc) and steroidogenic acute regulatory (StAR) protein were also investigated. Our preparation was found to contain approximately 87% Leydig cells and very few macrophages. The results demonstrated that H2O2 (>1 x 10(-4) M) significantly inhibited the basal and hCG-stimulated testosterone release. H2O2 abolished forskolin- or 8-Br-cAMP-evoked testosterone release. In the presence of pregnenolone, progesterone, or androstenedione, the inhibitory effect of H2O2 on testosterone release was prevented. H2O2 also inhibited pregnenolone production in the presence of trilostane (an inhibitor of 3beta-hydroxysteroid dehydrogenase), therefore diminished the activity of P450scc in Leydig cells. In addition to the inhibition of hormone secretion, H2O2 also regulated steroidogenesis by diminishing protein expression of StAR. These results suggest that H2O2 acts directly on rat Leydig cells to diminish testosterone production by inhibiting P450scc activity and StAR protein expression.     

Stimulation of the secretion of luteinizing hormone by ginsenoside-Rb1 in male rats

Ginsenoside-Rb1 is one of the pharmacologically active components of ginseng, the dry root of Panax ginseng C. A. Meyer (Araliaceae), a well-known traditional Chinese medicine. Ginseng enhanced mounting behaviour of male rats and increased sperm counts in rabbit testes. Some experimental results suggested no sex hormone-like function in ginseng but probably gonadotropin-like action. The present study was to explore the effect of ginsenoside-Rb1 on the secretion of luteinizing hormone (LH) both in vivo and in vitro. Male rats were orchidectomized (Orch) for 2 weeks or subjected to swim training for 1 week before catheterization via the right jugular vein. They were intravenously injected with ginsenoside-Rb1 (10 microg/kg) or saline at 15 min prior to a challenge of gonadotropin-releasing hormone (GnRH) or 10 min-swim. Blood samples were collected at several time intervals following intravenous injection of ginsenoside-Rb1. In the in vitro experiment, male rats were decapitated and their anterior pituitary gland (APs) were either bisceted or enzymatically dispersed. The hemi-APs were preincubated with Locke's medium at 37 degrees C for 90 min and then incubated with Locke's medium containing ginsenoside-Rb1 (10(-7) - 10(-4) M) for 30 min. The dispersed AP cells (1 x 1(5) cells per well) were primed with dihydrotestosterone (DHT, 10(-8) M) for 3 days, and then challenged with ginsenoside-Rbl (10(-6) and 10(-5) M, n = 8) for 3 h. The concentrations of LH or testosterone in samples were measured by radioimmunoassays. Administration of ginsenoside-Rb1 did not alter the levels of plasma LH in both intact and Orch rats but significantly increased plasma LH concentration at the termination of the 10 min swimming exercise. Administration of ginsenoside-Rb1 resulted in a lower testosterone response to GnRH challenge or swimming exercise as compared with saline-treated rats. Ginsenoside-Rb1 dose-dependently increased the release of LH from both hemi-AP tissues and the DHT-primed dispersed AP cells in vitro. These results suggest that ginsenoside-Rb1 increases LH secretion by acting directly on rat AP cells.     

Altered sinus nodal and atrioventricular nodal function in freely moving mice overexpressing the A1 adenosine receptor

To investigate whether altered function of adenosine receptors could contribute to sinus node or atrioventricular (AV) nodal dysfunction in conscious mammals, we studied transgenic (TG) mice with cardiac-specific overexpression of the A1 adenosine receptor (A1AR). A Holter ECG was recorded in seven freely moving littermate pairs of mice during normal activity, exercise (5 min of swimming), and 1 h after exercise. TG mice had lower maximal heart rates (HR) than wild-type (WT) mice (normal activity: 437 +/- 18 vs. 522 +/- 24 beats/min, P < 0.05; exercise: 650 +/- 13 vs. 765 +/- 28 beats/min, P < 0.05; 1 h after exercise: 588 +/- 18 vs. 720 +/- 12 beats/min, P < 0.05; all values are means +/- SE). Mean HR was lower during exercise (589 +/- 16 vs. 698 +/- 34 beats/min, P < 0.05) and after exercise (495 +/- 16 vs. 592 +/- 27 beats/min, P < 0.05). Minimal HR was not different between genotypes. HR variability (SD of RR intervals) was reduced by 30% (P < 0.05) in TG compared with WT mice. Pertussis toxin (n = 4 pairs, 150 microg/kg ip) reversed bradycardia after 48 h. TG mice showed first-degree AV nodal block (PQ interval: 42 +/- 2 vs. 37 +/- 2 ms, P < 0.05), which was diminished but not abolished by pertussis toxin. Isolated Langendorff-perfused TG hearts developed spontaneous atrial arrhythmias (3 of 6 TG mice vs. 0 of 9 WT mice, P < 0.05). In conclusion, A1AR regulate sinus nodal and AV nodal function in the mammalian heart in vivo. Enhanced expression of A1AR causes sinus nodal and AV nodal dysfunction and supraventricular arrhythmias.     

In vitro splicing of erythropoietin by the Mycobacterium tuberculosis RecA intein without substituting amino acids at the splice junctions

Protein splicing is a self-catalyzed process involving the excision of an intervening polypeptide sequence, the intein, and joining of the flanking polypeptide sequences, the extein, by a peptide bond. We have studied the in vitro splicing of erythropoietin (EPO) using a truncated form of the Mycobacterium tuberculosis RecA mini-intein in which the homing endonuclease domain was replaced with a hexahistidine sequence (His-tag). The intein was inserted adjacent to cysteine residues to assure that the spliced product had the natural amino acid sequence. When expressed in Escherichia coli, intein-containing EPO was found entirely as inclusion bodies but could be refolded in soluble form in the presence of 0.5 M arginine. Protein splicing of the refolded protein could be induced with a reducing agent such as DTT or tris(2-carboxyethyl)phosphine and led to the formation of EPO and mini-intein along with some cleavage products. Protein splicing mediated by the RecA intein requires the presence of a cysteine residue adjacent to the intein insertion site. We compared the efficiencies of protein splicing adjacent to three of the four cysteine residues of EPO (Cys29, Cys33 and Cys161) and found that insertion of intein adjacent to Cys29 allowed far more efficient protein splicing than insertion adjacent to Cys33 or Cys161. For ease of purification, our experiments involved a His-tagged EPO fusion protein and a His-tagged intein and the spliced products (25 kDa EPO and 24 kDa mini-intein) were identified by Western blotting using anti-EPO and anti-His-tag antibodies and by mass spectroscopy. The optimal splicing yield at Cys29 (40%) occurred at pH 7.0 after refolding at 4 degrees C and splicing for 18 h at 25 degrees C in the presence of 1 mM DTT.     

Involvement of Cdk5/p25 in digoxin-triggered prostate cancer cell apoptosis

Cardiac digitalis has been considered to be a treatment for breast cancer. Our previous study indicates that digoxin, one member in digitalis, decreases the proliferation of prostate cancer cells, but the mechanisms remain unclear. In the present study, Ca(2+) proved to be an important factor in digoxin-triggered prostate cancer cell death. Because cyclin-dependent kinase (Cdk)5 and p35 cleavage (p25 formation) have been reported to be targets of intracellular Ca(2+), and subsequently correlated to apoptosis, we not only demonstrated first that Cdk5, p35, and p25 proteins were all expressed in prostate cancer cells (including lymph node carcinoma of the prostate (LNCaP) and DU-145 cells), but also showed where p25 formation and Cdk5 kinase activity were affected by treatment with digoxin. The inhibitor of p35 cleavage (calpeptin) was used to reduce p25 formation, and the result suggested that p25 accumulation might be the major cause of digoxin-triggered LNCaP cell death. Butyrolactone-I and roscovitine, two Cdk5 kinase inhibitors, were also found to prevent digoxin-triggered LNCaP cell death. In addition, treatment of siRNA-Cdk5 diminished digoxin-triggered cell death, as compared with the treatments of siRNA-Cdk1 or siRNA-Cdk2, which implies the specific involvement of Cdk5 in digoxin-triggered cell death. Caspase inhibitor set and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay were used to demonstrate that digoxin-triggered LNCaP cell apoptosis through Cdk5 activation. These results suggest that Cdk5/p35 and p25 are novel players in digoxin-triggered prostate cancer cell apoptosis and, therefore, become potential therapeutic targets.     

Accumulation of 1,3-beta-D-glucans, in response to aluminum and cytosolic calcium in Triticum aestivum

One of the most rapid responses to aluminum (Al) stress in plants is enhanced synthesis and deposition of 1,3-beta-D-glucans (callose) in root tips. Ironically, Al-induced synthesis and deposition of callose occurs in vivo, despite evidence from in vitro systems that suggests that Al is a powerful inhibitor of 1,3-beta-D-glucan synthase. We set out to test the hypothesis that an Al-induced increase in the activity of free calcium in the cytoplasm ([Ca(2+)](cyt)) is the trigger for enhanced synthesis of callose in in vivo systems, an effect that would not be observed in in vitro systems. Root tips of an Al-sensitive cultivar of Triticum aestivum were treated with Al (0-100 microM) or the Ca ionophore A23187 (0-3 micro M) for 3-24 h, and the effects on [Ca(2+)](cyt) and synthesis of callose were measured using confocal laser scanning microscopy. Treatment with Al induced a rapid increase in both [Ca(2+)](cyt) (4.7-fold) and synthesis of callose (30-fold). Treatment with the Ca ionophore, A23187, also elicited an increase in [Ca(2+)](cyt) (6.6-fold). Despite a greater increase in [Ca(2+)](cyt) in the presence of A23187, this increase was accompanied by a smaller increase in callose deposition (11-fold) than was observed in the presence of Al. These data suggest that an increase in [Ca(2+)](cyt) is not the only factor modulating increases in callose synthesis and deposition in the presence of Al.     

5-HT induces duodenal mucosal bicarbonate secretion via cAMP- and Ca2+-dependent signaling pathways and 5-HT4 receptors in mice

In previous studies, we have found that 5-hydroxytryptamine (5-HT) is a potent stimulant of duodenal mucosal bicarbonate secretion (DMBS) in mice. The aim of the present study was to determine the intracellular signaling pathways and 5-HT receptor subtypes involved in 5-HT-induced DMBS. Bicarbonate secretion by murine duodenal mucosa was examined in vitro in Ussing chambers. 5-HT receptor involvement in DMBS was inferred from pharmacological studies by using selective 5-HT receptor antagonists and agonists. The expression of 5-HT(4) receptor mRNA in duodenal mucosa and epithelial cells was analyzed by RT-PCR. cAMP-dependent signaling pathway inhibitors MDL-12330A, Rp-cAMP, and H-89 and Ca(2+)-dependent signaling pathway inhibitors verapamil and W-13 markedly reduced 5-HT-stimulated duodenal bicarbonate secretion and short-circuit current (I(sc)), whereas cGMP-dependent signaling pathway inhibitors NS-2028 and KT-5823 failed to alter these responses. Both SB-204070 and high-dose ICS-205930 (selective 5-HT(4) receptor antagonists) markedly inhibited 5-HT-stimulated bicarbonate secretion and I(sc), whereas methiothepine (5-HT(1) receptor antagonist), ketanserin (5-HT(2) receptor antagonist), and a low concentration of ICS-205930 (5-HT(3) receptor antagonist) had no effect. RS-67506 (partial 5-HT(4) receptor agonist) concentration-dependently increased bicarbonate secretion and I(sc), whereas 5-carboxamidotryptamine (5-HT(1) receptor agonist), alpha-methyl-5-HT (5-HT(2) receptor agonist), and phenylbiguanide (5-HT(3) receptor agonist) did not significantly increase bicarbonate secretion or I(sc). RT-PCR analysis confirmed the expression of 5-HT(4) receptor mRNA in murine duodenal mucosa and epithelial cells. These results demonstrate that 5-HT regulates DMBS via both cAMP- and Ca(2+)-dependent signaling pathways and 5-HT(4) receptors located in the duodenal mucosa and/or epithelial cells.     

Role of histamine and acid back-diffusion in modulation of gastric microvascular permeability and hemorrhagic ulcers in Salmonella typhimurium-infected rats

Documentation concerning the pathogenesis of gastric hemorrhagic ulcer in Salmonella typhimurium (Salmonella typhi)-infective disease is lacking. This research first proposed that alterations of mast cell histamine release, gastric acid back-diffusion and mucosal microvascular permeability are important in modulating gastric ulcer and hemorrhage in Salmonella typhi-infected rats. Additionally, effects of several histamine-related drugs on this ulcer model were evaluated. Male Wistar rats were deprived food for 36 h. Live cultures of Salmonella typhi (OU 5045, 1 x 10(10) CFU in 1.0 mL of sterilized phosphate buffer saline) were challenged, intrajejunally to rats just before withdrawal of food. Control rats received the same volume of sterilized vehicle only. Rat stomachs were irrigated for 3 h with either normal saline or simulated gastric juice. Gastric acid back-diffusion, mucosal histamine concentration, microvascular permeability as well as luminal hemoglobin content and ulcer areas were determined. Severe gastric hemorrhage and mucosal ulcerations, particularly in acidic stomachs, were observed in Salmonella typhi-infected rats. A positive correlation of histamine to gastric hemorrhage and ulcer was found in those rats with Salmonella typhi-infection. This hemorrhagic ulcer in Salmonella typhi-infected rats was effectively ameliorated by intraperitoneal ketotifen, diphenhydramine and ranitidine but was worsen by exogenous histamine or diamine oxidase. In conclusion, enhancement of acid back-diffusion, mast cell histamine release and microvascular permeability is important in modulating gastric hemorrhage and ulcer in Salmonella typhi-infected rats.