Melatonin-mediated regulation of human MT(1) melatonin receptors expressed in mammalian cells

In mammals, the pineal hormone melatonin activates G protein-coupled MT(1) and MT(2) melatonin receptors. Acute exposure of recombinant MT(1) and MT(2) melatonin receptors to supraphysiological concentrations of melatonin differentially regulates these two receptors with the MT(2), but not the MT(1), exhibiting rapid desensitization and internalization. In the present study, we sought to determine whether prolonged exposure to supraphysiological and physiological concentrations of melatonin desensitized and/or internalized the MT(1) melatonin receptor. Using a Chinese hamster ovary (CHO) cell line stably expressing MT(1)-FLAG or transiently expressing MT(1)-green fluorescent protein (GFP) melatonin receptors, we found that prolonged exposure (8h) to supraphysiological concentrations of melatonin (100 nM) significantly increased the number of MT(1) melatonin receptors and decreased the affinity (K(i)) of melatonin for competition for 2-[125]iodomelatonin. A similar treatment also desensitized the MT(1) melatonin receptor-mediated stimulation of [(35)S]GTPgammaS binding, but did not internalize the receptor. In contrast, prolonged exposure to a concentration of melatonin mimicking nocturnal levels (400 pM) did not affect the number of MT(1) melatonin receptors, the affinity for melatonin, or the functional sensitivity of the receptor. We conclude that in vivo endogenous melatonin does not significantly affect the functional sensitivity of MT(1) melatonin receptors, however, exogenous melatonin taken therapeutically at doses above physiological levels could desensitize the receptor thereby affecting physiological responses mediated following activation of MT(1) melatonin receptors.     

Temporal and spatial expression patterns of canonical clock genes and clock-controlled genes in the suprachiasmatic nucleus

In mammals, the suprachiasmatic nuclei (SCN) of the hypothalamus control endogenous circadian rhythms and entrainment to the environment. A core SCN region of calbindin (CalB)-containing cells is retinorecipient and the cells therein lack rhythmic expression of clock genes and electrical activity. The core is surrounded by a 'shell' of rhythmic oscillator cells. In the present experiments, we studied the spatial arrangement of oscillator cells by examining the spatial and temporal patterns of expression of the canonical clock genes Per1, Per2 and vasopressin mRNA, a clock-controlled gene. Surprisingly, in the SCN shell, the dorsomedial cells were the first to rhythmically express both Per1 and VP mRNA, with gene expression then spreading very slowly through much of the nucleus for the next 12 h then receding to baseline levels. Following a light pulse, Per expression increased after 1 h in the core SCN and after 1.5 h in the shell. Although expression in the shell occurred earlier in light-pulsed animals than in those housed in constant darkness, it still followed the same spatial and temporal expression pattern as was observed in constant darkness. The results suggest that not only is the SCN organized into light-responsive and rhythmic regions but also that the rhythmic region of the SCN itself has an ordered arrangement of SCN oscillator cells.     

Oscillatory transient inward currents in ventricular myocytes of healthy versus myopathic Syrian hamster

The present experiments were performed in order to study abnormal action potential configuration and ion channel activity in ventricular myocytes obtained from 23 male myopathic Syrian hamsters (Biobreeders strain 14.6, 32-52 weeks old) compared with 10 age-matched healthy control hamsters (Biobreeders F1B) by means of whole-cell patch-clamp techniques. The results show that the myopathic myocytes had a longer action potential duration, a reduced transient outward K(+) current on depolarization and a smaller transient inward current on repolarization after prolonged depolarizing pulses (> 500 msec). However, the L-type Ca(2+) current and the inwardly rectifing K(+) current were not significantly different from those of healthy myocytes. The oscillatory transient inward currents could be diminished by treatment with ryanodine (0.01-1 micromol/L), a sarcoplasmic reticulum (SR) Ca(2+) release channel blocker, or with Na(+)-free superfusate. We conclude that the hereditary myopathic hamsters are less likely to develop delayed after depolarization-related transient inward currents and triggered arrhythmias owing to a smaller SR Ca(2+) content.     

Anthrax toxin receptor proteins

Anthrax toxin is a key virulence factor for Bacillus anthracis, the causative agent of anthrax. Here we discuss what is known about the anthrax toxin receptor (ATR), the cellular receptor for anthrax toxin, and how this information is being used to develop treatments for anthrax as well as to understand aspects of cancer. ATR was identified recently as a type I transmembrane protein with unknown function that contains an extracellular integrin-like inserted (I) domain. The ATR I domain contains the toxin binding site, and a soluble form of this domain was shown to serve as an effective antitoxin to protect cultured cells from toxin action. ATR is encoded by the tumor endothelial marker 8 (TEM8) gene, which is selectively up-regulated during blood vessel formation and in tumor vasculature, raising the possibility that this protein normally functions in angiogenesis. Therefore, identification of the cellular receptor for anthrax toxin has made possible new avenues of research in the areas of anthrax pathogenesis, antitoxin development, and cancer biology.     

The effect of β-blocker on hamster model BIO 53.58 with dilated cardiomyopathy determined using<Superscript>123</Superscript>I-MIBG myocardial scintigraphy

OBJECTIVE: 123I-metaiodobenzylguanidine (MIBG) myocardial scintigraphy is currently used to evaluate cardiac sympathetic nerve function, but MIBG also has the capacity to evaluate dilated cardiomyopathy (DCM) severity and therapeutic effectiveness. In this study, we administered beta-blockers to a DCM hamster model and evaluated the effect of therapy using MIBG. We also pathologically compared the effects of myocardial fibrosis suppression. METHODS: BIO 53.58 hamsters were divided into the following five groups based on beta-blocker administration: vehicle (COT), 2 mg/kg/day carvedilol (CLT), 20 mg/kg/day (CHT) carvedilol, 4 mg/kg/day (MLT) metoprolol, 40 mg/kg/day (MHT) metoprolol. F1B hamsters were administered a vehicle (COF). Plasma catecholamine, noradrenaline (p-NADR), adrenaline (p-ADR), and dopamine (p-DOPA) were assayed, and MIBG was performed. The count ratio of the heart to the mediastinum (H/M) and left ventricle myocardial washout ratio (WR) were calculated. We then performed an autopsy and calculated the percent change in fibrotic area from myocardial sections. RESULTS: H/M of the initial image in the COT group was significantly lower at 2.4 +/- 0.2 than the 2.9 +/- 0.7 in the COF group (p < 0.05). The CLT and CHT groups had higher H/M values compared to the COT group (3.1 +/- 0.6, 3.0 +/- 0.6 versus 2.4 +/- 0.2: p < 0.05). Significant correlations were evident between the H/M of the delayed image and p-NADR and p-DOPA (p < 0.05, p < 0.01, respectively) as well as between WR and p-NADR and p-DOPA (p < 0.05). Percent change in fibrotic area was significantly lower in the beta-blocker groups than in the COT group (p < 0.05). Significant negative correlations were seen between the H/M of the delayed image and the percent change in fibrosis area. CONCLUSIONS: The delayed image H/M and WR acutely reflected cardiac disorder and sympathetic nerve function disorder in BIO 53.58 hamsters. In the carvedilol-administered groups, there was improvement compared to the initial H/M image, indicating the efficacy of the beta-blocker in DCM.     

Cytosolic beta-glycosidases for activation of glycoside prodrugs of daunorubicin

Human cytosolic beta-glycosidase is a small monomeric enzyme that is active under physiological conditions, which might be ideal for enzyme-prodrug therapy. We have previously reported the synthesis of a galactoside (DNR-GlA3) and a glucoside (DNR-GsA3) prodrug of daunorubicin. In the present study, we established that cellular uptake of DNR-GlA3 and DNR-GsA3 was low in contrast to that of daunorubicin. Recombinant human beta-glycosidase converted both prodrugs to daunorubicin as shown by liquid chromatography. The kinetics of the conversion of DNR-GlA3 and DNR-GsA3 by human beta-glycosidase, however, was unfavorable as the K(m) values were, respectively, 3- and 6-fold higher than those of another mammalian beta-glycosidase of bovine origin. The V(max) values were, respectively, 3.3 and 8.5nmol/hr/mg as compared to 158.3 and 147.8nmol/hr/mg of the bovine enzyme. Treatment of OVCAR-3 cells with human beta-glycosidase (0.5U/mL) and 0.5 microM DNR-GlA3 or DNR-GsA3 resulted in, respectively, 86 and 81% cell growth inhibition, while the prodrugs alone inhibited growth to only 19 and 1%. Treatment of cells with the bovine enzyme and the prodrugs inhibited cell growth more efficiently. We conclude that the endogenous intracellular beta-glycosidase is not available for extracellular prodrug activation. Thus, the incorporation of the enzyme in enzyme-prodrug therapy might be an elegant approach to achieve tumor-specific prodrug conversion. The efficiency of glycoside prodrug conversion might be improved by design of a prodrug that is more readily activated by human beta-glycosidase or by evolution of the enzyme into a mutant form that displays high activity towards these prodrugs.     

Expression of the murine CB2 cannabinoid receptor using a recombinant Semliki Forest virus

A recombinant Semliki Forest virus (SFV) RNA construct, SFV1-mCB(2) RNA, was employed for the high-level expression of the murine CB(2) (mCB(2)) cannabinoid receptor in baby hamster kidney cells. Biosynthetic radiolabel incorporation studies in concert with urea-sodium dodecylsulfate-polyacrylamide gel electrophoresis (urea-SDS-PAGE) and western immunoblotting revealed that two major proteins of approximately 26 and 40kDa were produced by the construct. The 40kDa product, but not the 26kDa product, was glycosylated as determined by 2-deoxy-D-glucose incorporation and peptide-N-glycosidase F digestion analysis. Assessment of [3H]CP55940 ([3H]-(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol) binding data for membranes of cells transfected with SFV1-mCB(2) RNA indicated a K(d) of 0.35+/-0.04nM and a B(max) of 24.4+/-2.7pmol/mg. A rank order of binding affinities for cannabinoids, which paralleled that reported for native mCB(2) receptors, was observed. The CB(2) receptor-specific antagonist SR144528 (N-[(1S)-endo-1,3,3-trimethyl bicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide) blocked binding of [3H]CP55940, while the CB(1) receptor-specific antagonist SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride] had a minimal effect. These results indicate that the recombinant receptor expressed from SFV1-mCB(2) RNA exhibits properties, including ligand binding features, that are consistent with those for the native mCB(2) receptor. However, the presence of both 26 and 40kDa receptor species is consistent with alternative translation from two AUG start sites using the SFV1-mCB(2) RNA expression system.     

Prostaglandin E2 Release by Human and Syrian Hamster Tumor Cells and Their Sensitivity to Cytostatic Activity of Natural Killers

The release of prostaglandin E₂ by human and Syrian hamster tumor cells in response to contact with natural killers and their sensitivity to cytostatic activity of natural killers were studied. A previously reported correlation between prostaglandin E₂ release by Syrian hamster tumor cells and their resistance to cytostatic activity of natural killers was confirmed. This resistance can be cancelled by inhibition of prostaglandin E₂ release with indomethacin. Unlike malignant tumor cells of Syrian hamsters, only few human tumor cells resistant to cytostatic activity of natural killers secrete prostaglandin E₂. The resistance of these cell strains to cytostatic activity of natural killers could be cancelled by indomethacin treatment. Possible role of prostaglandin E₂ release as a mechanism of tumor cell protection from effector cells of this type of natural (congenital) immunity is discussed.     

Zinc salts precipitate unconjugated bilirubin in vitro and inhibit enterohepatic cycling of bilirubin in hamsters

BACKGROUND: We have evidence for enterohepatic cycling of bilirubin experimentally and in vivo in humans. This study was designed to investigate whether Zn salts might inhibit such cycling of bilirubin. MATERIALS AND METHODS: Micellar bile salt solutions with unconjugated bilirubin were prepared, appropriate concentrations of Zn salts were added, and unconjugated bilirubin precipitation was measured. Hamsters and Wistar rats were fed a chow diet or a chow diet enriched with 1% ZnSO4, and bilirubin secretion rates were monitored. RESULTS: Unconjugated bilirubin was precipitated maximally (90%) after a 10-min incubation with 5 mM Zn salts in the pH range of 6.8-9.0. In control hamsters, total bilirubin secretion rates into bile were 36.0 +/- 2.8 nmol h(-1) 100g(-1) body weight, whereas they were 25.0 +/- 3.3 nmol h-1 100(-1) g in the ZnSO4 group (P < 0.05). CONCLUSIONS: Zn salts that flocculate at physiological pH adsorb unconjugated bilirubin almost completely from unsaturated micellar BS solutions. In addition, Zn salts administered orally suppress biliary bilirubin secretion rates in hamsters. These findings suggest that the administration of Zn salts may inhibit the enterohepatic cycling of unconjugated bilirubin in humans who are predisposed to pigment gallstone formation due to diet, disease or drugs.     

Reduction of human recombinant type II phospholipase A2 and prostaglandin F2 alpha release by microtubule depolymerizing agents

1. The present study examines the effects of the microtubule depolarizing agent colchicine on secretory type II phospholipase A2 (PLA2) function in Chinese hamster ovary (CHO) cells that specifically overexpress human type II PLA2 and the effect of both colchicine and tubulazole on the release of type II PLA2 and prostaglandin (PG) F2 alpha from human placental explants. 2. Significant suppression by colchicine (0.01-10 mumol/L) of PLA2 activity (P < 0.00001), immunoreactive type II PLA2 (irPLA2; P < 0.00001) and PGF 2 alpha release (P < 0.01) was observed in medium from overexpressing CHO cells. These effects were significantly reduced (P < 0.0001) in the presence of 10 mumol/L taxol, an agent that prevents depolymerization of microtubules. The addition of 30 mumol/L arachidonic acid significantly reduced (P < 0.0001) the inhibition of PGF2 alpha production in CHO cell lines. 3. The addition of 1 mumol/L colchicine to human placental explants for 24 h significantly reduced irPLA2 (P < 0.00001) and PGF2 alpha production (P < 0.00001). Similarly, 1 mumol/L tubulazole significantly blocked irPLA2 (P < 0.001) and PGF2 alpha (P < 0.0001). 4. At 10 mumol/L, taxol significantly reduced irPLA2 inhibition by colchicine (n = 8; P < 0.05) and tubulazole (n = 8; P < 0.05). Similarly, taxol significantly reduced the reduction in PGF2 alpha production caused by colchicine (P < 0.001) and by tubulazole (P < 0.001). 5. These results suggest that integrity of the microtubule system is required for PLA2 function and the subsequent production of pro-inflammatory mediators.