ABSTRACT
Nocistatin and nociceptin/orphaninFQ (N/OFQ) are the two new peptides which may have roles in nociception, memory, anxiety, and other biological functions. Nocistatin acts as a functional antagonist to N/OFQ in several functions, but their neuro-anatomical sites of interaction are unknown. We investigated the effect of combined intracerebroventricular (i.c.v.) injection of nocistatin with N/OFQ, on N/OFQ induced c-Fos expression in the mouse hippocampus, using c-Fos immunohistochemistry. We found that co-injection of nocistatin with N/OFQ significantly attenuated N/OFQ induced c-Fos expression in the hippocampus.
Subject(s)
Analgesics, Opioid/pharmacology , Hippocampus/drug effects , Opioid Peptides/pharmacology , Proto-Oncogene Proteins c-fos/metabolism , Animals , Hippocampus/cytology , Hippocampus/metabolism , Humans , Injections, Intraventricular , Mice , NociceptinABSTRACT
Rat fetuin, which is the rat counterpart of human alpha 2-HS glycoprotein and bovine fetuin, is only detectable in calcified tissues such as bone matrices and dentin, and bone cells such as osteoblasts and osteocytes immunohistochemically. The effect of this protein on bone resorption was examined to study its physiological role in bone metabolism. Rat fetuin increased bone resorption in the presence of low concentrations of parathyroid hormone (PTH), but it had no activity on bone resorption without PTH. The increase in bone resorption by PTH and PTH plus rat fetuin was inhibited by the addition of chymostatin, an inhibitor for cathepsin L. Moreover, we found that when type I collagen from rat was preincubated with rat fetuin, the digestion of rat type I collagen by cathepsin L was increased. These findings suggest that rat fetuin present in bone matrix is important in bone resorption.
Subject(s)
Bone Resorption/chemically induced , Endopeptidases , Parathyroid Hormone/pharmacology , alpha-Fetoproteins/pharmacology , Animals , Bone Resorption/physiopathology , Cathepsin L , Cathepsins/metabolism , Collagen/metabolism , Culture Media, Conditioned , Cysteine Endopeptidases , Dose-Response Relationship, Drug , Drug Synergism , Female , Mice , Mice, Inbred ICR , Phosphorylation , Pregnancy , Stimulation, Chemical , alpha-Fetoproteins/physiologyABSTRACT
In this study, we show that N-acetylcysteine (NAC), a precursor of glutathione and an intracellular free radical scavenger, almost completely prevented hepatocyte growth factor (HGF)-suppressed growth of Sarcoma 180 and Meth A cells, and HGF-induced apoptosis, assessed by DNA fragmentation, and increase in caspase-3 activity, in Sarcoma 180 cells. The reduced form of glutathione also prevented HGF-suppressed growth of the cells as effective as NAC. Ascorbic acid partially prevented the effect of HGF, but other antioxidants such as superoxide dismutase, catalase, and vitamin E, and the free radical spin traps N-t-butyl-alpha-phenylnitrone and 3,3,5, 5-tetramethyl-1-pyrroline-1-oxide did not have protective effects. HGF caused morphological changes of the cells, many cells showing condensation and rounding, and enhanced the generation of intracellular reactive oxygen species (ROS) as judged by flow cytometric analysis using 2',7'-dichlorofluorescein diacetate. NAC completely prevented both HGF-induced morphological changes and the enhancement of ROS generation in the cells. However, NAC did not prevent the HGF-induced scattering of Madin-Darby canine kidney cells. To our knowledge, this is the first report that HGF stimulates the production of ROS, and our results suggest the involvement of oxidative stress in the mechanism by which HGF induces growth suppression of tumor cells.
Subject(s)
Cell Division/physiology , Hepatocyte Growth Factor/physiology , Sarcoma, Experimental/pathology , Acetylcysteine/pharmacology , Animals , Antioxidants/pharmacology , Apoptosis , Cell Line , Dogs , Oxidative Stress , Reactive Oxygen Species , Tumor Cells, CulturedABSTRACT
Rat fetuin, a counterpart of human alpha2-HS glycoprotein and bovine fetuin, shows strong intermolecular binding and association with other serum proteins. Therefore, to measure its concentration in rat serum, we pretreated serum samples with 1% SDS plus 5% (ca. 0.7 M) 2-mercaptoethanol at 100 degrees C for 3 min, and then subjected them to SDS-PAGE under reducing conditions followed by Western blotting. We found that the fetuin concentrations in normal rat serum determined by Western blotting were 2.5-4.5 mg/ml. These concentrations were three orders of magnitude higher than the previously reported concentrations. We also tried to measure the fetuin concentration in rat serum by means of an enzyme-linked immunosorbent assay after treatment of the samples with 0.1% sodium dodecyl sulfate (SDS) plus 10 mM 2-mercaptoethylamine at 100 degrees C for 3 min, but it gave a value of about 1/4 of that on Western blotting. Rat fetuin is expressed mainly in the liver, with a peak 2-4 weeks after birth, as determined by Northern blot analysis. The fetuin mRNA level in the liver changes almost in parallel with its serum concentration. The tibia also expresses fetuin, but much less than the liver.
Subject(s)
Aging/blood , RNA, Messenger/genetics , alpha-Fetoproteins/metabolism , Animals , Blotting, Northern , Blotting, Western , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Rats , alpha-Fetoproteins/geneticsABSTRACT
Hepatocyte growth factor, which is now known to be the same protein as scatter factor, induced oligonucleosomal fragmentation of nuclear DNA of Sarcoma 180 cells and increased the activity of caspase-3, a key component in control of the apoptotic cell death pathway to about 2.6 times that in control cells on 48 hr incubation, but did not increase the activity of caspase-1. Both HGF-induced DNA fragmentation and caspase-3 activity were completely inhibited by co-incubation with an inhibitor of caspase-3, Ac-DEVD-H. In contrast, HGF did not affect the expression of the apoptosis suppressors Bcl-2 and Bcl-x. These results indicate that HGF activates the apoptosis signaling pathway by increasing caspase-3 activity in Sarcoma 180 cells.
