OBJECTIVETo investigate the role of endothelin (ET) in the proliferation and collagen synthesis of human scar-derived fibroblasts and the modulation of its antagonists such as nitric oxide (NO), tetrandrine (Tet).

METHODSWith the cultured fibroblasts from the scarring tissue, the cell proliferation was determined by [3H]-TdR incorporation, while the collagen synthesis was evaluated by [3H]-proline incorporation.

RESULTSThe ET-1 was significantly increasing the proliferation and collagen synthesis of human scar-derived fibroblasts. The values of [3H]-TdR absorption in the 2.5 ng/ml, 25 ng/ml and 100 ng/ml of ET-1 groups were 1.8 times, 4 times and 4.9 times more than in the control group, respectively (P < 0.01), while the values of the [3H]-proline incorporation were 1.1 times, 3.1 times and 3.8 times respectively (P < 0.01). The fibroblasts, treated with 50 micrograms/ml of S-nitroso-N-acetyl penicillamine(SNAP), were no detectable effect on the basal level of DNA synthesis, but produced decreasing effect on the [3H]-TdR absorption (the rate of inhibition was 22.89%, P < 0.05). It was found that the SNAP inhibited the [3H]-proline incorporation in cultured fibroblasts, but the rate of [3H]-proline incorporation induced by ET-1 was unaltered. The Tet with 3 micrograms/ml, in which does not inhibit the basal level of DNA synthesis, was significantly decreasing the collagen synthesis and decreasing the ET-mediated DNA synthesis (the rate of inhibition was 33.21% (P < 0.01).

CONCLUSIONThese results indicate that the ET can obviously increase the proliferation and collagen synthesis of human scar-derived fibroblasts, but it can be partially antagonized by NO and Tet.

Benzylisoquinolines ; pharmacology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cicatrix ; pathology ; Collagen ; biosynthesis ; DNA ; biosynthesis ; Endothelins ; antagonists & inhibitors ; pharmacology ; Fibroblasts ; cytology ; radiation effects ; Humans ; Nitric Oxide ; metabolism ; pharmacology ; Proline ; metabolism ; S-Nitroso-N-Acetylpenicillamine ; pharmacology

To investigate the effect of nitric oxide (NO) on the proliferation of trabecular meshwork (TM) cells, primarily cultured porcine TM cells were exposed to NO donor (SNAP, -nitroso-N-acetyl-D, L-penicillamine) with and without its inhibitor (L-NAME, N (w) -Nitro-L-arginine methyl ester). The proliferation of TM cells was quantified by a rapid colorimetric assay. Acridine orange/Hoechest 33342 staining and flow cytometry with annexin-PI were done. As a result, NO inhibited the proliferation of TM cells significantly in a dose-dependent manner and this inhibitory effect was abolished by L-NAME. Fluorescent microscopy and flow cytometric analysis revealed that NO induced apoptotic cell death. The current results suggest that NO inhibit the proliferation of TM cells and apoptosis may be involved in some degree.
Acridine Orange ; Animals ; Benzimidazoles ; Cell Division/drug effects ; Cell Survival/drug effects ; Cells, Cultured ; Flow Cytometry ; Fluorescent Dyes ; Nitric Oxide/*pharmacology ; Nitric Oxide Donors/pharmacology ; S-Nitroso-N-Acetylpenicillamine/pharmacology ; Swine ; Trabecular Meshwork/*cytology/physiology

BACKGROUNDNitric oxide (NO) is a biologically active molecule which has been reported to protect the heart against ischemia and reperfusion injury in different species. This study aimed to test the hypothesis that nitric oxide may induce the expression of heat shock protein 72 (HSP72) which may protect the heart against ischemia.

METHODSRabbits were given intravenous saline or S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide donor, or Zaprinast, an inhibitor of cyclic guanosine monophosphate (GMP)-phosphodiesterase, which may increase myocardial cyclic GMP content. Twenty-four hours later, the rabbits were either sampled to measure HSP72, or induced with a 30-minute coronary occlusion followed by a 120-minute reperfusion, and then the infarct size was measured. Meanwhile, chelerythrine (CHE, an inhibitor of protein kinase C) was given intravenously 5 minutes before SNAP injection and the effect on HSP72 expression and infarct size was determined.

RESULTSTwenty-four hours after pretreatment, immunoblotting showed HSP72 expression increased in the SNAP group compared with control groups, and this was blocked by CHE. Myocardial infarct size in the SNAP group was smaller than that of the control group ((32.4 +/- 5.8)% vs (51.1 +/- 4.7)%, P < 0.05). Pretreated with CHE abolished the infarct size-limiting effect of SNAP ((46.0 +/- 5.1)%). Pretreatment with Zaprinast neither induced HSP72 expression nor reduced infarct size ((55.4 +/- 5.4)%).

CONCLUSIONNO induced HSP72 expression and a delayed protection to the heart via the activities of protein kinase C by a cyclic GMP-independent pathway.

Animals ; Benzophenanthridines ; pharmacology ; Cyclic GMP ; metabolism ; HSP72 Heat-Shock Proteins ; biosynthesis ; Hemodynamics ; Male ; Myocardial Infarction ; metabolism ; physiopathology ; prevention & control ; Myocardial Ischemia ; metabolism ; physiopathology ; prevention & control ; Nitric Oxide ; metabolism ; Nitric Oxide Donors ; pharmacology ; Phosphodiesterase Inhibitors ; pharmacology ; Protein Kinase C ; metabolism ; Purinones ; pharmacology ; Rabbits ; S-Nitroso-N-Acetylpenicillamine ; pharmacology

We evaluated DNA protection effect of heat shock protein (HSP) against cytotoxic effects of exogenous nitric oxide (NO) and reactive oxygen intermediate (ROI). Cultured human corneal fibroblasts were divided into 4 groups. Control (Group I) was not exposed to a sub-lethal heat treatment. Other 3 groups were exposed to 43 degrees C for 1 hr, then incubated at 37 degrees C during different duration (1, 6, 24 hr, Group II, III, IV, respectively). Expression pattern of HSP 70 was analyzed by Western blot. Cell viability was measured by MTT assay and the relationship between HSP 70 expression and DNA damage was examined by terminal deoxyribonucleotidyl transferase mediated dUTP-digoxigenin nick and labeling (TUNEL) stain and single cell gel electrophoresis. Expression pattern of HSP 70 was dependent on recovery times. Cell viability following heat treatment was significantly increased and the TUNEL positive cell number was decreased at 6 hr. In single cell gel electrophoresis, tail moments were increased in a dose-dependent manner by SNAP and X/XO. Following heat treatment, tail moments showed decreased significantly at 6 hr. These results suggest that induction of HSP 70 by sub-lethal heat treatment is closely related with cytoprotective effects against oxidative stresses in human corneal fibroblasts.
Cell Survival ; Cells, Cultured ; Cornea/*cytology ; DNA Damage ; Dose-Response Relationship, Drug ; Fibroblasts/cytology/drug effects/*metabolism ; Heat ; Heat-Shock Proteins 70/genetics/*metabolism ; Humans ; In Situ Nick-End Labeling ; Nitric Oxide/metabolism ; Nitric Oxide Donors/pharmacology ; *Oxidative Stress ; Reactive Oxygen Species/metabolism ; Research Support, Non-U.S. Gov't ; S-Nitroso-N-Acetylpenicillamine/pharmacology ; Xanthine/pharmacology ; Xanthine Oxidase/pharmacology

PURPOSE: To determine the effect of exogenous nitric oxide (NO) on the migration of trabecular meshwork (TM) cells and its association with expression of matrix metalloproteinases (MMPs). METHODS: Primary human TM cells treated with 1 or 10 microM S-nitroso-N-acetyl-penicillamine (SNAP) and examined for changes in adherence. TM cells were seeded onto transwell culture inserts, and changes in their migratory activity were quantified. Reverse transcription polymerase chain reaction was performed to determine the relative changes in mRNA expression of MMPs and tissue inhibitor of metalloproteinases (TIMPs). RESULTS: Treatment with SNAP did not significantly suppress TM cell adhesion or migration (p > 0.05). Treatment of TM cells with 10 microM SNAP decreased expression of MMP-2 and increased expression of membrane type MMP-1 and TIMP-2. Treatment with interleukin-1alpha triggered MMP-3 expression but did not exert significant effects on MMP-3 activation in response to SNAP. CONCLUSIONS: These data suggest that NO revealed no significant effect on the migration of TM cells because NO decreased MMP-2 and increased TIMP-2 expression. Although expression of certain MMPs and TIMPs change in response to NO donors, NO may modulate trabecular outflow by changing the cellular production of extracellular matrix without having a significant effect on the migration of TM cells.
Cell Movement/*drug effects ; Cell Survival/drug effects ; Cells, Cultured ; DNA Primers/chemistry ; Gene Expression Regulation, Enzymologic/*physiology ; Humans ; Matrix Metalloproteinases/*genetics ; Nitric Oxide Donors/*pharmacology ; RNA, Messenger/genetics ; Real-Time Polymerase Chain Reaction ; S-Nitroso-N-Acetylpenicillamine/*pharmacology ; Tissue Inhibitor of Metalloproteinase-2/*genetics ; Trabecular Meshwork/cytology/*drug effects/enzymology