Search

Matrix metalloproteinases (MMPs) are a multigene family of enzymes secreted by a variety of cells, including human umbilical vein endothelial cells (HUVECs). Because metalloproteinases are potentially destructive agents, their production is tightly controlled at several levels. Rather little is known about the presence and regulation of MMPs in endothelial cells. In this study, we investigated the expression and regulation of MMP-2 and membrane type-matrix metalloproteinase (MT-MMP1), a membrane metalloproteinase strictly related to MMP-2 activation. Zymographic analysis of conditioned medium (CM) of HUVECs showed the presence of gelatinolytic activity mainly at 72 and 64 and 62 kD. The 64- and 62-kD bands, respectively, represent the intermediate and the completely active forms of MMP-2. When HUVECs were treated with forskolin (FK) (100 and 25 mumol/l), there was a decrease in the appearance of the 64 to 62 kDa doublet, suggesting an inhibition of the fully activated form of MMP-2. FK raises intracellular cAMP in HUVECs. The same data were obtained using dibutyryl-cAMP. Northern analysis revealed that the expression of MMP-2 increased slightly after treatment with FK, in contrast with gelatin zymography results. Taking into consideration the mechanism of activation of MMP-2, we tested the hypothesis that this compound could modulate MT-MMP1. As expected, FK was able to decrease MT-MMP1 expression. These data correlate with experiments using membranes of FK-treated HUVECs and incubated with control CM. Zymography revealed that when CM was incubated with membranes prepared from FK-treated HUVECs, there was a decrease in the appearance of the 64-kDa band, suggesting that the expression of MT-MMP1 was negatively modified. These results correlate with the MT-MMP1 protein level, negatively modified after FK treatment.

Interleukin 1 beta (IL-1 beta) and platelet-derived growth factor (PDGF) induced proliferation in many cell types. Both peptides are released by activated macrophages and other cells in response to injury and are thought to play a crucial role in a number of pathological processes. We found that IL-1 beta stimulates proliferation of rabbit articular chondrocytes and induces synthesis and release of PDGF into their culture medium. This effect, which is time- and dose-dependent (0.05-5 ng/ml), is restricted to PDGF-AA, one of the three PDGF isoforms; IL-1 beta effect on PDGF is inhibited by actinomycin D and alpha-amanitin, suggesting a transcriptional regulation of PDGF-A chain. IL-1 beta stimulates PDGF-AA synthesis also in the presence of indomethacin, a prostaglandin synthesis inhibitor. Transforming growth factor beta 1 (TGF-beta 1), a dimeric polypeptide which displays multiple biological activities, inhibits in a dose-dependent manner (1-10 ng/ml) PDGF-AA production induced by IL-1 beta. In a binding assay, TGF-beta 1 induces 45% decrease in specific binding sites for 125I-IL-1 beta, with no change in affinity.

The roles of local intestinal and centrally mediated opiate-specific mechanisms underlying gastrointestinal transit inhibiton by five typical narcotic analgesics were assessed by the rat charcoal meal test. The doses (mg kg-1 s.c.) reducing the progression of charcoal to 50% of drug-free controls in 5 min (ID50) were approximately 1 for morphine and methadone, 0.5 and 40 for diamorphine and pethidine (all given 25 min before charcoal) and 0.001 for etorphine (10 min before charcoal). The delay in test meal travel caused by these ID50 doses was completely prevented by i.p. naloxone. Intracerebroventricular (i.c.v.) naloxone fully antagonized pethidine and etorphine but had no effect on morphine. Morphine, but either pethidine nor etorphine, was antagonized by i.p. N-methyl naloxone (a peripheral antagonist). Diamorphine and methadone were partially antagonized by i.c.v. naloxone or i.p. N-methyl naloxone.

Propranolol, a beta-adrenergic blocker agent widely used in a number of cardiovascular disorders, increases plasminogen activator (PA) activity in confluent bovine aortic endothelial cells (BAEC). This effect is time and dose dependent (10-100 microM). Hybridization studies with specific cDNA showed that propranolol was able to induce an increase in urokinase mRNA expression in a dose and time dependent manner. The propranolol effect seems to be specific for urokinase mRNA, because it does not affect a-actin mRNA expression. Cycloheximide, similarly to propranolol, also increases urokinase mRNA, indicating that the gene expression may be regulated by some rapidly turning over protein. When compounds were used in combination, a superinduction phenomenon was observed.