cAMP involvement in the expression of MMP-2 and MT-MMP1 metalloproteinases in human endothelial cells.

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.

Effects of thrombin and thrombin peptide activating receptor (SFLLRN) on proteoglycan synthesis and distribution in human endothelial cells.

In the present study we compared the effect of alpha-thrombin and the active receptor peptide (SFLLRN) on proteoglycan (PG) release from human endothelial cells (HUVEC) exposed for 24 h in the presence of 35S-sulfate and 3H-glucosamine. The effect of thrombin (5U/ml) and receptor peptide (100 microM) was evaluated on the distribution of PGs in the conditioned medium, cellular extract and extracellular matrix. In the conditioned medium, thrombin increased the amount of PGs. In extracellular matrix thrombin decreased radioactive PGs measured by both isotopes; no difference was observed in cell extracts. SFLLRN peptide showed a consistent increase in PG content in the medium. At variance with thrombin, in cell extracts the peptide increased the amount of PGs. No relevant differences were observed in PG content of extracellular matrix. These results indicate that thrombin and the peptide act differently on PG distribution and underline the different role of the serine protease and of the receptor binding activities of thrombin.

Molecular analysis of human beta-arrestin-1: cloning, tissue distribution, and regulation of expression. Identification of two isoforms generated by alternative splicing.

The cDNA for human beta-arrestin-1 was cloned by polymerase chain reaction (PCR) and identified based on its remarkably high amino acid identity (98.6%) with the bovine sequence. Two alternatively spliced isoforms of human beta-arrestin-1, differing only in the presence or absence of 24 base pairs/8 amino acids within the sequence, were identified and called beta-arrestin-1A and beta-arrestin-1B, respectively. Both isoforms were found in all tissues tested. Southern blot analysis revealed the existence of a single gene for beta-arrestin-1, suggesting that the two isoforms are generated by alternative mRNA splicing. The possible presence of similar isoforms was investigated for the other members of the arrestin/beta-arrestin gene family by PCR. Two isoforms of arrestin were revealed in bovine peripheral blood leukocytes. The expression of beta-arrestin-1 was studied in several human tissues and cell types. High levels of beta-arrestin-1 mRNA and immunoreactivity were found in peripheral blood leukocytes. The possible regulation of the expression of beta-arrestin-1 was also investigated. Our work documents for the first time that the expression of beta-arrestin-1 is modulated by intracellular cAMP. Using two cell types, human endothelial cells and smooth muscle cells, we found that 6-8-h treatments with the cAMP-inducing agents cholera toxin, forskolin, iloprost, and isoproterenol raised beta-arrestin-1 mRNA by 2-4-fold. Forskolin preferentially increased beta-arrestin-1A expression in smooth muscle cells, as assessed by PCR. beta-Arrestin-1 immunoreactivity was 2-3-fold higher in smooth muscle cells exposed to forskolin for 8 h, compared with untreated controls. We conclude that (i) the finding of alternatively spliced isoforms of beta-arrestin-1 and arrestin documents a novel mechanism to generate diversity within the arrestin/beta-arrestin gene family; (ii) the abundant expression of beta-arrestin-1 in peripheral blood leukocytes further supports our previous suggestion of a major role for the beta ARK/beta-arrestin system in regulating receptor-mediated immune functions; (iii) the increased expression of beta-arrestin-1 by cAMP suggests a new mechanism for the regulation of receptor-mediated responses.

IL-1 beta-induced expression of PDGF-AA isoform in rabbit articular chondrocytes is modulated by TGF-beta 1.

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.

Increased expression of urokinase mRNA in bovine aortic endothelial cells treated with propranolol.

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.

Catecholamines do not induce fibrinolytic activity increase in cultured bovine endothelial cells.

This study has investigated the catecholamine involvement in the fibrinolytic modulation of cultured bovine aortic endothelial cells (BAEC). Adrenaline and isoproterenol, at concentrations ranging from 10 to 100 microM, were unable to modulate the fibrinolytic response of these cells. beta-Adrenergic binding studies using 3H-CGP 12177 as radioligand evidenced the presence of about 23,113 +/- 2,065 sites/cell, with a KD of 1.23 +/- 0.29 nM. Isoproterenol stimulated cAMP accumulation at concentrations ranging from 1 to 100 microM, with a maximal accumulation of 30 pmol/10(6) cell. Hence, in our experimental conditions BAEC, although possessing functional beta-adrenergic receptors, were unable to increase any fibrinolytic activity in response to catecholamines.

Central and peripheral inhibition of gastrointestinal transit in rats: narcotics differ substantially by acting at either or both levels.

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.

The peripheral narcotic antagonist N-allyl levallorphan-bromide (CM 32191) selectively prevents morphine antipropulsive action and buprenorphine in-vivo binding in the rat intestine.

The assumed low ability of the quaternary narcotic antagonist N-allyl levallorphan-bromide (CM 32191) to cross the blood-brain barrier and its selectivity in relieving the peripherally-elicited antipropulsive action of morphine while preserving analgesia has been tested. To ascertain the extent of penetration of CM 32191 into the cns, its relative potency in preventing the in-vivo binding of high specific activity [3H]buprenorphine in the rat cns and small intestine was compared. Pretreatment was with CM 32191 at 16, 30 or 60 mg kg-1 s.c., 20, 60 or 120 min before buprenorphine, the concentrations of which in cerebrum and spinal cord were comparable with control values, but were consistently reduced in the intestine (longitudinal muscle with attached myenteric plexus). Pretreatment with naloxone (20 min, 0.5 or 1 mg kg-1 s.c.) lowered buprenorphine binding in intestine and cns. Neither narcotic antagonist produced significant changes in buprenorphine plasma concentrations. The peripheral selectivity of CM 32191, methyl naloxone and naloxone was examined by investigating in the same rats nociception in the hot plate (central opiate-sensitive mechanism) and the transit of a charcoal meal along the small intestine (local opiate-sensitive mechanism). Both effects were inhibited by morphine (5 mg kg-1 i.v.). Naloxone (10 min pretreatment, 0.5 or 1 mg kg-1 s.c.) did not selectively antagonize intestinal action of the morphine since the relief of charcoal transit inhibition was consistently associated with complete loss of analgesia.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of gastrointestinal transit by morphine and FK 33-824 in the rat and comparative narcotic antagonist properties of naloxone and its N-methyl quaternary analog.

Morphine and the enkephalin-like peptide FK 33-824 given i.p. to rats potently inhibit gastrointestinal transit of a charcoal test meal, the doses inhibiting transit by 50% being respectively about 10 and 1 microgram/kg. These intestinal effects are opiate-specific, locally elicited and presumably involve the same action site since they are reversed by the "peripherally selective" narcotic antagonist N-methyl naloxone with apparent pA2 values of 7.34 and 7.10 respectively against morphine and FK 33-824. The calculated pA2 value for naloxone/morphine with the same in vivo test procedure (8.83) and the relative ability of naloxone and N-methyl naloxone to prevent 3H-etorphine binding to rat brain membranes (IC50 43 and 541 nM), indicate different potency ratios between the two narcotic antagonists in the in vitro binding cell-free system (about 1:10) and in vivo (about 1:30), but the latter ratio agrees with published findings in the isolated guinea pig ileum.