endothelin-1 and 6-anilino-5-8-quinolinedione

The mechanism of endothelin-1 (ET-1)-induced nitric oxide (NO) production, MMP-1 production and MMP-13 production was investigated in human osteoarthritis chondrocytes. The cells were isolated from human articular cartilage obtained at surgery and were cultured in the absence or presence of ET-1 with or without inhibitors of protein kinase or LY83583 (an inhibitor of soluble guanylate cyclase and of cGMP). MMP-1, MMP-13 and NO levels were then measured by ELISA and Griess reaction, respectively. Additionally, inducible nitric oxide synthase (iNOS) and phosphorylated forms of p38 mitogen-activated protein kinase, p44/42, stress-activated protein kinase/Jun-N-terminal kinase and serine-threonine Akt kinase were determined by western blot. Results show that ET-1 greatly increased MMP-1 and MMP-13 production, iNOS expression and NO release. LY83583 decreased the production of both metalloproteases below basal levels, whereas the inhibitor of p38 kinase, SB202190, suppressed ET-1-stimulated production only. Similarly, the ET-1-induced NO production was partially suppressed by the p38 kinase inhibitor and was completely suppressed by the protein kinase A kinase inhibitor KT5720 and by LY83583, suggesting the involvement of these enzymes in relevant ET-1 signalling pathways. In human osteoarthritis chondrocytes, ET-1 controls the production of MMP-1 and MMP-13. ET-1 also induces NO release via iNOS induction. ET-1 and NO should thus become important target molecules for future therapies aimed at stopping cartilage destruction.

Topics: Aminoquinolines; Apoptosis; Carbazoles; Cartilage, Articular; Cells, Cultured; Chondrocytes; Collagenases; Cyclic AMP-Dependent Protein Kinase Type II; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Endothelin-1; Enzyme Induction; Female; Guanylate Cyclase; Humans; Imidazoles; Indoles; Male; MAP Kinase Signaling System; Matrix Metalloproteinase 1; Matrix Metalloproteinase 13; Middle Aged; Nitric Oxide; Nitric Oxide Synthase Type II; Osteoarthritis, Knee; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Processing, Post-Translational; Pyridines; Pyrroles; Signal Transduction

In order to explore the role of nitric oxide (NO) in the control of fetoplacental vascular tone in normal pregnancy we have examined the effects of NO donors on uteroplacental arteries pre-contracted with the vasoconstrictor endothelin-1 (ET-1) or serotonin (5-HT). We have furthermore examined the effects of guanylate cyclase inhibitors on the NO-induced relaxation. Segments of placental arteries (n=102) obtained from 39 placentas immediately after delivery were mounted in organ baths and superfused with Krebs-Ringer solution at 37 degrees C. The vessel segments were exposed to drugs for various intervals and the tension was recorded isometrically and registered on a polygraph. Cyclic guanosine monophosphate (cGMP) analysis was performed after extraction of vessel segments using a specific radioimmunoassay. The placental artery segments responded to ET-1 and 5-HT with a dose-dependent vasoconstriction. After pre-contraction with ET-1 (10(-7) M) or 5-HT (10(-6) M), the vessels relaxed in response to the NO donors glyceryltrinitrate (GTN) (10(-6) M) and S-nitroso-N-acetyl-penicillamine (SNAP) (10(-5) M). In the presence of the non-specific guanylate cyclase inhibitor LY 83583 (10(-6) M), the vessels responded with a small contraction. In the presence of the soluble guanylate cyclase (sGC) inhibitor 1H[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) the non-treated vessels responded with a relaxation. 1H[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one gave no obvious relaxation in pre-contracted vessels. Addition of 8-Br-cGMP, the cell-permeant analogue of cGMP, with or without pre-contraction had no effect on the vessels. Cyclic guanosine monophosphate analysis showed that GTN treatment caused an increase in cGMP after 12 min. Our results indicate that NO acts as a vasodilator in placental vessels. The cGMP-dependent mechanisms may be involved in NO-induced relaxation but cGMP-independent mechanisms appear also to be involved.

Topics: Adult; Aminoquinolines; Cyclic GMP; Dose-Response Relationship, Drug; Endothelin-1; Female; Humans; Muscle, Smooth, Vascular; Nitric Oxide; Nitric Oxide Donors; Nitroglycerin; Placenta; Placental Circulation; Pregnancy; S-Nitroso-N-Acetylpenicillamine; Serotonin; Signal Transduction; Superoxide Dismutase; Thionucleotides; Vasoconstriction; Vasoconstrictor Agents; Vasodilation

The aim of this study was to determine the effects of endothelin-1 (ET-1) on proteoglycan (PG) and collagen synthesis by rat articular chondrocytes (RAC). PG and collagen synthesis was measured by [(35)S]-sulphate and [(3)H]-glycine incorporation, respectively into monolayers of confluent RAC exposed to ET-1 (10(-11) M-10(-7) M). ET-1 stimulated PG and collagen synthesis in these cells in a concentration-dependent manner during the first 24 h of incubation. Prolonged contact of the cells with ET-1 resulted in a gradual decrease, and finally, inhibition of ET-1 effects. This inhibition is mediated by nitric oxide (NO) released in response to ET-1 since: (1) nitric oxide synthase inhibitor, nitro-L-arginine-methyl ester (L-NAME), enhanced both basal and ET-1-induced [(35)S]-sulphate and [(3)H]-glycine incorporations; (2) sodium nitroprusside (SNP), which spontaneously releases NO, inhibited both basal and ET-1-induced incorporations, and was also able to suppress the effects of L-NAME; (3) NO levels in the culture media were also correlated with the inhibition of [(35)S]-sulphate and [(3)H]-glycine incorporation; and (4) SNP also inhibited aggrecan and collagen II transcriptions, probably via cGMP. This effect was mimicked with 8-bromo-cGMP. Interestingly, the LY83583, which blocks the NO-dependent production and release of cGMP, inhibited PG-collagen synthesis but had no effect on their mRNA expressions. Thus, normal levels of cGMP appeared to be necessary for PG-collagen synthesis, whereas decreased levels are detrimental. In conclusion, NO, produced by rat AC in response to ET-1, counteracts the stimulation and finally induces inhibition of PG-collagen synthesis by ET-1 in these cells but NO-induced cGMP is only partially responsible for this inhibition.

Topics: Aminoquinolines; Animals; Cells, Cultured; Chondrocytes; Collagen; Collagen Type II; DNA; Dose-Response Relationship, Drug; Endothelin-1; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitrites; Nitroprusside; Prostaglandins; Proteoglycans; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors

The secretagogue effect of endothelins (ETs) on the rat adrenal cortex is mediated by the ETB receptor. ETB receptors are coupled with nitric oxide (NO) synthase (NOS), and NO is known to inhibit steroid-hormone secretion from adrenal cortex. We investigated whether ETB-mediated NO production interferes with the stimulatory action of ETs on rat adrenal cortex. The selective agonist of ETB receptor BQ-3020 concentration-dependently increased aldosterone secretion from dispersed zona glomerulosa (ZG) cells and corticosterone secretion from dispersed zona fasciculata-reticularis (ZF/R) cells, and the NOS inhibitor NG-nitro-L-arginine methylester (L-NAME) potentiated the effect of BQ-3020 in a concentration-dependent manner. The guanylate cyclase inhibitor Ly-83583, at a concentration suppressing guanylin- and L-arginine-induced cyclic-GMP release from dispersed adrenocortical cells, did not affect the secretory response of ZG and ZF/R cells to BQ-3020. ET-1, an agonist of both ETA and ETB receptors, stimulated the release of both aldosterone and corticosterone by in situ perfused rat adrenal gland. This effect was potentiated by L-NAME and unaffected by Ly-83583. Collectively, our findings allow us to suggest that endogenous NO exerts in vivo and in vitro a cyclic-GMP-independent buffering action on the ETB receptor-mediated adrenocortical secretagogue action of ETs.

Topics: Adrenal Cortex; Aldosterone; Aminoquinolines; Animals; Arginine; Dose-Response Relationship, Drug; Endothelin-1; Endothelins; Gastrointestinal Hormones; Male; Natriuretic Peptides; NG-Nitroarginine Methyl Ester; Nitric Oxide; Peptide Fragments; Peptides; Rats; Rats, Sprague-Dawley; Zona Glomerulosa

We have proposed that endothelin-1 (ET-1), formed through the activation of a cytochrome P450 (CYP450)-based monooxygenase reaction, is important for generation of contractile tone in the ductus arteriosus and, consequently, for closure of the vessel at birth. The present investigation was undertaken to ascertain, using an isolated ductus preparation from near-term fetal lambs, whether carbon monoxide (CO) and nitric oxide (NO) qualify as regulators of the CYP450/ET-1 system. Preparations released ET-1 at rest and its amount showed no significant reduction following removal of the endothelium. Basal release was not changed by the NO synthesis inhibitor, N(G)-nitro-L-arginine methylester (L-NAME, 100 microM), nor by agents altering cyclic GMP content (i.e. increase; ONO-1505, 1 microM) and action (i.e. decrease; LY-83583, 10 microM). These findings extend previous work showing no effect of the CO synthesis inhibitor zinc protoporphyrin IX (ZnPP, 10 microM) under the same conditions (10). Conversely, both CO (65 microM) and the NO donor, sodium nitroprusside (SNP, 10 microM), curtailed ET-1 release. ET-1 release was increased by oxygen and reduced by pyrogens (endotoxin and IL-1, both at 100 ng mL(-1)). The endotoxin effect tended to be reversed by L-NAME and ZnPP, used singly or in combination. We conclude that ET-1 is formed naturally in the ductus and that its formation may change in response to physiological (oxygen) and pathophysiological (pyrogens) stimuli. Endogenous CO and NO, however, appear to have little or no role as ET-1 regulators.

Topics: Aminoquinolines; Animals; Carbon Monoxide; Cyclic AMP; Ductus Arteriosus; Endothelin-1; Endotoxins; Imidazoles; Interleukin-1; Mesylates; Muscle Relaxation; NG-Nitroarginine Methyl Ester; Nitric Oxide; Oligopeptides; Oxygen; Piperidines; Sheep

Endothelin-1 (ET-1) is a potent mitogen for rat articular chondrocytes (AC) in short term culture (24 h). Prolonged incubation (72 h) of AC with ET-1 resulted in inhibition of [3H]thymidine incorporation. This inhibition seemed to be mediated by prostaglandins (PGs) released in response to ET-1, since indomethacin (INDO) enhanced ET-1-induced [3H]thymidine incorporation. In agreement with this hypothesis, exogenous prostaglandins (PGE2, PGF2alpha and TxB2) blocked all basal, ET-1-induced and ET-1 induced-INDO-enhanced [3H]thymidine incorporation and ET-1 stimulated PGE2 release in a time and concentration-dependent manner. INDO also blocked cGMP production and 6-anilino-5,8-quinolinedione, a relatively specific inhibitor of cGMP formation, enhanced the stimulation and suppressed the inhibition of ET-1-induced DNA synthesis. In addition, 8-bromo-cGMP, an analogue of cGMP, blocked at all time periods studied, both basal and ET-1-induced incorporations of [3H]thymidine. Thus, PGs produced in response to ET-1 counteract the ET-1-induced stimulation of [3H]thymidine incorporation into rat AC by increasing cGMP production.

Topics: Aminoquinolines; Animals; Cell Division; Cells, Cultured; Chondrocytes; DNA Replication; Endothelin-1; Enzyme Inhibitors; Guanosine Monophosphate; Rats; Rats, Wistar; Thymidine; Tritium

Prostaglandins (PG)E2 and prostacyclin (PGI2) can cause vasodilation in selective vascular beds and could act in part by inhibiting the production of the vasoconstrictor endothelin-1 (ET-1). We recently reported that these prostanoids inhibit ET-1 production/secretion from cultured endothelial cells via the generation of guanosine 3'-5'-cyclic monophosphate (cGMP). It is unclear whether this results from the stimulation of the particulate (membrane) of soluble (cytosolic) form of guanylate cyclase, and whether these effects are through an intermediate, such as nitric oxide. PGE2 and PGI2 each caused a three- to fourfold increase in both membrane and whole bovine aortic endothelial cell guanylate cyclase activity. The stimulations were significantly reversed (80-90%) by the compound LY-83583, an antagonist to cGMP generation, but were unaffected by methylene blue (MB), an inhibitor of nitric oxide-induced soluble guanylate cyclase. In contrast, the prostaglandins did not generate cGMP in cytosolic fractions. The prostaglandins inhibited ET-1 secretion from the intact cells, which was significantly prevented by LY-83583, but not by MB. Neither prostaglandin stimulated NO synthase activity, an indicator of nitric oxide generation. We conclude that PGE2 and PGI2 are likely to inhibit ET-1 secretion through the activation of the particulate guanylate cyclase, identifying a novel mechanism by which the prostanoids signal in the endothelial cell.

Topics: Aminoquinolines; Animals; Cattle; Cyclic GMP; Dinoprostone; Endothelin-1; Endothelium, Vascular; Epoprostenol; Guanylate Cyclase; Nitric Oxide Synthase