OBJECTIVE: Calcitonin has been suggested to have chondroprotective effects. One signaling pathway of calcitonin is via the second messenger cAMP. We undertook this study to investigate whether increased cAMP levels in chondrocytes would be chondroprotective. METHODS: Cartilage degradation was induced in bovine articular cartilage explants by 10 ng/ml oncostatin M (OSM) and 20 ng/ml tumor necrosis factor (TNF). In these cultures, cAMP levels were augmented by treatment with either forskolin (4, 16, or 64 microM) or 3-isobutyl-1-methyl xanthine (IBMX; 4, 16, or 64 microM). Cartilage degradation was assessed by 1) quantification of C-terminal crosslinking telopeptide of type II collagen fragments (CTX-II), 2) matrix metalloproteinase (MMP)-mediated aggrecan degradation by (342)FFGV- G2 assay, 3) aggrecanase-mediated degradation by (374)ARGS-G2 assay, 4) release of sulfated glycosaminoglycans (sGAG) into culture medium, 5) immunohistochemistry with a monoclonal antibody recognizing the CTX-II epitope, and 6) toluidine blue staining of proteoglycans. MMP expression and activity were assessed by gelatin zymography. RESULTS: OSM and TNF induced an 8,000% increase in CTX-II compared with control (P 80%). OSM and TNF stimulated MMP expression as visualized by zymography, and MMP expression was dose-dependently inhibited by forskolin and IBMX. The highest concentration of IBMX lowered cytokine-induced release of sGAG by 72%. CONCLUSION: Levels of cAMP in chondrocytes play a key role in controlling catabolic activity. Increased cAMP levels in chondrocytes inhibited MMP expression and activity and consequently strongly inhibited cartilage degradation. Specific cAMP modulators in chondrocytes may be potential treatments for cartilage degenerative diseases.
We studied the in vivo mechanism of beta-adrenergic receptor (beta-AR) hyporesponsiveness induced by intratracheal instillation of interleukin-1beta (IL-1beta, 500 U) in Brown-Norway rats. Tracheal and bronchial smooth muscle responses were measured under isometric conditions ex vivo. Contractile responses to electrical field stimulation and to carbachol were not altered, but maximal relaxation induced by isoproterenol (10(-6)-10(-5) M) was significantly reduced 24 h after IL-1beta treatment in tracheal tissues and to a lesser extent, in the main bronchi. Radioligand binding using [125I]iodocyanopindolol revealed a 32+/-7% reduction in beta-ARs in lung tissues from IL-1beta-treated rats, without any significant changes in beta2-AR mRNA level measured by Northern blot analysis. Autoradiographic studies also showed significant reduction in beta2-AR in the airways. Isoproterenol-stimulated cyclic AMP accumulation was reduced by IL-1beta at 24 h in trachea and lung tissues. Pertussis toxin reversed this hyporesponsiveness to isoproterenol but not to forskolin in lung tissues. Western blot analysis revealed an IL-1beta-induced increase in Gi(alpha) protein expression. Thus, IL-1beta induces an attenuation of beta-AR-induced airway relaxation through mechanisms involving a reduction in beta-ARs, an increase in Gi(alpha) subunit, and a defect in adenylyl cyclase activity.
Sphingolipids, especially as elements of the sphingomyelin signal transduction cycle, are thought to play a significant role as second messengers and modulators of events in heart muscle cells. A possible modulatory role of sphingosine in signal transduction in the beta-adrenergic pathway in the heart was examined. Neonatal rat cardiomyocytes were incubated with sphingosine and/or other agents after which cAMP levels and contraction rates were measured. Heart rate in anaesthetized rats was also measured before and after sphingosine injection in the jugular vein. Sphingosine caused a decrease in basal cAMP levels and diminished isoproterenol-induced increase in cAMP levels. These changes were dose- and time-dependent and showed a significant negative effect on signal transmission in the beta-adrenergic pathway in cardiomyocytes. Increase in cAMP intracellular levels by forskolin, which activates adenylcyclase, was not inhibited by sphingosine. A phosphodiesterase inhibitor was used in all experiments in which cAMP was measured excluding effects on cAMP breakdown. It was also demonstrated that sphingosine caused reduction in the beating rate of cultured cardiomyocytes and a dose-dependent reduction in heart rate of anaesthetized rats. The sphingosine-induced inhibition of bradycardic response of anaesthetized rats reached a maximum about 5-10 min after the onset of sphingosine administration and returned to normal within 60 min. Sphingosine may modulate the signal transmission of the beta-adrenoceptor pathway upstream of adenylcyclase in rat cardiomyocytes. This may contribute to the sphingosine-induced decrease in heart rate of rats in vivo.