Cyclic adenosine monophosphate-stimulating agents induce ecto-5?-nucleotidase activity and inhibit DNA synthesis in rat cultured mesangial cells (original) (raw)
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Adenosine stimulates 5'-nucleotidase activity in rat mesangial cells via A2 receptors
Febs Letters, 1993
Because A, adenosine receptor activation stimulates adenylate cyclase and cyclic AMP induces 5'-nucleotidase expresslon m rat mesanglal cells. we examined the effect of adenosine and its analogs on 5'-nucleotidase activity m these cells. Az adenosine receptors were characterized usmg ['HIS'-N-ethylcarboxamidoadenosine (NECA) as a tracer. There was a single group of receptor sites with a k;, value of 0.53 PM and a number of sites of 1,317 fmol/mg. ['HINECA binding was inhlblted preferentially by A1 adenosme analogs and antagomsts Slmllarlq, the order of potency for CAMP stimulation was m favour of A2 adenosine analogs. Rat mesangial cells expressed surface 5'-nucleotidase actlvlty Exposure of cells for 48 h to adenosine analogs showed that at low concentrations A, analogs stimulated 5'-nucleotidase actlvlty These results mdlcate that adenosmr upregulates activity of 5'-nucleotidase, the enzyme responsible for Its local formatlon. via AZ receptor stlmulatlon and increase m CAMP productlon.
Journal of Cellular Physiology, 1989
We have studied the effect of increased intracellular levels of cyclic AMP on the growth response to platelet-derived growth factor (PDGF) of human foreskin fibroblasts in culture. It was found that forskolin, a potent stimulator of adenylate cyclase activity, inhibits the stimulatory effect of PDGF on 3H-thymidine incorporation with a dose dependence similar to that observed with regard to cyclic AMP formation. A time-course study indicated that forskolin has no effect on ongoing DNA synthesis but affects events in the prereplicative phase. The cell-cycle block induced by forskolin was found to be reversible; after removal of the drug, DNA synthesis was initiated after a lag period, similar to that of the prereplicative phase of control cells. Forskolin had no effect on PDGF binding, receptor autophosphorylation, or c-fos mRNA expression. However, a reduction in PDGF-induced c-myc mRNA expression was observed in cultures given forskolin. Forskolin was also found to have a marked stimulatory effect on the expression of interferon-p2 mRNA expression. However, we were unable to demonstrate that the growth-inhibitory effect of forskolin i s mediated by interferon+. In conclusion, an increase in CAMP levels leads to a reversible inhibition of PDGF-induced DNA synthesis in human fibroblasts, which may be related to an inhibition of c-myc mRNA expression. Cells were cultured in Eagle's minimum essential medium, supplemented with 10% newborn calf serum (GIBCO) and antibiotics (100 U penicillin and 50 pg streptomycin per ml) in a humidified atmosphere containing 5% COz. For experiments, subconfluent cell cultures were serum-starved for 2-4 days in MCDB 104 medium containing 0.5 mM Ca2+.
Journal of Clinical Investigation, 1995
protein kinase (MAPK) * isozymes * cAMP-phosphodies- terase We studied interactions between the mitogen-activated protein kinase (MAPK) signalling pathway and cAMP-protein kinase (PKA) signaling pathway in regulation of mitogenesis of mesangial cells (MC) determined by [3H]thymidine incorporation, with or without added EGF. Forskolin or dibutyryl cAMP strongly (by 60-70%) inhibited [3H]thymidine incorporation into MC. Cilostamide, lixazinone or cilostazol selective inhibitors of cAMP-phosphodiesterase (PDE) isozyme PDE-L, inhibited mitogenesis to similar extent as forskolin and DBcAMP and activated in situ PKA, but without detectable increase in cAMP levels. Cilostamide and cilostazol were more than three times more effective at inhibiting mesangial mitogenesis than rolipram and denbufylline, inhibitors of isozyme PDE-IV, even though PDE-IV was two times more abundant in MC than was PDE-M. On the other hand, when incubated with forskolin, rolipramenhanced cAMP accumulation was far greater (10-10Ox) than with cilostamide. EGF increased MAPK activity (+300%); PDE isozyme inhibitors which suppressed mitogenesis also inhibited MAPK. PDE isozyme inhibitors also suppressed PDGF-stimulated MC proliferation. We conclude that cAMP inhibits the mitogen-dependent MAPKsignaling pathway probably by decreasing the activity of Raf-1 due to PKA-catalyzed phosphorylation. Further, we surmise that minor increase in the cAMP pool metabolized by PDE-l is intimately related to regulation of mesangial proliferation. Thus, PDE isozyme inhibitors have the potential to suppress MC proliferation by a focused effect upon signaling pathways. (J. Clin. Invest. 1995. 96:401-410.)
Journal of cellular …, 1989
We have studied the effect of increased intracellular levels of cyclic AMP on the growth response to platelet-derived growth factor (PDGF) of human foreskin fibroblasts in culture. It was found that forskolin, a potent stimulator of adenylate cyclase activity, inhibits the stimulatory effect of PDGF on 3H-thymidine incorporation with a dose dependence similar to that observed with regard to cyclic AMP formation. A time-course study indicated that forskolin has no effect on ongoing DNA synthesis but affects events in the prereplicative phase. The cell-cycle block induced by forskolin was found to be reversible; after removal of the drug, DNA synthesis was initiated after a lag period, similar to that of the prereplicative phase of control cells. Forskolin had no effect on PDGF binding, receptor autophosphorylation, or c-fos mRNA expression. However, a reduction in PDGF-induced c-myc mRNA expression was observed in cultures given forskolin. Forskolin was also found to have a marked stimulatory effect on the expression of interferon-p2 mRNA expression. However, we were unable to demonstrate that the growth-inhibitory effect of forskolin i s mediated by interferon+. In conclusion, an increase in CAMP levels leads to a reversible inhibition of PDGF-induced DNA synthesis in human fibroblasts, which may be related to an inhibition of c-myc mRNA expression.
1974
To observe the possible role of cAMP on the DNA synthesis during specialization-division of myelogenous precursor cells, the authors observed the DNA and RNA synthesis of the cells by in vitro autoradiography. And it is concluded that cAMP or its dibutyryl derivative added to the media penetrated into myelogenous precursor cells and metamyelocytes of mice and enhanced the DNA synthetic capacity of them. cAMP hardly enhanced RNA synthesis. Discussion is made on relation between enhancement of DNA synthesis of metamyelocytes and their possible rejuvenation.</p
Journal of Cellular Physiology, 1989
Analogs of cyclic adenosine monophosphate (CAMP) (N6benzoyl CAMP and N'monobutyryl CAMP) as well as agents that increased the intracellular level of CAMP (glucagon and isobutylmethylxanthine) inhibited the ECF-stimulated DNA replication of adult rat hepatocytes in primary culture independently of cell density. This inhibition was strongly potentiated by the glucocorticoid dexamethasone. The effect of LAMP (and dexamethasone) was not due to toxicity, because the inhibition was reversible and the cell ultra~tructure preserved. cAMP acted by decreasing the rate of transition from GIto S-phase, the duration of GI-and S-phase of the hepatocyte cell cycle being unaffected. DNA replication started in the extranucleolar compartment of the nucleus and ended in the nucleolar compartment as described earlier for cells grown in the absence of cAMP (O.K. Vintermyr and S.O. Dmkeland, J. Cell. Physiol., 1987, 132:12-21). The action of cAMP was very rapid: significant inhibition of the transition was noted 2 hr after the addition of glucagonABMX and half-maximal inhibition after 4 hours. The determination of extranucleolarly labelled nuclei in cells pulse-labelled with ['Hlthymidine allowed precise analysis of rapid change, in the probability of transition from G Ito S-phase. The extranucleolar labelling index could also be determined in cells continuously exposed to [3H]thymidine.
Effect of dipyridamole on glomerular mesangial cell ecto-5′-nucleotidase expression
Experientia, 1994
Although dipyridamole has been extensively studied as an anti-aggregating agent, its mechanism of action has not been elucidated. Cultured mesangial cells were treated with dipyridamole 1-100 gM from 6-72 h. Ecto-5'-nucleotidase activity approximately doubled (from 115 +__ 11 to 226 • 14nmol/min/mg) after treatment with 100 gM dipyridamole for 72 h. This effect was concentration-and time-dependent Cycloheximide, an inhibitor of protein synthesis, did not alter basal 5'-nucleotidase activity. However, it prevented stimulation by 5 gM dipyridamole. Adenosine availability at the receptor sites was increased by dipyridamole and S-(p-nitrobenzyl)-6-thioinosine (NBTI), which inhibit adenosine uptake into the cell. Addition of dipyridamole or NBTI to the adenosine-treated mesagial cells produced an additive increase in ecto-5'-nucleotidase activity. Dipyridamole, through its effect on extracellular adenosine and ecto-5'-nucleotidase, may have an influence upon regulation of the glomerular microcirculation.
The Biochemical journal, 1990
1. The rate of [3H]thymidine incorporation into DNA was measured in phytohaemagglutinin-stimulated lymph-node lymphocytes of the rat. 2. Addition of nucleobases or nucleosides to culture medium that already contained 0.2 mM-glutamine had a small stimulatory effect on incorporation. At lower concentrations of glutamine, adenosine (even at 1 microM) caused a marked increase in the rate of incorporation. 3. In the absence of added glutamine, addition of nucleosides or nucleobases markedly increased the rate of incorporation: nucleosides were more effective than nucleobases; and the rate of proliferation in the presence of 10 microM-adenosine plus 10 microM-uridine was similar to that in the presence of optimal concentrations of glutamine. 4. The rate of incorporation was dramatically decreased by an inhibitor of the pathway of pyrimidine nucleotide synthesis de novo. Addition of the pyrimidine nucleosides completely overcame the inhibition; addition of the pyrimidine nucleobases was mu...