Endothelin and Isoproterenol Counter-Regulate cAMP and Mitogen-Activated Protein Kinases

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Abstract
Mitogen-activated protein kinases (MAPK) and cAMP are important components of the intracellular signaling pathways. We studied the effects of endothelin-1 (ET-1) and isoproterenol (ISO) on the intracellular cAMP level in human pericardial smooth-muscle cells and investigated how these two ligands regulate the activity of MAPK (p42/p44 MAPK). ET-1 or ET-3 alone did not exhibit any effect on the cAMP level in these cells. In contrast, ISO at 10 μM caused a 12-fold increase in the accumulation of cAMP (370± 70 pmol/ml vs. 31 ± 5 pmol/ml). Addition of ET-1 attenuated ISO-stimulated cAMP accumulation by 50% in a dose-dependent manner, with an IC50 of 0.12 nM. ET-3 was 100-fold less potent (IC50 = 15 nM). The attenuating effect of ET-1 was completely blocked by 1 μM FR139317, suggesting that the effect is primarily mediated by the ETA receptor. In serum-deprived cells, the basal MAPK activity was low (0.07 ± 0.01 nmoles Pi/mg/min). Addition of 10 nM ET-1 stimulated MAPK 15-fold within 5 min at 37°C(1.08 ± 0.02 nmoles Pi/mg/min). ISO alone (10 μM) had no significant effect on MAPK. However, ISO markedly attenuated ET-1-stimulated MAPK activity; a ∼50% decrease in MAPK activity was observed in the presence of 10μM ISO. Similar results were obtained when forskolin was tested. The effects of ISO and forskolin on attenuating ET-1-stimulated MAPK activity could be reversed by treating cells with H89, an inhibitor of protein kinase A. These results show that ET-1 partially attenuated the accumulation of cAMP induced by ISO, and that ISO attenuated the MAPK activity induced by ET-1, possibly via activation of protein kinase A. This study suggests that counter-regulation among various ligands and cross-talk among different signaling pathways may be required to modulate biologic functions in a living cell.