MECHANISMS OF THE RECEPTOR-DEPENDENT CA2- THE ROLE OF CALMODULIN AND PROTEIN-KINASES( ENTRY REGULATION IN THYMOCYTES )

It has been shown that Ca2+ influx in thymocytes and some - other cell types is activated after intracellular Ca2+ stores mobilization by re ceptor agonists or reticular Ca2+-ATPase inhibitors. However, the rela tionship between Ca2+ release from the internal pool and activation of the external Ca2+ influx is still not exactly understood. The present study has been carried out in order to elucidate the mechanisms of re ceptor-dependent Ca2+ entry in rat thymocytes. Measurements of the int racellular Ca2+ concentration were conducted by Ca2+-sensitive dye Qui n 2/AM. Pretreatment of the cells with calmodulin inhibitors R24571 (1 -3 muM), W7 (50 muM) or 48/80 (4 mug/ml) completely prevented the Ca2 entry initiated by Ca2+-mobilizing agents with different mechanisms o f action namely by receptor agonist concanavalin A (ConA) (10 mug/ml), reticular Ca2+-ATPase inhibitor 2,5-di-(tert-butyl)-1,4-benzohydroqui non (BHQ) (1 muM) or Ca2+-ionophore ionomycin (10 nM). At the same tim e the calmodulin inhibitors used did not block the Ca2+-response when were added during sustained phase. That fact excluded the direct actio n of calmodulin or of its inhibitors on plasma membrane Ca2+-channels. We have also found that staurosporine, protein kinase inhibitor, at 2 50 nM completely blocks Ca2+-response to ConA but has no effect on BHQ -activated cytosolic Ca2+-level increase. Similarly cAMP-increasing ag ents decrease the Ca2+-response of the cells to ConA by 50% but do not affect the BHQ-activated Ca2+ rise. These results indicate that tyros ine and cAMP-dependent protein kinases are not directly involved in th e Ca2+ influx regulation process in thymocytes and act on the stages p receding the Ca2+-mobilization from intracellular stores. Calmodulin p articipation in Ca2+-entry induction confirms that-exactly the cytosol ic Ca2+ level rise due to the mobilization from intracellular stores ( but not the store Ca2+-concentation decrease) activates the plasma mem brane Ca2+-channels. Calmodulin effects are most probably mediated by calmodulin-dependent protein phosphatase.