Studies of the intracellular Ca²⁺ levels in human adult skin mast cells activated by the ligand for the human c-kit receptor and anti-IgE

The human c-kit receptor ligand, rhSCF, is the only cytokine known to be active on human mast cells, but its intracellular signal transduction pathway is still unknown. We compared the effect of rhSCF on intracellular Ca²⁺ levels in purified ( > 70% pure) adult skin mast cells with two other immunologic stimuli, namely, anti-IgE and substance P. Both rhSCF (1 μg/mL) and anti-IgE (3 μg/ mL) induced a rapid (<20sec) and sustained (T _{1 2} for decay > 10 min) increase in free cytosolic Ca²⁺ concentration. In contrast, substance P (5 μM) elicited a very rapid (<1 sec) and transient (T _{1 2} for decay ∼-5 sec) rise in intracellular Ca²⁺ levels. Intracellular cAMP levels were then increased by pharmacologic means to examine the role of the cyclic nucleotide in controlling the Ca²⁺ response in skin mast cells. A combination of the general phosphodiesterase inhibitor, isobutylmethylxanthine (IBMX) (200 μM) and the adenylate cyclase activator, forskolin (30 μM) was effective in inhibiting the Ca²⁺ response induced by rhSCF or anti-IgE (82 and 68% inhibition, respectively), while IBMX and forskolin alone were much less effective. The phosphodiesterase isozyme IV inhibitor, rolipram (10 μM), variably affected the increase in Ca²⁺ levels induced by anti-IgE, but it exerted a significant inhibitory activity on anti-IgE- or rhSCF-induced response in the presence of forskolin (30 μg/mL) (33 and 67%, respectively). Two different protein kinase C (PKC) activators TPA (200 nM) and bryostatin 1 (200 nM) similarly inhibited rhSCF- (22 and 32%, respectively) and anti-IgE-induced (24 and 32%) Ca²⁺ response. Finally, the kinase inhibitor genistein (30 μg/mL) was a somewhat more effective inhibitor of the rise in intracellular Ca²⁺ induced by rhSCF (100%) than that activated by anti-IgE (54%) (P < 0.05). These data indicate that rhSCF and anti-IgE may act on human mast cells through a common pathway to increase free cytosolic Ca²⁺ levels and this effect is similarly modulated by various drugs.