In the case of EGF, a resensitizing effect was observed at the higher concentrations tested (100 and 200 ng/mL) but not at 50 ng/mL, although the latter concentration was sufficient to elicit a calcium response
(Fig. 4E) . Accordingly, PDGF also evoked a receptor resensitization in most cases in which no apparent calcium response was triggered
(Fig. 4C) . Together, these observations suggest that the growth factor–evoked resensitization is independent of any growth factor–evoked calcium response. To elucidate the intracellular signaling pathways mediating the resensitizing effect of EGF, we tested a series of selective pathway blockers. The selective inhibitor of the EGF receptor tyrosine kinase, tyrphostin AG1478,
25 completely blocked both the EGF-induced calcium response and the P2Y receptor resensitization
(Figs. 5A 5D)whereas the PDGF-induced resensitization remained unaffected by AG1478 (not shown), suggesting that the activation of the specific receptor tyrosine kinase is a necessary step in the signaling chain of the EGF effect. Concanavalin A, an inhibitor of receptor internalization,
26 27 had no effect on either the amplitude of the EGF-evoked intracellular calcium transient or the EGF-evoked resensitization
(Fig. 5D) . Monensin, a fungal sodium ionophore that has been shown to inhibit EGF receptor recycling from endosomes,
28 was observed to cause transient increases of intracellular free calcium but was without effect on the EGF-evoked P2Y receptor resensitization
(Fig. 5D) . The lack of effect of monensin and concanavalin A suggests that receptor internalization and subsequent recycling, induced by binding of EGF, were not involved in the P2Y receptor resensitization. However, an inhibitor of microtubular polymerization, nocodazole (which also induced a transient calcium response) significantly decreased the EGF-evoked P2Y receptor resensitization, with decreases in both the percentage of cells displaying resensitization and the amplitude of the ATP-evoked calcium responses (not shown). Nocodazole had no effect on the ATP-evoked calcium transients in control cells (not shown). It is concluded that the EGF-evoked resensitization is dependent on an activation of the EGF receptor tyrosine kinase, and requires an intact cytoskeleton.