Fig 2.

Calcium signaling in HEK-MrgA1 (A–C) and HEK-MrgC11 (D–F). Cells loaded with Fura-2/AM were stimulated with each agonist, and fluorescence was recorded. Graphs represent an average plot of [Ca²⁺]_i measurements versus time (in s) in a minimum of 8 cells from representative experiments. Individual data points represent images taken at 0.8-s intervals. (A and D) U73122 (○), the active phospholipase C inhibitor blocked agonist-induced rise in [Ca²⁺]_i. However, U73343 (•), the inactive analogue, did not affect FLRFa or γ2-MSH-induced Ca²⁺ mobilization. After a 10-min pretreatment with U73122 and U73343, each agonist was added. (B and E) Extracellular [Ca²⁺] dependency of Ca²⁺ mobilization. Cells were preincubated for 2 min with 2 mM EGTA (○) or normal medium containing 1.2 mM calcium (•), and then 3 μM FLRFa or 1 μM γ2-MSH were added. (C and F) TG prevents the agonist-evoked increase of [Ca²⁺]_i in HEK-MrgA1 (C) and HEK-MrgC11 (F). In the presence of 2 mM EGTA, TG (1 μM final concentration) was added to deplete internal Ca²⁺ store.