Fig. 1.

Calcium signaling through stably expressed D1 and D2 dopamine receptors is caused by G_q/11-mediated activation of PLC. (a) Changes in fluorescence corresponding to changes in intracellular calcium levels on treatment of D1–D2_HEK cells with SKF81297 (1 μM) or SKF81297 and quinpirole (1 μM each). The time of agonist addition is indicated with open arrow. AFU, arbitrary fluorescence units. (b) Dose–response curves of peak calcium levels in response to agonist (EC_{50(SKF81297+quinpirole)}, 50.8 ± 8.8 nM, n = 8; EC_50(SKF81297), 147.6 ± 46.9 nM, n = 8). (c) Treatment of cells with 10 μM SCH23390 (SCH) or raclopride (Rac) abolished agonist (1 μM)-mediated rises in calcium (n = 5). (d) Rises in calcium in response to SKF81297 and quinpirole (1 μM each) were eliminated by the IP₃ receptor blocker 2-aminoethoxydiphenyl borate (2-APB; 100 μM) as well as by depletion of intracellular calcium stores with thapsigargin (TG; 1 μM) or inhibition of PLC with U71322 (50 μM) (n = 6 for all). The inactive isomer of U71322, U73343, did not abolish the calcium signal, although the effect of SKF81297 and quinpirole was reduced by 18.9 ± 7.9% (n = 4). The G_q/11 inhibitor YM-254890 (YM; 100 nM) blocked increases in calcium in D1–D2_HEK cells in response to SKF81297 and quinpirole (n = 5). Background levels of fluorescence were qualitatively determined from individual fluorescence profiles but were generally considered to be below 3,500 AFU. +, P < 0.05; ∗∗, P < 0.0001; Student's t test compared with corresponding control.