Fig. 6. Dopamine-mediated intracellular calcium release in D1-D2 dopamine receptor-expressing cells. (a) Sample curve illustrating change in fluorescence corresponding to changes in intracellular calcium levels upon treatment of D1-D2 cells with 1 mM dopamine (DA). The rise in fluorescence could be blocked by incubation of cells with either 10 mM SCH23390 (SCH) or 10 mM eticlopride (ETIC). 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 dopamine (EC_50(DA), 33.8 ± 4.1 nM; n = 4).

Fig. 7. D1-D2 receptor-mediated intracellular calcium release does not occur through modulation of adenylyl cyclase. (a) In D1-D2 cells treated with SKF 81297 and quinpirole (1 mM each), pretreatment with the adenylyl cyclase inhibitor SQ22536 (500 m M) slightly attenuated the calcium signal compared with control (-11.22 ± 0.03%, n = 6), whereas inhibitors for protein kinase A (H89; 10 mM) and protein kinase C [bisindolmaleimide (bis-indol; 0.5 mM)] increased the signal (31.4 ± 10.0% and 22.9 ± 6.5%, respectively; n = 6 for both). In D1-D2 cells treated with 1 mM SKF81297, SQ22536 and H89 increased the signal compared with control (64.0 ±0.06% and 80.9 ± 0.04%, respectively; n = 3 for both), whereas bisindolmaleimide had no effect (n = 3). (b) Pertussis toxin (PTX) treatment of D1-D2 cells decreased the calcium signal in response to SKF81297 and quinpirole (1 mM each) by 26.8 ± 0.03% (n = 10) and by 17.3 ± 0.05% (n = 6) in response to SKF81297 (1 mM), but it did not abolish the signal. The effects of PTX were likely nonspecific because it has been shown that PTX similarly attenuates a purinergic receptor--Gq/11-mediated calcium signal in HEK 293 cells (1). *, P < 0.05; **, P < 0.005; Student's t test compared with corresponding control.

Fig. 8. D1 agonist specificity for adenylyl cyclase activation. (a) SKF83959 does not activate adenylyl cyclase in a dose-dependent manner in D1 or D1-D2 cells. Treatment with SKF81297 or SKF83822, however, resulted in dose-dependent increases in cAMP in D1 cells. In D1-D2 cells, these drugs stimulated cAMP, although there was a biphasic effect. Treatment of D1-D2 cells with raclopride (Rac; 10 mM) abolished the inhibition of cAMP observed with higher concentrations of SKF81297 or SKF83822, indicating that the inhibitory effect was caused by agonist action on D2 receptors. Values for cAMP production were normalized to the maximal stimulation obtained from SKF81297 treatment of D1 cells. The methods were as follows: D1 or D1-D2 receptor cells were incubated for 20 min with D1 receptor agonists (10 nM-10 mM) in medium lacking FBS. Cells were then washed with PBS and lysed in 0.1 N HCl. The supernatant was collected and assayed for cAMP accumulation using a cAMP ELISA (Cayman Chemical, Ann Arbor, MI) according to the manufacturer's instructions.

Fig. 9. Activation of G_q/11-coupled D1-D2 dopamine receptor complexes increases calcium/calmodulin-dependent kinase IIa(CaMKIIa) levels in the nucleus accumbens of wild-type but not D1 or D2 dopamine receptor-deficient, mice. (a and b) Injection of adult mice with SKF83959 and quinpirole (1 mg/kg each) caused more than an 8-fold increase in the number of neurons expressing P-CaMKIIa and a 54% increase in the intensity of immunolabel per cell compared with saline-injected control animals. (c and d) In mice deficient for functional D1 dopamine receptors (D1-/-), the number of P-CaMKIIa-positive cells and the intensity of immunolabel/cell were both elevated in saline-injected controls compared with saline-injected wild-type mice. In response to SKF83959 and quinpirole (1 mg/kg each), there was no significant increase in the number of immunolabeled cells and only a slight increase in immunolabel intensity (15.8 ± 6.3%). (e and f) In mice deficient for functional D2 dopamine receptors (D2-/-), the number of P-CaMKIIa-positive cells was elevated in saline-injected controls compared with saline-injected wild-type mice. However, in response to SKF83959 and quinpirole, there was no change in the number of immunolabeled cells or in immunolabel intensity. (g and h) Quantification of data from three independent experiments. In each experiment, two animals were used for each condition, and two to four coronal slices were prepared and analyzed from each. *, P < 0.005. Methods were as described in the text. Experiments were also performed with 0.5 mg/kg and 3 mg/kg agonists, with similar results (data not shown).

1. So CH, Varghese G, Curley KJ, Kong MM, Alijaniaram M, Ji X, Nguyen T, O'Dowd BF, George SR (2005) Mol Pharmacol 68:568-578.