Article Figures & Data
Figures
- Fig. 1.
Effect of dopamine on the level of phospho-DARPP-32 in neostriatum. Slices were incubated with dopamine (100 μm) in the presence of the dopamine uptake inhibitor nomifensine (10 μm) for the indicated times.A, Phospho-DARPP-32 was detected at a molecular mass of ∼32 kDa using mAb-23 against thr34-phospho-DARPP-32. Note that the phospho-DARPP-32 mAb also detected a cross-reactive protein band at a molecular mass of ∼75 kDa, the levels of which were not affected by dopamine. B, Total DARPP-32 was detected in the same membrane as shown in A using mAb C24–5a against DARPP-32. C, The amount of phospho-DARPP-32 was quantitated by densitometry, and the data were normalized to values obtained with untreated tissue. Data represent mean ± SEM for the number of experiments shown in parentheses. * p < 0.05, ** p < 0.01 compared with 0 min; †p < 0.05 compared with 2 min.
- Fig. 2.
Effects of D1 agonist (SKF82526) and D2 agonist (quinpirole) on the basal level of phospho-DARPP-32 in neostriatum. Slices were incubated with (A) SKF82526 (1 μm) or (B) quinpirole (1 μm) for the indicated times. The amount of phospho-DARPP-32 was quantitated by densitometry, and the data were normalized to values obtained with untreated tissue. Data represent mean ± SEM for 4–12 experiments. * p < 0.05, ** p < 0.01 compared with 0 min; †p < 0.05 compared with 5 min.
- Fig. 3.
Opposing effects of D1 agonist (SKF82526) and D2 agonist (quinpirole) on the level of phospho-DARPP-32 in neostriatum. Slices were preincubated with the indicated concentrations of quinpirole (1 nm to 1 μm) for 5 min and then incubated with quinpirole plus SKF82526 (1 μm) for an additional 5 min. The amount of phospho-DARPP-32 was quantitated by densitometry, and the data were normalized to values obtained with SKF82526 alone. Data represent mean ± SEM for four to five experiments. * p < 0.01 compared with SKF82526 alone.
- Fig. 4.
Effect of the antipsychotic drug raclopride on the level of phospho-DARPP-32 in (A) neostriatum and (B) nucleus accumbens. Slices were incubated for a total of 20 min. Raclopride (1 μm) was added at 0 min, quinpirole (100 nm) at 10 min, and SKF82526 (1 μm) at 15 min of incubation. The amount of phospho-DARPP-32 was quantitated by densitometry, and the data were normalized to values obtained with SKF82526 alone. A, Data represent mean ± SEM for six to nine experiments. *p < 0.01 compared with no addition; †p < 0.01 compared with SKF82526 alone; §p < 0.05 compared with quinpirole alone; ¶p < 0.01 compared with SKF82526 plus quinpirole.B, Data represent mean ± SEM for five to seven experiments. * p < 0.05 compared with no addition; † p < 0.01 compared with SKF82526 alone; §p < 0.01 compared with quinpirole alone; ¶p < 0.02 compared with SKF82526 plus quinpirole.
- Fig. 8.
Postulated pathways by which dopamine may regulate DARPP-32 phosphorylation. Activation of D1 receptors increases cAMP, leading to the activation of PKA and the phosphorylation of DARPP-32 on thr34, converting it into a potent inhibitor of protein phosphatase-1. Activation of D2 receptors decreases DARPP-32 phosphorylation by two mechanisms (which might occur in the same or in different groups of neurons): one involves an inhibition of adenylyl cyclase, a decrease in cAMP, a decrease in activity of PKA and a decreased phosphorylation of DARPP-32; the other involves an increase in intracellular Ca2+, an activation of calcineurin, and an increased dephosphorylation of thr34-phospho-DARPP-32. This scheme is supported by evidence showing that NMDA receptor activation (Halpain et al., 1990) and thapsigargin (present study), both of which raise intracellular Ca2+, cause the dephosphorylation of thr34-phospho-DARPP-32.
- Fig. 5.
Effect of D2 agonist (quinpirole) on stimulated levels of phospho-DARPP-32 in neostriatum. Slices were preincubated with quinpirole (1 μm) for 5 min and then incubated with quinpirole plus either (A) SKF82526 (1 μm), (B) forskolin (10 μm), or (C) 8-bromo-cAMP (1 mm) for an additional 5 min. The amount of phospho-DARPP-32 was quantitated by densitometry, and the data were normalized to values obtained with SKF82526, forskolin, or 8-bromo-cAMP alone. Data represent mean ± SEM for three to four experiments. *p < 0.01 compared with SKF82526, forskolin, or 8-bromo-cAMP alone.
- Fig. 6.
Effect of Ca2+-free/EGTA medium on the level of phospho-DARPP-32 in neostriatum. Slices were incubated in control or Ca2+-free/EGTA medium for 20 min.A, Phospho-DARPP-32 was detected using a phosphorylation state-specific mAb (mAb-23). B, The amount of phospho-DARPP-32 was quantitated by densitometry, and the data were normalized to values obtained with control. Data represent mean ± SEM for four experiments. * p < 0.01 compared with control.
- Fig. 7.
Absence of effect of D2 agonist (quinpirole) on the level of phospho-DARPP-32 in Ca2+-free/EGTA medium. Neostriatal slices were incubated in Ca2+-free/EGTA medium for a total of 20 min. Buffer was replaced by Ca2+-free/EGTA medium at 0 min, quinpirole (1 μm) was added at 10 min, and (A) SKF82526 (1 mm), (B) forskolin (10 μm), or (C) 8-bromo-cAMP (1 mm) was added at 15 min of incubation. The amount of phospho-DARPP-32 was quantitated by densitometry, and the data were normalized to values obtained with SKF82526, forskolin, or 8-bromo-cAMP alone. Data represent mean ± SEM for three to four experiments.
Tables
Cyclosporin A (−) Cyclosporin A (+) A. D1 agonist SKF82526 0 min 1.00 16.0 ± 1.6* 5 min 7.14 ± 0.97* 31.6 ± 2.81-160 30 min 3.07 ± 0.66*,1-165 28.0 ± 3.11-160 B. D2 agonist quinpirole 0 min 1.00 13.50 ± 1.74* 2 min 0.525 ± 0.051* 5.98 ± 1.281-160 10 min 0.580 ± 0.058* 5.60 ± 1.051-160 C. D1 + D2 agonist Control 1.00 11.0 ± 2.5* SKF82526 10.61 ± 2.28* 21.1 ± 2.31-160 SKF82526 + quinpirole 4.78 ± 1.20*,1-165 20.3 ± 2.51-160 Slices were preincubated in the absence or presence of cyclosporin A (CyA) (5 μm) for 60 min, followed by the addition of SKF82526 and/or quinpirole. The amount of phospho-DARPP-32 was quantitated by densitometry, and the data were normalized to values obtained with control. A, Slices were incubated with SKF82526 (1 μm) for the indicated times. Data represent means ± SEM for five to nine experiments.
↵* p < 0.01 compared with 0 min/CyA (−);
p < 0.01 compared with 0 min/CyA (−);
p < 0.01 compared with control/CyA (−);
↵F1-160 p < 0.01 compared with 0 min/CyA (+);
p < 0.01 compared with 0 min/CyA (+). C, Slices were preincubated with quinpirole (1 μm) for 5 min and then incubated with quinpirole plus SKF82526 (1 μm) for an additional 5 min. Data represent means ± SEM for six to nine experiments.
p < 0.01 compared with control/CyA (+);
↵F1-165 p < 0.01 compared with 5 min/CyA (−). B, Slices were incubated with quinpirole (1 μm) for the indicated times. Data represent means ± SEM for five to seven experiments.
p < 0.05 compared with SKF82526/CyA (−).
- Table 2.
Effect of D2 agonist on the level of phospho-DARPP-32 is not mediated through the NMDA receptor
MK801 (−) MK801 (+) Control 0.103 ± 0.021* 0.247 ± 0.0442-160 SKF82526 1.000 1.115 ± 0.078 SKF82526 + NMDA 0.042 ± 0.009* 1.042 ± 0.085 SKF82526 + quinpirole 0.598 ± 0.137* 0.616 ± 0.0842-165 Neostriatal slices were incubated for a total of 20 min in the absence or presence of the NMDA receptor antagonist MK801 (100 μm). MK801 was added at 0 min, NMDA (100 μm) or quinpirole (1 μm) at 10 min, and SKF82526 (1 μm) at 15 min of incubation. The amount of phospho-DARPP-32 was quantitated by densitometry, and the data were normalized to values obtained with SKF82526 alone. Data represent means ± SEM for four to seven experiments.
↵* p< 0.01 compared with SKF82526/MK801 (−);
↵F2-160 p < 0.05 compared with control/MK801 (−);
↵F2-165 p < 0.01 compared with SKF82526/MK801 (+).
- Table 3.
Effect of D2 agonist on the level of phospho-DARPP-32 is not reduced by tetrodotoxin (TTX)
TTX (−) TTX (+) Control 0.686 ± 0.109 1.0003-150 SKF82526 3.282 ± 0.5753-160 5.492 ± 0.7803-165,3-168 SKF82526 + quinpirole 1.716 ± 0.4093-150,3-165 1.968 ± 0.5593-168,3-150 Neostriatal slices were incubated for a total of 20 min in the absence or presence of TTX (1 μm). TTX was added at 0 min, quinpirole (1 μm) at 10 min, and SKF82526 (1 μm) at 15 min of incubation. The amount of phospho-DARPP-32 was quantitated by densitometry, and the data were normalized to values obtained with TTX alone. Data represent means ± SEM for six to seven experiments.
↵F3-160 p < 0.01,
↵F3-150 p < 0.05 compared with control/TTX (−);
↵F3-165 p < 0.05 compared with SKF82526/TTX (−);
↵F3-168 p < 0.01 compared with control/TTX (+);
* p < 0.01 compared with SKF82526/TTX (+).
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