Role of adrenoceptors in the regulation of dopamine/DARPP-32 signaling in neostriatal neurons.

Studies in animal models of Parkinson's disease have revealed that degeneration of noradrenaline neurons is involved in the motor deficits. Several types of adrenoceptors are highly expressed in neostriatal neurons. However, the selective actions of these receptors on striatal signaling pathways have not been characterized. In this study, we investigated the role of adrenoceptors in the regulation of dopamine/dopamine- and cAMP-regulated phosphoprotein of M(r) 32 kDa (DARPP-32) signaling by analyzing DARPP-32 phosphorylation at Thr34 [protein kinase A (PKA)-site] in mouse neostriatal slices. Activation of beta(1)-adrenoceptors induced a rapid and transient increase in DARPP-32 phosphorylation. Activation of alpha(2)-adrenoceptors also induced a rapid and transient increase in DARPP-32 phosphorylation, which subsequently decreased below basal levels. In addition, activation of alpha(2)-adrenoceptors attenuated, and blockade of alpha(2)-adrenoceptors enhanced dopamine D(1) and adenosine A(2A) receptor/DARPP-32 signaling. Chemical lesioning of noradrenergic neurons mimicked the effects of alpha(2)-adrenoceptor blockade. Under conditions of alpha(2)-adrenoceptor blockade, the dopamine D(2) receptor-induced decrease in DARPP-32 phosphorylation was attenuated. Our data demonstrate that beta(1)- and alpha(2)-adrenoceptors regulate DARPP-32 phosphorylation in neostriatal neurons. G(i) activation by alpha(2)-adrenoceptors antagonizes G(s)/PKA signaling mediated by D(1) and A(2A) receptors in striatonigral and striatopallidal neurons, respectively, and thereby enhances D(2) receptor/G(i) signaling in striatopallidal neurons. alpha(2)-Adrenoceptors may therefore be a therapeutic target for the treatment of Parkinson's disease.

Regulation of DARPP-32 Thr75 phosphorylation by neurotensin in neostriatal neurons: involvement of glutamate signalling.

Neurotensin is a neuropeptide involved in dopaminergic signalling. We have recently reported that neurotensin stimulates the phosphorylation of DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of Mr 32 kDa) at Thr34 (PKA-site) by activating dopamine D1-type receptors in neostriatal neurons. DARPP-32 is also phosphorylated by cyclin-dependent kinase 5 on Thr75, and the phosphorylated form of DARPP-32 at Thr75 inhibits protein kinase (PKA) activity. In this study, we examined the effect of neurotensin on DARPP-32 Thr75 phosphorylation using mouse neostriatal slices. Neurotensin decreased the level of phospho-Thr75 DARPP-32 at 2 min of incubation, maximally to about 50% of control at a concentration of 1 micro m. Pretreatment with a combined neurotensin receptor type 1 (NTR1)/type 2 (NTR2) antagonist, SR142948, reduced the basal level of phospho-Thr75 DARPP-32 and abolished the ability of neurotensin to decrease DARPP-32 Thr75 phosphorylation. However, neither an NTR1 antagonist, SR48692, an NTR2 antagonist, levocabastine, nor the two combined affected the basal level and the neurotensin-mediated decrease in DARPP-32 Thr75 phosphorylation. The effect of neurotensin was abolished by tetrodotoxin (TTX) or MK801 plus CNQX, but not by SCH23390 or raclopride. These results indicate that neurotensin stimulates the release of glutamate by activating a hypothesized unidentified neurotensin receptor, resulting in the dephosphorylation of DARPP-32 at Thr75 by activating NMDA and AMPA receptors expressed at medium spiny neurons. Thus, neurotensin, by removing the inhibition of PKA by phospho-Thr75 DARPP-32, potentiates its signalling via the dopamine/D1 receptor/PKA/phospho-Thr34 DARPP-32/PP-1 cascade.

Effect of methylphenidate on dopamine/DARPP signalling in adult, but not young, mice.

Methylphenidate (MPH), a dopamine uptake inhibitor, is the most commonly prescribed drug for the treatment of attention-deficit/hyperactivity disorder (ADHD) in children. We examined the effect of MPH on dopamine- and cAMP-regulated phosphoprotein, Mr 32 kDa (DARPP-32) phosphorylation at Thr34 (PKA-site) and Thr75 (Cdk5-site) using neostriatal slices from young (14-15- and 21-22-day-old) and adult (6-8-week-old) mice. MPH increased DARPP-32 Thr34 phosphorylation and decreased Thr75 phosphorylation in slices from adult mice. The effect of MPH was blocked by a dopamine D1 antagonist, SCH23390. In slices from young mice, MPH did not affect DARPP-32 phosphorylation. As with MPH, cocaine stimulated DARPP-32 Thr34 phosphorylation in slices from adult, but not from young mice. In contrast, a dopamine D1 agonist, SKF81297, regulated DARPP-32 phosphorylation comparably in slices from young and adult mice, as did methamphetamine, a dopamine releaser. The results suggest that dopamine synthesis and the dopamine transporter are functional at dopaminergic terminals in young mice. In contrast, the lack of effect of MPH in young mice is likely attributable to immature development of the machinery that regulates vesicular dopamine release.