Stoichiometry of Tyrosine Hydroxylase Phosphorylation in the Nigrostriatal and Mesolimbic Systems In Vivo

Abstract

Electrical stimulation of the medial forebrain bundle increases 32 P incorporation into striatal tyrosine hydroxylase (TH) at Ser 19 , Ser 31 , and Ser 40 . In the present studies, the effects of acute haloperidol and related drugs on sitespecific TH phosphorylation stoichiometry (PS) in the nigrostriatal and mesolimbic systems were determined by quantitative blot immunolabeling using phosphorylation statespecific antibodies. The striatum (Str), substantia nigra (SN), nucleus accumbens (NAc), and ventral tegmental area (VTA) from Sprague-Dawley rats were harvested 30 -40 min after a single injection of either vehicle, haloperidol (2 mg/kg), raclopride (2 mg/kg), clozapine (30 mg/kg), or SCH23390 (0.5 mg/kg). In vehicle-injected control rats, Ser 19 PS was 1.5-to 2.5-fold lower in Str and NAc than in SN and VTA, Ser 31 PS was two-to fourfold higher in Str and NAc than in SN and VTA, and Ser 40 PS was similar between the terminal field and cell body regions. After haloperidol, Ser 40 PS increased twofold in Str and NAc, whereas a smaller increase in SN and VTA was observed. The effects of haloperidol on Ser 19 PS were similar to those on Ser 40 in each region; however, haloperidol treatment increased Ser 31 PS at least 1.6-fold in all regions. The effects of raclopride on TH PS were comparable to those of haloperidol, whereas clozapine treatment increased TH PS at all sites in all regions. By contrast, the effects of SCH23390 on TH PS were relatively small and restricted to the NAc. The stoichiometries of site-specific TH phosphorylation in vivo are presented for the first time. The nigrostriatal and mesolimbic systems have common features of TH PS, distinguished by differences in TH PS between the terminal field and cell body regions and by dissimilar increases in TH PS in the terminal field and cell body regions after acute haloperidol.

The KCNQ Channel Opener Retigabine Inhibits the Activity of Mesencephalic Dopaminergic Systems of the Rat

“…9A). The short-term effect of haloperidol on TH phosphorylation is in agreement with previous reports (Salvatore et al, 2000;Håkansson et al, 2004). Hence, because of the clear time-dependent response on haloperidol-induced Ser40 phosphorylation, the effect of retigabine was monitored 30 min after haloperidol administration.…”

The neuropeptide galanin modulates behavioral and neurochemical signs of opiate withdrawal

“…It has previously been shown that Ser-40 in TH is specifically phosphorylated by cAMP-dependent protein kinase (PKA) (25). Thus, TH Ser-40 phosphorylation can be used as a measure of cellular cAMP levels in noradrenergic neurons.…”

“…Each blot was cut at the 77-kDa protein marker and blocked in 5% BSA. The bottom portion of each blot was incubated overnight at 4°C in 1:200 Ser-40P-TH antibody (25), and the top portion was incubated in 1:250 PYK2 antibody (Upstate Biotechnology, Lake Placid, NY). After washing with Tris-buffered saline͞0.1% Triton X-100, blots were incubated with peroxidase-labeled 1:2,000 anti-rabbit IgG (Vector Laboratories) for 1 h at room temperature.…”

Regulation of tyrosine hydroxylase by stress‐activated protein kinases

“…This may also explain the activation of TH observed when assays are performed on cell extracts, even when Ser19 seems to be the only site phosphorylated, while most groups find no effect on enzyme activity resulting from the phosphorylation of TH at Ser19 in vitro. Recently, it was reported that even low incorporation of phosphate into Ser40 was sufficient to increase the TH activity in vivo (Salvatore et al 2000). Thus, the increased TH activity found in chromaffin cells may originate from Ser40 phosphorylation rather than Ser19 or from the presence of 14-3-3 proteins.…”

“…Ser19, Ser31 and Ser40, by a variety of protein kinases, indicating that its regulation may involve multiple signalling pathways. Studies on TH from several species suggest that Ser40 is the main site involved in direct activation of TH (Sutherland et al 1993;Lew et al 1999;Salvatore et al 2000). This serine residue is phosphorylated in vitro by cAMP-dependent kinase (PKA), protein kinase C (PKC), mitogen-activated protein kinase-activated protein kinase 1 (MAPKAP-K1) and Ca 2+ /calmodulin-dependent protein kinase II (CaM-KII; Kappock and Caradonna 1996).…”