Human Dopamine D3 Receptors Mediate MitogenActivated Protein Kinase Activation Via a Phosphatidylinositol 3Kinase and an Atypi

However, if the contrary is true, effective external control of mitogenesis, such as that being done in cancer therapy, cannot be attained by modulating steps of D2 pathway alone. It can thus be agreed upon that D3 pathway warranted investigation. Comments on the methods The researchers thus wanted to determine 1) the effect of D3 ligands on the phosphorylation state of MAPK, and2) the signal transduction factors involved in this stimulation. To do this, D3-expressing Chinese hamster ovary (CHO) cells were exposed to different substances which inhibit proposed key factors in MAPK activation. The use of CHO is probably due to the fact that it is mammalian (and thus closely similar to that of man) and it can be inserted with genes from other organisms to determine their function. Currently, however, human cell lines that can be transfected are already commercially available, and they could have been used in this study in order for its results to be more translatable to human function. The substances used were summarized in table 1. A range of concentrations could have been used, instead of only one, since substances may not be able to function if applied in amounts less than their effective concentration. Table 1. Inhibitory substances used to determine signal transduction factors in D3-mediated MAPK activation Activity Substances used D3 stimulant 100 nM dopamine (DA), at which maximal MAPK phosphorylation was observed 20 ng/ml fibroblast growth factor (FGF) Kinase inhibitors PI-3 kinase inhibitors: 1 – 10 µM Wortmannin, 30 µM LY 294002 Protein tyrosine kinase (PTK) inhibitors: geninstein, lavendustin A MAPK Kinase (MEK) inhibitor: 50 µM PD 98059 Protein kinase C (PKC) inhibitors 1 mM phorbol-12-myristate- 13-acetate (PMA) 10 µM Ro 31-8220 Go 6976 Go 6983 Gi/Go inhibitor 100 ng/ml Pertussis toxin (PTX) D3 agonists 100 nM (+)7-OH-DPAT 100 nM PD 128,907 DA antagonists Haloperidol GR218,231 S14297 After this, activation of MAPK was revealed using a monoclonal antibody specific for phosphorylated and unphosphorylated forms of MAPK. Secondary antibody with horse radish peroxidase (HRP) was also used for chemiluminescence. This is a standard immunoblotting technique still being used until now. It was commendable that immunoblots were repeated at least three times. Comments on the data Dopamine was able to phosphorylate both MAPK, with that of pp42 stronger than the other, and amount phosphorylated did not wane for at least 20 min. DA was then used in the subsequent experiments. Dopaminergic antagonists also attenuated DA-induced MAPK activation, with S14297 less than the others, further establishing the MAPK-activating abilities of D3. S14297 was also found to have partial MAPK stimulatory activity. In D3-containing CHO exposed to DA, pretreatment of PTX blocked DA- and D3 agonist-mediated MAPK phosphorylation, indicating that the pathway between D3 and MAPK implicated Gi/Go proteins. In contrast, all kinase inhibitors, except PD 98059, did not affect D3 and MAPK interaction. The researchers also did overnight exposure of CHO to PMA to supposedly decrease PKC. With or without PKC, MAPK did not seem to be activated without DA, and this was confirmed when DA was added