Absence of D2S dopamine receptor in the prolactin-secreting MMQ pituitary clone: characterization of a wild D2L receptor coupled to native transduction mechanisms.
We used the PCR amplification technique in an attempt to characterize further the dopamine D2L receptor expressed in the prolactin-secreting pituitary MMQ cell clone, derived from the prolactin- and ACTH-secreting Buffalo rat 7315 alpha pituitary tumour. By semiquantitative PCR amplification we were unable to detect the mRNA encoding the D2S receptor isoform, which derives from the well-known process of alternative splicing, producing two D2 receptor subtypes (D2L and D2S) in such tissues as the anterior pituitary and the corpus striatum. Although the pharmacology of the D2 receptor has been established in many studies on both native receptors and transfected receptor isoforms, because of the lack of tissues naturally expressing only one receptor isoform, MMQ cells represent the first example of cells uniquely or prevalently expressing only the D2L receptor, conceivably coupled to its native transduction mechanisms. These considerations prompted us to evaluate the pharmacology and the second messenger systems known to be modulated by dopamine. Scatchard analysis of [3H]spiperone binding resulted in a linear plot, consistent with the existence of a single class of binding sites, with a Kd of 0.055 +/- 0.002 nM and a Bmax of 27 +/- 3.5 fmol/mg protein. Competition experiments confirmed the GTP-dependence and the order of potency for agonist and antagonist ligands consistent with binding to a D2 receptor. The inhibitory effects of dopamine on adenylyl cyclase activity, inositol phosphate production and intracellular free calcium concentrations, the latter presumably via the opening of K+ channels, and prolactin secretion, as well as the reversal of the effect by the D2-selective antagonist (-)sulpiride and pretreatment with pertussis toxin, are consistent with the known biological actions of dopamine at D2 receptors. Based on our observations, the MMQ cell line can be considered a useful tool for investigating ligand-receptor interactions to develop new selective dopaminergic D2L ligands for the therapy of dopamine-related disorders such as schizophrenia, depression, Parkinson's disease and drug addiction.
Ventra
C
C
Dipartimento di Neuroscienze e della Comunicazione Interumana, II Facoltà di Medicina e Chirurgia, Università degli Studi di Napoli, Federico II, Napoli, Italy.
Meucci
O
O
Grimaldi
M
M
Scorziello
A
A
Porcellini
A
A
Schettini
G
G
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000502:physiology; D002999:Clone Cells / Q000737:chemistry; D003001:Cloning, Molecular; D018491:Dopamine Agonists / Q000494:pharmacology; D018492:Dopamine Antagonists / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000502:physiology; D006462:Hemolytic Plaque Technique; D007295:Inositol Phosphates / Q000502:physiology; D015640:Ion Channel Gating; D008564:Membrane Potentials / Q000187:drug effects; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000172:deficiency* / Q000235:genetics; D010911:Pituitary Neoplasms / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D016133:Polymerase Chain Reaction; D015221:Potassium Channels / Q000502:physiology; D011388:Prolactin / Q000378:metabolism*; D011869:Radioligand Assay; D051381:Rats; D011915:Rats, Inbred BUF; D017448:Receptors, Dopamine D2 / Q000145:classification / Q000172:deficiency* / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D015290:Second Messenger Systems; D015398:Signal Transduction / Q000502:physiology*; D014407:Tumor Cells, Cultured
D018491:Dopamine Agonists; D018492:Dopamine Antagonists; D007295:Inositol Phosphates; D009363:Neoplasm Proteins; D015221:Potassium Channels; D017448:Receptors, Dopamine D2; D006160:Guanosine Triphosphate; D011388:Prolactin; D000262:Adenylyl Cyclases; D002118:Calcium