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ABSTRACT

The effect of adenosine and its analogue ( − )-N₆-R-phenylisopropyladenosine (PIA) on both anterior pituitary adenylate cyclase activity and prolactin secretion was examined in the rat. Adenosine inhibited basal adenylate cyclase activity in a dose-dependent manner and also reduced the stimulation of the enzyme by vasoactive intestinal peptide (VIP). Likewise, in primary cultures of anterior pituitary cells, adenosine decreased prolactin secretion in both basal and VIP-stimulated conditions. In perifusion experiments, adenosine also inhibited prolactin release in both basal and TRH-stimulated conditions. PIA produced a biphasic pattern of response of basal adenylate cyclase activity, being inhibitory at low and stimulatory at high concentrations. In VIP-stimulated conditions, low concentrations of PIA inhibited both adenylate cyclase activity and prolactin release from primary cultures of pituitary cells, while no additive stimulatory effect was seen at high concentrations. Similarly, low concentrations of PIA reduced both basal and TRH-stimulated prolactin release from perifused pituitaries, while increasing PIA concentrations restored prolactin release. These data show that adenosine affects basal and stimulated prolactin secretion from anterior pituitary cells. Adenosine receptors seem to be coupled to the adenylate cyclase system in the anterior pituitary gland, suggesting a possible relationship between the effect of adenosine on adenylate cyclase activity and prolactin secretion.

ABSTRACT

We used the PCR amplification technique in an attempt to characterize further the dopamine D_2L receptor expressed in the prolactin-secreting pituitary MMQ cell clone, derived from the prolactinand ACTH-secreting Buffalo rat 7315a pituitary tumour. By semiquantitative PCR amplification we were unable to detect the mRNA encoding the D_2S receptor isoform, which derives from the wellknown process of alternative splicing, producing two D₂ receptor subtypes (D_2L and D_2S) 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 D_2L 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 re-suited in a linear plot, consistent with the existence of a single class of binding sites, with a K _d of 0·055±0·002 nm and a B_max 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 D_2L ligands for the therapy of dopamine-related disorders such as schizophrenia, depression, Parkinson's disease and drug addiction.

ABSTRACT

We studied the effect of adenosine on prolactin secretion by the anterior pituitary, and the transduction mechanisms whereby the purine exerts its action. Adenosine inhibited prolactin release in basal and in vasoactive intestinal peptide (VIP)- or TRH-stimulated conditions. Pertussis toxin pretreatment reduced the inhibition of VIP-stimulated prolactin secretion which was induced by adenosine, while it completely abolished the effect of the purine on TRH-evoked prolactin release. In membrane preparations of anterior pituitary cells, adenosine reduced the adenylate cyclase activity stimulated by VIP. Such an inhibition was not blocked by pertussis toxin pretreatment.

Furthermore, the purine reduced TRH-stimulated inositol phosphate production in cultured anterior pituitary cells, an effect that was reversed by pretreatment with pertussis toxin. In addition, the nucleoside did not significantly affect the TRH-induced rise in intracellular calcium.

In conclusion, our data show that adenosine inhibits prolactin secretion, acting on purinergic receptors coupled to the adenylate cyclase enzyme and phospholipase C. The effect of the nucleoside on adenylate cyclase seems to be achieved either by the involvement of an adenosine receptor coupled to the catalytic subunit of the enzyme via a pertussis toxin-sensitive G protein, or by the activation of a site directly coupled to the catalytic subunit of the adenylate cyclase (the P site). Its effect on phospholipase C seems to be mediated by a purinergic receptor coupled to the intracellular effector via a pertussis toxin-sensitive G protein.