It is well documented that prolactin (PRL) release and PRL gene expression in birds are controlled by the tonic stimulation of hypothalamic vasoactive intestinal peptide (VIP). However, there is good evidence that dopamine (DA) exerts both stimulatory (at the hypothalamic level) and inhibitory (at the pituitary level) effects on PRL secretion. The interactions between VIP and DA in the regulation of PRL gene transcription are not known. This study was designed to examine the effects of a D 2 DA receptor agonist (D 2 AG; R(−)-propylnorapomorphine HCl) on basal and VIP-stimulated PRL gene transcription rate, PRL mRNA steady-state levels, PRL mRNA stability and PRL release from cultured turkey anterior pituitary cells. The D 2 AG (10 −10 M) completely inhibited the stimulatory effect of VIP (10 −7 M) upon nascent PRL mRNA as determined utilizing a nuclear run-on transcription assay. To examine further the effect of the D 2 AG on PRL mRNA post-transcriptional events, anterior pituitary cells were treated with different concentrations of D 2 AG (10 −12 -10 −4 M). Semi-quantitative RT-PCR and RIA were performed to determine the levels of PRL mRNA and PRL content in the medium respectively. The results show that D 2 AG inhibited VIP-stimulated PRL mRNA steady-state levels as well as basal and VIP-stimulated PRL release, effects which were diminished by the D 2 DA receptor antagonist, S(−)-eticlopride HCl (10 −10 M). Actinomycin D (5 µg/ml), an inhibitor of mRNA synthesis, was used to assess the effect of D 2 AG on PRL mRNA stability in response to VIP. The stimulatory effect of VIP on PRL mRNA stability was completely negated by the D 2 AG (from a half-life of 53·0±2·3 h in VIP-treated cells to 25·5±1·6 h in D 2 AG+VIP-treated cells, P≤0·05). These results support the hypothesis that VIP and DA play a major role in the regulation of PRL gene expression in avian species, at both the transcriptional and post-transcriptional levels. In addition, these findings suggest that the DAergic system inhibits PRL release and synthesis by antagonizing VIP at the pituitary level via D 2 DA receptors.

Our recent work has demonstrated that dopamine, acting through D2 dopamine receptors on pituitary cells, inhibits the stimulatory effects of vasoactive intestinal peptide (VIP) on prolactin release and prolactin gene transcription. It is hypothesised that the stimulatory and inhibitory roles of VIP and dopamine, respectively, on prolactin synthesis and release are mediated by their opposite effects on intracellular Ca2+ concentration ([Ca2+]i) in lactotrophs. The present study aimed: (i) to investigate the effect of VIP and dopamine on [Ca2+]i of cultured turkey anterior pituitary cells and (ii) to examine the role of Ca2+ signalling in mediating the regulatory effects of VIP and dopamine on prolactin mRNA levels and prolactin release. Changes in [Ca2+]i were measured spectrofluorometrically using Fura-2/AM as a fluorescent Ca2+ indicator. Semi-quantitative reverse transcription-polymerase chain reaction and radioimmunoassay were used to determine prolactin mRNA levels and prolactin release, respectively. VIP or the L-type Ca2+ channel activator, Bay K8644 (Bay) increased [Ca2+]i in a concentration- and time-dependent fashion, an effect abolished by preincubating the cells with R(-)-propylnorapomorphine HCl, a D2 dopamine receptor agonist (D2AG) or Verapamil (VR), a specific L-type Ca2+ channel blocker. Similarly, either VR or D2Ag diminished the VIP/Bay stimulatory effect on prolactin expression and release. On the other hand, pretreatment of pituitary cells with thapsigargin (TG) or neomycin (NEO), to deplete the intracellular Ca2+ stores, showed no effect on basal or VIP-stimulated prolactin mRNA levels; although VIP-induced prolactin release was partially inhibited by NEO but not TG. These results suggest that intracellular Ca2+ represents a common signal transduction pathway through which VIP and dopamine can exert antagonistic control on prolactin synthesis and release in avian lactotrophs.