Ethanol and Estradiol Modulate Alternative Splicing of Dopamine D2 Receptor Messenger RNA and Abolish the Inhibitory Action of Bromocriptine on Prolactin Release From the Pituitary Gland

Chronic drinking of alcohol has been shown to increase blood levels of prolactin (PRL), which leads to hyperprolactinemia and various reproductive dysfunctions in both humans and animals. Using Fischer-344 female rats as an animal model, we have shown that ethanol increases and potentiates estradiol’s stimulatory action on plasma levels of PRL and lactotrophic cell proliferation (De et al., 1995). Furthermore, ethanol stimulates both basal and estradiol-induced PRL secretion and lactotrophic cell proliferation in primary cultures of rat pituitary cells (De et al., 2002). However, the mechanism by which ethanol induces hyperprolactinemia is not well understood. Dopamine secreted from the hypothalamus into hypophysial portal vessels is the major inhibitor of PRL expression and secretion (Ben-Jonathan and Hnasko, 2001). Dopamine’s inhibitory action of PRL is mediated by the dopamine D2 receptor, which belongs to the pertussis toxin-sensitive Gi/Go protein-coupled receptor family (Missale et al., 1998). Recent studies have provided some indirect evidence of alcohol’s effect on dopaminergic neurotransmission (Robbins and Everitt, 1999). Dopamine D2 receptors in the brain are decreased in alcoholic patients (Blum et al., 1991; Hietala et al., 1994; Tupala et al., 2001; Volkow et al., 1996). These reports suggest that dopamine D2 receptors in the brain may be a target for ethanol’s action. However, little is known about the action of alcohol on the D2 receptor system that regulates the function of the pituitary lactotropes. The dopamine D2 receptor exists as two alternatively spliced isoforms, short (D2S) and long (D2L). The D2L isoform has an insertion of 29 amino acids in the third intercellular loop. Both isoforms of the dopamine D2 receptor are expressed in lactotropes (Missale et al., 1998). It has been reported that the D2L receptor displays a lower affinity than the D2S receptor (Dal Toso et al., 1989). In addition, each receptor isoform individually couples to a specific Gα protein because the third intercellular loop, which has a deletion in D2S, seems to play a central role in G-protein coupling (Albert, 2002; Montmayeur et al., 1993; Senogles, 1994; Wolfe and Morris, 1999). A D2S receptor-specific signaling pathway has also been reported (Senogles, 2000). Hence, each isoform of the dopamine D2 receptor may have its own specific physiologic function. The D2L receptor is predominant in the pituitary gland and in the striatum, but it is no more abundant than D2S in the substantia nigra and the hypothalamus (Guivarc’h et al., 1995). These observations suggest that a tissue-specific factor could modulate the messenger RNA (mRNA) splicing. In addition, it was observed that changes in the physiologic concentrations of sex steroid hormones (estradiol, progesterone, or testosterone) were able to modify the ratio of these two D2 receptor isoform expressions (Guivarc’h et al., 1995). Such an effect was also observed in primary cultures of pituitary cells and lactotrophic tumor-derived MMQ cells. The relative amounts of each isoform were modified by estradiol, progesterone, and testosterone. In particular, estradiol increased the D2L:D2S ratio in primary cultures of pituitary cells and the MMQ cell line (Guivarc’h et al., 1998). Furthermore, estrogen treatment alters the lactotropes’ responsiveness to dopamine and reduces dopamine’s inhibitory action on PRL secretion (Livingstone et al., 1998). This evidence suggests that alteration of the ratio of the two D2 receptor isoforms impairs the inhibitory effect of dopamine on PRL secretion. In this study, we tested whether ethanol alters the expression and splicing of the dopamine D2 receptor by determining the D2 receptor expression and alternative splicing in vivo and in vitro, using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) techniques. In addition, we examined ethanol’s effect on dopamine’s inhibitory action of PRL secretion. We show here that ethanol administration increased the D2L receptor percentage but decreased the D2S receptor percentage in vivo and in vitro and reduced the dopaminergic agent’s inhibition of PRL release.