D1- and D2-dopamine receptors in the bovine pineal gland were previously identified and characterized. The data indicate that the density of D1-dopamine receptors far exceeded that of D2-dopamine receptors. In our previous study, the mRNA for both the D1- and D2-dopamine receptors which elucidated the status of dopamine and its possible involvement in the pineal function was identified. A selective D1-agonist enhanced N-acetyltransferase (NAT) activity and increased the melatonin level, whereas, a selective D2-agonist inhibited NAT activity and decreased the melatonin level. An attempt has been made in the present study to clarify the mechanism of dopamine in controlling melatonin production in bovine pineal. The level of intracellular cyclic 3',5'-adenosine monophosphate (cAMP) was determined after a 2-hr incubation of bovine pinealocytes with selected combinations of drugs. SKF 38393, a selective D1-agonist, enhanced intracellular level of cAMP, and its effect was blocked by SCH 23390, a D1-selective antagonist. In contrast quinpirole, a selective D2-agonist, inhibited forskolin-stimulated intracellular level of cAMP while its effect was blocked by a D2-selective antagonist, spiperone. In addition, the dopamine-dependent phosphorylation of the transcription factors, cAMP responsive element-binding protein (CREB) was investigated. Immunoblots showed that SKF 38393 enhanced CREB phosphorylation and the stimulatory effect was abolished by SCH 23390 whereas quinpirole inhibited forskolin-stimulated phosphorylated CREB production and the inhibitory effect was prevented by spiperone. Taken together with our previous data, the results indicate that activation of D1-dopamine receptor in bovine pinealocyte stimulates NAT activity and enhances melatonin level whereas activation of D2-dopamine receptor leads to an inhibitory effect and these stimulatory and inhibitory effects act, in part, via a cAMP-dependent transcription mechanism.

Previous studies have identified and characterized D1- and D2-dopamine receptors in bovine pineal glands. The data indicate that the density of D1-dopamine receptors (974 fmol/mg protein) far exceed that of D2-dopamine receptors (37 fmol/mg protein). The objective of this study was to identify the mRNAs for both D1- and D2-dopamine receptors and to elucidate the status of dopamine and its possible involvement in the pineal function, particularly on melatonin synthesis. The expression of these dopamine receptor subtypes were determined by using a reverse transcriptase-polymerase chain reaction technique with specific pairs of primers to amplify D1- and D2-dopamine receptor mRNAs. Amplification of RNAs from bovine striatum (positive control) and bovine pineal gland resulted in products of the predicted lengths of 231 bp for D1- and 333 bp for D2-dopamine receptors. The results indicate that both D1- and D2-dopamine receptor mRNAs are present in the bovine pineal gland. The role of dopamine receptors was investigated by studying the effects of selective D1- and D2-dopamine agonists and antagonists on the N-acetyltransferase (NAT) activity of cultured bovine pinealocytes. The data showed that SKF-38393, a selective D1-agonist, enhanced NAT activity, and increased melatonin level, and the stimulatory effect was blocked by SCH-23390, a D1-selective antagonist, whereas quinpirole, a selective D2-agonist, inhibited NAT basal activity and decreased the melatonin basal level. Furthermore the inhibitory effect was blocked by D2-selective antagonists, spiperone, haloperidol, and domperidone. The present results indicate that the pineal dopamine receptors have a distinct effect on pineal function. The precise mechanism whereby activation of dopamine receptors altered the NAT activity and melatonin level needs to be further delineated.