The bovine mammary gland expresses multiple functional isoforms of serotonin receptors

Abstract

Recent studies in dairy cows have demonstrated that serotonergic ligands affect milk yield and composition. Correspondingly, serotonin (5-HT) has been demonstrated to be an important local regulator of lactational homeostasis and involution in mouse and human mammary cells. We determined the mRNA expression of bovine 5-HT receptor (HTR) subtypes in bovine mammary tissue (BMT) and used pharmacological agents to evaluate functional activities of 5-HT receptors. The mRNAs for five receptor isoforms (HTR1B, 2A, 2B, 4, and 7) were identified by conventional real-time (RT)-PCR, RT quantitative PCR, and in situ hybridization in BMT. In addition to luminal mammary epithelial cell expression, HTR4 was expressed in myoepithelium, and HTR1B, 2A, and 2B were expressed in small mammary blood vessels. Serotonin suppressed milk protein mRNA expression (alpha-lactalbumin and beta-casein mRNA) in lactogen-treated primary bovine mammary epithelial cell (BMEC) cultures. To probe the functional activities of individual receptors, caspase-3 activity and expression of alpha-lactalbumin and beta-casein were measured. Both SB22489 (1B antagonist) and ritanserin (2A antagonist) increased caspase-3 activity. Expression of alpha-lactalbumin and beta-casein mRNA levels in BMEC were stimulated by low concentrations of SB224289, ritanserin, or pimozide. These results demonstrate that there are multiple 5-HT receptor isoforms in the bovine mammary gland, and point to profound differences between serotonergic systems of the bovine mammary gland and the human and mouse mammary glands. Whereas human and mouse mammary epithelial cells express predominately the protein for the 5-HT(7) receptor, cow mammary epithelium expresses multiple receptors that have overlapping, but not identical, functional activities.

Figure 1

5-HT receptor expression in bovine hypothalamic tissue, lactating mammary tissue, and primary bovine mammary epithelial cells. The mRNA expression of the HTR was detected by RT-PCR and electrophoresed through a 2% agarose gel and stained with EtBR (primers listed in Table 1). Lane (A) Holstein hypothalamic tissue, Lane (B) lactating Holstein mammary gland tissue from a first trimester pregnant animal, and Lane (C) primary bovine mammary epithelial cells isolated from three multiparous, non-pregnant (∼30 days dry), Holstein cows treated for 7 days with serum-free proliferation media (IGF-I, EGF and INS).

Figure 2

Morphological distribution of HTR in lactating bovine mammary tissue. In situ hybridizations of DIG-labeled anti-sense cRNA probes for HTR1B, HTR2A, HTR2B, HTR4, and HTR7, as well as representative anti-sense cRNA DIG-labeled probe in the mammary gland. Anti-sense cRNA-labeled probe for HTR7 in the bovine hypothalamus is also provided. A representative section that has been stained for HTR7 and has been counterstained for smooth muscle α-actin and GS-I is shown. Black arrows (→) indicate epithelial cells. Arrowheads (^) represent myoepithelial cells, and asteriks (*) correspond to vascular endothelium.

Figure 3

Effects of selective 5-HT receptor antagonists on α-lactalbumin mRNA expression in primary bovine mammary epithelial cells. Mean with s.e.m. of (A) α-lactalbumin mRNA expression in lactogenic BMEC cultures treated with SB224289, a 5-HT-1B receptor antagonist; (B) α-lactalbumin mRNA expression in lactogenic BMEC cultures treated with ritanserin, a 5-HT-2A receptor antagonist; and (C) α-lactalbumin mRNA expression in lactogenic BMEC cultures treated with pimozide, a 5-HT-7 receptor antagonist. Columns with * indicate statistical significance of *P<0·05, **P<0·001, ***P<0·0001 compared with lactogenic control.

Figure 4

Effects of selective 5-HT receptor antagonists on β-casein mRNA expression in primary bovine mammary epithelial cells. Mean with s.e.m. of (A) β-casein mRNA expression in lactogenic BMEC cultures treated with SB224289, a 5-HT-1B antagonist; (B) β-casein mRNA expression in lactogenic BMEC cultures treated with ritanserin, a 5-HT-2A receptor antagonist; and (C) β-casein mRNA expression in lactogenic BMEC cultures treated with pimozide, a 5-HT-7 receptor antagonist. Columns with *Indicate statistical significance of *P<0·05, **P<0·001, ***P<0·0001 compared with lactogenic control.

Figure 5

Caspase-3 activity in primary bovine mammary epithelial cells treated with 5-HT receptor antagonists. Mean with s.e.m. of caspase-3 activity in lactogenic cultures of BMEC treated with 1·0 μM SB224289, 1·0 μM ritanserin, 0·1 μM SB204741, 0·0001 μM SB204070, and 0·1 μM pimozide. Columns with * indicate statistical significance of *P<0·05; ***P<0·0001.