|Miles, E -|
|Xue, Y -|
|Boling, J -|
|Matthews, J -|
Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 25, 2011
Publication Date: July 26, 2011
Citation: Miles, E.D., Xue, Y., Strickland, J.R., Boling, J.A., Matthews, J.C. 2011. Ergopeptines bromocriptine and ergovaline and the dopamine type-2 receptor inhibitor domperidone inhibit bovine equilibrative nucleoside transporter 1-like activity. J. Agric. Food Chem. 59(17):9691-9699. Interpretive Summary: ”: Inhibition of Equilibrative Nucleoside Transporter 1 Activity by Ergopeptines. By Miles et al., page 000. Cattle grazing endophyte-infected tall fescue have reduced productive and reproductive capacities. Using a bovine kidney cell line, this research found that both a model ergopeptine, and indigenous ergopeptine of endophyte-infected tall fescue, inhibit the function of a nucleoside transporter (ENT1) that is expressed by gastrointestinal tract, peripheral, and neuronal tissues. The manner of ENT1 inhibition has both competitive and non-competitive binding components. These results indicate that impaired nucleoside transport by a variety of cell types may contribute to the wide-spread physiologic effects of fescue toxicosis.
Technical Abstract: Forages infected with Neotyphodium coenophialum produce ergot alkaloids that alter the systemic physiology of cattle such that reproduction, lactation, and growth are decreased. Ergopeptines are one predominant class of ergot alkaloids. However, aside from their interactions with biogenic amine receptors (e.g., dopamine type-2 receptor, D2R), little is known about how ergopeptines affect animal metabolism. The goal of this study was to evaluate the potential effect(s) of ergopeptines on bovine nucleoside transporters (NT) using cultured Madin-Darby Bovine Kidney (MDBK) cells. The first objective of this research was to characterize the NT activity of MDBK cells. The second objective was to test the hypothesis that NT activity would be inhibited by ergopeptines and, if so, determine the type of inhibition. Uridine (12 µM) uptake (pmol.mg-1 protein.4 min-1) assays, using nitrobenzylthioinosine as a biochemical discriminator of NT, revealed that ENT1-like activity accounted for 94% of total uridine uptake by MDBK cells, with high-affinity (Km = 51.6 µM) recognition of uridine. Inhibitory competition (IC50) experiments revealed that ENT1-like uridine uptake was inhibited by both bromocriptine (synthetic model ergopeptine; IC50 = 31.5 µM) and ergovaline (a predominant ergopeptine in endophyte-infected tall fescue; IC50 54.4 µM). Subsequent bromocriptine kinetic inhibition analysis of uridine uptake demonstrated a 2-fold increase in calculated apparent Km. In addition, the Vmax of uridine uptake was decreased up to 24.8%, indicating that bromocriptine inhibited ENT1-like activity through both competitive and a non-competitive mechanisms. Because immunoblot analysis determined that D2R was expressed in MDBK cells, the potential role of D2R-mediated inhibition of ENT1 activity was evaluated by measuring the effect of domperidone (D2R antagonist) on ENT1 activity in the presence and absence of bromocriptine (D2R agonist). Instead of alleviating a 64% bromocriptine-induced inhibition of ENT1 activity, domperidone inhibited ENT1 activity 65% in the absence of bromocriptine - and 81% in the presence of bromocriptine. Subsequent competitive inhibition experiments demonstrated an IC50 value of 7.2 µM for domperidone inhibition of uridine uptake. These findings suggest that cattle consuming ergopeptines may have reduced ENT1 activity, resulting in impaired nucleoside transport and metabolism by a variety of cell types.