mRNA expression and binding sites for α2-adrenergic receptor subtypes in muscles layers of the ileum and spiral colon of dairy cows

Abstract

Objective - To measure maximum binding capacity (Bmax) and levels of mRNA expression for α2-adrenergic receptor (AR) subtypes in ileal and colonic muscle layers of healthy dairy cows. Sample Population - Ileal and colonic muscle specimens from 6 freshly slaughtered cows. Procedures - Ileal and colonic muscle layers were obtained by scraping the mucosa and submucosa from full-thickness tissue specimens. Level of mRNA expression for α2-AR subtypes was measured by realtime reverse transcriptase-PCR analysis and expressed relative to the mean mRNA expression of glyceraldehyde phosphate dehydrogenase, ubiquitin, and 18S ribosomal RNA. Binding studies were performed with tritiated RX821002 (3H-RX821002) and subtype-selective ligands as competitors. Results - mRNA expression for (α 2AD-, α2B-, and (α 2C-AR subtypes was similar in ileal and colonic muscle layers. The mRNA expression for α2AD-AR was significantly greater than that for α2B,- and α 2C-AR subtypes, representing 92%, 6%, and 2%, respectively, of the total mRNA. Binding competition of 3H-RX821002 with BRL44408, imiloxan, and MK-912 was best fitted by a 1-site model. The Bmax of α2AD-and 2C-AR subtypes was greater than that of α 2B-AR. The Bmax and level of mRNA expression were only correlated (r = 0.8) for α2AD-AR. Ratio of Bmax to mRNA expression for α2C-AR was similar to that for α2B-AR, but significantly greater than for α2AD-AR. Conclusions and Clinical Relevance - Subtypes of α2-AR in bovine intestinal muscle layers are represented by a mixture of α2AD- and α 2C-ARs and of α2B-AR at a lower density. Information provided here may help in clarification of the role of AR subtypes in α2- adrenergic mechanisms regulating bovine intestinal motility.