EXAMINING THE FUNCTIONAL ROLES OF AQUAPORINS AND UREA TRANSPORTERS IN THE MOVEMENT OF UREA ACROSS THE RUMINAL EPITHELIUM

Abstract

Ussing chamber studies of ruminal epithelial tissue have provided insight into the mechanisms that regulate serosal-to-mucosal urea transport. Of these mechanisms, urea transport (UT-B) proteins have been shown to facilitate urea movement across the ruminal epithelium; however, other mechanisms may be involved as well because inhibiting UT-B does not completely eliminate urea transport. Of the aquaporins (AQP), which are a family of membrane-spanning proteins that are predominantly involved in the movement of water, AQP-3, -7, and -10 are also permeable to urea, but it is not clear if they contribute to urea transport across the ruminal epithelium. My objectives were to determine the relative functional roles of UT- and AQP-mediated serosal to mucosal urea flux (Jsm-urea) in response to changes in dietary carbohydrate fermentability, as well as ruminal ammonia and blood urea concentrations. The objectives of the Chapter 2 studies were: 1) to evaluate if there are differences in the magnitude of serosal-to-mucosal urea transfer in ruminal epithelium obtained from the caudal-dorsal or ventral sacs; 2) to determine the optimum mucosal buffer pH for maximal urea transport across the bovine ruminal epithelium; 3) to determine the time that is required for steady-state isotope equilibration with bovine ruminal epithelium; and 4) to determine if NiCl2 and HgCl2 are suitable inhibitors of aquaporin-mediated urea transport in bovine ruminal epithelium. Steady-state Jsm-urea and Jsm-mannitol fluxes were observed by 45 min following isotopic additions to the serosal buffer. Epithelia collected from the caudal-dorsal sac had higher Jsm-urea (P = 0.03) and lower Jsm-mannitol (P < 0.01) than that collected from the ventral sac. Reducing mucosal buffer pH from 7.0 to 5.2 increased Jsm-urea quadratically, where Jsm-urea increased from pH 7.0 to 6.4 and thereafter decreased (P = 0.01). Both HgCl2 and NiCl2 inhibited Jsm-urea (P < 0.01); however, the addition of HgCl2 increased Tissue Conductance (Gt) when compared to NiCl2. The objectives of Chapter 3were to determine: 1) the functional roles of AQP and UT-B in the serosal-to-mucosal urea flux (Jsm-urea) across rumen epithelium; and 2) whether functional adaptation occurred in response to increased diet fermentability. Serosal addition of phloretin. The addition of phloretin or NiCl2 reduced the Jsm-urea from 116.5 to 54.0 and 89.5 nmol·cm-2·h-1, respectively across all dietary treatments. When both inhibitors were added simultaneously, Jsm-urea was further reduced to 36.8 nmol·cm-2·h-1. Phloretin-sensitive and NiCl2-sensitive Jsm-urea III were not affected by diet. The Jsm-urea tended to increase linearly as the duration of adaptation to MGD increased, with the lowest Jsm-urea being observed in animals fed CON (107.7 nmol·cm-2·h- 1) and the highest for those fed the MGD for 21 d (144.2 nmol·cm-2·h-1). Phloretin-insensitive Jsm-urea tended to increase linearly as the duration of adaptation to moderate grain diet increased, whereas there was a tendency for NiCl2-insensitive Jsm-urea to be affected by diet. Gene transcript abundance for AQP-3 and UT-B in ruminal epithelium increased linearly as the duration of MGD adaptation increased. For AQP-7 and AQP-10, gene transcript abundance in animals that were fed the MGD was greater when compared to CON animals. The objective of Chapter 4 was to determine the effect of an acute dose of NH3 on total and aquaporin (AQP)-mediated urea flux across the ruminal epithelium in Angus cross bulls and Plains bison bulls. Ruminal NH3 was not affected by species (P = 0.60) or diet (P = 0.27) while PUN tended (P = 0.055) to be greater for BIS (12.5 mg/dL) than BOV (10.8 mg/dL), but was not affected by diet (P = 0.22). The Jsm-urea tended to decrease with addition of NiCl2 (P = 0.065), while mucosal NH3 had no effect on Jsm-urea (P = 0.41). Jsm-urea and Jsm-mannitol were not affected by species (P = 0.41) or dietary treatment (P = 0.29). I evaluated the effects of mucosal NH3 and serosal urea concentrations on total and phloretin sensitive Jsm-urea in Chapter 5. High Ammonia (HA) tended to inhibit total Jsm-urea with HU, but there was no effect of either NH3 concentration on total Jsm-urea with Low Urea (LU; interaction, P = 0.055). Addition of phloretin in the presence of serosal urea or mucosal NH3 had no effect on Jsm-urea. The Jsm-mannitol was not affected by serosal urea (P = 0.86) or mucosal NH3 (P = 0.22) concentration. The Jsm-urea and Jsm-mannitol tended to be positively correlated for HA (R2 = 0.301, P = 0.08), but not Low Ammonia (LA; R2 = 0.027, P = 0.70) in combination with LU. The same pattern was observed with High Urea (HU) treatments where Jsm-urea and Jsm-mannitol tended to be positively correlated for HA (R2 = 0.329, P = 0.08), but not LA (R2 = 0.111, P = 0.32). This research provides evidence that both AQP and UT-B play an important role in the Jsm-urea. In the future, additional research will be required to determine the mechanisms involved in NH3 inhibition of Jsm-urea, as this key step is critical in the role of urea-N recycling in ruminants.