Oertzenweg 19 b
Tel.+49 30 838 62600 Fax.+49 30 838-62610
The impact of the livestock industry on the globally increasing emissions of nitrogen is profound. Particularly in ruminants, a large percentage of the protein that is fed does not lead to the desired increases in meat and milk production, but is microbially broken down into ammonia, absorbed across the epithelia of the gastro-intestinal tract, hepatically converted into urea in an energy-consuming process and finally emitted into the environment via the kidney.
Countless studies attempting to reduce nitrogen emissions by ruminal acidification or synchronisation of the carbohydrate and protein content of the diet have not resolved this issue. In vitro studies on the sheep rumen show that epithelial efflux of ammonia does not only involve simple diffusion of the uncharged form (NH3), but also a transport of the protonated ammonium ion (NH4+). So far, there is very little information on the underlying mechanisms. The aim of the present study was to characterize the mechanisms for the absorption of ammonia across the bovine ruminal epithelium using the patch-clamp-technique, flux studies in the Ussing chamber and pH-selective microelectrodes. The experiments yielded the following results:
- The absorption of ammonia across the ruminal epithelium of cows induced a positive short-circuit current in the Ussing chamber (10 mmol·l-1; *40 mmol·l-1 *p < 0.05).
- A 2.5-hour incubation in the Ussing chamber exposing the mucosal side of the tissue to 10 mmol·l-1 NH4Cl led to a serosal concentration of 3.08 ± 0.17 mmol·l-1 NH4+. Increasing the mucosal concentration to 40 mmol·l-1 NH4Cl resulted in an increased serosal concentration of 8.72 ± 0.65 mmol·l-1 NH4+. No significant alkalinization of the only lightly buffered serosal solution was observed, as should have been the case if NH3 were the primary form transported (p = 0.18; N/ n = 8/40).
- In microelectrode measurements, the replacement of 40 mmol·l-1 NMDG+ by an equimolar amount of NH4+ (pH 6.4 and 7.4) resulted in a significant depolarization of the transepithelial and apical potential (pH 6.4: ΔPDt 2.78 ± 0.37 mV; ΔPDa 2.13 ± 0.5 mV; pH 7.4: ΔPDt 2.53 ± 0.46 mV; ΔPDa 1.0 ± 1.8 mV N/n = 6/12, p < 0.05). Simultaneously the intracellular pH dropped significantly at both values of pH (pH 6.4: ΔpHi -0.13 ± 0.02; pH 7.4: ΔpHi -0.16 ± 0.05; N/n = 6/12, p < 0.05). These results suggest a transcellular pathway. The acidification observed also implies that the apical influx of NH4+ slightly exceeded the basolateral efflux at pH 6.4 as well as 7.4.
- The NH4+ conductance of the ruminal epithelium was blocked by divalent cations (Ca2+ and Mg2+). This was shown in patch clamp measurements of isolated cells filled with Na-gluconate solution or K-gluconate solution (n = 8, p = 0.008 and n = 7, p = 0.016), as well as in intact epithelia measured in the Ussing Chamber (microelectrode-technique) (N/n = 3/4, p = 0.005). Verapamil (1 mmol·l-1) had an inhibitory effect (N/n = 3/7, p = 0.008).
- Various specific modulators of the TRP-channel-family had an effect on the NH4+ induced current. The mucosal application of menthol (200 μmol·l-1; 1 mmol·l-1) resulted in a clear but transient increase of Isc (ΔIsc 5.35 ± 0.97 μAcm-2 (N/n = 4/11, p = 0.02) and ΔIsc 4.53 ± 0.94 μAcm-2 (N/n = 3/12, p = 0.022), respectively). In contrast, the application of capsaicin (100 μmol·l-1) led to a significant decrease of the transepithelial current (ΔIsc -3.25 ± 1.16 μAcm-2, N/n = 4/6, p = 0.02). Thymol (100 μmol·l-1 and 1 mmol·l-1) showed clearly visible effects on the NH4+ induced current, although with great variability depending on the tissue studied (N/n = 2/4 and N/n = 6/9). The application of the solvent alone had no effect (N/n = 3/6). Similar effects could be demonstrated on ovine ruminal epithelium.
- Apical replacement of chloride by gluconate led to a hyperpolarization of the apical potential, suggesting a basolateral localisation of the anion-channels expressed by ruminal epithelial cells. A participation of these channels in the currents induced by TRP-modulators appears unlikely.
In summary, the results suggest an involvement of non-selective cation channels belonging to the TRP-family in the efflux of ammonia from the rumen. A participation of these channels in the efflux of protons across the ruminal epithelium to maintain pH-homeostasis is discussed. A targeted manipulation of ammonium transport across the ruminal epithelium of cows through breeding or the application of TRP-channel modulators appears possible.