Protein kinase C is involved in the regulation of Na + transport across rumen epithelium

Electrogenic Na+-transport across rumen epithelium occurs via a non-selective cation channel, blocked by luminal Ca2+ and Mg2+ and by cytosolic Mg2+. The role of protein kinase C (PKC) in the regulation of this pathway was studied with isolated epithelia from goat rumen incubated in Ussing chambers. Omission of Ca2+ and Mg2+ from the luminal side induced an increase in short circuit current followed by a decline. This down-regulation of Na+-current was delayed after activation of PKC with 6-[N-decylamino]-4-hydroxymethylinole. Na+-current declined again after inhibition of PKC with chelerythrine, showing that PKC contributes to the regulation of ruminal electrogenic Na+-transport.


INTRODUCTION
Rumen epithelium exhibits a non-selective cation conductance, which permits the passage of Na + and other monovalent cations in the absence of divalent cations.This conductance is blocked by Ca 2+ and Mg 2+ ions from the luminal side and by Mg 2+ ions from the cytosolic side (Leonhard-Marek, 2002;Stumpff et al., 2004).In other epithelia, protein kinase C (PKC) is involved in the regulation of Na + transport via the epithelial Na + channel ENaC.PKC stimulates Na + transport across frog skin (Civan et al., 1991), whereas it decreases Na + channel activity in epithelia from the urinary tract (e.g., Ling and Eaton, 1989).We wanted to know whether protein kinase C is involved in the regulation of electrogenic Na + transport across the rumen.

MATERIAL AND METHODS
Isolated epithelia from the rumen of slaughtered goats were incubated in Ussing chambers under short circuit conditions.Epithelia were bathed in a standard bicarbonate buffer, which contained indomethacine (10 -5 mol l -1 ) in order to avoid a prestimulation of the tissues via prostaglandines.Divalent free solutions contained 0.5 mmol l -1 EGTA.We used 6-[N-decylamino]-4-hydroxymethyl-inole (DHI) as an activator of PKC and chelerythrine chloride (CC) as a specifi c inhibitor of PKC.DHI and CC were added to both sides of the epithelia in a concentration of 2 µmol l -1 .

RESULTS
The omission of Ca 2+ and Mg 2+ ions from the luminal side opened the nonselective cation conductance which could be measured as an increase in short circuit current (Isc) and transepithelial conductance.Isc remained on a plateau value for about 15 min and then declined slowly (Figure 1).This time-dependent decrease in Isc measured over 30 min without any additions was -0.52±0.05µeq cm -2 h -1 (n=12).When stimulating PKC with DHI shortly after the Isc plateau, as shown in fi gure 2, Isc decreased much less (-0.34±0.05µeq cm -2 h -1 in 30 min, n=9; P<0.05).
When we then added CC in the presence of DHI, thereby blocking the protein kinase C again, the Isc also started to decrease again (Figure 2, ∆ Isc 0-15 min PKC AND RUMINAL NA + TRANSPORT Figure 1.Omission of divalent cations from the luminal side increased the short circuit current (Isc) across rumen epithelium (example from goat rumen).The Isc plateau was followed by a downregulation.Re-addition of Ca and Mg at the end of the experiment induced an immediate decline in Isc towards baseline levels after CC addition: -0.20±0.02µeq cm -2 h -1 , n=8).This was signifi cantly different (P<0.05) from the time-dependent decrease in the only presence of DHI as measured simultaneously in other pieces of rumen epithelia (-0.06±0.01µeq cm -2 h -1 , from 30 to 45 min after DHI addition, n=3).
The addition of CC shortly after the Isc plateau resulted in an Isc decrease (∆ Isc) of -0.60±0.05µeq cm -2 h -1 (n=9) measured over the next 30 min which was not signifi cantly different from the mere time-dependent decrease of -0.52±0.05µeq cm -2 h -1 (n=12) shown above.
DHI and CC given in the presence of divalent cations on the luminal side had no effect on the Isc (n=10).

DISCUSSION
The increase in Isc after luminal omission of Ca 2+ and Mg 2+ ions refl ects an increase in Na + transport as shown earlier (Rübbelke, 1998).We opened this pathway for a longer time and observed that the increase in Na + current was followed by a decline (Figure 1).The decline in Isc from the peak value indicates that autoregulatory mechanisms exist to regulate Na + uptake to the activity of the Na + /K + -ATPase.This is in agreement with the classical Na + transport via ENaC (Turnheim, 1994) and with observations in the omasum of sheep (Schultheiss and Martens, 1999).
One of the factors that regulate Na + entry has been shown to be the ubiquitous protein kinase C.However, observations in this fi eld were contradictory.While LEONHARD-MAREK S. ET AL.PKC increases transepithelial Na + transport across frog and toad skin, PKC inhibited Na + transport across epithelia from the urinary tract.This means that regulation of epithelial Na + permeability by protein kinase C is tissue specifi c (Chalfant et al., 1996).
The contribution of protein kinase C to Na + transport across the rumen resembles its action on frog and toad skin, albeit the pathways for Na + uptake are different between skin and rumen.

CONCLUSIONS
We have shown that protein kinase C can be activated in rumen epithelia, and that it is involved in the regulation of electrogenic Na + transport processes across the rumen wall.

Figure 2 .
Figure 2. DHI inhibited and chelerythrine (CC) increased the down-regulation of the divalent cation sensitive current (example from goat rumen)