Role of Na+/H+ exchange and HCO3- transport in pHi recovery from intracellular acid load in cultured epithelial cells of sheep rumen

This study sought to investigate effects of short-chain fatty acids and CO2 on intracellular pH (pH(i)) and mechanisms that mediate pHi recovery from intracellular acidification ill cultured ruminal epithelial cells of sheep. pH(i) was studied by spectrofluorometry using the DH-sensitive fluorescent indicator 2',7'-bis (carboxyethyl)-5(6')-carboxyfluorescein acetoxymethyl ester (BCECF/AM). The resting pH(i) in N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solution was 7.37 +/- 0.03. in HEPES-buffered solution, a NH4+/NH3-prepulse (20 mM) or addition of butyrate (30 mM) led to a rapid intracellular acidification (P < 0.05). Addition of 5-(N-ethyl-N-isopropyl)amiloride (EIPA; 10 mu M) or HOE-694 (200 mu M) inhibited pH(i) recovery from an NH4+/NH3-induced acid load by 58% and 70%, respectively. pH(i) recovery from acidification by butyrate was reduced by 62% and 69% in the presence of EIPA (10 mu M) and HOE-694 (200 mu M), respectively. Changing from HEPES(20 mM) to CO2/HCO3--buffered (5%/20 mM) solution caused a rapid decrease of pH(i) (P < 0.01), followed by an effective counter-regulation. 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS; 100 mu M) blocked the pH(i) recovery by 88%. The results indicate that intracellular acidification by butyrate and CO2 is effectively counter-regulated by an Na+/H+ exchanger and by DIDS-sensitive, HCO3--dependent mechanism(s). Considering the large amount of intraruminal weak acids in vivo, both mechanisms are of major importance for maintaining the pH(i) homeostasis of ruminal epithelial cells.