Müller F, Aschenbach J R, Gäbel G
Department of Veterinary Physiology, Leipzig University, An den Tierkliniken, Germany.
J Comp Physiol B. 2000 Jun;170(4):337-43. doi: 10.1007/s003600000107.
This study sought to investigate effects of short-chain fatty acids and CO2 on intracellular pH (pHi) and mechanisms that mediate pHi recovery from intracellular acidification in cultured ruminal epithelial cells of sheep. pHi was studied by spectrofluorometry using the pH-sensitive fluorescent indicator 2',7'-bis (carboxyethyl)-5(6')-carboxyfluorescein acetoxymethyl ester (BCECF/AM). The resting pHi in N-2-hydroxyethylpiperazine-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 (20 mM) led to a rapid intracellular acidification (P < 0.05). Addition of 5-(N-ethyl-N-isopropyl)-amiloride (EIPA: 10 microM) or HOE-694 (200 microM) inhibited pHi recovery from an NH4+/NH3-induced acid load by 58% and 70%, respectively. pHi recovery from acidification by butyrate was reduced by 62% and 69% in the presence of EIPA (10 microM) and HOE-694 (200 microM), respectively. Changing from HEPES-(20 mM) to CO2/HCO3(-)-buffered (5%/20 mM) solution caused a rapid decrease of pHi (P < 0.01), followed by an effective counter-regulation. 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS; 100 microM) blocked the pHi 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 pHi homeostasis of ruminal epithelial cells.
本研究旨在探讨短链脂肪酸和二氧化碳对绵羊瘤胃上皮细胞内pH值（pHi）的影响，以及介导细胞内酸化后pHi恢复的机制。使用pH敏感荧光指示剂2',7'-双（羧乙基）-5(6')-羧基荧光素乙酰氧基甲酯（BCECF/AM），通过荧光分光光度法研究pHi。在N-2-羟乙基哌嗪-N'-2-乙烷磺酸（HEPES）缓冲溶液中，静息pHi为7.37±0.03。在HEPES缓冲溶液中，NH4+/NH3预脉冲（20 mM）或添加丁酸盐（20 mM）导致细胞内迅速酸化（P<0.05）。添加5-（N-乙基-N-异丙基）-amiloride（EIPA：10 μM）或HOE-694（200 μM）分别抑制NH4+/NH3诱导的酸负荷后pHi恢复的58%和70%。在存在EIPA（10 μM）和HOE-694（200 μM）的情况下，丁酸盐酸化后pHi的恢复分别降低了62%和69%。从HEPES-（20 mM）缓冲溶液改为CO2/HCO3(-)-缓冲（5%/20 mM）溶液导致pHi迅速下降（P<0.01），随后是有效的反向调节。4,4'-二异硫氰酸根合芪-2,2'-二磺酸（DIDS；100 μM）使pHi恢复受阻88%。结果表明，丁酸盐和二氧化碳引起的细胞内酸化可通过Na+/H+交换器和对DIDS敏感的、依赖HCO3(-)的机制有效反向调节。考虑到体内瘤胃内大量的弱酸，这两种机制对于维持瘤胃上皮细胞的pHi稳态都至关重要。
