Relationship between intra- and extracellular pH in primary cultures of rat astrocytes

We studied the influence of extracellular pH (pHe) on the mechanisms regulating intracellular pH (pHi) in astrocytes cultured from neonatal rat cortex, using single cell microspectrofluorometry and the pH-sensitive fluorophore 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. When pHe was maintained at control values of 7.35 during acid transients caused by an increased CO2 tension, pHi was rapidly regulated back to normal. However, at pHe 6.9 or below, there was no recovery of pHi. Steady-state pHi was also strongly dependent on pHe (pHi = 1.14 + 0.80 pHe). The pHi recovery after normalization of pHe was very rapid, indicating that a prolonged exposure to a low pH stimulates pH-regulating mechanisms, and was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and amiloride or the removal of Na+. Recovery was also slowed down by Cd2+. The pHe-dependent acidification was not critically influenced by DIDS or amiloride and was not inhibited by tetrodotoxin, tetraethylammonium, Ba2+,2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)-quinoxaline, kynurenic acid, 5-hydroxysaclofen, bicuculline, Cd2+, or albumin. The results emphasize the importance of pHe for pHi regulation and suggest that in astrocytes pHi is not regulated back to normal levels until pHe is normalized.