Intracellular pH regulation in cultured astrocytes from rat hippocampus. I. Role Of HCO3-.

We studied the regulation of intracellular pH (pH) in single cultured astrocytes passaged once from the hippocampus of the rat, using the dye 2',7'-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) to monitor pH. Intrinsic buffering power (beta) was 10.5 mM (pH unit) at pH 7.0, and decreased linearly with pH; the best-fit line to the data had a slope of -10.0 mM (pH unit). In the absence of HCO, pH recovery from an acid load was mediated predominantly by a Na-H exchanger because the recovery was inhibited 88% by amiloride and 79% by ethylisopropylamiloride (EIPA) at pH 6.05. The ethylisopropylamiloride-sensitive component of acid extrusion fell linearly with pH. Acid extrusion was inhibited 68% (pH 6.23) by substituting Li for Na in the bath solution. Switching from a CO/HCO-free to a CO/HCO-containing bath solution caused mean steady state pH to increase from 6.82 to 6.90, due to a Na-driven HCO transporter. The HCO-induced pH increase was unaffected by amiloride, but was inhibited 75% (pH 6.85) by 400 microM 4, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and 65% (pH 6.55-6.75) by pretreating astrocytes for up to approximately 6.3 h with 400 microM 4-acetamide-4'-isothiocyanatostilbene-2, 2'-disulfonic acid (SITS). The CO/HCO-induced pH increase was blocked when external Na was replaced with -methyl--glucammonium (NMDG). In the presence of HCO, the Na-driven HCO transporter contributed to the pH recovery from an acid load. For example, HCO shifted the plot of acid-extrusion rate vs. pH by 0.15-0.3 pH units in the alkaline direction. Also, with Na-H exchange inhibited by amiloride, HCO increased acid extrusion 3.8-fold (pH 6.20). When astrocytes were acid loaded in amiloride, with Li as the major cation, HCO failed to elicit a substantial increase in pH. Thus, Li does not appear to substitute well for Na on the HCO transporter. We conclude that an amiloride-sensitive Na-H exchanger and a Na-driven HCO transporter are the predominant acid extruders in astrocytes.

Motor responses of cultured rat cerebral vascular smooth muscle cells to intra- and extracellular pH changes.

Alkalizing perivascular fluid constricts, whereas acidification dilates, cerebral arterioles. It is not known whether vascular smooth muscle cells (VSMCs), endothelium, or neuronal elements sense pH changes. We hypothesized that VSMCs themselves transduce extracellular pH (pHo) changes. We examined the motor responses of cultured adult rat middle cerebral arterial VSMCs during pHo and intracellular pH (pHi) changes. Motor responses were inferred from the deformation pattern of a silicone substratum, dimethylpolysiloxane, which wrinkles as cells contract. pHi was measured with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Cultured VSMCs retained motor responses to vasoconstrictors (5-hydroxytryptamine and K+), and to sodium nitroprusside, which were typical of intact arterioles. VSMCs contracted with increasing and relaxed with decreasing pHo. Hypocapnia contracted VSMCs when the pHo increased, and hypercapnia relaxed VSMCs when the pHo decreased. However, at a constant pHo, changes in PCO2 caused opposite responses despite equivalent changes in pHi. Thus VSMCs contract with increased pHo and relax with decreased pHo just as intact arterioles do. These responses do not reflect changes in pHi or PCO2. pHi changes paradoxically alter VSMC tone in the direction opposite that caused by pHo changes.