INTRACELLULAR PH REGULATION IN CULTURED RAT ASTROCYTES IN CO2 HCO3--CONTAINING MEDIA/

We studied the regulation of intracellular pH (pH(i)) and the mechanis ms of pH(i) regulation in cultured rat astrocytes using microspectrofl uorometry and the pH-sensitive fluorophore 2',7'-bis(carboxyethyl-)-5, 6-carboxyfluorescein. Control pH(i) was 7.00+/-0.02 in HCO3--containin g solutions at an extracellular pH of 7.35. Addition of 4,4'-diisothio cyanatostilbene-2,2'-disulphonic acid (DIDS) or amiloride decreased pH (i), as did removal of extracellular Na+, while removal of extracellul ar Cl-was followed by an increase in pH(i). Following exposure to an a cid transient induced by increasing the CO2 content from 5 to 15%, pH( i) rapidly returned to base line, with an average initial rate of reco very of 0.10 pH units min-1 (corresponding to a mean acid extrusion ra te of 6.3+/-0.36 mmolo l-1 min-1). Regulation of pH(i) was impaired wh en either amiloride or DIDS was added or Cl-was removed. This inhibiti on was enhanced when both DIDS and amiloride were present, and pH(i) r egulation was completely blocked in the absence of extracellular Na+. The rapid regulation of pH(i) normally seen following a transient alka linisation was not inhibited by amiloride or removal of Na+, but was p artially inhibited by DIDS and by the absence of extracellular Cl-. Th e results are compatible with the presence of at least three different pH(i)-regulating mechanisms: a Na+/H+ antiporter, a Na+-dependent HCO 3-/Cl- exchanger (both regulating pH(i) during a transient acidificati on), and a passive Cl-/HCO3- exchanger (regulating pH(i) during transi ent alkalinisation). The results fail to provide firm evidence of the presence of an electrogenic Na+/HCO3-symporter.