Comparison of simultaneous pH measurements made with 8-hydroxypyrene-1,3,6-trisulphonic acid (HPTS) and pH-sensitive microelectrodes in snail neurones

 We have evaluated the pyrene-based ratiometric fluorescent dye, 8-hydroxypyrene-1,3,6-trisulphonic acid (HPTS), by using it in conjunction with glass pH-sensitive microelectrodes to measure intracellular pH (pH_i) in voltage-clamped snail neurones. Intracellular acidification with propionic acid, and alkalinization following the activation of H⁺ channels allowed the calibration of the dye to be compared with that of the pH microelectrode over the pH range 6.50–7.50. HPTS calibrated in vitro and glass pH-sensitive microelectrodes produced similar absolute resting pH_i values, 7.16±0.05 (n=10) and 7.17±0.06 (n=9) respectively in nominally CO₂/HCO₃ ^–-free saline. At both extremes of the pH range there were small discrepancies. At acidic pH_i, 6.87±0.09 (n=5), the intracellular HPTS measurement differed by –0.08±0.03 pH units from the pH-sensitive microelectrode measurement. At alkaline pH_i,7.32±0.10 (n=5), HPTS measurements produced pH values that differed by +0.07±0.04 pH units from those of the pH-sensitive microelectrode. Some of the discrepancy could be accounted for by the slow response of the recessed-tip pH-sensitive microelectrode (time constant 77±15 s, n=3). Further experiments showed that HPTS, used at an intracellular concentration of 200 μM to 2 mM, did not block activity-dependent pH_i changes. The intracellular HPTS concentration was calculated by measurement of intracellular chloride during a series of HPTS-KCl injections. Comparison of HPTS with 2′,7′-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF), at the same concentration, showed that HPTS produces a larger change in ratio over the pH range 6.00–8.00. Received: 19 February 1998 / Received after revision and accepted: 22 April 1998