COMPARISON OF SIMULTANEOUS PH MEASUREMENTS MADE WITH 8-HYDROXYPYRENE-1,3,6-TRISULFONIC ACID (HPTS) AND PIT-SENSITIVE MICROELECTRODES IN SNAIL NEURONS

We have evaluated the pyrene-based ratiometric fluorescent. dye, 8-hyd roxypyrene-1,3,6-trisulphonic acid (HPTS), by using it in conjunction with glass pH-sensitive microelectrodes to measure intracellular pH (p H(i)) in voltage-clamped snail neurones. Intracellular acidification w ith propionic acid, and alkalinization following the activation of Hchannels allowed the calibration of the dye to be compared with that o f the pH microelectrode over the pH range 6.50-7.50. HPTS calibrated i n vitro and glass pH-sensitive microelectrodes produced similar absolu te resting pH(i) values, 7. 16+/-0.05 (n=10) and 7.17+/-0.06 (n=9) res pectively in nominally CO2HCO3--free saline. At both extremes of the p H 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 u nits from the pH-sensitive microelectrode measurement. At alkaline pH( i), 7.32+/-0.10 (n=5), HPTS measurements produced pH values that diffe red by +0.07+/-0.04 pH units from those of the pH-sensitive microelect rode. Some of the discrepancy could be accounted for by the slow respo nse of the recessed-tip pi-I-sensitive microelectrode (time constant 7 7+/-15 s, n=3). Further experiments showed that HPTS, used at an intra cellular concentration of 200 mu M to 2 mM, did not block activity-dep endent pH(i) changes. The intracellular HPTS concentration was calcula ted by measurement of intracellular chloride during a series of HPTS-K Cl injections. Comparison of HPTS with 2',7'-bis(2-carboxyethyl)-5, 6- carboxyfluorescein (BCECF), at the same concentration, showed that HPT S produces a larger change in ratio over the pH range 6.00-8.00.