REGULATION OF PH IN RAT-BRAIN SYNAPTOSOMES .2. ROLE OF CL-

1. We have previously shown that rat brain synaptosomes exhibit a very dynamic Na+/H+ exchanger. We have also observed that although synaptosomes lack HCO3--based transport mechanisms, they do respond with changes in pH(i) upon Cl-o(-) removal. 2. Here we show that when synaptosomes are transferred from Ringer solution (RS)to Cl--free RS, there is a cytosolic alkalinization of similar to 0.22 pH units. This phenomenon is DIDS (4,4'-diisothiocyanostilbene- 3,3'-disulfonic acid) inhibitable. The alkalinization is completely reversed when Cl- ions are reintroduced. The presence of HCO3- or Ca-2(+) does not modify the response to Cl--removal or replenishment. 3. In acid-loading experiments, the initial rate of pH(i) recovery is higher in Cl--free RS than in RS. The final resting pH, after the recovery in Cl--free RS is similar to 0.22 pH units higher than that obtained in media containing Cl-. The magnitude of the NaOAc-induced acidification is 2.5-fold larger in the presence than in the absence of Cl-. Similar results are obtained in the presence of HCO3-. 4. These data suggest that H+ movements may be coupled to Cl- movements. To study this possibility further, we developed a technique to simultaneously measure H+ and Cl- by using the fluorescence of 5' (and 6')-carboxy-10-dimethylamino-3-hydroxy-spyro-[H-7 benzo[c]xanthene-7,1'(3'H)-isobenzofuran]3'-one (SNARF-1) and MQAE [N-(6-methoxyquinolyl)acetoxy ester], respectively. 5. Our results indicated that the steady-state [Cl-](i) in synaptosomes is similar to 56 mM, thus indicating that Cl-i(-); is not passively distributed. 6. Preincubation of synaptosomes in Cl--free RS for 30 min results in a 10-fold decrease in [Cl-](i). These [Cl-](i)-depleted synaptosomes exhibit a decrease in the magnitude of the alkalinization and/or the rate of pH(i) recovery upon Cl- removal when compared with non-Cl--depleted preparations. 7. Simultaneous measurements of H+ and Cl- extrusion using extrasynaptosomal SNARF-1 and MQAE, respectively, indicated that Cl- removal results in a sustained release of H+ and Cl- to the external media. Because HCO3- transport systems are not involved in the alkalinization triggered by Cl- removal or in the regulation of pH(i) in synaptosomes, then the activity of HCO3-/Cl- exchanger cannot explain these observations. 8. Therefore, our results indicate that Cl--H+ co-transport activity is involved in pH(i) regulation in synaptosomes.