STIMULUS-EVOKED CHANGES OF EXTRACELLULAR AND INTRACELLULAR PH IN THE LEECH CENTRAL-NERVOUS-SYSTEM .2. MECHANISMS AND MAINTENANCE OF PH HOMEOSTASIS

1. We have studied extracellular pH (pH(e)) and intracellular pH (pH(i)) changes evoked by repetitive electrical side nerve stimulation (20 Hz, 1 min) in segmental ganglia of the leech Hirudo medicinalis using double-barreled, pH-sensitive microelectrodes to elucidate the involvement of neurotransmitters, of carbonic anhydrase, and of active acid/base transport on the extracellular H+ homeostasis. In saline buffered with 5% CO2-24 mM HCO3-, the stimulation induced a small and brief alkalinization followed by an acidification in the extracellular spaces (ECS), whereas neurons acidified and glial cells alkalinized (see previous paper). 2. Blocking synaptic transmitter release by superfusion with 20 mM Mg2+ saline (CO2/HCO3--free) led to a reversible reduction of both activity-induced pH(e) changes by similar to 90% and to a complete suppression of the intracellular acidification of neurons. After application of the glutamate/kainate receptor blocker 6-cyano-7-dinitroquinozaline- 2.3-dione (CNQX, 50 mu M) to CO2/HCO3--free saline, the stimulus-evoked pH(e) changes were reversibly reduced. The gamma-aminobutyric acid-A (GABA(A)) receptor antagonist picrotoxin (50 mu M) led to an amplification of the extracellular alkalinization in the presence of CO2/HCO3-. Bath application of the excitatory transmitter agonists carbachol or kainate to CO2/HCO3--free saline induced biphasic alkaline-acid transients in the ECS; the inhibitory transmitters GABA and serotonin had no detectable effects on the pH(e), (saline buffered with CO2/HCO3-). 3. Amiloride (2 mM), a blocker of the Na+-H- exchanger, reduced the stimulus-evoked extracellular acidification by nearly 70% and slowed the pH(i) recovery of pressure neurons from the stimulus-induced intracellular acidification. 4. Inhibition of carbonic anhydrase by ethoxyzolamide (EZA. 2 mu M) potentiated the stimulus-evoked alkalinization in the ECS. In contrast, the pH(i) transients in neurons and glial cells were not significantly altered by EZA. 5. 4,4-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, 0.5 mM), an inhibitor of glial Na+/HCO3- co-transport, resulted in an enhancement of the extracellular stimulus-evoked alkalinization and turned the glial alkalinization into an acidification. 6. The results suggest that the activity-induced extracellular alkalinization is partly due to a transmitter-induced flux of H-/OH- equivalents across the cell membranes. Cellular acid secretion via the Na+-H+ exchanger contributes to the stimulus-evoked extracellular acidification. Our study also indicates a significant amount of carbonic anhydrase activity in the ECS of the leech CNS mediating,o the fast supply of acid/base equivalents fi om the CO2/HCO3- buffer system, thereby reducing the activity-induced pH(e) changes. Transport of bicarbonate into the glial cells after activation of the membrane potential-dependent Na+/HCO3-, co-transporter contributes to the muffling of the extracellular alkalinization evoked by the neuronal stimulation.