Changes in pHi associated with activation of ion secretion in avian nasal salt gland cells

The fluorescent pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)- carboxyfluorescein (BCECF) was used to determine changes in intracellular pH (pHi) associated with activation of secretion in isolated cells from the salt-secreting avian nasal gland. A correction procedure overcoming artifacts due to BCECF leakage is described. Resting pHi averaged 7.15 +/- 0.03 and was unaffected by the nominal removal of medium HCO3- or by the addition of the anion-exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) but was significantly reduced by amiloride (7.07 +/- 0.02). Muscarinic activation of secretion resulted in a rapid intracellular acidification that was compensated by mechanisms which raised pHi to restore approximately resting levels within 5 min. The principal mechanism involved was amiloride-sensitive and independent of any sustained intracellular Ca2+ concentration change. Recovery of pHi was also aided by HCO3(-)-dependent and DIDS-sensitive mechanisms not seen in the resting cell. The direction of the latter was pHi-dependent, with DIDS further decreasing pHi in acidified cells and increasing pHi in alkalinized cells. This suggests that the DIDS-sensitive pathways are activated under conditions where pHi has been shifted away from resting levels in either direction and act primarily to restore resting pHi.