Regulation of pH in rat brain synaptosomes. I. Role of sodium, bicarbonate, and potassium

Abstract

1. We investigated the regulation of intracellular pH (pHi) in rat brain isolated nerve terminals (synaptosomes), using fluorescence pH indicators and time-resolved fluorescence spectroscopy. 2. The resting pHi was not significantly affected by the presence or absence of HCO3-. Removal of external Na+, in the absence or presence of HCO3- caused a rapid acidification of pHi. The recovery from acid loads was primarily due to the activity of the Na+/H+ exchanger, confirming the relevance of this transport system in synaptosomes. 3. Our data revealed that in synaptosomes the activity of the Na+/H+ exchanger was not regulated by either protein kinase C or kinase A. In contrast, Ca2+ played an important role in the regulation of Na+/H+ exchanger. This was supported by the observation that 4Br-A23187 induced a Na(+)-dependent alkalinization of the resting pHi and greatly enhanced the initial rate and the degree of the recovery from acid loads. 4. In most eukaryotic cells, HCO3(-)-based transport mechanisms play an important role in pHi regulation. In synaptosomes, however, HCO3- transport is not significantly involved in pHi regulation, because the presence or absence of HCO3- does not affect resting pHi nor the rate of pHi recovery to acid loads. Further studies to address the role of Cl- and HCO3- in pHi regulation in synaptosomes are discussed in the companion paper. 5. Increasing the concentration of Ko+ also resulted in a rise of steady-state pHi by a processes that is Ca2+ and HCO3- independent. This alkalinization could be due to either K+/H+ exchanger activity, K(+)-induced depolarization, reduction of delta microH+, or a direct reduction of delta microK+. Calculated H+ driving forces suggest that the reduction in the inwardly directed H+ leak is sufficient to explain this K(+)-induced alkalinization because it changes the delta microH+ by virtue of setting the membrane potential difference (Em) to the K+ equilibrium potential (EK+).

Inhibition of Synaptosomal Uptake of 3H-L-glutamate and 3H-GABA by Hyperforin, a Major Constituent of St. John's Wort The Role of Amiloride Sensitive Sodium Conductive Pathways

“…An important system regulating intracellular Na ϩ concentrations is the Na ϩ -H ϩ -exchanger, which can be specifically inhibited by amiloride and several of its analogues (Grinstein and Rothstein 1986;Frelin et al 1988). Na ϩ -H ϩ -exchangers have also been identified on brain cell membranes and synaptosomes (Kalaria et al 1998;Sauvaigo et al 1984;Sanchez-Armass et al 1994). Inhibition by amiloride or amiloride derivatives re- duces synaptosomal Na ϩ uptake leading to a reduced free intracellular Na ϩ concentration (Sauvaigo et al 1984;Liu et al 1996).…”

Anionic Selectivity Sequence of the Cl−–H+ Symporter in the Synaptosomal Preparation from Rat Brain Cortex

“…Cytosolic pH was determined by fluorescence of intracellular Pyranine (8-hydroxy pyrene-1,3,6,trisulfonic acid) as previously described [3]. The dye was loaded in the resealed membrane vesicles during the first homogenization step in 0.32 M sucrose solution containing 10 mM Pyranine.…”

“…In mammalian isolated presynaptic nerve terminals (synaptosomes), the Na + /H + exchanger has been the only unequivocally demonstrated H + -transporting mechanism in the synaptosomal preparation [1][2][3][4]. Regarding the Cl -/ HCO 3 -exchanger, previous data [2,5] indicated that there was no experimental evidence supporting the participation of HCO 3 --based transport mechanisms in synaptosomes, even though the neuronal somas posses one or more types of Cl -/HCO 3 -exchangers [6][7][8][9], in addition to the Na + /H + exchanger.…”

“…It is possible that this symporter is confined to the nerve terminals, considering that Baxter and Chesler [9] reported that under nominally HCO 3 --free conditions, the removal of external Cl -did not alter the steady state pH cyt in cultured fetal rat hippocampal neurons.…”

Mag-Fura-2 (Furaptra) Exhibits both Low (µM) and High (nM) Affinity for Ca²⁺

“…These buffers were supplemented with 6.8 ÌM of nigericin and 2 ÌM valinomycin to collapse the H + and K + gradients. Collapsing of the H + and K + gradients is essential, since it has been shown that certain cell types exhibit changes in pH in upon A23187 treatment [17,26]. To evaluate the in situ sensitivity of MagFura-2, cells were co-loaded with high-and low-affinity Ca 2+ probes.…”