pH dependence of transmission at electrotonic synapses of the crayfish septate axon

Gap junctions between segments of the crayfish septate axon mediate electrotonic transmission of impulses propagating along the length of the nerve cord. We simultaneously measured intracellular pH (pHi) and gap junctional conductance (gj) while axons were exposed to saline equilibrated with CO2, weak acids, and the weak base ammonium chloride. Normal pHi is about 7.1. When pHi is elevated, gj is unaffected. When pHi is reduced, gj declines with an apparent pK of about 6.7 and a Hill coefficient of about 2.7. We also measured effects of pHi on non-junctional conductance (gnj) and on the coupling coefficient, k. Over the pHi range 6.2-8, gnj increases approximately linearly with pHi. Since k is a function of gj and gnj, it reached a maximum at about pHi 7.1, decreasing at higher and lower pHi. The pHi dependence of gj in crayfish septate axon is less steep and has a lower apparent pK than the gj-pHi relation in two vertebrate embryos previously examined. This finding illustrates a difference in gating among analogous and possibly homologous membrane channels.     

Acidic regulation of junction channels between glomus cells in the rat carotid body. Possible role of [Ca]i

The purpose of this work was to characterize the gap junctions between cultured glomus cells of the rat carotid body and to assess the effects of acidity and accompanying changes in [Ca²⁺]_i on electric coupling. Dual voltage clamping of coupled glomus cells showed a mean macrojunctional conductance (G_j) of 1.16 nS±0.6 (S.E.), range 0.15–4.86 nS. At normal pH_o (7.43), a steady transjunctional voltage (ΔV_j=100.1±10.9 mV) showed multiple junction channel activity with a mean microconductance (g_j) of 93.98±0.6 pS, range 0.3–324.5 pS. Single-channel conductances, calculated as variance/mean g_j, gave a mean value of 16.7±0.2 pS, range 5.13–39.38 pS. Manual measurements of single-channel activity showed a mean g_j of 22.03±0.2 pS, range 1.3–160 pS. Computer analysis of the noise spectral density distribution gave a channel mean open time of 12.7±1.5 ms, range 6.37–23.42 ms. The number of junction channels, estimated in each experiment from G_j/single-channel g_j, showed a range of 7 to 258 channels (mean, 107.2). Optical measurements of [Ca²⁺]_i gave a mean value of 80.2±4.27 nM at pH_o of 7.43. Acidification of the medium with lactic acid (1 mM, pH 6.3) induced: 1) Variable changes in G_j (decreases and increases); 2) A significant decrease in mean g_j (to 80.36±0.34 pS) and in single-channel conductance (g_j=12.8±0.2 pS in computer analyses and 17.23±0.2 pS when measured by hand); 3) Variable changes in open times, resulting in a similar mean (12.8±1.5 ms) and 4) No change in the number of junction channels. When pH_o was lowered to 6.3 [Ca²⁺]_i did not change significantly (there were increases and decreases). However, when pH_o was lowered to 4.4, [Ca²⁺]_i increased significantly to 157.1±8.1 nM. It is concluded that saline acidification to pH 6.3 depresses the conductance of junction channels and this effect may be either a direct effect on channel proteins or synergistically enhanced by increases in [Ca²⁺]_i. However, there are no studies correlating changes of [Ca²⁺]_i and intercellular coupling in glomus cells. Stronger acidification (pH_o 4.4), producing much larger changes in [Ca²⁺]_i, may enhance this synergism. But, again, there are no studies correlating these effects.     

Octanol,a gap junction uncoupling agent,changes intracellular [H+] in rat astrocytes

Octanol rapidly closes gap junction channels but its mechanism of action is not known. Because intracellular [H⁺], pH_i, also affects the conductance of gap junctions, we studied octanol's effects on pH_i in cultured rat astrocytes, which are highly coupled cells. Octanol (1 mM) caused an acid shift in the pH_i of 90% of rat hippocampal astrocytes which averaged −0.19 ± 0.09 pH units in magnitude. In 58% of the cells tested, a biphasic change in pH_i was seen; octanol produced an initial acidification lasting ∼10 min that was followed by a persistent alkalinization. The related gap junction uncoupling agent, heptanol, had similar effects on pH_i. Octanol-induced changes in pH_i were similar in nominally HCO₃⁻-free and HCO₃⁻-containing solutions, although the rate of initial acidification was significantly greater in the presence of HCO₃⁻. The initial acidification was inhibited in the presence of the stilbene DIDS, an inhibitor of Na⁺/HCO₃⁻ cotransport, indicating that octanol caused acidification by blocking this powerful acid extruder. The alkalinization was inhibited by amiloride which blocks the Na⁺/H⁺ exchanger (NHE), an acid extruder, suggesting that the alkaline shift induced by octanol was caused by stimulation of NHE. As expected, octanol's effects on astrocytic pH_i were prevented by removal of external Na⁺, which blocks both Na⁺/HCO₃⁻ cotransport and NHE. Octanol had only small effects on intracellular Ca²⁺ (Ca²⁺_i) in astrocytes. Hepatocytes which, like astrocytes, are strongly coupled to one another, showed no change in pH_i with octanol application. Fluorescence recovery after photobleaching (FRAP) was used to study the effect of changes in astrocyte pH_i on degree of coupling in hippocampal astrocytes. Coupling was decreased by intracellular acid shifts ∼−0.2 pH units in size. Octanol's effects on astrocyte pH_i were complex but a prompt initial acidification was nearly always seen and could contribute to the uncoupling action of this drug in astrocytes. Because octanol uncouples hepatocytes without changing their pH_i, this compound clearly can influence gap junctional conductance independent of changes in pH_i. © 1996 Wiley-Liss, Inc.     

Variation of gap junction sensitivity to H ions with time of day

Conductance of gap junctions between segments of the lateral axons of the crayfish nerve cord is reduced by cytoplasmic acidification. Simultaneous measurements of axoplasmic pH and junctional conductance at 09.00 and 12.00 h revealed a shift of about 0.25 pH units in the apparent pK of the crayfish electrotonic synapse. These findings indicate that gating mechanisms of gap junctions are not fixed properties, but may rather be modulated by other, possibly humoral, factors.     

Intracellular acidification of the leech giant glial cell evoked by glutamate and aspartate

Glutamate is an excitatory receptor agonist in both neurones and glial cells, and, in addition, glutamate is also a substrate for glutamate transporter in glial cells. We have measured intracellular and extracellular pH changes induced by bath application of glutamate, its receptor agonist kainate, and its transporter agonist aspartate, in the giant neuropile glial cell in the central nervous system of the leech Hirudo medicinalis, using double-barrelled pH-sensitive microelectrodes. The giant glial cells responded to glutamate and aspartate (100–500 μM), and kainate (5–20 μM) with a membrane depolarization or an inward current, and with a distinct intracellular acidification. Glutamate and aspartate (both 500 μM) evoked a decrease in intracellular pH (pH_i) by 0.187 ± 0.081 (n = 88) and 0.198 ± 0.067 (n = 86) pH units, respectively. With a resting pH_i of 7.1 or 80 nM H⁺, these acidifications correspond to a mean increase of the intracellular H⁺ activity by 42 nM and 45 nM. Kainate caused a decrease of pH_i by 0.1 − 0.35 pH units (n = 15). The glutamate/aspartate-induced decrease in pH_i was not significantly affected by the glutamate receptor blockers kynurenic acid (1 mM) and 6-cyano-7-dinitroquinoxaline-2,3-dione (CNQX, 50–100 μM), which greatly reduced the kainate-induced change in pH_i. Extracellular alkalinizations produced by glutamate and aspartate were not affected by CNQX. Reduction of the external Na⁺ concentration gradually decreased the intracellular pH change induced by glutamate/aspartate, indicating half maximal activation of the acidifying process at 5–10 mM external Na⁺ concentration. When all external Na⁺ was replaced by NMDG⁺, the pH_iresponses were completely suppressed (glutamate) or reduced to 10% (aspartate). When Na⁺ was replaced by Li⁺, the glutamate- and aspartate-evoked pH_i responses were reduced to 18% and 14%, respectively. Removal of external Ca²⁺ reduced the glutamate- and aspartate-induced pH_i responses to 93 and 72%, respectively. The glutamate/aspartate-induced intracellular acidifications were not affected by the putative glutamate uptake inhibitor amino-adipidic acid (1 mM). DL-aspartate-β-hydroxamate (1 mM), and dihydrokainate (2 mM), which caused some pH_i decrease on its own, reduced the glutamate/aspartate-induced pH_i responses by 40 and 69%, respectively. The putative uptake inhibitor DL-threo-β-hydroxyaspartate (THA, 1 mM) induced a prominent intracellular acidification (0.36 ± 0.05 pH units, n = 9), and the pH_i change evoked by glutamate or aspartate in the presence of THA was reduced to less than 10%. The results indicate that glutamate, aspartate, and kainate produce substantial intracellular acidifications, which are mediated by at least two independent mechanisms: 1) via activation of non-NMDA glutamate receptors and 2) via uptake of the excitatory amino acids into the leech glial cell. © 1997 Wiley-Liss Inc.     

NMDA induces a biphasic change in intracellular pH in rat hippocampal slices

As alterations in intracellular pH (pH_i) tend to exert a profound effect on the properties of cells, this study was undertaken to examine NMDA-induced changes in pH_i in rat hippocampal slices using the BCECF fluorescent technique. The ‘resting' pH_i in the CA1 pyramidal cell layers was 6.93±0.07 (mean±S.D., n=72 slices) in 25 mM HCO₃⁻/5% CO₂-buffered solution at 37°C. Exposure of hippocampal slices to NMDA in the range of 10–1000 μM produced a biphasic change in pH_i: an initial transient alkaline shift was followed by a long-lasting acid shift. Dizocilpine (10 μM) but not CNQX (40 μM) blocked the NMDA-induced changes in pH_i. In 0 Ca medium (0 mM Ca²⁺ supplemented 1 mM EGTA, referred to as 0 Ca), pH_i acid shift caused by NMDA (20 μM) declined by about 11%, whereas the initial alkaline shift almost completely disappeared. In an independent experiment, the NMDA-induced increase in intracellular Ca²⁺ ([Ca²⁺]_i) was reduced by more than 80% in 0 Ca medium. Glucose substitution using equimolar pyruvate (as an energy-yielding substrate) suppressed this NMDA-induced pH_i acid shift by two-thirds, while the NMDA-induced pH_i alkaline shift was enhanced. Fluoride (10 mM), a glycolytic inhibitor, abolished NMDA-induced pH_i acid shift. Furthermore, the lactate content of hippocampal slices was markedly increased following exposure to NMDA. In conclusion, activation of NMDA receptors in rat hippocampal slices evokes a biphasic change in pH_i. The initial alkaline shift is suggested to be associated with calcium influx, and the following acid shift may be caused by an increase in lactate production through the acceleration of glycolysis, as well as the increased [Ca²⁺]_i. The pH_i acid shift produced by the increased lactate may contribute to proton modulation of the NMDA receptor and NMDA-induced cell injury or death.     

Electrical coupling between cultured glomus cells of the rat carotid body: observations with current and voltage clamping

Electrically coupled pairs of cultured rat glomus cells were used. In one group of experiments, both cells were current-clamped. Delivery of positive or negative pulses to Cell 1 elicited appreciable voltage noise in this cell and large action potentials (probably Ca²⁺ spikes) in about 10% of them. Both passive and active electrical events spread to Cell 2, presumably through the gap junctions between them. The coupling coefficient (K_c) was larger for the spikes than for non-regenerative voltage noise. In another group of experiments, Cell 1 was current-clamped and Cell 2 was voltage-clamped at Cell 1 E_M. Pulses of either polarity, delivered to Cell 1, produced current flow through the intercellular junction and allowed direct measurement of junctional currents (I_j) and total conductances (G_j). I_j had a mean value of about 12.5 pA and G_j of 391 pS. Unitary (presumably single channel) conductance (g_j) was about 78 pS.     

Interregional differences in brain intracellular pH and water compartmentation during acute normoxic and hypoxic hypocapnia in the anesthetized dog

Interregional differences in intracellular pH (pH_i) in brain tissue, and its regulation following 1 and 5 h of respiratory alkalosis (with and without hypoxemia) were determined in N₂O anesthetized dogs. Two techniques for pH_i estimation were used (Tco₂ and¹⁴C-DMO) and included corrections for measured extracellular fluid (³⁵SO₄²⁻) space (ECS). Cortical pH_i by the two techniques agreed closely in control and in 3 of the 4 experimental conditions, suggesting: (a) our estimation of extracellular fluid (ECF) [HCO₃⁻] from measured CSF [HCO₃⁻] was a valid ssumption; and (b) our method had sufficient resolution to determine the magnitude of brain pH_i regulation during respiratory acid-base disturbances.When moderate normoxic respiratory alkalosis (PaCO₂ ≈ 25 mm Hg) was imposed for 5 h, pH_i (in most brain regions) was well regulated and always exceeded the incomplete regulation noted in bulk CSF. When moderate hypoxemia (PaO₂ ≈ 45 mm Hg) accompanied hypocapnia, pH_i was more closely regulated during the early phase (1 h) of respiratory alkalosis.Increased levels of metabolic acids (especially lactic acid) were critical to brain pH_i regulation during the initial hour of respiratory alkalosis and accounted for much of the independent effect of hypoxemia on pH_i regulation. However, these metabolic acids remained unchanged as pH_i was more completely regulated between 1 and 5 h of continued hypocapnia or hypoxic hypocapnia. This time-dependent tregulation of pH_i may involve some regulatory role for changed transmembrane fluxes of H⁺ and/or HCO₃⁻.Significant interregional differences were observed in both pH_i and in ECS; with tendencies toward more alkaline pH_i and lower ECS in brain stem and white matter. With respiratory alkalosis ECS fell and intracellular fluid increased in both cortex and caudate nucleus, possibly reflecting an osmotic effect of increased metabolic acid levels or reduction in cell membrane ion pumping.     

The influence of calcium transients on intracellular pH in cortical neurons in primary culture

The objective of this study was to assess the influence of Ca²⁺ influx on intracellular pH (pH_i) of neocortical neurons in primary culture. Neurons were exposed to glutamate (100–500 μM) or KCl (50 mM), and pH_i was recorded with microspectroflurometric techniques. Additional experiments were carried out in which calcium influx was triggered by ionomycin (2 μM) or the calcium ionophore 4-Br-A23187 (2 μM). Glutamate exposure either caused no, or only a small decrease in pH_i (ΔpH ≈ 0.06 units). When a decrease was observed, a rebound rise in pH_i above control was observed upon termination of glutamate exposure. In about 20% of the cells, the acidification was more pronounced (ΔpH ≈ 0.20 units), but all these cells had high control pH_i values, and showed gradual acidification. Exposure of cells to 50 mM KCl consistently increased pH_i. Since this increase was similar in the presence and nominal absence of HCO₃⁻, it probably did not reflect influx of HCO₃⁻ via a Na⁺-HCO₃⁻ symporter. Furthermore, since it occurred in the absence of external Ca²⁺ (or a measurable rise in Ca_i²⁺) it seemed independent of Ca²⁺ influx. It is tentatively concluded that the rise in pH_i was due to reduced passive influx of H⁺ along the electrochemical gradient, which is reduced by depolarization. In Ca²⁺-containing solutions, depolarization led to a rebound increase in pH_i above control. This, and the rebound found after glutamate transients, may reflect Ca²⁺-triggered phosphorylation and upregulation of the Na⁺/H⁺ antiporter which extrudes H⁺ from the cell. Ionomycin and 4-Br-A23187 gave rise to a large rise in Ca_i²⁺ and to alkalinization of the cell (ΔpH ≈ 0.5). Since amiloride or removal of Na⁺ from the external solution did not alter the rise in pH_i, it was probably not due to accelerated H⁺ extrusion. However, removal of Ca²⁺ from extracellular fluid prevented the rise, suggesting that it was secondary to Ca²⁺/2H⁺ exchange across plasma membranes.     

Intracellular acidification and Ca2+ transients in cultured rat cerebellar astrocytes evoked by glutamate agonists and noradrenaline

The effect of different neurotransmitters on the intracellular pH (pH_i) and intracellular calcium (Ca²⁺_i) was studied in cultured astrocytes from neonatal rat cerebellum, using the fluorescent dyes 2,7′-bis(carboxyethyl)-5,6-carboxy-fluorescein (BCECF) and Fura-2. Application of glutamate or kainate (100 μM) in a HEPES-buffered, CO₂/HCO₃^?-free saline induced a decrease in pH_i and an increase in Ca²⁺_i. Amplitude and time course of the pH_i and Ca²⁺_i transients were different. Glutamate and kainate evoked a mean acidification of 0.22 ± 0.05 (n = 29) and 0.20 ± 0.04 (n = 12) pH units, respectively. The changes in pH_i and Ca²⁺_i induced by kainate, but not by glutamate, were inhibited by 6-cyano-7-dinitroquinozalin-2,3-dion (CNQX; 50 μM). In order to elucidate the mechanism of the agonist-induced acidification, whether the pH_i changes were secondary to the Ca²⁺ rises was tested. In the absence of extracellular Ca²⁺, the kainate-induced Ca²⁺_i transient was suppressed, while the intracellular acidification was only reduced by 13%. Removal of extracellular Ca²⁺ reduced the glutamate-induced pH_i change by 8%, while the second component of the Ca²⁺_i transient was abolished. Application of trans-(±)-1-amino-(1S,3R)-cyclopentadicarboxylic acid (t-ACPD, 100 μM), a metabotropic glutamate receptor agonist, and of noradrenaline (20 μM) evoked a Ca²⁺_i increase, but no change of pH_i. D-aspartate, which has a low affinity to glutamate receptors, but is known to be transported by the glutamate uptake system in some astrocytes, evoked an intracellular acidification, similar to that induced by glutamate, but no Ca²⁺_i transient. The results suggest that the kainate-induced acidification is only partly due to the concomitant Ca²⁺_i rise, while the glutamate/aspartate-induced acidification is mainly due to the activation of the glutamate uptake system. © 1995 Wiley-Liss, Inc.     

CO interact with intracellular [H] with and without CO2–HCO3 in the cat carotid chemosensory discharge

To test the hypothesis whether CO₂–HCO₃⁻ buffer is essential for the expression of chemoreception and to distinguish between pH_i and pH_o interaction with pCO in the carotid chemosensory response, we superfused-perfused in vitro cat carotid bodies using HEPES-Tyrode's solution with and without CO₂–HCO₃⁻, and compared the responses at the same pH_o in the absence and presence of light. In the absence of light, pCO (>138 Torr) stimulated the carotid body chemoreceptors in CO₂–HCO₃⁻ buffer at pH_o of 7.40, whereas pCO (69–550 Torr) did not stimulate the neural discharge in HEPES buffer at the pH_o of 7.4–7.1 but did so below pH_o 7.1. In the presence of light, all the responses were diminished proportionately.     

Ammonium‐evoked alterations in intracellular sodium and pH reduce glial glutamate transport activity

The clearance of extracellular glutamate is mainly mediated by pH‐ and sodium‐dependent transport into astrocytes. During hepatic encephalopathy (HE), however, elevated extracellular glutamate concentrations are observed. The primary candidate responsible for the toxic effects observed during HE is ammonium (NH₄⁺/NH₃). Here, we examined the effects of NH₄⁺/NH₃ on steady‐state intracellular pH (pH_i) and sodium concentration ([Na⁺]_i) in cultured astrocytes in two different age groups. Moreover, we assessed the influence of NH₄⁺/NH₃ on glutamate transporter activity by measuring D ‐aspartate‐induced pH_i and [Na⁺]_i transients. In 20–34 days in vitro (DIV) astrocytes, NH₄⁺/NH₃ decreased steady‐state pH_i by 0.19 pH units and increased [Na⁺]_i by 21 mM. D ‐Aspartate‐induced pH_i and [Na⁺]_i transients were reduced by 80–90% in the presence of NH₄⁺/NH₃, indicating a dramatic reduction of glutamate uptake activity. In 9–16 DIV astrocytes, in contrast, pH_i and [Na⁺]_i were minimally affected by NH₄⁺/NH₃, and D ‐aspartate‐induced pH_i and [Na⁺]_i transients were reduced by only 30–40%. Next we determined the contribution of Na⁺, K⁺, Cl^?‐cotransport (NKCC). Immunocytochemical stainings indicated an increased expression of NKCC1 in 20–34 DIV astrocytes. Moreover, inhibition of NKCC with bumetanide prevented NH₄⁺/NH₃‐evoked changes in steady‐state pH_i and [Na⁺]_i and attenuated the reduction of D ‐aspartate‐induced pH_i and [Na⁺]_i transients by NH₄⁺/NH₃ to 30% in 20–34 DIV astrocytes. Our results suggest that NH₄⁺/NH₃ decreases steady‐state pH_i and increases steady‐state [Na⁺]_i in astrocytes by an age‐dependent activation of NKCC. These NH₄⁺/NH₃‐evoked changes in the transmembrane pH and sodium gradients directly reduce glutamate transport activity, and may, thus, contribute to elevated extracellular glutamate levels observed during HE. © 2008 Wiley‐Liss, Inc.     

Independent changes of intracellular calcium and pH in identified leech glial cells

The intracellular Ca²⁺ (Ca²⁺_i) and the intracellular pH (pH_i) were measured in identified neuropile glial cells in the central nervous system of the leech Hirudo medicinalis, using the fluorescent dye fura-2, and double-barrelled, neutral carrier, pH-sensitive microelectrodes. Different stimuli were used to elicit Ca²⁺_i and/or pH_i changes, such as application of ammonium, high external K⁺-concentration, and low external pH. Ammonium (20 mM) and high external K⁺ (20 mM), which depolarized the glial membrane by 20–30 mV, evoked rapid and large rises of Ca²⁺_i. In contrast to the Ca²_i changes, amplitude and direction of the pH_i changes were dependent on the presence of CO₂/HCO₃^? in the saline. The addition of CO₂/HCO₃^?, and the subsequent reduction of external pH from 7.4 to 7.0, had no effect on Ca²⁺_i, but caused significant changes of pH_i. The results suggest that the ammonium- and K⁺-induced Ca²⁺_i rises were due to the membrane depolarization leading to a Ca²⁺ influx through voltage-gated Ca²⁺ channels in the glial membrane, while the pH_i changes resulted from movements of ammonia and from the activation or inhibition of the Na⁺-HCO₃^? cotransporter. This indicates that changes of intracellular Ca²⁺ and pH can occur independently of each other, suggesting that the homeostasis of these ions is not necessarily interrelated in these glial cells.     

Hypoxia affects differently the intracellular pH of clustered and isolated glomus cells of the rat carotid body

Clustered and isolated glomus cells, cultured from rat carotid bodies, were exposed to hypoxia (pO₂ 2–30 torr) induced by applications of sodium-dithionite (Na₂S₂O₄). Hypoxia decreased or increased intracellular pH (pH_i) of clustered cells about equally, but lowered it in most isolated cells. The levels of intracellular acidification were similar in both groups whereas alkalinization was more pronounced in the clusters. The H⁺ equilibrium potential (E_H) and its changes during hypoxia (ΔE_H), were determined almost exclusively by pH_i. Seventy-five percent of clustered cells became depolarized whereas 80% of isolated cells underwent hyperpolarization. In both groups, changes in the resting potential (ΔE_M) were directly and significantly correlated with ΔE_H, thus ΔpH_i. These observations support the view that clustered and isolated rat glomus cells behave differently. This difference may occur because of the presence (in the clusters) or absence (in isolated cells) of enveloping sustentacular cell processes.     

Impulse conduction in inhomogeneous axons: Effects of variation in voltage-sensitive ionic conductances on invasion of demyelinated axon segments and preterminal fibers

Conduction in inhomogeneous axons may be blocked by several mechanisms. Conduction in demyelinated axons may fail since normal internodal membrane is inexcitable, because values of sodium conductance are too low to support impulse conduction. In addition, focal loss of myelin causes increased current leakage which slows or blocks invasion of impulses into the demyelinated zone due to inadequate current density. Similar considerations apply to the invasion of non-myelinated preterminal axons from myelinated parent fibers, where conduction can be blocked as a result of inadequate current density. A cable model of an axon is presented which allows myelinated regions, regions without myelin, and variable length transition zones of redistributed channel densities, to be studied. Action potentials and membrane currents were studied. Computer simulations using this model show that the safety factor for invasion is dependent on temperature. These studies also show that small changes in axon membrane properties, at the transition region between the myelinated zone and the region without myelin, may promote invasion of the region without myelin. In particular, increasing sodium conductance (g_Na) or decreasing potassium conductance (g_K) promotes invasion. Because of the non-linear behavior of excitable membranes the spatial distribution of channels is shown also to have significant effects on invasion. Thus, relatively small degrees of membrane reorganization may lead to functional changes with respect to the invasion of demyelinated axon regions. Similarly, the properties of the heminode at the distal part of the parent myelinated fiber may determine the invasion characteristics of non-myelinated terminal axons.     

Intracellular pH regulation in single cultured astrocytes from rat forebrain

We used the fluorescent pH-sensitive dye 2′,7′-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) to monitor intracellular pH (pH_i) in single astrocytes cultured from the forebrain of neonatal rats. When exposed to a nominally CO₂/HCO₃^? -free medium buffered to pH 7.40 with HEPES at 37^°C, the cells had a mean pH_i of 6.89. Switching to a medium buffered to pH 7.40 with 5% CO₂ and 25 mM HCO₃^? caused the steady-state pH_i to increase by an average of 0.35, suggesting the presence of a HCO₃^? -dependent acid-extrusion mechanism. The sustained alkalinization was sometimes preceded by a small transient acidification. In experiments in which astrocytes were exposed to nominally HCO₃^?-free (HEPES-buffered) solutions, the application and withdrawal of 20 mM extracellular NH₄⁺ caused pH_i to fall to a value substantially below the initial one. pH_i spontaneously recovered from this acid load, stabilizing at a value ～ 0.1 higher than the one prevailing before the application of NH₄⁺. In other experiments conducted on cells bathed in HEPES-buffered solutions, removing extracellular Na⁺ caused pH_i to decrease rapidly by 0.5. Returning the Na⁺ caused pH_i to increase rapidly, indicating the presence of an Na⁺-dependent/HCO₃^?-independent acid-extrusion mechanism; the final pH_i after returning Na⁺ was ～ 0.08 higher than the initial value. This pH_i recovery elicited by returning Na⁺ was not substantially affected by 50 μM ethylisopropylamiloride (EIPA), but was speeded up by 50 μM 4,4′-diisothiocyanostilbene-2,2′-disulfonate (DIDS). Increasing [K⁺]_? from 5 to 25 mM caused pH_i to increase reversibly by ～ 0.2 in nominally CO₂/HCO₃^?-free solutions, and by ～ 0.1 in CO₂/HCO₃^?-containing solutions, although the initial pH_i was ～ 0.17 higher in the presence of CO₂/HCO₃^-. These results suggest the presence of a depolarization-induced alkalinization. Our results suggest the presence of both HCO₃^? dependent and -independent acid-base transport systems in cultured mammalian astrocytes, and indicate that astrocyte pH_i is sensitive to changes in either membrane voltage or [K⁺]₀ per se. © 1993 Wiley-Liss, Inc.     

Intracellular pH regulation in rat Schwann cells

We examined H⁺ and HCO₃^? transport mechanisms that are involved in the regulation of intracellular pH of Schwann cells. Primary cultures of Schwann cells were prepared from the sciatic nerves of 1–3-day-old rats. pH_i of single cells attached to cover slips was continuously monitored by measuring the absorbance spectra of the pH-sensitive dye dimethylcarboxyfluorescein incorporated intracellularly. The average pH_i of neonatal Schwann cells bathed in HEPES mammalian solution was 7.17 ± 0.02 (n = 32). In the nominal absence of HCO₃^?, pH_i spontaneously recovered from an acute acid load induced by exposing the Schwann cells to 20 mM NH₄⁺ (NH₄⁺ prepulse). This pH_i recovery from the acute acid load was totally inhibited in the absence of external Na⁺ or in the presence of 1 mM amiloride. In both cases, the pH_i recovery was readily restored upon readdition of external Na⁺ or removal of amiloride. In the steady-state, addition of amiloride caused a small and slow decrease in pH_i which was readily reversed upon removal of amiloride. In the presence of HCO₃^?, removal of external Cl- caused pH_i to rapidly and reversibly increase by 0.23 = 0.03 (n = 15) and the initial rate of alkalinization was 20.6 ± 2.7 × 10-4 pH/sec. In the absence of external Na⁺, removal of bath Cl^? still caused pH_i to increase by 0.15 ± 0.02 and the initial rate of pH_i increase was not significantly altered. In the nominal absence of HCO₃^?, removal of bath Cl- caused pH_i to increase very slightly (0.05 ± 0.01) with an initial dpH_i/dt of only 4.4 ± 0.2 × 10^?4 pH/sec (n = 4). Addition of 100 μM DIDS did not inhibit the pH_i increase caused by removal of bath Cl^?. These data indicate that (1) Rat Schwann cells regulate their pH_i via an Na-H exchange mechanism which is moderately active at steady-state pH_i. (2) In the presence of HCO₃^?, there is a Na-independent Cl-HCO₃ (base) exchanger which also contributes to regulation of intracellular pH in Schwann cells. © 1994 Wiley-Liss, Inc.     

Presynaptic pH and vesicle fusion in Drosophila larvae neurones

Both intracellular pH (pH_i) and synaptic cleft pH change during neuronal activity yet little is known about how these pH shifts might affect synaptic transmission by influencing vesicle fusion. To address this we imaged pH‐ and Ca²⁺‐sensitive fluorescent indicators (HPTS, Oregon green) in boutons at neuromuscular junctions. Electrical stimulation of motor nerves evoked presynaptic Ca²⁺_i rises and pH_i falls (～0.1 pH units) followed by recovery of both Ca²⁺_i and pH_i. The plasma‐membrane calcium ATPase (PMCA) inhibitor, 5(6)‐carboxyeosin diacetate, slowed both the calcium recovery and the acidification. To investigate a possible calcium‐independent role for the pH_i shifts in modulating vesicle fusion we recorded post‐synaptic miniature end‐plate potential (mEPP) and current (mEPC) frequency in Ca²⁺‐free solution. Acidification by propionate superfusion, NH₄⁺ withdrawal, or the inhibition of acid extrusion on the Na⁺/H⁺ exchanger (NHE) induced a rise in miniature frequency. Furthermore, the inhibition of acid extrusion enhanced the rise induced by propionate addition and NH₄⁺ removal. In the presence of NH₄⁺, 10 out of 23 cells showed, after a delay, one or more rises in miniature frequency. These findings suggest that Ca²⁺‐dependent pH_i shifts, caused by the PMCA and regulated by NHE, may stimulate vesicle release. Furthermore, in the presence of membrane permeant buffers, exocytosed acid or its equivalents may enhance release through positive feedback. This hitherto neglected pH signalling, and the potential feedback role of vesicular acid, could explain some important neuronal excitability changes associated with altered pH and its buffering. Synapse 67:729–740, 2013 . © 2013 Wiley Periodicals, Inc.     

Influence of Intra- and Extracellular Acidification on Free Radical Formation and Mitochondria Membrane Potential in Rat Brain Synaptosomes

Brain ischemia is accompanied by lowering of intra- and extracellular pH. Stroke often leads to irreversible damage of synaptic transmission by unknown mechanism. We investigated an influence of lowering of pH_i and pH_o on free radical formation in synaptosomes. Three models of acidosis were used: (1) pH_o 6.0 corresponding to pH_i decrease down to 6.04; (2) pH_o 7.0 corresponding to the lowering of pH_i down to 6.92: (3) 1 mM amiloride corresponding to pH_i decrease down to 6.65. We have shown that both types of extracellular acidification, but not intracellular acidification, increase 2′,7′-dichlorodihydrofluorescein diacetate fluorescence that reflects free radical formation. These three treatments induce the rise of the dihydroethidium fluorescence that reports synthesis of superoxide anion. However, the impact of amiloride on superoxide anion synthesis was less than that induced by moderate extracellular acidification. Superoxide anion synthesis at pH_o 7.0 was almost completely eliminated by mitochondrial uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone. Furthermore, using fluorescent dyes JC-1 and rhodamine-123, we confirmed that pH_o lowering, but not intracellular acidification, led to depolarization of intrasynaptosomal mitochondria. We have shown that pH_o but not pH_i lowering led to oxidative stress in neuronal presynaptic endings that might underlie the long-term irreversible changing in synaptic transmission.     

Effects of pH on membrane resistance in normal and dystrophic mouse skeletal muscle fibers

Membrane cable properties of skeletal muscle fibers of dystrophic mice (Rej-129) and their littermate controls were examined using a conventional two-microelectrode recording technique. Fibers from dystrophic mice had a decreased membrane resistivity (R_m) compared with those from normal mice (517 ± 27 vs 642 ± 34 Ω ? cm²), while the internal resistivities (R_i) did not differ significantly. The increase in membrane specific conductance was due to an increased Cl^? conductance (g_Cl) (2304 vs 1346 μS/cm² for normal fibers), although the K⁺ conductance (g_K) was actually decreased (234 vs 369 μS/cm² for normal fibers). With changes in pH, membrane conductances of normal and dystrophic skeletal muscle fibers varied differently, mainly due to differences in effects on the Cl^? conductance. This contrast may be due to altered regulation of internal pH in dystrophic muscle.