Elevated lysosomal pH in neuronal ceroid lipofuscinoses (NCLs).

We report here the intracellular (pHi) and lysosomal pH in fibroblasts of six forms of neuronal ceroid lipofuscinoses (NCLs). Acid extrusion rate and pH(i) values were measured by the membrane-permeant acetoxymethyl ester of the indicator dye, 2',7'-bis(carboxyethyl)-5-(and-6)-carboxy-fluorescein (BCECF) and lysosomal pH by a spectrofluorometric assay utilizing a novel acidotropic probe, Lysosensor yellow/blue. Intracellular pH was normal in all NCLs. Elevated lysosomal pH was detected in all NCL forms except CLN2 and CLN8. Elevated pH most probably disturbs the catalytic activity of lysosomes and is one important factor in explaining accumulation of ceroid and lipofuscin-like autofluorescent lipopigments characteristic of NCLs. Using the novel spectrofluorometric assay introduced in this study provides a fast and repeatable technique to measure intralysosomal pH from cell suspensions.

Regulation of intracellular pH in salamander retinal rods.

1. We measured intracellular pH (pHi) in rods isolated from the retina of the axolotl salamander, Ambystoma mexicanum, using the fluorescent indicator 2',7'-bis(carboxyethyl)-5(and -6)-carboxyfluorescein (BCECF). 2. The light exposures associated with data acquisition had no marked effect on pHi. There was no sharp change between the value obtained from the first exposure of dark-adapted rods and subsequent readings. Increasing the acquisition frequency from 1 to 10 min-1 either had no effect, or brought about a slow acidification, which was stopped or reversed when the low frequency was restored. 3. In nominally HCO3(-)-free solution at pH 7.5, the rods had a steady-state pHi of 7.09 +/- 0.02 (n = 46) and a buffering power (beta i) of 24 +/- 1 mM (pH unit)-1 (n = 48). The buffering power was virtually constant in the pH range 6.6-8.0. In the same range, pHi dependent linearly on perfusion pH (pHo) with regression coefficients of 0.4-0.5. 4. There were no significant differences between the inner and outer segment of intact rods as regards steady-state pHi or responses to experimental treatments. 5. Recovery from an intracellular acid load imposed by sodium propionate or an NH4Cl prepulse in nominally bicarbonate-free perfusate was completely blocked by decreasing the extracellular Na+ concentration to 7 mM, and slowed by 86% by applying 1 mM amiloride. 6. Introduction of 2% CO2-13 mM HCO3- caused an alkalinization that was often preceded by a transient acidification. Steady-state pHi was on average 0.1 pH units higher than in nominally bicarbonate-free solution. The mean acid extrusion rate, calculated on the assumption that CO2-HCO3- behaves as an open system, was 19% higher (31 +/- 2 mM h-1) than in a solution buffered only by Hepes (26 +/- 2 mM h-1). 7. In the presence of CO2-HCO3-, 100 microM 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) decreased the acid extrusion rate by 20% on average. Lowering the extracellular Cl-concentration to 7 mM raised pHi, but did not significantly affect the acid extrusion rate. 8. We conclude that retinal rods regulate pHi by both Na(+)-H+ exchange and mechanism(s) involving HCO3(-)-Cl- exchange. In the present conditions, the Na(+)-H+ exchanger appears as the dominant mechanism for acid extrusion.

The absorption of hydrophobic chemicals across perfused rainbow trout gills: methodological aspects.

The absorption rates of 4-bromophenol, 2,4-dibromophenol, 3,4,5-trichloroguaiacol, and tetrachloroveratrol across fish gill epithelium have been measured in a perfused gill preparation from rainbow trout (Oncorhynchus mykiss). In additional experiments the absorption rate of tetrachloroveratrol was measured in free swimming fish. The absorption rates were perfusion limited in gills perfused with a saline containing 2% polyvinylpyrrolidone MW 40,000 (PVP-40) as a plasma protein substitute. Replacement of PVP-40 with 2% bovine serum albumin increased the absorption rates 5-10 times, and in this case rates were not perfusion limited. Furthermore, they were not ventilation limited at ventilatory water flow rates between 0.45 and 1.5 liter/(min 100 g). Our observations suggest that the absorption rates in the presence of albumin are limited by diffusion in the epithelial cell layers. The rates measured in the perfused gills are in close agreement with those measured in vivo. We conclude that the preparation is well suited to investigate the mechanisms behind the absorption of hydrophobic compounds across fish gills and how the absorption rate is related to the physicochemical properties of the compounds, as well as to environmental factors.

Simultaneous measurement of intracellular and extracellular carbonic anhydrase activity in intact muscle fibres.

The presence and properties of membrane-bound carbonic anhydrases have been difficult to establish with conventional enzymological and immunohistochemical techniques. We have therefore studied carbonic anhydrase (CA) activity in single intact crayfish muscle fibres by superfusing them alternately with a 4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid (HEPES)-buffered and a 5% CO2/HCO3(-)-buffered solution (pH of both solutions 7.4) while recording the intracellular pH (pHi) and extracellular surface pH (pHs) with H(+)-selective microelectrodes. In order to prevent regulation of pHi, Na+ ions were replaced with N-methyl-D-glucamine. Application of the CO2-containing solution produced a fast fall in pHi coupled with a marked (0.5-0.8 pH units) transient increase in pHs. Submicromolar concentrations of acetazolamide (AA) and benzolamide (BA) immediately blocked the pHs transients. A concentration of 8 x 10(-8) M (both compounds) reduced the response by 50%. A more prolonged application of BA and AA at concentrations of 10(-7) M and higher slowed the CO2-induced fall in pHi, which attained a rate corresponding to uncatalysed intracellular CO2 hydration at an AA concentration of 10(-4) M. The effect of BA and AA on the pHi changes developed with a time constant of 25 +/- 4 min and 7.6 +/- 1.5 min respectively, indicating that BA is less permeant than AA. CNO- ions (5 x 10(-4) M) had little effect on the CO2-induced pHs and pHi changes.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of action of GABA on intracellular pH and on surface pH in crayfish muscle fibres.

1. The mode of action of gamma-aminobutyric acid (GABA) on intracellular pH (pHi) and surface pH (pHs) was studied in crayfish muscle fibres using H(+)-selective microelectrodes. The extracellular HCO3- concentration was varied (0-30 mM) at constant pH (7.4). 2. GABA (5 x 10(-6)-10(-3) M) produced a reversible fall in pHi which showed a dependence on the concentrations of both GABA and HCO3-. The fall in pHi was associated with a transient increase in pHs and it was inhibited by a K(+)-induced depolarization. 3. In the presence of 30 mM-HCO3-, a near-saturating concentration of GABA (0.5 mM) produced a mean fall in pHi of 0.43 units. This change in pHi accounted for about two-thirds of the GABA-induced decrease (from -66 to -29 mV) in the sarcolemmal H+ driving force, while the rest was due to the simultaneous depolarization. 4. The apparent net efflux of HCO3- (JHCO3e) produced by a given concentration of GABA was estimated on the basis of the instantaneous rate of change of pHi. In the presence of 30 mM-HCO3-, JHCO3e following exposure to 0.5 mM-GABA had a mean value of 8.0 mmol l-1 min-1. Under steady-state conditions (at plateau acidosis), the intracellular acid load produced by 0.5 mM-GABA was about 25% of that seen at the onset of the application. 5. The GABA-induced HCO3- permeability, calculated on the basis of the flux data, showed a concentration dependence similar to that of the GABA-activated conductance described in previous work. 6. The GABA-induced increase in pHs was immediately blocked by both a membrane-permeant inhibitor of carbonic anhydrase (acetazolamide, 10(-6) M) and by a poorly permeant inhibitor (benzolamide, 10(-6) M). 7. Application of acetazolamide (10(-4) M) for 5 min or more produced a decrease of up to 60% in the maximum rate of fall of pHi at GABA concentrations higher than 20 microM. 8. The recovery of the GABA-induced acidosis was associated with a fall in pHs. The recovery was completely blocked in solutions devoid of Na+ or of Cl-, as well as by DIDS (4,4'-diisothiocyanostilbene-2,2'-disulphonic acid, 10(-5) M). This indicates that the maintenance of a non-equilibrium H+ gradient at plateau acidosis and the recovery of pHi are attributable to Na(+)-dependent Cl(-)-HCO3- exchange. 9. We conclude that the effects of GABA on pHi and pHs are due to electrodiffusion of HCO3- across postsynaptic anion channels.(ABSTRACT TRUNCATED AT 400 WORDS)

Influence of GABA-gated bicarbonate conductance on potential, current and intracellular chloride in crayfish muscle fibres.

1. The effects of gamma-aminobutyric acid (GABA) on membrane potential and conductance as well as on the intracellular Cl- activity (aiCl) and intracellular pH (pHi) were studied in crayfish muscle fibres using a three-microelectrode voltage clamp and ion-selective microelectrodes. In the presence of CO2-HCO3-, the intracellular HCO3- activity (aiHCO3) was estimated from pHi. 2. In a nominally HCO3(-)-free solution, a near-saturating concentration of GABA (0.2 mM) produced a marked increase in membrane conductance but little change in potential. In a solution containing 30 mM-HCO3- (equilibrated with 5% CO2 + 95% air; pH 7.4), the GABA-induced increase in conductance was associated with a depolarization of about 15 mV, with an increase in aiCl and with a decrease in aiHCO3. All these effects were blocked by picrotoxin (PTX). The depolarizing action of GABA was augmented following depletion of extracellular and intracellular Cl-. 3. The GABA-induced increase in aiCl which took place in the presence of HCO3- was blocked by clamping the membrane potential at its resting level. This indicates that the increase in aiCl was due to passive redistribution of Cl-. In both the presence and absence of HCO3-, the GABA-activated transmembrane flux of Cl- showed reversal at the level of the resting potential, which indicates that under steady-state conditions the Cl- equilibrium potential (ECl) is identical to the resting potential. 4. In a Cl(-)-free, 30 mM-HCO3(-)-containing solution, 0.5 mM-GABA produced a PTX-sensitive increase in conductance which amounted to 15% of the conductance activated in the presence of Cl-. In the absence of both Cl- and HCO3-, the respective figure was 2.8%. Assuming constant-field conditions, the conductance data yielded a permeability ratio PHCO3/PCl of 0.42 for the GABA-activated channels. 5. In a Cl(-)-containing, HCO3(-)-free solution, the reversal potential of the GABA-activated current (EGABA) was, by about 1 mV, less negative than the resting membrane potential (RP). In a solution containing Cl- and 30 mM-HCO3-, EGABA-RP was 12 mV. Simultaneous measurements of EGABA, aiCl and aiHCO3 (pHi) gave a PHCO3/PCl value of 0.33. 6. In a Cl(-)-free, HCO3(-)-containing solution EGABA was close to the HCO3- equilibrium potential (EHCO3) and an experimental acidosis which produced a negative shift in EHCO3 was associated with a similar shift in EGABA.(ABSTRACT TRUNCATED AT 400 WORDS)

Factors affecting the absorption of phenolics and carboxylic acids in the guppy (Poecilia reticulata).

The rate of absorption of 17 phenols, anisoles, and carboxylic acids in the guppy (Poecilia reticulata Peters) was measured at pH levels from 3 to 9. The rate was directly proportional to the concentration, and the compounds did not interfere with each other's absorption. The primary route of uptake was across the gill epithelium, but 25-40% of the total amount penetrated across the skin. When the fish were exposed at a pH low enough to prevent ionization of acidic compounds, the initial rate of uptake increased with lipophilicity up to a partition coefficient of about 10(4). Above this point, the absorption rate did not correlate with lipophilicity indicating that the uptake rate of the most lipophilic compounds was limited by the unstirred water layers. The effect of lipophilicity on absorption accounts for about half of the effect found in toxicity and bioconcentration. The absorption rate of acids declined with pH rising, but did not follow changes in the concentration of nonionized acid. The slopes of the absorption rate vs pH curves were distinctly less steep than the slope of the dissociation curve. In addition, the curves of the most lipophilic acids showed a shift to the right, which can be ascribed to the unstirred layers on the surface of the epithelia.

Relation between physicochemical properties of phenols and their toxicity and accumulation in fish.

The 96-hr LC50 values of 21 substituted phenols for the guppy (Poecilia reticulata) were determined at pH levels 6-8 and related to the lipophilicity defined as log P from the 1-octanol/water system, and to the delta pKa value (pKa of phenol-pKa). Log P was the more important parameter and exhibited a good correlation with log(1/LC50) at pH levels. The contribution of delta pKa when introduced as a second parameter into the regression equation was dependent on the pH of water: at pH 6 it was positive but turned negative as the pH was raised to 8. If the LC50 values were corrected for ionization using an empirically formulated relation between toxicity and pH, the resulting regression equation could be used to predict the toxicity at any pH from 6 to 8. When corrected for ionization, log BCF (the bioconcentration factor) of 8 phenols was highly correlated with log P but not with delta pKa. The regression of log BCF on log P sufficed to explain the regression of toxicity on lipophilicity.

Influence of pH on the toxicity of substituted phenols to fish.

The 96-hr LC50 values of six chloro-, bromo-, and nitro- substituted phenols to the guppy (Poecilia reticulata Peters) were determined by a semistatic method in the pH range of 5 to 8. The pH did not affect appreciably the toxicity of 4-chlorophenol, which is primarily nonionized over the whole range, but the toxicity of more acidic phenols decreased as the pH increased. The changes in toxicity were substantially smaller than they would be, if only the nonionized phenol form were toxic. The results could be explained by assuming that the phenate ion also contributes to the toxicity, but its molar toxicity decreases with rising pH.