pH regulation in barnacle muscle fibers: dependence on intracellular and extracellular pH

1979 ◽  
Vol 237 (3) ◽  
pp. C185-C193 ◽  
Author(s):  
W. F. Boron ◽  
W. C. McCormick ◽  
A. Roos
Keyword(s):  
Ph Regulation ◽  
Muscle Fibers ◽  
Extracellular Ph ◽  
Disulfonic Acid ◽  
Extrusion Rate ◽  
Barnacle Muscle ◽  
Buffering Power ◽  
Acid Extrusion ◽  
Barnacle Muscle Fibers ◽  
Very High

Intracellular pH (pHi) regulation was studied in acid-loaded barnacle muscle fibers by monitoring recovery of pHi with a pH-sensitive microelectrode. By multiplying the rate of pHi recovery by total intracellular buffering power, the acid extrusion rate was obtained. The acid extrusion rate was greatest at low values of pHi, and declined toward zero as pHi approached normal levels. It increased as the extracellular pH (pHo) was raised either by increasing external [HCO3] ([HCO3]o) at constant PCO2 or by decreasing PCO2 at constant [HCO3]o, but more so in the former case than in the latter. These observations suggest that pHo per se is an important determinant of the acid extrusion rate, but that raising [HCO3]o by itself also stimulates acid extrusion. This would be expected if acid extrusion involves the inward movement of HCO3. When fibers were exposed to HCO3-containing solutions at very low or very high pHo, pHi drifted downward or upward, respectively; thbe drifts were inhibited by 4-acetamido-4' isothiocyanostilbene-2,2' disulfonic acid (SITS). Our results are discussed in terms of possible mechanisms of acid extrusion.

pH regulation in barnacle muscle fibers: dependence on extracellular sodium and bicarbonate

1981 ◽  
Vol 240 (1) ◽  
pp. C80-C89 ◽  
Author(s):  
W. F. Boron ◽  
W. C. McCormick ◽  
A. Roos
Keyword(s):  
Kinetic Data ◽  
Ph Regulation ◽  
Muscle Fibers ◽  
Extrusion Rate ◽  
Barnacle Muscle ◽  
Rate Of Recovery ◽  
Extracellular Sodium ◽  
Acid Extrusion ◽  
Barnacle Muscle Fibers

Intracellular pH (pHi) regulation was studied in barnacle muscle fibers with pH-sensitive microelectrodes. The cells were acid loaded, and the subsequent recovery of pHi was monitored. The rate of recovery was reduced by one-third when external Na+ ([Na+]o) was replaced by Li+, but recovery was completely abolished when Na+ was replaced by choline or N-methyl-D-glucamine. In other experiments, varying amounts of Na+ were replaced by choline, and the acid extrusion rate, derived from the recovery rate of pHi, was calculated at a single value of pHi, 6.80. The dependence of the acid extrusion rate on [Na+]o could be described by Michaelis-Menten kinetics; at pHo (extracellular) = 8.0 and [HCO3-]o (extracellular) = 10 mM, the apparent Km and Vmax were 59 mM and 1.3 mmol x l(-1) x min-1. When [HCO3-]o was reduced to 2.5 mM at the same pHo, Km did not change significantly, but Vmax was substantially reduced. On the other hand, when pHo was reduced to 7.4 at constant [HCO3-]o, Vmax changed only slightly, but Km increased substantially. In similar experiments, we examined the dependence of the acid extrusion rate on [HCO3-]o. At pHo = 8.0 and [Na+]o = 440 mM, the apparent Km and Vmax were 4.1 mM and 2.1 mmol x 1-1 x min-1. When pHo was reduced to 7.4, Vmax was not altered, but Km substantially increased. The kinetic data are discussed in terms of the role of pHo, [Na+]o, and [HCO3-]o in the pHi-regulating system.

Shrinkage-induced activation of Na(+)-H+ exchange in barnacle muscle fibers

1994 ◽  
Vol 266 (6) ◽  
pp. C1744-C1753 ◽  
Author(s):  
B. A. Davis ◽  
E. M. Hogan ◽  
W. F. Boron
Keyword(s):  
Intracellular Ph ◽  
Muscle Fibers ◽  
Single Muscle ◽  
Extrusion Rate ◽  
Barnacle Muscle ◽  
Hypertonic Solutions ◽  
Acid Extrusion ◽  
Ph Microelectrodes ◽  
Barnacle Muscle Fibers ◽  
Exchange Activity

We examined the effect of shrinkage on Na(+)-H+ exchange in single muscle fibers at intracellular pH (pHi) values of 6.8, 7.2, and 7.6 using pH microelectrodes and internal dialysis. Under normotonic conditions (975 mosmol/kgH2O) at pHi 6.8, the amiloride-sensitive acid-extrusion rate (JAmil/s) averaged 17 microM/min. Exposure to hypertonic solutions (1,600 mosmol/kgH2O) increased JAmil/s to 304 microM/min at pHi 6.8. At pHi approximately 7.2 and 7.6, hypertonicity increased JAmil/s from approximately 0 to approximately 172 microM/min (pHi 7.2) and approximately 0 to approximately 90 microM/min (pHi 7.6). Thus, under normotonic conditions, Na(+)-H+ exchange activity is slight at pHi approximately 6.8 and virtually nil at higher pHi values. Shrinkage stimulated Na(+)-H+ exchange, more at low pHi values. We also examined the Cl- dependence of the Na(+)-H+ exchanger's response to shrinkage. Our results indicate that shrinkage-induced activation of Na(+)-H+ exchange requires Cl-, specifically intracellular Cl-. These results establish that shrinkage is both pHi dependent and requires intracellular Cl-.

scholarly journals The interaction of intracellular Mg2+ and pH on Cl- fluxes associated with intracellular pH regulation in barnacle muscle fibers.

1988 ◽  
Vol 91 (4) ◽  
pp. 495-513 ◽  
Author(s):  
J M Russell ◽  
M S Brodwick
Keyword(s):  
Ph Regulation ◽  
Muscle Fibers ◽  
Acid Value ◽  
Inhibitory Effect ◽  
Disulfonic Acid ◽  
Anion Transporter ◽  
Stilbene Derivatives ◽  
Acid Extrusion ◽  
Barnacle Muscle Fibers ◽  
Dialysis Technique

The intracellular dialysis technique was used to measure unidirectional Cl- fluxes and net acid extrusion by single muscle fibers from the giant barnacle. Decreasing pHi below normal levels of 7.35 stimulated both Cl- efflux and influx. These increases of Cl- fluxes were blocked by disulfonic acid stilbene derivatives such as SITS and DIDS. The SITS-sensitive Cl- efflux was sharply dependent upon pHi, increasing approximately 20-fold as pHi was decreased from 7.35 to 6.7. Under conditions of normal intracellular Mg2+ concentration, the apparent pKa for the activation of Cl- efflux was 7.0. We found that raising [Mg2+]i, but not [Mg2+]o, had a pronounced inhibitory effect on both SITS-sensitive unidirectional Cl- fluxes as well as on SITS-sensitive net acid extrusion. Increasing [Mg2+]i shifted the apparent pKa of Cl- efflux to a more acid value without affecting the maximal flux that could be attained. This relation between pHi and [Mg2+]i on SITS-sensitive Cl- efflux is consistent with a competition between H ions and Mg ions. We conclude that the SITS-inhibitable Cl- fluxes are mediated by the pHi-regulatory transport mechanism and that changes of intracellular Mg2+ levels can modify the activity of the pHi regulator/anion transporter.

scholarly journals Intracellular pH and Na fluxes in barnacle muscle with evidence for reversal of the ionic mechanism of intracellular pH regulation.

1983 ◽  
Vol 82 (1) ◽  
pp. 47-78 ◽  
Author(s):  
J M Russell ◽  
W F Boron ◽  
M S Brodwick
Keyword(s):  
Intracellular Ph ◽  
Transport Mechanism ◽  
Ph Regulation ◽  
Forward Direction ◽  
Extrusion Process ◽  
Acid Uptake ◽  
Barnacle Muscle ◽  
Na Efflux ◽  
Acid Extrusion ◽  
Barnacle Muscle Fibers

The ion transport mechanism that regulates intracellular pH (pHi) in giant barnacle muscle fibers was studied by measuring pHi and unidirectional Na+ fluxes in internally dialyzed fibers. The overall process normally results in a net acid extrusion from the cell, presumably by a membrane transport mechanism that exchanges external Na+ and HCO-3 for internal Cl- and possibly H+. However, we found that net transport can be reversed either by lowering [HCO-3]o and pHo or by reducing [Na+]o. This reversal (acid uptake) required external Cl-, was stimulated by raising [Na+]i, and was blocked by SITS. When the transporter was operating in the net forward direction (acid extrusion), we found a unidirectional Na+ influx of approximately 60 pmol . cm-2 . s-1, which required external HCO-3 and internal Cl- and was stimulated by cyclic AMP and blocked by SITS or DIDS. These properties of the Na+ influx are all shared with the net acid extrusion process. We also found that under conditions of net forward transport, the pHi-regulating system mediated a unidirectional Na+ efflux, which was significantly smaller than the simultaneous Na+ influx. These data are consistent with a reversible transport mechanism which, even when operating in the net forward direction, mediates a small amount of reversed transport. We also found that the ouabain-sensitive Na+ efflux was sharply inhibited by acidic pHi, being totally absent at pHi values below approximately 6.8.

scholarly journals Intracellular pH Regulation in Cultured Astrocytes from Rat Hippocampus

1997 ◽  
Vol 110 (4) ◽  
pp. 453-465 ◽  
Author(s):  
Mark O. Bevensee ◽  
Regina A. Weed ◽  
Walter F. Boron
Keyword(s):  
Intracellular Ph ◽  
Ph Regulation ◽  
Disulfonic Acid ◽  
Bath Solution ◽  
Major Cation ◽  
Cultured Astrocytes ◽  
Buffering Power ◽  
Acid Load ◽  
Acid Extrusion ◽  
Best Fit

We studied the regulation of intracellular pH (pHi) in single cultured astrocytes passaged once from the hippocampus of the rat, using the dye 2′,7′-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) to monitor pHi. Intrinsic buffering power (βI) was 10.5 mM (pH unit)−1 at pHi 7.0, and decreased linearly with pHi; the best-fit line to the data had a slope of −10.0 mM (pH unit)−2. In the absence of HCO3−, pHi recovery from an acid load was mediated predominantly by a Na-H exchanger because the recovery was inhibited 88% by amiloride and 79% by ethylisopropylamiloride (EIPA) at pHi 6.05. The ethylisopropylamiloride-sensitive component of acid extrusion fell linearly with pHi. Acid extrusion was inhibited 68% (pHi 6.23) by substituting Li+ for Na+ in the bath solution. Switching from a CO2/HCO3−-free to a CO2/HCO3−-containing bath solution caused mean steady state pHi to increase from 6.82 to 6.90, due to a Na+-driven HCO3− transporter. The HCO3−-induced pHi increase was unaffected by amiloride, but was inhibited 75% (pHi 6.85) by 400 μM 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), and 65% (pHi 6.55–6.75) by pretreating astrocytes for up to ∼6.3 h with 400 μM 4-acetamide-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS). The CO2/HCO3−-induced pHi increase was blocked when external Na+ was replaced with N-methyl-d-glucammonium (NMDG+). In the presence of HCO3−, the Na+-driven HCO3− transporter contributed to the pHi recovery from an acid load. For example, HCO3− shifted the plot of acid-extrusion rate vs. pHi by 0.15–0.3 pH units in the alkaline direction. Also, with Na-H exchange inhibited by amiloride, HCO3− increased acid extrusion 3.8-fold (pHi 6.20). When astrocytes were acid loaded in amiloride, with Li+ as the major cation, HCO3− failed to elicit a substantial increase in pHi. Thus, Li+ does not appear to substitute well for Na+ on the HCO3− transporter. We conclude that an amiloride-sensitive Na-H exchanger and a Na+-driven HCO3− transporter are the predominant acid extruders in astrocytes.

Effect of weak acids on pH regulation and anion transport in barnacle muscle fibers

1981 ◽  
Vol 241 (5) ◽  
pp. C193-C199 ◽  
Author(s):  
D. W. Keifer
Keyword(s):  
Anion Exchange ◽  
Ph Regulation ◽  
Muscle Fibers ◽  
External Solution ◽  
Weak Acid ◽  
Partial Recovery ◽  
Weak Acids ◽  
Barnacle Muscle ◽  
Initial Drop ◽  
Barnacle Muscle Fibers

The intracellular pH (pHi) of barnacle muscle fibers was measured with microelectrodes while the fibers were exposed to the weak acids, propionic acid (98 mM), or 5,5-dimethyloxazolidine-2,4-dione (DMO) (100 mM), at extracellular pH (pH0) 7.8. Both propionate and DMO caused an initial drop in pHi, followed by a partial recovery to the final steady pHi of 7.16. In the presence of 6 mM HCO3(-) (pH0 7.8), the final pHi was 7.31 for either weak acid. In other experiments, pHi was initially lowered by temporarily exposing the fiber to NH4Cl-containing solution. The rate of subsequent pHi recovery at pHi 6.84 was expressed as an H+ equivalent flux. In the absence of HCO3(-), the H+ equivalent flux was stimulated (two- to threefold) by both propionate and DMO. Part of this stimulation was due to the reduced Cl- concentration in the external solution when the anion of the weak acid is substituted for Cl-. Another part of the stimulation may have been due to the increased buffering in the intracellular unstirred layer. HCO3(-) greatly stimulated (ninefold) the H+ equivalent flux, but in the presence of HCO3(-), propionate and DMO had no additional effect. There is no evidence from the present work indicating that either propionate or DMO anions acted as substrates for operation of the anion exchange mechanism (Cl-/Cl- and HCO3(-)/Cl-). Since exposure of the cell to either of the weak acids lowered pHi and since the rate of anion exchange fluxes is known to increase when pHi is lowered, propionate most likely stimulated anion fluxes indirectly by lowering pHi.

Activation of Na-H exchange by intracellular lithium in barnacle muscle fibers

1992 ◽  
Vol 263 (1) ◽  
pp. C246-C256 ◽  
Author(s):  
B. A. Davis ◽  
E. M. Hogan ◽  
W. F. Boron
Keyword(s):  
Volume Regulation ◽  
Muscle Fibers ◽  
Cell Volume Regulation ◽  
Low Ph ◽  
Dialysis Fluid ◽  
Barnacle Muscle ◽  
Extrusion Mechanism ◽  
Acid Extrusion ◽  
Barnacle Muscle Fibers

We internally dialyzed single barnacle muscle fibers (BMF) for 90 min with a dialysis fluid (DF) containing no Na+ and either 0 or 100 mM Li+ and measured intracellular pH (pHi) with a microelectrode. During dialysis, the pH 8.0 artificial seawater (ASW) contained neither Na+ nor HCO3-. After we halted dialysis with a Li(+)-free/low-pH DF and allowed pHi to stabilize at approximately 6.8, adding 440 mM Na(+)-10 mM HCO3- to the ASW caused pHi to recover rapidly and stabilize at 7.32. In contrast, when the DF contained 100 mM Li+, pHi stabilized at 7.49. In fibers dialyzed to a pHi of approximately 7.2, Li+ stimulated a component of acid extrusion that was dependent on Na+ but not affected by SITS. Thus Li+ activates a Na(+)-dependent acid-extrusion mechanism other than the well characterized Na(+)-dependent Cl-HCO3 exchanger. To study the Li(+)-activated mechanism, we minimized Na(+)-dependent Cl-HCO3 exchange by raising pHDF to 7.35 and pretreated BMFs with SITS. We found that dialysis with Li+ elicits a Na(+)-dependent pHi increase that is largely blocked by amiloride, consistent with the hypothesis that Li+ activates a latent Na-H exchanger even at a normal pHi. In the absence of Li+, the Na-H exchanger is relatively inactive at pHi 7.35 (net acid-extrusion rate, Jnet = 9.5 microM/min) but modestly stimulated by reducing pHi to 6.8 (Jnet = 64 microM/min). In the presence of Li+, the Na-H exchanger is very active at pHi values of both 7.35 (Jnet = 141 microM/min) and 6.8 (Jnet = 168 microM/min). Thus Li+ alters the pHi sensitivity of the Na-H exchanger. Because the Na-H exchanger is only approximately 6% as active as the Na(+)-dependent Cl-HCO3 exchanger in the absence of Li+ at a pHi of approximately 6.8, we suggest that the major role of the Na-H exchanger may not be in pHi regulation but in another function such as cell-volume regulation.

Permeability of barnacle muscle fibers to water and nonelectrolytes

1976 ◽  
Vol 30 (1) ◽  
pp. 197-212
Author(s):  
Daniel F. Wolff ◽  
Osvaldo A. Alvarez ◽  
Fernando F. Vargas
Keyword(s):  
Muscle Fibers ◽  
Barnacle Muscle ◽  
Barnacle Muscle Fibers

Effects of calcium on membrane potential and sodium influx in barnacle muscle fibers

1983 ◽  
Vol 244 (3) ◽  
pp. C297-C302 ◽  
Author(s):  
S. S. Sheu ◽  
M. P. Blaustein
Keyword(s):  
Membrane Potential ◽  
Muscle Fibers ◽  
Cation Channels ◽  
Channel Blockers ◽  
Sodium Influx ◽  
Permeable Channel ◽  
Barnacle Muscle ◽  
Nonselective Cation Channels ◽  
Flux Measurements ◽  
Barnacle Muscle Fibers

The influence of internal and external Ca2+ on membrane potential and 22Na influx were tested in internally perfused giant barnacle muscle fibers. The fibers depolarized by about 2-3 mV, and Na+ influx increased when external Ca2+ was removed. These effects were inhibited and reversed by adding 2 mM La3+ externally but not by tetrodotoxin (TTX). Ca2+ channel blockers did not prevent the depolarization. Increasing internal free Ca2+ ([Ca2+]i) from 10(-7) to 10(-5) M also stimulated Na+ influx and depolarized the fibers by a few millivolts. Neither external La3+ nor TTX prevented the effects of raising [Ca2+]i; however, internal tetrabutylammonium ions depolarized the fibers and attenuated the internal Ca2+-dependent effects. These data are consistent with the idea that removal of external Ca2+ activates a La3+-sensitive channel that is permeable to Na+; raising [Ca2+]i activates a La2+-insensitive, Na+-permeable channel that may be similar to the internal Ca2+-activated nonselective cation channels observed in cardiac muscle. The results demonstrate that all Na+ (and Ca2+) fluxes that do not contribute to Na-Ca exchange must be carefully identified before the exchange stoichiometry can be determined from Na+ and Ca2+ flux measurements.

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