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.

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.

pH regulation and response to AVP in A10 cells differ markedly in the presence vs. absence of CO2-HCO3-

1990 ◽  
Vol 259 (3) ◽  
pp. C471-C483 ◽  
Author(s):  
D. Kikeri ◽  
M. L. Zeidel ◽  
B. J. Ballermann ◽  
B. M. Brenner ◽  
S. C. Hebert
Keyword(s):  
Steady State ◽  
Ph Regulation ◽  
Disulfonic Acid ◽  
Potential Contribution ◽  
Vasoactive Agents ◽  
Cell Responses ◽  
Extrusion Mechanism ◽  
Acid Extrusion ◽  
Ambient Co2 ◽  
Stimulation Of

The fluorescent pH-sensitive dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) was used to determine the effect of ambient CO2-HCO3- on the regulation of intracellular pH (pHi) and the pHi response to arginine vasopressin (AVP) in A10 vascular smooth muscle (VSM) cells. Steady-state pHi averaged 7.04 +/- 0.02 in the absence and 7.25 +/- 0.01 in the presence of CO2-HCO3-. In the absence of CO2-HCO3-, virtually all (greater than 96%) of the acid extrusion from acidification occurred by amiloride-sensitive Na(+)-H+ exchange. However, in the presence of CO2-HCO3-, acid extrusion after acidification occurred by both Na(+)-H+ exchange and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-sensitive Na(+)-dependent Cl(-)-HCO3- exchange. In CO2-HCO3(-)-containing media, amiloride-sensitive Na(+)-H+ exchange mediated 85% of acid extrusion at a pHi of 6.48, but the DIDS-sensitive acid extrusion mechanism (NA(+)-dependent Cl(-)-HCO3- exchange) was the dominant acid extrusion mechanism at a pHi of 6.94. Base exited A10 cells by a DIDS-sensitive process consistent with Na(+)-independent Cl(-)-HCO3- exchange. Both amiloride- and DIDS-sensitive processes regulated steady-state pHi in CO2-HCO3-. AVP (10(-7) M) alkalinized steady-state pHi in the absence of CO2-HCO3- (delta pHi = 0.08 +/- 0.01 pH units) by stimulating Na(+)-H+ exchange; however, AVP did not alter pHi of untreated cells in CO2-HCO3- (delta pHi = -0.01 +/- 0.01 pH units) because of concomitant stimulation of Na(+)-independent Cl(-)-HCO3-exchange. We conclude that the steady-state pHi, the mechanisms of pHi regulation, and the pHi response to AVP in A10 cells are critically influenced by the presence of extracellular CO2-HCO3-. Thus the potential contribution of pHi changes to VSM cell responses to vasoactive agents should be evaluated in the presence of CO2-HCO3-.

scholarly journals Cyclic AMP-stimulated chloride fluxes in dialyzed barnacle muscle fibers.

1981 ◽  
Vol 78 (5) ◽  
pp. 499-520 ◽  
Author(s):  
J M Russell ◽  
M S Brodwick
Keyword(s):  
Sulfonic Acid ◽  
Chloride Transport ◽  
Muscle Fibers ◽  
Electrical Conductance ◽  
Adenosine Monophosphate ◽  
Dialysis Fluid ◽  
Barnacle Muscle ◽  
Stilbene Derivatives ◽  
High Concentrations ◽  
Barnacle Muscle Fibers

Unidirectional chloride efflux and influx were studied in giant barnacle muscle fibers that were internally dialyzed. When cyclic 3'5'-adenosine monophosphate (cAMP) was included in the dialysis fluid, both unidirectional fluxes were stimulated by about the same amount. This stimulation was not associated with measurable changes either in membrane electrical conductance or with net movements of chloride. The stimulation required the trans-side presence of chloride. The stimulated flux was inhibited by the sulfonic acid stilbene derivatives 4-acetamido-4'-isothiocyanostilbene-2',2'-disulfonate (SITS) and 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) or by furosemide. When cAMP was presented in high concentrations (10-5 M), the effect on chloride fluxes was characterized by a desensitization phenomenon. This desensitization was not the result of an increased amount of phosphodiesterase activity, but may be related to ATP and/or intracellular calcium levels. These results further support the hypothesis that the barnacle sarcolemma possesses a specialized chloride transport mechanism that largely engages in Cl-Cl exchange under conditions of normal intracellular pH.

scholarly journals Properties of chloride transport in barnacle muscle fibers.

1979 ◽  
Vol 73 (3) ◽  
pp. 343-368 ◽  
Author(s):  
J M Russell ◽  
M S Brodwick
Keyword(s):  
Chloride Transport ◽  
Muscle Fibers ◽  
Chloride Concentration ◽  
The Other ◽  
Resting Potential ◽  
Disulfonic Acid ◽  
Amino Group ◽  
Intact Muscle ◽  
Membrane Resting Potential ◽  
Barnacle Muscle Fibers

Unidirectional chloride-36 fluxes were measured in internally dialyzed barnacle giant muscle fibers. About 50--60% of the Cl efflux was irreversibly blocked by the amino-group reactive agent, 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonic acid (SITS), when it was applied either intra- or extracellularly. Similarly, Cl influx was also blocked by SITS. No significant effect on [Cl]i of SITS was noted in intact muscle fibers. However, the rate of net Cl efflux from muscle fibers which were Cl-loaded by overnight storage at 6 degrees C could be slowed by SITS treatment. Two classes of anions were defined based upon their effects on Cl efflux. Methanesulfonate and nitrate inhibited Cl efflux either when they replaced external chloride or when they were added to a constant external chloride concentration. The other group of anions (propionate, formate) stimulated both Cl efflux and influx and such stimulation could be blocked by SITS. Propionate influx was not nearly as large as the stimulated Cl efflux and was unaffected by SITS. Neither the effects of SITS nor those of the anion substitutes could be simply accounted for by changes in the membrane resting potential or conductance. These results suggest a mediated transport system for chloride across the barnacle sarcolemma.

pH regulation in the vertebrate central nervous system: microelectrode studies in the brain stem of the lamprey

1987 ◽  
Vol 65 (5) ◽  
pp. 986-993 ◽  
Author(s):  
Mitchell Chesler
Keyword(s):  
Brain Stem ◽  
Ph Regulation ◽  
Disulfonic Acid ◽  
Small Rise ◽  
Free Media ◽  
Acid Extrusion ◽  
Acid Loading ◽  
The Brain ◽  
Vertebrate Central Nervous System ◽  
Dependent Mechanism

Studies of intracellular pH (pHi) in nervous tissue are summarized and recent investigation of intracellular and extracellular pH (pHo) in the isolated brain stem of the lamprey is reviewed. In the lamprey, pHi regulation was studied in single reticulospinal neurons using double-barrel ion-selective microelectrodes (ISMs). In nominally [Formula: see text]-free HEPES-buffered media, acute acid loading was followed by a spontaneous recovery of pHi requiring 10–20 min and was associated with a prolonged rise in intracellular Na+. The recovery of pHi was blocked by 1–2 mM amiloride. Amiloride also caused a small rise in pHo. Substitution of external Na+ caused a slow intracellular acidification and extracellular alkalinization. Return of external Na+ reversed these effects. Transition from HEPES to [Formula: see text]-buffered media increased the rate of acid extrusion during recovery of pHi. Recovery in [Formula: see text]-buffered media was inhibited by 4,4′-diisothio-cyanostilbene-2,2′-disulfonic acid and was slowed after exposure to Cl−-free media. Following inhibition of acid extrusion by amiloride, transition to [Formula: see text] media restored pHi recovery. These data indicate that lamprey neurons recover from acute acid loads by both Na+–H+ exchange and an independent [Formula: see text]-dependent mechanism. Evidence for [Formula: see text]-dependent acid extrusion in other vertebrate cells and the protocols of pHi studies using ISMs are discussed.

pH regulation in single glomerular mesangial cells. II. Na+-dependent and -independent Cl(-)-HCO3- exchangers

1988 ◽  
Vol 255 (6) ◽  
pp. C857-C869 ◽  
Author(s):  
G. Boyarsky ◽  
M. B. Ganz ◽  
R. B. Sterzel ◽  
W. F. Boron
Keyword(s):  
Mesangial Cells ◽  
Ph Regulation ◽  
Large Fraction ◽  
Preceding Paper ◽  
Disulfonic Acid ◽  
Glomerular Mesangial Cells ◽  
Total Acid ◽  
Small Component ◽  
Rate Of Recovery ◽  
Acid Extrusion

We used the pH-sensitive dye 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) to further characterize the mechanisms of intracellular pH (pHi) regulation in renal mesangial cells. In the accompanying paper [Am. J. Physiol. 255 (Cell Physiol. 24): C844-C856, 1988], we showed that acid extrusion from mesangial cells is mediated by both an ethylisopropylamiloride (EIPA)-sensitive Na+-H+ exchanger and a 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS)-sensitive-HCO3(-)-dependent mechanism. In this study, we examined the ionic dependencies of pHi-regulatory mechanisms in the presence of CO2-HCO3-. We found that in CO2-HCO3-, approximately 90% of the net acid extrusion occurring during recovery from an acid load is blocked by removing external Na+. Short-term (less than 15 min) removal of external Cl- has little effect on the rate of recovery in CO2-HCO3-. In contrast longer periods of external Cl- removal (1-2 h) blocks 40-60% of the rate of recovery, which is consistent with the hypothesis that a large fraction of the SITS-sensitive-HCO3(-)-dependent recovery mechanism described in the preceding paper is also Na+- and Cl(-)-dependent. Therefore, this Cl(-)-dependent component is probably mediated by a Na+-dependent Cl(-)-HCO3- exchanger. As much as 16% of total acid extrusion is insensitive to EIPA and long-term Cl- removal but is blocked by SITS. Thus either 1-2 h of Cl- removal is insufficient to wash out all internal Cl-, or a small component of acid extrusion is mediated by a Cl(-)-independent mechanism, such as the electrogenic Na+/HCO3- cotransporter. We also studied the effect on pHi of the removal and readdition of external Cl-, observing pHi changes consistent with the existence of a Na+-independent Cl(-)-HCO3- exchanger, which would presumably function as an acid loader. In contrast to the Na+-H+ exchanger and Na+-dependent Cl(-)-HCO3- exchanger, which are stimulated at low pHi, the Cl(-)-HCO3- exchanger is stimulated at high pHi. Thus the acid-extruding and acid-loading mechanisms have opposite pHi dependencies.

Apical ammonium inhibition of cAMP-stimulated secretion in T84 cells is bicarbonate dependent

2005 ◽  
Vol 289 (4) ◽  
pp. G768-G778 ◽  
Author(s):  
Roger T. Worrell ◽  
Alison Best ◽  
Oscar R. Crawford ◽  
Jie Xu ◽  
Manoocher Soleimani ◽  
...  
Keyword(s):  
Mole Fraction ◽  
Anion Exchanger ◽  
Inhibitory Effect ◽  
Disulfonic Acid ◽  
Apical Surface ◽  
Rt Pcr ◽  
T84 Cells ◽  
Western Blots ◽  
Anion Transporter ◽  
Normal Human

Normal human colonic luminal (NH4+) concentration ([NH4+]) ranges from ∼10 to 100 mM. However, the nature of the effects of NH4+ on transport, as well as NH4+ transport itself, in colonic epithelium is poorly understood. We elucidate here the effects of apical NH4+ on cAMP-stimulated Cl− secretion in colonic T84 cells. In HEPES-buffered solutions, 10 mM apical NH4+ had no significant effect on cAMP-stimulated current. In contrast, 10 mM apical NH4+ reduced current within 5 min to 61 ± 4% in the presence of 25 mM HCO3−. Current inhibition was not simply due to an increase in extracellular K+-like cations, in that the current magnitude was 95 ± 5% with 10 mM apical K+ and 46 ± 3% with 10 mM apical NH4+ relative to that with 5 mM apical K+. We previously demonstrated that inhibition of Cl− secretion by basolateral NH4+ occurs in HCO3−-free conditions and exhibits anomalous mole fraction behavior. In contrast, apical NH4+ inhibition of current in HCO3− buffer did not show anomalous mole fraction behavior and followed the absolute [NH4+] in K+-NH4+ mixtures, where K+ concentration + [NH4+] = 10 mM. The apical NH4+ inhibitory effect was not prevented by 100 μM methazolamide, suggesting no role for apical carbonic anhydrase. However, apical NH4+ inhibition of current was prevented by 10 min of pretreatment of the apical surface with 500 μM DIDS, 100 μM 4,4′-dinitrostilbene-2,2′-disulfonic acid (DNDS), or 25 μM niflumic acid, suggesting a role for NH4+ action through an apical anion exchanger. mRNA and protein for the apical anion exchangers SLC26A3 [downregulated in adenoma (DRA)] and SLC26A6 [putative anion transporter (PAT1)] were detected in T84 cells by RT-PCR and Northern and Western blots. DRA and PAT1 appear to associate with CFTR in the apical membrane. We conclude that the HCO3− dependence of apical NH4+ inhibition of secretion is due to the action of NH4+ on an apical anion exchanger.

pH regulation in single glomerular mesangial cells. I. Acid extrusion in absence and presence of HCO3-

1988 ◽  
Vol 255 (6) ◽  
pp. C844-C856 ◽  
Author(s):  
G. Boyarsky ◽  
M. B. Ganz ◽  
R. B. Sterzel ◽  
W. F. Boron
Keyword(s):  
Steady State ◽  
Loading Rate ◽  
Mesangial Cells ◽  
Ph Regulation ◽  
Disulfonic Acid ◽  
Glomerular Mesangial Cells ◽  
Fluorescence Excitation ◽  
Dependent Transport ◽  
Acid Extrusion ◽  
Acid Loading

We have developed a technique to measure the fluorescence of a pH-sensitive dye (2,7-biscarboxyethyl-5(6)-carboxyfluorescein) in single glomerular mesangial cells in culture. The intracellular fluorescence excitation ratio of the dye was calibrated using the nigericin-high-K+ approach. In the absence of CO2-HCO3-, mesangial cells that are acid loaded by an NH+4 prepulse exhibit a spontaneous intracellular pH (pHi) recovery that is blocked either by ethylisopropylamiloride (EIPA) or removal of external Na+. This pHi recovery most probably reflects the activity of a Na+-H+ exchanger. When the cells are switched from a N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solution to one containing CO2-HCO3-, there is an abrupt acidification due to CO2 entry, which is followed by a spontaneous recovery of pHi to a steady-state value higher than that prevailing in HEPES. Both the rate of recovery and the higher steady-state pHi imply that the application of CO2-HCO3- introduces an increase in net acid extrusion from the cell. One third of total net acid extrusion in CO2-HCO3- is EIPA sensitive and most likely is mediated by the Na+-H+ exchanger. The remaining two thirds of acid extrusion could be caused by a decrease in the background acid-loading rate and/or the introduction of a new, HCO3- -dependent acid-extrusion mechanism. The HCO3- -induced alkalinization cannot be accounted for by a HCO3- -induced reduction in the acid-loading rate. The latter can be estimated by applying EIPA in the absence of HCO3- and observing the rate of pHi decline. We found that this acid-loading rate is only about one fifth as great as the total net acid extrusion rate in the presence of HCO3-. Indeed, two thirds of net acid extrusion in HCO3- is blocked by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), an inhibitor of HCO3- -dependent transport. Furthermore, the effects of EIPA and SITS were additive. Thus, in the presence of CO2-HCO3-, a SITS-sensitive-HCO3- -dependent transporter is the dominant mechanism of acid extrusion. This mechanism also accounts for the increase in steady-state pHi on addition of CO2-HCO3-.

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.

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