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  • 1
    Electronic Resource
    Electronic Resource
    Regulation of cytoplasmic pH of cultured bovine corneal endothelial cells in the absence and presence of bicarbonate (1988)
    Springer
    The journal of membrane biology 103 (1988), S. 29-40 
    Details
    ISSN: 1432-1424
    Keywords: intracellular pH ; sodium bicarbonate cotransport ; Na+/H+ antiport ; Cl−/HCO 3 − exchange ; amiloride ; DIDS ; cornea ; endothelium ; cell culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Intracellular pH (pH i ) in confluent monolayers of cultured bovine corneal endothelial cells was determined using the pH-dependent absorbance of intracellularly trapped 5(and 6)carboxy-4′,5′-dimethylfluorescein. Steady-state pH was 7.05±0.1 in the nominal absence of bicarbonate, and 7.15±0.1 in the presence of 28mm HCO 3 − /5% CO2. Following an acid load imposed by a NH4Cl prepulse, pH i was regulated in the absence of HCO 3 − by a Na+-dependent process inhibitable to a large extent by 1mm amiloride and 0.1mm dimethylamiloride. In the presence of 28mm HCO 3 − /5% CO2, this regulation was still dependent on Na+, but the inhibitory potency of amiloride was less. DIDS (1mm) partially inhibited this regulation in the presence, but not in the absence of bicarbonate. With cells pretreated with DIDS, amiloride was as effective in inhibiting recovery from acid load as in the absence of HCO 3 − . The presence of intracellular Cl− did not appreciably affect this recovery, which was still sensitive to DIDS in the absence of Cl−. Removal of extracellular Na+ led to a fall of pH i , which was greatly attenuated in the absence of HCO 3 − . This acidification was largely reduced by 1mm DIDS, but not by amiloride. Cl removal led to an intracellular alkalinization in the presence of HCO 3 − . The presence of a Cl−/HCO 3 − exchanger was supported by demonstrating DIDS-sensitive36Cl− uptake into confluent cell monolayers. Thus, bovine corneal endothelial cells express three processes involved in intracellular pH regulation: an amiloride-sensitive Na+/H− antiport, a Na−−HCO 3 − symport and a Cl−/HCO 3 − exchange, the latter two being DIDS sensitive.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01871930
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  • 2
    Electronic Resource
    Electronic Resource
    Evidence for coupled transport of bicarbonate and sodium in cultured bovine corneal endothelial cells (1984)
    Springer
    The journal of membrane biology 81 (1984), S. 189-204 
    Details
    ISSN: 1432-1424
    Keywords: corneal endothelium ; cell culture ; intracellular potential ; pH regulation ; bicarbonate ; bicarbonate-sodium cotransport ; lithium ; stilbenes ; amiloride ; ouabain
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Usin gintracellular microelectrode technique, the response of the voltageV across the plasma membrane of cultured bovine corneal endothelial cells to changes in sodium and bicarbonate concentrations was investigated. (1) The electrical response to changes in [HCO 3 − ] o (depolarization upon lowering and hyperpolarization upon raising [HCO 3 − ] o ) was dependent on sodium. Lithium could fairly well be substituted for sodium, whereas potassium or choline were much less effective. (2) Removal of external sodium caused a depolarization, while a readdition led to a hyperpolarization, which increased with time of preincubation in the sodium-depleted medium. (3) The response to changes in [Na+] o was dependent on bicarbonate. In a nominally bicarbonate-free medium, its amplitude was decreased or even reversed in sign. (4) Application of SITS or DIDS (10−3 m) had a similar effect on the response to sodium as bicarbonate-depleted medium. (5) At [Na+] o =151mm and [HCO 3 − ] o =46mm, the transients ofV depended, with 39.0±9.0 (sd) mV/decade, on bicarbonate and, with 15.3±5.8 (sd) mV/decade, on sodium. (6) After the preincubation of cells with lithium, replacement of Li by choline led to similar effects as the replacement of sodium by choline, though the response ofV was smaller with Li. This response could be reduced or reversed by the removal of bicarbonate or by the application of SITS. (7) Amiloride (10−3 m) caused a reversible hyperpolarization of the steady-state potential by 8.5±2.6 mV (sd). It did not affect the immediate response to changes in [Na+] o or [HCO 3 − ] o , but reduced the speed of regaining the steady-state potential after a change in [HCO 3 − ] o . (8) Ouabain (10−4 m) caused a fast depolarization of −6.8±1.1 (sd) mV, which was followed by a continuing slower depolarization. The effect was almost identical at 10−5 m. (9) It is suggested, that corneal endothelial cells possess a cotransport for sodium and bicarbonate, which transports net negative charage with these ions. It is inhibitable by stilbenes, but not directly affected by amiloride or ouabain. Lithium is a good substitute for sodium with respect to bicarbonate transport and is transported itself. In addition, the effect of amiloride provides indirect evidence for the existence of a Na+/H+-antiport. A model for the transepithelial transport of bicarbonate across the corneal endothelium is proposed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01868713
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  • 3
    Electronic Resource
    Electronic Resource
    Na+-dependent HCO 3 − transport and Na+/H+ exchange regulate pH i in human ciliary muscle cells (1992)
    Springer
    The journal of membrane biology 127 (1992), S. 215-225 
    Details
    ISSN: 1432-1424
    Keywords: ciliary muscle ; intracellular pH ; cell culture ; sodium-bicarbonate cotransport ; sodium/proton exchange
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary We investigated intracellular pH (pH i ) regulation in cultured human ciliary muscle cells by means of the pH-sensitive absorbance of 5(and 6)-carboxy-4′,5′-dimethylfluorescein (CDMF). The steady-state pH i was 7.09±0.04 (n = 12) in CO2/ HCO 3 − -buffered and 6.86±0.03 (n = 12) in HEPES-buffered solution. Removal of extracellular sodium for 6 min acidified the cells by 1.11±0.06 pH units (n = 12) in the presence of CO2/ HCO 3 − and by 0.91±0.05 pH units (n = 8) in its absence. Readdition of external sodium resulted in a rapid pH i recovery, which was almost completely amiloride-sensitive in the absence of CO2/ HCO 3 − but only slightly influenced by amiloride in its presence. Application of DIDS under steady-state conditions significantly acidified the ciliary muscle cells by 0.25±0.02 (n = 4) in 6 min, while amiloride had no effect. The pH i recovery after an intracellular acid load was completely dependent on extracellular sodium. In HEPES-buffered solution the pH i recovery was almost completely mediated by Na+/H+ exchange, since it was blocked by amiloride (1 mmol/liter). In contrast, a marked amilorideinsensitive pH i recovery was observed in CO2/HCO 3 − -buffered solution which was mediated by chloride-independent and chloride-dependent Na+ HCO 3 − cotransport. This recovery, inhibited by DIDS (0.2 mmol/liter). was also observed if the cells were preincubated in chloride-free solution for 4 hr. Analysis of the sodium dependence of the pH i recovery after NH4Cl prepulse revealed V max = 0.57 pH units/min, K m= 39.7 mmol/liter extracellular sodium for the amiloride-sensitive component and V max = 0.19 pH units/min, K m= 14.3 mmol/liter extracellular sodium for the arniloride-insensitive component. We conclude that Na+/H+ exchange and chloride-independent and chloride-dependent Na+HCO 3 − cotransport are involved in the pH i regulation of cultured human ciliary muscle cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00231509
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  • 4
    Electronic Resource
    Electronic Resource
    Effect of calcium, furosemide and chlorothiazide on net volume reabsorption and basolateral membrane potential of the distal tubule (1981)
    Springer
    Pflügers Archiv 389 (1981), S. 121-126 
    Details
    ISSN: 1432-2013
    Keywords: Distal renal tubule ; Amphiuma ; Basolateral membrane potential ; Volume reabsorption ; Calcium ; Diuretics
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract In the distal tubule of the isolated kidney of Amphiuma net volume reabsorption (split-oil droplet method) and basolateral membrane potential (Ψ b ) were measured. Luminal perfusion solution could be changed rapidly from 108 mmol·l−1 NaCl plus 0.1 mmol·l−1 calcium to solutions containing 103 or 97 mmol·l−1 NaCl plus 3.6 or plus 7.2 mmol·l−1 calcium. Furthermore, 10−4 mol·l−1 furosemide or chlorothiazide were applied luminally. (1) Addition of 7.2 mmol·l−1 calcium hyperpolarized Ψ b from −73.4 mV to −108.3 mV and inhibited net volume reabsorption. (2) Similarly, when furosemide was injected, Ψ b was hyperpolarized and net volume reabsorption reduced. Application of both high calcium and furosemide further inhibited volume reabsorption. (3) The effects of chlorothiazide were similar to those of furosemide. However, when both high calcium and chlorothiazide were administered Ψ b and volume reabsorption were almost normalized. (4) The data are consistent with the hypothesis that calcium and the diuretics interfere primarly with chloride uptake across the luminal membrane and thus reduce sodium chloride transport. When chlorothiazide in the presence of high luminal calcium almost normalized chloride transport, it is likely that its effects were by stimulating calcium transport and thus increasing intracellular calcium activity.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00582101
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  • 5
    Electronic Resource
    Electronic Resource
    Anion dependence of electrical effects of bicarbonate and sodium on cultured bovine corneal endothelial cells (1985)
    Springer
    Pflügers Archiv 403 (1985), S. 175-185 
    Details
    ISSN: 1432-2013
    Keywords: Bicarbonate ; Intracellular potentials ; Bicarbonate sodium cotransport ; Chloride ; Anion exchanger ; Cell culture ; Cornea ; Endothelium ; Furosemide ; Barium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Using intracellular microelectrode technique, the effect of anion substitution on the voltage responses to extracellular bicarbonate and sodium was explored in cultured bovine corneal endothelial cells. 1. The overall amplitude of voltage changes induced by periodic changes of [HCO 3 − ]0 (depolarization upon removal of HCO 3 − and hyperpolarization upon readdition) was reduced when Cl− was replaced by organic anions (cyclamate, methylsulfate, benzenesulfonate) or by SO 4 2− , and to a lesser extent by substitution with Br−. 2. There was a similar effect of anion substitution on the response to changes of [Na+]0. 3. In both cases, in the absence of Cl, the voltage V returned at a slower rate to baseline levels after it had been transiently changed by either an imposed Na- or HCO3-gradient, indicating a slower dissipation of these gradients. The direct response of V to these imposed gradients was affected only to a minor degree. 4. Replacement of Cl− by SO 4 2− or organic anions led to a slow, reversible depolarization of the cell, while substitution with Br− had only a slight effect. 5. The effect of anion substitution on the voltage responses to HCO 3 − or Na+ could not be mimicked by a depolarization induced by Ba2+ (1 mM). 6. Furosemide (10−3 M) led to a slight reduction of the voltage responses to HCO 3 − , but could not suppress the effect of anion substitution on these reactions. It could neither suppress the depolarization induced by anion substitution and had no effect on steady-state PD. 7. It is suggested, that cultured bovine corneal endothelial cells, in addition to a previously demonstrated electrogenic HCO 3 − −Na+-cotransport, which is probably not dependent on Cl, possess an electroneutral mechanism for HCO 3 − and/or Na+-movement, which depends on Cl. No evidence for a Cl-conductance could be obtained.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00584097
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  • 6
    Electronic Resource
    Electronic Resource
    Membrane potentials and intracellular chloride activity in the ciliary body of the shark (1986)
    Springer
    Pflügers Archiv 407 (1986), S. S112 
    Details
    ISSN: 1432-2013
    Keywords: Ciliary epithelium ; Shark ; Intracellular membrane potential ; Intracellular chloride activity ; Ouabain ; Furosemide
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract We have found that membrane potential in the isolated ciliary epithelium of the shark,Squalus acanthias, is −53 mV. High extracellular potassium or ouabain (10−5 mol·l−1) decrease the potential, and furosemide (10−4 mol·l−1) hyperpolarizes it. There is no difference in membrane potential between the cells of the non-pigmented and pigmented layers. Intracellular chloride activity (64 mmol·l−1) was significantly higher than could be predicted from the equilibrium distribution (26 mmol·l−1) across the cell membranes. When furosemide was applied to the aqueous side of the epithelium, intracellular chloride activity decreased to 35 mmol·l−1 and approached electrochemical equilibrium. The data indicate that the ciliary epithelium possesses an active, furosemide-sensitive chloride transport mechanism which could be a Na−Cl or a 1 Na-1 K-2 Cl symport.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00584939
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  • 7
    Electronic Resource
    Electronic Resource
    The effects of extracellular potassium, ouabain, and prostaglandins on intracellular potassium activity in sheep cardiac Purkinje fibers (1980)
    Springer
    Pflügers Archiv 388 (1980), S. 169-175 
    Details
    ISSN: 1432-2013
    Keywords: Sheep Purkinje fiber ; Intracellular potassium activity ; Extracellular potassium concentration ; Ouabain ; Prostaglandins
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract (1) Intracellular K activity (a K i ) of sheep heart Purkinje fibers was measured using K-selective microelectrodes (liquid ion exchanger).a K i in the resting state with an extracellular K of 4 mmol·l−1 was 112.9±6.1 mmol·l−1 (n=47) for a membrane potential (V M) of −73.3±0.9 mV.V M deviated from the calculated potassium equilibrium potential (E K=−93 mV). (2) When extracellular K was decreased to 2 mmol·l−1 or increased to 6 and 10 mmol·l−1 E K changed from −114 to −84 and −73 mV, with little change ina K i . (3)a K i andV M significantly decreased after administration of 10−6 mol·l−1 ouabain. (4) Prostaglandins (PGI2 10–100 μg·l−1 and PGE2 0.01–1 μg·l−1) decreaseda K i without greatly changingV M. The differences betweenV M andE K became smaller. These effects indicate an increase in K permeability and may explain the antiarrhythmic action of prostaglandins.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00584124
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  • 8
    Electronic Resource
    Electronic Resource
    Electrogenic sodium-bicarbonate symport in cultured corneal endothelial cells (1985)
    Springer
    Pflügers Archiv 405 (1985), S. S167 
    Details
    ISSN: 1432-2013
    Keywords: Corneal endothelium ; Cell culture ; Intracellular potential ; Sodium-bicarbonate cotransport ; pH regulation ; Stilbenes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Intracellular potential measurements on confluent monolayers of cultured bovine corneal endothelial cells were used to define passive ion transport processes in these cells. Previous studies [11, 12] have provided the experimental basis for a cellular model, is which bicarbonate entry across the basolateral membrane in indirectly driven by a Na+/H+-exchanger, which is inhibitable by amiloride (1 mmol/l). Na+ and HCO 3 − leave the cell via an electrogenic bicarbonate sodium cotransport, which is inhibitable by the disulfonic stilbene derivates SITS or DIDS. This model is also compatible with transepithelial work from other groups. In this paper, we briefly review the evidence we have obtained for this model.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00581801
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  • 9
    Electronic Resource
    Electronic Resource
    Evidence for Na/H exchange and Cl/HCO3 exchange in A10 vascular smooth muscle cells (1988)
    Springer
    Pflügers Archiv 412 (1988), S. 29-36 
    Details
    ISSN: 1432-2013
    Keywords: Vascular smooth muscle cells ; A10 cell line ; Intracellular pH ; Na/H exchange ; Cl/HCO3 exchange
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract In the present study we used the pH sensitive absorbance of 5(and6)-carboxy-4′,5′-dimethylfluorescein to investigate intracellular pH (pHi) regulation in A10 vascular smooth muscle cells: (1) The steady state pHi in A10 cells averaged 7.01±0.1 (mean±SEM,n=26) at an extracellular pH of 7.4 (28 mM HCO3/5% CO2). (2) Removal of extracellular sodium led to an intracellular acidification of 0.36±0.07 pH-units (mean±SEM,n=8). (3) pHi-Recovery after an acute intracellular acid load (by means of NH4Cl-prepulse) was reversibly blocked by 1 mM amiloride and was dependent on the presence of sodium. The velocity of pHi recovery increased with increasing sodium concentrations with an apparentK m for external sodium of about 30 mM and aV max of about 0.35 pH units/min. These findings are compatible with a Na/H exchanger being responsible for pHi recovery after an acid load. (4) Removal of extracellular chioride induced an intracellular alkalinization of 0.23±0.03 pH-units (mean±SEM,n=10). The alkalinization was dependent on the presence of extracellular bicarbonate (5) Removal of chloride during pHi recovery from an alkaline load (imposed by acetate prepulse) stopped and reversed pHi backregulation. Chloride removal had no effect in the absence of bicarbonate or in the presence of 10−4 M DIDS, suggesting that the effects were mediated by a Cl/HCO3 exchanger. In conclusion we have demonstrated evidence for a Na/H exchanger and a Cl/HCO3 exchanger in A10 vascular smooth muscle cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00583728
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  • 10
    Electronic Resource
    Electronic Resource
    Kinetic properties of Na+/H+ exchange in cultured bovine pigmented ciliary epithelial cells (1988)
    Springer
    Pflügers Archiv 412 (1988), S. 80-85 
    Details
    ISSN: 1432-2013
    Keywords: Ciliary epithelium ; Tissue culture ; Na+/H+ exchange ; Amiloride
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Uptake studies with22Na were performed in cultured bovine pigmented ciliary epithelial cells, in order to characterize mechanisms of Na+ transport. A large part of Na+ uptake was sensitive to amiloride, quinidine and harmaline. Na+ uptake was stimulated by intracellular acidification (using the NH 4 + prepulse technique), and was inhibited with increasing extracellular proton concentration. Decreasing extracellular pH from 7.5 to 7.0 increased the apparentK M for Na+ from 38 to 86 mM without considerable changes inV max. In the presence of 5 mM Na+ half maximal inhibition of amiloride sensitive Na+ uptake by extracellular protons was observed at a hydrogen concentration of 50 nM. In the presence of 50 mM Na+ the proton concentration necessary for 50% inhibition was 139 nM. Thus, the mode of inhibition of extracellular H+ seemed to be competitive with aK i of 20–40 nM. 10 μM amiloride increased the apparentK M for Na+ from 33 mM to 107 mM, whileV max remained nearly unchanged. IC50 for amiloride was 6 μM at 5 mM Na+ and 36 μM in the presence of 150 mM Na+. Thus, amiloride behaves as a competitive inhibitor with aK i of about 5 μM. The affinities of Na+ to the transport site (K M≈16 mM), to the inhibitory site for protons (K M≈21 mM), and to the inhibitory site for amiloride (K M≈26 mM) were in the same order of magnitude. In summary, we have presented evidence for the presence of a Na+/H+ exchanger in cultured bovine pigmented ciliary epithelial cells. The kinetic data suggest the presence of only one common extracellular binding site for Na+, H+ and amiloride.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00583734
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