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M Wiederholt

Publications and source records attributed to M Wiederholt.

At least 91 records · Page 5Linked to original sources
PubMed1988

Evidence for Na/H exchange and Cl/HCO3 exchange in A10 vascular smooth muscle cells.

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 apparent Km for external sodium of about 30 mM and a Vmax 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 chloride 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.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1988

Kinetic properties of Na+/H+ exchange in cultured bovine pigmented ciliary epithelial cells.

Uptake studies with 22Na 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 apparent KM for Na+ from 38 to 86 mM without considerable changes in Vmax. 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 a Ki of 20-40 nM. 10 microM amiloride increased the apparent KM for Na+ from 33 mM to 107 mM, while Vmax remained nearly unchanged. IC50 for amiloride was 6 microM at 5 mM Na+ and 36 microM in the presence of 150 mM Na+. Thus, amiloride behaves as a competitive inhibitor with a Ki of about 5 microM. The affinities of Na+ to the transport site (KM approximately 16 mM), to the inhibitory site for protons (KM approximately 21 mM), and to the inhibitory site for amiloride (KM approximately 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗
PubMed1988

Regulation of cytoplasmic pH of cultured bovine corneal endothelial cells in the absence and presence of bicarbonate.

Intracellular pH (pHi) 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 28 mM HCO3-/5% CO2. Following an acid load imposed by a NH4Cl prepulse, pHi was regulated in the absence of HCO3- by a Na+-dependent process inhibitable to a large extent by 1 mM amiloride and 0.1 mM dimethylamiloride. In the presence of 28 mM HCO3-/5% CO2, this regulation was still dependent on Na+, but the inhibitory potency of amiloride was less. DIDS (1 mM) 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 HCO3-. 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 pHi, which was greatly attenuated in the absence of HCO3-. This acidification was largely reduced by 1 mM DIDS, but not by amiloride. Cl removal led to an intracellular alkalinization in the presence of HCO3-. The presence of a Cl-/HCO3- exchanger was supported by demonstrating DIDS-sensitive 36Cl- 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+-HCO3- symport and a Cl-/HCO3- exchange, the latter two being DIDS sensitive.

Acid-Base Equilibrium↗
PubMed1988

Characterization of Cl-/HCO3- exchange in cultured bovine pigmented ciliary epithelium.

Many recent data indicate that transport of Cl- across the ciliary epithelium plays an important role in aqueous humor formation. We used 36Cl to investigate the pathways for Cl- transport in confluent monolayers of cultured bovine pigmented ciliary epithelial cells. Cl- uptake mainly occurred via a mechanism with typical characteristics of an anion exchanger, and could be stimulated by an outwardly directed HCO3- gradient. One mM SITS and 1 mM DIDS inhibited Cl- uptake by some 80-90%, the latter with an IC50 of about 20 microM. HCO3- stimulated Cl- uptake could be partly inhibited for furosemide and to a lesser extent by bumetanide, indicating an action of loop-diuretics on the anion exchanger. 36Cl- uptake was cis-inhibited by the halides Cl-, I- and Br-, by NO3-, formate and acetate. Inhibition of Cl- uptake by extracellular HCO3- was less effective in the absence of extracellular Na+, suggesting that not only HCO3- but also NaCO3- binds to the carrier. SO2/4-, cyclamate and gluconate did not significantly reduce Cl- uptake via the anion exchanger. DIDS-senstive Cl- uptake showed saturation kinetics with respect to the Cl- concentration with an apparent Km of 8 mM. Cl- efflux could be stimulated by external Cl- and HCO3- and was inhibited by DIDS. Thus, cultured bovine pigmented ciliary epithelial cells express a Cl-/HCO3- exchanger. A possible role of this carrier system for aqueous humor formation is discussed [corrected].

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1988

Evidence for Na+/H+ exchange and pH sensitive membrane voltage in cultured bovine corneal epithelial cells.

Two methods were used to investigate cellular ion transport processes in confluent monolayers of cultured bovine corneal epithelial cells: measurements of membrane voltage (V) using conventional microelectrodes, and intracellular pH (pHi) measurements using the pH sensitive absorbance of intracellularly trapped 5 (and 6)-carboxy-4', 5'dimethyl-fluorescein. (1) V averaged -39.2 +/- 0.9 mV (mean +/- SEM, n = 71) with a range of -30 to -59 mV. Increasing extracellular potassium depolarized the cell membrane with a K+-slope of 43.3 mV/decade [K+] (for [K+] between 20 and 80 mM). Intracellular as well as extracellular acidification reversibly depolarized the cell membrane. Depolarization induced by 40 mM K+-pulses was smaller at extracellular pH (pHo) of 6.9 as compared to pHo = 7.9. These findings are compatible with a pH-sensitive K+ conductance. (2) During steady state pHi was 6.96 +/- 0.05 (mean +/- SEM, n = 7). After intracellular acidification, induced by NH4Cl-prepulse technique, pHi was regulated back towards normal steady state pHi. Application of 1 mM amiloride reversibly inhibited pHi recovery. Furthermore, pHi backregulation was inhibited by removing sodium from the extracellular solution. The effect was reversible after readdition of sodium. These findings suggest that a Na+/H+ exchange is present in corneal epithelial cells and participates in pHi backregulation after an intracellular acid load.

Acids↗
PubMed1987

K+-conductance and electrogenic Na+/K+ transport of cultured bovine pigmented ciliary epithelium.

Using intracellular microelectrode technique, we investigated the changes in membrane voltage (V) of cultured bovine pigmented ciliary epithelial cells induced by different extracellular solutions. (1) V in 213 cells under steady-state conditions averaged -46.1 +/- 0.6 mV (SEM). (2) Increasing extracellular K+ concentration [( K+]o) depolarized V. Addition of Ba2+ could diminish this response. (3) Depolarization on doubling [K+]o was increased at higher [K+]o (or low voltage). (4) Removing extracellular Ca2+ decreased V and reduced the V amplitude on increasing [K+]o. (5) V was pH sensitive. Extra- and intracellular acidification depolarized V; alkalinization induced a hyperpolarization. V responses to high [K+]o were reduced at acidic extracellular pH. (6) Removing K+o depolarized, K+o readdition after K+ depletion transiently hyperpolarized V. These responses were insensitive to Ba2+ but were abolished in the presence of ouabain or in Na+-free medium. (7) Na+ readdition after Na+ depletion transiently hyperpolarized V. This reaction was markedly reduced in the presence of ouabain or in K+-free solution but unchanged by Ba2+. It is concluded that in cultured bovine pigmented ciliary epithelial cells K+ conductance depends on Ca2+, pH and [K+]o (or voltage). An electrogenic Na+/K+-transport is present, which is stimulated during recovery from K+ or Na+ depletion. This transport is inhibited by ouabain and in K+- or Na+-free medium.

Animals↗
PubMed1987

Effects of ouabain and furosemide on transepithelial electrical parameters of the isolated shark ciliary epithelium.

Sections of the ciliary epithelium of adult sharks (Squalus acanthias) were mounted in Ussing-type chambers (area 0.2 cm2) for measurements of transepithelial potential difference (PD), short circuit current (SCC) and calculation of transepithelial resistance (R). In 15 preparations PD was aqueous side negative (-0.51 +/- 0.12 mV; SCC 18.3 +/- 2.5 microA cm-2; R 30.7 +/- 3.1 Ohm cm2). However, in 15 other preparations incubated in identical Ringer's solution PD was aqueous side positive (0.53 +/- 0.09 mV; SCC -19.6 +/- 2.3 microA cm-2; R 27.9 +/- 2.8 Ohm cm2). 10(-5) M ouabain or 10(-4) M furosemide were applied either to the aqueous or blood side of the isolated ciliary epithelium at transepithelial negative or positive PD. When the transepithelial PD was positive on the aqueous side ouabain decreased PD and SCC within 15 to 45 min. When the spontaneous PD was negative both PD and SCC decreased when ouabain was applied to the blood side. When the drug was given to the aqueous side a biphasic response (first stimulation, then inhibition) of PD and SCC was observed. Furosemide when given to the blood side (with aqueous side PD positive) or to the aqueous side (with aqueous side PD negative) decreased PD and SCC. However, a transient stimulation of both electrical parameters was observed when furosemide was applied to either the blood side (with aqueous side PD negative) or to the aqueous side (with aqueous side PD positive). The polarity and magnitude of PD and SCC probably depend on the relative activity of sodium and chloride pumps across the cell membranes of the non-pigmented and/or pigmented cell layer. However, additional transport mechanisms cannot be excluded.

Animals↗
PubMed1986

The regulation of intracellular pH in monkey kidney epithelial cells (BSC-1). Roles of Na+/H+ antiport, Na+-HCO3(-)-(NaCO3-) symport, and Cl-/HCO3- exchange.

Using the pH-sensitive absorbance of 5 (and 6)-carboxy-4',5'- dimethylfluorescein, we investigated the regulation of cytoplasmic pH (pHi) in monkey kidney epithelial cells (BSC-1). In the absence of HCO3-, pHi is 7.15 +/- 0.1, which is not significantly different from pHi in 28 mM HCO3-, 5% CO2 (7.21 +/- 0.07). After an acid load, the cells regulate pHi in the absence of HCO3- by a Na+ (or Li+)-dependent, amiloride-inhibitable mechanism (indicative of Na+/H+ antiport). In 28 mM HCO3-, while still dependent on Na+, this regulation is only blocked in part by 1 mM amiloride. A partial block is also observed with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) (1 mM). With cells pretreated with DIDS, 1 mM amiloride nearly totally inhibits this regulation. Cl- had no effect on pHi regulation in the acidic range. In HCO3(-)-free saline, Na+ removal leads to an amiloride-insensitive acidification, which is dependent on Ca2+. In 28 mM HCO3-, Na+ (and Ca2+) removal led to a pronounced reversible and DIDS-sensitive acidification. When HCO3- was lowered from 46 to 10 mM at constant pCO2 (5%), pHi dropped by a DIDS-sensitive mechanism. Identical changes in pHo (7.6 to 6.9) in the nominal absence of HCO3- led to smaller changes of pHi. In the presence but not in the absence of HCO3-, removal of Cl- led to a DIDS-sensitive alkalinization. This was also observed in the nominal absence of Na+, which leads to a sustained acidification. It is concluded that in nominally bicarbonate-free saline, the amiloride-sensitive Na+/H+ antiport is the predominant mechanism of pHi regulation at acidic pHi, while being relatively inactive at physiological values of pHi. In bicarbonate saline, two other mechanisms effect pHi regulation: a DIDS-sensitive Na+-HCO3- symport, which contributes to cytoplasmic alkalinization, and a DIDS-sensitive Cl-/HCO3- exchange, which is apparently independent of Na+.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1986

Kinetic properties of the sodium bicarbonate (carbonate) symport in monkey kidney epithelial cells (BSC-1). Interactions between Na+, HCO-3, and pH.

BSC-1 kidney epithelial cells derived from the African green monkey are known to express a Na+HCO3- symport (Jentsch, T. J., Schill, B. S., Schwartz, P., Matthes, H., Keller, S. K., and Wiederholt, M. (1985) J. Biol. Chem. 260, 15554-15560). In the present work, 4,4-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-sensitive 22Na+ uptake into confluent monolayers of BSC-1 is measured in the presence of ouabain (10(-4) M) and amiloride (10(-3) M) to define the interactions between Na+ and HCO3- binding and pH. Dependence of DIDS-sensitive 22Na+ fluxes on either Na+ or HCO3- can be described by Michaelis-Menten kinetics. External apparent Km for HCO3- decreases with increasing Na+ concentration (Km app (HCO3-) = 36 +/- 10, 18 +/- 5, and 9 +/- 3 mM at 20, 45, and 151 mM Na+o, respectively (pHo = 7.4)). Similarly, external apparent Km for Na+ decreases with increasing HCO3- concentration (Km app (Na+) = 73 +/- 22, 28 +/- 8, and 14 +/- 4 mM at 6, 17, and 56 mM HCO3o-, respectively (pHo = 7.4)). Vmax app remains constant within the experimental error. When data are replotted as a function of calculated NaCO3- concentration, they can be approximated by a single Michaelis-Menten equation. DIDS-sensitive uptake at constant Na+ and HCO3- displays a broad pH optimum in the range between 7.2 and 7.6. The data are compatible with the ion pair model in which the transported species, NaCO3-, binds to the transport site with Km = 15.3 +/- 4 microM. However, the data may also be fitted by either a random or ordered bireactant system. Sets of parameters necessary for these fits are given.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1986

Membrane potentials and intracellular chloride activity in the ciliary body of the shark.

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 X l-1) decrease the potential, and furosemide (10(-4) mol X 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 X l-1) was significantly higher than could be predicted from the equilibrium distribution (26 mmol X l-1) across the cell membranes. When furosemide was applied to the aqueous side of the epithelium, intracellular chloride activity decreased to 35 mmol X 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.

Animals↗
PubMed1986

[Effect of cyclosporin A, dexamethasone and various preservatives on epithelial wound healing in the denervated guinea pig cornea].

In order to examine a possible connection between punctate keratopathy following keratoplasty and the various drugs used in postoperative treatment, denervation was performed in white Pirbright guinea pigs by corneal trepanation with a 6 mm trephine to a depth of 0.25 mm. One week later an iodine gas applicator was used to produce erosion in the cornea, which had healed in the meantime. Until the erosions healed, the corneas were photographed four times daily after fluorescein staining; the negatives were enlarged by projection and the erosions measured planimetrically. The linear regression method was used to calculate a compensating straight line and thus the hourly epithelial wound-healing surface (mm2/h) for each eye. This was 0.76 mm2/h in control eyes. Dexamethasone 0.1%, benzalconium chloride 0.01%, and chlorobutanol 0.2% did not significantly inhibit wound healing, while Spersadex (dexamethasone 0.1% + benzalconium chloride 0.01%) and Totocortin (dexamethasone 0.1 + chlorobutanol 0.2%) did. Polyvinyl alcohol (1.4% without preservative) significantly delayed epithelial wound healing. Cyclosporin A 2% (in castor oil) had no influence on epithelial wound healing.

Animals↗
PubMed1986

Effect of calcium antagonist compound nisoldipine on transepithelial electrical parameters in the isolated frog cornea.

The new calcium antagonist, nisoldipine, inhibited short circuit current and transcorneal potential difference in the isolated frog cornea (a chloride transporting epithelium). Transepithelial resistance increased. The effects of nisoldipine were dose-dependent. Nisoldipine did not change the stimulation of short circuit current induced by epinephrine. Intracellular membrane potential across both the apical and basolateral cell membrane were decreased by nisoldipine. The data indicate that intracellular calcium seemed to modulate chloride transport of the cornea by changing cell membrane permeability.

Animals↗