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H Helbig

Publications and source records attributed to H Helbig.

68 records · Page 4Linked to original sources
PubMed1989

Effect of acetylcholine on membrane potential of cultured human nonpigmented ciliary epithelial cells.

Human nonpigmented ciliary epithelial cells (NPE) were grown in tissue culture after transformation with an origin-defective mutant of SV-40 DNA. In these cells membrane potentials (V) were measured using the microelectrode technique. Addition of 10(-4) M acetylcholine led to a bisphasic voltage response. An immediate, transient hyperpolarization was followed by a sustained depolarization below the steady state level. These responses were irreversibly blocked by 10(-5) M atropine. In Ca2+-free media the initial addition of acetylcholine resulted in an unchanged voltage response. A second application of acetylcholine in Ca2+-free solution evoked only an abortive response of V, and further addition had no effect on V. In the presence of Ca2+ channel blockers (10(-5) M verapamil, 1 mM Co2+) the acetylcholine-induced response of the membrane potential was not changed. The initial hyperpolarization induced by acetylcholine was reduced by 33 +/- 3% (n = 6) in the presence of 2 mM Ba2+ and by 79 +/- 6% (n = 6) in the presence of 1 mM quinidine. Moreover, the amplitude of the hyperpolarization was dependent on the extracellular K+ concentration. With increasing extracellular K+ concentration (and decreasing transmembrane K+ gradient) the acetylcholine-induced hyperpolarization was reduced. To further elucidate the role of Ca2+ in the acetylcholine-induced responses, we measured cytoplasmic Ca2+ activity using the fluorescence of intracellularly trapped Fura-2. Cytoplasmic Ca2+ activity increased immediately and transiently upon addition of acetylcholine. We conclude that acetylcholine transiently hyperpolarizes V in cultured human NPE by activation of K+ channels mediated by mobilization of Ca2+ from intracellular stores.

Acetylcholine↗
PubMed1989

Effect of hypotonic media on the membrane voltage of cultured bovine corneal endothelial cells.

The effects of hypotonicity on cultured bovine corneal endothelial cells were investigated using standard microelectrode and superfusion techniques. Confluent monolayers of cells were superfused with an isotonic (305 +/- 5 mosm/kg) control solution until a stable membrane voltage (V) was obtained, then with a hypotonic (240 +/- 5 mosm/kg) solution. Under control conditions, V was - 51.4 +/- 0.8 mV (means +/- SEM, n = 154). Decreasing solution osmolality resulted in an immediate depolarization: mean maximal delta V = 18.7 +/- 0.9 mV at 2.6 +/- 0.2 minutes with a gradual recovery to a new but still depolarized steady-state V (delta v = 11.1 +/- 0.9 mV at 8.2 +/- 0.3 minutes, n = 25). The depolarizing response to hypotonicity persisted in the presence of amiloride (10(-3)M), DIDS (10(-3)M), bumetanide (10(-4)M) or ouabain (10(-4)M) as well as in the absence of extracellular Cl-, Na+, HCO3- or Ca2+. Relative K+ conductance was estimated by the effect on V of increased extracellular [K+] - this was significantly reduced at 5, 10 and 20 mM K+ under hypotonic conditions. The depolarization induced by 1mM Ba2+ was also reduced from 19.6 +/- 0.5 mV (n = 8) under isotonic conditions to 15.4 +/- 0.4 mV (n = 6) under hypotonic conditions (p less than 0.001). The conductive HCO3- pathway - as judged by the hyperpolarization of V induced by increasing extracellular [HCO3-] from 28 to 60 mM, was also reduced under hypotonic conditions (delta V = 17.2 +/- 0.8 mV, n = 13 (isotonic) compared to delta V = 9.5 +/- 0.3 mV, n = 15 (hypotonic].(ABSTRACT TRUNCATED AT 250 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1989

[Measuring intracellular pH in cultivated corneal epithelial cells].

Electrolyte and fluid transport across the corneal epithelium plays an important role for the maintenance of corneal transparency. Little is known about the cellular mechanisms underlying the transepithelial transport phenomena. We have therfore started to investigate the transmembranal transport processes in cultured corneal epithelial cells. Studies were performed in an established corneal epithelial cell line from the rabbit (SIRC cells) and in primary cultured bovine corneal epithelial cells. In these cells, intracellular pH was continuously measured using the pH-sensitive absorbance of intracellularly trapped dimethylcarboxyfluorescein. In both cell types, SIRC cells and cultured bovine corneal epithelial cells, evidence was found in favor of a sodium-dependent and amiloride-sensitive Na/H exchange. In cultured bovine corneal epithelial cells, Na/H exchange was shown to play an important role in intracellular pH recovery after an acid load. Furthermore, as has been demonstrated in other epithelia, Na+/H+ exchange may also be important for transepithelial corneal transport.

Acid-Base Equilibrium↗
PubMed1988

Characterization of Na+/H+ exchange in a rabbit corneal epithelial cell line (SIRC).

Continuous intracellular pH (pHi) measurements were performed in SIRC rabbit corneal epithelial cells using the pH-sensitive absorbance of intracellularly trapped 5(and 6)-carboxy-4',5'-dimethylfluorescein. Steady-state pHi in nominally bicarbonate free Ringer's solution averaged 6.87 +/- 0.02 (mean +/- S.E., n = 53). After intracellular acidification induced by the NH4Cl-prepulse technique, there was a sodium-dependent pHi recovery towards the normal steady-state pHi. The initial pHi recovery rate was a saturable function of extracellular sodium concentration with an apparent Km for external sodium of about 25 mM and a Vmax of about 0.28 pH units/min. Virtually no pHi recovery was observed in the absence of extracellular sodium. Sodium removal during steady state acidified the cells by 0.36 +/- 0.05 pH units (mean +/- S.E., n = 13) within 5 min. There was a dose-dependent inhibition of pHi recovery after NH4Cl prepulse by amiloride with an IC50 of about 15 microM. Amiloride in a concentration of 1 mM almost completely abolished pHi recovery. Amiloride (1 mM) applied during steady state induced an intracellular acidification of 0.2 +/- 0.03 pH units (mean +/- S.E., n = 7) within 5 min. These findings suggest that a Na+/H+ exchange is present in SIRC rabbit corneal epithelial cells. Na+/H+ exchange seems to be the major process involved in pHi recovery in SIRC cells after an intracellular acid load. Na+/H+ exchange also plays a role in the maintenance of steady-state pHi.

Amiloride↗
PubMed1988

Na+/H+ exchange regulates intracellular pH in a cell clone derived from bovine pigmented ciliary epithelium.

The regulation of intracellular pH (pHi) was monitored in a virus-transformed cell clone derived from bovine ciliary body exhibiting characteristics of pigmented ciliary epithelium. Data were obtained from confluent monolayers grown on plastic coverslips in nominally bicarbonate-free media using the pH-sensitive absorbance of 5- (and 6-) carboxy-4',5'-dimethylfluorescein. Under resting conditions, pHi averaged 6.98 +/- 0.01 (SEM; n = 57). When cells were acid loaded by briefly exposing them to Ringer containing NH4+ and then withdrawing the NH4+, pHi spontaneously regained its initial value. In the presence of 1 mM amiloride or in the absence of Na+, this process was blocked, indicating the involvement of an Na+/H+ exchanger in the regulation of pHi after an acid load. Removing Na+ during resting conditions decreased cytoplasmatic pH. This acidification could be slowed by amiloride, which is evidence for reversal of the Na+/H+ countertransport exchanging intracellular Na+ for extracellular protons. Application of 1 mM amiloride during steady state led to a slow acidification. Thus the Na+/H+ exchanger is operative during resting conditions extruding protons, derived from cellular metabolism, or from downhill leakage into the cell. Addition of Na+ to Na+ -depleted cells led to an alkalinization, which was sensitive to amiloride, with an IC50 of about 20 microM. This alkalinization was attributed to the Na+/H+ exchanger and exhibited saturation kinetics with increasing Na+ concentrations, with an apparent KM of 29.6 mM Na+. It is concluded that Na+/H+ exchange regulates pHi during steady state and after an acid load.

Amiloride↗
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↗
PubMedDate not supplied

Traumatic rupture of the globe caused by cow horns.

PURPOSE: We investigated the epidemiology, clinical findings and functional outcome of open-globe injuries caused by cow horns over a 50-year period in eastern Switzerland. METHODS: We retrospectively evaluated the files of cases with ruptures of the globe caused by cow horns between 1950 and 1999. RESULTS: We found 59 cases with ruptures of the globe by cow horns, accounting for 5% of all open-globe injuries. The incidence of these accidents did not change during the observation period. Twenty-two eyes (37%) were enucleated. Only 7 eyes (12%) retained a vision of > or = 0.1. Between 1950 and 1989 only 2/43 eyes (5%) reached a vision of > or = 0.1. In the 1990's, with the introduction of vitreous surgery 5/16 eyes (31%) had a vision of > or = 0.1. Four patients (7%) had blinding eye disease in the partner eye, and three (5%) had a second open-globe trauma. CONCLUSIONS: In rural regions, with cattle breeding, open-globe injuries by cow horns are relatively common and the frequency is still the same as 50 years ago. The visual prognosis of these eyes is still guarded, but functional results have improved with the introduction of vitreous surgery. Patients who have had a rupture of the globe appear to have an increased risk for partner eye trauma. Therefore, all efforts are justified to preserve even limited vision in severely injured eyes.

Adolescent↗