Biomedical subjects

M Wiederholt

Publications and source records attributed to M Wiederholt.

At least 73 records · Page 4Linked to original sources

PubMed1989

Mechanically stripped pigmented and non-pigmented epithelium of the shark ciliary body: morphology and transepithelial electrical properties.

Sections of intact ciliary epithelium and mechanically stripped non-pigmented (NPE) and pigmented (PE) cell layers of adult sharks (Squalus acanthias) were mounted in Ussing-type chambers (area 0.1 cm2). Addition of 10(-5) M forskolin to the aqueous side of intact epithelium significantly increased short-circuit current (Isc) within 15 min and a maximum of approx. 30 microA cm-2 was reached after 45-60 min. Transepithelial potential difference (V) increased from -0.8 mV (aqueous side negative as compared with blood/stromal side) to -1.5 mV, whereas resistance (R) was unchanged (50 omega cm2). Forskolin was without effect when applied to the blood side. In stripped PE preparations (R 15 omega cm2), 10(-5) M forskolin applied to the apical side induced a qualitatively similar change of Isc and V compared with the intact tissue. The forskolin-induced effects were fully reversed by 10(-4) M bumetanide and were not dependent on pretreatment of the tissue with 10(-3) M BaCl2. In stripped NPE preparations resistance was usually less than 10 omega cm2 and was not stable. This is consistent with the morphologic observation that although tight junctions were still demonstrable in stripped NPE cells, the apical membranes were damaged. In preparations taken for light and electron microscopy the stripped PE layer revealed intact epithelial cells. In particular, the basal thirds of the stripped PE cells were in very close contact with each other. These attachment zones may have the appearance of tight junctions. Thus the PE cells of the shark ciliary epithelium can be successfully isolated for transepithelial transport studies. The adenylate cyclase system is present in PE cells, and transepithelial transport of chloride may be regulated by intracellular cAMP.

Animals↗

PubMed1989

Regional differences in the morphology and enzyme distribution of the spiny dogfish (Squalus acanthias) ciliary epithelium.

The ciliary epithelium of spiny dogfish eyes has previously been used for studies of epithelial electrolyte transport and the results are found to be comparable to those obtained in mammals. In this study we report the ultrastructure and enzyme histochemistry of the ciliary epithelium of spiny dogfish in comparison with that of mammals. In contrast to the mammals, the lens in spiny dogfish is connected to the anterior and middle part of the ciliary epithelium by a broad zonula-like suspensory ligament and by a short ligament to the ventral papilla, while the posterior region is mainly free of zonula-like fibers. The non-pigmented epithelium (NPE) of the posterior region shows numerous membrane infoldings, interdigitations and many mitochondria in the cytoplasm as well as histochemical staining for Na+ K(+)-ATPase and carbonic anhydrase (CA). These findings are similar to those seen in the anterior pars plicata of mammals which is mainly involved in aqueous secretion. In the anterior and middle part, the NPE cells have only a few membrane infoldings and few mitochondria in the cytoplasm, but abundant surfaces of rough endoplasmic reticulum, numerous ribosomes and Golgi material, indicating protein synthesis. Histochemically the cells stain for Na+, K(+)-ATPase but not for CA. These findings are comparable to the pars plana in mammals. In contrast to mammals, where the pigmented epithelium (PE) shows nearly no staining for Na+, K(+)-ATPase but an intensive one for CA, the PE cells of spiny dogfish are heavily stained for Na+, K(+)-ATPase in all regions of the eye, but show nearly no staining for CA.

Animals↗

PubMed1989

A randomised prospective study of treatment of non-ischaemic central retinal vein occlusion by isovolaemic haemodilution.

In a randomised study 25 patients over 50 years of age with non-ischaemic central retinal vein occlusion (CRVO) were assigned to either a treatment (14 patients, isovolaemic haemodilution) or a control group (11 patients, no treatment). After three months eight eyes in patients with haemodilution improved, whereas none of the untreated eyes had better visual acuity (p less than 0.01). Thirteen haemodiluted and 11 control patients could be observed for one year. Six eyes of the haemodiluted patients retained a better visual acuity, whereas no improvements had occurred in the control group (p less than 0.025). In fluorescein angiography the lowering of the packed cell volume to 35-32% accelerated the time of maximal venous filling (tmvf) from 17.4 (SEM 1.4) s to 11.4 (SEM 0.9) s (p less than 0.005). In patients with non-ischaemic CRVO without treatment the passage time did not change. The shortened tmvf reflects a higher blood velocity. Thus isovolaemic haemodilution improves the visual prognosis in non-ischaemic CRVO probably by inducing a higher blood fluidity, which results in higher blood velocity, at least in areas of compromised retinal microcirculation.

Aged↗

PubMed1989

Electrogenic Na+-ascorbate cotransport in cultured bovine pigmented ciliary epithelial cells.

The high level of ascorbic acid (AA) in the aqueous humor of many mammals suggests an active transport of AA across the double-layered ciliary epithelium from blood to aqueous humor. We used [14C]AA to study AA uptake in bovine pigmented ciliary epithelial cells in tissue culture. We observed a 40-fold intracellular accumulation of AA, which was dependent on extracellular Na+. With labeled dehydroascorbate (DHA, the oxidized form of the vitamin) in the medium, there was a 20-fold intracellular accumulation of the label. However, the time course of DHA uptake was different compared with AA uptake and was not Na+ dependent, suggesting different transport systems for AA and DHA. AA uptake was inhibited by 1 mM phloretin and in the presence of isoascorbate. Furthermore, AA uptake was markedly reduced when intracellular Na+ was elevated by preincubation with ouabain or amphotericin B. With increasing AA concentration, Na+-dependent AA uptake exhibited first-order saturation kinetics with half-maximal uptake at 76 microM AA. Na+ dependence of AA uptake revealed a sigmoidal curve of Na+-dependent AA uptake vs. Na+ concentration with a half-maximal AA uptake at 45.4 mM Na+. The slope of the Hill plot from these data was 1.94, suggesting a transport system translocating two or more Na+ for one AA. This stoichiometry implies electrogenicity of the transporter. We, therefore, measured membrane potentials using conventional microelectrodes. Addition of 200 microM AA resulted in a depolarization of the membrane voltage by 4.9 +/- 0.5 mV (n = 22), which was absent in Na+ free medium and was markedly reduced by phloretin.(ABSTRACT TRUNCATED AT 250 WORDS)

Amphotericin B↗

PubMed1989

Continuous membrane voltage recordings in A10 vascular smooth muscle cells: effect of AVP.

Continuous membrane voltage (V) recordings were obtained in A10 vascular smooth muscle cells (rat aorta) using glass microelectrodes. Resting membrane voltage in 262 impalements averaged 54.0 +/- 0.4 (SE) mV. Relative K+ conductance was characterized, and the contribution of electrogenic Na+-K+-ATPase to membrane voltage was investigated. Action potentials could be induced by application of 1 mM barium or 10(-4) M acetylcholine. In a few recordings, spontaneous spike activity occurred, and this could be abolished by 5 mM MgCl2 or by removal of extracellular Ca2+. Barium-induced action potentials were not dependent on the presence of extracellular Na+ and not inhibitable by 10(-6) M tetrodotoxin. Application of 10(-6) M [Arg8] vasopressin (AVP) for 30 s caused a typical biphasic membrane voltage response with an initial transient hyperpolarization of -9.5 +/- 1.1 mV and a more sustained subsequent depolarizing response averaging 28.2 +/- 1.3 mV (mean +/- SE, n = 58). The effect of AVP on membrane voltage was blocked by the V1-antagonist [beta-mercapto-beta,beta-cyclopentamethylenepropionyl1,O-Me- Tyr2,Arg8]vasopressin. The initial hyperpolarizing component of the membrane voltage response to AVP became more prominent when V was predepolarized, for example, by a preceding AVP application. However, when AVP was applied during high K+ depolarization or in the presence of quinidine (1 mM), the initial hyperpolarizing response was practically abolished. The time course of the initial hyperpolarization was shown to be similar to the calcium transient observed in fura-2-loaded A10 cell suspensions after the application of AVP. We conclude that the initial AVP-induced hyperpolarization in A10 cells corresponds to an activation of Ca2+-activated K+ channels.

Acetylcholine↗

PubMed1989

Role of HCO3- in regulation of cytoplasmic pH in ciliary epithelial cells.

Cytoplasmic pH (pHi) was monitored using the pH-sensitive absorbance of 5(6)carboxy-4',5'-dimethylfluorescein in monolayers of a cell clone derived from bovine pigmented ciliary epithelium (PE) transformed with the simian virus 40. 1) Changing extracellular media from a nominally HCO3(-)-free solution to a solution containing 28 mM HCO3(-)-5% CO2 at constant extracellular pH (7.4) resulted in a delayed alkalinization of pHi, which was 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) sensitive and was inhibited in Na+-free medium and in Cl(-)-depleted cells. 2) DIDS pretreatment acidified pHi in HCO3(-)-containing media. 3) Replacing extracellular Cl- resulted in a DIDS-sensitive, HCO3(-)-dependent, and Na+-independent alkalinization. 4) Replacing extracellular Na+ in HCO3(-)-containing media led to a partly DIDS-sensitive intracellular acidification. 5) Recovery of pHi after an alkali load (acetate prepulse) had a HCO3(-)-dependent and DIDS-sensitive component. 6) Two Na+-dependent components participated in pHi regulation after an acid load (NH4+ prepulse) in HCO3(-)-containing solution. One was amiloride sensitive, the other was DIDS sensitive and was inhibited in HCO3(-)-free media and after Cl- depletion. We conclude that in cultured PE, in addition to Na+-H+ exchange, two HCO3-transporters participate in pHi regulation. Cl(-)-dependent Na+-HCO3-symport regulates pHi during steady state and after an acid load, and Na+-independent Cl(-)-HCO3-exchange is involved in pHi recovery after an alkali load.

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

PubMed1989

Effect and compatibility of isovolaemic haemodilution in the treatment of ischaemic and non-ischaemic central retinal vein occlusion.

In a prospective study, 48 eyes with non-ischaemic and 35 with ischaemic central retinal vein occlusion (CRVO) were treated by isovolaemic haemodilution (IHD). Two or more cardiovascular risk factors were present in 42% of patients with non-ischaemic and in 69% of patients with ischaemic CRVO (p less than 0.025). Nevertheless, IHD in no case caused serious cardiovascular complications. Minor problems were short fainting spells in 5% and a general weakness in 16% of the haemodiluted patients. The effect of IHD was measured by determining the time of maximal venous filling (tmvf) in fluorescein angiographies. Lowering the packed cell volume to 32-35% accelerated the tmvf from 18.4 +/- 1.61 to 13.1 +/- 1.0 s (p less than 0.001) in eyes with non-ischaemic CRVO and from 24.5 +/- 1.2 to 14.8 +/- 1.3 s (p less than 0.001) in eyes with ischaemic CRVO. After 3 months, an increase in visual acuity had occurred in 27% of eyes with non-ischaemic CRVO and in 48.5% with ischaemic CRVO. These improvement rates could nearly be maintained after 1 year. 59% of eyes with non-ischaemic and 25% with ischaemic CRVO were able to read (visual acuity greater than or equal to 6/15) after 1 year.

Cardiovascular Diseases↗

PubMed1989

Sodium bicarbonate cotransport in cultured pigmented ciliary epithelial cells.

Uptake of 22Na+ was studied in cultured bovine pigmented ciliary epithelial cells (PE) in HCO3-containing media. Two components of Na+-uptake were stimulated by intracellular acidification (NH4+-prepulse): One was amiloride-sensitive, the other DIDS-sensitive. The amiloride-sensitive component of Na+-uptake probably represents Na+/H+-exchange, which has previously been characterized in PE. The second, DIDS-sensitive component stimulated by intracellular acidification, was Cl- and HCO3--dependent. We conclude that a stilbene-sensitive, Cl--dependent Na+-HCO3--cotransport is present in PE. This transport could play an important role in aqueous humor formation.

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

PubMed1989

Coupling of 22Na and 36Cl uptake in cultured pigmented ciliary epithelial cells: a proposed role for the isoenzymes of carbonic anhydrase.

Uptake studies with 22Na and 36Cl were performed in cultured bovine pigmented ciliary epithelial cells (PE) to investigate interdependence of Na+ and Cl- transport. (1) 22Na uptake into NaCl depleted cells was stimulated by Cl-. This stimulation was abolished by the simultaneous application of amiloride (1 mM) and bumetanide (0.1 mM), indicating two independent mechanism for Cl- stimulated Na+ uptake: loop diuretic sensitive Na+/Cl- symport and an indirect stimulation of Na+/H+ exchange by Cl-. The latter component of Cl- stimulated Na+ uptake was HCO3- dependent. (2) 36Cl uptake was increased by extracellular Na+. Na+-stimulated Cl- uptake also consisted of two components. One was bumetanide sensitive and the other was blockable by amiloride and partly inhibited by the carbonic anhydrase (CA) inhibitor methazolamide (0.1 mM). (3) Homogenized PE cells were tested for biochemical CA activity using an electrometric method. The cytoplasmic as well as the membrane fraction contained specific CA activity. (4) A model is presented for Na+ and Cl- transport into PE: in addition to Na+/Cl- symport, Na+/H+ and Cl-/HCO3- double exchange may operate in the ciliary epithelium. The latter mechanism provides NaCl uptake into the cell in exchange for H+ and HCO3-, which recycle as CO2 across the membrane. This recycling of CO2 and HCO3-/H+ (and hence indirectly NaCl uptake) is facilitated by the cooperation between membrane bound and cytoplasmic CA.

Amiloride↗

PubMed1989

Intracellular voltage recordings in bovine non-pigmented ciliary epithelial cells in primary culture.

Bovine non-pigmented ciliary epithelial cells (NPE) have been isolated by a technique of selective adhesion to tissue culture plastic. NPE cells in primary culture proliferated and maintained epithelial-like morphology for about 4 weeks in tissue culture medium containing 10% fetal calf serum. If grown for longer than 4 weeks in serum-containing medium, cells changed their morphology and became elongated and spindle-shaped. Membrane potentials were measured using conventional microelectrodes. In NPE cells of epithelial-like shape, replacing extracellular Na+ induced a transient hyperpolarization of the membrane potential, while in elongated cells of spindle-shaped morphology an immediate depolarization was observed. We therefore only used epithelial-like NPE for further experiments. In these cells the mean membrane potential was -40.3 +/- 0.5 mV (n = 36). Relative K+ conductance was increased by extracellular alkalinization. Removing extracellular K+ led to a depolarization and readdition of K+ to K+ depleted cells resulted in a hyperpolarization. Both voltage responses were sensitive to ouabain, indicating that Na+/K+ ATPase is inhibited by K+ replacement, and that there is overshoot-activation of the pump when K+ is readded. Extracellular Cl- replacement led to a DIDS sensitive, transient depolarization, which is compatible with a stilbene-sensitive Cl(-)-conductance. Removing HCO3- led to a Na+ dependent and DIDS-sensitive depolarization. However, the electrical response on replacement of extracellular Na+ was not influenced by DIDS or the extracellular HCO3(-)-concentration.

Acetylcholine↗

PubMed1989

Electrical membrane properties of a cell clone derived from human nonpigmented ciliary epithelium.

Intracellular potentials were measured in a SV-40 virus-transformed cell clone derived from human nonpigmented ciliary epithelium using the microelectrode technique. (1) Membrane potential averaged -50.2 mV (+/- 0.6, n = 207). (2) Increasing the extracellular K+ concentration depolarized the membrane voltage. The amplitude of this potential response was reduced in the presence of 1 mM Ba2+. (3) Superfusing the cells with a Ca2+-free solution containing 1 mM EGTA depolarized the intracellular potential and diminished the voltage response upon increasing extracellular K+. (4) Extracellular alkalinization hyperpolarized the membrane potential and increased the voltage amplitude on increasing extracellular K+. (5) Addition of ouabain immediately reduced the intracellular potential. Removing extracellular K+ depolarized membrane voltage, readdition of K+ after K+ depletion transiently hyperpolarized intracellular voltage. Both potential responses were inhibited in the presence of ouabain. (6) Replacing extracellular Cl- by cyclamate resulted in a transient depolarization followed by a hyperpolarization. In the presence of SITS or DIDS (greater than or equal to 0.1 mM) the electrical responses of the cell membrane to Cl- replacement were blocked. We conclude that cultured human nonpigmented ciliary epithelial cells possess an electrogenic Na+/K+-ATPase, a K+ conductance modulated by Ca2+ and pH, and a Cl- conductance sensitive to stilbene derivatives.

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

PubMed1989

[Carbonic anhydrase and its function in ion transport in cultivated pigmented ciliary body epithelial cells].

The effect of inhibitors of the carbonic anhydrase on aqueous humor formation suggests that carbonic anhydrase has an important role in ion transport across the ciliary epithelium. We therefore investigated the role of carbonic anhydrase in Na+ and Cl- transport in cultured bovine pigmented ciliary epithelial cells (PE) using the radioactive isotopes 22Na and 36Cl. Our findings can be summarized as follows. (1) Na+ uptake into the cell occurs via an amiloride-sensitive Na+/H+ exchanger. (2) Cl- is transported into the cell in exchange for bicarbonate ions. (3) PE cells contain biochemical carbonic anhydrase activity. (4) Na+ uptake into NaCl-depleted cells is markedly stimulated by Cl-. This stimulation is HCO3- dependent and is completely blocked by amiloride and partly inhibited by the carbonic anhydrase inhibitor methazolamide. (5) A model is introduced for transport of NaCl in the ciliary epithelium: carbonic anhydrase is responsible for coupling of Na+/H+ exchange and Cl-/HCO3- countertransport. This model could explain the effect of carbonic anhydrase inhibitors on intraocular pressure.

Animals↗

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

Regulation of intracellular pH in cultured bovine retinal pigment epithelial cells.

Regulation of intracellular pH (pHi) in bovine retinal pigment epithelium (RPE) was investigated in cell culture. pHi was measured using the pH-sensitive absorbance of intracellularly trapped 5 (and 6)-carboxy-dimethyl-fluorescein (CDMF). (1) Regulation of pHi after induction of an acid load by removal of NH4Cl could be blocked either totally by removal of extracellular sodium, or subtotally (about 90%) by application of amiloride (1 mmol/l). Additional flux measurements revealed a dose-dependent, amiloride-sensitive 22Na+-uptake into Na+-loaded cells. Both results suggest the presence of a Na+/H+ antiport. (2) When alkalinization of the cells was induced by preincubation with 50 mmol/l acetate in HCO3(-)-Ringer's and subsequent removal of the weak acid, the following regulation was dependent on the presence of extracellular chloride. This process could be blocked with DIDS (1 mmol/l), suggesting the presence of a Cl-/HCO3- exchange mechanism. (3) We found no evidence for a Na+/HCO3(-)-cotransport, which had been postulated to be present in RPE by others. We conclude that two processes are involved in regulation of pHi in RPE: A Na+/H+ antiport responsible for recovery of pHi from acid load, and a DIDS-sensitive Cl-/HCO3- exchange mechanism responsible for recovery of pHi after alkalinization.

Animals↗