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E Frömter

Publications and source records attributed to E Frömter.

At least 55 records · Page 3Linked to original sources
PubMed1992

Pathways of NH3/NH4+ permeation across Xenopus laevis oocyte cell membrane.

While acid loading with extracellular NH4Cl solutions usually first alkalinizes the cells through NH3 influx, and acidifies only when NH4Cl is removed, Xenopus oocytes became immediately acidic upon NH4Cl addition and the cells did not acidify further upon its removal. Since NH4Cl solutions also collapsed the membrane potential (Vm) and resistance (Rm), we conclude that primarily NH4+ entered the cells where it liberated H+, with NH3 being trapped in intracellular lipid stores. To identify the NH4+ permeation pathway we have used K+ channel blockers (Ba2+, Cs+, tetraethylammonium, quinidine), various cation transport inhibitors (ouabain, bumetanide, amiloride) and other inhibitors, some of which block non-selective cation channels (La3+, diphenylamine-2-carboxylate, and p-chloromercuribenzoate). However, only the latter substances partially prevented the collapse of Vm and Rm. This suggests, that NH4+ passes through non-selective cation channels. In accordance with the voltage dependence and/or stretch activation of such channels NH4+ fluxes appeared to be asymmetric. NH4+ influx, which depolarized and swelled the cells, was large and acidified rapidly, while the efflux, which repolarized and shrank the cells, was slow and alkalinized only slowly.

Ammonia↗
PubMed1992

Acetazolamide inhibition of basolateral Cl-/HCO3- exchange in rabbit renal proximal tubule S3 segment.

Cell pH (pH(i)) and cell membrane potential (Vb) were measured in isolated S3 segments of rabbit renal proximal tubule with double-barrelled microelectrodes to search for a possible effect of the carbonic anhydrase inhibitor, acetazolamide (ACZ), on Cl-/HCO3- exchange in the basolateral cell membrane. ACZ was found to retard and reduce the pH(i) response to bath Cl- removal reversibly with half-maximal inhibition at 0.42 mmol/l and a rather flat concentration dependence (Hill coefficient approximately 0.36). To determine whether the retardation resulted from inhibition of cytoplasmic carbonic anhydrase, which might have delayed the attainment of HCO3-/CO2 equilibrium, we have measured the response of pH(i) to step changes in PCO2 in the presence and absence of ACZ. ACZ greatly retarded the pH(i) response to CO2 steps; however, the concentration dependence differed (half-maximal inhibition at 18 mumol/l) and even at maximal ACZ concentrations the response to CO2 steps was more than twice as fast as the response to Cl- replacement. Since, in addition, the ACZ inhibition of Cl-/HCO3- exchange could not be overcome by increasing PCO2 we conclude that the ACZ effect on Cl-/HCO3- exchange in rabbit proximal tubule S3 segments does not result from inhibition of cytosolic or membrane-bound carbonic anhydrase, but from a direct interaction with the exchanger molecule.

Acetazolamide↗
PubMed1992

Acetazolamide inhibition of basolateral base exit in rabbit renal proximal tubule S2 segment.

The influence of the carbonic anhydrase inhibitor acetazolamide (ACZ) was investigated on HCO3- transport mechanisms in the basolateral cell membrane of rabbit renal proximal tubule. Experiments were performed on isolated S2 segments using double-barrelled microelectrodes to measure cell membrane potential (Vb) and cell pH (pH(i)) during step changes in bath perfusate ion concentrations. Peritubular application of ACZ (1 mmol/l) reduced the initial Vb response to 10:1 reduction of bath HCO3- concentration only slightly, from +53.8 +/- 4.2 mV to +49.1 +/- 0.3 mV (n = 5), but caused an intermittent overshooting repolarization in the secondary Vb response. In conjunction with these effects it left the initial pH(i)) response virtually unchanged but induced a secondary slow acidification. These observation indicate that--under the present experimental conditions--ACZ does not block the Na(+)-HCO3- cotransporter but acts via inhibition of cytosolic carbonic anhydrase. This was confirmed by studying the effect of elevated intracellular HCO3- concentrations under reduced flux conditions and by comparing the concentration dependence of the Vb response with the inhibition kinetics of cytosolic carbonic anhydrase. In contrast, peritubular ACZ inhibited Na(+)-independent Cl-/HCO3- exchange in the basolateral cell membrane of S2 segments directly in a similar way to that described in the preceding publication for S3 segments.

Acetazolamide↗
PubMed1992

C-type potassium channels in the lateral cell membrane of guinea-pig outer hair cells.

The basolateral cell membrane of outer hair cells (OHC) from the mammalian cochlea is known to contain K(+)-channels. The prevailing type, a high-conductance K(+)-channel was further characterized in the present study in order to support its classification as C channel. OHC were isolated from the 3rd and 4th turn of the guinea-pig cochlea. Cell-attached and excised inside-out patches of the lateral cell membrane were investigated. The C-type channel had a selectivity for K+ over Na+ of 12:1 to 20:1 and displayed Goldman-type rectification and voltage-dependence of the open probability. The kinetics of both opening and closing could be described by time constants in the range of ms. The channel provides a calcium- and voltage-activated pathway through OHC lateral membranes for passive K+ transport.

Animals↗
PubMed1991

Sodium dependence of the epithelial sodium conductance expressed in Xenopus laevis oocytes.

The epithelial Na+ conductance was expressed in Xenopus laevis oocytes by injection of size-fractionated mRNA of bovine tracheal epithelium. Fractionation was achieved by sucrose density gradient centrifugation. Successful expression was analysed by recording current/voltage (I/V) curves in the presence and absence of amiloride (10 mumol/l). The newly expressed conductance was half-maximally inhibited by 44 nmol/l amiloride and exhibited a selectivity for Na+ over K+ of 140:1. I/V curves obtained at different extracellular Na+ concentrations ([Na+]o) were subjected to a Goldman-fit analysis to obtain the relation between Na+ permeability (PNa) and [Na+]o. The data show that decreasing [Na+]o from 85 mmol/l to 0.85 mmol/l increased PNa by more than threefold, which is thought to reflect Na+ channel inhibition by increasing [Na+]o. This effect clearly exceeded what can be attributed to concentration saturation of single Na+ channel conductance (Palmer and Frindt (1986) Proc Natl Acad Sci USA 83:2767). No correlation of inhibition with intracellular Na+ concentration was observed. Preservation of the [Na+]o-dependent self-inhibition by the newly expressed Na+ conductance suggests that it is an intrinsic property of the Na+ channel protein, probably mediated by an extracellular Na+ binding site.

Amiloride↗
PubMed1991

Evidence for a cytosolic inhibitor of epithelial chloride channels.

It has been known for several years that the outwardly rectifying 30-pS chloride channel, the regulation of which has been reported to be defective in cystic fibrosis, can be activated by excision of a membrane patch from a cell. This suggested that the cytosol contains an inhibitory factor, which diffuses away after excision, thereby releasing the channel block. To test for such an inhibitor we have isolated cytosol from two epithelial cell lines, and in larger quantities from pig kidney cortex. Kidney cortex was chosen because published and unpublished evidence suggested that proximal tubular cells might also have a tonically suppressed Cl- conductance in the brush-border membrane, which is activated during isolation of membrane vesicles. The inhibitory effect of the cytosol preparations was assessed by: (a) measuring conductive Cl- fluxes on renal proximal tubular brush-border membrane vesicles preloaded with or without cytosol, and (b) recording single Cl- channel currents from excised membrane patches of nasal polyp epithelia and CFPAC-1 cells in the presence and absence of cytosol. All cytosol preparations tested were found to inhibit both conductive Cl- flux in membrane vesicles and single Cl- channels in patch-clamp experiments. In the latter case a type of flicker block was observed with a reduction of channel open probability. Stepwise dilution of the cytosol consistently reduced the inhibitory potency. Since the inhibition was preserved after boiling the cytosol for 10 min, we conclude that the inhibitor is a heat-stable substance. Whether it is identical with the postulated intracellular regulator that couples the defective function of the cystic fibrosis gene product to Cl- channel inhibition cannot be decided at present.

Animals↗
PubMed1990

The chloride/base exchanger in the basolateral cell membrane of rabbit renal proximal tubule S3 segment requires bicarbonate to operate.

Isolated microperfused S3 segments of rabbit renal proximal tubule were investigated with pH-sensitive double-barrelled intracellular microelectrodes to determine whether the Cl-/base exchanger, which we have previously identified in the basolateral cell membrane of this segment requires HCO3- or can also work in CO2/HCO3- free conditions. Cell pH (pHi) was measured in response to sudden substitution of bath Cl- by gluconate. In control solutions containing 25 mmol/l HCO3 pHi increased initially by 5.0 +/- 0.3 x 10(-3) unit/s but after perfusion with CO2/HCO3(-)-free solutions pHi of the same cells increased only by 1.3 +/- 0.2 x 10(-3) unit/s in response to Cl- substitution. From measurements of the cellular buffering power it was calculated that the control base flux had fallen drastically from 3.7 +/- 0.3 to 0.3 +/- 0.1 x 10(-12) mol/s.cm tubule length. To test whether the remaining flux might have resulted from metabolic CO2, oxidative metabolism was poisoned with cyanide (5 mmol/l). This abolished the pH change (delta pHi) in CO2/HCO3(-)-free solutions, but did not affect the pH shift in the presence of HCO3-. The data indicate that basolateral Cl-/base exchange in S3 segment requires HCO3- to operate. A model in which HCO3- absorption proceeds in form of OH- and CO2 can be largely excluded.

Animals↗
PubMed1990

Barium blocks cell membrane and tight junction conductances in Necturus gallbladder epithelium. Experiments with an extended impedance analysis technique.

The site and concentration dependence of the blocking effect of Ba2+ on Necturus gallbladder epithelium has been investigated. A new approach was used which combines time-dependent electrical cell coupling analysis with intermittently performed measurements of transepithelial and apparent intracellular impedance. From the coupling pulse data the sum of apical and basolateral membrane conductances is obtained, which is then held constant during fitting of the impedance data. This combination technique yields more reliable estimates of apical and basolateral membranes resistances (Ra, Rbl) and of tight junction resistance (Rj) than our previous impedance analysis technique. Using the new approach we have found that luminal Ba2+ concentrations between 0.5 and 1.0 mmol/l increase Ra with saturation-type kinetics without affecting Rbl and Rj, while higher luminal Ba2+ concentrations progressively increase Rj. Corresponding effects were observed under serosal Ba2+. The results validate the new impedance analysis approach and demonstrate that millimolar concentrations of Ba2+ block tight junction conductances. Accordingly, Ba2+ can no longer be considered a tool to exclusively alter cell membrane resistances in epithelia.

Animals↗
PubMed1990

Evidence of chloride/bicarbonate exchange mediating bicarbonate efflux from S3 segments of rabbit renal proximal tubule.

The mechanism of HCO3- exit from rabbit renal proximal tubule S3 segments was investigated. Isolated tubules were perfused luminally and peritubularly with test solutions and cell pH (pHi), cell Cl- activity ([Cl-]i) and cell Na+ activity ([Na+]i) were measured with ion-selective microelectrodes. From the response of pHi and [Cl-]i to changes in bath Cl- or HCO3- concentrations a Cl-/HCO3- exchanger was identified in the basolateral cell membrane. It was reversibly inhibited by millimolar concentrations of the disulfonic stilbene SITS (4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid). Cell potential measurements and preliminary determinations of initial ion flux rates suggested a stoichiometry of Cl- to HCO3- flux near 1.0. The transport rate appeared to saturate already at low bath Cl- concentrations (approximately 30 mmol/l), but it was independent of bath pH in the range of 7.4-6.4. Cl-/HCO3- exchange was not directly coupled to Na+ flux although in approximately half of the experiments long-term incubation in Na(+)-free solutions indirectly inhibited the exchanger. Sudden application of SITS under control conditions revealed that the exchanger normally facilitates the exit of HCO3- from cell to interstitium at the expense of Cl- uptake into the cell. How Cl- ions recirculate towards the peritubular surface is presently not known.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1990

Comparison of the basolateral K+ conductance of the epithelial cells in frog gastric fundus mucosa.

Frog stomach transepithelial potential difference (Vt) and resistance (Rt) as well as the voltage divider ratio (VDR) and serosal membrane potential (VS) of surface epithelial (SEC) and oxyntic (OC) cells were recorded at rest and during stimulation with histamine. Serosal membrane K+ permeability was tested by sudden elevation of serosal K+ concentration from 4 to 13 mmol l-1. Stimulation decreased both Vt and Rt and increased VDR of the OC (from 9.4 +/- SD 3.0 to 14.4 +/- 4.1, n = 10, P less than 0.001), while VS remained virtually unchanged (-66.3 +/- 4.5 mV, n = 10); in SEC, however, VDR as well as VS increased, the latter from -67.3 +/- 5.9 to -75.7 +/- 7.3 mV, n = 9, P less than 0.001. Elevation of serosal K+ reversibly diminished Vt and Vs in both cell types. The transepithelial response to K+ increased after stimulation. However, the cell potential response delta Vs,K increased only in the SEC (from +16.0 +/- 2.9 to +18.5 +/- 2.6 mV, n = 9, P less than 0.001) but not significantly in the OC. We conclude that in frog stomach both OC and SEC are stimulated by histamine: the SEC respond with a hyperpolarization, which reflects an increase in their basolateral K+ conductance; the OC do not respond with a hyperpolarization, possibly because histamine increases the basolateral membrane K+ conductance as well as other ion conductances which have not yet been identified.

Animals↗
PubMed1990

Effect of histamine on the basolateral K+ conductance of frog stomach oxyntic cells and surface epithelial cells.

The transepithelial potential difference (Vt) and resistance (Rt) and the basolateral cell membrane potential (Vs) of oxyntic cells (OC) and surface epithelial cells (SEC) were measured in isolated stomachs of Rana esculenta. At rest, Vs of OC and SEC was virtually identical [-66.3 +/- 4.5 (SD) (n = 10) and -67.3 +/- 5.9 mV (n = 9)] and both cells responded to increasing serosal K+ concentration from 4 to 13 mmol/l with virtually the same depolarization (delta Vs,K) of +16.2 +/- 2.0 and +16.0 +/- 2.9 mV, respectively, while Vt declined by approximately half as much. Histamine (0.1 mmol/l) reduced Vt and Rt and increased the voltage divider ratio in both cell types, indicating a fall in basolateral membrane resistance. In the OC, this increase was neither associated with a significant alteration of Vs nor with a change in delta Vs,K. In the SEC, however, histamine markedly increased Vs to -75.5 +/- 7.3 mV (n = 9) as well as delta Vs,K to +18.5 +/- 2.6 mV, which was paralleled by an increase in delta Vt,K from 9.8 +/- 3.9 to +12.8 +/- 4.2 mV. The data indicate that 1) both OC and SEC respond to histamine, 2) both OC and SEC contain a basolateral K+ conductance that increases under histamine (in OC probably, in parallel with other ion conductances), and 3) in Rana esculenta the SEC contribute substantially to Vt.

Animals↗