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E Frömter

Publications and source records attributed to E Frömter.

At least 19 recordsLinked to original sources
PubMed1995

Hypertonicity activates nonselective cation channels in mouse cortical collecting duct cells.

We investigated the effect of cell shrinkage on whole-cell currents of M-1 mouse cortical collecting duct cells. Addition of 100 mM sucrose to an isotonic NaCl bath solution induced cell shrinkage and increased whole-cell currents within 5-10 min by approximately 12-fold. The effect was reversible upon return to isotonic solution and could also be elicited by adding 100 mM urea or 50 mM NaCl. Replacement of bath Na+ by K+, Cs+, Li+, or Rb+ did not significantly affect the stimulated inward current, but replacement by N-methyl-D-glucamine reduced it by 88.1 +/- 1.3% (n = 34); this demonstrates that hypertonicity activates a nonselective alkali cation conductance. The activation was independent of extra- and intracellular Ca2+, but 1 or 10 mM ATP in the pipette suppressed it in a concentration-dependent manner, indicating that intracellular ATP levels may modulate the degree of channel activation. Flufenamic acid (0.1 mM) and gadolinium (0.1 mM) inhibited the stimulated current by 68.7 +/- 5.9% (n = 9) and 32.4 +/- 11.7% (n = 6), respectively, whereas 0.1 mM amiloride had no significant effect. During the early phase of hypertonic stimulation single-channel transitions could be detected in whole-cell current recordings, and a gradual activation of 30 and more individual channels with a single-channel conductance of 26.7 +/- 0.4 pS (n = 29) could be resolved. Thus, we identified the nonselective cation channel underlying the shrinkage-induced whole-cell conductance that may play a role in volume regulation.

Adenine Nucleotides↗
PubMed1995

A calcium-activated and nucleotide-sensitive nonselective cation channel in M-1 mouse cortical collecting duct cells.

We recently reported that M-1 mouse cortical collecting duct cells show nonselective cation (NSC) channel activity (Proc. Natl. Acad. Sci. USA 89:10262-10266, 1992). In this study, we further characterize the M-1 NSC channel using single-channel current recordings in excised inside-out patches. The M-1 NSC channel does not discriminate between Na+, K+, Rb+, Cs+, and Li+. It has a linear I-V relation with a conductance of 22.7 +/- 0.5 pS (n = 78) at room temperature. The Pcation/P(anion) ratio is about 60 and there is no measurable conductance for NMDG, Ca2+, Ba2+, and Mn2+. Cytoplasmic calcium activates the M-1 NSC channel at a threshold of 10(-6) M and depolarization increases channel activity (NPo). Cytoplasmic application of adenine nucleotides inhibits the M-1 NSC channel. At doses of 10(-4) M and 10(-3) M, ATP reduces NPo by 23% and 69%, respectively. Furthermore, since ADP (10(-3) M) reduces NPo by 93%, the inhibitory effect of adenine nucleotides is not dependent on the presence of a gamma-phosphoryl group and therefore does not involve protein phosphorylation. The channel is not significantly affected by 8-Br-cGMP (10(-4) M) or by cGMP-dependent protein kinase (10(-7) M) in the presence of 8-Br-cGMP (10(-5) M) and ATP (10(-4) M). The NSC channel is not sensitive to amiloride (10(-4) M cytoplasmic and/or extracellular) but flufenamic acid (10(-4) M) produces a voltage-dependent block, reducing NPo by 35% at depolarizing voltages and by 80% at hyperpolarizing voltages. We conclude that the NCS channel of M-1 mouse cortical collecting duct cells belongs to an emerging family of calcium-activated and nucleotide-sensitive nonselective cation channels. It does not contribute to amiloride-sensitive sodium absorption and is unlikely to be a major route for calcium entry. The channel is normally quiescent but may be activated under special physiological conditions, e.g., during volume regulation.

Animals↗
PubMed1995

Cl- channel inhibition by glibenclamide is not specific for the CFTR-type Cl- channel.

As long as the question of which channels are responsible for cAMP-mediated epithelial Cl- secretion remains unsolved, it is still important to search for specific inhibitors that might help to relate macroscopic to microscopic events. Following the report by Sheppard and Welsh (J Gen Physiol 100: 573, 1992) that glibenclamide inhibits whole-cell Cl- currents in genetically manipulated fibroblasts expressing the cystic fibrosis transmembrane conductance regulator (CFTR), we have studied the effect of glibenclamide on different types of Cl- channels of HT29 and T84 cells at the single-channel level. Our results confirm that micromolar concentrations of glibenclamide inhibit the linear, low-conductance Cl-channel, which appears to represent CFTR and show that the inhibition results from a typical flicker block. However, the same concentrations of glibenclamide inhibit also the outwardly rectifying intermediate conductance Cl- channel which, potentially, may contribute to transepithelial Cl- secretion.

Chloride Channels↗
PubMed1995

Characterization of two distinct Cl- conductances in fused human respiratory epithelial cells. I. Anion selectivities, stimulation and intermeshing signal transduction pathways.

With the aim of further elucidating the role of the epithelial Cl- conductance and its defect in cystic fibrosis (CF) patients we studied the properties and regulation of the Cl- conductance in primary cultures of human nasal polyp epithelia. To facilitate microelectrode punctures and to gain access to the cytoplasmic compartment for injection of antibodies, we prepared giant cells using a polyethylene-glycol fusion technique. The membrane potential (Vm) and resistance (Rm) and their responses to ionic substitutions in the bath were measured under control conditions and in the presence of different secretagogues. In non-CF cells Vm averaged-12.5 mV (SD +/- 6.6 mV, n = 69) and was independent of time after fusion, while Rm dropped from 12.4 +/- 7.3 M omega (n = 51) to 3.5 +/- 5.5 M omega (n = 24) in the 2nd week after fusion. The low Vm values reflected a vanishing K+ conductance in the presence of a dominating Cl- conductance that increased with time. In young cells, a Cl- conductance prevailed which could be stimulated by application of the Ca2+ ionophore, A23187, or of carbachol. As determined in CF cells, it had an outwardly rectifying current/voltage (ilV) relationship and exhibited the selectivity sequence I- > Br- > Cl- > F- > isethionate (ISE-) both in Vm and Rm measurements. With increasing age after fusion, a Cl- conductance prevailed in non-CF cells which could be stimulated by cyclic adenosine monophosphate (cAMP) or forskolin and which was downregulated by A23187. It had a linear ilV relationship and exhibited the selectivity sequence Br- > Cl- > I- > F- > ISE- if determined from Vm measurements, but a sequence of Cl- > Br- > F- = ISE- > I- if determined from Rm measurements. This points to multiple-ion pore behaviour of the respective Cl- channel. In agreement with observations described in the following publication, the results suggest that the cAMP-regulated Cl- conductance corresponds to the CF-gene product while the molecular nature of the Ca(2+)-regulated Cl conductance is not yet known.

Anions↗
PubMed1995

Characterization of two distinct Cl- conductances in fused human respiratory epithelial cells. II. Relation to cystic fibrosis gene product.

The present microelectrode experiments on fused respiratory epithelial cells of cystic fibrosis (CF) origin and non-CF origin aim at characterizing the molecular basis of the Cl- conductances regulated by cyclic adenosine monophosphate (cAMP) or respectively Ca2+, as described in the preceding publication. Cell membrane potential (Vm) and resistance (Rm) were recorded as well as their response to substitution of 90% of bath Cl- by isethionate (delta Vm,ISE), by I- (delta Vm,I), or by other halide anions. Fused CF cells had significantly (P < 0.05) higher control Vm values (-18.0 +/- 9.4 mV, +/- SD, n = 68) than fused non-CF cells (-12.5 +/- 6.6 mV, n = 69) and responded to the Ca2+ ionophore A23187 with an increase in the Vm response to Cl- substitution, but did not respond to forskolin. This indicates that CF cells express only the Ca(2+)-stimulated Cl- conductance. Injection of the antibody M3A7 against a fusion protein containing amino acids 1195 to 1480 of the CF gene product into young, forskolin-stimulated or old non-CF cells decreased delta Vm,ISE and delta Vm,I within 15 min to values observed in CF cells. This indicates inhibition of the cAMP-stimulated Cl- conductance and supports the molecular identity of this conductance with the CF gene product. However, the slow onset of inhibition does not allow secondary effects to be excluded and a slight fall in Rm remains unexplained. Stimulation of the Ca(2+)-regulated Cl- conductance was not impaired. Injection of M3A7 into CF cells or of a control antibody in non-CF cells had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies↗
PubMed1994

Electrophysiological investigation of microdissected gastric glands of bullfrog. I. Basolateral membrane properties in the resting state.

In the present experiments we have made a new attempt to characterize the ion transport properties of H(+)-secreting cells of the gastric mucosa using electrophysiological techniques. Individual gastric glands of bullfrog fundus mucosa were manually dissected, mounted in holding pipettes and superfused with various test solutions while individual cells were punctured with conventional or H(+)-sensitive double-barrelled microelectrodes. All measurements were performed in the resting state (0.1 mmol/l cimetidine). In HCO3(-)-containing control Ringer solution the cell membrane potential (Vb) averaged -45.6 +/- 0.9 mV (+/- SEM, n = 54). From the fast initial Vb responses to changing bath K+, Na+, Cl- or HCO3- concentrations we deduced that the basolateral cell membrane contains conductances for K+, Na+, and Cl- but not for HCO3-, and that a Na(+)-HCO3- cotransporter is not present. The K+ conductance was inhibited by Ba2+ (3 mmol/l), but the Cl- conductance was not inhibited by 4,4' diisothiocyanato-stilbene-2,2' disulphonic acid (DIDS, 0.3 mmol/l), nor selectively inhibited by 5-nitro-2-(3)- phenylpropyl-aminobenzoate (NPPB, 10 mumol/l). In a great number of cells the Vb response to Cl- substitution revealed two components: an initial spiking depolarization which reflected conductive Cl- efflux and a secondary slow hyperpolarization, the origin of which was not immediately evident. Since the latter response could be mimicked by CO2-free perfusion, strongly depressed by Ba2+ and eliminated by DIDS, we conclude that it reflects HCO3- uptake into the cells via a DIDS sensitive Cl-/HCO3- exchanger which alkalinizes the cells and stimulates the basolateral K+ conductance. Our results confirm, revise and extend the results of previous, less direct, investigations of gastric cell ion transport.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1994

Electrophysiological investigation of microdissected gastric glands of bullfrog. II. Basolateral membrane properties in the presence of histamine.

Following the technical approach described in the preceding publication we have investigated if, and how, stimulation of gastric HCl secretion affects the basolateral ion transport properties of oxyntopeptic cells of Rana catesbeiana stomach. To this end microdissected gastric glands were punctured with conventional or H(+)-sensitive glass microelectrodes and the effects of changing bath ion concentrations on the cell membrane potential (Vb) and cell pH (pHi) were determined. Except for a transient alkalinization, histamine (0.5 mmol/l) did not significantly affect Vb or pHi. The latter averaged 7.18 +/- 0.03 (mean +/- SEM, n = 5) under resting conditions (0.1 mmol/l cimetidine) and 7.21 +/- 0.07 (n = 5) in the presence of histamine. In addition, neither the initial velocity nor the final steady-state value of the cell alkalinization following a 10:1 reduction of bath Cl- concentration changed in the presence of histamine, and the same holds true for the cell acidification following a 10:1 reduction of bath HCO3- concentration. These observations indicate that the basolateral Cl-/HCO3- exchanger was not stimulated by histamine, and that no other base transporters were activated. By contrast, the Vb response to elevation of bath K+ concentration decreased, and so did the initial depolarizing Vb response to bath Cl- substitution, while the secondary hyperpolarizing response increased. The latter observations are compatible with the notion that stimulation by histamine reduced a pH-insensitive part of the basolateral K+ conductance and reduced also the basolateral Cl- conductance.

Animals↗
PubMed1994

Model of bicarbonate secretion by resting frog stomach fundus mucosa. I. Transepithelial measurements.

In the present in vitro experiments on gastric fundus mucosa of Rana esculenta we try to define the mechanism of alkaline secretion that is observed in summer frogs in the resting stomach (blockage of HCl secretion by ranitidine, 10(-5) mol/l). The transepithelial voltage and the rate of alkalinization (ASR) of an unbuffered gastric lumen perfusate was measured as a function of serosal (and mucosal) fluid composition. ASR was high (0.88 +/- S.E. 0.09 microEq.cm-2.h-1, n = 11) during serosal bath perfusion with HCO(3-)-Ringer solution, decreased slightly to 0.50 +/- 0.07 microEq.cm-2.h-1 (n = 6) in HCO(3-)-free HEPES-buffered Ringer solution of the same pH, and decreased to approximately 20% when carbonic anhydrase was inhibited by acetazolamide. While replacement of mucosal or serosal Cl- did not--within 1 h--significantly alter ASR, replacement of serosal Na+ in the presence or absence of HCO3- strongly reduced ASR, and a similar reduction was observed after serosal application of the anion transport inhibitor DIDS (4,4-diisothiocyanatostilbene-2,2-disulphonate, 2.10(-4) mol/l), the metabolic poison rotenone (10(-5) mol/l), the uncoupler dinitrophenol (10(-4) mol/l), and the Na+ pump inhibitor ouabain (10(-4) mol/l), while serosal amiloride (10(-4) mol/l) had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1994

Model of bicarbonate secretion by resting frog stomach fundus mucosa. II. Role of the oxyntopeptic cells.

In the present publication we report mainly electrophysiological studies on oxyntopeptic cells of frog gastric mucosa which aim at clarifying a possible involvement of these cells in the process of resting gastric alkali (HCO3-) secretion, described in the preceding publication. The experiments were performed on intact gastric fundus mucosa of Rana esculenta mounted in Ussing chambers. After removal of the muscle and connective tissue layer oxyntopeptic cells were punctured from the serosal surface with conventional or pH-sensitive microelectrodes to measure, besides transepithelial voltage and resistance, the basolateral cell membrane potential, the voltage divider ratio, and the cell pH in response to secretagogues and/or changes in serosal ion concentration. Carbachol (10(-4) mol/l), which transiently stimulated HCO3- secretion by 0.22 mumol.cm-2.h-1, transiently acidified the cells by 0.09 +/- SEM 0.03 pH units (n = 6) and transiently induced an apical cell membrane anion conductance. According to the model of gastric HCO3- secretion presented in the preceding publication, this anion conductance could be involved in gastric HCO3- secretion, mediating, besides Cl- efflux, also apical HCO3- efflux. In addition carbachol stimulated basolateral Na+(HCO3-)n-cotransport, which according to the results from the preceding publication mediates basolateral HCO3- uptake for secretion. By contrast, cAMP-mediated secretagogues, such as histamine or others, which stimulate HCl secretion and transiently alkalinize the oxyntopeptic cells, were found to down-regulate the basolateral Na+(HCO3-)n-cotransporter.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

An electrophysiological study of angiotensin II regulation of Na-HCO3 cotransport and K conductance in renal proximal tubules. I. Effect of picomolar concentrations.

The effect of picomolar concentrations of angiotensin II (AII) was investigated in isolated perfused rabbit renal proximal tubules using conventional or pH-sensitive intracellular microelectrodes. Under control conditions cell membrane potential (Vb) and cell pH (pHi) averaged -53.8 +/- 1.9 mV (mean +/- SEM, n = 49) and 7.24 +/- 0.01 (n = 10), respectively. AII (at 10(-11) mol/l), when applied from the bath (but not when applied from the lumen perfusate), produced the following effects: approximately 85% of the viable tubules responded with a small depolarization (+5.5 +/- 0.4 mV, n = 43) which was accompanied in half of the pHi measurements by a slow acidification (delta pHi = -0.03 +/- 0.01, n = 5). The remaining 15% responded with a small hyperpolarization (delta Vb = -3.1 +/- 0.4 mV, n = 6). All changes were fully reversible and repeatable. Experiments with fast changes in bath HCO3 or K concentrations, as well as measurements of the basolateral voltage divider fraction in response to transepithelial current flow, explain these observations as stimulation of a basolateral Na-HCO3 cotransporter and of a basolateral K conductance. Both counteract in their effect on Vb, but can be individuated by blocker experiments with 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and barium. Both the stimulation of Na-HCO3 cotransport and the stimulation of the K conductance may result from down-regulation of the level of cyclic adenosine monophosphate in the cell.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1994

An electrophysiological study of angiotensin II regulation of Na-HCO3 cotransport and K conductance in renal proximal tubules. II. Effect of micromolar concentrations.

In the first part of our study, we described the effect of picomolar concentrations of angiotensin II (AII) on cell membrane potential (Vb) and cell pH (pHi) of isolated perfused rabbit renal proximal tubules. In the present publication we summarize respective observations with micromolar concentrations of AII. With a few exceptions nearly all experiments showed mirror-image-like results. In the majority of the experiments 10(-6) mol/l AII, when applied from the bath (but not when applied from the lumen), slightly hyperpolarized the cells by -3.4 +/- 0.3 mV (mean +/- SEM, n = 20) and alkalinized them by up to 0.06 pH units, while the lower AII concentrations, which were applied in the previous study, depolarized and acidified. The present observations suggest that micromolar concentrations of AII inhibit basolateral Na-HCO3 cotransport. This conclusion was confirmed by a decreasing Vb response to step changes of basolateral HCO3 concentration. In addition, there was a tendency of the Vb response to K concentration steps to decrease, but measurements of the voltage divider ratio did not point to a significant inhibition of a basolateral K conductance. In spite of the almost perfect reciprocity of the results with 10(-6) and 10(-11) mol/l AII, some specific observations suggest that micromolar concentrations of AII do not simply cause mirror-image-like effects, but influence still further transport systems compared to picomolar concentrations.

Angiotensin II↗
PubMed1994

[The strategy for research on tubular transport in the kidney].

The present publication summarises the experimental strategies that have been developed to identify, localize and characterize renal tubular transport mechanisms on the nephron, cellular and molecular level. 1) Identification and localization of transporters can be achieved on single ne (mRNA hybridization) techniques. 2) The functional properties of transporters and their regulation can be investigated on individual nephron segments in vivo or in vitro, on cell cultures, isolated cells, or on isolated membrane vesicles, applying different analysis techniques: net flux and tracer take-up studies, microscopic volume determinations, microelectrode measurements (conventional and ion-selective), patch-clamp measurements, microspectrofluorometry, X-ray microanalysis of frozen tissues, nuclear magnetic resonance, etc. 3) The primary structure (amino acid sequence) of transport proteins can be determined by protein purification with subsequent amino acid sequencing. More powerful, however, is expression cloning, e.g. screening of cDNA libraries for expression of a given transport protein in a model cell system (e. g. Xenopus laevis oocytes) with subsequent base sequence analysis. 4) The molecular function of transport properties can best be studied in heterologous expression experiments (overexpression, easy technical, e. g. in oocytes). This also allows monomeric and multimeric transporters to be distinguished and is a prerequisite for mutagenic studies in which the function of individual amino acid constituents of the protein can be determined. Such studies eventually allow detailed molecular models of transport function to be developed.

Animals↗
PubMed1993

The Na(+)-HCO3- cotransporter operates with a coupling ratio of 2 HCO3- to 1 Na+ in isolated rabbit renal proximal tubule.

All the relevant literature reports indicate that net rates of salt and water absorption and cell membrane potentials (Vb) are lower, but intracellular Na+ concentration is higher in rabbit renal proximal tubule in vitro than in rat proximal tubule in vivo. Since the different driving forces should influence basolateral Na(+)-HCO3- cotransport we have studied the operation of the cotransporter in isolated rabbit renal proximal tubule in vitro with special emphasis on the stoichiometry of flux coupling (q). Using conventional and ion-selective intracellular microelectrodes three series of experiments were performed: (a) we determined the Vb response to a 2:1 reduction of bath HCO3- or Na+ concentration, (b) we determined initial efflux rates of HCO3- or Na+ ions in response to a sudden 10:1 reduction of bath HCO3- concentration, and (c) we collapsed the tubules and determined electrochemical driving forces of Na+ and HCO3- across the basolateral cell membrane under conditions approaching zero net flux in the control state in the presence of Ba2+- and in Cl(-)-free solutions. All measurements concurrently yielded a coupling ratio of approximately two HCO3- ions to one Na+ ion (q = 2). This result contrasts with the ratio q = 3, which we have previously observed in similar experiments on rat renal proximal tubule in vivo [Yoshitomi et al. (1985) Pflügers Arch 405:360] and which was also observed on rabbit renal basolateral cell membrane vesicles in vitro [Soleimani et al. (1987) J Clin Invest 79:1276].(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Tight-junction tightness of Necturus gall bladder epithelium is not regulated by cAMP or intracellular Ca2+. I. Microscopic and general electrophysiological observations.

Following the publications by Duffey et al. [Nature 294:451 (1981)] and Palant et al. [Am J Physiol 245: C203 (1983)] it is generally accepted that tight-junction tightness of Necturus gall bladder epithelium is up-regulated by cAMP-mediated and Ca(2+)-mediated stimulation. This conclusion was mainly based on observed increases in transepithelial resistance (Rt). However, since in leaky epithelia Rt cannot be simply equated with the tight junction resistance (Rj), but may include large contributions from the lateral space resistance (Rlis), we asked whether the observed increases in Rt resulted indeed from Rj or whether Rlis also increased. The experiments were performed on Necturus gall bladders using forskolin or the Ca2+ ionophore A23187 as stimulants. Forskolin (2 mumol/l) had a biphasic effect. In the first 5 min Rt decreased from 128 +/- 13 to 119 +/- 14 omega cm2 (P < 0.05, n = 10) which probably reflects stimulation of an apical cell membrane Cl- conductance (see accompanying paper). Subsequently Rt increased in approximately 30 min to 184 +/- 20 omega cm2 and then remained fairly constant. Simultaneously the lateral spaces collapsed. If the spaces were now transiently opened by passing mucosa-positive direct current across the epithelium, Rt fell transiently to 111 +/- 7 omega cm2, but returned gradually to its elevated level when the spaces collapsed again. When the spaces were constantly dilated by a serosa-positive hydrostatic pressure of 1 cm H2O, forskolin neither affected the space width nor increased Rt, and current passage was virtually ineffective, although the cells depolarized in response to forskolin as usual.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Tight-junction tightness of Necturus gall bladder epithelium is not regulated by cAMP or intracellular Ca2+. II. Impedance measurements.

In the preceding publication we have reported that, contrary to the prevailing opinion in the literature, the tight-junction tightness of Necturus gall bladder epithelium is not up-regulated by cAMP-mediated or by Ca(2+)-mediated stimulation. This conclusion was based on our observation that the stimulant-induced increase in transepithelial resistance (Rt) occurred only when the lateral intercellular spaces were allowed to collapse, which suggested that the increase reflected primarily or exclusively the increasing resistance of the lateral spaces (Rlis) rather than the postulated increase in tight-junction resistance (Rj). An alternative explanation could have been that the constancy of Rt after space dilatation reflected an increase Rj that was masked by a concomitant fall in apical and basolateral cell membrane resistances Ra and Rbl. To decide between those possibilities we have performed impedance measurements with transepithelial and intracellular microelectrodes on Necturus gall bladder epithelium. Applying previously developed analysis procedures, the measurements readily showed that elevation of intracellular Ca2+ concentration increased Rlis, but left Rj as well as Ra and Rbl quasi constant. Experiments with forskolin, theophylline or isobutylxanthine, on the other hand, were less clear. These stimulants activated an apical Cl- conductance, which drastically reduced Ra and apparently caused low-frequency polarization effects that could not be accounted for by the classical epithelial equivalent circuit. After elimination of the polarization phenomena by uni- or bilateral substitution of Cl- by isethionate or sulphate, however, we were able to demonstrate that Rj remains constant under cAMP-mediated stimulation irrespective of whether the lateral spaces are kept open or are allowed to collapse. We conclude that the tight-junction resistance of Necturus gall bladder epithelium is not controlled by intracellular Ca2+ or by cAMP-mediated stimulation.

Animals↗
PubMed1993

Hypertensive sodium-proton exchanger phenotype persists in immortalized lymphoblasts from essential hypertensive patients. A cell culture model for human hypertension.

An enhancement of sodium-proton exchange activity is a frequently observed ion transport abnormality in essential hypertension. The cellular basis for this has not yet been elucidated. Due to the lack of a specific cell culture system it has been impossible to distinguish between intrinsic cellular abnormalities and influences exerted by the hypertensive neurohumoral milieu. Using Epstein-Barr virus we have immortalized lymphocytes from controls and from patients with essential hypertension that exhibited enhanced sodium-proton exchanger activity. Sodium-proton exchanger activity was determined in cells loaded with the fluorescent cytosolic pH indicator 2'7'-biscarboxyethyl-5,6-carboxyfluorescein acetoxymethylester (BCECF) after pretreatment with 250 nM of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate for 10 min. Cell lines from hypertensive patients displayed higher Vmax values of sodium-proton exchange than those from normotensive controls (129.6 +/- 30.0 vs. 77.1 +/- 13.2 mmol H+/min.; P < 0.001). Hill coefficients for H+ were distinctly lower in hypertension compared to normotension (1.12 +/- 0.12 vs. 1.50 +/- 0.14; P < 0.0001). The enhanced antiporter activity in cell lines from hypertensive patients was not accompanied by a corresponding increase in steady-state NHE-1 mRNA transcript levels, which argues against overexpression of antiporter protein in hypertension. The cells from hypertensive patients with high sodium-proton exchange activity proliferated distinctly faster than those from normotensive controls. These human cell lines represent a novel model to study the mutual interaction between sodium-proton exchange and cell proliferation, and may provide insights into the alterations in ion transport observed in a group of patients with essential hypertension.

Adult↗
PubMed1992

Microelectrode determination of oxyntic cell pH in intact frog gastric mucosa. Effect of histamine.

Intracellular pH (pHi) of acid-secreting cells was measured in intact gastric fundus mucosa of Rana esculenta with double-barrelled pH microelectrodes. Tissues were mounted, serosal side up, between two half chambers and individual cells were impaled after microsurgical removal of the serosal muscle layer. Transepithelial potential difference (Vt) and resistance (Rt) as well as serosal cell membrane potential (Vs) and pHi were continuously recorded at rest (0.1 mmol/l cimetidine) or during stimulation (0.5 mmol/l histamine). During chamber perfusion with HCO3-/CO2-buffered Ringer solution of pHo = 7.36, Vt and Rt were -21.7, SD +/- 6.0 mV and 229 +/- 83 omega cm2 (n = 17) while Vs and pHi averaged -57.3 +/- 6.9 mV and 7.4 +/- 0.11 (n = 25). The latter value is considerably more alkaline than all recent pHi measurements obtained with microspectrofluorometric techniques on isolated cells, glands or intact tissue. The difference may in part be explained by use of HCO3(-)-free solutions in most of the previous studies because we observed that such solutions decrease pHi to 6.89 +/- 0.18 (n = 4). Again, in contrast to recent literature, application of histamine in HCO3-/CO2-buffered solution led to further transient alkalinization by 0.12 +/- 0.05 pH unit (n = 8). Since in accidental punctures of the gastric gland lumen we noticed that H+ secretion only began approximately 5 min after histamine application, we conclude that the histamine-induced initial alkalinization does not reflect stimulation of the H+/K+ ATPase pump.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗