The electrophysiological analysis of tubular transport.
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Animals↗
Biomedical subjects
E Frömter
Publications and source records attributed to E Frömter.
Membrane potential and ion channels in isolated outer hair cells of guinea pig cochlea.
Single outer hair cells from guinea pig cochlea were prepared by microdissection and cultured for several hours in a modified Hanks' medium. Cells, which were viable, judged by morphological criteria, were investigated with the patch-clamp technique. Membrane potentials up to -70 mV could be measured in the cultured cells. Ion channels were found in the basolateral membrane and in the cuticular region of the hair cells. These ion channels could play an important role in transducing sound-induced sterociliary motion into membrane depolarizations, thereby regulating the contractile state of the outer hair cells and in turn the resonance properties of the cochlear amplifier.
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How big is the electrochemical potential difference of Na+ across rat renal proximal tubular cell membranes in vivo?
The intracellular Na+ concentration of surface loops of proximal tubules was measured with double-barrelled Na+ -sensitive microelectrodes in micropuncture experiments on rat kidneys in situ and in vivo. With the help of a fast recording system, it was possible to select only valid measurements, by discarding all records in which the Na+ concentration rose after the impalement presumably as a result of sodium ion inflow via puncture leaks. Under free-flow conditions cell Na+ activity averaged 13.1, S.D. +/- 2.1 mmol/l which, assuming an activity coefficient of 0.75, corresponds to a Na+ concentration of 17.5 mmol/l. The simultaneously recorded cell membrane potential was -73.5 mV in agreement with best estimates derived previously. The comparison of cell sodium activity and cell pH (Yoshitomi and Frömter [17]) demonstrates that the electrochemical potential difference of Na+ is greater than that of H+, as required for the operation of Na+/H+ counter transport in the brushborder membrane but that bicarbonate efflux via the sodium bicarbonate cotransport system at the peritubular cell membrane requires a HCO3- to Na+ stoichiometry of greater than 2.0 or the involvement of other ions at least under free-flow conditions.
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Identification of mitochondria-rich cells in unstained vital preparations of epithelia by autofluorescence.
To recognize mitochondria-rich cells in unstained vital preparations of heterogeneously composed epithelia we report here the use of an autofluorescence technique. Upon illumination with wavelengths of 400-440 or 450-490 nm mitochondria-rich cells emit fluorescence light of wave-lengths of greater than 470 or greater than 520 nm respectively, which probably arises from flavoproteins. Illumination does not seem to impair cell function and the fluorescence does not show any sign of fading over observation times of 20 min or greater. The method allows us to identify parietal cells from chief cells in gastric glands and obviously also intercalated cells from principal cells in renal collecting ducts. It might also be useful in other epithelia.
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Electrophysiological studies on lateral intercellular spaces of Necturus gallbladder epithelium.
The lateral intercellular spaces of Necturus gallbladder epithelium were punctured with double-barrelled ion selective microelectrodes in order to determine the ion concentrations of lateral space fluid and the contribution of the lateral spaces to transepithelial resistance. Neither under control conditions, nor after diluting the bathing fluids to increase the rate of volume absorption, nor during passage of direct current of 200 microA/cm2, were any reliable concentration differences observed between lateral space fluid and external bathing fluids. These observations suggest that water can follow salt transport without requiring osmotic concentration gradients of greater than 1 or 2 mosmol/l and indicate that recently observed high values of water permeability must still be considered as underestimates. After developing a test to recognize and exclude leaky punctures, the contribution of the lateral spaces to transepithelial resistance could be determined. It amounted to around 29%. This value agrees well with results from recent impedance measurements which were performed under control conditions in the same preparation.
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Is the voltage divider ratio a reliable estimate of the resistance ratio of the cell membranes in tubular epithelia?
The length dependence of the voltage divider ratio (VDR) was investigated in a double cable model of tubular epithelia with point source current injection into the tubular lumen in order to find out, whether there is a region, in which the VDR - as in flat sheet epithelia - is an appropriate measure of the relative magnitude of the apical (ra) and basal (rb) cell membrane resistances. Irrespective of the choice of the cable parameters, we find that VDR, defined as luminal over cellular voltage deflection, overestimates the resistance ratio (ra + rb):rb near the origin, but underestimates it at distances (chi) greater than 1 luminal length constant (beta). In the region chi less than beta there is a crossover point, where VDR is an accurate estimate of the resistance ratio. If the difference between VDR at the origin and at large distances (chi greater than beta) is small, then VDR is a good estimate of the resistance ratio. This is also true, if VDR is constant between chi approximately 0.5 beta and chi greater than beta, (with the exception of some cases, in which the longitudinal resistance in the cell column is exceedingly high). If the latter conditions do not apply, we find that VDR, as measured at chi = beta, underestimates the resistance ratio at worst only by 8.8%, provided the cable properties are such that the luminal voltage attenuation exhibits only one single exponential (with maximum tolerable amplitude deviation of 5% at the origin).(ABSTRACT TRUNCATED AT 250 WORDS)
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Membrane potentials of individual cells of isolated gastric glands of rabbit.
Individual glands of rabbit gastric mucosa were prepared for measurements of cell membrane potentials. In the first experiments a collagenase isolation technique was used which produced gland fragments that were fixed on agarose. In later experiments a microdissection technique was used which allowed whole glands to be isolated that were held in suction pipettes. Individual parietal or chief cells could be recognized and impaled with microelectrodes, however, the yield of reliable recordings was small and the distinction from artifacts sometimes difficult. In acceptable recordings the membrane potentials of both cell types varied between around -20 and -35 mV or exceptionally -50 mV in both preparations, with mean values being around -26 mV. The significance of the recordings was tested by ion substitution experiments. Substitution of all chloride by sulfate increased the membrane potential to values ranging up to -60 and -80 mV that are commonly observed in other cells.
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Rheogenic sodium-bicarbonate cotransport in the peritubular cell membrane of rat renal proximal tubule.
The mechanism of bicarbonate transport across the peritubular cell membrane was investigated in rat kidney proximal tubules in situ by measuring cell pH and cell Na+ activity in response to sudden reduction of peritubular Na+ and/or HCO3-. The following observations were made: 1. sudden peritubular reduction of either ion concentration produced the same transient depolarizing potential response; 2. bicarbonate efflux in response to peritubular reduction of bicarbonate was accompanied by sodium efflux; 3. sodium efflux in response to peritubular sodium removal was accompanied by cell acidification indicating bicarbonate efflux; 4. all aforementioned phenomena were inhibited by SITS (10(-3) mol/l) except for a small SITS-independent sodium efflux and depolarization which occurred in response to peritubular sodium removal and was not accompanied by cell pH changes; 5. bicarbonate efflux and accompanying potential changes in response to reduction of peritubular bicarbonate virtually vanished in sodium-free solutions. From these observations we conclude that bicarbonate efflux proceeds as rheogenic sodium-bicarbonate cotransport with a stoichiometry of bicarbonate to sodium greater than 1. The question which of the charged species of the bicarbonate buffer system moves cannot yet be decided. Attempts to determine the stoichiometry from the SITS-inhibitable initial cell depolarization and from the SITS-inhibitable initial fluxes suggest a stoichiometry of 3 HCO3-: 1 Na+. In addition to sodium-dependent bicarbonate flux, evidence was obtained for a sodium-independent transport system of acids or bases which is able to regulate cell pH even in sodium-free solutions.
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Electrophysiological evidence for Cl secretion in shark renal proximal tubules.
The electrophysiology of shark proximal tubules (Squalus acanthias) was investigated using conventional microelectrodes and cable analysis. Under in vitro perfusion with symmetrical Ringer solutions, tubule transepithelial resistance was 36.3 +/- 2.3 omega X cm2 (means +/- SE, n = 44). Other electrophysiological variables varied widely under control conditions. In unstimulated tubules (n = 16) the transepithelial voltage (VT,o) was lumen positive (1.2 +/- 0.2 mV), the basolateral membrane potential (Vbl,x) was -61.3 +/- 1.6 mV, and the fractional resistance of the apical membrane (fRa) was 0.67 +/- 0.02. Spontaneously stimulated tubules (n = 28) had lumen-negative VT,o values (-1.5 +/- 0.4 mV), low Vbl,x values (-41.3 +/- 1.7 mV), and low fRa values (0.30 +/- 0.02). The stimulated state can be induced in unstimulated tubules via treatment with cAMP. Multiple microelectrode impalements in a single tubule revealed epithelial cells sharing similar electrophysiological properties. Selective ion substitutions in the tubule lumen and peritubular bath uncovered an increased Cl conductance in the apical membrane of spontaneously and cAMP-stimulated tubules. Anthracene-9-carboxylic acid tended to reverse the stimulated state, and furosemide hyperpolarized Vbl,x. These results constitute the first evidence for secretory Cl transport in a renal proximal tubule. The electrophysiological responses to ion substitutions, stimulators, and inhibitors are strikingly similar to those of known Cl-transporting epithelia.
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Renal tubular transport mechanisms of amino acids.
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Amino Acids↗
[The isolated living hair cell. A new model for the study of hearing function].
Living, predominantly outer hair cells were prepared from the guinea pig cochlea using a non-enzymatic, microsurgical approach. Viability of hair cells was demonstrated by dye-exclusion as well as by electrophysiogical patch-damp procedures. This allowed to measure a mammalian hair cell potential of -70 mV under direct visualisation. Furthermore, hair cells could be kept under cell culture conditions for 6-9 hrs. Isolated, mammalian cochlea hair cells are a new, useful model to investigate the molecular basis of hearing and hearing disorders located in sensory cells. As example this report shows experimental approaches to analyse membraneous and cytoplasmic functions of hair cells.
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Electrophysiological analysis of bicarbonate permeation across the peritubular cell membrane of rat kidney proximal tubule. I. Basic observations.
The membrane potential response of proximal tubular cells to changing HCO3- concentrations was measured in micro-puncture experiments on rat kidney in vivo. No significant effect was noticed when luminal bicarbonate concentration was changed. Changing peritubular HCO3- by substitution with Cl- resulted in conspicuous membrane potential transients, which reached peak values after 100-200 ms and decayed towards near control with time constants of approximately 2 s. The polarity of the potential changes and the dependence of the initial potential deflections on the logarithm of HCO3- concentration suggest a high conductance of the peritubular cell membrane for HCO3- buffer, but not for Cl-, SO4(2-) or isethionate. At constant pH, tHCO3- was estimated to amount to approximately 0.68. At constant pCO2, tHCO3- was even greater because of an additional effect of OH- or respectively H+ gradients across the cell membrane. The secondary repolarization may be explained by passive net movements of K+ and HCO3- across the peritubular cell membrane, which result in a readjustment of intracellular HCO3- to the altered peritubular HCO3- concentration. Application of carbonic anhydrase inhibitors in the tubular lumen reduced the initial potential response by one half and doubled the repolarization time constant. The same effect occurred instantaneously when the inhibitor was applied - together with the HCO3- concentration step - in the peritubular perfusate. This observation demonstrates that membrane bound carbonic anhydrase is somehow involved in passive rheogenic bicarbonate transfer across the peritubular cell membrane, and suggests that HCO3- permeation might occur in form of CO2 and OH- (or H+ in opposite direction).
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Electrophysiological analysis of bicarbonate permeation across the peritubular cell membrane of rat kidney proximal tubule. II. Exclusion of HCO3(-)-effects on other ion permeabilities and of coupled electroneutral HCO3(-)-transport.
Cell membrane potentials of rat kidney proximal tubules were measured in response to peritubular ion substitutions in vivo with conventional and Cl- sensitive microelectrodes in order to test possible alternative explanations of the bicarbonate dependent cell potential transients reported in the preceding paper. Significant direct effects of bicarbonate on peritubular K+, Na+, and Cl- conductances could be largely excluded by blocking K+ permeability with Ba2+ and replacing all Na+ and Cl- by choline or respectively SO4(2-) isethionate, or gluconate. Under those conditions the cell membrane response to HCO3- was essentially preserved. In addition it was observed that peritubular Cl- conductance is negligibly small, that Cl-/HCO3- exchange - if present at all - is insignificant, and that rheogenic HCO3- flow with coupling to Na+ flow is also absent or insignificant. A transient disturbance of the Na+ pump or a transient unspecific increase of the membrane permeability was also excluded by experiments with ouabain and by the observation that SITS (4-acetamido-4'-isothiocyano-2,2' disulphonic stilbene) blocked the HCO3- response instantaneously. The data strongly support the notion that the potential changes in response to peritubular HCO3- concentration changes arise from passive rheogenic bicarbonate transfer across the peritubular cell membrane, and hence that this membrane has a high conductance for bicarbonate buffer.
4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
Rapid determination of intraepithelial resistance barriers by alternating current spectroscopy. I. Experimental procedures.
A method has been developed to determine rapidly and simultaneously the resistance of the tight junctions, the resistance of the lateral intercellular space, and the resistances and capacitances of the apical and basolateral cell membrane in leaky epithelia by alternating current spectroscopy. The present paper describes the experimental procedures. Multi-frequency sine-wave currents are simultaneously applied across the tissue and the transepithelial and intracellular voltage responses are recorded with shielded microelectrodes, digitized, and stored in a computer. Using Fast Fourier Transform techniques the frequency-dependent transepithelial impedance and an apparent basal cell membrane impedance are then calculated from the voltage and current waveform. By fitting appropriate model circuits to the data the above listed individual resistances can be deduced. The model calculations and the fit results are described in the subsequent paper [14]. In the present design the analysis covers the frequency range between 2.5 Hz and 12.5 kHz. The minimal measuring time is in the order of 1-2s.
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Rapid determination of intraepithelial resistance barriers by alternating current spectroscopy. II. Test of model circuits and quantification of results.
The impedance of Necturus gallbladder epithelium was measured with transepithelial and intracellular microelectrodes in different transport states. The data are analysed with five electrical equivalent circuits, which differ with respect to the configuration of the paracellular shunt path (lumped vs distributed model of the lateral space), and of the apical cell membrane (non-ideal capacitance or surface amplification by micro-tubular infoldings). Least square fits indicate: 1. that the lumped model cannot represent the epithelium properly, even under control conditions; 2. that the distributed model, which considers the lateral intracellular space separately as a cable-like structure, describes the data well, both under control conditions and during collapse of the lateral spaces; and 3. that the above indicated variations of the apical membrane configuration improve the fits, but have little effect on the magnitude of the calculated circuit parameters. Quantitatively the analysis of 214 measurements on 25 gallbladders under control conditions yields the following results: The resistances of the tight junctions, of the lateral intercellular space, and of the apical and basal cell membrane are Rj = 123, Rlis = 35.5, Ra approximately 3,500, and Rb = 225 (all in omega cm2), and the capacitances of the cell membranes are Ca = 4.95 and Cbl = 26.5 (mu F/cm2). In oxygen deficiency and after cessation of chamber perfusion transport decreased, the spaces collapsed, and Rlis increased to approximately 130 omega cm2. Although the accuracy of the estimated Ra values is still limited, the analysis shows that it is possible to determine Rj and Rlis separately and to follow their changes in response to experimental maneuvers.
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Cell pH of rat renal proximal tubule in vivo and the conductive nature of peritubular HCO3- (OH-) exit.
Intracellular pH (pHc) was measured on surface loops of rat kidney proximal tubules under free-flow conditions in vivo using fine tip double-barrelled pH microelectrodes based on a neutral H+ ligand. The microelectrodes had Nernstian slopes and a resistance of the order of 10(12) omega. By using a driven shield feed back circuit the response time to pH jumps was lowered to around 1 s. At a peritubular pH of 7.42 and a luminal pH of 6.68 +/- 0.13 (n = 27), pHc was 7.17 +/- 0.08 (n = 19). Perfusing the peritubular capillaries suddenly with bicarbonate Ringer solutions of plasma-like composition which were equilibrated with high or low CO2 pressures, acidified or respectively alkalinized the cells rapidly as expected from the high CO2 permeability of the cell membranes. Such data allowed us to calculate the cytoplasmic buffering power of the tubular cells. Sudden peritubular perfusion with Ringer solution containing only 3 mmol/l of HCO3- at constant physiological CO2 pressure led to a similar fast cell acidification which indicated that the peritubular cell membrane is also highly permeable for bicarbonate or OH- (H+). The latter response was completely blocked by the stilbene derivative SITS at the concentration of 10(-3) mol/l. The observations indicate first that pHc of rat proximal tubule is more acidic than was previously thought on the basis of distribution studies of weak acids, second that intracellular bicarbonate concentration is around 13 mmol/l and third that bicarbonate exit across the peritubular cell membrane is a passive rheogenic process via a conductive pathway which can be inhibited by SITS.(ABSTRACT TRUNCATED AT 250 WORDS)
Acid-Base Equilibrium↗
Viewing the kidney through microelectrodes.
Electrical measurements with microelectrodes have proven of great value in the investigation of renal ion transport mechanisms. By virtue of their fine tips (less than 1 micron in diameter) and because of the ease of recording electrical transients, they provide measurements with unparallelled space and time resolution. Early electrophysiological work was done largely in amphibians, because of their larger cell size, whereas studies in mammals were restricted to transepithelial electrical parameters, which provided no insight into individual membrane mechanisms. However, recently techniques have been developed to impale individual tubular cells of mammalian kidneys successfully, both in vivo and in vitro. Such experiments allow us to identify ion transport properties of individual cell membranes from the response of the electrical potential to fast pertubations of the luminal and/or peritubular fluid composition or to applied currents. The power of this approach was greatly increased by the development of ion-selective microelectrodes, allowing us to measure intracellular concentrations of Na+, K+, Cl-, Ca2+, and HCO3- or pH directly and to follow quantitatively their changes in response to different experimental maneuvers. In the present paper our knowledge of ion transport mechanisms of mammalian proximal tubular cell membranes is summarized, with emphasis on the transport of Na+, K+, HCO3-, and Cl-. With the exception of transcellular transport of Cl-, which is quantitatively less important, the major transport mechanisms of all other ions have been identified in the brush border and in the peritubular cell membrane. Emphasis is given to the description of HCO3- exit across the peritubular cell membrane, which has not thus far been studied with other than electrophysiological techniques. Microelectrode techniques will probably continue to provide new insight when regulatory phenomena are studied on the cellular level and individual conductance channels in renal cell membranes are identified with the newly developed patch-clamp technique.
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The intracellular chloride activity of rat kidney proximal tubular cells.
The intracellular Cl- activity was determined in rat kidney proximal tubular cells in vivo, using single-barreled Cl- sensitive microelectrodes filled with Corning no. 477913 liquid ion exchanger resin to measure VCl and using - in separate experiments - conventional KCl-filled microelectrodes to measure the membrane potential, Vm. After correction for interference from other anions on VCl the intracellular Cl- activity averaged 13.1 mmol X l-1 SD +/- 4.5 mmol X l-1 (n = 96). This value is approximately two-fold higher than the intracellular equilibrium activity which can be calculated from the extracellular Cl- activity of 90-103 mmol X l-1 and Vm of -71.2 mV, SD +/- 4.9 mV (n = 23) to amount to 6.3 to 6.7 mmol X l-1. Since both cell membranes are permeable for Cl- ions, as concluded from luminal and/or peritubular Cl- substitution experiments, we conclude that the cellular Cl- accumulation above equilibrium results from transcellular active Cl- transport, the detailed mechanism of which is presently not known. From the slow decline of intracellular Cl- concentration after substitution of luminal Cl- by gluconate, however, we deduce that transcellular Cl- absorption is of minor importance in surface tubules of rat kidney under free flow and that the major part of transtubular Cl- flux is passive and paracellular.
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