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PubMed1983

Intracellular chloride activity and apical membrane chloride conductance in Necturus gallbladder.

Open-tip and Cl--selective microelectrodes were used to study the effect of external pH on apical membrane potential (Va) and intracellular chloride activity (aiCl) in epithelial cells of Necturus gallbladder. Increasing the pH from 7.2 to 8.2 in the mucosal, the serosal, or in both bathing solutions simultaneously, hyperpolarized Va (control value -60 +/- 5 mV) by about -6, -10 and -17 mV, respectively, but did not significantly change the transepithelial potential (VT = 0.3 +/- 0.5 mV). Identical hyperpolarizations were recorded with Cl--selective microelectrodes, even 40 min after changing external pH. Thus, aiCl (12 +/- 2 mM) remained essentially constant. The ratio fVa between the deflections in Va and VT produced by transepithelial current pulses, which is an approximate measure of the fractional resistance of the apical membrane, decreased when mucosal pH was increased, and increased when serosal pH was raised. The changes in Va and fVa are due, in part at least, to the known pH dependence of cell membrane K+ conductance (PK) in this tissue. The constancy of aiCl, despite significant increases in Va, indicates that cell membrane Cl- conductance (PCl) is virtually zero or decreases, with increased external pH, in a way that compensates for the increased driving force for Cl- exit. Experiments in which 90 mM gluconate or 90 mM methylsulfate were substituted for an equivalent amount of luminal Cl- did not provide any evidence for a significant contribution of Cl- ions, per se, to the emf or conductance of the apical membrane. They suggested, rather, a dependence of apical membrane cation permeability on luminal Cl- concentration. Since basolateral membrane PCl is known to be very low, the insensitivity of aiCl to Va is the consequence of a negligible electrodiffusive Cl- permeability at both cell membranes. Thus, overall, transcellular Cl- transport in Necturus gallbladder is, in large measure, effected by electroneutral processes.

Animals↗
PubMed1983

Microelectrode study of K+ accumulation by tight epithelia: II. Effect of inhibiting transepithelial Na+ transport on reaccumulation following depletion.

The effects of restoring serosal potassium to potassium-depleted toad urinary bladders have been re-examined using double-barrelled microelectrodes. The data confirm the existence of a time-lag phenomenon, a dissociation between potassium reaccumulation and restoration of short-circuit current. Returning serosal potassium stimulates an increase in intracellular potassium activity 21-26 min before any increase can be detected in short-circuit current. The reaccumulation of potassium has been further studied using split frog skin, a far more suitable preparation for electrophysiologic study than toad bladder. Under baseline short-circuited conditions, potassium is accumulated against an electrochemical gradient of 22 +/- 4 mV. Reaccumulation of potassium by potassium-depleted tissues can be blocked by inhibiting the Na,K-exchange pump with high concentrations of ouabain. On the other hand, blocking apical sodium entry by the addition of 10(-4) M amiloride to the outer bathing medium does not interfere with reaccumulation of potassium. The data support the concept that the time-lag phenomenon of toad bladder reflects stimulation of potassium reaccumulation by the sodium pump in exchange for the extrusion of excess cell sodium collected during the period of potassium depletion. This reaccumulation of potassium can proceed before the entry of significant added amounts of sodium across the apical plasma membrane.

Animals↗
PubMed1991

Na+ channel activity in cultured renal (A6) epithelium: regulation by solution osmolarity.

Solution osmolarity is known to affect Na+ transport rates across tight epithelia but this variable has been relatively ignored in studies of cultured renal epithelia. Using electrophysiological methods to study A6 epithelial monolayers, we observed a marked effect of solution tonicity on amiloride-sensitive Na+ currents (I(sc)). I(sc) for tissues bathed in symmetrical hyposmotic (170 mOsm), isosmotic (200 mOsm), and hyperosmotic (230 or 290 mOsm) NaCl Ringer's solutions averaged 25 +/- 2, 9 +/- 2, 3 +/- 0.4, and 0.6 +/- 0.5 microA/cm2, respectively. Similar results were obtained following changes in the serosal tonicity: mucosal changes did not significantly affect I(sc). The changes in I(sc) were slow and reached steady-state within 30 min. Current fluctuation analysis measurements indicated that single-channel currents and Na+ channel blocker kinetics were similar for isosmotic and hyposmotic conditions. However, the number of conducting Na+ channels was approximately threefold higher for tissues bathed in hyposmotic solutions. No channel activity was detected during hyperosmotic conditions. The results suggest that Na+ channels in A6 epithelia are highly sensitive to relatively small changes in serosal solution tonicity. Consequently, osmotic effects may partly account for the large variability in Na+ transport rates for A6 epithelia reported in the literature.

Amiloride↗
PubMed1985

Protamine reversibly decreases paracellular cation permeability in Necturus gallbladder.

Protamine, a naturally occurring arginine-rich polycationic protein (pI 9.7 to 12), was tested in Necturus gallbladder using a transepithelial AC-impedance technique. Protamine sulfate or hydrochloride (100 micrograms/ml = 20 microM), dissolved in the mucosal bath, increased transepithelial resistance by 89% without affecting the resistance of subepithelial layers. At the same time, transepithelial voltage (psi ms) turned from slightly mucosa-positive values to mucosa-negative values of approximately +1 to -5 mV. The effect of protamine on transepithelial resistance was minimal at concentrations below 5 micrograms/ml but a maximum response was achieved between 10 and 20 micrograms/ml. Resistance started to increase within 1 min and was maximal after 10 min. These effects were not inhibited by serosal ouabain (5 X 10(-4) M) but could be readily reversed by mucosal heparin. The sequence of protamine effect and heparin reversal could be repeated several times in the same gallbladder. Mucosal heparin, a strong negatively charged mucopolysaccharide, or serosal protamine were without effect. Mucosal protamine reversibly decreased the partial ionic conductance of K and Na by a factor of 3, but did not affect Cl conductance. Net water transport from mucosa to serosa was reversibly increased by 60% by protamine. We conclude that protamine reversibly decreases the conductance of the cation-selective pathway through the tight junction. Although this effect is similar to that reported for 2,4,6-triamino-pyrimidinium (TAP), the mechanism of action may differ. We propose that protamine binds to the apical cell membrane and induces a series of intracellular events which leads to a conformational alteration of the tight junction structure resulting in decreased cationic permeability.

Animals↗
PubMed1984

Apical sodium entry in split frog skin: current-voltage relationship.

Apical Na+ entry into frog skin epithelium is widely presumed to be electrodiffusive in nature, as for other tight epithelia. However, in contrast to rabbit descending colon and Necturus urinary bladder, the constant field equation has been reported to fit the apical sodium current (INa)-membrane potential (psi mc) relationship over only a narrow range of apical membrane potentials or to be inapplicable altogether. We have re-examined this issue by impaling split frog skins across the basolateral membrane and examining the current-voltage relationships at extremely early endpoints in time after initiating pulses of constant transepithelial voltage. In this study, the rapid transient responses in psi mc were completed within 0.5 to 3.5 msec. Using endpoints to 1 to 25 msec, the Goldman equation provided excellent fits of the data over large ranges in apical potential of 300 to 420 mV, from approximately -200 to about +145 mV (cell relative to mucosa). Split skins were also studied when superfused with high serosal K+ in order to determine whether the INapsi mc relationship could be generated purely by transepithelial measurements. Under these conditions, the basolateral membrane potential was found to be -10 +/- 3 mV (cell relative to serosa, mean +/- SE), the basolateral fractional resistance was greater than zero, and the transepithelial current was markedly and reversibly reduced. For these reasons, use of high serosal K+ is considered inadvisable for determining the INa-psi mc relationship, at least in those tissues (such as frog skin) where more direct measurements are technically feasible. Analysis of the INa-psi mc relationships under baseline conditions provided estimates of intracellular Na+ concentration and of apical Na+ permeability of 9 to 14 mM and of approximately 3 X 10(-7) cm X sec-1, respectively, in reasonable agreement with estimates obtained by different techniques.

Amiloride↗
PubMed1982

Single-file diffusion multi-ion mechanism of permeation in paracellular epithelial channels.

The transepithelial fluxes, conductances and permeabilities of Li+, Na+, K+, Cs+, NH4+ and H3CNH3+ were studied under ionic concentrations ranging from 12 to 250 mM in Bufo arenarum gallbladders. When these measurements are carefully corrected in order to get only the component due to the paracellular cation channels, the following results are obtained: (1) The permeability ratios (cationic/anionic) are a decreasing function of salt concentration. (2) The partial conductances through paracellular cationic channels show nonlinear saturable concentration kinetics. (3) Moreover, partial conductance kinetics of K+, Cs+ and NH4+ present a maximum followed, at higher concentrations, by a negative-slope region. (4) The selectivity sequences obtained from biionic potentials do not agree with those obtained from partial conductance measurements. (5) The unidirectional 22Na tracer flux (serosal to mucosal) is inhibited by 63% when the K+ symmetrical concentration in the bathing solutions is raised from 25 to 200 mM. (6) When the unidirectional 42K fluxes (serosal to mucosal) at 200 mM KCl Na-free solutions are compared with K+ partial conductance by means of the Hodgkin and Keynes (Hodgkin, A.L., Keynes, R.D. 1955, J. Physiol London 128:61-88) expression, the n' factor is 2.0. These results indicate that cations do not follow the independence principle and behave as in single-file diffusion multi-ion pores when crossing the paracellular cation channels of Bufo arenarum gallbladder epithelium.

Animals↗
PubMed1989

Distribution of ion channels on taste cells and its relationship to chemosensory transduction.

The presence and regional localization of voltage-gated ion channels on taste cells in Necturus maculosus were studied. Lingual epithelium was dissected from the animal and placed in a modified Ussing chamber such that individual taste cells could be impaled with intracellular microelectrodes and the chemical environment of the apical and basolateral membranes of cells could be strictly controlled. That is, solutions bathing the mucosal and serosal surfaces of the epithelium could be exchanged independently and the effects of pharmacological agents could be tested selectively on the apical or basolateral membranes of taste cells. In the presence of amphibian physiological saline, action potentials were elicited by passing brief depolarizing current pulses through the recording electrode. Action potentials provided a convenient assay of voltage-gated ion channels. As in other excitable tissues, blocking current through Na+, K+, or Ca2+ channels had predictable and consistent effects on the shape and magnitude of the action potential. A series of experiments was conducted in which the shape and duration of regenerative action potentials were monitored when the ionic composition was altered and/or pharmacological blocking agents were added to the mucosal or to the serosal chamber. We have found the following: (i) voltage-gated K+ channels (delayed rectifier) are found predominately, if not exclusively, on the chemoreceptive apical membrane; (ii) voltage-gated Na+ and Ca2+ channels are found on the apical (chemoreceptive) and basolateral (synaptic) membranes; (iii) there is a K+ leak channel on the basolateral membrane which appears to vary seasonally in its sensitivity to TEA. The nonuniform distribution of voltage-gated K+ channels and their predominance on the apical membrane may be important in taste transduction: alterations in apical K+ conductance may underlie receptor potentials ellicted by rapid stimuli.

Animals↗
PubMed1981

Mechanisms for the effects of acetylcholine on sodium transport in frog skin.

In frog skin (Rana temporaria) acetylcholine applied to the serosal surface produces either a sustained inhibiton or sustained stimulation of short-circuit current (SCC). The former effect is accompanied by a reduction and the latter by an increase in total tissue conductance. Both effects of acetylcholine can be accounted for, within experimental error, by changes in net sodium flux across the tissue. By use of selective agonists and antagonists it is concluded that acetylcholine interacts with muscarinic receptors in the serosal membrane. The effects of cholinoceptor agents are also seen with isolated epithelium. The stimulatory effect of acetylcholine is potentiated by theophylline and blocked by inhibitors of prostaglandin synthetase and by mepacrine. It is suggested that acetylcholine stimulates transport by liberating prostaglandins which may then activate adenylcyclase. The inhibitory effect of acetylcholine is correlated with a reduction in cyclic AMP content of the epithelium. Calcium appears to be an important determinant of the type of response seen eith acetylcholine, but the mechanism is not known.

Acetylcholine↗
PubMed1985

Cyclic AMP-induced changes in membrane conductance of Necturus gallbladder epithelial cells.

Enhanced cellular cAMP levels have been shown to increase apical membrane Cl- and HCO3- conductances in epithelia. We found that the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) increases cAMP levels in Necturus gallbladder. We used conventional open-tip and double-barreled Cl- -selective microelectrodes to study the effects of IBMX on membrane conductances and intracellular Cl- activities in gallbladders mounted in a divided chamber and bathed with Ringer's solutions at 23 degrees C and pH 7.4. In HCO3- -free media, 0.1 mM IBMX added to the mucosal medium depolarized the apical membrane potential Va, decreased the fractional resistance FR, and significantly reduced intracellular Cl- activity (aCli). Under control conditions, aCli was above the value corresponding to passive distribution across the apical cell membrane. In media containing 25 mM HCO3-, IBMX caused a small transient hyperpolarization of Va followed by a depolarization not significantly different from that observed in HCO3- -free Ringer's. Removal of mucosal Cl-, Na+ or Ca2+ did not affect the IBMX-induced depolarization in Va. The basolateral membrane of Necturus gallbladder is highly K+ permeable. Increasing serosal K+ from 2.5 to 80 mM, depolarized Va. Mucosal IBMX significantly reduced this depolarization. Addition of 10 mM Ba2+, a K+ channel blocker, to the serosal medium depolarized Va and, essentially, blocked the depolarization induced by IBMX. These results indicate that mucosal IBMX increases apical HCO3- conductance and decreases basolateral K+ conductance in gallbladder epithelial cells via a cAMP-dependent mechanism. The latter effect, not previously reported in epithelial tissues, appears to be the major determinant of the IBMX-induced depolarization of Va.

1-Methyl-3-isobutylxanthine↗
PubMed1991

Electrophysiological studies of forskolin-induced changes in ion transport in the human colon carcinoma cell line HT-29 cl.19A: lack of evidence for a cAMP-activated basolateral K+ conductance.

Forskolin (i.e., cAMP)-modulation of ion transport pathways in filter-grown monolayers of the Cl(-)-secreting subclone (19A) of the human colon carcinoma cell line HT29 was studied by combined Ussing chamber and microimpalement experiments. Changes in electrophysiological parameters provoked by serosal addition of 10(-5) M forskolin included: (i) a sustained increase in the transepithelial potential difference (3.9 +/- 0.4 mV), (ii) a transient decrease in transepithelial resistance with 26 +/- 3 omega.cm2 from a mean value of 138 +/- 13 omega.cm2 before forskolin addition, (iii) a depolarization of the cell membrane potential by 24 +/- 1 mV from a resting value of -50 +/- 1 mV and (iv) a decrease in the fractional resistance of the apical membrane from 0.80 +/- 0.02 to 0.22 +/- 0.01. Both, the changes in cell potential and the fractional resistance, persisted for at least 10 min and were dependent on the presence of Cl- in the medium. Subsequent addition of bumetanide (10(-4) M), an inhibitor of Na/K/2Cl cotransport, reduced the transepithelial potential, induced a repolarization of the cell potential and provoked a small increase of the transepithelial resistance and fractional apical resistance. Serosal Ba2+ (1 mM), a known inhibitor of basolateral K+ conductance, strongly reduced the electrical effects of forskolin. No evidence was found for a forskolin (cAMP)-induced modulation of basolateral K+ conductance. The results suggest that forskolin-induced Cl- secretion in the HT-29 cl.19A colonic cell line results mainly from a cAMP-provoked increase in the Cl- conductance of the apical membrane but does not affect K+ or Cl- conductance pathways at the basolateral pole of the cell. The sustained potential changes indicate that the capacity of the basolateral transport mechanism for Cl- and the basal Ba(2+)-sensitive K+ conductance are sufficiently large to maintain the Cl- efflux across the apical membrane. Furthermore, evidence is presented for an anomalous inhibitory action of the putative Cl- channel blockers NPPB and DPC on basolateral conductance rather than apical Cl- conductance.

Biological Transport↗
PubMed1985

Effects of intracellular sodium and potassium iontophoresis on membrane potentials and resistances in toad urinary bladder.

Glass microelectrodes were used to measure membrane potentials and the ratio of apical to basolateral membrane resistances before and after the passage of current from the potential-recording microelectrode to ground, in toad urinary bladder epithelium, in order to iontophorese cations into the cell. After application of the current, there was a transient change in the tip potential of the microelectrode. This artifact was measured with the microelectrode in the mucosal medium and was subtracted from the potential recorded in the cell. The serosal medium was bathed by Ringer's solution containing 51.5 mM K+ to minimize any current-induced increase of K+ in the unstirred layer. Under those conditions, both Na+ and K+ iontophoresis caused a significant hyperpolarization of basolateral membrane potential (Vcs) and a significant increase in the ratio of apical to basolateral membrane resistances (Ra/Rb). When bladders were exposed to amiloride in the mucosal solution, Na+ iontophoresis caused the basolateral membrane to hyperpolarize, but no significant changes were observed in Ra/Rb. When Na+ was injected in the presence of serosal ouabain, Vcs depolarized and Ra/Rb increased. K+ iontophoresis caused the basolateral membrane potential to hyperpolarize in the presence of ouabain but Ra/Rb did not change significantly. These results indicate that the Na+ pump in toad bladder is rheogenic, that apical Na+ conductance is sensitive to the cell levels of Na+ and K+ and that the basolateral membrane is K+ permeable.

Amiloride↗
PubMed1983

Influx mechanisms for Na+ and Cl- across the brush border membrane of leaky epithelia: a model and microelectrode study.

This paper presents a numerical model for the movement of Na+, K+, Cl-, H+ and HCO3- in a leaky epithelium. The model describes the active transport of Na+ and K+ at the serosal membrane and electrodiffusive permeation across the mucosal, serosal and junctional pathways. The model accounts for H+ and HCO3- production in the cell. The influx of Na+ and Cl- is assumed to occur mainly via Na/H and Cl/HCO3 exchange. The behavior of the cell, with this influx mechanism, is compared to a cell with an obligatory neutral coupled influx of Na+ and Cl-. All parameters are obtained from the literature, primarily from studies utilizing the Necturus gallbladder. The analysis shows (i) that it is virtually impossible in steady-state experiments to distinguish between cells with Na/H - HCO3/Cl transport and cells with Na/Cl transport mechanisms. (ii) That nonsteady-state experiments can decide whether Na/H - HCO3/Cl or Na/Cl transport mechanisms mediate the influx of salt. A comparison between studies with ion-selective microelectrodes and the model predictions indicates that the influx of Na+ and Cl- is mediated by Na/H - HCO3/Cl transport when the external solutions contain CO2 and HCO3. (iii) The model also explains the diuretic effects of furosemide and carbonic anhydrase inhibitor, as well as the stimulatory effects on salt transport of elevated levels of HCO3- at a constant pH. (iv) The model fails to explain some experiments performed in HCO3/Co2-free media and some experiments using inhibitors.

Animals↗
PubMed1978

Effect of sodium transport inhibition on active phosphate transport by toad bladder.

The relationship between active Na transport (estimated by the short-circuit (SCC)) and active inorganic phosphate (Pi) transport was studied in the toad bladder. When SCC was inhibited by amiloride, ouabain, or removal of K from the serosal bathing solution, active Pi transport was totally inhibited. When Na was replaced isotonically by choline in either the mucosal bathing solution or both the mucosal and serosal bathing solutions, there was no measurable SCC or active Pi transport. These experiments are compatible with the hypothesis that active Pi transport occurs only in the presence of active Na transport.

Amiloride↗
PubMed1988

Constitutive and transport-related endocytotic pathways in turtle bladder epithelium.

Proton secretion in the urinary bladder of the freshwater turtle is mediated by a proton pump located in the apical membrane of a population of cells characteristically rich in carbonic anhydrase. Earlier studies have demonstrated that these cells exhibit apical-membrane endocytotic and exocytotic processes which are thought to be involved in the regulation of the rate of proton transport via alterations in the number of pumps within the apical membrane. In this study, we sought to characterize these processes using two different methods. Analysis of transepithelial impedance yielded estimates of membrane capacitance which could be related to membrane area, thereby allowing one to monitor net changes in apical-membrane area resulting from changes in the net rates of endo- and exocytosis. Uptake of the fluid-phase marker FITC-dextran provided a measure of net extracellular volume uptake which was related to net rates of endocytosis. Our major conclusions are summarized as follows. The bladder cells exhibit a high baseline rate of endocytosis which appears to be a constitutive process similar to pinocytosis. This process is completely inhibited when ambient temperature is reduced to 15 degrees C. In addition, serosal application of 0.5 mM acetazolamide causes a transient increase in the rate of endocytosis, concomitant with a decrease in the rate of transport. Reduction of ambient temperature to 15 degrees C reduces the rate of acetazolamide-induced endocytosis, but does not abolish it. Addition of 1 mM serosal azide not only prevents the acetazolamide-induced increase in endocytosis, but also prevents the decrease in transport caused by acetazolamide. Azide has no effect on the baseline rate of endocytosis, nor does it prevent inhibition of carbonic anhydrase by acetazolamide. The specificity of azide, coupled with the different temperature sensitivities, demonstrate that the constitutive and transport-dependent endocytotic pathways are distinct processes. The observation that azide prevents both the acetazolamide-induced increase in endocytosis and the decrease in transport strongly supports the notion that endocytosis of proton-pump-containing membrane is requisite for the inhibition of transport by acetazolamide. Finally, the results also demonstrate that acetazolamide does not inhibit proton secretion simply by inhibiting carbonic anhydrase.

Acetazolamide↗
PubMed1975

Sodium fluxes through the active transport pathway in toad bladder.

To assess the active components of sodium flux across toad bladder as a function of transepithelial potential, unidirectional sodium fluxes between identical media were measured before and after adding sufficient ouabain (1.89 X 10(-3)M) to eliminate active transport, while clamping transepithelial potential to 0, 100 or 150 mV. Evidence was adduced that ouabain does not alter passive fluxes, and that fluxes remain constant if ouabain is not added. Hence, the ouabain-inhibitable fluxes represent fluxes through the active path. Results were analyzed by a set of equations, previously shown to describe adequately passive fluxes under electrical gradients in this tissue, here modified by the insertion of E, the potential at which bidirectional sodium fluxes (beta E, and theta E) through the active pathway are equal. According to these equations, beta E and theta E are the logarithmic mean of bidirectional fluxes through the active path at any potential, and the flux ratio in this path is modified by a constant factor Qia, which represents the ratio of the bulk diffusion coefficient to the tracer diffusion coefficient in this pathway. The data are shown to conform closely to these equations. Qia averages 2.54. Hence, serosal-to-mucosal flux vanishes rapidly as potential falls below E. Mean E in these experiments was 158 +/- 1 mV. Thus, linear dependence of net flux in both active and passive pathways on potential is present, even though the sodium fluxes in both paths fail to conform to the Ussing flux ratio equation. Qip less than 1 in the passive path (qualitatively similar to exchange diffusion) and Qia greater than 1 in the active path (as in single file pore diffusion). Both of these features tend to reduce the change in serosal-to-mucosal sodium flux induced by depolarization from spontaneous potential to zero potential ("short-circuiting").

Animals↗
PubMed1982

Anti-IgE and Con A-induced histamine release from mast cells of four rat strains: correlation with total serum IgE.

Anti-IgE- and Con A-induced histamine release from serosal mast cells were compared to each other and to total serum levels of IgE in non-immunized, alum-injected, and Silica gel-injected rats of the BN, Fischer, PVG, and SD strains. The results indicate that the degree of anti-IgE- and Con A-induced release is strain-dependent and varies with immunization conditions. Furthermore, there is a gross but not complete correlation between the degree of serosal mast cell histamine release induced by the two secretagogues. However, Con A- or anti-IgE-induced release could significantly be correlated to serum levels of total IgE only in the Fischer strain but not in the BN or the PVG strains. In the SD strain, Con A-induced release correlated to serum IgE levels in Silica gel-injected but not in alum-injected animals.

Animals↗
PubMed1978

Basolateral membrane potential of a tight epithelium: ionic diffusion and electrogenic pumps.

The contribution of specific ions to the conductance and potential of the basolateral membrane of the rabbit urinary bladder has been studied with both conventional and ion-specific microelectrode techniques. In addition, the possibility of an electrogenic active transport process located at the basolateral membrane was studied using the polyene antibiotic nystatin. The effect of ion-specific microelectrode impalement damage on intracellular ion activities was examined and a criterion set for acceptance or rejection of intracellular activity measurements. Using this criterion, we found (K+) = 72 mM and (Cl-) = 15.8 mM. Cl- but not K+ was in electrochemical equilibrium across the basolateral membrane. The selective permeability of the basolateral membrane was measured using microelectrodes, and the data analyzed using the Goldman, Hodgkin-Katz equation. The sodium to potassium permeability ratio (PNa/PK) was 0.044, and the chloride to potassium permeability ratio (PCl/PK) was 1.17. Since K+ was not in electrochemical equilibrium, intracellular (K+) is maintained by active metabolic processes, and the basolateral membrane potential is a diffusion potential with K+and C1- the most permeable ions. After depolarizing the basolateral membrane with high serosal potassium bathing solutions and eliminating the apical membrane as a rate limiting step for ion movement using the polyene antibiotic nystatin, we found that the addition of equal aliquots of NaCl to both solutions caused the basolateral membrane potential to hyperpolarize by up to 20mV (cell interior negative). This potential was reduced by 80% within 3 min of the addition of ouabain to the serosal solution. This hyperpolarization most probably represents a ouabain sensitive active transport process sensitive to intracellular Na+. An equivalent electrical circuit for Na+ transport across rabbit urinary bladder is derived, tested, and compared to previous results. This circuit is also used to predict the effects that microelectrode impalement damage will have on individual membrane potentials as well as time-dependent phenomena; e.g., effect of amiloride on apical and basolateral membrane potentials.

Animals↗
PubMed1981

Effects of bicarbonate on fluid and electrolyte transport by guinea pig and rabbit gallbladder: stimulation of absorption.

The effect of bicarbonate (HCO3) on fluid absorption by guinea pig gallbladder was investigated in vitro. Stimulation of fluid absorption was concentration dependent resulting in a fourfold increase in transport over the range 1 to 50 mM. Phosphate, Tris, glycodiazine and glutamine buffers failed to substitute for HCO3 in stimulating absorption. Unidirectional 22Na fluxes were measured across short-circuited sheets of guinea pig and rabbit gallbladders mounted in Ussing-type chambers. In both species the net Na flux was unaffected by serosal HCO3 alone but was stimulated by addition of HCO3 to the mucosal bathing solution. Transepithelial electrical potential difference in rabbit gallbladder was about 1.4 mV (lumen positive) when HCO3 was present in the mucosal or in both compartments. This fell to 0.2 mV under HCO3-free conditions or when HCO3 was present only in the serosal solution. The respective values for guinea pig gallbladder were -1.6 and -0.6 mV (lumen negative). HCO3 stimulation of Na absorption by guinea pig gallbladder was abolished by increasing the bathing pH from 7.4 to 7.8, an effect resulting mainly from a reduction in JNams. Tris buffer (25 mM) inhibited HCO3-dependent fluid absorption in this species completely at pH 8.5 and partially at 7.5. These results indicate that HCO3 stimulates gallbladder transport in both species by an action from the mucosal side. This effect cannot be attributed to simple buffering of H+ but may be explained by the participation of HCO3 in the maintenance of intracellular H+ for a Na/H-exchange.

Animals↗