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PubMed1989

Electrophysiological characteristics of the Necturus proximal duodenal mucosa: effects of ion substitutions.

Voltage-sensitive glass micro-electrodes were used to determine the electrical characteristics of Necturus proximal duodenal epithelium. Some comparative experiments with amiloride were performed with gastric antrum. The apical and the basolateral cell membrane potential differences in duodenum averaged -32 mV and -34 mV (cell negative) respectively. The transepithelial potential difference was -2 mV (lumen negative). The EMF across the apical cell membrane was -29 mV and that across the basolateral cell membrane -39 mV. The transepithelial resistance (Rt) of 63 omega cm2 and the paracellular pathway resistance (Rs) of 80 omega cm2 are of magnitudes similar to that previously reported for more distal amphibian small intestine. The apical and basolateral cell membrane resistances, however, were lower than those reported for distal small intestine. Ion permeabilities for Na+, K+ and Cl- across the apical cell membrane were calculated from ion substitution experiments. The permeability sequence across the apical cell membrane was PK:PCl:PNa 3.02:1.31:1.00. Luminal amiloride (10(-4)M) was without significant effect, further indicating a low duodenal membrane conductance for Na+. The low conductances for K+, Na+ and Cl- suggest that the major ion transport modes across the apical duodenal cell membrane are electroneutral in nature. In contrast, amiloride caused a marked increase in the transmembrane potentials in the antrum.

Amiloride↗
PubMed1989

Alkaline secretion by Necturus proximal duodenal mucosa.

Proximal duodenum from the amphibian Necturus was stripped of muscle layers and the mucosa was mounted as a tube for studies of alkali transport or as a flat sheet for intracellular impalement by voltage-sensitive glass micro-electrodes. The mucosa alkalinized the unbuffered luminal perfusate at a high rate (3.4 muequiv. cm-1 h-1) and developed a transepithelial electric potential difference of 5.7 mV (lumen negative). Transport was inhibited by 2,4-dinitrophenol (10(-4) M) and by furosemide (10(-3) M) and SITS (10(-3) M) on the seros but not on the mucosal side, indicating dependence on tissue metabolism and on serosal membrane Cl-/HCO3- exchange. Prostaglandin E2 (10(-7)-10(-5) M) and dibutyryl cyclic AMP (10(-6)-10(-4) M) had no effects on the secretion or transepithelial electrical potential difference. removal of serosal HCO3- decreased luminal alkalinization by 75%, indicating a contribution by passive migration of HCO3- and/or a dependence of transcellular transport on the nutrient supply of this ion. Administration of HCO3- (17.8 mM) to the luminal perfusate affected neither the transepithelial nor transmembrane electrical potential differences nor the resistance ratio. It is thus unlikely that the luminal membrane possesses any major HCO3- conductance.

2,4-Dinitrophenol↗
PubMed1981

Mechanism of the effect of cyanide on cell membrane potentials in Necturus gall-bladder epithelium.

1. Addition of sodium cyanide to the mucosal or the serosal medium bathing the isolated gall-bladder of Necturus maculosus causes hyperpolarization of both apical and basolateral membrane of the epithelial cells. The effect of cyanide is practically immediate, reversible (if exposure is brief), and long-lasting (greater than 30 min). 2. The hyperpolarization is accompanied by: (a) reduction of the equivalent resistance of the cell membranes, as shown by cable analysis and input resistance measurements, and (b) increase of the potassium selectivity of both cell membranes, as evidenced by the effects of external substitutions of potassium for sodium on cell membrane potentials. We conclude that the cyanide-induced hyperpolarization is caused mainly or exclusively by an increase of the potassium permeability of the cell membranes. 3. Addition of the calcium ionophore A23187 (5 microM) to the mucosal medium in the presence of 1 mM-calcium caused similar effects to those produced by cyanide. After either cyanide or A23187, addition of the other agent did not cause further membrane potential changes. 4. Quinine (100 microM, mucosal medium) reduced the potassium permeability of the apical membrane both under control conditions and during exposure to cyanide. 5. We suggest that the cyanide-induced increase of the potassium permeability of the cell membrane is mediated by an elevation of intracellular calcium ion activity, attributable to release from mitochondrial sources.

Absorption↗
PubMed1982

A micro-electrode study of oligopeptide absorption by the small intestinal epithelium of Necturus maculosus.

1. The effects of some amino acids and oligopeptides on the electrical properties of the brush-border membrane of the small intestine of the mudpuppy, Necturus maculosus were studied in vitro using micro-electrodes. 2. A number of amino acids (glycine, L-proline and L-leucine) and small peptides (carnosine, glycyl-L-proline, L-leucyl-L-leucine, glycylglycine and glycylglycylglycine) depolarized the brush-border membrane. This was associated with a reduction of input resistance. Tetraglycine did not appreciably reduce the membrane potential. 3. Evidence is presented that the electrical effects caused by application of the peptides are not solely due to the effects of the products of peptide hydrolysis. Furthermore there appears to be more than a single system available to the peptides. 4. Stereospecificity is found for both amino acids and peptides but appears to be more marked for the peptides. 5. Peptide-induced depolarizations are less markedly reduced in the absence of external Na+ than are the depolarizations caused by the amino acids. 6. These results are discussed with respect to the mechanism and significance of amino acid and peptide transport in the intestinal tract.

Amino Acids↗
PubMed1984

Active sodium transport and fluid secretion in the gall-bladder epithelium of Necturus.

Intracellular Na, K and Cl activities (acNa, acK and acCl) and membrane potentials were measured in Necturus gall-bladder epithelium using double-barrelled ion-sensitive micro-electrodes. Mucosal membrane potential was about -55 mV and the mean control activities were acNa = 14.7 mM, acK = 91.6 mM and acCl = 20.3 mM. Replacing mucosal Na by K caused a fall in acNa that followed an exponential time course. The rate of change in acNa was linearly related to acNa above a certain value (congruent to 3 mM). acK and acCl both increased in K Ringer solution. From the change in all three ions the cell was estimated to swell at an initial rate of 0.13% s-1. From the initial rate of change in acNa, a net cell efflux of Na of 405 pmol cm-2 s-1 was calculated. Replacement of Na by Tris or choline led to a similar result. The transepithelial Na transport rate was for this group of animals 346 pmol cm-2 s-1. Ouabain (10(-3) M) produced an increase in acNa and acCl, whereas acK decreased. The cells were estimated to swell at an initial rate of 0.06% s-1. The initial Na influx after Na-pump inhibition was calculated to be 162 pmol cm-2 s-1. The parallel measure of the transepithelial rate of transport of Na gave a value of 189 pmol cm-2 s-1. Ouabain inhibited the decrease in acNa after replacement of Na by K by about 80%. A fast depolarization, ranging from 2 to 7 mV, occurred after the perfusion with ouabain. Em then slowly decreased from about 53 to 32 mV in 1 h. It is concluded that (a) the major fraction of the transepithelial transport of Na is transcellular and mediated by the Na pump, (b) the pumping rate is linearly dependent on internal Na within a certain range and (c) the Na pump is electrogenic under normal circumstances.

Animals↗
PubMed1987

Steady-state analysis of ion fluxes in Necturus gall-bladder epithelial cells.

1. The steady-state fluxes of Na, K and Cl ions have been measured in Necturus gall-bladder epithelium by a technique that involves labelling the cells with tracer ions from the mucosal bath only, whilst the serosa is kept at low specific activity. After removing tracer, the efflux is followed into the serosal bath, revealing two exponential components. 2. The time constant of the fast component lies between 0.03 and 0.04 s-1 and corresponds to that of the extracellular space. The slow component closely matches the cellular efflux, with constants which lie between 0.14 and 0.46 X 10(-2) s-1. 3. Full unstirred-layer calculations have been performed to determine the specific activities in the mucosal solution, the cell and the corium (subepithelium). These involved measuring the diffusion coefficients of Na and Cl in the isolated corium: they are restricted by factors of 0.17 and 0.11. 4. The partial flux equations for this double-membrane system have been solved to obtain the cellular fluxes for all three ions. The results indicate that: (i) the net transcellular Na flux is 190 pmol cm-2 s-1, equivalent to the transepithelial salt flux during fluid secretion; (ii) the net transcellular K flux is effectively zero because this ion recirculates across the serosal membrane; (iii) the net transcellular Cl flux is 27 pmol cm-2 s-1, or 15% of the net transepithelial salt flux. 5. The permeability of the paracellular pathway to Cl is 1.65 X 10(-5) cm s-1 and the available driving forces will allow a maximum net electrodiffusive Cl transport of 10% through the shunt pathway. 6. 1:1 coupling of Na and Cl net fluxes at the mucosal membrane of this epithelium cannot be present, and processes other than simple electrodiffussion are required to effect net Cl transport by another route. 7. The serosal fluxes of K and Cl do not obey the flux-ratio equation. A component of these fluxes must be present which is neither active (pumped) nor passive (electrodiffusive and independent). If they are symmetrical in the steady state then the ratio of these exchange fluxes lies between 2:1 and 3:1 depending upon the size of the pump flux. They support the view that a mode of coupled K and Cl transport may be operating at the basolateral membrane of these cells.

Animals↗
PubMed1987

Cell and luminal activities of chloride, potassium, sodium and protons in the late distal tubule of Necturus kidney.

1. Double-barrelled (selective vs. conventional) microelectrodes were used to assess the steady-state activities (a) of the ions Cl-, K+, Na+ and H+ in peritubular blood capillaries (abld) and in cell (acell) and lumen (alum) of the late distal tubule (l.d.t.) of Necturus. 2. a(cell)cl, a(lum)cl and a(bld)cl were 5.5 +/- 0.3, 11.8 +/- 1.0 and 70.5 +/- 0.1 mM, respectively. They were used to compute the chemical potentials for Cl- across the three diffusive barriers of the tissue. Basolateral and apical membrane potentials were -74.3 +/- 1.1 and -60.1 +/- 2.0 mV, respectively (cell negative); the lumen was thus negative with respect to blood, by 13.6 +/- 1.5 mV. The electrochemical potential difference (e.p.d.) for Cl- of 42 mV across the apical membrane opposes Cl- absorption, implying active apical Cl- uptake, since Cl- is known to be absorbed in the l.d.t. Basolateral Cl- exit is favoured by an e.p.d. of 10 mV. 3. a(cell)K, a(lum)K and a(bld)K were 65.8 +/- 0.8, 2.5 +/- 0.1 and 2.5 +/- 0.1 mm, respectively. The electrochemical distribution of K+ indicates that K+ absorption, if present, proceeds against an adverse apical e.p.d. of 18 mV. Basolateral K+ distribution is close to its electrochemical equilibrium, suggesting high K+ permeability at this membrane. 4. a(cell)Na was 9.0 +/- 0.4 mM, a(bld)Na 71.0 +/- 0.3 mM, and a(lum)Na was approximated at about 9 mM. Diffusive Na+ entry from lumen to cell is favoured by an e.p.d. close to 65 mV. Basolateral Na+ exit must be active, since it proceeds against an e.p.d. of 130 mV. 5. Cell, luminal and blood pH were 7.14 +/- 0.03, 6.52 +/- 0.08 and 7.37 +/- 0.04, respectively. The luminal electrochemical potential of H+ is higher than that of cell (by 91 mV) and blood (by 34 mV) indicating that proton secretion into the lumen must be active. 6. The e.p.d. of each ion across the epithelium opposes, by its orientation, the established direction of net transepithelial ion transport, suggesting that the shunt pathway may serve only for back-diffusion.

Absorption↗
PubMed1989

Characterization of a phosphorylation-activated Cl-selective channel in isolated Necturus enterocytes.

1. The cell-attached and excised inside-out configurations of the patch-clamp technique were employed to probe isolated enterocytes of Necturus maculosus for the presence of Cl(-)-selective channels. 2. Chloride-selective channels were rarely observed unless cells were previously stimulated by agonists that raise cyclic AMP. In cell-attached patches forskolin (20 microM) or dibutyryl cyclic AMP 2 mM) evoked single-channel activity that reversed, depending on the cell, between 9 and 27 mV positive to the spontaneous membrane potential. This is close to the Cl- equilibrium potential in those cells; the single-channel current-voltage relationship was linear with a unitary slope conductance between 17 and 25 pS (pipettes filled with 100 mM-NaCl). 3. Large depolarizing voltage steps also activated Cl- channels in excised inside-out membrane patches that were previously quiescent. This mode of activation produced a distinctive single-channel current-voltage relationship with strong outward rectification at depolarizing membrane potentials. Single-channel cord conductance at negative potentials was 15-18 pS and increased to 45 pS at + 100 mV. 4. Altering the Cl- concentration in the bathing solution of excised inside-out patches displaced the observed reversal potential (Erev) to values predicted for Cl- equilibrium potential. Replacement of K+ for Na+ was without effect. 5. The effect of different anions upon Erev was used to determine the channel anion selectivity in excised inside-out patches. The permeability sequence was SCN- greater than I- greater than Br- greater than Cl- greater than F- greater than HCO3- greater than gluconate which corresponds to Eisenman's sequence 1. Neither ionic size nor diffusion rates determine the permeation of ions through the channel. 6. In channels activated by depolarization the open probability (Po) was insensitive to changes in the Ca2+ concentration (less than 10(-8)-10(-3) M) bathing the cytoplasmic face of excised inside-out patches. Depolarization was also without marked effect on Po. 7. Chloride channels in excised inside-out patches were inhibited by stilbene and diphenylamine-2-carboxylate derivatives. 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS, 5 x 10(-5) M) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB, 1 x 10(-5) M) caused an irreversible 'flickery' blockade without altering single-channel current. 3'5-Dichlorodiphenylamine-2-carboxylic acid (DDPC, 5 x 10(-5) M) reduced the currents at every voltage without apparent effects on gating properties of the channel.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Potassium currents of isolated Necturus enterocytes: a whole-cell patch-clamp study.

1. The whole-cell recording mode of the patch-clamp technique was used to investigate the ionic currents of enterocytes isolated from the small intestine epithelium of Necturus maculosus. 2. When enterocytes were bathed in a physiological Ringer solution and dialysed with a K(+)-rich intracellular solution containing 1.5 x 10(-7) M intracellular Ca2+, strongly outwardly rectifying currents were observed. These currents were absent from enterocytes internally dialysed with K(+)-free solutions. 3. The kinetics of activation of the outwardly rectifying current was monoexponential with rate constants decreasing with depolarization from 160 ms at 20 mV to 40 ms at 60 mV. Similar voltage dependence of the relaxations after activation were observed. 4. Strongly buffering intracellular Ca2+ with EGTA inhibited outward currents, while increasing Ca2+ increased both their magnitude and rate of activation. 5. Bath application of the K+ channel inhibitors Ba2+ and TEA greatly attenuated outwardly rectifying currents. This observation plus the fact that tail currents reverse near EK points to K+ as the charge carrier in these currents. 6. Outside-out patches showed maxi K+ channels and lower conductance K+ channels. Averaging fluctuations of the maxi K+ channels gave a kinetic behaviour similar to the whole-cell currents.

Animals↗
PubMed1991

Cytosolic pH regulates maxi K+ channels in Necturus gall-bladder epithelial cells.

1. The patch clamp technique was used to study the effects of internal and external pH on the Ca(2+)- and voltage-activated maxi K+ channel present in the apical membrane of Necturus gall-bladder epithelial cells. 2. When the pH of the solution bathing the cytosolic side of inside-out patches (pHi) was lowered from 7.9 to 6.9, with internal free Ca2+ concentration ([Ca2+]i) buffered below saturation levels for the channel gating sites, channel open probability (Po) decreased. At saturating Ca2+ concentrations, Po was near 1.0, and unaffected by pHi. The results are consistent with a competitive interaction between Ca2+ and H+ at regulatory binding sites. Kinetic analysis assuming competitive binding yields a Hill coefficient for H+ of 1.3. 3. At sub-maximal [Ca2+]i, changing the pH of the solution bathing the extracellular surface of the patch (pHo) between 8 and 7, had no effect on maxi K+ channel Po, but lowering pHo to 6 or 5 significantly reduced Po. At saturating [Ca2+]i, Po was independent of pHo. 4. There were no effects of either pHi or pHo on single-channel conductance. 5. Inasmuch as reductions in either pHo or pHi decrease maxi K+ channel Po, changes in maxi K+ channel activity account in part for the reduction of apical membrane K+ conductance elicited by acidification of the bathing medium. The dominant effect of pH on maxi K+ channels is on the cytosolic surface of the membrane. 6. The change in Po elicited by small changes in [H+]i (delta Po/delta [H+]i) is -7.6 microM-1, compared to delta Po/delta [Ca2+]i = 2.6 microM-1, both at Vm = -30 mV and at physiological intracellular [H+] and [Ca2+]. This implies that [H+]i and [Ca2+]i have opposite effects on channel Po at physiological levels and underlines the importance of pHi in channel gating.

Action Potentials↗
PubMed1991

Water permeability of ventricular cell membrane in choroid plexus epithelium from Necturus maculosus.

1. The osmotic water permeability Lp and the relations between the flows of H2O, K+ and Cl- were studied in the ventricular membrane of the epithelium from the choroid plexus of Necturus maculosus. 2. The flows were induced by abrupt changes in external osmolarity of the ventricular solution. Solution changes were convective and no effects of unstirred layers could be detected on measured parameters. 3. The initial rate of change in intracellular concentrations of K+ and Cl- was monitored by double-barrelled ion-selective microelectrodes. 4. The initial rate of flux of H2O could be monitored as the changes in the concentration of intracellular choline ions (Ch+i). When 0.5 mmol l-1 of choline chloride was added to the external solutions, Ch+i attained values of 1-5 mmol l-1. The dilution or concentration of Ch+i could be recorded by K+ electrodes since the sensitivity of these to Ch+ is more than 50 times greater than to K+. 5. The Lp of the ventricular membrane of the epithelium was 1.4-2.1 x 10(-4) cm s-1 (osmol l-1)-1 and independent of the direction of the induced water flux. Lp was unchanged in tissues adapted to osmolarities of half the physiological value. 6. The efflux of H2O induced by mannitol was associated with an instantaneous efflux of K+ which was inhibited by furosemide. The fluxes had a ratio of 40 mmol l-1. The influx of H2O induced by the removal of NaCl from the ventricular solution was associated with an instantaneous influx of K+. The H2O influx had a ratio to the flux of K+ of 70 mmol l-1. 7. The efflux of H2O induced by mannitol was associated with an efflux of Cl- which was inhibited by furosemide. The ratio of the two fluxes was in the range 15-44 mmol l-1. 8. The conclusion is that the Ch+ method gives a reliable measure of the movement of H2O across the ventricular membrane. The magnitude of the Lp and its relevance to transepithelial transport are discussed. The osmotically induced H2O movement is accompanied by furosemide-sensitive fluxes of K+ and Cl- of the same magnitude. This suggests that co-transport between H2O and KCl can take place in the membrane.

Animals↗
PubMed1991

Secondary active transport of water across ventricular cell membrane of choroid plexus epithelium of Necturus maculosus.

1. The interaction between Cl-, K+ and H2O fluxes were studied in the ventricular membrane of the choroid plexus epithelium from Necturus maculosus by means of ion-selective microelectrodes. The flux of H2O was measured by means of K+ electrodes as the dilution or concentration of intracellular choline ions, Ch+i. 2. In one series of experiments Cl- was readministered to the ventricular solution of tissues incubated in media with low Cl- concentrations. The resulting influx of Cl- was associated with an instantaneous influx of K+ and H2O. 3. Both the Cl- and the K+ influxes were reduced by the diuretic furosemide but were unaffected by inhibitors of Na+, K(+)-ATPase or changes in membrane potentials induced by Ba2+. Since the influx of K+ proceeds against its electrochemical gradient and is unaffected by changes in membrane potentials, the membrane exhibits secondary active, electroneutral transport of K+. 4. The influx of water, initiated simultaneously with the influx of K+ and Cl-, commenced before these ions had changed the osmolarity of the intracellular solution significantly. The influx of H2O could proceed against an osmotic gradient. The influx stopped when 100 mmol l-1 of mannitol was added to the ventricular solution at the same time as the Cl- ions. The influx of H2O was inhibited by K+ removal, furosemide or high external Ba2+ (10 mmol l-1), but not by strophanthidin, ouabain or low concentrations of Ba2+ (0.5 mmol l-1). The influx could not continue with other permeable anions, NO3-, acetate- or SCN-, replacing Cl-. 5. In another series of experiments Cl- was removed from the ventricular solution of tissues bathed in saline solutions with normal concentrations of Cl-. The resulting efflux of Cl- was associated with an instantaneous efflux of K+ and H2O. This efflux of H2O could proceed against an osmotic gradient of up to 70 mosmol l-1. This effect was inhibited by furosemide, in which case the water fluxes were entirely dependent on the osmotic gradients and the osmotic water permeability Lp of the ventricular membrane. 6. The data suggest that there is a coupling between the flux of KCl and of water in the ventricular membrane, which implies that the reflection coefficient sigma for KCl under the given circumstances is less than one. I suggest that the ability of leaky epithelia to transport against osmotic gradients depends on such a coupling, which derives from the properties of the proteins through which K+, Cl- and H2O leave the cell.

Animals↗
PubMed1993

Convective fluid flow through the paracellular system of Necturus gall-bladder epithelium as revealed by dextran probes.

1. Bidirectional paracellular fluxes using radioactive dextrans as inert molecular probes have been measured across Necturus gall-bladder epithelium during conditions of normal fluid absorption. There is a net flux at all radii analysed (0.4-2.2 nm) in the direction of fluid absorption. 2. The net flux is substantial at all radii within the range. The data extraplate to 2 x 10(-6) cm s-1 at zero probe radius, which is very close to the rate of epithelial fluid absorption. 3. The unstirred layers at the epithelial faces during transport have been determined; their contribution to the net fluxes is negligible. 4. Two possible mechanisms for the net flow of probes are considered: (i) that the probes diffuse across the junctions and are then entrained in a local osmotic flow along the interspaces and subepithelium; (ii) that the probes are entrained in volume flow across the junctions and the emergent solution subsequently passes through the interspaces and subepithelium. Model calculations clearly rule out mechanism (i) in which the maximum net flow obtainable is less than 10% of that observed. In addition the presence of leak paths shunting the junctions is not compatible with the observed fluxes. With mechanism (ii) the net flows are correctly predicted with all the fluid flow being transjunctional. The fluid absorption is therefore entirely paracellular. 5. The slope of the net flow curve shows no apparent change in magnitude over the range of the probe radii, indicating that effectively only one population of convective channels is present with parallel walls separated by about 7.7 nm. This agrees with the width previously determined by electron microscopy. 6. If the fluid absorption is junctional then the cellular route offers little if any relative contribution. The hydraulic conductivity of the junctions is not high enough, or the osmotic permeability of the membranes low enough, to accommodate this by osmosis and therefore the junctional fluid absorption must be non-osmotic.

Absorption↗
PubMed1994

Cotransport of K+, Cl- and H2O by membrane proteins from choroid plexus epithelium of Necturus maculosus.

1. The interaction between K+, Cl- and H2O fluxes was studied in the ventricular membrane of the choroid plexus epithelium from Necturus maculosus by means of ion-selective microelectrodes. 2. Three experimental strategies were adopted: the osmolarity of the ventricular solution was increased abruptly by addition of (i) mannitol or (ii) KCl; (iii) Na+ in the ventricular solution was replaced isosmotically by K+. 3. The mannitol experiments showed that H2O had two pathways across the ventricular membrane. One was purely passive, with a water permeability, L'p, of 0.64 x 10(-4) cm s-1 (osmol l-1)-1. This operated in parallel with an ion-dependent pathway of similar magnitude which was abolished in Cl(-)-free solutions. 4. When KCl was added there was a flow of H2O into the cell. Surprisingly, this took place despite the osmotic gradient which favoured an efflux of H2O. The effect was blocked by frusemide (furosemide), in which case KCl had the same effects as applications of NaCl or mannitol. 5. Replacement of Na+ with K+ caused an influx of H2O. This flux could proceed against osmotic gradients implemented by mannitol. 6. The present data and those of earlier publications show that the interdependence of the fluxes of K+, Cl- and H2O in the exit membrane can be described as cotransport. The fluxes have a fixed stoichiometry of 1:1:500, the flux of one species is able to energize the flux of the two others, and the transport exhibits saturation and is specific for K+ and Cl-. 7. A molecular model based upon a mobile barrier in a membrane spanning protein gives an accurate quantitative description of the data.

Animals↗
PubMed1964

INULIN AND ALBUMIN ABSORPTION FROM THE PROXIMAL TUBULE IN NECTURUS KIDNEY.

In the kidney of the amphibian, Necturus, appreciable quantities of inulin and human serum albumin are transported from the tubular lumen of the proximal tubules into the blood. These findings suggest that inulin may not be a satisfactory indicator for measuring net water movement in the kidney of this species.

Albumins↗
PubMed1983

Reversible carbon dioxide-induced inhibition of dye coupling in Necturus gallbladder.

The cells of Necturus gallbladder epithelium are electrically coupled. This work used intracellular injection of the fluorescent dye Lucifer yellow to demonstrate that these cells are also dye coupled and that this coupling is rapidly and reversibly inhibited by high concentrations of carbon dioxide. Dye coupling is also inhibited by the calcium ionophore A23187.

Animals↗
PubMed1983

Ca2+ regulation of tight-junction permeability and structure in Necturus gallbladder.

To explore the role of Ca2+ in tight-junction permeability, the Necturus gallbladder was exposed to varying Ca2+ concentrations and to the Ca2+ ionophore A23187 added to the mucosal side (1.9 X 10(-6) to 6.8 X 10(-5) M). Electrophysiological parameters measured in an Ussing-type chamber were correlated with tight-junction morphology revealed by freeze-fracture electron microscopy. In Ca2+-free bathing media, transepithelial resistance decreases and tight-junctional ultrastructure is fragmented. In 1.8 mM Ca2+ media, A23187 induces an initial drop in transepithelial resistance, followed by an increase in transepithelial resistance to a value 20% above base line. At peak response to A23187, NaCl diffusion potentials decrease. Freeze-fracture replicas reveal that the number of junctional strands increase pari passu with junctional depth. Both physiological and morphological changes were partially reversible. The initial decrease in transepithelial resistance coincided with a persistent hyperpolarization of the mucosal cell membrane potential difference and a decrease in the mucosal-to-serosal cell membrane resistance ratio. Thus A23187 alters both the transcellular and paracellular pathway, resulting in opposing effects on transepithelial resistance.

Animals↗
PubMed1984

Chloride movement across basolateral membrane of Necturus gallbladder epithelium.

The relative Cl- and K+ sensitivity of the basolateral membrane potential of the in vitro Necturus gallbladder epithelium was determined. Tissues were punctured with two conventional glass microelectrodes to simultaneously measure the intracellular voltage (Vcs) and the voltage across the subepithelial connective tissue (Vse). Increasing the serosal K+ concentration from 2.5 to 25 mM caused a rapid monotonic depolarization of Vcs without changes of Vse. Reduction of serosal Cl- concentration (98 to 8 mM) caused a transient change of Vse. Thus the difference between Vcs and Vse more accurately reflected the basolateral membrane voltage (Vc) after Cl- concentration changes. The changes of Vc were small and biphasic in response to the decrease of serosal Cl- concentration. Perfusion of a low-ionic-strength solution in the mucosal chamber decreased the current that normally passes through the epithelium. Consistent with the notion that the basolateral voltage changes are attenuated by parallel pathways, the K+-induced depolarization increased by 80% under these conditions. The changes of Vc in response to Cl- substitutions were not different from those of tissue bathed in control solution. Thus the basolateral membrane voltage is relatively insensitive to changes of serosal Cl- concentration. I conclude that Cl- movement across the basolateral membrane is not attributable to simple electrodiffusion, and Cl- exit from these cells at this membrane must be electroneutral.

Animals↗