Ionic transfer across the isolated frog large intestine

The unidirectional fluxes of sodium, chloride, and of the bicarbonate and CO(2) pair were determined across the isolated large intestine of the bullfrog, Rana catesbiana. The isolated large intestine of the frog is characterized by a mean transmembrane potential of 45 mv., serosal surface positive with respect to mucosal. The unidirectional sodium flux from mucosal to serosal surface was found to be equal to the short-circuit current, thus the net flux was less than the simultaneous short-circuit current. This discrepancy between active sodium transport and short-circuit current can be attributed to the active transport of cation in the same direction as sodium and/or the active transport of anion in the opposite direction. The unidirectional fluxes of chloride and the bicarbonate and CO(2) pair revealed no evidence for active transport of either anion. A quantitative study of chloride fluxes at 45 mv. revealed a flux ratio of 1.8 which is considerably less than a ratio of 6 expected for free passive diffusion. It was concluded that a considerable proportion of the isotopic transfer of chloride could be attributed to "exchange diffusion." Study of the electrical properties of the isolated frog colon reveals that it can be treated as a simple D. C. resistance over the range of -20 to +95 mv.     

Ion transport by rabbit colon. I. Active and passive components.

Descending rabbit colon, stripped of muscularis externa, absorbs Na and Cl under short-circuit conditions and exhibits a residual ion flux, consistent with HCO3 secretion, whose magnitude is approximately equal to the rate of active Cl absorption. Net K transport was not observed under short-circuit conditions. The results of ion replacement studies and of treatment with ouabain or amiloride suggest that the short-circuit current ISC is determined solely by the rate of active Na transport and that the net movements of Cl and HCO3 are mediated by a Na-independent, electrically-neutral, anion exchange process. Cyclic AMP stimulates an electrogenic Cl secretion, abolishes HCO3 secretion but does not affect the rate of Na absorption under short-circuit conditions. Studies of the effect of transepithelial potential difference on the serosa-to-mucosa fluxes Jism of Na, K and Cl suggest that JNasm,JIsm and one-third of JCl-sm may be attributed to ionic diffusion. The permeabilities of the passive conductance pathway(s) are such that Pk:PNa:PCl= 1.0:0.07:0.11. Electrolyte transport by in vitro rabbit colon closely resembles that reported from in vivo studies of mammalian colon and thus may serve as a useful model for the further study of colonic ion transport mechanisms.     

Sodium-Sulfate Symport by Aplysia californica Gut

Sulfate transport across plasma membranes has been described in a wide variety of organisms and cell types including gastrointestinal epithelia. Sulfate transport can be coupled to proton, sodium symport or antiport processes involving chloride or bicarbonate. It had previously been observed in Aplysia gut that sulfate was actively absorbed. To understand the mechanism for this transport, short-circuited Aplysia californica gut was used. Bidirectional transepithelial fluxes of both sodium and sulfate were measured to see whether there was interaction between the fluxes. The net mucosal-to-serosal flux of Na(+) was enhanced by the presence of sulfate and it was abolished by the presence of serosal ouabain. Similarly, the net mucosal-to-serosal flux of sulfate was dependent upon the presence of Na(+) and was abolished by the presence of serosal ouabain. Theophylline, DIDS and bumetanide, added to either side, had no effect on transepithelial potential difference or short-circuit current in the Aplysia gut bathed in a Na2SO4 seawater medium. However, mucosal thiosulfate inhibited the net mucosal-to-serosal fluxes of both sulfate and Na(+) and the thiosulfate-sensitive Na(+) flux to that of sulfate was 2:1. These results suggest the presence of a Na-SO4 symporter in the mucosal membrane of the Aplysia californica foregut absorptive cell.     

The Electrical Characteristics of Active Sodium Transport in the Toad Bladder

The mechanism responsible for active sodium transport in the urinary bladder of the toad appears to be located at the serosal boundary of the epithelial cell layer of the bladder. Studies of the potential step observed at the serosal boundary in the open-circuited state were undertaken in an attempt to define the factors responsible for its production. Glass micropipettes were used to measure the serosal potential step in bladders exposed on the serosal side to solutions of high potassium or of high potassium and low chloride concentration. Observed potentials exceed the maximum values which would have been expected if the serosal potential step were a potassium or chloride diffusion potential. Measurements of net cation flux exclude the possibility of a diffusion potential at this border due to the passive movement of any anionic species. The observed independence of transbladder potential and short-circuit current from the pH of the serosal medium over a wide range of pH makes it unlikely that the observed serosal potential step is a hydrogen ion diffusion potential. We conclude that the active sodium transport mechanism in toad bladder is "electrogenic."     

Sodium-phosphate symport by Aplysia californica gut

Phosphate transport across plasma membranes has been described in a wide variety of organisms and cell types including gastrointestinal epithelia. Phosphate transport across apical membranes of vertebrate gastrointestinal epithelia requires sodium; whereas, its transport across the basolateral membrane requires antiport processes involving primarily chloride or bicarbonate. To decipher the phosphate transport mechanism in the foregut apical membrane of the mollusc, Aplysia californica, in vitro short-circuited Aplysia californica gut was used. Bidirectional transepithelial fluxes of both sodium and phosphate were measured to see whether there was interaction between the fluxes. The net mucosal-to-serosal flux of Na+ was enhanced by the presence of phosphate and it was abolished by the presence of serosal ouabain. Similarly, the net mucosal-to-serosal flux of phosphate was dependent upon the presence of Na+ and was abolished by the presence of serosal ouabain. Theophylline, DIDS and bumetande, added to either side, had no effect on transepithelial difference or short-circuit current in the Aplysia gut bathed in a Na2HPO4 seawater medium. However, mucosal arsenate inhibited the net mucosal-to-serosal fluxes of both phosphate and Na+ and the arsenate-sensitive Na+ flux to that of phosphate was 2:1. These results suggest the presence of a Na-PO4 symporter in the mucosal membrane of the Aplysia californica foregut absorptive cell.     

Ion Transport in Isolated Rabbit Ileum : II. The interaction between active sodium and active sugar transport

The addition of actively transported sugars to the solution bathing the mucosal surface of an in vitro preparation of distal rabbit ileum results in a rapid increase in the transmural potential difference, the short-circuit current, and the rate of active Na transport from mucosa to serosa. These effects are dependent upon the active transport of the sugar per se and are independent of the metabolic fate of the transported sugar. Furthermore, they are inhibited both by low concentrations of phlorizin in the mucosal solution and by low concentrations of ouabain in the serosal solution. The increase in the short-circuit current, ΔI_sc, requires the presence of Na in the perfusion medium and its magnitude is a linear function of the Na concentration. On the other hand, ΔI_sc is a saturable function of the mucosal sugar concentration which is consistent with Michaelis-Menten kinetics suggesting that the increase in active Na transport is stoichiometrically related to the rate of active sugar transport. An interpretation of these findings in terms of a hypothetical model for intestinal Na and sugar transport is presented.     

Intestinal glucose and galactose transport in the cultured gilthead bream (Sparus aurata)

1. Electrical parameters and transepithelial glucose and galactose transport were determined in vitro across anterior and posterior intestine of the culture fish Sparus aurata. 2. Electrical potential difference (PD) and short-circuit current (Isc) were serosa-positive in anterior intestine, while they were serosa-negative or near zero in posterior intestine. 3. Tissue conductance (Gt) was higher in posterior than in anterior intestine. In both parts it was decreased when the Na ion was omitted in mucosal and serosal reservoirs. 4. Addition of glucose or galactose to the mucosal side of intestine caused an increase in PD and Isc in posterior intestine but did not significantly change PD and Isc in anterior intestine. 5. Isotopic flux of glucose and galactose measurements in short-circuit conditions showed a net active glucose and galactose absorption in posterior intestine, while in anterior intestine active transport of glucose or galactose was not observed. 6. The net transport of glucose and galactose in posterior intestine was decreased to zero in the absence of Na in mucosal and serosal reservoirs or in the presence of ouabain (1 mM) in serosal solution.     

Active sodium transport by the isolated toad bladder

Studies were made of the active ion transport by the isolated urinary bladder of the European toad, Bufo bufo, and the large American toad, Bufo marinus. The urinary bladder of the toad is a thin membrane consisting of a single layer of mucosal cells supported on a small amount of connective tissue. The bladder exhibits a characteristic transmembrane potential with the serosal surface electrically positive to the mucosal surface. Active sodium transport was demonstrated by the isolated bladder under both aerobic and anaerobic conditions. Aerobically the mean net sodium flux across the bladder wall measured with radioactive isotopes, Na²⁴ and Na²², just equalled the simultaneous short-circuit current in 42 periods each of 1 hour's duration. The electrical phenomenon exhibited by the isolated membrane was thus quantitatively accounted for solely by active transport of sodium. Anaerobically the mean net sodium flux was found to be slightly less than the short-circuit current in 21 periods of observation. The cause of this discrepancy is not known. The short-circuit current of the isolated toad bladder was regularly stimulated with pure oxytocin and vasopressin when applied to the serosal surface under aerobic and anaerobic conditions. Adrenaline failed to stimulate the short-circuit current of the toad bladder.     

Components of Sodium and Chloride Flux Across Toad Bladder

The effect of transepithelial potential difference (ψ) on Na and Cl flux across toad bladder was assessed by measuring isotopic flux between identical media at various values of ψ. The contribution of edge damage to ionic permeability was eliminated, resulting in relatively high spontaneous ψ (-97 ±4 mv) and low electrical conductance g. Bidirectional Na fluxes were measured simultaneously. Unidirectional Cl fluxes were measured in paired hemibladders at ψ = 0 mv or -97 mv. Net Na flux J_Na, at ψ = 0 mv, was slightly less than short-circuit current (SCC). At ψ = -97 mv, J_Na averaged 17% of SCC, and was sometimes zero. ΔJ_Na/Δψ (= g₊) averaged 60% of g between -97 mv and +75 mv; at -150 mv, g₊ fell, indicating rectification. Analysis of unidirectional Na fluxes indicates low passive conductance (1.5 μmho/mg wet weight), a bidirectional, electrically neutral flux of approximately 0.13 μa/mg, and relatively large conductance of the active transport path at ψ ≥ -97 mv. The absence of appreciable transstimulation of serosal (S)-to-mucosal (M) Na flux (in response to increasing mucosal Na concentration) indicates that the electrically neutral flux is not exchange diffusion in the usual sense. Analysis of Cl fluxes indicates similar values for passive conductance and neutral flux, suggesting linked neutral flux of Na and Cl. Either the electromotive force of the Na pump E, its conductance g_a, or both are strong functions of ψ. The product of these two quantities, Eg_a, is a measure of the “transport capacity” at any given value of ψ, independent of the direct effect of ψ on J_Na through the pump path. Eg_a varies with ψ. Hence estimation of the net Na flux or current at any one value of ψ, including ψ = 0, fails to reveal the maximal transport capacity of the pump, its resting electromotive force (when J_Na = 0 through the pump), or the dependence of transport capacity on potential.     

Na and Cl transport across the isolated turtle colon: Parallel pathways for transmural ion movement

Summary Transmural fluxes of³H-mannitol and²²Na or³⁶Cl were measured simultaneously in portions of isolated turtle colon stripped of serosal musculature. The relationships between mannitol flux and the flux of Na or Cl are characteristic of simple diffusion and suggest that transmural mannitol flow is largely confined to a paracellular pathway where Na, Cl and mannitol move much as in free solution. The contribution of edge damage to the transmural mannitol flow appears to be minimal. Mucosal hyperosmolarity causes blisters in epithelial tight junctions and increases the diffusional permeability to Na and mannitol, suggesting that the rate-limiting barrier in the shunt path is the tight junction. If the total mucosa to serosa flux of Na is corrected for the portion traversing the shunt pathway it is apparent that changes in the short-circuit current are completely accounted for by the mucosa to serosal movement of Na through a cellular path. In addition, the serosa to mucosa flux of Na appears to be restricted to the shunt. These observations suggest that there is no appreciable backflux of Na through the active, cellular path. In the presence of 10^–4 m amiloride the short-circuit current is markedly reduced and the mucosa to serosa Na flux is restricted to the shunt, so that the net Na flux is abolished. The small amiloride-insensitive short-circuit current is consistent with HCO₃ secretion. Mucosa to serosa and serosa to mucosa fluxes of Cl appear to be largely restricted to the paracellular shunt path and there is no evidence for any net flow of Cl under short-circuit conditions. The total tissue conductance can be described as the sum of three components: a shunt conductance which is linearly related to the transmural mannitol flow, an active conductance which is linearly related to the short-circuit current and a small residual conductance. The shunt conductance is attributable to the diffusive movements of Na and Cl through the paracellular path. Variations in the active Na transport from tissue to tissue are largely attributable to variations in the apparent conductance of the active Na transport path. The driving force for active Na transport can be described as an apparent emf of approximately 130 mV. These results suggest that transmural mannitol flux provides a quantitative estimate of the ion permeability and electrical conductance of a paracellular shunt path across the isolated turtle colon and thereby facilitates the study of the transport characteristics and electrical properties of cellular paths for transepithelial solute movement.     

Na, Cl, and Water Transport by Rat Colon

Segments of the colon of anesthetized rats have been perfused in vivo with isotonic NaCl solutions and isotonic mixtures of NaCl and mannitol. Unidirectional and net fluxes of Na and Cl and the net fluxes of water and mannitol have been measured. Net water transport was found to depend directly on the rate of net Na transport. There was no water absorption from these isotonic solutions in the absence of net solute transport, indicating that water transport in the colon is entirely a passive process. At all NaCl concentrations studied, the lumen was found to be electrically negative to the surface of the colon by 5 to 15 mv. Na fluxes both into and out of the lumen were linear functions of NaCl concentration in the lumen. Net Na absorption from lumen to plasma has been observed to take place against an electrochemical potential gradient indicating that Na is actively transported. This active Na transport has been interpreted in terms of a carrier model system. Cl transport has been found to be due almost entirely to passive diffusion.     

Na transport across frog skin at low external Na concentrations

Isolated frog skin was bathed with a dilute solution containing 1 mm NaCl on the outside and with normal Ringer’s solution on the inner surface. Net Na flux was determined by simultaneous measurement of unidirectional fluxes with Na²² and Na²⁴ and intracellular electrical potentials were examined with microelectrodes. There was a net inward transport of Na under both open-circuit and short-circuit conditions. The short-circuit current was approximately 15% greater than the net Na flux; the discrepancy could be accounted for by a small outward flux of Cl. The electrical potential profile did not differ greatly from that observed in skins bathed on the outside with normal Ringer’s solution. Under open-circuit conditions, there were usually several potential steps and under short-circuit conditions the cells were negative relative to the bathing solutions. Estimates of epithelial Na concentrations utilizing radioactive Na suggested that if all epithelial Na were in a single compartment, an active entry step would be necessary to allow a net inward Na transport. The results could also be explained by a series arrangement of Na compartments without necessarily postulating an active Na entry. The behavior of the potential profile suggested that this latter alternative was more likely.     

Electrical characteristics of isolated Aplysia californica intestine

• 1.1. Transmural potential difference and short-circuit current of intestinal sheets of Aplysia califonica were stable up to 5 hr.
• 2.2. Transmural potential difference was serosa negative relative to the mucosa and the short-circuit current was consistent with a net active anion transport from mucosa to serosa.
• 3.3. Transmural potential difference and short-circuit current were dependent upon the presence of sodium and chloride in the bathing medium.
• 4.4. Transmural potential difference and short-circuit current were predominantly dependent upon aerobic metabolism, however a finite residual electrical component was dependent upon glycolytic energy.
• 5.5. The major portion of the short-circuit current is carried by a net active chloride transfer from the mucosal to serosal compartments while the minor portion is carried by a net active sodium transfer in the same direction.

     

Electrogenic Na(+)-dependent L-alanine transport in the lizard duodenum. Involvement of systems A and ASC

L-Alanine transport across the isolated duodenal mucosa of the lizard Gallotia galloti has been studied in Ussing chambers under short-circuit conditions. Net L-alanine fluxes, transepithelial potential difference (PD), and short-circuit current (Isc) showed concentration-dependent relationships. Na(+)-dependent L-alanine transport was substantially inhibited by the analog alpha-methyl aminoisobutyric acid (MeAIB). Likewise, MeAIB fluxes were completely inhibited by L-alanine, indicating the presence of system A for neutral amino acid transport. System A transport activity was electrogenic and exhibited hyperbolic relationships for net MeAIB fluxes, PD, and Isc, which displayed similar apparent K(m) values. Na(+)-dependent L-alanine transport, but not MeAIB transport, was partially inhibited by L-serine and L-cysteine, indicating the participation of system ASC. This transport activity represents the major pathway for L-alanine absorption and seemed to operate in an electroneutral mode with a negligible contribution to the L-alanine-induced electrogenicity. It is concluded from the present study that the active Na(+)-dependent L-alanine transport across the isolated duodenal mucosa of Gallotia galloti results from the independent activity of systems A and ASC for neutral amino acid transport.     

Effect of furosemide on unidirectional fluxes of sodium and chloride across the skin of the frog, Rana pipiens.

1. The diuretic furosemide, when added to the outside solution at a concentration of 5-10-4 M, increases the electrical potential difference (PD) across the isolated frog skin, but the short-circuit current (Isc) is unchanged. Lower concentrations had no significant effect on these electrical parameters. 2. When SO42- or NO3- are substituted for Cl- in the Ringer's solution furosemide has no effect on the PD or Isc. 3. Simultaneous unidirectional fluxes of Na+ and Cl- show that furosemide (5-10-4 M outside) reduces both the influx and outflux of Cl-, while the Na+ fluxes are not altered. 4. Furosemide (5-10-4 M) on the corium side of the frog skin had no significant effect on either PD, Isc or undirectional fluxes of Cl-. 5. It is suggested that furosemide reduces passive Cl- transfer, possibly by interacting with the Cl-/Cl- exchange diffusion mechanism which has been observed in this tissue. These observations further suggest that perhaps the diuretic action of furosemide may be mediated by such an effect on passive Cl- permeability which is linked to the active Cl- transport mechanism in the renal tubule.     

Ion Transport in Isolated Rabbit Ileum : III. Chloride fluxes

Unidirectional Cl fluxes across in vitro segments of rabbit ileum have been determined both in the absence and in the presence of an electrochemical potential gradient. The results indicate that Cl transport in this preparation can be attributed to purely passive forces uninfluenced by solvent drag or exchange diffusion. Furthermore, on the basis of this and previous studies, it has been demonstrated that the sum of the partial ionic conductances of Na and Cl accounts for at least 90 per cent of the total tissue conductance.     

Effect of amphotericin B and gestational age on sodium transport across the rat visceral yolk sac placenta in vitro

The transport properties of the rat visceral yolk sac placenta from Days 14.5 to 18.5 of gestation were studied in vitro. All tissues had a positive potential difference, fetal side relative to maternal side, and showed net Na transport towards the fetus. Basal short-circuit current and net Na flux increased rapidly with gestational age over the period studied. Amphotericin B applied to the maternal surface of the yolk sac stimulated current and net Na flux, indicating that the apical membrane Na permeability limited transport and revealing a reserve capacity for transport. Contrary to their basal values, current and Na flux following treatment with amphotericin were independent of gestational age.     

Analysis of the Components of Ionic Flux across a Membrane

The unidirectional flux of an ionic species may occur because of several mechanisms such as active transport, passive diffusion, exchange diffusion, etc. The contribution of such mechanisms to the total unidirectional flux across a membrane cannot be determined by only measuring that flux. It is shown that if the pertinent experimental data (the opposite unidirectional fluxes and the composite phenomenological resistance coefficient of the ionic species for a given electrochemical potential difference) obey a certain inequality, then the parameters of a model consisting of parallel, independent, active transport, and passive processes may be determined. Although the existence of "additional" processes including exchange diffusion, single-file pore diffusion, isotope interaction, etc. is not disproved, their existence is unnecessary if the inequality is satisfied. Two types of violations of the inequality may occur: (a) if the upper limit is disobeyed the presence of another substance contributing to the measured resistance and/or a constant affinity of the active transport process may be indicated; (b) if the lower limit is disobeyed it is necessary to postulate the existence of an additional process. For the latter type of violation, exchange diffusion is chosen as an example. Methods are given for determining the contribution of exchange diffusion, active transport, and passive diffusion to the unidirectional flux for some special cases.     

Transport of sodium and potassium in intestinal epithelial cells

Transport properties of rabbit small intestinal mucosa were investigated $\text{in vitro}$ to characterize the process by which epithelial cells maintain normal Na and K gradients across the cell membrane. Active transport of Na from the cell proceeded at a faster rate in the presence of K; and active transport of K into the cell was stimulated by the presence of Na. Following preincubation at 0°C to reduce tissue K content, a greater transmural electrical potential difference (PD) and short-circuit current (I_sc) developed as the temperature was raised to 37°C if K was present in the bathing solution. The PD and I_sc, which generally reflect the rate of active Na transport in ileum under control conditions, increased immediately upon raising the K concentration in the serosal solution from 0 to 10 nM.The results present the first direct indication in mammalian intestine of an interdependence of the Na and K active transport processes which regulate the intracellular content of these cations.     

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").