Thiocyanate inhibition of active chloride absortion in Aplysia intestine

This investigation was principally undertaken to examine the mechanism of active chloride absorption across the Aplysia californica intestine by using various inhibitors of ion transport. Isolated intestine, mounted between identical oxygenated sodium-free seawater solutions, maintained stable transmural potential differences (serosa negative) and short-circuit currents for several hours at 25°C. The metabolic inhibitors, 2,4-dinitrophenol and flouride, reduced both transmural potential difference and short-circuit current; however, the electrical characteristics were predominantly dependent upon glycolytic energy. The addition of thiocyanate to the mucosal solution inhibited both electrical characteristics in parallel, and this inhibition could be titrated according to the thiocyanate concentration. The short-circuit current was carried wholly by a net active chloride transfer from mucosa to serosa as determined by flux measurements. These results suggest that active chloride absorption may be mediated by a primary active transport process.     

Mechanisms of transport of Na+ and Cl- in the lizard colon

1. Ionic fluxes of sodium and chloride across lizard colon mucosa were measured and compared with the electrical characteristics of the tissue under voltage-clamped conditions. 2. In a Ringer-bicarbonate solution there was both a net sodium flux (JNanet) and a net chloride flux (JClnet) from mucosa to serosa. The net flux residual (JR) was near zero, indicating that net sodium and chloride transport is the result of an electrically neutral transport mechanism. 3. In the presence of sodium, the net chloride flux was abolished and the short-circuit current (Isc) and the electrical potential difference (PD) were unchanged. In the absence of chloride the net sodium flux was abolished and the short-circuit current and electrical potential difference were not modified. 4. From an analysis of the effects of the inhibitors, furosemide, amiloride and disulfonic stilbene (DIDS), a plausible model was developed to explain the characteristics of sodium and chloride absorption.     

Sodium transport by isolated bullfrog small intestine. Effect of prostaglandin E1.

Addition of 446 micron prostaglandin E1 (PGE1) to the serosal medium of isolated short-circuited bullfrog small intestine elicited small increases transmural potential difference and short-circuit current while addition of PGE1 to the mucosal medium caused no change in the electrical parameters. Addition of 100 micron indomethacin to the mucosal medium inhibited both potential difference and short-circuit current with a resultant increase in steady-state tissue resistance. In the presence of mucosal 100 micron indomethacin, serosal 60 micron PGE1 markedly stimulated transmural potential difference and short-circuit current with a resultant decrease in steady-state tissue resistance. Serosal arachidonic acid (330 micron) stimulated transmural potential difference and short-circuit current and this effect was abolished by the addition of 100 micron indomethacin to the mucosal medium. Serosal 60 micron PGE1 only stimulated the M (mucosa) leads to S (serosa) unidirectional flux of sodium. These results strongly suggest that the PGE1 action is mediated either via a series of metabolic reactions which possibly increase the permeability of the mucosal membrane to sodium or via direct stimulation of rheogenic sodium pump activity.     

Thiocyanate transport across fish intestine (Pleuronectes platessa)

Summary When bathed on both sides with identical chloride-containing salines thein vitro preparation of the plaice intestine maintains a negative (serosa to mucosa) short-circuit current of 107±11 A/cm², a transepithelial potential difference of 5.5±0.6 mV (serosa negative), and a mean mucosal membrane potential of –45.4±0.6 mV. Under these conditions the intracellular chloride activity is 32mm.If chloride in the bathing media is partially, or completely substituted by thiocyanate the measured electrical parameters do not change but transepithelial flux determinations show a reduction in chloride fluxes and the presence of a significant thiocyanate flux. The addition of piretanide (10^–4 m) reduced the short-circuit current and the mucosa-to-serosa fluxes of chloride and thiocyanate; this inhibition is similar to the effect of piretanide on chloride transport in this tissue.The results indicate that thiocyanate is transported in this tissue via the piretanide-sensitive chloride pathway and are compared with the effects of thiocyanate on other tissues reported in the literature.     

Sodium transport by isolated bullfrog small intestine. Effect of Prostaglandin E1

Addition of 446 μM prostaglandin E₁ (PGE₁) to the serosal medium of isolated short-circuited bullfrog small intestine elicited small increases in transmural potential difference and short-circuit current while addition of PGE₁ to the mucosal medium caused no change in the electrical parameters. Addition of 100 μM indomethacin to the mucosal medium inhibited both potential difference and short-circuit current with a resultant increase in steady-state tissue resistance. In the presence of mucosal 100 μM indomethacin, serosal 60 μM PGE₁ markedly stimulated transmural potential difference and short-circuit current with a resultant decrease in steady-state tissue resistance. Serosal arachidonic acid (330μM) stimulated transmural potential difference and short-circuit current and this effect was abolished by the addition of 100 μM indomethacin to the mucosal medium. Serosal 60 μM PGE₁ only stimulated the M (mucosa) → S (serosa) unidirectional flux of sodium. These results strongly suggest that the PGE₁ action is mediated either via a series of metabolic reactions which possibly increase the permeability of the mucosal membrane to sodium or via direct stimulation of rheogenic sodium pump activity.     

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.     

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.     

Ion Transport in Isolated Rabbit Ileum : I. Short-circuit current and Na fluxes

The transmural potential difference, short-circuit current, and Na fluxes have been investigated in an in vitro preparation of isolated rabbit ileum. When the tissue is perfused with a physiological buffer, the serosal surface is electrically positive with respect to the mucosal surface and the initial potential difference in the presence of glucose averages 9 mv. Unidirectional and net Na fluxes have been determined under a variety of conditions, and in each instance, most if not all of the simultaneously measured short-circuit current could be attributed to the active transport of Na from mucosa to serosa. Active Na transport is dependent upon the presence of intact aerobic metabolic pathways and is inhibited by low concentrations of ouabain in the serosal medium. A method is described for determining whether a unidirectional ionic flux is the result of passive diffusion alone, in the presence of active transport of that ion in the opposite direction. Using this method we have demonstrated that the serosa-to-mucosa flux of Na may be attributed to passive diffusion with no evidence for the presence of carrier-mediated exchange diffusion or the influence of solvent-drag.     

The conventional short-circuiting technique under-short-circuits most epithelia

Summary The relationships among ion current, membrane potential difference, and resistance of an epithelium are studied. The short-circuit technique introduced by Ussing and Zerahn does not completely short circuit the epithelium if the series resistance parallel to the cell layer between the voltage electrodes is not properly compensated. The residual potential difference across the epithelial cell layer in the short-circuit state is proportional to both the measured short-circuit current and the resistance of the diffusion barriers not compensated. In the conventionally short-circuited small intestinal mucosa the villus and crypt areas are hypo-polarized to different degrees rather than simultaneously hyper- and hypo-polarized. Short-circuiting the whole tissue reduces but does not abolish the passive net ion movement across the tissue. Measurements of the electrical properties of the whole and denuded rat distal small intestine in HCO₃-Ringer solution containing 10mm glucose reveal that the measured short-circuit current has under-estimated approximately 33% of the true short-circuit current and that the passive net Na flux from serosa to mucosa and Cl flux from mucosa to serosa are not negligible in the short-circuit state.     

Transmural potential differences and short-circuit current intensity in the posterior intestine of Blennius parvicornis

Simultaneous measurements of the transmural potential difference (PD) and the short-circuit current intensity (Isc) in the posterior intestine of the fish Blennius parvicornis were made in normal Ringer and in solutions of different ionic composition. The ouabain effects on these two parameters were also tested in normal Ringer solution. The absence of K+ from the Ringer solution on both the mucosal and serosal sides has no apparent effect on the PD and Isc within the first 15 min, but it makes them null after 30 min. When Na+ is substituted in both compartments, using Tris as substitute, a serosal negativity increase is initially observed, but it gradually decreases to zero after 30 min of experimentation. Similarly the PD and Isc drop to zero in the absence of Cl- (sulfate as substitute). Ouabain diminishes the serosa negative potential difference to zero after 30 min presenting a lineal relation to the Isc. A likely transport mechanism for Cl- dependent on the Na+ - K+ pump, is discussed.     

Effects of acetylcholine, serotonin and noradrenalin on ion transport in the middle and posterior part of Anguilla anguilla intestine

The effects of acetylcholine analogues, serotonin and catecholamines on ion transport were studied in both the middle and the posterior intestine of Anguilla anguilla, mounted in an Ussing chamber, with the aim of understanding whether these regulators affect different mechanisms in the different tracts. In the middle intestine, acetylcholine analogues and serotonin decreased the serosa negative transepithelial potential and short-circuit current without altering the transepithelial resistance; catecholamines reversed the inhibitory effects of both regulators. Similar opposite effects were produced by both the acetylcholine analogues and noradrenalin in the posterior intestine. However, the lowering of the short-circuit current elicited by serotonin was paralleled by the decrease of the transepithelial resistance, whilst noradrenalin had the opposite effects on both parameters. These observations, together with the results of experiments performed by measuring the dilution potential in the control condition and in the presence of either serotonin or serotonin plus noradrenalin, led us to hypothesize that serotonin increases the anion conductance of the paracellular pathway while noradrenalin decreases it. In both the middle and posterior intestine, these regulators probably affect transcellular transport mechanisms by acting on the Na-K-Cl transporter; both acetycholine and serotonin decrease its activity while noradrenalin increases it. Accepted: 22 May 1999     

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.     

Alterations in electrophysiology of isolated amphibian small intestine produced by removing the muscle layers

Isolated segments of Amphiuma small intestine bathed in chloride or sulfate buffer generate a greater short-circuit current and a larger change in current in response to galactose when the serosal muscle layers are stripped from the mucosa. Intact (unstripped) segments are not apparently anoxic since stripped segments exposed to serosal N₂ for 3 h display normal short-circuit currents but a reduced potential response to galactose, while the presence of muscle layers tends to reduce the short-circuit current but does not alter the potential response to galactose. Bullfrog small intestine also generates greater short-circuit current following removal of the muscle layers. The enhancing effect of stripping appears to be related to removal of a resistance to ion flow across the tissue.     

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.     

Effects of caffeine on transport, metabolism and ultrastructure of isolated rat colon

The effect of caffeine on the transport, metabolism and ultrastructure of the colon were determined. Segments of proximal colon were excised from the anesthetized rat and prepared for radioisotopic tracing of ion transport in the flux chambers or oxidative metabolism in an incubator. Other segments were fixed before or after caffeine administration for electron microscopy. The isolated rat colon actively transported both Na+ and Cl- in the absorptive direction, mucosa to serosa. Serosal addition of 10 mmol/l caffeine abolished the smaller Na+ transport but did not significantly affect the larger Cl- transport. The electrical potential difference and the short-circuit current rose accordingly. Although the oxidation of glucose was inhibited by 35%, caffeine had no significant effect on the oxidation of the fatty acid, butyric acid. Comparable metabolic responses were obtained using the isolated terminal ileum of the rat. Neither the height nor the density of the microvilli in the proximal colon were affected significantly by caffeine. It may be concluded that caffeine, unlike theophylline, effectively preserves the normal absorptive condition of the colon. Thus, caffeine may have actions other than inhibition of phosphodiesterase in the distal intestine.     

Chloride-dependent transmural potential in the rectal wall of Schistocerca gregaria

Rectum transmural potential (PD) and short-circuit current (I_sc) of the desert locust, Schistocerca gregaria, have been studied in vitro, with everted rectal wall preparations in solutions of different ionic composition. Initially, a PD of about 35 mV (lumen positive) and a I_sc of about 300 μA cm^?2 were recorded. Omission of sodium or potassium (Tris as substitute), from the luminal side or from both sides led to an increase of 4 to 6 mV in PD (lumen more positive) together with an increase in I_sc. In the absence of chloride alone (sulphate as substitute) the PD quickly dropped to nearly zero. In each case the control values were recovered on replacing the corresponding ions. Neither the PD nor the I_sc changed when substitutions affected only the haemocoelic solution. These findings corroborate the assumption that active transport of chloride ions from lumen to haemolymph is the major factor for transmural PD and account for the short-circuit current in the rectal wall of desert locust. A working scheme is given to explain the influence of sodium, potassium, and chloride ions on the PD.     

Actions of carbaryl on the ionic transport across the isolated skin of Rana esculenta

1. Carbaryl, a carbamate used as a pesticide, increases the short-circuit current (SCC) across the isolated frog skin in a dose-dependent manner. 2. This effect is due to the stimulation of sodium absorption and chloride secretion. 3. Carbaryl action on short-circuit current is unrelated to its inhibitory power on cholinesterase; this statement is supported by two experimental results: (a) carbaryl is equally active on both sides of the skin, (b) atropine pretreatment does not inhibit the carbaryl action on SCC.     

Imidazoline Receptor Contributes to Ion and Water Transport across the Intestine of the Eel Acclimated to Sea Water

Guanabenz, an I2-imidazoline-related compound with high affinity for intestinal membrane of the eel (), enhanced the transepithelial potential difference (PD) and short-circuit current (Isc) from serosa to mucosa after pretreatment with isobutylmethylxanthine (IBMX), serotonin (5-HT) and methacholine (MCh). The mucosal effect of guanabenz was not mimicked by adrenaline, indicating that the mucosal guanabenz binding site is not adrenoceptors. The mucosal guanabenz enhanced the Isc in a concentration-dependent manner. Similar enhancement in the Isc was also obtained after addition of other imidazoline derivatives such as ST93, clonidine, ST91, naphazoline and UK14,304 into the mucosal fluid. On the other hand, the effect of guanabenz was completely blocked by mucosal RX821002 or efaroxan, another imidazoline derivatives. Since some imidazoline derivatives act as agonists and others as antagonist, there must exist imidazoline receptor on the mucosal side of the eel intestine. Accompanied by an increase in the PD, NaCl and water absorption across the intestine was also enhanced by mucosal guanabenz. To search for endogenous ligands for the imidazoline receptor, luminal fluid in the intestine of the seawater eels was collected. However, most luminal fluid was ineffective. Only one among 10 samples showed guanabenz-like activity, suggesting that the endogenous ligands is secreted into the lumen under restricted condition alone.     

Tonic activity of submucosal neurons influences basal ion transport

The influence of tonically active submucosal neurons on basal ion transport was studied using sheets of guinea pig ileum set up in flux chambers. Tetrodotoxin evoked an immediate and sustained decrease in short-circuit current that was sustained for 60 minutes compared with control tissues in which basal currents gradually decreased over time. Time-dependent changes in basal short-circuit currents in tissues treated with atropine were not significantly different from control tissues. The decrease in short-circuit current after tetrodotoxin resulted from a greater increase in net chloride absorption than sodium absorption. Changes in net sodium and chloride transport were due to an increase in the mucosal-to-serosal fluxes of these ions. The results suggest that tonic activity of submucosal neurons limits the absorptive capacity of the guinea pig ileum.