How to overcome osmotic stress? Marine crabs conquer freshwater. New insights from modern electrophysiology

In the present article we review our findings on split lamella preparations of crab gills mounted in modified Ussing-chambers with respect to mechanistic and ecophysiological aspects. The leaky gill epithelium of shore crabs adapted to brackish water absorbs Na⁺ and Cl^? in a coupled mode, and shows similarities to other salt-absorbing epithelia exposed to moderately diluted media. The results so far obtained for NaCl uptake across the gills of the shore crab are compatible with a transport model where two cell types operate in parallel, one displaying cotransport-like NaCl absorption, similar to that in the thick ascending limb of Henle's loop of the mammalian mephron, and the other one with characteristics of amiloride-sensitive, channel-mediated Na⁺ uptake by frog skin. Although there is no clear evidence for the apical mechanisms in this model, it may serve as a good basis for more detailed studies in the future. The moderately tight gill epithelium of freshwater adapted Chinese crabs absorbs Na⁺ and Cl^? independently from each other, and shows similarities to other salt-absorbing epithelia exposed to freshwater. The characteristics of a positive, Na⁺-dependent short-circuit current with externally Cl^?-free saline indicate that active Na⁺ uptake proceeds in a frog-skin-like mode via apical Na⁺-channels and the basolateral Na⁺/K⁺-pump. The nature of a negative short-circuit current with external Cl^?-saline indicates that active and Na⁺-independent Cl^? uptake is driven by an apical V-type H⁺-pump and proceeds via apical Cl^?/ HCO₃ ^?-exchange and basolateral Cl^?-channels.     

Effects of ouabain on frog gastric mucosa in vitro

The effect of the addition of ouabain to the nutrient solution was determined on resistance, potential difference (p.d.) and H⁺ secretion rate. In NaCl media, 10^?3 M ouabain decreased significantly the p.d. from 25.6 mV to 16.1 mV in 30 min and to 11.0 mV in 60 min. NO significant changes occurred in resistance and H⁺ secretion rate. In Na₂SO₄ (Cl^?-free) media, ouabain produced a biphasic effect on p.d. The p.d. changed from ?28.0 mV (nutrient-negative) to a nadir of ?37.4 mV in 7 min and then increased to ?16.4 mV in 60 min. At the nadir there was no significant change in resistance or H⁺ secretion rate but at 60 min, unlike Cl^? media, resistance increased by 36% and H⁺ secretion rate decreased by 43%. To decide whether the ouabain-caused decrease in H⁺ rate in Na₂SO₄ media was due to an effect on the H⁺ pump or on resistance of the return pathways, the voltage was clamped at 0 and 40 mV. Clamping the voltage showed that in the case of a marked decrease in the H⁺ secretion rate, the H⁺ transport mechanism itself was inhibited (and not the parallel pathway). The decrease in p.d. due to ouabain in Cl^? and SO₄^2? media indicates that the (Na⁺ + K⁺)-ATPase mechanism may be electrogenic.     

The inhibitory effect of strophanthidin on secretion by the isolated gastric mucosa

The unidirectional fluxes of Na⁺ and Cl^- were measured across the isolated gastric mucosa of the bullfrog (R. catesbiana). The addition of strophanthidin, a cardiac aglycone, resulted in marked reductions of the spontaneous potential and short-circuit current. Associated with these changes, the isolated gastric mucosa ceased secreting chloride and hydrogen ion. Although the active component of chloride transfer was inhibited, the exchange diffusion component seemed to increase. No significant changes in membrane conductance or sodium flux were noted. Possible mechanisms of strophanthidin inhibition were discussed in view of its effect on chloride transport across the gastric mucosa and on sodium and potassium transfer in other tissues. It was concluded that the cardiac glycosides may not be specific inhibitors of sodium and potassium transport. This non-specific inhibition suggests that active chloride transport is affected by strophanthidin directly and/or anion secretion is dependent upon normal functioning of cation transport systems in the tissue.     

Some features of hydrogen (ion) secretion by the frog skin

We have studied the movements of H⁺ from the in vitro frog skin into the outside solution because it has been suggested that the movement of sodium from the outside solution into the skin may result from the forced exchange of Na⁺ by H⁺.Our main observations can be summarized as follows: (a) Hydrogen moves from the skin into the outside solution at a rate of 0.04 μequiv · cm^?2 · h^?1 while Na⁺ influx had a value of 0.49 μequiv · cm^?2 · h^?1. (b) The rate of H⁺ secretion is not significantly affected by substituting the Na⁺ in the outside solution by K⁺ nor by inhibiting Na⁺ influx with amiloride (5 · 10^?5 M). (c) Acetazolamide (5 · 10^?3 M) blocked H⁺ secretion without altering the potential difference across the skin. (d) The rate of H⁺ production is not underestimated because it may have been neutralized by HCO₃^? secreted into the outside solution in exchange for Cl^?. Substituting all the Cl^? by SO₄^2? in the outside solutions does not result in an increase in the rate of H⁺ production. (e) The steady-state rate of H⁺ secretion is not affected by large changes in electrochemical potential gradients for H⁺. Neither abolishing the potential difference across the skin nor a 10-fold change in H⁺ concentration in the outside solution affected significantly the steady-state rate of H⁺ secretion. (f) The H⁺ secretion was abolished by the metabolic inhibitors dinitrophenol (1 · 10^?4 M) and Antimycin A (1.5 · 10^?6 M) which also markedly reduced the potential difference across the skin.Observations (a), (b), and (c) suggest that H⁺ and Na⁺ movements across the outer border of the isolated frog skin are not coupled. The ratio of Na⁺ to H⁺ movements is very different from unity and Na⁺ movements can be abolished without any effects on H⁺ secretion and conversely H⁺ movements can be abolished without interruption of Na⁺ uptake.A second conclusion suggested by these results is that the H⁺ secretion does not result from movement of H⁺ following its electrochemical potential gradient since that rate of secretion is not affected by marked changes in either potential or [H⁺]. Furthermore, the effects of metabolic inhibitors suggest that H⁺ secretion requires the expenditure of energy by the cell.     

Effect of quinidine on Na,H+, and water transport by the turtle and toad bladders

Summary The effect of quinidine on Na and H⁺ transport by the turtle bladder and water transport by the toad bladder was examined. Quinidine inhibited the short-circuit current and the potential difference in a dose-dependent fashion. The effect of quinidine on the short-circuit was not dependent on extracellular calcium concentration and was not reversible with removal of the drug. Quinidine inhibited H⁺ secretion in a dose-dependent fashion. The effect of quinidine on H⁺ secretion also was not dependent on extracellular calcium concentration and was not reversible, either with removal of the drug or with stimulation of H⁺ secretion with 5% CO₂. The effect of quinidine on Na or H⁺ transport could not be elicited by an equivalent dose of tetracaine, suggesting that the inhibitory effect of quinidine is not dependent on its anesthetic properties. Quinidine also inhibited vasopressin and cyclic AMP stimulated water flow in the toad bladder. Quinidine did not alter calcium uptake by the turtle bladder but increased calcium efflux by the turtle and toad bladders. These observations suggest that a rise in cytosolic calcium is responsible for the inhibitory effect of quinidine on Na, H⁺, and water transport.     

Catecholamine-stimulation of Cl− secretion in MDCK cell epithelium

Cultured epithelial monolayers of MDCK cells grown upon Millipore filter supports and mounted in Ussing chambers for transport studies respond to addition of $\text{5 · 10}{}^{\mathrm{?7}}\text{M}$ adrenalin from only the basal bathing solution by an increased short-circuit current, due both to an increased transmonolayer potential difference (basal solution electropositive) and an increased transmonolayer conductance. Measurement of tracer Na⁺, K⁺ and Cl^? fluxes demonstrate that the adrenalin-stimulated short-circuit current results primarily from basal to apical net Cl^? secretion. Half-maximal stimulation of the short-circuit current was observed at ($\text{3.1 ± 0.3) · 10}{}^{\mathrm{?8}}\text{M adrenalin}$; the order of potency of adrenergic agonists for short-circuit current stimulation was $\text{isoprenalin}\text{>}\text{adrenalin}\text{>}\text{noradrenalin}$, consistent with adrenalin action being mediated by a β-adrenergic receptor. The adrenalin-stimulated short-circuit current was sensitive to inhibition (75%) by basal additions of furosemide ($\text{1 · 10}{}^{\mathrm{?4}}\text{M}$); phloretin inhibition (54%, 57%) was observed from both epithelial surfaces. Amiloride (10^?4 M) and 4-acetamido-4-isothiocyanostilbene-2, 2′-disulphonic acid (SITS) (10 μM) were ineffective as inhibitors of the adrenalin response. The increased short-circuit current was sensitive to replacement of medium Na⁺ by choline (87%) and Tris (93%). Li⁺ was a partially effective substitute cation for Na⁺ · NO₃^?, and isethionate were ineffective substitutes for Cl^? whereas Br^? was partially effective. Partial replacement of medium Na⁺ by choline gave an upward-curving non-saturable dependence of the adrenalin-stimulated short-circuit current upon [Na]; partial replacement of Cl^? by NO₃^? in contrast gave a saturable increase with a $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of approx. 65 mM Cl^?.     

Effect of inhibitors on chloride-dependent transmural potential in the rectal wall of Schistocerca gregaria

Previous experiments in vitro revealed a transmural potential difference (PD) and a short-circuit current (Isc) across the rectal wall of Schistocerca gregaria, which were dependent on chloride ions in lumen. The present report shows that anoxia, dinitrophenol and cyanide inhibit DP and Isc, proving that the required energy derives from oxidative metabolism. Acetazolamide also inhibits DP and Isc. Ouabain, when in haemocoele, has also an inhibitory effect, not ascribable to a Na⁺K⁺ pump blocking action. It is suggested that HCO₃^? plays an important rôle in the active transport of Cl^? from lumen to haemocoele and that ouabain may in some way inhibit chloride pumping.     

Fetal rabbit gastric epithelial cells cultured on floating collagen gels

Summary Cells were isolated from ∼ 30 d fetal rabbit stomachs and cultured on floating collagen gels. Electron microscopy showed monolayers in which only one cell type persisted. These columnar cells were joined at apical borders by tight junctions and contained an extensive endoplasmic reticular network with an occasional intracellular canaliculus. They also occasionally contained what appeared to be secretory granules (mucus?), and therefore had some characteristics of all the cell types of the intact fetal stomachs, which showed oxyntic, mucous, and undifferentiated cells. In Ussing chambers with Ringer's solution on both sides, cultures developed transepithelial potential (potential difference [PD], mV, mucosa ground)=13, resistance (resistance [R], Ω-cm²)=285, and short-circuit current (I _sc , μA/cm²)=45 (n=7), clearly indicating that cellular polarity and junctional integrity were maintained. These transport parameters were somewhat different for intact fetal stomachs (PD=20, R=70, and I _sc =220 [n=4]), which may be due to extensive folding of intact fetal stomachs or the presence of only one cell type in culture, or both. Although gastric stimulants histamine, dibutyryl cycle AMP (dbcAMP), and isobutyl-methylxanthine (IMX) (a phosphodiesterase inhibitor) did not elicit H⁺ secretion or electrophysiological changes in monolayers or intact stomachs, 10⁻⁴ M apical amiloride caused a decrease in I _sc in cultured monolayers (27%) and intact stomachs (50%). Thus, Na⁺ transport seems to be a significant fraction of ion transport in both preparations. This culture system may allow the study of oxyntic cell differentiation and the development of H⁺, Na⁺, and Cl⁻ transport in the gastric mucosa. This work was supported by NIH Grant AM 19520. The electron microscope was purchased in part by NSF Grant PM 76-80300. C. Bisbee was supported by National Cancer Institute Grants CA-05388 and CA-09041. C. Logsdon received support from the Systems and Integrative Biology Training Grant.     

Abscisic acid effects on intracellular pH and ion fluxes in barley leaf segments

Changes in intracellular pH and in H⁺, K⁺ and Cl^? fluxes were evaluated in different experimental conditions in leaf segments of barley (Hordeum vulgare cv. Georgie) incubated in the dark, at pH 5.5, in the presence or absence of abscisic acid (ABA), and a comparison was made between the effects of ABA and those of erythrosin B (EB), a plasmalemma H⁺-pump inhibitor. In all conditions tested, ABA induced a cell sap acidification, an alkalinization of the external medium, a decrease in K⁺ intracellular contents, and an increase in the contents of Cl^?. The ABA-induced decrease in K⁺ content was chiefly due to the inhibition of K⁺ influx. On the contrary, ABA did not influence the uptake of Cl^?, but inhibited Cl^? efflux, the inhibition satisfactorily accounting for the larger Cl^? content observed in the presence of the hormone. The intracellular acidification and the decrease in apparent outward net transport of H⁺ observed with ABA were seemingly not associated with the activity of the proton pump, the transmembrane electrical potential difference, or K⁺ transport. On the contrary, a correlation was evident with the changes in Cl^? content. These results and, in particular, the similarity between the effects of ABA and those induced by 4,4 -diisothiocyano-2,2-disulfonic acid stilbene (DIDS), a Cl^? channel-blocking agent, suggest that the ABA-induced changes in intracellular pH and in H⁺ transport might depend on the capability of ABA to inhibit Cl^? efflux, more than on a primary inhibition of the H⁺ pump, and propose an important role for ABA in regulating the Cl^? channels.     

A Mathematical Model of Action Potential in Cells of Vascular Plants

A mathematical model of action potential (AP) in vascular plants cells has been worked out. The model takes into account actions of plasmalemma ion transport systems (K⁺, Cl^? and Ca²⁺ channels; H⁺- and Ca²⁺-ATPases; 2H⁺/Cl^? symporter; and H⁺/K⁺ antiporter), changes of ion concentrations in the cell and in the extracellular space, cytoplasmic and apoplastic buffer capacities and the temperature dependence of active transport systems. The model of AP simulates a stationary level of the membrane potential and ion concentrations, generation of AP induced by electrical stimulation and gradual cooling and the impact of external Ca²⁺ for AP development. The model supports a hypothesis about participation of H⁺-ATPase in AP generation.     

Effect of silver chloride on the short-circuit current across the isolated toad skin.

Since addition of 10^?4M AgNO₃ to either an inside or outside bathing medium containing sulfate had no effect on short-circuit current (SCC), a measure of net Na⁺ transport, or transmural potential difference (PD) in the isolated surviving toadskin, the effect of adding Ag⁺ to chloridebased Ringer solution was studied. Exposure of the outside bathing medium to 10^?4M AgNO₃ resulted in, after a 20 minute time lag, a 250 ± 51% (N=6) increase in SCC within 100 minutes as opposed to an immediate response which had a 350 ± 26% (N=8) increase in SCC by addition of 10^?4M AgNO₃ to the inside bathing solution. The dose response curve relating change in SCC to the Ag⁺ concentration added to the inside bathing medium was saturable at 10^?5M Ag⁺. The uptake of Ag⁺ by the tissue, as measured by atomic absorption spectrophotometry, showed no correlation to the relative change in SCC. Na⁺ flux experiments under short-circuited conditions showed that Ag⁺Cl^? stimulated only the unidirectional outside to inside Na⁺ flux. These results indicate that Ag⁺Cl^? enhances active sodium transport and that Ag⁺Cl^? binding to specific membrane groups is required for this effect.     

Na+-coupled Cl− transport in the gastric mucosa of the guinea pig

The Cl^–/HCO ₃ ^– exchange mechanism usually postulated to occur in gastric mucosa cannot account for the Na⁺-dependent electrogenic serosal to mucosal Cl^– transport often observed. It was recently suggested that an additional Cl^– transport mechanism driven by the Na⁺ electrochemical potential gradient may be present on the serosal side of the tissue. To verify this, we have studied Cl^– transport in guinea pig gastric mucosa. Inhibiting the (Na⁺, K⁺) ATPase either by serosal addition of ouabain or by establishing K⁺-free mucosal and serosal conditions abolished net Cl^– transport. Depolarizing the cell membrane potential with triphenylmethylphosphonium (a lipid-soluble cation), and hence reducing both the Na⁺ and Cl^– electrochemical potential gradients, resulted in inhibition of net Cl^– flux. Reduction of short-circuit current on replacing Na⁺ by choline in the serosal bathing solution was shown to be due to inhibition of Cl^– transport. Serosal addition of diisothiocyanodisulfonic acid stilbene (an inhibitor of anion transport systems) abolished net Cl^– flux but not net Na⁺ flux. These results are compatible with the proposed model of a Cl^–/Na⁺ cotransport mechanism governing serosal Cl^– entry into the secreting cells. We suggest that the same mechanism may well facilitate both coupled Cl^–/Na⁺ entry and coupled HCO ₃ ^– /Na⁺ exit on the serosal side of the tissue.     

The excretion of hydrogen ion by the isolated amphibian skin: Effects of antidiuretic hormone and amiloride

H⁺ extrusion by the isolated skins of two amphibia, Rana ridibunda and Bufo bufo was studied in order to test for the presence of exchange mechanisms of the type Na⁺/H⁺ and Cl^?/HCO₃^?, which have been described in several epithelial structures. The preparations were mounted in chambers of the Ussing type, so that the short-circuit current could be used as a function of Na⁺ transport and the pH-stat technique was utilized to determine the rates of H⁺ extrusion under different experimental conditions. These conditions were either the withdrawal of the ions intervening in the mentioned exchanges (Cl^- or Na⁺, or the addition of drugs with well-known effects on Na⁺ uptake and transport (antidiuretic hormone and amiloride).In the frog skin, H⁺ excretion was detected in solutions containing either Cl^? or SO₄^2?, with identical rates. Again, Na⁺ substitution by Mg²⁺ had no effect on H⁺ excretion rates, neither did the suppression of Na⁺ influx by amiloride or its stimulation by antidiuretic hormone. These experiments were repeated with similar results in gland-free preparations of the epidermis of frog skin separated from the corion by the action of collagenase.Experiments in toad skin showed that H⁺ excretion could not be detected when Cl^? was present in the outer medium, but became apparent if an impermeant anion, SO₄^2?, was used. This observation is compatible with the existence of an exchange mechanism of the type Cl^?/HCO₃^?. Secondly, in these preparations H⁺ extrusion increased after stimulation with antidiuretic hormone and decreased when amiloride was used or when Na⁺ was substituted by Mg²⁺, suggesting that at least a fraction of the total H⁺ efflux is linked to Na⁺ influx. In the isolated frog skin this mechanism does not seem to be operative.     

H+ and Cl− secretion in the stomach of the teleost fish, Anguilla anguilla : stimulation by histamine and carbachol

The fundus of an eel stomach was mounted in an Ussing chamber and bathed with control Ringer on the serosal side and with unbuffered solution on the mucosal side. The gastric mucosa exhibited a mucosa negative transepithelial voltage (V _t), a “short circuit” current (I _SC) and a small spontaneous acid secretion rate (J _H). All these parameters were abolished by cimetidine treatment. Bilateral ion substitution experiments in tissues lacking spontaneous acid secretion suggested that a net Cl⁻ transport from serosa to mucosa was responsible for the genesis of the I _SC in the absence of H⁺ secretion. Serosal application of histamine (10⁻⁴ mol · l⁻¹) or carbachol (10⁻⁴ mol · l⁻¹) stimulated both I _SC and J _H. The action of carbachol was independent of histamine. The control as well as the histamine-stimulated I _SC was sensitive to both serosal bumetanide (10⁻⁵ mol · l⁻¹), inhibitor of the Na⁺-K⁺-2Cl⁻ cotransport, and 4,4-diisothiocyano-stilbene-2,2-disulphonic acid (DIDS, 5 · 10⁻⁴ mol · l⁻¹), inhibitor of the Cl⁻-HCO⁻ ₃ exchange, while the I _SC stimulated by carbachol was nullified by serosal DIDS. These data suggested that the non-acidic Cl⁻ uptake across the serosal membrane was linked to the activity of both Na⁺-K⁺-2Cl⁻ cotransport and Cl⁻-HCO⁻ ₃ antiporter; histamine stimulated both transporters while carbachol was limited to the anion exchanger. The finding that the acid secretion was strictly dependent on serosal Cl⁻ and was completely blocked by serosal DIDS suggested that the Cl⁻ accompanying H⁺ secretion entered the cell through the serosal membrane by the Cl⁻-HCO⁻ ₃ exchange. In addition, the acid secretion stimulated by carbachol was also dependent on serosal Na⁺ and sensitive to the application of 5-N-N-dimethyl-amiloride in the serosal bath, suggesting that the increased activity of the Cl⁻-HCO⁻ ₃ during carbachol treatment was linked to the activation of serosal Na⁺-H⁺ exchange. The inhibitory effect of luminal omeprazole (10⁻⁴ mol · l⁻¹) on acid secretion suggested the presence of the H⁺-K⁺ pump on the luminal membrane. Accepted: 18 September 1997     

Role of Ca in stimulus-secretion coupling in the gastric oxyntic cell: Effect of A23187

The effects of Ca⁺⁺ ionophore A23187 on H⁺ secretion and histamine release were studied in the isolated gastric mucosa of the toad . A23187 added from the mucosal side stimulated H⁺ secretion. At high concentrations, A23187 also caused histamine release. This histamine was not sufficient to explain the effects of A23187 on H⁺ secretion. Metiamide, only partially inhibited the effect of ionophore. There was summation and/or potentiation of effects between A23187 and histamine. The results are consistent with the hypothesis that Ca⁺⁺ acts as a second messenger in stimulus-secretion coupling in the oxyntic cell. It is possible that Ca⁺⁺ and cAMP may interact as parallel second messengers in the control of gastric H⁺ secretion.     

The ascidian sperm reaction: evidence for Cl- and HCO3- involvement in acid release

Ascidia callosa sperm are triggered to undergo initiation of the sperm reaction (mitochondrial swelling) by increasing the pH or lowering the Na⁺ concentration of the medium. The optimal [Na⁺] for acid release is 20 mM with excellent correlation between acid release and initiation of morphological changes. Increasing the [K⁺] to around 20 mM inhibits acid release when applied up to 1 min after triggering the sperm but with less inhibition at 2 and 4 min, suggesting that K⁺ inhibits initiation of acid release rather than acid release itself. Acid release and the sperm reaction can also be triggered by Cl^?-free (NO^?₃ or glutamate substituted) seawater (SW). Cl^? efflux accompanies H⁺ efflux with twice as many Cl^? being released as H⁺. Both H⁺ and Cl^? release in Cl^?-free SW are dependent upon CO₂ being present in HCO^?₃-free medium, suggesting that H⁺ efflux is in part Cl^? and HCO^?₃-mediated. However, the chloride channel blocking agent SITS has no effect on H⁺ release and augments Cl^? release. Acid release results in a substantial increase in internal pH as determined by partitioning of 9-amino acridine. We envision acid release from ascidian sperm as involving two systems, the Na⁺-dependent acidification system of unreacted sperm and the Cl^?- and HCO^?₃-mediated H⁺ release at activation. The mechanism controlling acid release would then involve inactivation of the internal acidification process and activation of the chloride-bicarbonate-mediated alkalinization process.     

Energy sources for amino acid transport in animal cells

The existence of an electrogenic Na⁺ pump in Ehrlich cells which substantially contributes to the membrane potential, previously derived from the distribution of the lipid soluble cation tetraphenylphosphonium (TPP⁺), could be confirmed by an independent method based on the quenching of fluorescence of a cyanine dye derivative, after the mitochondrial respiration had been suppressed by appropriate inhibitors. The mitochondrial membrane potential, by adding to the overall potential as measured in this way is likely to cause an overestimation of the membrane potential difference (p.d.). But since this error tends to diminish with increasing pump activity, the true p.d. of the plasma membrane should easily account for the driving force to drive the active accumulation of amino acids in the absence of an adequate Na⁺ concentration gradient. Accordingly, the F₂-aminoisobutyric acid (AIB) uptake rises linearly with the distribution of TPP⁺ at constant Na⁺ concentrations, suggesting that each responds directly to membrane potential. There is evidence that the electrogenic (free) movement of Cl^? is slow, at least at normal p.d., whereas a major part of the Cl^? movement across the cellular membrane appears to occur by an electrically silent Cl^?-base exchange mechanism. By such a mode Cl^?, together with an almost stoichiometric amount of K⁺, may under certain conditions move into the cell against a high adverse electrical potential difference. This “paradoxical” movement of K⁺Cl^? contributing to the deviation of the Cl^? distribution from the electrochemical equilibrium distribution, is not completely understood. It is insensitive towards ouabain but can almost specifically be inhibited by furosemide. As a likely explanation a H⁺–K⁺ exchange pump was previously offered, even though unequivocal evidence of such a pump is so far lacking. According to available evidence the electrogenic movement of free Cl^? is too small, at least at normal orientation of the p.d., to significantly shunt the electrogenic pump potential so that the establishment of such a potential is plausible. The evidence presented is considered strong in favor of the gradient hypothesis since even in the absence of an adequate Na⁺ concentration gradient, the electrogenic Na⁺ pump will contribute sufficient extra driving force to actively transport amino acid into the cells.     

Bile salt alteration of ion transport across jejunal mucosa

The effect of conjugated dihydroxy and trihydroxy bile salts on electrolyte transport across isolated rabbit jejunal mucosa was studied. Both taurochenodeoxycholic acid and taurocholic acid increased the short-circuit current (I_sc) in bicarbonate-Ringer solution but not in a bicarbonate-free, chloride-free solution. Taurochenodeoxycholic acid was significantly more effective than taurocholic acid in increasing I_sc. The presence of theophylline prevented the taurochenodeoxycholic acid-and taurocholic acid-induced increase in I_sc. Transmural ion fluxes across jejunal mucosa demonstrated that 2 mM taurochenodeoxycholic acid decreased net Na⁺ absorption, increased net Cl⁻ secretion and increased the residual flux (which probably represents HCO₃⁻ secretion). These studies support the hypothesis that cyclic AMP may be a mediator of intestinal electrolyte secretion.     

Effect of 3-isobutyl-1-methylxanthine on HCO3− transport in turtle bladder: Evidence for electrogenic HCO3− secretion

Ouabain-treated turtle bladders bathed on both surfaces by identical HCO₃^?/CO₂-containing, Cl^?-free Na⁺ media exhibit a short-circuit current (I_sc) and transepithelial potential (p.d.) serosa electronegative to mucosa. Addition of 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of cyclic nucleotide phosphodiesterase, rapidly reverses the direction of the I_sc and p.d.. The IBMX-induced reversal of I_sc and p.d. is (1) dependent on the presence of HCO₃^? (and CO₂) in the serosal bathing fluid, (2) independent of Na⁺ and other ions in the bathing medium, (3) decreased by inhibitors of carbonic anhydrase or oxidative metabolism, (4) increased by the serosal addition of cyclic AMP or the disulfonic stilbene, SITS. The results constitute evidence that the reversed I_sc elicited by IBMX represents electrogenic secretion of HCO₃^?.