Proton pump activity is required for active uptake of chloride in isolated amphibian skin exposed to freshwater

Net proton secretion and unidirectional chloride fluxes were measured in isolated skin of toads ( Bufo bufo) and frogs ( Rana esculenta) mounted in an Ussing chamber and exposed to a Ringer's solution on the serosal side and a freshwater-like solution (1-3 mM Cl(-)) on the external side. Active proton secretion was 34.2+/-2.0 pmol.cm(-2).s(-1) ( n=18) in frog skin, and 16.7+/-1.7 pmol.cm(-2).s(-1) ( n=10) in toad skin. Proton secretion by toad skin was dependent on the transepithelial potential ( V(T)), and an amiloride-insensitive short-circuit current was stimulated by exogenous CO(2)/HCO(3)(-), indicating the presence of a rheogenic proton pump. Cl(-) influx was 37.4+/-7.5 pmol.cm(-2).s(-1) ( n=14) in frog skin and 19.5+/-3.5 pmol.cm(-2).s(-1) ( n=11) in toad skin. In toad skin, the mean Cl(-) flux ratio was larger than expected for simple electro-diffusion. In 8 of 11 sets of paired skins, influx was greater than the efflux indicating active uptake of Cl(-). Cl(-) influx in toad skin was unaffected by large perturbations (100-150 mV) of V(T), which was accomplished by adding amiloride to the outer bath under open circuit conditions. A component of the Cl(-) efflux seemed to be dependent on V(T). 4,4'-Diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS; 0.3 mM or 1.3 mM) inhibited Cl(-) influx and, surprisingly, increased Cl(-) efflux in toad skin. Influx and efflux of Cl(-) in toad skin were highly dependent on the external [Cl(-)] in the freshwater range (0.1-4 mM). (36)Cl(-) influx decreased whereas the total Cl(-) efflux increased as a function of external [Cl(-)]. These data indicate the presence of a DIDS-sensitive, electroneutral carrier mechanism with an external binding site for Cl(-). Ethoxzolamide (100 micro M), an inhibitor of carbonic anhydrase, reduced proton secretion and Cl(-) influx in frog skin. Concanamycin A (0.1-10 micro M), a specific vacuolar-type proton pump (V-ATPase) inhibitor, significantly reduced proton secretion in frog skin. In addition, concanamycin A (1 micro M) significantly reduced Cl(-) influx in frog skin. We suggest that the active proton secretion and Cl(-) influx are coupled. We hypothesise that an apical V-ATPase is capable of energising active Cl(-) uptake in fresh water by creating a favourable gradient for an apical HCO(3)(-) exit in exchange for external Cl(-). The data also suggest that a carbonic anhydrase activity provides H(+) and HCO(3)(-) for apically co-expressed proton pumps and Cl(-)/HCO(3)(-) exchangers.     

Na(+) and Cl(-) transport by the urinary bladder of the freshwater rainbow trout (Oncorhynchus mykiss)

Freshwater (FW) rainbow trout (Oncorhynchus mykiss) urinary bladders mounted in vitro under symmetrical saline conditions displayed electroneutral active absorption of Na(+) and Cl(-) from the mucosal side; the transepithelial potential (V(t)) was 0.1 mV, and the short-circuit current was less than 1 microA cm(-2). Removal of Na(+) from mucosal saline decreased Cl(-) absorption by 56% and removal of Cl(-) decreased Na(+) absorption by 69%. However, active net absorption of both Na(+) and Cl(-) was not abolished when Cl(-) or Na(+) was replaced with an impermeant ion (gluconate or choline, respectively). Under physiological conditions with artificial urine (?Na(+) = 2.12 mM, ?Cl(-) = 3.51 mM) bathing the mucosal surface and saline bathing the serosal surface, transepithelial potential (V(t)) increased to a serosal positive approximately +7.6 mV. Unidirectional influx rates of both Na(+) and Cl(-) were 10-20-fold lower but active absorption of both ions still occurred according to the Ussing flux ratio criterion. Replacement of Na(+) with choline, or Cl(-) with gluconate, in the mucosal artificial urine yielded no change in unidirectional influx of Cl(-) or Na(+), respectively. However, kinetic analyses indicated a decrease in maximum Na(+) transport rate (J(max)) of 66% with no change in affinity (K(m)) in the low Cl(-) mucosal solution relative to the control solution. Similarly, there was a 79% decrease in J(max) values for Cl(-), again with no change in K(m), in the low-Na(+) mucosal bathing. The mucosal addition of DIDS, amiloride or bumetanide (10(-4) M) had no effect on either Na(+) or Cl(-) transport, under either symmetrical saline or artificial urine/saline conditions. Addition of the three drugs simultaneously (10(-4) M), or chlorothiazide (10(-3) M), under symmetrical saline conditions also had no effect on Na(+) or Cl(-) transport rates. Cyanide (10(-3) M) addition to mucosal artificial urine caused a slowly developing decrease of Na(+) influx to 59% and Cl(-) influx to 50% in the period after drug addition. Na(+) and Cl(-) reabsorption appears to be a partially coupled process in the urinary bladder of O. mykiss; transport mechanisms are both dependent upon and independent of the other ion.     

Studies on the effect of the pyrethroid insecticide Decamethrin on ionic transport through the in vitro skin of Rana esculenta

The effect of the insecticide Decamethrin on ionic transport through the isolated skin of Rana esculenta was studied; the skins were bathed with 1-2 mEq Na2SO4 or choline-Cl solutions (exterior), and with Ringer normal (interior). Under open circuit (OC) conditions, mucosal Decamethrin (10(-6) M), did not provoke changes in Na+ fluxes. At 10(-5) M there was a slight inhibition of the JoNa+ after 30 min. The Cl- fluxes did not change. With longer treatments (60-90 min, OC, 10(-5) M) the JnNa+ was inhibited; at 10(-4) M it was augmented. The JnH+ was not affected. Serosal Decamethrin did not modify Na+ and H+ fluxes. In short circuit conditions, Decamethrin (10(-5) M) in the mucosal face inhibited the JnNa+; the JnH+ did not change in these conditions. The abscence of interaction of mucosal Decamethrin with Amiloride was shown.     

Effect of E. coli heat-stable enterotoxin on colonic transport in guanylyl cyclase C receptor-deficient mice

We studied the functional importance of the colonic guanylyl cyclase C (GCC) receptor in GCC receptor-deficient mice. Mice were anesthetized with pentobarbital sodium, and colon segments were studied in Ussing chambers in HCO3- Ringer under short-circuit conditions. Receptor-deficient mouse proximal colon exhibited similar net Na+ absorption, lower net Cl- absorption, and a negative residual ion flux (J(R)), indicating net HCO3- absorption compared with that in normal mice. In normal mouse proximal colon, mucosal addition of 50 nM Escherichia coli heat-stable enterotoxin (STa) increased the serosal-to-mucosal flux of Cl- (J(s-->m)(Cl)) and decreased net Cl- flux (J(net)(Cl)) accompanied by increases in short-circuit current (I(sc)), potential difference (PD), and tissue conductance (G). Serosal STa had no effect. In distal colon neither mucosal nor serosal STa affected ion transport. In receptor-deficient mice, neither mucosal nor serosal 500 nM STa affected electrolyte transport in proximal or distal colon. In these mice, 1 mM 8-bromo-cGMP produced changes in proximal colon J(s-->m)(Cl) and J(net)(Cl), I(sc), PD, G, and J(R) similar to mucosal STa addition in normal mice. We conclude that the GCC receptor is necessary in the mouse proximal colon for a secretory response to mucosal STa.     

Effects of Alkali Metal Gations on the Potential across Toad and Bullfrog Urinary Bladder

Isolated urinary bladders of the bullfrog (R. catesbeiana) and the toad (B. marinus) were mounted in an Ussing chamber. Potential differences up to 114 mv were observed in bullfrog bladder when the mucosal surface was bathed in dilute Na₂SO₄ and the serosal surface in sulfate Ringer's. In experiments with bullfrogs, K was used to replace Na in the mucosal solution and Na was used for K in the serosal solutions. The selectivity was judged in terms of the relative effectiveness of the replacement cation in maintaining the bladder potential. In experiments with toads, K and Rb were equally poor replacements for Na at the mucosal border, while Rb was a good replacement for K at the serosal border. Li in the mucosal solution appeared to depress the potential in part irreversibly. At the serosal border, Li was a partially effective substitute for K, more so than was Na. However, both were poor replacements compared to Rb. The mucosal surface of the urinary bladder of both frog and toad appears to be Na-selective and the serosal surface appears to be K-selective, consistent with the Koefoed-Johnsen-Ussing model for frog skin.     

Cyclic AMP-induced K⁺ secretion occurs independently of Cl⁻ secretion in rat distal colon

cAMP induces both active Cl(-) and active K(+) secretion in mammalian colon. It is generally assumed that a mechanism for K(+) exit is essential to maintain cells in the hyperpolarized state, thus favoring a sustained Cl(-) secretion. Both Kcnn4c and Kcnma1 channels are located in colon, and this study addressed the questions of whether Kcnn4c and/or Kcnma1 channels mediate cAMP-induced K(+) secretion and whether cAMP-induced K(+) secretion provides the driving force for Cl(-) secretion. Forskolin (FSK)-enhanced short-circuit current (indicator of net electrogenic ion transport) and K(+) fluxes were measured simultaneously in colonic mucosa under voltage-clamp conditions. Mucosal Na(+) orthovanadate (P-type ATPase inhibitor) inhibited active K(+) absorption normally present in rat distal colon. In the presence of mucosal Na(+) orthovanadate, serosal FSK induced both K(+) and Cl(-) secretion. FSK-induced K(+) secretion was 1) not inhibited by either mucosal or serosal 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34; a Kcnn4 channel blocker), 2) inhibited (92%) by mucosal iberiotoxin (Kcnma1 channel blocker), and 3) not affected by mucosal cystic fibrosis transmembrane conductance regulator inhibitor (CFTR(inh)-172). By contrast, FSK-induced Cl(-) secretion was 1) completely inhibited by serosal TRAM-34, 2) not inhibited by either mucosal or serosal iberiotoxin, and 3) completely inhibited by mucosal CFTR(inh)-172. These results indicate that cAMP-induced colonic K(+) secretion is mediated via Kcnma1 channels located in the apical membrane and most likely contributes to stool K(+) losses in secretory diarrhea. On the other hand, cAMP-induced colonic Cl(-) secretion requires the activity of Kcnn4b channels located in the basolateral membrane and is not dependent on the concurrent activation of apical Kcnma1 channels.     

Cultured monolayers of the dog jejunum with the structural and functional properties resembling the normal epithelium

The development of a culture of the normal mammalian jejunum motivated this work. Isolated crypt cells of the dog jejunum were induced to form primary cultures on Snapwell filters. Up to seven subcultures were studied under the electron microscope and in Ussing chambers. Epithelial markers were identified by RT-PCR, Western blot, and immunofluorescent staining. Confluent monolayers exhibit a dense apical brush border, basolateral membrane infoldings, desmosomes, and tight junctions expressing zonula occludens-1, occludin-1, and claudin-3 and -4. In OptiMEM medium fortified with epidermal growth factor, hydrocortisone, and insulin, monolayer transepithelial voltage was -6.8 mV (apical side), transepithelial resistance was 1,050 Omega.cm(2), and short-circuit current (I(sc)) was 8.1 microA/cm(2). Transcellular and paracellular resistances were estimated as 14.8 and 1.1 kOmega.cm(2), respectively. Serosal ouabain reduced voltage and current toward zero, as did apical amiloride. The presence of mRNA of alpha-epithelial Na(+) channel (ENaC) was confirmed. Na-d-glucose cotransport was identified with an antibody to Na(+)-glucose cotransporter (SGLT) 1. The unidirectional mucosa-to-serosa Na(+) flux (19 nmol.min(-1).cm(-2)) was two times as large as the reverse flux, and net transepithelial Na(+) flux was nearly double the amiloride-sensitive I(sc). In plain Ringer solution, the amiloride-sensitive I(sc) went toward zero. Under these conditions plus mucosal amiloride, serosal dibutyryl-cAMP elicited a Cl(-)-dependent I(sc) consistent with the stimulation of transepithelial Cl(-) secretion. In conclusion, primary cultures and subcultures of the normal mammalian jejunum form polarized epithelial monolayers with 1) the properties of a leaky epithelium, 2) claudins specific to the jejunal tight junction, 3) transepithelial Na(+) absorption mediated in part by SGLT1 and ENaC, and 4) electrogenic Cl(-) secretion activated by cAMP.     

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.     

Intracellular gradients of ion activities in the epithelial cells of theNecturus gallbladder recorded with ion-selective microelectrodes

In Necturus gallbladder epithelial cells the intracellular electrical potential, as recorded with microelectrodes, varied from -28 mV in the mucosal end to about -50 mV in the serosal end of the transporting cell. The Na+ activity varied concurrently from about 39 mM to between 8 and 19 mM. Thus, within the cell both the recorded electrical and chemical gradients caused Na+ to move towards the serosal end. Serosal addition of ouabain (5 X 10(-4) M) caused the intracellular Na+ activity to attain electrochemical equilibrium within 30 min. However, the intracellular electrical potential gradient was only slowly affected. In cells from animals stored at 5 degrees C, the Cl- activity varied from about 55 mM in the mucosal end to 28 mM in the serosal end, and the K+ activity from 50 mM to between 95 and 131 mM. Both ions were close to electrochemical equilibrium within the cytoplasm but were too concentrated to be in equilibrium with the mucosal solution. Bubbling CO2 through the mucosal solution caused the intracellular gradients to vanish. When Na+ in the bathing solutions was exchanged for K+, the intracellular electrical potential became roughly constant at about -5 mV. The Cl- activity became constant in 65 mM, and the K+ activity became constant at 109 mM, both close to equilibrium with the mucosal solution. The Na+ activity was reduced to about 1 mM. The ratio of cytoplasmic resistivities between cells bathed in K+-rich saline to cells bathed in Na+-rich saline was measured by means of triple-barreled electrodes and compared to the same ratio as assessed from the activity measurements. The two values were equal only if one assumes the mobility of Na+ inside the cell to be less than 1/10 of the mobility of K+ or Cl-. The same conclusion was reached by comparing the intracellular Na+ flux calculated from the gradient of electrochemical potential to that flux assess from the net solute absorption. Animals kept at 15 degrees C had lower intracellular Na+ activities, higher Cl- and K+ activities, and higher rates of absorption than animals stored at 5 degrees C. Finally, the degree to which the intracellularly recorded electrical and chemical potentials could reflect an electrode artefact is discussed.     

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.     

Thiazides stimulate calcium absorption in urinary bladder of winter flounder

Thiazides inhibit voltage-independent NaCl absorption in the urinary bladder of the winter flounder presumably by blocking an electroneutral mucosal Na/Cl co-transporter. As thiazides stimulate calcium absorption in mammalian distal convoluted tubule while inhibiting NaCl absorption, we studied the effects of hydrochlorothiazide (HCTZ) on unidirectional 45Ca fluxes and intracellular electrical potential in short-circuited bladders to examine possible mechanisms of HCTZ effects on calcium transport. Basal secretory calcium flux was, on average, slightly larger than absorptive flux, reflecting small net calcium secretion. Mucosal addition of HCTZ (10(-4) M) stimulated absorptive calcium flux by 46% while the secretory flux was unaltered. Thus, HCTZ tended to induce net calcium absorption. Pre-treatment with serosal ouabain (10(-4) M) attenuated the HCTZ-induced increase in absorptive calcium flux. Moreover, HCTZ hyperpolarized the mucosal membrane potential by 18% as measured by conventional open-tip microelectrodes. These effects of HCTZ are consistent with the hypothesis that HCTZ indirectly stimulates Na/Ca exchange located at the serosal membrane. In conclusion, HCTZ in flounder urinary bladder, as in mammalian distal convoluted tubule, simultaneously inhibits NaCl absorption and stimulates calcium absorption. This study expands on the functional similarities between the flounder urinary bladder and the mammalian distal convoluted tubule.     

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.     

Coupling of volume and Na+ transport in frog skin epithelium

Whole skins and isolated epithelia were bathed with isotonic media (congruent to 244 mOsm) containing sucrose or glucose. The serosal osmolality was intermittently reduced (congruent to 137 mOsm) by removing the nonelectrolyte. Transepithelial and intracellular electrophysiological parameters were monitored while serosal osmolality was changed. Serosal hypotonicity increased the short-circuit current (ISC) and the basolateral conductance, hyperpolarized the apical membrane (psi mc), and increased the intracellular Na+ concentration. The increases in apical conductance and apical Na+ permeability (measured from Goldman fits of the relationship between amiloride-sensitive current and psi mc) were not statistically significant. To verify that the osmotically induced changes in ISC were mediated primarily at the basolateral membrane, the basolateral membrane potential of the experimental area was clamped close to 0 mV by replacing the serosal Na+ with K+ in Cl--free media. The adjoining control area was exposed to serosal Na+. Serosal hypotonicity produced a sustained stimulation of ISC across the control, but not across the adjoining depolarized tissue area. The current results support the concept that hypotonic cell swelling increases Na+ transport across frog skin epithelium by increasing the basolateral K+ permeability, hyperpolarizing the apical membrane, and increasing the electrical driving force for apical Na+ entry.     

Uncoupled basal sodium absorption and chloride secretion in prairie dog (Cynomys ludovicianus) gallbladder.

1. Prairie dog gallbladders mounted in a Ussing-type chamber and bathed with symmetrical Ringer's solutions exhibited a transepithelial resistance (Rt) of 51 +/- 5 omega cm2, a lumen negative potential difference (Vms) of 11.5 +/- 0.7 mV and a short-circuit current (Isc) of 6.9 +/- 0.3 microEq/hr/cm2. 2. Radioisotopic ion flux experiments revealed that the basal Isc of 6.9 +/- 0.3 microEq/hr/cm2 was mostly accounted for by net Na+ absorption of 3.2 +/- 0.5 microEq/hr/cm2 and net Cl- secretion of 2.9 +/- 0.3 microEq/hr/cm2. 3. In HCO3- free Ringer's, net Na+ flux was virtually abolished, net Cl- flux decreased by 50% and Isc was reduced by 77%. 4. 10(-3) M mucosal amiloride and DIDS reduced Isc by 28 and 24%, respectively. 5. Mucosal NaCl diffusion potentials indicated that the paracellular pathway was cation selective. 6. Thin section electron micrographs showed a single cell population in this epithelium suggesting that net Na+ absorption and Cl- secretion may emerge from the same cells. 7. We conclude that prairie dog gallbladder epithelium is an electrogenic tissue and, in contrast to gallbladders of most other species, simultaneously but independently absorbs Na+ and secretes Cl-.     

Electrophysiological responses of frog skin to the peptides bombesin, caerulein, and physalaemin

Isolated skins from the frog Rana pipiens were mounted on Ussing-type chambers and bathed with Amphibian Ringer's solution on both sides. Electrical potential difference, resistance, and short-circuit current (SCC) were measured by using calomel electrodes, Ag-AgCl electrodes, Ringer's-agar bridges, and Tektronix digital multimeters. Under the conditions employed, SCC is a measure of net Na+ transport. The frog skin peptides bombesin, caerulein, and physalaemin were administered to the serosal side at concentrations of 0.5, 5.0, and 50 ng/ml. Control electrophysiological parameters were: potential difference, 23 +/- 2 mV; resistance, 738 +/- 59 ohms cm2; and SCC 32 +/- 3 microA/cm2. Although bombesin and caerulein had no significant, reversible effect on potential difference, resistance, or SCC, physalaemin significantly, and reversibly, depressed SCC by 22%. Caerulein did significantly depress SCC, but the response was not reversible.     

Chloride extrusion in the isolated perfused teleost gill

Summary Chloride extrusion is examined in the isolated perfused gill of the pinfish,Lagodon rhomboides. In both sea water and Ringer's baths, the Cl efflux from the isolated gill is 45% that of the intact animal. The transepithelial electrical potential (TEP) across the isolated gill in sea water is equal to that in vivo, in Ringer's the gill TEP is slightly less than in vivo. Cl efflux is linearly dependent upon afferent flow of the perfusate. Furosemide, added to the perfusate inhibits 57% of the Cl efflux in gills bathed bilaterally by Ringer's. Ouabain causes a marked vasoconstriction and increase in afferent pressure. Removal of Na from the perfusate produces an inhibition of the Cl efflux that is not potential mediated. Net extrusion of Cl is inhibited in isolated gills bathed bilaterally by sodium free Ringer's.     

Effect of sodium on amiloride- and triamterene-induced current fluctuations in isolated frog skin

The apparent association constants of two agents, amiloride and triamterene, that block the Na-selective channel of apical membrane of frog skin are shown to decrease as the Na concentration is increased in the apical bathing solution in isolated skin of the frog, Rana temporaria, Rana esculenta, and Rana pipiens. These results were obtained in "normally polarized" skins. These effects were independent of the anion used (chloride or methylsulfate) or the cation used as the Na substitute (Tris, DDA, or K ion). When NaCl was replaced with mannitol, the Na effect on the amiloride association rate constant persisted, which shows that ionic strength was not critically involved. The amiloride corner frequency was unaffected when the clamp potential was altered from +100 to -60 mV. The Na dependence was greatly attenuated or absent when the serosal surface was bathed in 120 mM K Ringer's, an effect that appears to be attributable to some pharmacological effect of high serosal K. A previously described three-state model is used to analyze the inhibitory effect of Na on the blocker association rate constant.     

Effect of divalent cations on ion fluxes and leaf photochemistry in salinized barley leaves

Photosynthetic characteristics, leaf ionic content, and net fluxes of Na(+), K(+), and Cl(-) were studied in barley (Hordeum vulgare L) plants grown hydroponically at various Na/Ca ratios. Five weeks of moderate (50 mM) or high (100 mM) NaCl stress caused a significant decline in chlorophyll content, chlorophyll fluorescence characteristics, and stomatal conductance (g(s)) in plant leaves grown at low calcium level. Supplemental Ca(2+) enabled normal photochemical efficiency of PSII (F(v)/F(m) around 0.83), restored chlorophyll content to 80-90% of control, but had a much smaller (50% of control) effect on g(s). In experiments on excised leaves, not only Ca(2+), but also other divalent cations (in particular, Ba(2+) and Mg(2+)), significantly ameliorated the otherwise toxic effect of NaCl on leaf photochemistry, thus attributing potential targets for such amelioration to leaf tissues. To study the underlying ionic mechanisms of this process, the MIFE technique was used to measure the kinetics of net Na(+), K(+), and Cl(-) fluxes from salinized barley leaf mesophyll in response to physiological concentrations of Ca(2+), Ba(2+), Mg(2+), and Zn(2+). Addition of 20 mM Na(+) as NaCl or Na(2)SO(4) to the bath caused significant uptake of Na(+) and efflux of K(+). These effects were reversed by adding 1 mM divalent cations to the bath solution, with the relative efficiency Ba(2+)>Zn(2+)=Ca(2+)>Mg(2+). Effect of divalent cations on Na(+) efflux was transient, while their application caused a prolonged shift towards K(+) uptake. This suggests that, in addition to their known ability to block non-selective cation channels (NSCC) responsible for Na(+) entry, divalent cations also control the activity or gating properties of K(+) transporters at the mesophyll cell plasma membrane, thereby assisting in maintaining the high K/Na ratio required for optimal leaf photosynthesis.     

The effect of ethanol on ion transport in frog skin.

Frog skin has been used as a model epithelial sodium-transporting system to study the effect of ethanol on ion transport. Treatment of the outside of frog skin with ethanol decreased the net sodium transport due to inhibition of 22Na+ influx. Ethanol did not alter sodium outflux when bathin the outside of the skin. The inhibition was in proportion to the concentration of ethanol, 0.25 M resulting in 50% inhibition. The chloride permeability of the skin was increased several-fold when the skin was exposed to ethanol in either bathing solution. With 0.4 M ethanol in the inner bathing solution, all the unidirectional fluxes of Na+ and C1- were increased. The movement of C1- was evaluated by comparison of C1- flux with urea flux, since urea is thought to move passively across frog skin via an extracellular (shunt) pathway. Chloride flux was increased to a greater extent than urea flux. These experiments indicate that ethanol affects chloride permeability beyond an increase in extracellular ion flow and independent of its effect of Na+ transport.     

Functional plasticity of mitochondrion-rich cells in the skin of euryhaline medaka larvae (Oryzias latipes) subjected to salinity changes

A noninvasive technique, the scanning ion-selective electrode technique (SIET) was applied to measure Na(+) and Cl(-) transport by the yolk-sac skin and individual mitochondrion-rich cells (MRCs) in intact medaka larvae (Oryzias latipes). In seawater (SW)-acclimated larvae, significant outward Na(+) and Cl(-) gradients were measured at the yolk-sac surface, indicating secretions of Na(+) and Cl(-) from the yolk-sac skin. With Na(+) pump immunostaining and microscopic observation, two groups of MRCs were identified on the yolk-sac skin of SW-larvae. These were single MRCs (s-MRCs), which do not have an accompanying accessory cell (AC), and multicellular complex MRCs (mc-MRCs), which usually consist of an MRC and an accompanying AC. The percentage of mc-MRC was ～60% in 30 parts per thousand of SW, and it decreased with the decrease of external salinity. By serial SIET probing over the surface of the MRCs and adjacent keratinocytes (KCs), significant outward fluxes of Na(+) and Cl(-) were detected at the apical opening (membrane) of mc-MRCs, whereas only outward Cl(-) flux, but not Na(+) flux, was detected at s-MRCs. Treatment with 100 μM ouabain or bumetanide effectively blocked the Na(+) and Cl(-) secretion. Following freshwater (FW) to SW transfer, Na(+) and Cl(-) secretions by the yolk-sac skin were fully developed in 5 h and 2 h, respectively. In contrast, both Na(+) and Cl(-) secretions downregulated rapidly after SW to FW transfer. Sequential probing at individual MRCs found that Na(+) and Cl(-) secretions declined dramatically after SW to FW transfer and Na(+)/Cl(-) uptake was detected at the same s-MRCs and mc-MRCs after 5 h. This study provides evidence demonstrating that ACs are required for Na(+) excretion and MRCs possess a functional plasticity in changing from a Na(+)/Cl(-)-secreting cell to a Na(+)/Cl(-)-absorbing cell.