Conductive chloride flux across amphibian skin: inhibition by indacrinone and cobalt ion.

When amphibian skin was incubated under conditions in which transepithelial sodium transport was abolished, a conductive transepithelial Cl- flux arose when Cl- was removed from one of the compartments. This flux was matched by short-circuit current and it accounted entirely for transepithelial conductance. Cl- influx was larger than efflux; it was linearly related to the magnitude of transepithelial Cl- concentration difference. When applied to the epithelial surface of the tissue, divalent metal cations such as Co2+, and the ethacrynic acid derivative, indacrinone, reduced rapidly and reversibly both transepithelial Cl- (in)flux and short-circuit current. Frog skin proved to be more sensitive to these inhibitors than toad skin. Further characterization of transepithelial Cl- pathway(s) should benefit from the fact that Cl- across amphibian skin can easily be monitored by the short-circuit current method, and from the availability of agents which inhibit this passive flux rapidly and reversibly.     

In vitro effects of tetrodotoxin and hexamethonium on electrolyte transport in rabbit ileum treated with cholera toxin.

To determine if there was a role for the submucosal nerves in cholera toxin (CT)-induced secretion, we studied the effects of serosal addition of two neurotoxins, the nerve conduction blocking agent, tetrodotoxin (TTX), and the nicotinic ganglionic blocking agent, hexamethonium (HXM), on electrolyte secretion in control isolated rabbit ileum and in that stimulated by CT. 1). In the absence of CT, the short circuit current (Isc) decreased after TTX (10(-7) M) (P less than 0.01) and was unaltered by HXM (10(-5) M). In the presence of CT, Isc increased but was not modified by 10(-7) M TTX or 10(-5) M HXM. 2) In control tissues the mean isotopic Na+ and Cl- fluxes were not significantly altered by TTX addition. Cl- absorption alone was significantly reduced by HXM (delta JCl- = 1.95 +/- 0.81 microEq.hr-1.cm-2; P less than 0.02). After stimulation with CT, TTX significantly inhibited Na+ and Cl- secretion (delta JNa+ = 2.15 +/- 0.61 and delta JCl- = 2.15 +/- 0.76 microEq.hr-1.cm-2; P less than 0.01). Similarly, HXM significantly inhibited CT-stimulated Na+ and Cl- secretion (delta JNa+ = 1.73 +/- 0.70 and delta JCl- = 1.46 +/- 0.62 microEq.hr-1.cm-2; P less than 0.02). 3) In TTX and HXM treated tissues there was no difference in the increase in Isc caused by cAMP (2 x 10(-3) M), calcium ionophore A 23187 (4 x 10(-6) M) and glucose (10(-3) M) compared to the untreated tissues in the presence or absence of CT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Paths of ion transport across canine fetal tracheal epithelium

Fluid secretion by the fetal sheep lung is thought to be driven by secretion of Cl- by the pulmonary epithelium. We previously demonstrated Cl- secretion by tracheal epithelium excised from fetal dogs and sheep. In this study we characterized the ion transport pathways across fetal canine tracheal epithelium. The transport of Na+ and Cl- across trachea excised from fetal dogs was evaluated from transepithelial electrical properties and isotope fluxes. Under basal conditions the tissues were characterized by a lumen-negative potential difference (PD) of 11 mV and conductance of 5.2 mS/cm2. The short-circuit current (Isc) was 43 microA/cm2 (1.6 mueq.cm-2.h-1). Basal Na+ flows were symmetrical, but net Na+ absorption (1.1 mueq.cm-2.h-1) could be induced by exposure of the luminal surface to amphotericin B (10(-6) M). Bilateral replacement of Na+ reduced Isc by 85%. Replacement of submucosal Na+ or exposure to submucosal furosemide (10(-4) M) reduced net Cl- secretion by 60-70%. Luminal exposure to indomethacin (10(-6) M) induced a 50% decrease in Isc, whereas isoproterenol (10(-6) M) increased Isc by 120%. The properties of the Cl- secretory pathway across fetal dog trachea are consistent with the model proposed for Cl- secretion across adult dog trachea and other Cl- -secreting tissues (e.g., bullfrog cornea and shark rectal gland). The absence of basal Na+ absorption by fetal dog trachea probably reflects limited apical membrane Na+ permeability.     

Interaction of heptanol and pressure on sodium and chloride transport by toad skin

We examined the interaction of heptanol and hydrostatic pressure on Na+ and Cl- transport in isolated toad skin. In the presence of Cl-, heptanol decreased short-circuit current (Isc) and total transepithelial resistance (Rt). However, in the absence of Cl- in the mucosal bath, heptanol increased Rt, although it retained the same inhibitory effect on Isc. When transepithelial active Na+ transport was blocked by amiloride, heptanol had no effect on Isc whether or not Cl- was present, whereas it decreased the shunt resistance (Rs) only in the presence of Cl- in the mucosal bath. Moreover, this effect of heptanol on Rs was significantly smaller in the presence of diphenylamine-2-carboxylate (DPC), a known Cl- channel blocker. Pressure also decreased Isc through inhibition of active Na+ transport, but it increased Rs. When heptanol and pressure were applied together, their inhibitory effects on Isc were additive, but their effects on Rs were antagonistic. Furthermore, when a transepithelial Cl- current was produced by reducing the Cl- concentration of the serosal bath, heptanol stimulated this current, which was reversibly inhibited by pressure or DPC addition to the mucosal bath. When the heptanol-stimulated Cl- current was first inhibited by pressure, subsequent DPC addition had less or no effect. These results suggest that one site of an antagonistic interaction of heptanol and pressure in toad skin is an apical membrane Cl- conductance.     

Electrical properties and fluxes of Na+ and Cl- across lizard intestine

Electrical parameters and unidirectional Na+ and Cl- fluxes were determined in vitro across the duodenum, ileum and colon of lizard (Gallotia galloti). Electrical potential difference (PD) and short circuit current (Isc) were low in the three segments studied, whilst tissue conductance (Gt) was high. A net active transport of Na+ and Cl- was observed in the three segments. Net Na+ absorption was higher across duodenum and ileum than across the colon, while net Cl- absorption was similar in duodenum, ileum and colon. Ouabain virtually abolished Isc, PD and net Na+ and Cl- fluxes in all the segments. Amiloride abolished net Cl- flux in duodenum, ileum and colon, whereas net Na+ flux was abolished in colon but decreased in duodenum and ileum. PD and Isc were not affected by the presence of the diuretic.     

Actions of gastrin-releasing peptide and related mammalian and amphibian peptides on ion transport in the porcine proximal jejunum

The 27-amino acid peptide gastrin-releasing peptide (GRP) and the decapeptide neuromedin B (NMB) are structurally related to bombesin (BB) and exist within the mammalian small intestine. We examined the actions of porcine GRP and NMB on ion transport in the porcine proximal jejunum in vitro and compared their activities to those of their respective C-terminal amphibian homologs BB and ranatensin (RT). The 4 peptides transiently increased potential difference and short-circuit current (Isc) in jejunal mucosal sheets after their serosal administration in subnanomolar concentrations with an order of potency: GRP approximately RT greater than or equal to NMB greater than BB. BB and RT were more effective in elevating Isc than GRP and NMB; all peptides had variable effects on tissue conductance. Mucosal Isc responses to GRP (1 nM) were due in part to a stimulation of net Cl- secretion. GRP-induced Isc increases were halved by serosal furosemide (0.3 mM) and reduced by 65% and 90% in tissue bathing solutions lacking Cl- or Cl- and HCO3-, respectively. Tetrodotoxin reduced Isc responses to the peptide by 40%; GRP activity remained unaffected after blockade of gut muscarinic or nicotinic cholinergic receptors by atropine or hexamethonium, respectively. These results suggest that GRP and its natural homologs stimulate active electrogenic Cl- secretion in the porcine jejunum through interactions with GRP receptors located in the intestinal mucosa and submucosa.     

The role of mitochondria-rich cells in the chloride current conductance across toad skin

In early studies of salt transport across frog and toad skin, it was assumed that chloride movement is extracellular. However, later studies suggested that chloride movement is largely transcellular. Chloride transport across toad skin is greatly diminished in skins of salt-acclimated toads (Bufo viridis) and was correlated with the number of mitochondria-rich (m.r.) cells in the epithelium. The activated chloride conductance could be recovered upon in vitro incubation with theophylline. It was found that the short-circuit current (Isc) and the chloride conductance (Gcl) in toad skin could be separated experimentally by selective use of synthetic oxytocin (Syntocinon) or theophylline, and by substituting impermeable anions for chloride. With the use of the vibrating probe we demonstrated directly that chloride-dependent peak currents are localized only over m.r. cells, under hyperpolarized (V = -100 mV) conditions. It is concluded that the m.r. cells form the principal site for passive chloride movement across amphibian skin. This cellular pathway is regulated through a cyclic AMP-mediated process. It is suggested that the spatial separation of the sodium and chloride channels is essential to maintain the granulosum cells which are engaged in sodium transport hyperpolarized, and thus providing the driving force for the sodium entry into the cells.     

Bioelectric properties and ion flow across excised human bronchi

Bioelectric properties and ion transport of excised human segmental/subsegmental bronchi were measured in specimens from 40 patients. Transepithelial electric potential difference (PD), short-circuit current (Isc), and conductance (G), averaged 5.8 mV (lumen negative), 51 microA X cm-2, and 9 mS X cm-2, respectively. Na+ was absorbed from lumen to interstitium under open- and short-circuit conditions. Cl- flows were symmetrical under short-circuit conditions. Isc was abolished by 10(-4) M ouabain. Amiloride inhibited Isc (the concentration necessary to achieve 50% of the maximal effect = 7 X 10(-7) M) and abolished net Na+ transport. PD and Isc were not reduced to zero by amiloride because a net Cl- secretion was induced that reflected a reduction in Cl- flow in the absorptive direction (Jm----sCl-). Acetylcholine (10(-4) M) induced an electrically silent, matched flow of Na+ (1.7 mueq X cm-1 X h-1) and Cl- (1.9 mueq X cm-12 X h-1) toward the lumen. This response was blocked by atropine. Phenylephrine (10(-5) M) did not affect bioelectric properties or unidirectional ion flows, whereas isoproterenol (10(-5) M) induced a small increase in Isc (10%) without changing net ion flows significantly. We conclude that 1) Na+ absorption is the major active ion transport across excised human bronchi, 2) Na+ absorption is both amiloride and ouabain sensitive, 3) Cl- secretion can be induced by inhibition of the entry of luminal Na+ into the epithelia, and 4) cholinergic more than adrenergic agents modulate basal ion flow, probably by affecting gland output.     

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-.     

The electrical potential difference, short circuit current and Na+ and Cl- transport across different segments of sheep colon

1. The electrical potential difference (pd) and short circuit current (Isc) across the sheep colon descendens was significantly higher than across the sheep colon ascendens. 2. The ion equivalent of the Isc and the net Na+ transport from the mucosal (m) to the serosal (s) side of the short-circuited sheep colon descendens were identical, while the net Na+ transport across the colon ascendens exceeded the ion equivalent of the Isc. 3. There was a net m-s Cl- transport across both short-circuited colon segments, indicating that Cl-, like Na+, is absorbed by active transport. 4. The results suggest that active Na+ transport across the sheep colon descendens occurs entirely by an electrogenic mechanism, whereas active Na+ transport across the sheep colon descendens occurs entirely by an electrogenic mechanism, whereas active Na+ transport across sheep colon ascendens probably occurs by both an electrogenic and an electrically silent mechanism.     

Electrolyte transport across rabbit late proximal colon in vitro

The second part of rabbit proximal colon was investigated in vitro under short circuit conditions. Unidirectional sodium and chloride fluxes were measured during the soft faeces period and during the hard faeces period. Rabbit late proximal colon has a potential difference (psi mS) of 4 mV, a tissue conductance (GT) of 10-11 mS/cm2 and a short circuit current (Isc) of 1.5 mueq/cm2 X hr. Under control conditions sodium (2.65 mueq/cm2 X hr) and chloride (0.67 mueq/cm2 X hr) are absorbed. Ouabain abolished psi ms,Isc and the net sodium flux totally, whereas 0.1 mM amiloride only slightly decreased the net sodium flux. No differences in electrical properties and Na,Cl-fluxes were found between the faeces periods. Removal of sodium abolished psi ms and Isc totally, and a high potassium solution depolarized the preparation (psi ms = 0). A linear current-voltage relation characterizes the tissue as an ohmic resistor between -40 and +50 mV, and reveals a slope conductance of 14 mS/cm2 under KCl conditions. We conclude that the transport functions under in vitro conditions differ markedly from the in vivo situation, and that the diurnal differences of electrolyte transport in vivo occur mainly by the involvement of ionic gradients.     

Ion transport in an immortalized rat submandibular cell line SMG-C6

The immortalized rat submandibular epithelial cell line, SMG-C6, cultured on porous tissue culture supports, forms polarized, tight-junction epithelia facilitating bioelectric characterization in Ussing chambers. The SMG-C6 epithelia generated transepithelial resistances of 956+/-84Omega.cm2 and potential differences (PD) of -16.9 +/- 1.5mV (apical surface negative) with a basal short-circuit current (Isc) of 23.9 +/- 1.7 microA/cm2 (n = 69). P2 nucleotide receptor agonists, ATP or UTP, applied apically or basolaterally induced a transient increase in Isc, followed by a sustained decreased below baseline value. The peak DeltaIsc increase was partly sensitive to Cl- and K+ channel inhibitors, DPC, glibenclamide, and tetraethylammonium (TEA) and was completely abolished following Ca2+ chelation with BAPTA or bilateral substitution of gluconate for Cl-. The major component of basal Isc was sensitive to apical Na+ replacement or amiloride (half-maximal inhibitory concentration 392 nM). Following pretreatment with amiloride, ATP induced a significantly greater Isc; however, the poststimulatory decline was abolished, suggesting an ATP-induced inhibition of amiloride-sensitive Na+ transport. Consistent with the ion transport properties found in Ussing chambers, SMG-C6 cells express the rat epithelial Na+ channel alpha-subunit (alpha-rENaC). Thus, cultured SMG-C6 cells produce tight polarized epithelia on permeable support with stimulated Cl- secretory conductance and an inward Isc accounted for by amiloride-sensitive Na+ absorption.     

The identity of the current carriers in canine lingual epithelium in vitro

Ion transport across the lingual epithelium has been implicated as an early event in gustatory transduction. The fluxes of isotopically labelled Na+ and Cl- were measured across isolated canine dorsal lingual epithelium under short-circuit conditions. The epithelium actively absorbs Na+ and to a lesser extent actively secretes Cl-. Under symmetrical conditions with Krebs-Henseleit buffer on both sides, (1) Na+ absorption accounts for 46% of the short-circuit current (Isc); (2) there are two transcellular Na+ pathways, one amiloride-sensitive and one amiloride-insensitive; (3) ouabain, added to the serosal solution, inhibits both Isc and active Na+ absorption. When hyperosmotic (0.25 M) NaCl is placed in the mucosal bath, both Isc and Na+ absorption increase; net Na+ absorption is at least as much as Isc. Ion substitution studies indicate that the tissue may transport a variety of larger ions, though not as effectively as Na+ and Cl-. Thus we have shown that the lingual epithelium, like other epithelia of the gastrointestinal tract, actively transports ions. However, it is unusual both in its response to hyperosmotic solutions and in the variety of ions that support a transepithelial short-circuit current. Since sodium ion transport under hyperosmotic conditions has been shown to correlate well with the gustatory neural response, the variety of ions transported may likewise indicate a wider role for transport in taste transduction.     

Effect of chloride on pH microclimate and electrogenic Na+ absorption across the rumen epithelium of goat and sheep

Active Na+ absorption across rumen epithelium comprises Na+/H+ exchange and a nonselective cation conductance (NSCC). Luminal chloride is able to stimulate Na+ absorption, which has been attributed to an interaction between Cl-/HCO3- and Na+/H+ exchangers. However, isolated rumen epithelial cells also express a Cl- conductance. We investigated whether Cl- has an additional effect on electrogenic Na+ absorption via NSCC. NSCC was estimated from short-circuit current (Isc) across epithelia of goat and sheep rumen in Ussing chambers. Epithelial surface pH (pHs) was measured with 5-N-hexadecanoyl-aminofluorescence. Membrane potentials were measured with microelelectrodes. Luminal, but not serosal, Cl- stimulated the Ca2+ and Mg2+ sensitive Isc. This effect was independent of the replacing anion (gluconate or acetate) and of the presence of bicarbonate. The mean pHs of rumen epithelium amounted to 7.47 +/- 0.03 in a low-Cl- solution. It was increased by 0.21 pH units when luminal Cl- was increased from 10 to 68 mM. Increasing mucosal pH from 7.5 to 8.0 also increased the Ca2+ and Mg2+ sensitive Isc and transepithelial conductance and reduced the fractional resistance of the apical membrane. Luminal Cl- depolarized the apical membrane of rumen epithelium. 5-Nitro-2-(3-phenylpropylamino)-benzoate reduced the divalent cation sensitive Isc, but only in low-Cl- solutions. The results show that luminal Cl- can increase the microclimate pH via apical Cl-/HCO3- or Cl-/OH- exchangers. Electrogenic Na+ absorption via NSCC increases with pH, explaining part of the Cl- effects on Na+ absorption. The data further show that the Cl- conductance of rumen epithelium must be located at the basolateral membrane.     

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.     

Ionic events during the volume response of human peripheral blood lymphocytes to hypotonic media. II. Volume- and time-dependent activation and inactivation of ion transport pathways

Hypotonic dilution of human peripheral blood lymphocytes (PBL) induces large conductive permeabilities for K+ and Cl-, associated with the capacity of the cells to regulate their volumes. When rapid cation leakage is assured by the addition of the ionophore gramicidin, the behavior of the anion conductance pathway can be independently examined. Using this technique it is demonstrated that the volume- induced activation of Cl- transport is triggered at a threshold of approximately 1.15 X isotonic cell volume. If the volume of a cell is increased to this level or above, the Cl- transport system is activated, whereas if the volume of a swollen cell is decreased below the threshold value, the Cl- transport is inactivated. Activation and inactivation are independent of the relative volume changes and of the actual cellular Na+, K+, or Cl- concentrations, as well as of the changes in membrane potential in PBL. When net salt movement and thus volume change are inhibited by specific blockers of K+ transport (e.g., quinine, or Ca2+ depletion), volume-induced Cl- conductance shows a time-dependent inactivation, with a half-time of 5-8 min. The Cl- conductance, when activated, appears to involve an all-or-none response. In contrast, volume-induced K+ conductance is a graded response, with the increase in K+ flux being roughly proportional to the hypotonicity-induced increase in cell volume. The data indicate that during lymphocyte volume response in hypotonic media, anion conductance increases by orders of magnitude, exceeding the K+ conductance, so that the rate of the volume decrease (KCl efflux) is determined by a graded alteration in K+ conductance. When the cell volume approaches the isotonic value, it is stabilized by the inactivation of the anion conductance pathway.     

Cationophore properties of the new polyether antibiotic Salinomycin investigated in distal rabbit colon in vivo and in vitro

The distal rabbit colon was used as a model to investigate the influence of the cationophore Salinomycin in vivo with a single-pass perfusion, and in vitro with a modified Ussing chamber technique. For in vivo experiments with labelled 14C-PEG as a volume marker in the perfusate, a dose of 10E-4 mol/l Salinomycin was used. Net water (53 microliters/h/cm), net chloride (3 mumol/h/cm) and net sodium (3.6 mumol/h/cm) absorption was not significantly influenced, but net potassium secretion (-3 mumol/h/cm) was decreased to zero and transepithelial potential (PD) reduced from -45 mV to -33 mV. 10E-4 mol/l Salinomycin, applied in vitro on the muscosal side, decreased PD in 80 min and 10E-3 mol/l in 30 min from 18 mV to zero. Both concentrations decreased the short-circuit current (Isc = 77 microA/cm2) in 60 min, respectively 30 min to 40 microA/cm2. After 60 min mucosal 10E-4 mol/l Salinomycin the Isc increased, resulting from a transepithelial conductance (Gt) increase from 3 to 40 mS/cm2. A dose-related effect was present on PD, Isc and Gt at concentrations between 10E-7 and 10E-6 mol/l. The unidirectional 22Na fluxes were increased to 20 times the control values and net Na transport disappeared. We conclude that Salinomycin when applied in usual doses (10E-4 mol/l) as a coccidiostatic feed additive, profoundly affects colonic electrolyte transport.     

Avian colonic ion transport: effects of corticosterone and dexamethasone

1. Corticosterone, a natural corticosteroid hormone in birds, when injected into domestic fowl (Gallus domesticus) (2000 micrograms.kg-1, 4-5 h before experiment) increases both the basal Isc (short-circuit current) and amiloride-sensitive Isc as well as the PD across the colon in vitro. Dexamethasone, a synthetic analogue (650 micrograms.kg-1, 4-5 h before experiment) also increases the basal and amiloride-sensitive Isc as well as PD in these preparations. 2. In marked contrast, longer term injection or infusion of dexamethasone (650 micrograms.kg-1) for 3 or more days caused a decline in basal Isc and PD (the PD often reversed with the serosal side becoming electronegative) and a drop in resistance. However in these preparations, the amiloride-sensitive Isc was significantly elevated which could be accounted for by an increase in net Na flux. 3. No significant change occurs in net flux of Cl or K although unidirectional fluxes in both directions were increased for both ions in birds given dexamethasone for 3 days. 4. A disparity between the basal Isc and the amiloride-sensitive Isc appeared in these preparations from dexamethasone injected birds reflecting the transport of other ions, possibly HCO3- or H+. The possible role of corticosterone in mineral metabolism of birds is discussed.     

Ammonia uptake and its effects on ionoregulation in the freshwater crayfish Pacifastacus leniusculus (Dana)

Exposure of adult crayfish Pacifastacus leniusculus to Artificial Freshwater (AFW) media containing 1.5 m and 0.15 mmol x l(-1) total ammonia [Tamm; 0.1 x acute lethal concentration (24 h LC50) and 0.01 x 24 h LC50] and adjusted to pH 6.5, pH 8.2 and pH 10.5 resulted in significant increases in haemolymph ammonia over a 24-h period. Ammonia accumulated most rapidly at pH 10.5. These media were chosen to expose animals to a range of different un-ionised ammonia (UIA) [NH3] and ionised ammonia [NH4+] concentrations. From comparisons of measured transepithelial potential differences (PDte) with calculated Nernst potentials (PDNH4+) for the known haemolymph-to-medium gradients of [NH4+], it was deduced that, in pH 8.2 and pH 6.5 AFW, NH4+ was not in thermodynamic equilibrium across the integument (presumably gill epithelium). In pH 10.5 AFW with 1.5 mmol x l(-1) Tamm (predominantly NH3), the accumulation of ammonia in the haemolymph was in the NH4+ form due to haemolymph pH regulation by the crayfish in this alkaline external medium. Measured net fluxes of ammonia (Jamm(net)) were inwardly directed and maximal when [NH3] was the main component externally, but were also significant at pH 8.2 with high [NH4+] ([NH4+]:[NH3] approximately 20:1). Haemolymph Na+ depletion was significant and, over the 24-h exposure period, most rapid in high [NH3] medium but [Cl-] was unaffected. However, paradoxically, sodium uptake (measured JNa(in) on immediate transfer to high Tamm medium) was not significantly inhibited when [NH3] was the predominant ammonia species. In 1.5 mmol x l(-1) Tamm (mainly [NH4+), VNa(in) (the active component of JNa(in)) was significantly inhibited, particularly at low external [Na+]. This inhibition could not be demonstrated as one of competition at an Na+/NH4+ apical gill exchange site. The resultant net efflux of sodium from the animal showed that the ability of the animals to balance sodium losses at low external [Na+] was severely affected. Longer exposure to pH 10.5 AFW with high [NH3] (12 h) resulted in significantly increased JNa(out), while not significantly affecting JNa(in). Analysis of urinary Na+ losses showed that, while urinary flow rate and water reabsorption was most likely unaffected by ammonia exposure, final urine [Na+] was significantly elevated. The resulting urinary Na+ loss accounted for 63% of the increased JNa(out) in high [NH3] medium.     

Potassium dependence of sodium transport in frog skin

22Na+ and 42K+ fluxes across the basolateral membrane of the isolated epithelium of frog skin were investigated with regard to dependence on K+ in the basolateral solution. When K+ was removed from the basolateral solution (K+-free Ringer), there was a transient rise in short circuit current (Isc) that could be eliminated by pretreatment with ouabain. Concurrently, the apparent sodium efflux across the basolateral membrane (JNa*13) showed either no change or an immediate (1-2 min) small decrease (approximately equal to 10%) that was followed by a small transient increase. K+ fluxes showed either no change or a small decrease under these conditions. JNa*13 was partially ouabain sensitive during all of the above treatments. Furosemide partially inhibited both sodium and potassium flux after K+-free treatment. The pump, as defined by ouabain sensitivity of Na+ flux, continued to work even after 20 minutes of K+-free treatment. Pump activity may be maintained by potassium leaking from the cells that is recycled by the pump. However, the ouabain-sensitive transient rise in Isc after K+-free treatment cannot readily be explained by changes in either Na+ or K+ flux. A change in pump coupling ratio provides one explanation for these data.