Effects of anion substitution on hydration behavior and water uptake of the red-spotted toad, Bufo punctatus: is there an anion paradox in amphibian skin?

Amphibians absorb water osmotically across their skins and rely on chemosensory information from the skin to assess the suitability of hydration sources. The time spent with skin in contact with a moist surface provides a quantitative measure of their ability to perceive the ionic and osmotic properties of aqueous solutions. Dehydrated toads given hyperosmotic (250 mM) solutions of NaCl or Na-gluconate showed significantly longer periods of hydration behavior on the gluconate solution, but they lost water osmotically when immersed in either solution. Similarly, dehydrated toads given 250 mM solutions of NaCl, Na-acetate, Na-phosphate or Na-gluconate showed a progressively greater length of hydration time on solutions with the larger mol. wt anions. These results are consistent with the chemosensory phenomenon previously described in mammalian tongue as 'anion paradox'. On dilute (50 mM) solutions of NaCl or Na-gluconate, the hydration time was not different between anions, despite toads gaining water more rapidly when immersed in dilute NaCl than in Na-gluconate solutions. The differing behavioral results with hyperosmotic and hypoosmotic salt solutions suggest that chemosensory transduction through toad skin involves both transcellular and paracellular pathways.     

Transcellular and paracellular elements of salt chemosensation in toad skin

Dehydrated toads absorb water by pressing a specialized (seat patch) area of the skin to moist surfaces. This behavior, the water absorption response (WR), is preceded by periods of more limited skin contact (seat patch down, SPD) in which the suitability of the rehydration source is evaluated. WR and SPD behaviors were suppressed on 250 mM NaCl and 200 mM KCl solutions. Ten micromolar amiloride partially restored SPD and WR on NaCl solutions. The addition of 5 mM La(3+) also partially restored the initiation of WR and this effect was additive to the effect of amiloride, suggesting transcellular and paracellular pathways exist in parallel. Similarly, 5 mM La(3+) partially restored the initiation of WR on KCl solutions, to levels comparable to those with K(+)gluconate, suggesting a paracellular pathway for detection of K(+). Hyperosmotic (250 mM) NaCl solutions bathing the mucosal surface rapidly and reversibly increased the paracellular conductance of isolated skin and this increase was partially inhibited by 5 mM La(3+). These results suggest that the regulation of tight junctions has a chemosensory role in toad skin.     

Vascular aspects of water uptake mechanisms in the toad skin: perfusion, diffusion, confusion

Blood cell flow (BCF) in the water absorbing "seat patch" region of toad skin was measured with laser Doppler flow cytometry. BCF of dehydrated toads increased by a factor of 6-8 when water contact was made and declined gradually as toads rehydrated. Water absorption was initially stimulated and declined in parallel with BCF. Water absorption measured during the initial rehydration period did not correlate with BCF and hydrated toads injected with AVT increased water absorption without an increase in BCF indicating the lack of an obligate relation between blood flow and water absorption. Aquaporins 1-3 were characterized by RT-PCR analysis of seat patch skin. AQP 1 was localized in the endothelium of subepidermal capillaries and serves as a pathway for water absorption in series with the apical and basolateral membranes of the epithelium. Dehydrated toads rehydrated more rapidly from dilute NaCl solutions than from deionized water despite the reduced osmotic gradient. BCF of toads rehydrating on 50 mM NaCl was not different than on deionized water and blocking Na+ transport with 100 microM amiloride did not reduce water absorption from 50 mM NaCl. Thus, neither circulation nor solute coupling explains the greater absorption from dilute salt solutions. Rehydration from 10 mM CaCl2 was stimulated above that of DI water by a similar degree as with 50 mM NaCl suggesting the anion might control water permeability of the skin.     

Behavioral and neural responses of toads to salt solutions correlate with basolateral membrane potential of epidermal cells of the skin

Dehydrated toads initiated water absorption response (WR) behavior and absorbed water from dilute NaCl solutions. With 200-250 mM NaCl, WR behavior and water absorption were both suppressed. With 200-250 mM Na-gluconate, WR initiation was significantly greater than with NaCl but water loss was greater. Neural recordings from spinal nerve #6 showed a greater integrated response to 250 mM NaCl than to 250 mM Na-gluconate, whereas a larger rinse response was seen with Na-gluconate. Studies with isolated epithelium showed a large increase in conductance (G(t)) when 250 mM NaCl replaced NaCl Ringer's as the apical bathing solution that was accompanied by depolarization of the transepithelial potential (V(t)) and basolateral membrane potential (V(b)). Depolarization of V(b) corresponded with the neural response to 250 mM NaCl. When 250 mM Na-gluconate replaced Ringer's as the apical solution G(t) remained low, V(b) transiently hyperpolarized to values near the equilibrium potential for K(+) and corresponded with the reduced neural response. These results support the hypothesis that chemosensory function of the skin is analogous to that of mammalian taste cells but utilizes paracellular ion transport to a greater degree.     

Comparison of dehydration and angiotensin II-stimulated cutaneous drinking in toads, Bufo punctatus

Toads (Bufo punctatus) use a sequence of two postures to place the ventral skin on a moist surface and absorb water osmotically. First, the skin contacts the surface (seat patch down, SPD), and then the hindlimbs are abducted to maximize skin contact area (water absorption response, WR). Toads modulated behavior in response to hydration status and osmotic content of the hydration source. Dehydrated toads placed on water displayed both SPD and WR. Hydrated toads injected with angiotensin II (AII) displayed SPD longer than Ringer-injected controls but did not initiate WR and absorbed less water than dehydrated toads. These results suggest that dehydration has a more robust dipsogenic effect than AII. Dehydrated toads placed on 250 mM NaCl briefly initiated SPD but not WR. The addition of amiloride to the hyperosmotic salt solution resulted in brief display of WR but no water loss. Hydrated toads placed on 250 mM NaCl showed shorter periods of SPD behavior. The combination of AII injection and amiloride addition to the salt solution increased SPD initiation but SPD duration was short and water loss was prevented. Neither AII nor dehydration overrides chemosensory mechanisms in the skin that suppress cutaneous drinking from hypertonic solutions.     

Volume regulation in salt-acclimated toad (Bufo viridis): the role of urea and the urinary bladder

Body water (weight) was studied in the euryhaline toad Bufo viridis during high salt (500 mOsm NaCl) acclimation. Plasma osmolality was greatly increased upon salt acclimation mainly by urea, and was always hyperosmotic to the ambient solution. Water content was regulated quite efficiently in slowly acclimated undisturbed toads. Repeatedly catheterized toads behaved like osmometers when transferred to hyperosmotic solutions. Total urea loss was greatly reduced in salt acclimated toads, suggesting urine was not voided under these conditions. It is concluded that urea accumulation, inhibition of the urine voiding response and the urine in the bladder are the principal factors involved in volume regulation under conditions of salt acclimation.     

Lymph osmolality and rehydration from NaCl solutions by toads, Bufo marinus

Toads, Bufo marinus, allowed to maintain an ad libitum state of hydration were dehydrated by 10 15% of their standard weight and allowed to rehydrate from either deionized water or from 10 or 50 mmol l(-1) NaCl solutions. Toads rehydrating from the dilute salt solutions recovered a larger fraction of their standard weight than did toads rehydrating from deionized water despite there being a reduced osmotic gradient. Amiloride did not reduce water gain from these solutions. Water uptake from 100 mmol l(-1) sucrose and 50 mmol l(-1) Na gluconate was reduced relative to deionized water by a fraction predicted from the osmotic gradient. Thus, the presence of both Na+ and Cl- are required for the augmentation of water gain from dilute salt solutions. Toads allowed to rehydrate from 120 mmol l(-1) NaCl for 180 min recovered nearly as much water as toads rehydrating from deionized water for 120 min and the lymph osmolality was not reduced relative to the dehydrated condition. The recovery of water from the salt solution was greater than that predicted from the reduced osmotic gradient and amiloride partially inhibited the rehydration from 120 mmol l(-1) NaCl. Solute coupled water transport can therefore be demonstrated in living animals but only from a NaCl solution that is nearly isoosmotic with the lymph. The mechanism for enhanced water gain from dilute salt solutions remains unresolved.     

The active ion transport properties of canine lingual epithelia in vitro. Implications for gustatory transduction

The electrophysiological properties of the dorsal and ventral canine lingual epithelium are studied in vitro. The dorsal epithelium contains a special ion transport system activated by mucosal solutions hyperosmotic in NaCl or LiCl. Hyperosmotic KCl is significantly less effective as an activator of this system. The lingual frenulum does not contain the transport system. In the dorsal surface it is characterized by a rapid increase in inward current and can be quantitated as a second component in the time course of either the open-circuit potential or short-circuit current when the mucosal solution is hyperosmotic in NaCl or LiCl. The increased inward current (hyperosmotic response) can be eliminated by amiloride (10(-4) M). The specific location of this transport system in the dorsal surface and the fact that it operates over the concentration range characteristic of mammalian salt taste suggests a possible link to gustatory transduction. This possibility is tested by recording neural responses in the rat to NaCl and KCl over a concentration range including the hyperosmotic. We demonstrate that amiloride specifically blocks the response to NaCl over the hyperosmotic range while affecting the KCl response significantly less. The results suggest that gustatory transduction for NaCl is mediated by Na entry into the taste cells via the same amiloride-sensitive pathway responsible for the hyperosmotic response in vitro. Further studies of the in vitro system give evidence for paracellular as well as transcellular current paths. The transmural current-voltage relations are linear under both symmetrical and asymmetrical conditions. After ouabain treatment under symmetrical conditions, the short-circuit current decays to zero. The increase in resistance, though significant, is small, which suggests a sizeable shunt pathway for current. Flux measurements show that sodium is absorbed under symmetrical conditions. Mucosal solutions hyperosmotic in various sugars also induce an amiloride-sensitive inward current. In summary, this work provides evidence that the sodium taste receptor is most probably a sodium transport system, specifically adapted to the dorsal surface of the tongue. The transport paradigm of gustation also suggests a simple model for electric taste and possible mechanisms for sweet taste.     

The spinal nerves innervate putative chemosensory cells in the ventral skin of desert toads, Bufo alvarius

Toads normally obtain water by absorption across their skin from osmotically dilute sources. When hyperosmotic salt solutions are presented as a hydration source to dehydrated desert toads, they place the ventral skin onto the source but soon afterwards escape to avoid dehydration. The escape behavior coincides with neural excitation of the spinal nerves that innervate putative chemosensory cells in the ventral skin. In the present study, fluorescent dye translocated through the spinal nerves to those receptor cells in the epidermis was photoconverted in the presence of 3, 3'-diaminobenzidine tetrahydrochloride for electron-microscopic observation of the cells and associated nerve terminals. Most of the photoconverted cells were located in the deepest layer of the epidermis, with some being in more intermediate layers. No labeled cell was seen in the outermost layer of living cells. In desert toads, flask cells and Merkel cells are occasionally seen in the epidermis. An association of nerve fibers with these epidermal cells has been reported in some species of the anurans. In the present study, however, the cytological features of the photoconverted cells are neither reminiscent of flask cells nor Merkel cells, but are similar to those of surrounding epithelial cells in each layer of the epidermis. We hypothesize a sensory function for these cells, because they have a close association with nerve fibers and participate in the transepithelial transport of salts that must pass through all cell layers of the skin.     

Cutaneous blood flow and water absorption by dehydrated toads

Blood cell flux (BCF) in ventral pelvic skin capillaries was measured in toads, Bufo woodhouseii and Bufo punctatus, using a chamber that allowed hydration behavior and water absorption to be observed concurrently in unrestrained animals. Dehydrated B. woodhouseii and B. punctatus placed on a rehydration solution significantly increased BCF relative to that on a dry surface in less than 2 min. Skin contact with a rehydration solution rather than dehydration alone is the primary stimulus for increased seat patch blood flow. In B. woodhouseii, the water absorption response was initiated after the increase in BCF had started but before maximum BCF was reached. BCF and water uptake across the ventral skin of both species placed on deionized water were not different from those of toads placed on 50 mM NaCl. Similarly, no significant correlation between BCF and rate of water uptake could be observed in dehydrated toads of either species. Angiotensin II (AII) injection in hydrated B. punctatus had no effect on BCF, suggesting that factors other than AII are responsible for the increase in blood flow upon water contact in dehydrated toads.     

The effect of salt acclimation of the water uptake and osmotic permeability of the skin of the toad (Bufo viridis, L.)

1. Water uptake in vivo, and water fluxes across the isolated skin were studied in salt (NaCl) acclimated toads. 2. Water uptake of acclimated toads maintained in the solution of acclimation, decreased with the environmental salinity. 3. The osmotic water permeability (Pos) of the skin increased upon salt (NaCl) acclimation, both in vivo and in vitro. 4. Pos of the skin of toads acclimated to non-permeant solutes such as sucrose (230 mmol/l) or mannitol (400 nmol/l), was greatly reduced. 5. Oxytocin (syntocinon) increased the Pos both in tap water and salt acclimated toads. In high salt (greater than 200 mmol/l NaCl) acclimated toads however, the increased Pos and water flux at larger osmotic gradients, could not be stimulated further by the hormone. 6. The adaptive nature of the selective changes in the permeability properties of the skin under salt acclimation conditions is discussed.     

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.     

Ionic conductance pathways in the mouse medullary thick ascending limb of Henle. The paracellular pathway and electrogenic Cl- absorption

Net Cl- absorption in the mouse medullary thick ascending limb of Henle (mTALH) involves a furosemide-sensitive Na+:K+:2 Cl- apical membrane symport mechanism for salt entry into cells, which occurs in parallel with a Ba++-sensitive apical K+ conductance. The present studies, using the in vitro microperfused mouse mTALH, assessed the concentration dependence of blockade of this apical membrane K+-conductive pathway by Ba++ to provide estimates of the magnitudes of the transcellular (Gc) and paracellular (Gs) electrical conductances (millisiemens per square centimeter). These studies also evaluated the effects of luminal hypertonicity produced by urea on the paracellular electrical conductance, the electrical Na+/Cl- permselectivity ratio, and the morphology of in vitro mTALH segments exposed to peritubular antidiuretic hormone (ADH). Increasing luminal Ba++ concentrations, in the absence of luminal K+, produced a progressive reduction in the transcellular conductance that was maximal at 20 mM Ba++. The Ba++-sensitive transcellular conductance in the presence of ADH was 61.8 +/- 1.7 mS/cm2, or approximately 65% of the total transepithelial conductance. In phenomenological terms, the luminal Ba++-dependent blockade of the transcellular conductance exhibited negative cooperativity. The transepithelial osmotic gradient produced by luminal urea produced blebs on apical surfaces, a striking increase in shunt conductance, and a decrease in the shunt Na+/Cl- permselectivity (PNa/PCl), which approached that of free solution. The transepithelial conductance obtained with luminal 800 mM urea, 20 mM Ba++, and 0 K+ was 950 +/- 150 mS/cm2 and provided an estimate of the maximal diffusion resistance of intercellular spaces, exclusive of junctional complexes. The calculated range for junctional dilution voltages owing to interspace salt accumulation during ADH-dependent net NaCl absorption was 0.7-1.1 mV. Since the Ve accompanying ADH-dependent net NaCl absorption is 10 mV, lumen positive, virtually all of the spontaneous transepithelial voltage in the mouse mTALH is due to transcellular transport processes. Finally, we developed a series of expressions in which the ratio of net Cl- absorption to paracellular Na+ absorption could be expressed in terms of a series of electrical variables. Specifically, an analysis of paired measurement of PNa/PCl and Gs was in agreement with an electroneutral Na+:K+:2 Cl- apical entry step. Thus, for net NaCl absorption, approximately 50% of Na+ was absorbed via a paracellular route.     

Mechanisms of hyperosmotic acclimation in Xenopus laevis (salt,urea or mannitol)

The acclimation of the clawed toad Xenopus laevis to hyperosmotic solutions of NaCl (balanced solution of sea salt), urea or mannitol was studied. The animals could not be acclimated to salt solutions more concentrated centrated than 400 mosm·l^-1. Urea was tolerated till 500 mmol·l^-1. Plasma osmolality was always hyperosmotic to the environmental solution, but with diminished osmotic gradient at the highest tolerated solutions. Plasma urea concentration approached 90 mmol·l^-1, similar in the three solutions of acclimation. Urine volume was very small under all conditions. Serum aldosterone and corticosterone did not differ significantly, although there was a slight tendency towards lower aldosterone in the NaCl solution. In vivo water uptake in tap water acclimated animals was very small, and was higher in the other groups. Only the salt- and urea-acclimated, but not the tap water and mannitol-acclimated groups responded with a clear increase following injection of oxytocin or theophylline. In vitro urea fluxes were similar and invariable in both directions under all conditions. No significant effect of theophylline was observed. Sodium transport measured by the short-circuit technique in vitro was lower in salt- and mannitol-acclimation conditions, and was stimulated significantly under all conditions in response to serosal oxytocin or theopylline. It is concluded that Xenopus laevis can osmoregulate at a limited range of external solutions. It is limited in the increase of its plasma urea concentration; the transport properties of the skin do not change very much upon acclimation, except for the hydroosmotic response to oxytocin.Abbreviations I _sc short circuit current - PD potential difference - SW balanced sea water - TW tap water     

Effects of hyperosmotic stress on cultured airway epithelial cells

Inhalation of hyperosmotic solutions (salt, mannitol) has been used in the treatment of patients with cystic fibrosis or asthma, but the mechanism behind the effect of hyperosmotic solutions is unclear. The relation between osmolarity and permeability changes was examined in an airway cell line by the addition of NaCl, NaBr, LiCl, mannitol, or xylitol (295–700 mOsm). Transepithelial resistance was measured as an indicator of the tightness of the cultures. Cell-cell contacts and morphology were investigated by immunofluorescence and by transmission electron microscopy, with lanthanum nitrate added to the luminal side of the epithelium to investigate tight junction permeability. The electrolyte solutions caused a significant decrease in transepithelial resistance from 450 mOsm upwards, when the hyperosmolar exposure was gradually increased from 295 to 700 mOsm; whereas the nonelectrolyte solutions caused a decrease in transepithelial resistance from 700 mOsm upwards. Old cultures reacted in a more rigid way compared to young cultures. Immuno-fluorescence pictures showed weaker staining for the proteins ZO-1, claudin-4, and plakoglobin in treated samples compared to the control. The ultrastructure revealed an increased number of open tight junctions as well as a disturbed morphology with increasing osmolarity, and electrolyte solutions opened a larger proportion of tight junctions than nonelectrolyte solutions. This study shows that hyperosmotic solutions cause the opening of tight junctions, which may increase the permeability of the paracellular pathway and result in increased transepithelial water transport. This study was supported by the Swedish Asthma and Allergy Association and the Swedish Heart Lung Foundation.     

Water potential receptors in the skin regulate blood perfusion in the ventral pelvic patch of toads

Blood cell flux (BCF) in ventral pelvic skin capillaries was measured in conscious unrestrained Bufo bufo, using a laser Doppler flowcytometer. Hydrated toads responded to water contact with a small but significant increase in BCF. Dehydration alone did not change the BCF in seat patch skin before water contact. However, water contact by dehydrated toads elicited a rapid 600% increase in BCF. The BCF and water uptake of dehydrated toads rehydrating in water declined over 2 h but remained significantly above the low, constant values measured in hydrated toads. Arginine vasotocin injection in hydrated toads did not change skin BCF, but water uptake increased, and urine production decreased. Injection of the beta -adrenergic agonist isoproterenol increased BCF in hydrated toads by 900% and also increased the rate of water uptake. These increases corresponded in magnitude and duration to the response to water contact observed in dehydrated toads. Injection of dehydrated toads with the beta -adrenergic antagonist propranolol significantly reduced both BCF and water uptake. These results are consistent with an autonomic reflex mediated by skin water potential receptors that regulate blood perfusion of ventral pelvic skin.     

Effect of temperature, light and salinity on seed germination and radicle growth of the geographically widespread halophyte shrub Halocnemum strobilaceum

BACKGROUND AND AIMS: The small leafy succulent shrub Halocnemum strobilaceum occurs in saline habitats from northern Africa and Mediterranean Europe to western Asia, and it is a dominant species in salt deserts such as those of north-west China. The effects of temperature, light/darkness and NaCl salinity were tested on seed germination, and the effects of salinity were tested on seed germination recovery, radicle growth and radicle elongation recovery, using seeds from north-west China; the results were compared with those previously reported on this species from 'salt steppes' in the Mediterranean region of Spain. METHODS: Seed germination was tested over a range of temperatures in light and in darkness and over a range of salinities at 25 degrees C in the light. Seeds that did not germinate in the NaCl solutions were tested for germination in deionized water. Seeds from which radicles had barely emerged in deionized water were transferred to NaCl solutions for 10 d and then back to deionized water for 10 d to test for radicle growth and recovery. KEY RESULTS: Seeds germinated to higher percentages in light than in darkness and at high than at low temperatures. Germination percentages decreased with an increase in salinity from 0.1 to 0.75 M NaCl. Seeds that did not germinate in NaCl solutions did so after transfer to deionized water. Radicle elongation was increased by low salinity, and then it decreased with an increase in salinity, being completely inhibited by > or = 2.0 M NaCl. Elongation of radicles from salt solutions < 3.0 M resumed after seedlings were transferred to deionized water. CONCLUSIONS: The seed and early seedling growth stages of the life cycle of H. strobilaceum are very salt tolerant, and their physiological responses differ somewhat between the Mediterranean 'salt steppe' of Spain and the inland cold salt desert of north-west China.     

Effect of salt acclimation on digitalis-like compounds in the toad

Digitalis-like compounds (DLC) were shown to be a normal constituent of the skin and plasma of toads. In order to assess the possible physiological role of these compounds in the toad, their levels were determined in the brain, plasma and skin following acclimation in different NaCl solutions. We demonstrate that an increase in salt concentrations in the animal medium from 0 to 1.2% decreased the levels of DLC in the brain by 50% without altering significantly its levels in the plasma and skin. An increase in medium salt concentration to 1.5% resulted in a 50% increase of DLC levels in the skin without changing its levels in the plasma or brain. These results suggest that skin and brain DLC may participate in the long-term salt and water homeostasis in the toad, while the plasma compound either participates in the short-term regulations of salt and water homeostasis or have some other, unknown, function.     

Hyperosmotic instillation of rat stomach.

Instillation of equally hyperosmotic solutions (1000 m0sm/kg) of NaCl, KCl, MgCl₂, or dextrose depressed the electrical potential difference and the spontaneous acid secretion of rat stomachs. Luminal osmolality declined only slightly after hyperosmotic dextrose instillation, but loss of luminal chloride followed hyperosmotic salt instillation. Comparable losses of luminal sodium or potassium were observed. These changes result from a hyperosmotic alteration of ion transport through the gastric mucosa.