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.     

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.     

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.     

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.     

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.     

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.     

Chemosensory function of salt and water transport by the amphibian skin

Solute and water transport mechanisms of anuran skin mediate chemosensory functions that permit evaluation of ionic and osmotic properties of hydration sources in a manner similar to taste receptors in the mammalian tongue. Histochemical observations demonstrated apparent connections between spinal nerve endings and epithelial cells of the skin and we used neural and behavioral responses as measures of coupling between transport and chemosensation. The inhibition of transcellular Na+ transport by amiloride partially reduced the neural response and the avoidance of hyperosmotic NaCl but not KCl solutions. Cetylpyridinium chloride (CPC) reduced the neural response to hyperosmotic salt solutions, suggesting a chemosensory role for vanilloid receptors in the skin. Avoidance of hyperosmotic salt solutions was reduced by impermeant anions suggesting paracellular conductance is important for chemosensation. The effects of blocking the transcellular and paracellular pathways was additive but did not eliminate the avoidance of osmotically unfavorable solutions by dehydrated toads. The timing of the neural response to deionized water was similar to the onset of water absorption behavior and increased blood flow to the pelvic skin. Water absorption from 50 mM NaCl was greater than from deionized water when toads were fully immersed, but not when contact was limited to the ventral surface.     

Effects of dehydration on plasma osmolality, thirst-related behavior, and plasma and brain angiotensin concentrations in Couch's spadefoot toad, Scaphiopus couchii

Under dehydrating conditions, many terrestrial vertebrates species exhibit increases in plasma osmolality and their drinking behavior. Under some circumstances, this behavioral change is accompanied by changes in plasma and central angiotensin concentrations, and it has been proposed that these changes in angiotensin levels induce the thirst-related behaviors. In response to dehydration, the spadefoot toad, Scaphiopus couchii, exhibits thirst-related behavior in the form of cutaneous drinking. This behavior has been termed water absorption response (WR) behavior. Spadefoot toads live in harsh desert environments and are subject annually to dehydrating conditions that may induce thirst-related behavior. We tested the hypothesis that an increase in WR behavior is associated with both an increase in plasma osmolality and an increase in plasma and brain angiotensin concentrations. First, we determined the degree of dehydration that was necessary to initiate WR behavior. Animals dehydrated to 85% of their standard bladder-empty weight via deprivation of water exhibited WR behavior more frequently than control toads left in home containers with water available. Next, using the same dehydration methods, we determined the plasma osmolality and sodium concentrations of dehydrated toads. Toads dehydrated to 85% standard weight also had a significant increase in plasma osmolality, but exhibited no overall change in plasma sodium concentrations, indicating that while an overall increase in plasma osmolality appears to be associated with WR behavior in S. couchii, changes in sodium concentrations alone are not sufficient to induce the behavior. Finally, plasma and brain angiotensin concentrations were measured in control toads and toads dehydrated to 85% standard weight. Plasma and brain angiotensin concentrations did not increase in dehydrated toads, indicating that dehydration-induced WR behavior that is associated with changes in plasma osmolality may not be induced by changes in endogenous angiotensin concentrations in S. couchii.     

Short-term changes in natripheric and hydrosmotic water fluxes across the skin and in urine production due to increases in the osmolarity of the external environment in the toad

1. Sudden decreases in the osmotic gradient across the skin due to the replacement of water of the bath by 115 mM NaCl had no effect on water uptake of intact or hypophysectomized toads. 2. A concomitant decrease in the urine production was observed in intact but not in hypophysectomized animals. 3. Addition of amiloride chlorydrate (0.25 mM) to the 115 mM NaCl bath induced a significant decrease in water uptake both in intact and in hypophysectomized toads. 4. The osmotic permeability coefficient (LPD) increased significantly during the osmotic gradient reduction with 115 mM NaCl plus 0.25 mM amiloride or 230 mM sucrose in both groups. 5. No changes in the plasmatic osmolarity were detected during the development of these responses to the osmotic gradient reduction. 6. These results are consistent with the hypothesis of short-term changes in the natripheric and hydrosmotic fluxes of water across the skin and in urine production triggered by the osmotic gradient reduction. The possible participation of arginine vasotocin in these responses is discussed.     

Elevated plasma osmotic concentration stimulates water absorption response in a toad.

The water-seeking behavior (WR) of toads (Bufo viridis) was investigated. Fully hydrated toads that are allowed free choice of wet or dry filter paper voluntarily and spontaneously select to sit on water-soaked paper at a regular frequency during trials. Dehydration of bladder-emptied toads by 14% elicits WR in all animals. Injection of aldosterone or angiotensin-I reduced the dehydration threshold to 7% weight loss. WR frequency increased when plasma osmolality was elevated by injection of NaCl or other solutes (both ionic and non-ionic). Only urea, to which cell membranes are highly permeable, was the exception that did not produce this response. The increase in WR frequency induced by elevated plasma osmolality was augmented by injection of aldosterone or angiotensin-I. In vivo water uptake, measured in a water bath, was increased by an NaCl or oxytocin injection, but not by aldosterone. It is concluded that elevated plasma osmolality induces an increase in WR frequency that is separate and prior to the water uptake process. Different hormones are involved in each step.     

Angiotensin II elicits water seeking behavior and the water absorption response in the toad Bufo bufo

Fully hydrated toads, Bufo bufo were acclimated to a simulated terrestrial habitat, with access to shelters and water. To get from the shelters to the water, the toads had to walk across the pan of an Ohaus balance and the body weights were recorded on a computer. Toads were placed inside shelters immediately following injection of human angiotensin II (A II), Thr(8)-saralasin, or Ringer's in the dorsal lymph sac, and their behavior was recorded continuously by video surveillance. The injection doses were 1-100 microg/100 g body weight A II and 100 microg/100 g body weight saralasin dissolved in 0.1 ml Ringer's; control animals received the same volume of Ringer's. The latency from injection to the initiation of water absorption behavior (WR) was significantly shorter in both A-II- and saralasin-injected toads, compared to controls. A-II- and saralasin-injected toads also spent significantly more time in the water than controls. The bladder depots when WR was terminated were significantly larger in A-II- or saralasin-injected toads than in controls. The stimulatory action of Thr(8)-saralasin, an antagonist of A II in mammals, on WR behavior in B. bufo suggests differences in receptor structure and/or receptor distribution between amphibians and mammals.     

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.     

Intra- and extracellular dehydration has no effect on plasma levels of angiotensin II in an amphibian

Previous studies have demonstrated that both dehydration (intra and extracellular) and treatment with angiotensin II (A-II) induce changes in thirst-related behavior in the spadefoot toad, Scaphiopus couchii. One of the steps in determining a causal relationship between a hormone and a behavior is to determine that there is association between an animal's performance of the behavior and changes in endogenous hormonal concentrations. The hypothesis tested that plasma levels of the peptide hormone A-II would change as a result of dehydration known to induce water absorption response (WR) behavior in the spadefoot toad. Plasma samples were taken from toads dehydrated intracellularly by injection of hypertonic solutions of NaCl or sucrose at levels known to induce WR behavior. As an osmotic control, a group of animals was injected with urea, which has been demonstrated to not induce WR behavior. In order to determine the effects of extracellular dehydration on plasma, A-II levels in toads dehydrated by plasma volume depletion via cardiac puncture were compared to sham-punctured controls. None of the treatments in any experiment resulted in significant differences in plasma levels of angiotensin II among groups sampled at the time when WR behavior occurs. These results do not support the hypothesis that dehydration-induced thirst is stimulated by changes in plasma A-II concentrations at the onset of WR behavior. J. Exp. Zool. 286:343-349, 2000.     

Effects of angiotensin II and bladder condition on hydration behavior and water uptake in the toad,Bufo woodhousei

• 1.1. Water absorption response (WR) behavior and water weight gain were examined in hydrated toads, Bufo woodhousei, treated with angiotensin II (All) or with a control Ringer's solution. The effects of urinary bladder condition (ad lib. bladder urine or empty bladder) were examined concurrently.
• 2.2. Toads treated with All (100μg/100g body weight), spent more time in WR posture and absorbed more water than Ringer's-injected toads.
• 3.3. Toads with empty bladders maintained WR posture for longer periods of time and gained more weight than toads whose bladders were not emptied.
• 4.4. The effects of All and bladder urine on water absorption by B. woodhousei appear to be separate and additive.

     

Behavioral, molecular and integrative mechanisms of amphibian osmoregulation

Amphibian water balance has been studied at many levels of biological order. Terrestrial species must react to environmental cues that relate to water availability while some arboreal species have cutaneous skin secretions that can reduce evaporative water loss. The Indian tree frog. Polypedates maculatus, uses cutaneous secretions and wiping behavior to lower evaporation but also relies on moist microclimates to endure prolonged survival away from water. The related species, P. leucomystax, inhabits wetter forest habitats. Preliminary studies with this species are unable to demonstrate the expression of wiping behavior, indicating that arid habitats may be a powerful selective force for this behavior. Laboratory experiments on rehydrating toads in the genus Bufo indicate that animals are able to detect changes in barometric pressure and humidity that might result in the availability of water under field situations. Experiments with Bufonid species and with spadefoot toads, Scaphiopus couchi, show that the peptide hormone, angiotensin II, stimulates cutaneous drinking in a similar manner seen for oral drinking by other vertebrate classes. Amphibian tissues have long been used as a model for the study of basic physiological principles of epithelial ion and water transport. Recent progress with tissue cultures has provided information on the molecular structure of ion and water channels that can be applied to obtain a better understanding, at the molecular level, of ion and water balance strategies used by the wide variety of amphibian species. Terrestrial amphibians are more tolerant of dehydration than are other vertebrates and are able to store dilute urine in their urinary bladder. Toads appear to be able to detect the presence of water in their bladders in addition to the availability of water in their environment. Dehydrated toads are able to rehydrate very rapidly by the coordination of behavioral and physiological mechanisms to enhance cutaneous water absorption. The integration of behavior with cutaneous water gain, renal handling of ions and water and the role of the lymphatic system in overall water balance involves complex interactions between neural and hormonal factors. Experiments are summarized that describe the contribution of individual factors however much more information is needed before the nature of these interactions are fully understood.     

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.     

Skin resistance to water gain and loss has changed in cane toads (Rhinella marina) during their Australian invasion

The water‐permeable skin of amphibians renders them highly sensitive to climatic conditions, and interspecific correlations between environmental moisture levels and rates of water exchange across the skin suggest that natural selection adapts hydroregulatory mechanisms to local challenges. How quickly can such mechanisms shift when a species encounters novel moisture regimes? Cutaneous resistance to water loss and gain in wild‐caught cane toads (Rhinella marina) from Brazil, USA (Hawai''i) and Australia exhibited strong geographic variation. Cutaneous resistance was low in native‐range (Brazilian) toads and in Hawai''ian populations (where toads were introduced in 1932) but significantly higher in toads from eastern Australia (where toads were introduced in 1935). Toads from recently invaded areas in western Australia exhibited cutaneous resistance to water loss similar to the native‐range populations, possibly because toads are restricted to moist sites within this highly arid landscape. Rates of rehydration exhibited significant but less extreme geographic variation, being higher in the native range than in invaded regions. Thus, in less than a century, cane toads invading areas that impose different climatic challenges have diverged in the capacity for hydroregulation.     

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.     

Toads in the backyard: why do invasive cane toads (Rhinella marina) prefer buildings to bushland?

Like many invasive species, cane toads (Rhinella marina) in Australia concentrate in the disturbed habitats created by human activity, rather than in pristine areas. We surveyed cane toads in the wet–dry tropics of the Northern Territory to assess the abundances, body sizes, sexes, behaviour, hydration state and feeding rates of toads around buildings compared to those in areas remote from buildings, and conducted experimental trials to assess the effects of building-related variables (lights and increased toad densities) on the foraging success of toads. Toads around buildings were smaller than bushland conspecifics, and adult sex-ratios were female-biased. Toads were more sedentary around buildings than in the bush, but their feeding rates (based on direct observations and faeces production post-capture) were similar. That similarity, despite twofold-higher densities of competing toads around building, reflected the strong enhancement of feeding rates due to artificial lights attracting insects (in our experimental trials, a threefold increase regardless of the number of competing toads). Toads collected from around buildings were apparently in better hydric condition. Thus, access to water also may attract toads to buildings. The relative scarcity of adult male toads around buildings likely reflects waterbody-centred reproductive activities, whereas the concentration of females and juveniles around buildings is driven largely by access to the insects attracted by artificial light. We conclude that buildings enhance the persistence of cane toad populations and may facilitate their spread.     

Experimental exposure of Canadian toads to Basidiobolus ranarum

Experimental transmission of the fungus Basidiobolus ranarum was induced in two treatment groups of Canadian toads (Bufo hemiophrys) and caused a fatal mycotic dermatitis. Seven of 10 (70%) toads that had their ventral skin mildly abraded and exposed to B. ranarum developed hyperemia, and sloughing of their ventral skin and died. Toads with abraded ventral skin or exposure to infected skin also were affected statistically at a higher rate than those with abraded skin and exposure to pure cultures of B. ranarum inoculated into their water source. Of toads showing clinical disease, B. ranarum was identified by both impression smears and histology in all cases, but not from toads that appeared clinically healthy. The organism was cultured from 5 of 7 (71%) toads with clinical disease but not from any toad that appeared clinically healthy (n = 28). This study documents methods of experimental transmission of B. ranarum, an organism responsible for causing a mycotic dermatitis that is fatal to toads.