Effect of oxytocin on transepithelial transport of water and Na+ in distinct ventral regions of frog skin (Rana catesbeiana)

Thoracic, abdominal, and pelvic fragments of ventral skin of Rana catesbeiana were analysed regarding the effect of oxytocin on: (1) transepithelial water transport; (2) short-circuit current; (3) skin conductance and electrical potential difference; (4) Na⁺ conductance and electrical potential difference; (4) Na⁺ conductance, the electromotive force of Na⁺ transport mechanism, and shunt conductance; (5) short-circuit current responses to fast Na⁺ by K⁺ replacement in the outer compartment, and (6) epithelial microstructure. Unstimulated water and Na⁺ permeabilities were low along the ventral skin. Hydrosmotic and natriferic responses to oxytocin increased from thorax to pelvis. Unstimulated Na⁺ conductance was greater in pelvis than in abdomen, the other electrical parameters being essentially similar in both skin fragments. Contribution of shunt conductance to total skin conductance was higher in abdominal than in pelvic skin. Oxytocin-induced increases of total skin conductance, Na⁺ conductance, and shunt conductance in pelvis were significantly larger than in abdomen. An oscillatory behaviour of the short-circuit current was observed only in oxytocin-treated pelvic skins. Decrease of epithelial thickness and increase of mitochondria-rich cell number were observed from thorax to pelvis. Oxytocin-induced increases of interspaces were more conspicuous in pelvis and abdomen than in thorax.Abbreviations E _Na electromotive force of sodium transport mechansim - G _KCI skin conductance with external KCI Ringer - G _Na sodium conductance (series conductance) - G _shunt shunt pathway conductance - G _total total skin conductance - J _v water flux (in units of volume per area per time) - MRC mitochondria-rich cells - PD potential difference across skin - R _shunt resistance of the shunt pathway - SCC short-circuit current     

Development of aldosterone-stimulation of short-circuit current across larval frog skin

Summary The short-circuit current (SCC) across isolated skin from bullfrog larvae in developmental stage XXI was small and insensitive to amiloride. Overnight incubation of this tissue with 10^-6 M aldosterone stimulated the SCC from 1.35±0.55 to 14.55±4.12 A·cm^-2 with 11.18±4.46 A·cm^-2 being blocked by 100 M amiloride. Histologic examination of aldosterone-treated skins revealed a separation of the apical cell layer from the underlying epidermis that was not seen in untreated preparations. The onset of amiloride-sensitive Na⁺ transport thus coincided with the exposure of the apical surface of newly differentiated epithelial cells. Similar results were obtained with skin from stage XXI larvae whose rate of metamorphosis had been stimulated by 10 g·l^-1 thyroxine (T₄) but not with skin from T₄-treated larvae in stages XIX and XX. Fluctuation analysis of the amiloride-sensitive SCC of the above preparations failed to show a consistent Lorentzian component in the power-density spectrum. Fluctuation analysis was possible on skins from larvae whose development had been accelerated by 7–9 days treatment with 10 g·l^-1 triiodothyronine (T₃). Aldosterone treatment of these tissues resulted in a significant increase in Na⁺ channel density.Abbreviations ASCC component of the short-circuit current (A·cm^-2) that is blocked by amiloride - fc frequency (Hz) at which the magnitude of the Lorenzian component of the power spectra is reduced by half - i current (pA) through individual amiloride-sensitive Na⁺ channels - I _Na+ amiloride-sensitive short-circuit current (A·cm^-2) that remains after treatment with a given amiloride concentration - k ₀₁ the rate constant (s^-1·M^-1) for the association of amiloride with Na⁺ channels - k ₁₀ rate constant (s^-1) for the dissociation of amiloride from Na⁺ channels - K _b magnitude of the power spectrum (A²·s·cm^-2) at a frequency of 1 Hz - KSCC short-circuit (A·cm^-2) current with K⁺ as the primary mucosal cation - M density of amiloride-sensitive Na⁺ channels in the apical cell membrane - SCC short-circuit current (A·cm^-2) - S _(f) magnitude of the power spectra (A²·s·cm^-2) at a given frequency - S ₀ the magnitude of the plateau region of the Lorentzian component of the power spectra (A²·s·cm^-2) - T ₃ Triiodothyronine - T ₄ Thyroxine     

Calcium regulation of nucleocytoplasmic transport

Bidirectional trafficking of macromolecules between the cytoplasm and the nucleus is mediated by the nuclear pore complexes (NPCs) embedded in the nuclear envelope (NE) of eukaryotic cell. The NPC functions as the sole pathway to allow for the passive diffusion of small molecules and the facilitated translocation of larger molecules. Evidence shows that these two transport modes and the conformation of NPC can be regulated by calcium stored in the lumen of nuclear envelope and endoplasmic reticulum. However, the mechanism of calcium regulation remains poorly understood. In this review, we integrate data on the observations of calcium-regulated structure and function of the NPC over the past years. Furthermore, we highlight challenges in the measurements of dynamic conformational changes and transient transport kinetics in the NPC. Finally, an innovative imaging approach, single-molecule super-resolution fluorescence microscopy, is introduced and expected to provide more insights into the mechanism of calcium-regulated nucleocytoplasmic transport.     

Energetics of metamorphic climax in the pickerel frog (Lithobates palustris)

Anuran metamorphosis, the transition from aquatic larvae to terrestrial juveniles, is accompanied by significant morphological, physiological, and behavioral changes. Timing of metamorphosis and final size, which can influence adult fitness, may depend on sufficient energy accumulated during the larval period to support metamorphosis. However, only two species of anurans have been examined for energetic costs of metamorphosis, Rana tigrina and Anaxyrus terrestris. Based on these species, it has been hypothesized that differences in energy expenditure are related to duration of metamorphosis. To compare energetic costs of metamorphosis among species and examine this hypothesis, we quantified the total energy required for metamorphosis of Lithobates palustris tadpoles by measuring oxygen consumption rates over the duration of metamorphic climax using closed-circuit respirometry. Total energy costs for L. palustris were positively related to tadpole mass and duration of metamorphic climax. However, larger tadpoles completed metamorphosis more efficiently because they used proportionally less total energy for metamorphic climax than smaller counterparts. Costs were intermediate to R. tigrina, a larger species with similar metamorphic duration, and A. terrestris, a smaller species with shorter metamorphic climax. The results supported the hypothesis that amphibian species with more slowly developing tadpoles, such as ranids, require more absolute energy for metamorphosis in comparison to more rapidly developing species like bufonids.     

Benzodiazepines stimulate sodium ion transport in frog skin epithelium

Benzodiazepine binding sites are present in a variety of non-neuronal tissues including the kidney where they are localized to distal nephron segments. It is postulated that renal binding sites are involved in modulating ion transport. This study examined the effects of two benzodiazepines on sodium transport in frog skin epithelium, a model system for sodium transport in renal collecting duct. Treatment of short-circuited frog skin with diazepam (a non-selective benzodiazepine agonist) stimulated amiloride-sensitive short-circuit current, reflecting stimulation of active sodium transport. The diazepam response was equally effective with either serosal or mucosal application of the drug. Maximal stimulation of the current (42 +/- 8%) was achieved with 10 microM diazepam (serosal). Short-circuit current was similarly augmented by serosal or mucosal addition of Ro5-4864, a benzodiazepine agonist with selective activity at peripheral (non-neuronal) receptors. The natriferic response to diazepam was additive to that of vasopressin or cyclic AMP suggesting that the mode of action of benzodiazepines is probably distinct from the cyclic AMP pathway. Thus, frog skin appears to be a useful model to examine the epithelial effects of benzodiazepines. Whether stimulation of sodium transport, however, involves peripheral-type benzodiazepine receptors in this tissue requires further studies.     

Calcium role in base-line and ADH-stimulated sodium transport in frog skin

Treatment of ventral frog skin with serosal A23187 calcium ionophore caused an initially transient increase in transepithelial sodium transport. After 60 min of treatment with A23187, a steady-state transport value was reached which was significantly lower than the initial one. Furthermore, it was found that ionophore treatment greatly inhibited the natriferic response to ADH and to 8br-cAMP. A further analysis on the possible ionophore action mechanism was carried out through pretreatment of the skin with indomethacin, very powerful prostaglandin synthesis inhibitor. In the experimental conditions reported, A23187 seems no longer capable of inducing a transient increase in sodium transport, although it does inhibit the natriferic response to ADH.     

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 bathing 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 Cl^? were increased. The movement of Cl^? was evaluated by comparison of Cl^? 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 on Na⁺ transport.     

Odd behaviour of Li+ in frog skin ion transport

1. Frog skin epithelium has basolateral K+ channels that normally define the basolateral membrane potential between 80 and 100 mV. 2. The membrane mentioned also has almost silent chloride channels and a [Na+, K+, 2Cl-] cotransport, the latter probably maintains the high Cl- in the capital (also called syncytium) cells. 3. If the K+ channels are blocked by Ba2+ (or Li+) it is possible to demonstrate potential gating of the chloride channels of the basolateral membrane. 4. When the normal K+ channels are blocked, a potential-dependent K+ conductance slowly emerges. 5. If Li+ is substituted for outside Na+ the skin shows potential oscillations of about 40 mV at a frequency of about six per hour. 6. The anion channel inhibitor Indacrinone stops these oscillations. 7. The role of Cl- and K+ channels in these oscillations is discussed. 8. The transepithelial inward transport of Li+ requires the presence of Na+ and seems to be due to exchange of cellular Li+ against inside Na+ via the basolateral Na+/H+ exchanger.     

Automatic recording of electric potential and ion transport in frog skin

Summary An apparatus for the automatic recording of the bioelectric potential of isolated frog skin and of the short-circuit current, which is a measure of active Na⁺ transport, is described. The equivalence of the uninterrupted short-circuit current and active Na⁺ transport has been checked. Current and Na⁺ transport agreed within 2%.The automatic ion transport recorder is particularly suited in studies where the exact time course of the effects of enzyme inhibitors, drugs and other chemicals on skin is of interest. This is illustrated by showing the effects of fluoroacetate, quinone and hydroquinone on spontaneous skin potential and short-circuit current.Attention is called to characteristic transients in the potential records which are probably related to changes in skin permeability to passively moving chloride ions.Supported by Public Health Grant RG 3545.     

Kinetics of ionic transport across frog skin: Two concentration-dependent processes

Summary Sodium and chloride influxes across the nonshort-circuited isolated skin ofRana esculenta were measured at widely varying external ionic concentrations.The curve describing sodium transport has two Michaelis-Menten components linked at an inflection point occurring at an external sodium concentration of about 7 meq. Chloride transport can also be represented by two saturating components. A possible explanation of these kinetics is discussed.At sodium concentrations lower than 4 meq it is possible to define a component of the sodium transport mechanism as having a high affinity for sodium and which is independent of the nature of the external anion. A high affinity for chloride of the chloride transport system functioning at low external concentrations is also found but is significantly different from that of sodium. These systems show the physiological characteristics of the countertransports (Na _ext ⁺ /H _int ⁺ ; Cl _ext ^– /HCO _3int ^– ) functioning at low external concentrations.At external concentrations higher than 4 meq a low affinity transporting system in which chloride and sodium are linked superimpose on the high affinity components.The physiological significance of these results is discussed.     

A fluctuation analysis study of the development of amiloride-sensitive Na+ transport in the skin of larval bullfrogs (Rana catesbeiana)

In the presence of the Na⁺-channel blocker amiloride, the short-circuit current across the skins of bullfrog tadpoles in metamorphic stages XIX–XXIV was subjected to fluctuation analysis. The resulting power spectra contained a Lorentzian component of which the plateau value ($\text{S}{}_0$) decreased while the corner frequency ($\text{f}{}_{\mathrm{<mtext>c</mtext>}}$) increased as the mucosal amiloride concentration was increased from 0.5 to 24 μM. From the linear relationship between the $\text{f}{}_{\mathrm{<mtext>c</mtext>}}$ values and the amiloride concentrations it was possible to determine the binding ($\text{k′}{}_{01}$) and unbinding ($\text{k}{}_{10}$) constants for amiloride to its receptor on the Na⁺ channel. With these parameters as well as short-circuit current and $\text{S}{}_0$ values, the current through the individual Na⁺ channels ($\text{i}$) was calculated (average 0.58 pA). It did not increase significantly during late metamorphosis. The density of Na⁺ channels ($\text{M}$) in the apical membrane, on the other hand, increased significantly. It would appear that the increase in short-circuit current which occurs at this time is due primarily to an increase in amiloride-blockable Na⁺ channels. Unexpectedly, a Lorentzian component could be fitted to power spectra in amiloride-treated skins (stages XIX–XXI) which showed no amiloride-sensitive short-circuit current. Moreover, the typical increase in $\text{f}{}_{\mathrm{<mtext>c</mtext>}}$ with the amiloride concentration did not occur in these animals.     

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.     

Effect of FeCl3 on ion transport in isolated frog skin

Summary The effect of addition of FeCl₃ to the media bathing the isolated skin ofRana pipiens was studied by measuring short-circuit current, transepithelial potential, and resistance, and by determining the influx and efflux of sodium (J ₁₃ ^Na andJ ₃₁ ^Na , respectively) and the influx and efflux of chloride (J ₁₃ ^Cl andJ ₃₁ ^Cl , respectively) across the epithelium. With normal Ringer's solution on both sides of the skin, addition of 10^–3 m FeCl₃ to the external medium resulted in nearly complete inhibition of active Na transport (J ₁₃ ^Na decreased from 1.30±0.14 to 0.10±0.04 eq/cm² hr (N=8)) and in appearance of active chloride transport in outward direction due to an 80% increase inJ ₃₁ ^Cl . Average (J ₃₁ ^Cl –J ₁₃ ^Cl ) obtained from means of 8 skins in 6 consecutive control and last 3 experimental periods was –0.17±0.04 and 0.38±0.05 eq/cm² hr, respectively. FeCl₃ added to external medium also induced substantial net chloride movement in outward direction when external medium contained Na-free choline chloride Ringer's or low ionic strength solution. Under the latter condition net Na movement was virtually eliminated by external FeCl₃. After addition of FeCl₃ to serosal medium there was delayed inhibition ofJ ₁₃ ^Na but no change in chloride fluxes. Immediate and profound changes in Na and Cl transport systems seen after external application of FeCl₃ indicate charge effects of Fe³⁺ on surface of apical cell membranes, possibly close to or in ion channels.     

Diphenylamine-2-carboxylate stimulates sodium ion transport in frog skin epithelium

1. Diphenylamine-2-carboxylate (DPC), added to the mucosal side of the frog skin, increased reversibly the short-circuit current (I0), even in SO2-(4) Ringer. Amiloride blocked this effect. 2. The maximal stimulation was 140% of the control value and the EC50 was 0.26 mM DPC. 3. The stimulatory effect of DPC was additive to that of oxytocin. 4. The dose-response curves for amiloride determined in the absence and in the presence of 1 mM DPC showed an IC50 of 1.0 microM and 0.8 microM amiloride, respectively. 5. Thus DPC, a blocker of Cl- channels in various Cl-transporting epithelia, exerts a stimulatory effect on the amiloride-sensitive Na+ transport in frog skin.     

Sodium transport through the amiloride-sensitive Na-Mg pathway of hamster red cells

Previous work showed that in hamster red cells the amiloride-sensitive (AS) Na⁺ influx of 0.8 mmol/liter cells/hr is not mediated by Na-H exchange as in other red cells, but depends upon intracellular Mg²⁺ and can be increased by 40-fold by loading cells with Mg²⁺ to 10 mm. The purpose of this study was to verify the connection of AS Na⁺ influx with Na-dependent, amiloride-sensitive Mg²⁺ efflux and to utilize AS Na⁺ influx to explore that pathway.Determination of unidirectional influx of Na⁺ and net loss of Mg²⁺ in parallel sets of cells showed that activation by extracellular [Na⁺] follows a simple Michaelis-Menten relationship for both processes with a K _m of 105–107 mm and that activation of both processes is sigmoidally dependent upon cytoplasmic [Mg²⁺] with a [Mg²⁺]_0.5 of 2.1–2.3 mm and a Hill coefficient of 1.8. Comparison of V_max for both sets of experiments indicated a stoichiometry of 2 Na: l Mg. Amiloride inhibits Na⁺ influx and Mg²⁺ extrusion in parallel (K _i = 0.3 mm). Like Mg²⁺ extrusion, amiloride-sensitive Na⁺ influx shows an absolute requirement for cytoplasmic ATP and is increased by cell swelling. Hence, amiloride-sensitive Na⁺ influx in hamster red cells appears to be through the Na-Mg exchange pathway.There was no amiloride-sensitive Na⁺ efflux in hamster red cells loaded with Na⁺ and incubated with high [Mg²⁺] in the medium with or without external Na⁺, nor with ATP depletion. Hence, this is not a simple Na-Mg exchange carrier.     

Effect of hibernation on sodium and chloride ion transport in isolated frog skin

The aim of the study was to evaluate the effect of hibernation on electrophysiological parameters of isolated frog skin under control incubation (Ringer solution) and after inhibition of Na+ and CI- transepithelial transport by application of amiloride and bumetanide. The transepithelial electrical potential difference (PD in mV) was measured before and after mechanical stimulation of isolated frog skin. The tissues were mounted in a modified Ussing chamber. The results revealed a reduced PD of frog skin during hibernation. In February, as compared with November, PD of frog skin incubated in Ringer solution decreased by about 50%. Hibernation also affected hyperpolarization (dPD) of frog skin after mechanical stimulation. In November and December, dPD was about 50% and 30% lower, respectively, compared with the subsequent two months of the experiment. The incubation of frog skin with amiloride, a sodium ion channel blocker, resulted in reduced values of all measured electrophysiological parameters irrespective of the phase of hibernation. After application of chloride ion transport inhibitor (bumetanide), the PD in November and December decreased compared with the control incubation by about 80% and 75%, while in January and February by about 40% and 25%, respectively. In January and February dPD increased by four times and three times as compared with November and December. Hibernation reduces net ion flow in isolated frog skin. During the initial period of hibernation the sensitivity of the skin to mechanical stimulation also decreases. Towards the end of hibernation, on the other hand, excitation of mechanosensitive ion channels takes place.     

K+ transport and capacitance of the basolateral membrane of the larval frog skin

Skin from larval bullfrogs was mounted in an Ussing-type chamberin which the apical surface was bathed with a Ringer solution containing 115 mM K⁺ and thebasolateral surface was bathed with a Ringer solution containing 115 mMNa⁺. Ion transport was measured asthe short-circuit current(I_sc) with alow-noise voltage clamp, and skin resistance(R_m) wasmeasured by applying a direct current voltage pulse. Membrane impedance was calculated by applying a voltage signal consisting of 53 sine wavesto the command stage of the voltage clamp. From the ratio of theFourier-transformed voltage and current signals, it was possible tocalculate the resistance and capacitance of the apical and basolateralmembranes of the epithelium(R_a andR_b,C_a and C_b,respectively). With as the anion,R_m decreasedrapidly within 5 min following the addition of 150 U/ml nystatin to theapical solution, whereasI_sc increasedfrom 0.66 to 52.03 µA/cm² over a60-min period. These results indicate that nystatin becomes rapidlyincorporated into the apical membrane and that the increase inbasolateral K⁺ permeabilityrequires a more prolonged time course. Intermediate levels ofI_sc were obtainedby adding 50, 100, and 150 U/ml nystatin to the apical solution. Thisproduced a progressive decrease in R_a andR_b whileC_a andC_b remainedconstant. With Cl as theanion, I_sc valuesincreased from 2.03 to 89.57 µA/cm² following treatment with150 U/ml nystatin, whereas with gluconate as the anionI_sc was onlyincreased from 0.63 to 11.64 µA/cm². This suggests that theincrease in basolateral K⁺permeability produced by nystatin treatment, in the presence of morepermeable anions, is due to swelling of the epithelial cells of thetissue rather than the gradient for apicalK⁺ entry. Finally,C_b was notdifferent among skins exposed toCl,, or gluconate, despite the largedifferences inI_sc, nor didinhibition of I_scby treatment with hyperosmotic dextrose cause significant changes inC_b. These resultssupport the hypothesis that increases in cell volume activateK⁺ channels that are alreadypresent in the basolateral membrane of epithelial cells.