Ion selectivity of the apical membrane Na channel in the toad urinary bladder

Summary The ion selectivity of the apical membrane Na channel in the toad urinary bladder was investigated. The electrical potential difference and resistance across the basal-lateral membrane were reduced using high concentrations of KCl in the serosal bathing medium, and gradients for various ions were imposed across the apical membrane by altering the composition of the mucosal bathing medium. Ion fluxes through the channel were measured as the transepithelial current inhibited by amiloride, a specific blocker of the channel's Na conductance. The selectivity sequence for alkali metal cations was H>Li>NaK. K, permeability was barely detectable; the selectivity for Na over K was about 1000:1. Ammonium, hydroxyl ammonium and hydrazinium ions were, like K, virtually impermeant. The results suggest that the size of the unhydrated ion is an important factor in determining permeability in this channel.     

Control of sodium permeability of the outer barrier in toad skin

Summary The²⁴Na efflux (J _eff ^Na ) (i.e., the rate of appearance of²⁴Na in the outer compartment) in the isolated short-circuited toad skin bathed by NaCl-Ringer's solution on both sides is composed of para- and transcellular components of almost equal magnitudes. This relies on the assumption that amiloride acts on the transcellular component only and could block it completely.Ouabain induces a large transient increase of the transcellular component. This increase, which starts within a few minutes after the addition of ouabain, is due to electrical depolarization of the outer barrier, rather than a consequence of blocking Na recirculation across the inner barrier. The subsequent decline ofJ _eff ^Na , which takes place after the ouabain-inducedJ _eff ^Na peak, is due to a progressive block of outer barrier Na channels with time, which can eventually be complete, depending on the duration of action of ouabain. As the external Na concentration was always kept high and constant in these experiments, the results indicate that a rise in cell Na concentration, and not in the outer bathing solution, is the signal that triggers the reduction of outer barrier Na permeability (P ₀ ^Na ).Ouabain has no effect uponJ _eff ^Na with Na-free solution bathing the outer and NaCl-Ringer's solution the inner skin surface, showing the importance of Na penetration across the outer barrier, and not across the inner barrier due to its low Na permeability, in the process of closing the Na channels of this structure.Step changes from Na 115mm to Na-free external solution, or vice-versa, may affect both the outer barrier electrical potential difference (PD₀) and cell Na concentration (Na) _c . Therefore, the behavior ofJ _eff ^Na depends on which variable (if PD₀ or (Na) _c regulated outer barrier Na permeability) is most affected by step changes in outer bathing solution Na concentration.Amiloride in the control condition blocks the transcellular component ofJ _eff ^Na . However, in the condition of approximate short-circuiting of the outer barrier and high cellular Na concentration induced by long term effects of ouabain, when the Na channels of the outer barrier are already blocked by elevated cell Na concentration, amiloride may induce the opposite effect, increasing Na permeability of the outer barrier.With outer barrier Na channels completely blocked by high cell Na concentration, PCMB in the outer bathing medium induces a large increase ofJ _eff ^Na , rendering these channels again amiloride sensitive.The results are consistent with the notion that Na efflux from cell compartment to the outer bathing solution goes through the amiloride-sensitive Na channels of the apical border of the superficial cell layer of toad skin, with an apparent Na permeability modulated by cell ionic environment, most probably the cell Na concentration.The ensemble of the present results are consistent with Na permeability regulation taking place at the outer barrier level. However, this precise location could only be made unambiguously by measurements across the individual outer cell membranes.     

Interactions of amiloride and other blocking cations with the apical Na channel in the toad urinary bladder

Summary A simple model of the action of amiloride to block apical Na channels in the toad urinary bladder was tested. According to the model, the positively charged form of the drug binds to a site in the lumen of the channel within the electric field of the membrane. In agreement with the predictions of the model: (1) The voltage dependence of amiloride block was consistent with the assumption of a single amiloride binding site, at which about 15% of the transmembrane voltage is sensed, over a voltage range of ±160 mV. (2) The time course of the development of voltage dependence was consistent with that predicted from the rate constants for amiloride binding previously determined. (3) The ability of organic cations to mimic the action of amiloride showed a size dependence implying a restriction of access to the binding site, with an effective diameter of about 5 angstroms. In a fourth test, divalent cations (Ca, Mg, Ba and Sr) were found to block Na channels with a complex voltage dependence, suggesting that these ions interact with two or more sites. at least one of which may be within the lumen of the pore.     

Roles of external and cellular Cl− ions on the activation of an apical electrodiffusional Cl− pathway in toad skin

Summary This study is concerned with the short-circuit current,I _sc, responses of the Cl^–-transporting cells of toad skin submitted to sudden changes of the external Cl^– concentration. [Cl]₀. Sudden changes of [Cl]₀, carried out under apical membrane depolarization, allowed comparison of the roles of [Cl]₀ and [Cl]_cell on the activation of the apical Cl^– pathways. Equilibration of shortcircuited skins symmetrically in K-Ringer's solutions of different Cl^– concentrations permitted adjustment of [Cl]_cell to different levels. For a given Cl^– concentration (in the range of 11.7 to 117mm) on both sides of a depolarized apical membrane, this structure exhibits a high Cl^– permeability,P _(Cl)apical. On the other hand, for the same range of [Cl]_cell but with [Cl]₀=0,P _(Cl)apical is reduced to negligible values. These observations indicate that when the apical membrane is depolarizedP _(Cl)apical is modulated by [Cl]₀; in the absence of external Cl^– ions, intracellular Cl^– is not sufficient to activateP _(Cl)apical. Computer simulation shows that the fast Cl^– currents induced across the apical membrane by sudden shifts of [Cl]₀ from a control equilibrium value strictly follow the laws of electrodiffusion. For each experimental group, the computer-generatedI _sc versus ([Cl]_cell–[Cl]₀) curve which best fits the experimental data can only be obtained by a unique pair ofP _(Cl)apical andR _b (resistance of the basolateral membrane), thus allowing the calculation of these parameters. The electrodiffusional behavior of the net Cl^– flux across the apical membrane supports the channel nature of the apical Cl^– pathways in the Cl^–-transporting cell. Cl^– ions contribute significantly to the overall conductance of the basolateral membrane even in the presence of a high K concentration in the internal solution.     

Electrolytes control flows of water across the apical barrier in toad skin: The hydrosmotic salt effect

Summary The reversible dependence of skin osmotic water permeability (L _PD ) upon the ionic concentration of the outer bathing solution — which we have called hydrosmotic salt effect (HSE) — was studied in the isolated skin of the toadBufo marinus ictericus. The skin osmotic water flow (J _V ) was measured as a function of outer bathing solution osmolality (O _e ).L _PD , calculated as (J _v /) _P=0 (where and P are the osmotic and hydrostatic pressure differences across the skin, respectively) was constant whenO _e was altered with sucrose, a nonelectrolyte. In contrast,L _PD increased continuously in the hypotonic range asO _e was raised from zero (distilled water) with NaCl or KCl. The HSE could also be evoked in the condition of reversed osmotic volume flow, with the outer bathing medium made hypertonic with sucrose.Diffusional¹⁴C-sucrose permeability, measured in theJ _v =0 condition to prevent solvent drag of sucrose in the paracellular pathways, indicate that the hydrosmotic salt effect cannot be explained by assuming a paracellular permeability increase, due to tight junction opening, but might be interpreted as due to changes in the osmotic water permeability of the apical membranes of the most superficial cells of the epithelium.The hydrosmotic salt effect can be elicited in control skins and in vasopressin-stimulated skins, on top of the hormonal response.The time course of the hydrosmotic salt effect is substantially different from that of the hydrosmotic response to vasopressin. Its half-time is 4 to 5 times faster than that of vasopressin action, with individual values as short as 1.5 min.The time courses of the hydrosmotic salt-effect onset and reversibility are exponential, clearly contrasting with the typical sigmoidal shape of vasopressin onset and washout time courses.Based on time course data and on speed of response we postulate that the mechanism underlying the hydrosmotic salt effect is due to modifications of existing water pathways in the apical membrane, rather than to incorporation and removal of water permeability units in this structure.     

An Amiloride-sensitive Na+ conductance in the basolateral membrane of toad urinary bladder

Summary Exposing the apical membrane of toad urinary bladder to the ionophore nystatin lowers its resistance to less than 100 cm². The basolateral membrane can then be studied by means of transepithelial measurements. If the mucosal solution contains more than 5mm Na⁺, and serosal Na⁺ is substituted by K⁺, Cs⁺, or N-methyl-d-glucamine, the basolateral membrane expresses what appears to be a large Na⁺ conductance, passing strong currents out of the cell. This pathway is insensitive to ouabain or vanadate and does not require serosal or mucosal Ca²⁺. In Cl-free SO ₄ ^2– Ringer's solution it is the major conductive pathway in the basolateral membrane even though the serosal side has 60mm K⁺. This pathway can be blocked by serosal amiloride (K _i=13.1 m) or serosal Na⁺ ions (K _i 10 to 20mm). It also conducts Li⁺ and shows a voltage-dependent relaxation with characteristic rates of 10 to 20 rad sec^–1 at 0 mV.     

Effects of antidiuretic hormone upon electrical potential and resistance of apical and basolateral membranes of frog skin

Summary The effect of ADH upon the intracellular potential and the resistance of inner and outer borders of the transport pathway was investigated on isolated skins ofRana temporaria. Within 40 min after ADH (100-300 mU/ml), the intracellular potential under short-circuit conditions decreased to about 40% of the control value (–79±4 mV), concomitant with an increase in the short-circuit current to about 160% of the control value. Amiloride, applied when steady values under ADH had been reached, caused an immediate rise of the intracellular potential to values typical for control conditions. This confirms (i) the intracellular location of the microelectrode and the absence of impalement artifacts, and (ii) the ineffectiveness of ADH upon the electromotive forces of the inner border. ADH had no effect upon the intracellular potential after blockage of the Na entry by Amiloride. The equilibrium potential of the outer border was estimated to be about +20 mV under the influence of ADH. As this value is considerably less positive than might be expected for the chemical potential of Na, a significant contribution of ions other than Na to the outer border conductance and equilibrium potential is implicated. The resistance of the outer border was more significantly decreased than that of the active transcellular pathway after ADH due to an increase in the inner border resistance, which exceeded that of the outer border after ADH. The effect of ADH upon the outer membrane characteristics would be underestimated by a factor of two, if the alterations of the electrical potential difference were not taken into consideration.     

Identification of apical membranes from tight epithelia using spin-labeled amiloride and electron paramagnetic resonance spectroscopy

Summary Apical cell membranes from Na⁺-transporting epithelia were identified in centrifugal fractions prepared from homogenates of rainbow trout kidney, gill and frog skin using a spinlabeled, nitroxide derivative of amiloride and electron paramagnetic resonance spectroscopy. Spin-labeled amiloride (ASp) is a potent inhibitor of Na⁺ transport. Frog skin shortcircuit current was inhibited by 50% in the presence of 7×10^–8 m ASp, whereas 4×10^–7 m amiloride was required to obtain the same effect. ASp is a suitable probe for the amiloride binding site based on analytical criteria: Unbound ASp produces an EPR signal linear with concentration and detectable at micromolar concentrations. Estimates of ASp binding can usually be made on less than 100 g of membrane protein. While ASp binds nonspecifically to many materials, amiloride- or benzamil-displaceable binding occurred only in trout gill and kidney, and in frog skin, but not in trout skeletal muscle. ASp binds to membrane fractions produced by differential centrifugation of trout gill, kidney and frog skin. In trout gill and kidney, 81% and 91%, respectively, of the amiloride-displaceable ASp binding is found in the 10,000 xg fraction. All of the ASp binding in frog skin is found in the 10,000 xg fraction. These data indicate that spin-labeled amiloride is a useful probe for the identification of the amiloride binding site, and electron paramagnetic resonance spectroscopy will allow the amiloride binding site to be used as a molecular marker for apical membranes.     

Effects of Pb<Superscript>2+</Superscript> ions on Na<Superscript>+</Superscript> transport in the isolated skin of the toad <Emphasis Type="Italic">Pleurodema thaul</Emphasis>

The effects induced by lead ions on the short-circuit current (SCC) and on the potential difference (V) of the toad Pleurodema thaul skin were investigated. Pb²⁺ applied to the outer (mucosal) surface increased SCC and V and when applied to the inner (serosal) surface decreased both parameters. The stimulatory effect, but not the inhibitory action, was reversible after washout of the metal ion. The amiloride test showed that the increase was due principally to stimulation of the driving potential for Na⁺ (V-E_Na+) and that inhibition was accompanied by a reduction in the V-E_Na+ and also by a significant decrease in skin resistance indicating possible disruption of membrane and/or cell integrity. The effect of noradrenaline was increased by outer and decreased by inner administration of Pb²⁺. The results suggest that mucosal Pb²⁺ activates toad skin ion transport by stimulating the V-E_Na+ and that serosal Pb²⁺, with easier access to membrane and cellular constituents, inactivates this mechanism, revealing greater toxicity when applied to the inner surface of the skin. Abbreviations: SCC – short-circuit current; V – potential difference; V-E_Na+– driving potential for Na⁺; ENaC – epithelial sodium channel; R_Na+– active sodium resistance; RS – passive or shunt resistance; G_Na– active sodium conductance; GS – passive or shunt conductance; Gmax – total conductance; EC₅₀– half-maximal excitatory concentration; IC₅₀– half maximal inhibitory concentration; NA – noradrenaline.     

Amiloride and its analogs as tools in the study of ion transport

Amiloride inhibits most plasma membrane Na+ transport systems. We have reviewed the pharmacology of inhibition of these transporters by amiloride and its analogs. Thorough studies of the Na+ channel, the Na+/H+ exchanger, and the Na+/Ca2+ exchanger, clearly show that appropriate modification of the structure of amiloride will generate analogs with increased affinity and specificity for a particular transport system. Introduction of hydrophobic substituents on the terminal nitrogen of the guanidino moiety enhances activity against the Na+ channel; whereas addition of hydrophobic (or hydrophilic) groups on the 5-amino moiety enhances activity against the Na+/H+ exchanger. Activity against the Na+/Ca2+ exchanger and Ca2+ channel is increased with hydrophobic substituents at either of these sites. Appropriate modification of amiloride has produced analogs that are several hundred-fold more active than amiloride against specific transporters. The availability of radioactive and photoactive amiloride analogs, anti-amiloride antibodies, and analogs coupled to support matrices should prove useful in future studies of amiloride-sensitive transport systems. The use of amiloride and its analogs in the study of ion transport requires a knowledge of the pharmacology of inhibition of transport proteins, as well as effects on enzymes, receptors, and other cellular processes, such as DNA, RNA, and protein synthesis, and cellular metabolism. One must consider whether the effects seen on various cellular processes are direct or due to a cascade of events triggered by an effect on an ion transport system.     

Dopaminergic inhibition of vasopressin-stimulated water flow in the toad bladder: Evidence for local formation of dopamine

Summary Dopamine administration increases renal excretion of water and Na. It remains uncertain whether these effects of dopamine are the result of a hemodynamic effect or the consequence of a direct cellular action. We investigated the effect of dopamine on water transport by the isolated toad bladderin vitro. Dopamine failed to alter baseline water flow but caused a significant inhibition of arginine vasopressin (AVP) or cyclic adenosine monophosphate (AMP) stimulated water flow. The effect of dopamine on stimulated water flow was not due to activation of adrenergic, adrenergic, or cholinergic receptors. The selective antagonists of dopamine, metoclopramide and apomorphine, prevented the effect of dopamine on AVP-stimulated water flow. These observations suggest the existence of a dopaminergic receptor in the toad bladder.l-Dopa also inhibited AVP-stimulated water flow. The effect ofl-Dopa could be prevented by metoclopramide, thus suggesting thatl-Dopa is converted to dopamine by an aromatic amino acid decarboxylase present in the toad bladder. To investigate this possibility we measured the effect of the decarboxylase inhibitor, carbidopa, on the¹⁴CO₂ production generated by decarboxylation of¹⁴Cl-Dopa in isolated toad bladder epithelial cells. Isolated toad bladder epithelial cells generated significant amounts of¹⁴CO₂ from¹⁴Cl-Dopa. This effect could be blocked by carbidopa, thus suggesting the existence of an aromatic amino acid decarboxylase system in the toad bladder. Carbidopa also prevented the inhibitory effect ofl-Dopa on AVP-stimulated water flow, suggesting thatl-Dopa needs to be converted to dopamine to inhibit water flow. These data suggest the existence of a dopaminergic receptor in the toad bladder. These data also suggest that dopamine can be formed locally in the toad bladder and can thus serve as a local modulator of water transport.     

The apical localization of SGLT1 glucose transporter is determined by the short amino acid sequence in its N-terminal domain

SGLT1, an isoform of Na+-dependent glucose cotransporters, is localized at the apical plasma membrane in the epithelial cells of the small intestine and the kidney, where it plays a pivotal role in the absorption and reabsorption of sugars, respectively. To search the domain responsible for the apical localization of SGLT1, we constructed an N-terminal deletion clone series of rat SGLT1 and analyzed the localization of the respective products in Madin-Darby canine kidney (MDCK) cells. The products of N-terminal deletion clones up to the 19th amino acid were localized at the apical plasma membrane, whereas the products of N-terminal 20- and 23-amino-acid deletion clones were localized along the entire plasma membrane. Since single-amino-acid mutations of either D28N or D28G in the N-terminal domain give rise to glucose/galactose malabsorption disease, we examined the localization of these mutants. The products of D28N and D28G clones were localized in the cytoplasm, showing that the aspartic acid-28 may be essential for the delivery of SGLT1 to the plasma membrane. These results suggest that a short amino acid sequence of the N-terminal domain of SGLT1 plays important roles in plasma membrane targeting and specific apical localization of the protein.     

Hydrosmotic salt effect in toad skin: Urea permeability and glutaraldehyde fixation of water channels

Summary The hydrosmotic salt effect (HSE), the reversible dependence of skin osmotic water permeability upon the ionic concentration of the outer bathing solution, is known to induce the appearance of sucrose-impermeable pathways in the apical membrane of the outermost epithelial cell layer. Diffusional¹⁴C-urea permeability, measured in theJ _v=0 condition to prevent solvent drag effects, indicates that the newly formed pathways induced by HSE are narrower than the size of the urea molecule, being therefore highly selective for water molecules. After mild glutaraldehyde (2% solution) fixation of the apical membrane structures, the water channels induced by the HSE are no longer affected by the ionic strength of the outer solution. This indicates that the channel-forming membrane protein can be fixed in different configurations with the water channels in the open or closed states.Escola Paulista de Medicina, Department of Biophysics.     

Intracellular electrolyte concentrations in the frog skin epithelium: Effect of vasopressin and dependence on the Na concentration in the bathing media

Summary The intracellular electrolyte concentrations of the frog skin epithelium have been determined in thin freeze-dried cryosections using the technique of electron microprobe analysis. Stimulation of the transepithelial Na transport by arginine vasopressin (AVP) resulted in a marked increase in the Na concentration and a reciprocal drop in the K concentration in all epithelial cell layers. The effects of AVP were cancelled by addition of amiloride. It is concluded from these results that the primary mechanism by which AVP stimulates transepithelial Na transport is an increase in the Na permeability of the apical membrane. However, also some evidence has been obtained for an additional stimulatory effect of AVP on the Na pump. In mitochondria-rich cells and in gland cells no significant concentration changes were detected, supporting the view that these cells do not share in transepithelial Na transport. Furthermore, the dependence of the intracellular electrolyte concentrations upon the Na concentration in the outer and inner bathing solution was evaluated. Both in control and AVP-stimulated skins the intracellular Na concentration showed saturation already at low external Na concentrations, indicating that the self-inhibition of transepithelial Na transport is due to a reduction of the permeability of the apical membrane. After lowering the Na concentration in the internal bath frequently a Na increase in the outermost and a drop in the deeper epithelial layers was observed. It is concluded that partial uncoupling of the transport syncytium occurs, which may explain the inhibition of the transepithelial Na transport and blunting of the AVP response under this condition.     

Direct inhibition of epithelial Na+ channels by a pH-dependent interaction with calcium,and by other divalent ions

Summary Direct inhibitory effects of Ca²⁺ and other ions on the epithelial Na⁺ channels were investigated by measuring the amiloride-blockable²²Na⁺ fluxes in toad bladder vesicles containing defined amounts of mono- and divalent ions. In agreement with a previous report (H.S. Chase, Jr., and Q. Al-Awqati,J. Gen. Physiol. 81:643–666, 1983) we found that the presence of micromolar concentrations of Ca²⁺ in the internal (cytoplasmic) compartment of the vesicles substantially lowered the channel-mediated fluxes. This inhibition, however, was incomplete and at least 30% of the amiloride-sensitive²²Na⁺ uptake could not be blocked by Ca²⁺ (up to 1mm). Inhibition of channels could also be induced by millimolar concentrations of Ba²⁺, Sr²⁺, or VO²⁺, but not by Mg²⁺. The Ca²⁺ inhibition constant was a strong function of pH, and varied from 0.04 m at pH 7.8 to >10 m at pH 7.0 Strong pH effects were also demonstrated by measuring the pH dependence of²²Na⁺ uptake in vesicles that contained 0.5 m Ca²⁺. This Ca²⁺ activity produced a maximal inhibition of²²Na⁺ uptake at pH7.4 but had no effect at pH7.0. The tracer fluxes measured in the absence of Ca²⁺ were pH independent over this range. The data is compatible with the model that Ca²⁺ blocks channels by binding to a site composed of several deprotonated groups. The protonation of any one of these groups prevents Ca²⁺ from binding to this site but does not by itself inhibit transport. The fact that the apical Na⁺ conductance in vesicles, can effectively be modulated by minor variations of the internal pH near the physiological value, raises the possibility that channels are being regulated by pH changes which alter their apparent affinity to cytoplasmic Ca²⁺, rather than, or in addition to changes in the cytoplasmic level of free Ca²⁺.     

Differential effects of aldosterone and ADH on intracellular electrolytes in the toad urinary bladder epithelium

Summary Quantitative electron microprobe analysis was employed to compare the effects of aldosterone and ADH on the intracellular electrolyte concentrations in the toad urinary bladder epithelium. The measurements were performed on thin freeze-dried cryosections utilizing energy dispersive x-ray microanalysis. After aldosterone, a statistically significant increase in the intracellular Na concentration was detectable in 8 out of 9 experiments. The mean Na concentration of granular cells increased from 8.9±1.3 to 13.2±2.2 mmol/kg wet wt. A significantly larger Na increase was observed after an equivalent stimulation of transepithelial Na transport by ADH. On average, the Na concentration in granular cells increased from 12.0±2.3 to 31.4±9.3 mmol/kg wet wt (5 experiments). We conclude from these results that aldosterone, in addition to its stimulatory effect on the apical Na influx, also exerts a stimulatory effect on the Na pump. Based on a significant reduction in the Cl concentration of granular cells, we discuss the possibility that the stimulation of the pump is mediated by an aldosterone-induced alkalinization.Similar though less pronounced concentration changes were observed in basal cells, suggesting that this cell type also participates in transepithelial Na transport. Measurements in mitochondria-rich cells provided no consistent results.     

Effect of ouabain,amiloride, and antidiuretic hormone on the sodium-transport pool in isolated epithelia from frog skin (Rana temporaria)

Summary When tracer Na⁺ is added to the solution bathing the apical side of isolated epithelia the observed transepithelial tracer influx increases with time until a steady state is reached. The build-up of the tracer flux follows a single exponential course. The halftime for this build-up under control conditions was 0.92 ±0.06 min, and in the presence of ouabain 4.51±0.7 min. It is shown that the calculated Na⁺-transport pool is located in the cells. The Na⁺-transport pool under control conditions was 35.6 ±3.4 nmol/cm², which corresponds to an intracellular Na⁺ concentration of 7.9mm. Activation of the active Na⁺ transport by addition of antidiuretic hormone resulted in a highly significant increase in the Na⁺ transport pool, and inhibition of the transcellular Na⁺ transport with amiloride resulted in a decrease in the Na⁺-transport pool.Furthermore, the active Na⁺ transport increased along anS-shaped curve with increasing intracellular Na⁺ concentration (Na⁺-transport pool). The Na⁺ pump was found to be half saturated at an intracellular Na⁺ concentration of 12.5mm.     

Isolation and characterization of specialized regions of toad urinary bladder apical plasma membrane involved in the water permeability response to antidiuretic hormone

Summary Antidiuretic hormone (ADH) increases the apical (external facing) membrane water permeability of granular cells that line the toad urinary bladder. In response to ADH, cytoplasmic vesicles called aggrephores fuse with the apical plasma membrane and insert particle aggregates which are visualized by freeze-fracture electron microscopy. Aggrephores contain particle aggregates within their limiting membranes. It is generally accepted that particle aggregates are or are related to water channels. High rates of transepithelial water flow during ADH stimulation and subsequent hormone removal decrease water permeability and cause the endocytosis of apical membrane and aggrephores which retrieve particle aggregates. We loaded the particle aggregate-rich endocytic vesicles with horseradish peroxidase (HRP) during ADH stimulation and removal. Epithelial cells were isolated and homogenized, and a subcellular fraction was enriched for sequestered HRP obtained. The HRP-enriched membrane fraction was subjected to a density shifting maneuver (Courtoy et al.,J. Cell Biol. 98:870, 1984), which yielded a purified membrane fraction containing vesicles with entrapped HRP. The density shifted vesicles were composed of approximately 20 proteins including prominent species of 55, 17 and 7 kD. Proteins of these molecular weights appear on the apical surface of ADH-stimulated bladders, but not the apical surface of control bladders. Therefore, we believe these density shifted vesicles contain proteins involved in the ADH-stimulated water permeability response, possibly components of particle aggregates and/or water channels.     

Current-voltage relations of the basolateral membrane in tight amphibian epithelia: Use of nystatin to depolarize the apical membrane

Summary Exposure of the mucosal side of toad(Bufo bufo) urinary bladder and frog(Rana ridibunda) skin to the polyene ionophore nystatin, resulted in stable preparations in which the apical resistance was negligible compared to the basolateral resistance. The preparations support passive K currents in both directions and an amiloride-insensitive Na current in the apicalserosal direction which is blocked by ouabain. The nystatintreated toad bladder was used to study the electrical properties of the basolateral membrane by means of current-voltage curves recorded transepithelially. The K current showed strong rectification at cellular potentials negative with respect to the interstitial space. The ouabain-sensitive current increased with membrane voltage at negative voltages but saturated above+20 mV.