Current-voltage relations of the apical and basolateral membranes of the frog skin

We determined the current-voltage (I-V) relations of the apical and basolateral barriers of frog skins by impaling the cells with an intracellular microelectrode and assuming that the current across the cellular pathway was equal to the amiloride-inhibitable current. We found that: (a) The responses in transepithelial current and intracellular potential to square pulses of transepithelial potential (VT) varied markedly with time. (b) As a consequence of these transient responses, the basolateral I-V relation was markedly dependent on the time of sampling after the beginning of each pulse. The apical I-V plot was much less sensitive to the time of sampling within the pulse. (c) The I-V data for the apical barrier approximated the I-V relations calculated from the Goldman constant field equation over a relatively wide range of membrane potentials (+/- 100 mV). (d) A sudden reduction in apical bath [Na+] resulted in an increase in apical permeability and a shift in the apical barrier zero-current potential (Ea) toward less positive values. The shift in Ea was equivalent to a change of 45 mV for a 10-fold change in apical [Na+]. (e) The transient responses of the skin to square VT pulses were described by the sum of two exponentials with time constants of 114 and 1,563 ms, which are compatible with the time constants that would be produced by an RC circuit with capacitances of 65 and 1,718 microF. The larger capacitance is too large to identify it comfortably with a true dielectric membrane capacitance.     

Inward-rectifier potassium channels in basolateral membranes of frog skin epithelium

Inward-rectifier K channel: using macroscopic voltage clamp and single- channel patch clamp techniques we have identified the K+ channel responsible for potassium recycling across basolateral membranes (BLM) of principal cells in intact epithelia isolated from frog skin. The spontaneously active K+ channel is an inward rectifier (Kir) and is the major component of macroscopic conductance of intact cells. The current- voltage relationship of BLM in intact cells of isolated epithelia, mounted in miniature Ussing chambers (bathed on apical and basolateral sides in normal amphibian Ringer solution), showed pronounced inward rectification which was K(+)-dependent and inhibited by Ba2+, H+, and quinidine. A 15-pS Kir channel was the only type of K(+)-selective channel found in BLM in cell-attached membrane patches bathed in physiological solutions. Although the channel behaves as an inward rectifier, it conducts outward current (K+ exit from the cell) with a very high open probability (Po = 0.74-1.0) at membrane potentials less negative than the Nernst potential for K+. The Kir channel was transformed to a pure inward rectifier (no outward current) in cell- attached membranes when the patch pipette contained 120 mM KCl Ringer solution (normal NaCl Ringer in bath). Inward rectification is caused by Mg2+ block of outward current and the single-channel current-voltage relation was linear when Mg2+ was removed from the cytosolic side. Whole-cell current-voltage relations of isolated principal cells were also inwardly rectified. Power density spectra of ensemble current noise could be fit by a single Lorentzian function, which displayed a K dependence indicative of spontaneously fluctuating Kir channels. Conclusions: under physiological ionic gradients, a 15-pS inward- rectifier K+ channel generates the resting BLM conductance in principal cells and recycles potassium in parallel with the Na+/K+ ATPase pump.     

Functional role of V1- and V2-receptors of the apical and basolateral membranes of the frog bladder epithelial cells

Arginine vasotocin, 0.02--1 nM, increases osmotic water permeability of frog urinary bladder, arginine vasotocin after a simultaneous addition to the mucosal and serosal Ringer solutions rises the water permeability to a lesser degree than on the hormone addition only to the serosal solution. 1 nM remestyp, an agonist of V1-receptors, from the apical membrane decreases the hydroosmotic effect of arginine vasotocin added to the serosal Ringer solution. When added to the mucosal solution, combination of the same concentrations of arginine vasotocin and SR 49059, an antagonist of V--receptors, or desmopressin, agonist of V2-receptor alone, increases the effect of the same concentration of arginine vasotocin added to the serosal solution. 1 nM arginine vasotocin at the luminal membrane increases secretion into the Ringer solution of prostaglandin E, and prostaglandin E1 but not of prostaglandin F2 alpha. The data obtained indicate the presence of the arginine vasotocin receptors responsible for the hydroosmotic effect only in the basolateral membranes, while arginine prostaglandin E, participation is shown in modulation of the arginine vasotocin effect.     

Isolation of radio-iodinated apical and basal-lateral plasma membranes of toad bladder epithelium

Summary The apical and basal-lateral plasma membranes of toad bladder epithelium were radio-iodinated with the glucose-glucose oxidase-lactoperoxidase system. The covalently bound radio-iodine was used as a marker during subcellular fractionation and membrane isolation. Homogenization conditions that ensured rupture of more than 80% of the cells without substantial nuclear damage were defined by Nomarski optics. The nuclei were separated by differential centrifugation and the apical and basal-lateral components were resolved by differential and sucrose density gradient centrifugation. The apical components yielded two radioactive bands that were identified as glycocalyx and plasma membrane labeled with¹²⁵I. The basal-lateral components yielded a heterodisperse pattern made up of at least 3 radioactive bands, but the bulk of the activity of ouabain-sensitive ATPase comigrated with only one of these bands. The mitochondia, identified by assays for cytochrome oxidase and NADH cytochromec reductase activities, were separated from the radio-iodine labeled components by centrifugation in sucrose density gradients under isokinetic conditions. The labeled glycocalyx and the slowly migrating components of basal-lateral labeling were separated from the radio-iodinated membranes by centrifugation at 100,000 × g × 1 hr after removal of the mitochondria by the isokinetic method. The labeled membranes were then subjected to ultracentrifugation in sucrose density gradients under isopycnic conditions; the basal-lateral membranes containing ouabain-sensitive ATP-ase were well resolved from the apical membranes by this method. These results provide a relatively rapid method of attaining partial purification of the apical and basal-lateral plasma membranes of toad bladder epithelium.     

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.     

Isolation, purification and characteristics of brush border and basolateral membranes from cattle intestinal epithelium cells

The surface membrane of cattle intestine epithelium cells is separated into vesicated membrane fractions of the brush border and of basolateral membranes. The brush border membrane fraction is deposited with centrifugation (15,000 g) and is localized in the layers of 45, 56.5 and 48% in the density gradient of sucrose (105,000 g). Basolateral membranes, obtained at 70,000 g, in the density gradient of sucrose are in the layers of 30 and 31.5%. The brush border membranes are 8.5 times purified, basolateral membranes--9.1 times with their insignificant contamination with subcellular elements. The both fractions are deprived of mitochondria impurities.     

A method of isolation of apical membranous sheets from frog urinary bladder epithelium by stripping with gelatin

We have developed a technique for recovering apical membranous sheets from amphibian urinary bladders by gelatin stripping. The tissue is mounted on a lucite support and the apical surface is first stuck onto a gelatin-coated glass slide at 30 degrees C. This sandwich is then chilled on ice and the bladder is pulled away from the slide. Preliminary results indicate that this simple technique could be used to remove membranous apical sheets of various sizes, almost devoid of cytoplasmic contamination and without significant damage to the underlying cell structures. The method could also be adapted to prepare perforated cells and to study the cohesive forces between the different layers of the tissue.     

Electrical characteristics of the apical and basal-lateral membranes in the turtle bladder epithelial cell layer

(1) The active transport of Na⁺ across the turtle bladder epithelial cell layer consists of a passive entry step through a Na⁺-selective path in the apical membrane and an active extrusion step through Na⁺ pump-containing path in the basal-lateral membrane together with some back-leakage through the paracellular spaces and tight junctions between the epithelial cells. This hypothesis has now been verified qualitatively and to some extent, quantitatively by the use of an intracellularly-located microelectrode in conjunction with a conventional assembly of extracellularly-located macroelectrodes mainly in short-circuited bladders bathed by Na⁺-rich Ringer media. Under these conditions, the intracellular potential (V_sc) averaged 38.4 mV with the cell electronegative; the fractional resistance of the apical membrane ($\text{?R}{}_{\mathrm{<mtext>a</mtext>}}$) averaged 0.55; while the concomitant transepithelial parameters, short circuiting current (I_sc) and electrical conductance (G_t), average 68.6 μA/cm² and 0.98 mS/cm², respectively. (2) The relation between these parameters and the transepithelial flow of Na⁺ (orI_sc) is evoked by blocking Na⁺ entry into the cell (by the mucosal addition of amiloride or removal of mucosal Na⁺). Amiloride-induced blockade of the Na⁺ entry step results in a rapid hyperpolarization of the cell interior during which V_sc = —79.1 mV and $\text{?R}{}_{\mathrm{<mtext>a</mtext>}}\text{= 0.92}$. I_sc and G_t (equivalent to the shunt conductance under these conditions) averaged 5 μA/cm² and 0.35 mS/cm², respectively. The entire process is reversible on re-admission of Na⁺ entry into the cell. (3) A slow depolarization of the cell interior in the period of blocked transapical Na⁺ entry is opposite to that expected from an electroneutral Na⁺-K⁺ exchanging pump; but instead is the predictable response of an electrogenic Na⁺ pump in parallel with a passive K⁺-selective conductance in the basal-lateral membrane. (4) The electrogenicity concept is substantiated after pretreatment of the bladder with serosal ouabain, which changes the response of V_sc to amiloride (from the aforementioned biphasic response) to a step-function response, attributable mainly to the development of a slowly dissipating K⁺ diffusion potential across the basallateral membrane. (5) Under open-circuit conditions, the electronegativity of cell to mucosa (V_a) is a linear inverse function of the electropositivity of serosa to mucosa (V_t). For V_t ? 100 mV, V_a is positive; and for V_t between ?30 and 90 mV, V_a is negative.     

Possible apical respiration in the olfactory epithelium of the frog Rana temporaria

Bioluminescent analysis has been made of the effect of oxygen supply on the content of ATP in the isolated olfactory epithelium of the frog. It was shown that storage of epithelium preparations in the air increases their ATP content. When preparations are kept in the atmosphere of an inert gas, ATP level in the epithelium rapidly decreases, being recovered after transition of preparations to the air medium. The data obtained indicate the existence of apical type of respiration in the olfactory epithelium of the frog.     

Cholesterol localization in the membranes of granular cells in the bladder epithelium of the frog during stimulated water transport

The polyene antibiotic filipin has been used to characterize the cholesterol distribution in the membranes of resting and ADH-stimulated frog urinary bladder in freeze-fracture replicas. In general, the intracellular membranes takes up filipin only insignificantly. An exception is the cholesterol rich granule membrane. Both density and polarity of filipin-induced deformations were evaluated, and the asymmetry in membrane cholesterol was analysed. Upon ADH-stimulation of water flow both density and polarity of filipin-induced deformations altered differently in apical and basolateral regions of the plasma membrane. This difference is presumably due to the stretching of the basolateral membrane as a result of swelling, on the one hand, and to incorporation of aggregate containing membranes into the apical membrane, on the other one. The results obtained may suggest that the appearance of ADH-induced intramembranous particle aggregates in the apical membrane be accompanied with a relative cholesterol decrease in this apical membrane.     

Vasopressin-induced transfer via light vesicles of receptors and water channels from basolateral to apical membrane of toad bladder

Toad bladder epithelial cells were homogenized and fractionated by Percoll density-gradient centrifugation. Binding of tritium-labeled vasopressin ([3H]AVP) was measured in surface membranes (SM), microsomes (M), and a 100,000 g (60-min) microsomal supernatant fraction (S). More than two-thirds of the total receptors were in S. Receptors in SM--but not in S--were tightly coupled to G-protein as suggested by inhibition of [3H]AVP binding by GTP. GTP-sensitivity of [3H]AVP binding was not altered by vasotocin (AVT) stimulation, although the distribution of receptors shifted from SM to S. Intact bladders, exposed on the serosal side to 1-desamino, 7-lysine-(4-azidobenzoyl), 8-arginine vasotocin (d7-N3-AVT) in the presence of UV light, exhibited a persistent hydroosmotic response compared to controls stimulated with photoaffinity analog in the dark. Cell fractions from the irradiated bladders showed a reduction in [3H]AVP binding in SM and S. Intact bladders, exposed on the mucosal side to d7-N3-AVT in the presence of UV light (while stimulated from the serosal side with AVP) exhibited a decrease in [3H]AVP binding in SM compared to controls without d7-N3-AVT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for vasoactive intestinal peptide receptors in apical membranes from tracheal epithelium

[125I]VIP (vasoactive intestinal peptide) bound to apical membranes isolated from the bovine tracheal epithelium with a half maximal inhibition by unlabeled VIP (IC50) of 0.6 x 10(-9)M and binding was reversible. Glucagon did not affect [125I]VIP binding to the membranes. [125I]VIP was covalently cross-linked to tracheal membrane proteins using disuccinimidyl suberate. SDS-polyacrylamide gel electrophoresis of labeled tracheal membranes revealed one major [125I]-receptor complex of Mr = 71,000 to which binding of [125I]VIP was inhibited by 10 microM unlabeled VIP. These results are consistent with the presence of a specific, high-affinity receptor for VIP, with a Mr = 71,000, in apical membrane vesicles isolated from the bovine tracheal epithelium.     

Structural and functional characteristics of xenavidin, the first frog avidin from Xenopus tropicalis

Background  

Avidins are proteins with extraordinarily high ligand-binding affinity, a property which is used in a wide array of life science applications. Even though useful for biotechnology and nanotechnology, the biological function of avidins is not fully understood. Here we structurally and functionally characterise a novel avidin named xenavidin, which is to our knowledge the first reported avidin from a frog.     

Distinct apical and basolateral membrane requirements for stretch-induced membrane traffic at the apical surface of bladder umbrella cells

Epithelial cells respond to mechanical stimuli by increasing exocytosis, endocytosis, and ion transport, but how these processes are initiated and coordinated and the mechanotransduction pathways involved are not well understood. We observed that in response to a dynamic mechanical environment, increased apical membrane tension, but not pressure, stimulated apical membrane exocytosis and ion transport in bladder umbrella cells. The exocytic response was independent of temperature but required the cytoskeleton and the activity of a nonselective cation channel and the epithelial sodium channel. The subsequent increase in basolateral membrane tension had the opposite effect and triggered the compensatory endocytosis of added apical membrane, which was modulated by opening of basolateral K⁺ channels. Our results indicate that during the dynamic processes of bladder filling and voiding apical membrane dynamics depend on sequential and coordinated mechanotransduction events at both membrane domains of the umbrella cell.     

Intracellular voltage of isolated epithelia of frog skin: apical and basolateral cell punctures

Isolated epithelia of frog skin were prepared with collagenase, and the cells were punctured with intracellular microelectrodes across their apical (outer) and basolateral (inner) surfaces. Regardless of the route of cell puncture, the intracellular voltage (Vosc) in short- circuited isolated epithelia was markedly negative, averaging -70.4 mV for apical punctures and -91.6 mV for basolateral punctures. As in intact epithelia, amiloride outside caused the Vosc to become more negative (means of -96.7 and -101.8 mV), with a concomitant increase in the resistance of the apical barrier. Increasing the [K)i of the basolateral solution from 2.4 to 8.0 or 14.4 mM caused rapid step depolarization (5-10 s) of the Vosc under transepithelial Na transporting and amiloride-inhibited conditions of Na transport, with the delta Vosc ranging between 23.9 and 68.3 mV per decade change of [K]i. The finding that the Vosc of isolated epithelia of frog skin is independent of the route of cell penetration is consistent with the notion that the cells of the stratified epithelium are electrically coupled (functional syncitium). Moreover, the isolated epithelium can serve as a useful preparation, especially in studies designed to investigate the properties of the basolateral surfaces of cells.