Simon A. Lewis

ADH ACTION: EVIDENCE FOR A MEMBRANE SHUTTLE MECHANISM*

It is now well established that the action of antidiuretic hormone (ADH) is mediated by cAMP and that ADH induces physiological changes in the luminal cell Less certain at this stage are the events that link an elevation of intracellular cAMP to an increase in luminal membrane permeability to water and solutes. In recent years, three observations on the toad bladder have provided important insights into these post-CAMP events: (1) The demonstration that by a variety of maneuvers the changes in water permeability can be dissociated from changes in solute Permeability and therefore that separate pathways are involved.5-7 (2) The observations demonstrating that microtubules and microfilaments are involved in the water permeability response.8-10 (3 ) The finding that intramembrane particle aggregates visualized by freeze-fracture electron microscopy are associated with the modulation of luminal-membrane water pem1eabi1ity.ll-l~ Observations reported by many laboratories clearly demonstrate that these organized membrane structures perform an important role in the action of ADH.ll-la Most workers in the field now strongly suspect that these structures are the site of transmembrane water channels. The evidence that aggregates play a role in the ADH response has been discussed in several review papers.l73 Here we will describe how freeze-fracture observations have led to a new proposal for the mechanism of action of ADH and discuss to what extent this proposal is supported by the available evidence.

Active and passive properties of rabbit descending colon: A microelectrode and nystatin study

SummaryThe electrical properties of the basolateral membrane of rabbit descending colon were studied with microelectrode methods in conjunction with the polyene antibiotic nystatin. Two problems were examined: (i) the relative distribution of tight junctional, apical membrane and basolateral membrane resistances, and (ii) the ionic basis of the basolateral membrane potential. Intracellular K+ activity (K+) was measured using liquid ion exchanger microelectrodes ((K+)=76±2mm) and was found not to be in equilibrium with the basolateral membrane potential. In order to measure membrane resistances and to estimate the selective permeability of the basolateral membrane, the apical membrane was treated with nystatin and bathed with a K2SO4 Ringers solution which was designed to mimic intracellular K+ composition. This procedure virtually eliminated the resistance and electromotive force of the apical membrane. Shunt resistance was calculated by two independent methods based on microelectrode and transepithelial measurements. Both methods produced similar results (Rs=691±63 Ω cm2 and 770±247 Ω cm2, respectively). These findings indicate that the shunt has no significant selectivity, contrary to previous reports. Native apical membrane resistance was estimated as 705±123 V cm2 and basolateral membrane resistance was 95±14 V cm2.To estimate basolateral membrane selectivity, the serosa was bathed in a NaCl Ringers solution followed by a series of changes in which all or part of the Na+ was replaced by equimolar amounts of K+. From measures of bi-ionic potentials and conductance during these replacements, we calculated potassium permeability and selectivity ratios for the nystatin-treated colon by fitting these results to the constant field equations. By correcting for shunt conductance, it was then possible to estimate the selective permeability of the basolateral membrane alone. Selectivity estimates were as follows:PNa/PK=.08 andPCl/PK=.07 (uncorrected for shunt) andPNa/PK=.04 andPCl/PK=.06 (basolateral membrane alone).In a second set of experiments, evidence for an electrogenic Na+ pump in the basolateral membrane is presented. A small ouabain-sensitive potential could be generated in the nystatin-treated colon in the absence of chemical or electrical gradients by mucosal, but not serosal, addition of NaCl. We conclude that this electrogenic pump may contribute to the basolateral membrane potential; however, the primary source of this potential is “passive”: specifically, a potassium gradient which is maintained by an “active” transport process.An appendix compares the results of nystatin experiments to amiloride experiments which were conducted separately on the same tissues. The purpose of this comparison was to develop a comprehensive model of colonic transport. The analysis reveals a leak conductance in the apical membrane and the presence of an amiloride-insensitive conductance pathway.

Apical membrane area of rabbit urinary bladder increases by fusion of intracellular vesicles: an electrophysiological study.

SummaryMammalian urinary bladder undergoes, in a 24-hour period, a series of slow fillings and rapid emptying. In part the bladder epithelium accommodates volume increase by stretching the cells so as to eliminate microscopic folds. In this paper we present evidence that once the cells have achieved a smooth apical surface, further cell stretching causes an insertion of cytoplasmic vesicles resulting in an even greater apical surface area per cell and an enhanced storage capacity for the bladder. Vesicle insertion was stimulated by application of a hydrostatic pressure gradient which caused the epithelium to bow into the serosal solution. Using capacitance as a direct and nondestructive measure of area we found that stretching caused a 22% increase in area. Removal of the stretch caused area to return to within 8% of control. An alternate method for vesicle insertion was swelling the cells by reducing mucosal and serosal osmolarity. This perturbation resulted in a 74% increase in area over a 70-min period. Returning to control solutions caused area to decrease as a single exponential with an 11-min time constant. A microtubule blocking agent (colchicine) dit not inhibit the capacitance increase induced by hypoosmotic solutions, but did cause an increase in capacitance in the absence of a decreased osmolarity. Microfilament disrupting agent (cytochalasin B, C, B.) inhibited any significant change in capacitance after osmotic challenge. Treatment of bladders during swelling with C.B. and subsequent return, to control solutions increased the time constant of the recovery to control values (22 min). The Na+-transporting ability of the vesicles was determined and found to be greater than that of the apical membrane. Aldosterone increased the transport ability of the vesicles. We conclude that some constituent of urine causes a loss of apical membrane permeability. Using electrophysiological methods we estimated that the area of cytoplasmic vesicles is some 3.3 times that of the apical membrane area. We discuss these results in a general model for vesicle translocation in mammalian urinary bladder.

Evaluation by capacitance measurements of antidiuretic hormone induced membrane area changes in toad bladder

A technique for estimating effective transepithelial capacitance in vitro was used to investigate changes in epithelial cell membrane area in response to antidiuretic hormone (ADH) exposure in toad bladder. The results indicate that transepithelial capacitance increases by about 30% within 30 min after serosal ADH addition and decreases with ADH removal. This capacitance change is not blocked by amiloride and occurs whether or not there is a transepithelial osmotic gradient. It is blocked by methohexital, a drug which specifically inhibits the hydro-osmotic response of toad bladder to ADH. We conclude that the hydro-osmotic response of toad bladder to ADH is accompanied by addition of membrane to the plasmalemma of epithelial cells. This new membrane may contain channels that are permeable to water. Stimulation of Na+ transport by ADH is not related to membrane area changes, but appears to reflect activation of Na+ channels already present in the cell membrane before ADH challenge.

Studies of sodium channels in rabbit urinary bladder by noise analysis

SummarySodium channels in rabbit urinary bladder were studied by noise analysis. There are two components of short-circuit current (Isc) and correspondingly two components of apical Na+ entry, one amiloride-sensitive (termedIA and the A channel, respectively) and one amiloride-insensitive (IL and the leak pathway, respectively). The leak pathway gives rise tol/f noise, while the A channel in the presence of amiloride gives rise to Lorentzian noise. A two-state model of the A channel accounts well for how the corner frequency and plateau value of Lorentzian noise vary with amiloride concentration. The single-channel current is 0.64 pA, and the conducting channel density is on the order of 40 copies per cell. Triamterene blocks the A channel alone, and increasing external Na+ decreases the number but not the single-channel permeability of the A channel. Hydrostatic pressure pulses (“punching”) increase the number of both pathways. Repeated washing of the mucosal surface removes most of the leak pathway without affecting the A channel.Properties of the A channel revealed by noise analysis of various tight epithelia are compared, and the mechanism ofl/f noise is discussed. It is suggested that the A channel is synthesized intracellularly, stored in intracellular vesicles, transferred with or from vesicular membrane into apical membrane under the action of microfilaments, and degraded into the leak pathway, which is washed out into urine or destroyed. The A channel starts withPNa/PK∼30 and loses selectivity in stages untilPNa/PK reaches the free-solution mobility ratio (∼0.7) for the leak pathway. This turnover cycle functions as a mechanism of repair and regulation for Na+ channels, analogous to the repair and regulation of most intracellular proteins by turnover. Vesicular delivery of membrane channels may be operating in several other epithelia.

Apical and basolateral membrane ionic channels in rabbit urinary bladder epithelium.

This paper reviews the properties and regulation of single amiloride-sensitive Na+ channels in the apical membrane, and Cl− and K+ channels in the basolateral membrane of rabbit urinary bladder. According to fluctuation analysis, there is an average of one amiloride-sensitive Na+ channel for every 40 μm2 of apical membrane. Each Na+ channel passes 0.7 pA of current under normal, short-circuit conditions. Apical channels are hydrolysed by the endogenous enzyme urokinase, which is released into the urine by the kidney. After exposure to urokinase, the Na+ channel loses its amiloride sensitivity, and eventually becomes unstable in the membrane. The selectivity and kinetic properties of single anion and K+ channels in the basolateral membrane were also studied using the patch clamp technique. The properties of these channels are discussed in terms of the regulation of transepithelial Na+ transport.

The electrical basis for enhanced potassium secretion in rat distal colon during dietary potassium loading

Previous studies in rat distal colon provide evidence for an active absorptive process for potassium under basal conditions, and for active potassium secretion during chronic dietary potassium loading. The present studies were performed with conventional and potassium-selective microelectrodes to determine the electrical basis for the increase in transcellular (active) potassium secretion observed during potassium loading. Compared to control tissues, potassium loading resulted in a 5-fold increase in transepithelial voltage (VT) and a 52% decrease in total resistance (RT) in the distal colon. The rise inVT was due to a decrease in apical membrane resistance and an increase in basolateral membrane voltage from −45±2 mV (cell interior negative) in control to −56±2 mV (P<0.001) in potassium loaded tissues. This difference in basolateral membrane voltage reflected in increase in intracellular potassium activity from 86±4 mM to 153±12 mM (P<0.001). In control tissues, the sequential mucosal addition of the sodium channel blocker amiloride (0.1 mM) and the potassium channel blocker tetraethylammonium chloride (TEA: 30 mM) produced no effect on the electrical measurements. However, in potassium loaded tissues, amiloride and TEA produced transepithelial changes consistent with inhibition of apical membrane conductances for sodium and potassium, respectively, reflected by increases in the resistance ratio, α (ratio of apical to basolateral membrane resistances). These data indicate that the decrease in apical membrane resistance during potassium loading was caused by an increase in apical membrane conductance for both potassium and sodium.

Channels across Epithelial Cell Layers

Publisher Summary Electrolytes and hydrophilic nonelectrolytes require a hydrophilic environment for movement through membrane lipid bilayers, such as is offered by integral membrane proteins. These proteins include two important classes: the channels and the carriers. This chapter defines the channel as a selective pore through which substances can move passively without requiring a shuttlelike movement of the protein within the lipid bilayer. To date, four types of channels have been clearly demonstrated in epithelial cell membranes: (1) the amiloridesensitive Na + channel which is present in the apical membrane of many tight epithelia; (2) K + channels, which have been found in apical and basolateral membranes of both tight and leaky epithelia; (3) nonselective cation channels; and (4) vasopressin-stimulated H 2 0 channels. The chapter reviews the properties of these channels and evidence for their regulation by hormones, ions, and voltage.

LOCALIZATION OF THE ALDOSTERONE RESPONSE IN RABBIT URINARY BLADDER BY ELECTROPHYSIOLOGICAL TECHNIQUES

Sodium absorption by epithelia involves two steps, Na+ movement into the cell, usually thought to be a passive process, i.e. movement down a net electrochemical gradient, and active extrusion from the cell across the basolateral membrane against a net electrochemical gradient. The latter step, occurring against a net electrochemical gradient, uses the energy available from ATP via the catalytic transporting enzyme, (Na-+K-) -ATPase. In epithelia the actions of aldosterone have the ultimate goal of increasing the net rate of Na+ transport. In tight (or high electrical resistance) epithelia a number of sites have been hypothesized for the action of this hormone. Lewis, Eaton, and Diamond1 demonstrated that one of the sites of aldosterone action is to increase the apical membrane Napermeability. Specifically they found an increase in the entry of Na+ into the cell across the apical membrane of the rabbit urinary bladder. Such an increase in apical Na+ permeability can be from either insertion of newly synthesized channels or activation of preexisting channels in the apical membrane. Other hypothesized actions are (1 ) an increase in energy; (2) an increase in the number of transport pumps by either increasing pump density per unit area or increasing the membrane area at constant pump density; (3) an alteration in the binding affinity (ZQ or turnover rate (V,,,,,) of the pump; and (4) an alteration in the stoichiometry of the pump, i.e. the fixed coupling ratio of the pump could be altered so that more Na+ ions are transported per cycle or less Kions are transported per cycle. In this report we demonstrate, at least for the rabbit urinary bladder, that the action of aldosterone does not involve alterations in pump kinetics, density, membrane area changes, or stoichiometry, and that there is more than an adequate reserve in the above aspects of the pump to accommodate any increment in apical Na+ permeability. The methods used do not rely on the use of radio-tracers2s3 but rather on electrical methodology. Such methods not only allow a determination of the stoichiometry of the pump but in addition allow a discrimination between kinetics and density alterations.

Electrical properties of the rabbit urinary bladder assessed using gramicidin D

SummaryRecently, antibiotics have enjoyed widespread usage as tools in studies of epithelial transport. In the present study we assess the usefulness of the pore-forming antibiotic gramicidin D as a means for probing the electrical properties of the tight epithelium rabbit urinary bladder. Addition of 50 μM gramicidin to the mucosal bath (either a NaCl or KCl Ringers solution) led to a large irreversible increase in the transepithelial conductance (GT) within 800 sec.GT increased by approximately 1200% and 500% in KCl and NaCl Ringers solutions, respectively. Microelectrode measurements of the resistance ration (the ration of apical membrane resitance to basolateral membrane resistance) showed that apical membrane resistance is dereased by the drug. Measurements of the basolateral membrane resistance (Rbl) and tight junctional resistance (Rj) using a new and independent method (based on the perturbation of basolateral membrane electrogenic Na+ pump) demonstrated thatRbl andRj were unaffected, suggesting that the effects of gramicidin are restricted to the apical membrane for periods of at least 2 hours after drug addition. The selectivity of the gramicidin-induced permeability in the apical membrane was calculated from measurements of the apical membrane potential after ion substitutions using a modified version of the constant field equation. The selectivity sequence for cations was Cs+>K+>Na+>Li+>choline. Unlike the commonly used polyene antibiotics nystatin and amphotericin B, gramicidin did not induce a significant Cl− permeability. In addition, the dose-response curve had a slope of 1. A method is described for calculating membrane resistances directly from transepithelial measurements under some conditions of gramicidin use, without requiring the use of microlectrode measurements.