N. K. Wills

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.

Active potassium transport by rabbit descending colon epithelium

SummaryPrevious studies of rabbit descending colon have disagreed concerning potassium transport across this epithelium. Some authors reported active K+ secretion underin vitro short-circuited conditions, while others suggested that K+ transport occurs by passive diffusion through a highly potassium-selective paracellular route. For this reason, we re-examined potassium fluxes across the colon in the presence of specific and general metabolic inhibitors. In addition, electrochemical driving forces for potassium across the apical and basolateral membranes were measured using conventional and ion-sensitive microelectrodes. Under normal conditions a significant net K+ secretion was observed (JnetK=−0.39±0.081 μeq/cm2hr) with42K fluxes, usually reaching steady-state within approximately 50 min following isotope addition. In colons treated with serosal addition of 10−4m ouabain,JsmK was lowered by nearly 70% andJmsK was elevated by approximately 50%. Thus a small but significant net absorption was present (JnetK=0.12±0.027 μeq/cm2hr). Under control conditions, the net cellular electrochemical driving force for K+ was 17 mV, favoring K+ exit from the cell. Cell potential measurements indicated that potassium remained above equilibrium after ouabain, assuming that passive membrane permeabilities are not altered by this drug. Net K+ fluxes were abolished by low temperature.The results indicate that potassium transport by the colon may occur via transcellular mechanisms and is not solely restricted to a paracellular pathway. These findings are consistent with our previous electrical results which indicated a nonselective paracellular pathway. Thus potassium transport across the colon can be modeled as a paracellular shunt pathway in parallel with pump-leak systems on the apical and basolateral membranes.

Recent advances in the characterization of epithelial ionic channels

Physiologists have long recognized the importance of channels in the functioning of neurons and excitable membranes. This brief review has been an attempt to illustrate how channel properties are also essential to an understanding of epithelial transport physiology. Among their more important functions, channels influence membrane potentials and serve as conduits for ion movements. As the need to understand the molecular basis for ion transport continues to develop, it is crucial to be able to distinguish between different channel properties. For example, apparent voltage-dependent properties can arise because of a voltage-dependent gating process, or alternatively, because of a rectification of channel conductance. Voltage-dependent effects can also be only indirect, mediated by changes in cell volume, intracellular ion levels, the levels of secondary intracellular messengers such as Ca2+ (perhaps through voltage-dependent membrane Ca2+ channels), or possibly even by morphological changes. An important area for future research is to differentiate mechanisms which modulate the activity of open channels. For example, a decrease in channel number, a reduction in open-channel conductance or a decline in the probability of channel opening can all underlie changes in macroscopic permeability. The factors which mediate hormonal activation of epithelial channels particularly need to be understood. Specifically, the mechanisms of aldosterone and anti-diuretic hormone activation of apical membrane Na+ channels need to be identified. In conclusion, we are witnessing a new era in epithelial electrophysiology which promises to resolve many issues concerning the cellular regulation of ion transport and open new, unanticipated avenues of inquiry.

Noise analysis reveals K+ channel conductance fluctuations in the apical membrane of rabbit colon.

SummaryIn this paper we describe current fluctuations in the mammalian epithelium, rabbit descending colon. Pieces of isolated colon epithelium bathed in Na+ or K+ Ringers solutions were studied under short-circuit conditions with the current noise spectra recorded over the range of 1–200 Hz. When the epithelium was bathed on both sides with Na+ Ringers solution (the mucosal solution contained 50 μm amiloride), no Lorentzian components were found in the power spectrum. After imposition of a potassium gradient across the epithelium by replacement of the mucosal solution by K+ Ringers (containing 50 μm amiloride), a Lorentzian component appeared with an average corner frequency,fc=15.6±0.91 Hz and a mean plateau valueSo=(7.04±2.94)×10−20 A2 sec/cm2. The Lorentzian component was enhanced by voltage clamping the colon in a direction favorable for K+ entry across the apical membrane. Elimination of the K+ gradient by bathing the colon on both sides with K+ Ringers solutions abolished the noise signal. The Lorentzian component was also depressed by mucosal addition of Cs+ or tetraethylammonium (TEA) and by serosal addition of Ba2+. The one-sided action of these K+ channel blockers suggests a cellular location for the fluctuating channels. Addition of nystatin to the mucosal solution abolished the Lorentzian component. Serosal nystatin did not affect the Lorentzian noise. This finding indicates an apical membrane location for the fluctuating channels. The data were similar in some respects to K+ channel fluctuations recorded from the apical membranes of amphibian epithelia such as the frog skin and toad gallbladder. The results are relevant to recent reports concerning transcellular potassium secretion in the colon and indicate that the colon possesses spontaneously fluctuating potassium channels in its apical membranes in parallel to the Na+ transport pathway.

Na+ channels and amiloride-induced noise in the mammalian colon epithelium

(1) The effects of the Na+-channel blocker, amiloride, on the short-circuit current carried by Na+ was studied with fluctuation analysis, in rabbit descending colon epithelium. (2) In the presence of mucosal amiloride, the power spectrum of the Na+-current noise showed a Lorentzian component. When the Na+ current was reduced by increasing the blocker concentrations, the Lorentzian plateau decreased and corner frequency increased. Macroscopic short-circuit current and current-noise data are evidence for a two-state mechanism of the blocker interaction with the Na+ channel. (3) On- and off-rate constants for the blocker-receptor reaction, single-channel currents and Na+-channel density were calculated at room temperature and at 37 degrees C. Also, the activation energy for the amiloride-receptor reaction was estimated. The microscopic parameters obtained for the Na+ channel in the colon were similar to those found for Na+ channels in other tight epithelia.

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.

Transport-Dependent Alterations of Membrane Properties of Mammalian Colon Measured Using Impedance Analysis

SummaryDirect current (DC) measurement methods have been commonly used to characterize the conductance properties of the mammalian colon. However, these methods provide no information concerning the effects of tissue morphology on the electrophysiological properties of this epithelium. For example, distribution of membrane resistances along narrow fluid-filled spaces such as the lateral intercellular spaces (LIS) or colonic crypts can influence DC measurements of apical and basolateral membrane properties. We used impedance analysis to determine the extent of such distributed resistance effects and to assess the conductance and capacitance properties of the colon. Because capacitance is proportional to membrane area, this method provides new information concerning membrane areas and specific ionic conductances for these membranes.We measured transepithelial impedance under three conditions: (1) control conditions in which the epithelium was opencircuited and bathed on both sides with NaCl−HCO3 Ringers solutions, (2) amiloride conditions which were similar to control except that 100 μm amiloride was present in the mucosal bathing solution, and (3) mucosal NaCl-free conditions in which mucosal Na and Cl were replaced by potassium and sulfate or gluconate (“K+ Ringers”). Three morphologically-based equivalent circuit models were used to evaluate the data: (1) a lumped model (which ignores LIS resistance), (2) a LIS distributed model (distributed basolateral membrane impedance) and (3) a crypt-distributed model (distributed apical membrane impedance). To estimate membrane impedances, an independent measurement of paracellular conductance (Gs) was incorporated in the analysis. Although distributed models yielded improved fits of the data, the distributed and lumped models produced similar estimates of membrane parameters. The predicted effects of distributed resistances on DC microelectrode measurements were largest for the LIS-distributed model. LIS-distributed effects would cause a 12–15% underestimate of membrane resistance ratio (Ra/Rb) for the control and amiloride conditions and a 34% underestimate for the “K Ringers” condition. Distributed resistance effects arising from the crypts would produce a 1–2% overestimate ofRa/Rb.Apical and basolateral membrane impedances differed in the three different experimental conditions. For control conditions, apical membrane capacitance averaged 21 μF/cm2 and the mean apical membrane specific conductance (Ga-norm) was 0.17 mS/μF. The average basolateral membrane capacitance was 11 μF/cm2 with a mean specific conductance (Gb-norm) of 1.27 mS/μF.Ga-norm was decreased by amiloride or “K+ ringers” to 0.07 mS/gmF and 0.06 mS/μF, respectively. Basolateral conductance was also reduced by amiloride, whereas capacitance was unchanged (Gb-norm=0.97 mS/μF). For the “K+ Ringers condition, both basolateral conductance and capacitance were greatly increased such thatGb-norm was not significantly different from the control condition.

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.

Chapter 4 Mechanisms of Ion Transport by the Mammalian Colon Revealed by Frequency Domain Analysis Techniques

Publisher Summary This chapter presents frequency domain analysis methods, which are powerful tools for resolving the mechanisms of ion transport in epithelia. An overview of the basic features of the rabbit descending colon are provided. The ion transport properties of the mammalian colon provide numerous issues for investigators using frequency domain techniques. Results concerning potassium transport by this epithelium have been more controversial. A possible model for these transport systems is described. In choosing an appropriate equivalent-circuit model for the colon, two morphological features are taken into consideration: (1) the lateral intercellular space and (2) the crypt lumen. The dimensions of these parameters are important because a narrow or constricted intercellular space or crypt lumen is expected to have a relatively high electrical resistance. Initial attempts to fit the impedance data for the colon by a model, which ignores distributed resistance effects gave reasonable results. Moreover, use of a model, which includes the distributed effects caused by crypt lumen resistance (R p ) , greatly reduced the misfit of the theory to the data.

Chapter 20 Electrophysiology of Active Potassium Transport across the Mammalian Colon

Publisher Summary This chapter focuses on the electrophysiology of active potassium (K) transport across the mammalian colon. The chapter proposes the two basic models that can account for net K + transport. First, for active absorption, there must be an uptake mechanism that uses metabolic energy to drive K across the apical membrane against its net electrochemical gradient. Second, there must be a sufficient net driving force and conductance to support passive exit of this ion across the basolateral membrane. For active secretion, there must be a significant net electrochemical driving force to support K exit across the apical membrane. The apical membrane K + uptake mechanism is limited, but K + uptake are known to occur against the net electrochemical gradient for K + . Net K + absorption is inhibited by metabolic inhibitors, such as dinitrophenol or by cooling the epithelium. The chapter also focuses on the K + secretory system, as this is the mode of net transport most typically observed in the epithelium.