Effect of aldosterone and dexamethasone pretreatment on sodium transport in rat distal colon in vitro

Aldosterone and dexamethasone stimulate sodium absorption in the rat colon in vivo. In vitro, increased amiloride inhibitable short-circuit current (I_SC) has been demonstrated following aldosterone or dexamethasone treatment. Since I_SC bears no relationship to sodium flux (J_Na) in the untreated rat colon, we measured J_Na in partially stripped voltage clamped segments of rat distal colon. Our results demonstrate directly that continuous infusion of aldosterone or dexamethasone for 4–7 days stimulated amiloride inhibitable J_Na by stimulating J_NaM-S. The amiloride inhibitable portion of J_NaM-S was highly correlated with and approximately equal to the amiloride inhibitable I_SC. Amiloride had no effect in controls. We conclude that J_NaM-S in the rat distal colon is only sensitive to mucosal amiloride after treatment with aldosterone or dexamethasone. the amiloride sensitive I_SC in these treated tissues was a good measure of the amiloride sensitive J_Na. Small differences between aldosterone and dexamethasone treatment were noted in the effect on transepithelial resistance, potential difference, and the I_SC after amiloride.     

Effects of aldosterone and dexamethasone on apical membrane properties and Na-transport of rabbit distal colon in vitro

The effects of pre-treatment in vivo with aldosterone and dexamethasone were investigated on rabbit distal colon. Apical Na-permeability and net sodium transport were measured in vitro. In this epithelium, Natransport is entirely electrogenic. It can therefore be measured electrically as the fraction of short circuit current which is blockable by amiloride. The epithelia were studied in an Ussing chamber and the electrical values recorded by a computerized digital voltage clamp. Transepithelial parameters, and the transapical membrane parameters (in preparations depolarized from the serosal side) were investigated after treatment with the two hormones.Under transepithelial conditions, aldosterone and dexamethasone stimulated the short circuit current (I _sc) from control (17.4 A/cm²) to a similar degree (86.6 and 93.8 A/cm²). However, whereas aldosterone did not alter the transepithelial resistance (R _T) significantly, dexamethasone reducedR _T from 357 to 167 · cm². The stimulation of the potential difference (V _T) under control condition (6.6 mV) was therefore significantly different between aldosterone (28.7 mV) and dexamethasone (16 mV). Mucosal amiloride (0.1 mM) inhibitedI _sc andV _T completely under all conditions.Steady state current-voltage relations were obtained by voltage clamping the tissues in staircase increments before and after mucosal treatment with amiloride. As measured by the difference between these two states, Na-currents were calculated both for the transepithelial and the transapical condition. Intracellular Na-activity and apical Na-permeability were then calculated by the Nernst and Goldman-Hodgkin-Katz equations. These values were found to be increased after treatment with both hormones. Dexamethasone was a more potent stimulator of both values. The result suggest different effects on other cellular properties in addition to the effect on the presumably common pathway across the apical membrane.     

Amiloride and the mode of action of aldosterone on sodium transport across toad bladder and skin

Summary The characteristics of the inhibitory influence exerted by amiloride on active sodium transport by toad bladder and skin were taken advantage of to shed additional light on the mechanism whereby hormones-chiefly aldosterone-stimulate sodium transport across such epithelia.Toad skin treated with amiloride was as responsive to vasopressin and to aldosterone as the matched control preparation, while the response of toad skin to insulin was blunted under similar circumstances.When aldosterone-stimulated toad skin was exposed to amiloride, the resulting depression of sodium-transporting activity was less pronounced than was the case with matched control; on the other hand, insulin-treated membranes were as sensitive to amiloride as matched controls. Similar observations were made with triamterene.This is taken to indicate that the density of the permeases apparently required for sodium to cross the apical cell border of these specialized epithelia, is increased under the influence of aldosterone.The affinity of amiloride for toad bladder tissue was not influenced by aldosterone, as indicated by tissue labelling experiments.On leave of absence from the University of Chicago, Department of Medicine. Recipient of a U.S.P.H.S. Career Development Award K2-GM21.842.     

Electrical properties of amphibian urinary bladder epithelia

Summary Necturus urinary bladders were mounted in a two-compartment chamber filled with Ringer's solution and individual cells were punctured with microelectrodes from the serosal surface. By measuring the changes of transepithelial resistance and potential in response to luminal application of amiloride, and measuring the effects of amiloride on the voltage divider ratio and on the serosal cell membrane potential, two independent methods were obtained to calculate the cell membrane resistances and the resistance of the shunt path. The results from both methods agreed favourably. Th mucosal and serosal membrane resistances were found to vary respectively from 9–65 k cm² and from 1–7 k cm² under control conditions. Significant correlations were observed between the luminal membrane resistance as well as the luminal membrane zero current potential and the short circuit current, which indicate that the luminal membrane conductance consists predominantly of Na⁺-selective elements, whose density varies from bladder to bladder and determines the rate of transpithelial Na⁺ transport. Amiloride which blocks these elements increased the luminal resistance to 220 k cm². Increasing short circuit current was also correlated with increasing conductance of the serosal membrane (at fairly constant zero current potential) which points to an electrogenic mechanism of active Na⁺ transport across the serosal cell surface. The paracellular shunt path across the terminal bars plays a minor role in urinary bladder epithelium. Its resistance was estimated to be in the range of 50–125 k cm² or greater.     

The effect of aldosterone on sodium transport and membrane conductances in toad skin (Bufo viridis)

The effect of aldosterone (1 mol/l for 4–6 h) on Na⁺ transport across toad skin (Bufo viridis) was studied in skins preincubated in vitro. Short-circuit current (I _sc) was consistently and reproducibly elevated in skins from NaCl(100 and 200 mmol/l)-acclimated toads, where the baseline I _sc was greatly reduced. The effect of aldosterone was tested in NaCl and NaNO₃ Ringer's and also after oxytocin (50 mU/ml) in the latter conditions. Apical membrane conductance of the principal cells increased consistently after aldosterone in all skins and was linearly correlated with the I _sc under all conditions. This confirms that the stimulation of Na⁺ transport originates from the effect of the aldosterone on apical Na⁺ channels. Basolateral membrane conductance was also significantly elevated compared with control pieces in those tissues that were preincubated with aldosterone for 4–6 h. The increase, however, did not correlate with the magnitude of the I _sc. It is therefore concluded that aldosterone specifically stimulates, in addition to the apical effect, the basolateral membrane conductance. This stimulation appears to be direct and not a secondary response to the elevation of transepithelial transport rate.     

Segmental heterogeneity of basal and aldosterone-induced electrogenic Na transport in human colon

Recent in vitro studies in human colon have demonstrated marked segmental differences in electrogenic Na transport. In the present study, the Na channel blocker amiloride was used further to characterise basal and aldosterone-induced electrogenic Na transport in isolated human distal and proximal colon. Bathed in NaCl Ringer solution, distal and proximal colon exhibited similar basal electrical properties, but the amiloride-sensitive short-circuit current (I _sc) was 200% greater in the distal than in the proximal segment. Bathed in choline-Cl Ringer solution, totalI _sc decreased by 97% in distal colon and by 88% in proximal colon, indicating that Na dependent transport process(es) account almost entirely for theI _sc in both segments. Substituting Na₂SO₄ for NaCl Ringer solution (i) increased amiloride-sensitiveI _sc by 56% (p<0.01) in distal colon but had no effect on amiloride-sensitiveI _sc in proximal colon, and (ii) decreased amiloride-insensitiveI _sc in distal and proximal colon by 52% (p<0.05) and 81% (p<0.001) respectively. After the addition of nystatin to the apical membrane, the relationship between totalI _sc and mucosal Na concentration indicated that the activity of the basolateral membrane Na pump was similar in both colonic segments. In a further series of experiments, exposure of distal colon to 1 mol/l aldosterone for 5 h increased totalI _sc by 52% (p<0.05), which reflected stimulation of its amiloride-sensitive component; in contrast, aldosterone had no effect on proximal colon. These results indicate that Na-dependent electrogenic processes (with electrogenic Na transport predominant) are present throughout human colon, but there is marked segmental variability in the Na conductive properties of the apical membrane. Apical Na entry in distal colon occurs mainly through classical amiloride and aldosterone-sensitive Na channels. In contrast, the predominant apical Na entry mechanism in proximal colon is an amiloride and aldosterone-insensitive path-way.     

Effect of NPPB on chloride (CI) transport in distal colon of potassium (K+) adapted rats

Secondary hyperaldosteronism enhances the rate of K secretion in distal colon, at least in part, through the stimulation of Na⁺-K⁺-Cl^- cotransport across the basolateral membrane. To maintain a constant intracellular Cl^- activity an increase in Cl^- transport out of the cell must be assumed. We explored, under amiloride 10^-4 M and short circuited conditions, conductive pathways for Cl^- exit in the distal colon of K⁺-adapted rats by means of a putative Cl^- channel blocker, NPPB (5-nitro-2(3-phenyl-propylamino-benzoate. Results prior to NPPB showed an increase in JCl^ras after K⁺ loading from 5.84 + 0.66 to 8.33 ±0.86 and JCl^8m from 4.77 ±0.55 to 8.16±0.96/μEq h“¹ cm”² (P < 0.001), when compared with controls. Net fluxes were not different between groups. Luminal NPPB in K⁺ adaptation resulted in a decrease of JCl^sm, from 7.85 ±1.5 to 6.69 ± 1.5 μEq h^-1 cm^-2 (P < 0.05). There were no changes in both unidirectional Cl^- fluxes in controls under luminal NPPB and in potential difference (V) and short-circuit current (I^-.) under any condition. Finally, K⁺ adaptation resulted in an increase of luminal cyclic AMP (cAMP) concentration (0.09 ±0.02 to 0.20 ±0.03 pmol 100 μ^-1, P < 0.05), when compared with control rats. The data may suggest a transcellular recycling of Cl^- and an activated NPPB inhibitable serosal to mucosal Cl^- pathway on luminal membrane in the K⁺ adapted state, possibly mediated by an increase in cAMP production.     

Determination of cell membrane resistance in cultured renal epithelioid (MDCK) cells: effects of cadmium and mercury ions

Previous studies have indicated that the cell membrane of Madin Darby Canine Kidney (MDCK) cells is hyperpolarized by a number of hormones and trace elements, in parallel with an enhancement of potassium selectivity. Without knowledge of the cell membrane resistance (R _m), however, any translation of potassium selectivity into potassium conductance remains equivocal. The present study was performed to determine the R _m of MDCK cells by cellular cable analysis. To this end, three microelectrodes were impaled into three different cells of a cell cluster; current was injected via one microelectrode and the corresponding voltage deflections measured by the other two microelectrodes. In order to extract the required specific resistances, the experimental data were analysed mathematically in terms of an electrodynamical model derived from Maxwell's equations. As a result, a mean R _m of 2.0±0.2 kcm² and an intercellular coupling resistance (R _c) of 6.1±0.8 M were obtained at a mean potential difference across the cell membrane of -47.0±0.6 mV. An increase of the extracellular K⁺ concentration from 5.4 to 20 mmol/l depolarized the cell membrane by 16.2±0.5 mV and decreased R _m by 30.6±3.0%; 1 mmol/l barium depolarized the cell membrane by 20.1±1.1 mV and increased R _m by 75.9±14.3%. Omission of extracellular bicarbonate and carbon dioxide at constant extracellular pH caused a transient hyperpolarization (up to –60.4±1.4 mV), a decrease of R _m (by 75±4.5%) and a decrease of R _c (by 23.1±8.4%). The changes in R _m and R _c were probably the result of intracellular alkalosis. Cadmium ions (1 mol/l) led to a sustained, reversible hyperpolarization (to –64.8±1.3 mV) and to a decrease of R _m (by 77.0±2.7%); mercury ions (1 mol/l) cause a sustained hyperpolarization (to –60.1±1.2 mV) and a decrease of R _m (by 76.3±3.9%). Neither manoeuvre significantly altered R _c. We have previously shown that both cadmium and mercury hyperpolarize the cell membrane potential and increase its potassium selectivity; the decrease of the R _m observed in the present study indicates that these effects are due to an increase of the potassium-selective conductance of the cell membrane.     

Sodium pump quantity and turnover in rabbit descending colon at different rates of sodium absorption

³H-Ouabain binding to isolated epithelia and basolateral membrane vesicles of Na⁺-transporting epithelial cells of rabbit descending colon was determined to quantify the number of operative Na⁺-pump sites at different rates of transcellular Na⁺ transport which was varied over a wide range by chronic dietary Na⁺ restriction or Na⁺ loading. Both in intact epithelia and in basolateral membrane vesicles the maximal number of specific ouabain binding sites was higher in preparations from animals transporting Na⁺ at high rates than in preparations from animals transporting Na⁺ at low rates. The affinity of ouabain to its binding site and the association and dissociation rate constants were not dependent on the rate of Na⁺ transport. In intact epithelia the Na⁺ turnover rate per pump unit was twice as high in tissues with high Na⁺ transport than in tissues with low Na⁺ transport. In basolateral membrane vesicles the Na⁺ turnover rate was considerably higher than in intact epithelia and there was no difference in turnover rate between vesicle preparations obtained from tissues transporting Na⁺ at high or low rates. Hence, factors within the intact cell appear to control the turnover rate of the Na⁺-pump.     

Transcellular bicarbonate transport in rabbit gallbladder epithelium: mechanisms and effects of cyclic AMP

HCO₃ permeation through rabbit gallbladder epithelium has been investigated in vitro using voltage-clamp, pH-stat and microelectrode techniques. Mucosa-to-serosa flux of HCO₃ (4.9 mol cm^–2h^–1) was dependent on luminal Na and inhibited by amiloride (1 mmol/l, luminal bath), methazolamide (0.1 mmol/l, both sides), and ouabain (30 mol/l, serosal bath). Maximal rates of serosa-to-mucosa flux of HCO₃ (2.8 mol cm^–2h^–1) required serosal Na and mucosal Cl. This flux was inhibited by ouabain, 4-acetamido-4-isothiocyanato-stilbene-2,2-disulfonic acid (1 mmol/l, serosal bath), and 5-nitro-2-(3-phenylpropylamino)-benzole acid (0.1 mmol/l, luminal bath). Ineffective were methazolamide (0.1 mmol/l, both sides) and amiloride (1 mmol/l, serosal bath). 8-Br-cAMP (1 mmol/l, serosal bath) largely inhibited the absorptive and moderately stimulated the secretory flux. In tissue conductance, short-circuit current, and transmural voltage prostaglandin E₁ (1 mol/l, serosal bath) and 8-Br-cAMP caused moderate to negligible increases. No significant alterations of apical membrane potential ( –65 mV) and the apparent ratio of membrane resistances (R_a/R_b;1.9) were found. Cell membranes responded to luminal Cl removal mostly with a slow hyperpolarization that was mitigated by 8-Br-cAMP or, in some cases, converted into a small, transient depolarization. Our results are best explained by transcellular HCO₃ transport in both directions. In secretion, basolateral HCO₃ entry occurs by some form of co-transport with Na, and apical exit by Cl/HCO₃ exchange. cAMP opens no major electrodiffusive pathway for apical anion efflux. In absorption, HCO₃ import from the lumen into the cell is secondary to cAMP-sensitive Na/H exchange.     

Square wave pulse analysis of cellular and paracellular conductance pathways in necturus gallbladder epithelium

In search for a rapid and reliable method to identify and quantitatively determine cell membrane resistances and paracellular shunt resistances in epithelia we have developed appropriate techniques to measure transepithelial and intracellular potential transients in response to transepithelially applied square wave constant current pulses. Model considerations indicate that in a unilayered, homogeneous epithelium with open lateral spaces the transient potential response across each cell membrane should obey a single exponential function in case the tight junction resistance is high, as in a tight epithelium, whereas in a leaky epithelium it should consist of a superposition of two exponentials with equal sign at the membrane with the higher intrinsic time constant and of two exponentials of different sign (overshoot with recline) at the membrane with the lower intrinsic time constant. The latter predictions were experimentally verified in a study on Necturus gallbladder epithelium and equivalent circuit parameters for the cell membrane resistances and capacitances as well as for the resistance of the shunt path were calculated from the data by curve fitting procedures. The resistances of the apical and basal cell membrane and of the shunt path averaged 1220, 201 and 91 cm² respectively while the apical and basal cell membrane capacitances were 8.0 and 26.3 F/cm² respectively. The fact that the resistance values are 4–15 times lower than estimates derived previously from 2D-cable analysis relates to a better preservation of the transport function under the present incubation conditions as verified by a new series of cable analysis data. The capacitances agree well with estimates of the surface amplification of the cell membranes from electronmicrographs, thus confirming the validity of the interpretation of the observed voltage transients.     

Mechanisms of oxalate absorption and secretion across the rabbit distal colon

To further evaluate the mechanisms of oxalate (Ox^2–) transport in the intestine the following studies were performed using isolated, short-circuited segments of the rabbit distal colon (DC). In control buffer, the DC absorbed Ox^2– (net Ox^2– flux, J _Net ^Ox =5.4±0.7 pmol · cm^–2 · h^–1). Replacement of Na⁺ with N-methyl-d-glucamine (NMDG⁺) abolished Ox^2– absorption by decreasing mucosal to serosal Ox^2– flux (J _ms ^Ox ), without affecting Cl^– transport, while gluconate substitution for Cl^– did not affect J _Net ^Ox or net Na⁺ flux (J _Net ^Na ). Addition of Na⁺ to the serosal side of tissues bathed by NMDG⁺ buffer increased J _ms ^Ox 40% without altering mucosal to serosal Cl^– flux (J _ms ^Cl ). Serosal amiloride or dimethyl amiloride (10^–3 M) abolished J _Net ^Ox by decreasing J _ms ^Ox , it increased serosal to muscosal Cl^– flux (J _sm ^Cl ) and it gradually inhibited short-circuit current (I _sc). Mucosal amiloride (10^–4 M) abolished I _sc but had no effect on Ox^2– or Cl^– fluxes. Serosal 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS, 10^–6 M) reduced J _ms ^Ox by 20% and J _Net ^Ox by 43% without affecting J _ms ^Cl or J _Net ^Cl . Dibutyryl cyclic adenosine monophosphate (dB-cAMP, 5×10^–4 M, both sides) stimulated Ox^2– secretion (J _Net ^Ox = –12.6±3.3 pmol · cm^–2 · h^–1). The dB-cAMP-induced secretion of Ox^2– and Cl^– were fully abolished by serosal furosemide (10^–4 M) and partially inhibited (35%) by 5×10^–4 M mucosal NPPB [5-nitro-2-(3-phenylpropylamino)-benzoic acid], a putative Cl^– channel blocker. It is proposed that: (1) basal absorption of Ox^2–, but not Cl^–, is dependent upon a previously undescribed basolateral Na⁺-H⁺ exchanger that may be coupled to a DIDS-sensitive, basolateral anion exchange system that mediates Ox^2– flux; (2) the DC secretes Ox^2– in response to dB-cAMP by a mechanism that is indistinguishable from the pathway for Cl^– secretion.     

The effects of amiloride,ouabain and osmolality on sodium transport across bovine cornea

Electrical parameters across different parts of bovine cornea were measured after varying osmolality, temperature, pH and sodium concentration of bathing solutions, with and without amiloride or ouabain. It is concluded, that the transcorneal potential difference represents the sum of the transendothelial and transepithelial potential differences. Amiloride completely and reversibly inhibited the potential difference and the short circuit current originating in the epithelium (K _i=2·10^–6 mol/l). Furthermore, the transcorneal electrical resistance increased by 28±8%. The results were used to calculate the cellular and paracellular pathway resistances in the corneal epithelium. An equivalent electrical circuit for sodium transport across the bovine cornea is proposed, which simulates the results obtained.     

Dual-frequency method for synchronous measurement of cell capacitance,membrane conductance and access resistance on single cells

A dual-frequency method was developed to monitor changes of membrane capacitance, membrane conductance and serial resistance between patch pipette and cytoplasm of the cell in the whole-cell configuration. Measurement of real and imaginary components of cell admittance during excitation with two superimposed sinusoidal voltages of different frequencies provides mathematical solutions for all three variables. The validity of the method was verified with experiments on mast cells and exocrine pancreatic acinar cells. During degranulation of mast cells, induced by GTPS in the pipette solution, a stepwise increase in membrane capacitance could be observed, indicating that the resolution of the method is below 10 fF. Precalibration of the setup allows all calculated data to be expressed as absolute values. The capacitance measurement proved to be rather independent of changes in the access resistance and in the cell membrane resistance over a wide range. The huge changes in membrane conductance of mouse pancreatic acinar cells during hormonal stimulation with acetylcholine produced a relative error of less than 0.3% in the capacitance trace. This allows a clear distinction between changes of membrane conductance and cell capacitance. The method therefore offers great advantages in the study of exocytosis as well as endocytosis in cell types, such as exocrine gland cells, with major changes in membrane conductance during hormonal stimulation.     

Proliferation of goblet cells and vacuolated cells in the rabbit distal colon

Previous studies of colonic epithelial cell kinetics in mice and rats revealed a pattern similar to small intestine, where basally located stem cells proliferate, differentiating as they migrate towards the surface epithelium. Vacuolated and goblet cells are assumed to co-migrate at the same rate. The present study indicates that rabbit distal colon has more complicated epithelial cell kinetics. The zone of proliferation was detected immunohistochemically using proliferating cell nuclear antigen (PCNA) and confirmed with the use of colchicine to arrest dividing cells in metaphase. Migrating cells were tracked from the zero-hour position (PCNA labeling, mitosis) to positions 24, 48, 72 hrs by monitoring cell migration with the thymidine analog 5-Bromo-2-Deoxyuridine (BrdU). PCNA revealed a major proliferative zone in the upper third of the crypt column and the presence of mitotic figures after colchicine corroborated these results. Differentiated vacuolated cell proliferation was detected at three crypt sites: base, middle, and top of the crypt, while columnar cells arose from a population of dividing cells at the top of the crypt. Turnover of columnar and vacuolated cells occurred within 72 hrs. Goblet cells exhibited maximal proliferation at the crypt base and migrated at a much slower rate than the other cell types. In rabbit distal colon, populations of proliferating cells exist at multiple levels of the crypt column. Vacuolated and goblet cells differ in their labeling indices and migration rates, suggesting that the two cell types arise and migrate independently. Anat. Rec. 252:41–48, 1998. © 1998 Wiley-Liss, Inc.     

Procaine effects on sodium and chloride transport in frog skin

Procaine, a tertiary amine, has previously been shown to stimulate reversibly transepithelial Na transport across frog skin after application from the epithelial side. In the present study with intracellular recording from principal, i.e. amiloride-sensitive cells, we demonstrate that the stimulation results from increase in apical membrane Na permeability. A second effect of procaine (10–25 mmol/l) in the outside perfusion solution is a reversible increase of transepithelial conductance which drastically exceeds the predicted response of the transcellular Na pathway. It requires presence of chloride on the epithelial side and depends on the non-ionized molecule of procaine. Abolition of apical membrane Na uptake by amiloride or Na-free mucosal inbubation decreases the magnitude but does not prevent the stimulatory effect of procaine. The origin of this gain in conductance from stimulation of a Cl-specific pathway is demonstrated by a highly significant correlation between the increases in electrically determined tissue conductance and partial Cl conductance, obtained from measurements of influx and efflux of Cl-36. Measurements with microelectrodes indicate that the stimulated Cl-specific pathway is distinct from the principal cells. Since procaine activates a conductive pathway with similar response pattern as spontaneously existing Cl conductance, it might be a valuable tool for investigating mode and way of Cl movement across epithelial tissues.     

Ion transport and electrophysiology of the early proximal colon of rabbit

The ion transport properties of the mammalian descending colon have been the subject of numerous investigations during the last decade. In contrast, relatively few studies have investigated proximal segments of this organ. In the present study, we assessed transepithelial transport of Na⁺, K⁺ and Cl^– in the isolated initial segment (P1) of rabbit colon in vitro using radioisotopic tracer fluxes and electrophysiological techniques. Like the rabbit descending colon, the proximal colon actively absorbs sodium and chloride, howeveer, its transport systems are markedly different. In vivo, this segment absorbs potassium, however in vitro active potassium secretion was observed. Unlike the descending colon, Na⁺ absorption is relatively insensitive to amiloride and only a slight inhibition was obtained even at 1 mM concentrations of this drug. Na⁺ and Cl^– absorption appeared to be coupled (directly or indicrectly) since the absorption of each ion was inhibited by the removal of the other. Serosal ouabain also inhibited Na⁺ and Cl^– absorption and net K⁺ secretion. Unlike the descending colon, the proximal P1 segment did not have a net absorptive K⁺ transport system that was detectable in the presence of ouabain. Electrically, the early proximal colon has a low transepithelial resistance compared to descending colon (R _T=133±7 cm²) but a larger short-circuit current (l _sc=178±12 A/cm²). The transepithelial potential averaged –21±1 mV, in excellent agreement with values measured in vivo. The apical and basolateral membrane potentials averaged –21±1 mV and –42±1 mV and intracellular potassium activity was 70±2 mM. The findings indicate active K⁺ uptake across the basolateral membrane and passive exit across the apical membrane. The basolateral membrane conductance may be a potassium conductance that is blockable by barium. It is likely that K⁺ transport normally occurs by both cellular and paracellular routes in this epithelium. Because of the numerous differences between this segment and the descending colon, we conclude that the P1 segment of proximal colon has a distinct function in colonic electrolyte transport     

On the nature of delayed repolarization during sustained sodium coupled transport in frog proximal tubules

In proximal tubules of the frog kidney, stimulation of coupled transport of sodium with phenylalanine leads to depolarization of the cell membrane, followed by repolarization within a few minutes. The repolarization is due to a delayed increase of potassium conductance at the peritubular cell membrane. The present study was designed to test for the role of depolarization, of calmodulin and of arachidonic acid metabolites for the delayed increase of potassium conductance. To this end, the potential difference across the peritubular cell membrane of proximal convoluted tubules (PD_pt) has been recorded continuously during exposure of the lumen to phenylalanine or during galvanic current injection into a neighbouring cell. During control conditions, PD_pt averages –68.6±1.0 mV (n=45). Phenylalanine leads to a depolarization of the peritubular cell membrane by +31.5±1.3 mV (n=20), followed by a repolarization by –12.9±1.1 mV (n=20) within 3 min. Injection of currents from 10 to 80 nAmps leads to a depolarization by + 0.83±0.01 mV/nAmps which is again followed by repolarization. A linear correlation is observed between the magnitude of depolarization (dep) and repolarization (rep)within 3 min: rep (mV)= –(0.24±0.01) dep (mV) + (2.45±0.12) mV (r=0.09). Thus, depolarization is capable to trigger delayed repolarization. The extent of repolarization is a function of the magnitude of depolarization. The possible involvement of calmodulin or arachidonic acid metabolites has been tested for by inducing sodium coupled transport in the presence of 100 mol/l mepacrine, 10 mol/l indomethacin or 10 mol/l trifluoperazine. As a result, indomethacin and trifluoperazine do not significantly interfere with either depolarization or repolarization of the cell membrane during stimulation of sodium coupled transport while mepacrine significantly reduces repolarization.     

The effect of aldosterone and the renin-angiotensin system on sodium, potassium and chloride transport by proximal and distal rat colon in vivo.

1. The roles of aldosterone and angiotensin in the direct control of epithelial sodium transport in vivo have been investigated by measurement of electrical p.d. changes and of the fluxes of sodium, potassium and chloride in rat colon, an organ actively involved in electrolyte homoeostasis. Exogenous angiotensin and aldosterone were given by both short- and long-term infusions and endogenous secretion of the hormones was varied by dietary sodium variation and by nephrectomy and/or adrenalectomy. 2. In vitro angiotensin has been shown to influence colonic salt and water absorption but in the present in vivo experiments administered angiotensin had no significant action on p.d. or on the ionic fluxes of the proximal or distal colon. The increase in p.d. produced by infusing aldosterone was unaffected by giving angiotensin concurrently. The effect of sodium depletion in stimulating sodium absorption and potassium secretion was completely abolished by adrenalectomy but was unaffected by nephrectomy. 3. During prolonged infusion of angiotensin into adrenalectomized rats, a small fall in faecal fluid and sodium content was observed, but this change would have little significance in sodium homoeostasis. 4. Aldosterone and sodium depletion stimulated sodium absorption in both proximal and distal colon but significant increase in potassium secretion was demonstrable only in the distal colon. Bicarbonate secretion (by calculation) was unaffected. In the proximal colon, the increased sodium absorption appeared to be accompanied by increased chloride absorption while in the distal colon it was principally the sodium-potassium exchange that was increased. 5. Adrenalectomy reduced potassium secretion in both proximal and distal colon but sodium absorption was only significantly reduced in the proximal colon. 6. It was concluded that there is no evidence that angiotensin in the living animal has a role as an important salt retaining hormone by direct epithelial action. Aldosterone has a considerable effect which is independent of the presence of angiotensin, and which differs in proximal and distal colon in regard to the relative effects on chloride absorption and potassium secretion.