Ion transport by rabbit colon: II. Unidirectional sodium influx and the effects of amphotericin B and amiloride

Summary The unidirectional influx of Na from the mucosal solution into the epithelium ofin vitro descending rabbit colon (J _me ^Na ) determined under short-circuit conditions, is comprised of two components: one represents entry of Na into transporting epithelial cells and is abolished by amiloride which also abolishes Na absorption (J _net ^Na ). The other represents diffusional Na entry into paracellular pathways traversing the epithelium. In all instances, exposure of the mucosal surface to amphotericin B increased tissue conductance andJ _me ^Na and elicited K secretion. Tissues showing a spontaneousI _sc of approximately 4 eq/cm²hr did not respond to amphotericin B with increasedI _sc andJ _net ^Na . However, in tissues characterized by a lowerI _sc under control conditions, amphotericin B increasedI _sc andJ _net ^Na to approximately 4eq/cm²hr. These findings suggest that amphotericin increasesJ _net ^Na and elicits K secretion by disrupting the normal permselectivity of the mucosal membrane. Under these conditions the extrusion of Na from cell-to-serosal solution becomes the rate limiting step in transepithelial Na transport. Finally, a close correlation betweenJ _me ^Na andJ _net ^Na was observed when the rate of Na absorption varied either spontaneously or experimentally with amiloride, suggesting that the backflux of Na from cell-to-mucosal solution is undetectably small.     

Basolateral potassium channels of rabbit colon epithelium: role in sodium absorption and chloride secretion

In order to assess the role of different classes of K(+) channels in recirculation of K(+) across the basolateral membrane of rabbit distal colon epithelium, the effects of various K(+) channel inhibitors were tested on the activity of single K(+) channels from the basolateral membrane, on macroscopic basolateral K(+) conductance, and on the rate of Na(+) absorption and Cl(-) secretion. In single-channel measurements using the lipid bilayer reconstitution system, high-conductance (236 pS), Ca(2+)-activated K(+) (BK(Ca)) channels were most frequently detected; the second most abundant channel was a low-conductance K(+) channel (31 pS) that exhibited channel rundown. In addition to Ba(2+) and charybdotoxin (ChTX), the BK(Ca) channels were inhibited by quinidine, verapamil and tetraethylammonium (TEA), the latter only when present on the side of the channel from which K(+) flow originates. Macroscopic basolateral K(+) conductance, determined in amphotericin-permeabilised epithelia, was also markedly reduced by quinidine and verapamil, TEA inhibited only from the lumen side, and serosal ChTX was without effect. The chromanol 293B and the sulphonylurea tolbutamide did not affect BK(Ca) channels and had no or only a small inhibitory effect on macroscopic basolateral K(+) conductance. Transepithelial Na(+) absorption was partly inhibited by Ba(2+), quinidine and verapamil, suggesting that BK(Ca) channels are involved in basolateral recirculation of K(+) during Na(+) absorption in rabbit colon. The BK(Ca) channel inhibitors TEA and ChTX did not reduce Na(+) absorption, probably because TEA does not enter intact cells and ChTX is 'knocked off' its extracellular binding site by K(+) outflow from the cell interior. Transepithelial Cl(-) secretion was inhibited completely by Ba(2+) and 293B, partly by quinidine but not by the other K(+) channel blockers, indicating that the small (<3 pS) K(V)LQT1 channels are responsible for basolateral K(+) exit during Cl(-) secretion. Hence different types of K(+) channels mediate basolateral K(+) exit during transepithelial Na(+) and Cl(-) transport.     

Vanadium-induced Cl(-)-secretion in rabbit descending colon is mediated by prostaglandins.

Vanadium in the 4+ (vanadyl-ion) and 5+ (vanadate-ion) oxidation state stimulates furosemide-sensitive electrogenic Cl- secretion in isolated epithelia of rabbit descending colon. This effect is associated with an increased release of prostaglandin E2 from the tissue. Inhibitors of phospholipase A2 or cyclooxygenase abolish both vanadium-induced release of prostaglandin E2 and Cl- secretion. Neuronal mechanisms are not likely to be involved, as tetrodotoxin does not affect the vanadate induced Cl- secretion. Although vanadate is known to inhibit Na+,K(+)-ATPase activity, no inhibition of active Na+ transport was observed in intact colonic epithelia suggesting a rapid intracellular reduction of vanadate ions to vanadyl ions which have no inhibitory effect on the Na+,K(+)-ATPase. The present findings therefore indicate that vanadate stimulated colonic Cl- secretion involves intracellular conversion of vanadate to vanadyl and release of prostaglandin E2.     

Interaction between cell sodium and the amiloride-sensitive sodium entry step in rabbit colon

Summary Ouabain abolishes the short-circuit current (I _sc ) and decreases the transepithelial conductance (G _t ) of rabbit colon. In contrast, amphotericin B elicits a maximumI _sc and markedly increasesG _t . However, inboth instances the amiloride-sensitive Na entry step is completely blocked, presumably due to an increase in cell Na. Conversely, when Na-depleted tissues are suddenly exposed to 140mm Na, the amiloride-sensitiveI _sc and the amiloride-sensitive component ofG _t ( _a G _Na ) increase abruptly to their maximum values and the decline to steady-state plateaus with a half time of 6 min; throughout the decline (I _sc/a G _Na)=E _Na is constant at a value of 95 mV. In the presence of amphotericin B, theI _sc abruptly rises to the same maximum but does not decline. These findings indicate that in the presence of 140mm Na the conductance of the amiloride-sensitive Na entry step can vary from a maximum value of approximately 1.6 mmhos/cm² when cell Na is depleted, to zero when cell Na is abnormally elevated (e.g., in the presence of ouabain or amphotericin B). Our findings are consistent with a system in which the pathway responsible for transcellular Na transport parallels another cellular compartment with which it communicates. The Na capacity of the active transport pathway appears to be very small so that this compartment fills rapidly after exposure of Na-depleted cells to 140mm Na, and active transepithelial Na transport is initiated and reaches steady-state levels quickly. The Na capacity of the second compartment is much larger; the Na content of this compartment appears to be responsible for the negative feedback effect on the permeability of the amiloride-sensitive entry step.     

P-chloromercuribenzene sulfonate blocks and reverses the effect of amiloride on sodium transport across rabbit colon in vitro.

The addition of 10(-3) M p-chloromercuribenzene sulfonate (PCMBS) to the solution bathing the mucosal surface of rabbit colon has no effect on the rate of active Na transport but blocks or reverses the inhibitory action of amiloride. The tissue must be exposed to PCMBS for 20-30 min for a complete blocking effect, and removal of PCMBS from the mucosal solution after this period of exposure does not restore the sensitivity of the tissue to amiloride. The slow time-courses of the blocking and reversal effects suggest that PCMBS does not irreversibly interact with groups directly involved in the binding of amiloride.     

Rabbit distal colon epithelium: I. Isolation and characterization of basolateral plasma membrane vesicles from surface and crypt cells

Summary A method has been developed for the simultaneous isolation of basolateral plasma membrane vesicles from surface and crypt cells of rabbit distal colon epithelium by sequential use of differential sedimentation, isopycnic centrifugation and Ficoll 400 barrier centrifugation. The protein yield was high (total 0.81 mg/g mucosa) and surface and crypt cell-derived basolateral membrane fractions have been purified 34- and 9-fold with respect to the homogenate. The pattern of marker enzyme enrichments revealed only minor contamination by subcellular organelles. Latency of ouabain-sensitive (Na⁺, K⁺)-ATPase activity prior and after trypsin treatment of membranes indicated a vesicle configuration of sealed right side-out: sealed inside-out: leaky of approximately 211. The presence of sealed vesicles was also evident from the osmotic sensitivity of thed-[1-¹⁴C] mannitol equilibrium space determined with either fraction. Although considerably different in protein profile, surface and crypt basolateral membranes were similar in cholesterol to phospholipid molar ratio and membrane fluidity as determined by steady-state fluorescence polarization.Stopped-flow light scattering experiments revealed a rather low water permeability of the membranes with a permeability coefficient of 6 m/sec at 35°C, which is one order of magnitude lower than reported for small intestinal plasma membranes. Both membrane fractions have been shown to effectively generate outward uphill potassium ion gradients, a process that is energized by ATP and inhibited by the membrane-permeant cardiacglycoside digitoxin. These characteristics are consistent with the activity of a (Na⁺, K⁺) pump operating in inside-out vesicles.     

Effect of anions on amiloride-sensitive,active sodium transport across rabbit colon,in Vitro

Summary replacement of Cl in the solutions bathing partial mucosal strips of rabbit descending colon with sulfate, isethionate, hydroxypropane-sulfonate and, to a lesser degree, ethanesulfonate stimulates active Na absorption (J _net ^Na ) when the baso-lateral pump mechanism is not saturated. These effects are rapid in onset and are readily reversible. Our findings indicate that these stimulatory anions decrease the resistance of the amiloridesensitive Na entry step at the mucosal membrane (R _Na ^m ). However, when the active Na pump mechanism at the baso-lateral membrane is saturated these stimulatory anions do not decrease the resistance of the Na entry process. These findings suggest the presence of a negative feedback between the activity of the pump mechanism and the resistance of the Na entry step which may be mediated by the size of the intracellular Na transport pool. In other words, it seems that when the baso-lateral pump is operating at its maximal rate the resistance to Na entry across the mucosal membrane through the amiloride-sensitive pathway is at a minimum and cannot be further decreased.     

Membrane phospholipid composition affects function of potassium channels from rabbit colon epithelium

We tested the effects of membrane phospholipids on the functionof high-conductance,Ca²⁺-activatedK⁺ channels from the basolateralcell membrane of rabbit distal colon epithelium by reconstituting thesechannels into planar bilayers consisting of different 1:1 mixtures ofphosphatidylethanolamine (PE), phosphatidylcholine (PC),phosphatidylserine (PS), and phosphatidylinositol (PI). At low ambientK⁺ concentrations single-channelconductance is higher in PE/PS and PE/PI bilayers than in PE/PCbilayers. At high K⁺concentrations this difference in channel conductance is abolished. Introducing the negatively charged SDS into PE/PC bilayersincreases channel conductance, whereas the positively chargeddodecyltrimethylammonium has the opposite effect. All these findingsare consistent with modulation of channel current by the charge of thelipid membrane surrounding the channel. But theK⁺ that permeates the channelsenses only a small fraction of the full membrane surface potential ofthe charged phospholipid bilayers, equivalent to separation of theconduction pathway from the charged phospholipid head groups by 20 Å. This distance appears to insulate the channel entrancefrom the bilayer surface potential, suggesting large dimensions of thechannel-forming protein. In addition, in PE/PC and PE/PI bilayers, butnot in PE/PS bilayers, the open-state probability of the channeldecreases with time ("channel rundown"), indicating thatphospholipid properties other than surface charge are required tomaintain channel fluctuations.

     

Relation between intracellular sodium and active sodium transport in rabbit colon: Current-voltage relations of the apical sodium entry mechanism in the presence of varying luminal sodium concentrations

The current-voltage relations of the amiloride-sensitive Na entry pathway across the apical membrane of rabbit descending colon, exposed to a high K serosal solution, were determined in the presence of varying mucosal Na activities, (Na)m, ranging from 6.2 to 99.4 mM. These relations could be closely fit to the "constant field" flux equation yielding estimates of the permeability of the apical membrane to Na, PmNa, and the intracellular Na activity, (Na)c. The following empirical relations emerged: (Na)c increased hyperbolically with increasing (Na)m; PmNa decreased hyperbolically with increasing (Na)m and linearly with increasing (Na)c; spontaneous variations in Na entry rate at constant (Na)m could be attributed entirely to parallel, spontaneous variations in PmNa; the rate of Na entry increased hyperbolically with increasing (Na)m obeying simple Michaelis-Menten kinetics; the relation between (Na)c and "pump rate," however, was sharply sigmoidal and could be fit by the Hill equation assuming strong cooperative interactions between Na and multiple sites on the pump; the Hill coefficient was 2-3 and the value of (Na)c at which the pump-rate is half-maximal was 24 mM. The results provide an internally consistent set of relations among Na entry across the apical membrane, the intracellular Na activity and basolateral pump rate that is also consistent with data previously reported for this and other Na-absorbing epithelia.