Stanley G. Schultz

Sodium-coupled amino acid and sugar transport byNecturus small intestine

SummaryNecturus small intestine actively absorbs sugars and amino acids by Na-coupled mechanisms that result in increases in the transepithelial electrical potential difference (ψms) and the short-circuit current (Isc) which can be attributed entirely to an increase in the rate of active Na absorption. Studies employing conventional microelectrodes indicate that the addition of alanine or galactose to the mucosal solution is followed by a biphasic response. Initially, there is a rapid depolarization of the electrical potential difference across the apical membrane (ψms) which reverses polarity (i.e. cell interior becomes positive with respect to the mucosal solution) and a marked decrease in the ratio of the effective resistance of the mucosal membrane to that of the serosal membrane (Rm/Rs); these events do not appear to be dependent on the availability of metabolic energy. These initial, rapid events are followed by a slow increase in (Rm/Rs) toward control values which is paralleled by a repolarization ofψms and increases inψms andIsc; this slow series of events is dependent upon the availability of metabolic energy.The results of these studies indicate that: (i) the Na-coupled mechanisms that mediate the entry of sugars and amino acids across the apical membrane are “rheogenic” (conductive) and result in a decrease inRm and a depolarization ofψms; and (ii) the subsequent increase in (Rm/Rs) and repolarization ofψms are the results of a decrease inRs which is associated with an increase in the activity of the Na pump at the basolateral membrane.The physiologic implications of these findings are discussed and an equivalent electrical circuit model for “rheogenic” Na-coupled solute transport processes is analyzed.

Effects of Na-coupled alanine transport on intracellular K activities and the K conductance of the basolateral membranes ofNecturus small intestine

SummaryIntracellular electrical potentials and K activity, (K)c, were determined simultaneously inNecturus small intestine before and after the addition of alanine to the mucosal solution. As noted previously (Gunter-Smith, Grasset & Schultz, 1982), the addition of alanine to the mucosal solution resulted in a prompt depolarization of the electrical potential difference across the apical membrane (ψmc) and a decrease in the slope resistance of that barrier (rm). This initial response was followed by a slower repolarization of ψmc associated with a decrease in the slope resistance of the basolateral membrane (rs) so that when the steady state was achieved (rm/rs) did not differ significantly from control values in the absence of alanine.In the absence of alanine, ψmc averaged −32 mV and(K)c averaged 67mm. When a steady state was achieved in the presence of alanine these values averaged −24 mV and 50mm, respectively. The steady-stateelectrochemical potential differences for K across the basolateral membrane in the absence and presence of alamine did not differ significantly.Inasmuch as the rate of transcellular active Na transport or “pump activity” was increased two-to threefold in the presence of alanine, it follows that,if active Na extrusion across the basolateral membrane is coupled to active K uptake across that barrier with a fixed stoichiometry then, the decrease inrs must be due to an increase in the conductance of the basolateral membrane to K that parallels the increase in “pump activity”. This “homocellular” regulatory mechanism serves to (i) prevent an increase in (K)c due to an increase in pump activity; and (ii) repolarize ψmc and thus restore the electrical driving force for the rheogenic Na-coupled entry processes.

The electrophysiology of rabbit descending colon

SummaryA method is described for determining the “instantaneous” transepithelial current-voltage (I-V) relations across rabbit descending colon and deriving theI-V relations of the amiloride-sensitive Na-entry step across the apical membrane. The latter conforms closely to the predictions of the Goldman-Hodgkin-Katz “constant-field” flux equation over a wide range of values of the transapical electrical potential difference (−120 to +50 mV), suggesting that Na entry is the result of simple electrodiffusion through homogeneous pores or channels. The permeability of the apical membrane to Na averaged 0.012 cm/hr, and the intracellular Na activity averaged 10mm. In the studies, the rate of Na entry across the apical membrane varied, spontaneously, over a fourfold range; this variation is entirely attributable to parallel variations in the partial conductance of the apical membrane to Na with no change in the driving force for this movement.Bathing the serosal surface of the tissue with a high-K solution abolishes the electrical potential difference across the basolateral membrane and markedly reduces the resistance of that barrier. Under these conditions, theI-V relations of the amiloride-sensitive Na-entry step across the apical membrane also conform closely to the predictions of the “constant-field” flux equation.Finally, the significance of the point at which the transepithelialI-V relations in the absence and presence of amiloride intersect (“ENa”) and the origin of the “bends” in theseI-V relations at or around this point are discussed. We demonstrate that the point of intersection is simply that value of the transepithelial electrical potential difference at which Na entry is abolished and has no direct bearing on the energetics of the basolateral pump. The “bend” in theI-V relations appears to be due to an increase in the conductance of a pathway in the apical membrane that parallels the Na-entry pathway in the apical membrane that parallels the Na-entry pathway as well as an increase in the conductance of the paracellular pathway; thus, this “bend” does not appear to be directly related to changes in the “active Na transport pathway”.

Some properties of KCl-filled microelectrodes: Correlation of potassium “leakage” with tip resistance

SummaryThis study was undertaken in order to determine directly the rates of K leakage (JK) out of the tips of microelectrodes into a solution of 100 mM KCl (approximating the K concentration of the cell interior) and to relate these rates to the concentration of the filling solution and the tip resistance. The values ofJK for electrodes filled with 3m KCl having resistances of 16 and 30 MΩ (when measured in 3m KCl) were 10 and 5.5 fmol/sec, respectively. When the same electrodes were filled with 0.5m KCl, the resistances (measured in 0.5m KCl) increased to 62 and 115 MΩ, respectively, andJK fell to 1.8 and 1.0 fmol/sec, respectively. These values are in reasonable agreement with what would be expected from theoretical considerations if leakage of KCl were the result of diffusion plus convective flow due to the hydrostatic pressure of the filling solution.We conclude that K leakage out of microelectrodes filled with 3m KCl is unnecessarily high; leakage can be reduced fivefold by filling electrodes with 0.5m KCl without incurring significant increases in tip or diffusion potentials or unmanageable tip resistances.Finally, the lowest rate of K leakage observed (1 fmol/sec) is still very considerable for the case of animal cells with an intracellular volume of approximately 1 pl and a K content of approximately 100 fmol. The finding of stable intracellular potentials, often for many minutes, in some tissues suggests that K which enters the cell rapidly diffuses into neighboring cells via high conductance intercellular communications.

Enteral glutamine but not alanine maintains small bowel barrier function after ischemia/reperfusion injury in rats.

We previously demonstrated that glucose and glutamine, solutes metabolized by the gut, replenish ATP and enhance gut function compared with alanine, a solute not metabolized by the gut, following mesenteric ischemia/reperfusion (I/R). The purpose of the present study was to determine if the nonmetabolizable solute alanine differentially modulates cytoskeletal organization and paracellular small intestinal permeability compared with the metabolizable solutes glucose and glutamine following mesenteric I/R. At laparotomy, rats had jejunal sacs filled with 10 mM glucose, glutamine, alanine, or magnesium sulfate (5 mm, osmotic control) followed by superior mesenteric artery clamping for 60 min and 30 min of reperfusion or sham laparotomy. Jejunum was harvested for evaluation by deconvolution microscopy, fluorescent measurement of F:G actin ratio, or mounted in an Ussing chamber for determination of intestinal permeability. Deconvolution microscopy revealed that the actin cytoskeleton was preserved by enteral glutamine, comparable to shams, but disrupted by enteral alanine. Glucose and controls resulted in comparable disruption, which was less than that with alanine. The F:G actin ratio was highest for glutamine and lowest for alanine; glucose was comparable to controls. Intestinal permeability was highest for alanine and lowest for glutamine, which was comparable to shams. Permeability following glucose and controls was higher than that following glutamine but lower than that following alanine. The nonmetabolizable solute alanine resulted in disruption of the actin cytoskeleton and enhanced intestinal permeability under conditions of mesenteric I/R. The metabolizable solute glutamine was protective under these conditions, whereas glucose exerted minimal effect on the integrity of the cytoskeleton and intestinal permeability. The individual components of enteral diets may differentially modulate intestinal barrier function, which could have important implications when administered to critically injured patients.

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.

SummaryThe 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 99.4mm. These relations could be closely fit to the “constant field” flux equation yielding estimates of the permeability of the apical membrane to Na,PNam, and the intracellular Na activity, (Na)c. The following empirical relations emerged: (i) (Na)c increased hyperbolically with increasing (Na)m; (ii)PNam decreased hyperbolically with increasing (Na)m and linearly with increasing (Na)c; (iii) spontaneous variations in Na entry rate at constant (Na)m could be attributed entirely to parallel, spontaneous variations inPNam; (iv) the rate of Na entry increased hyperbolically with increasing (Na)m obeying simple Michaelis-Menten kinetics; (v) 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 24mm. 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.

Reconstitution of a calcium-activated potassium channel in basolateral membranes of rabbit colonocytes into planar lipid bilayers

SummaryA highly enriched preparation of basolateral membrane vesicles was isolated from rabbit distal colon surface epithelial cells employing the method described by Wiener, Turnheim and van Os (Weiner, H., Turnheim, K., van Os, C.H. (1989)J. Membrane Biol.110:147–162) and incorporated into planar lipid bilayers. With very few exceptions, the channel activity observed was that of a high conductance, Ca2+-activated K+ channel. This channel is highly selective for K+ over Na+ and Cl−, displays voltage-gating similar to “maxi” K(Ca) channels found in other cell membranes, and kinetic analyses are consistent with the notion that K+ diffusion through the channel involves either the binding of a single K+ ion to a site within the channel or “single-filling” (“multi-ion occupancy”). Channel activity is inhibited by the venom from the scorpionLeiurus quinquestriatus, Ba2+, quinine, and trifluoperazine. The possible role of this channel in the function of these cells is discussed.

The immune-enhancing enteral agents arginine and glutamine differentially modulate gut barrier function following mesenteric ischemia/reperfusion.

BACKGROUNDnImmune-enhancing enteral diets have been shown to improve patient outcome. One contributing mechanism may be via maintenance of gut barrier function. While recent data has shown that glutamine is beneficial, arginine may be harmful. We therefore hypothesized that the immune-enhancing agents, glutamine and arginine, differentially modulate gut barrier function.nnnMETHODSnAt laparotomy, rats had jejunal sacs filled with 10 mmol/L glutamine, arginine, fructose, or magnesium sulfate (osmotic control) followed by 60 minutes of superior mesenteric artery occlusion and 2 hours of reperfusion. Jejunum was harvested for histology, deconvolution microscopy, F:G actin, ATP, and permeability measurements.nnnRESULTSnGlutamine and fructose minimized mucosal injury compared with controls and arginine. Deconvolution microscopy confirmed that glutamine and fructose preserved the actin cytoskeleton but there was disruption by arginine which correlated with F:G actin ratios and tissue ATP levels. Permeability was enhanced by arginine compared with the other groups.nnnCONCLUSIONnArginine resulted in worsened mucosal injury, disruption of the actin cytoskeleton, decreased tissue ATP and enhanced permeability compared with glutamine which appeared protective. The immune-enhancing agent arginine results in breakdown of gut barrier function which may have important implications for critically injured patients.

Potassium transport across rabbit descending colonin Vitro: Evidence for single-file diffusion through a paracellular pathway

SummaryThe results of previous studies indicate that the bidirectional fluxes of K across short-circuited rabbit descending colon are attributable to passive diffusion through paracellular pathways and that this route is ten times more permeable to K than to Na and Cl. However, transepithelial diffusion potentials in the presence of large transepithelial Na and K concentration differences are much lower than those predicted by the “constant field equation” and appear to be inconsistent with this high K selectivity.The results of the present studies, designed to resolve this apparent contradiction, indicate that:n (a)The ratios of the bidirectional transepithelial fluxes of K determined over a wide range of combined chemical and electrical potential differences conform reasonably well with those predicted by the Ussing flux-ratio equation.(b)The permeability coefficient of K (PK), determined from the net fluxes in the presence of concentration differences and from unidirectional fluxes under short-circuit conditions, decreases with increasing K concentration; in the presence of low K concentrations,PK is approximately ten-timesPNa, but it approachesPNa in the presence of high K concentrations.PNa is not affected under these conditions.n These results provide an explanation for the failure to observe large transepithelial diffusion potentials in the presence of large transepithelial Na and K concentration differences. In addition, these results are consistent with the notion that K diffuses across this preparation through two parallel pathways, one that does not discriminate among K, Na and Cl (a “free-solution” shunt) and another that is highly K selective and involves an interaction with one, or at most two, sites along the route.

The electrophysiology of rabbit descending colon. II. Current-voltage relations of the apical membrane, the basolateral membrane, and the parallel pathways.

SummaryIn this paper we employ the data described in the previous paper (I) to derive the current-voltage (I-V) relations of the basolateral membrane, the amiloride-insensitive “leak” pathway across the apical membrane, and the parallel pathways across rabbit descending colon. The results indicate that:a)The resistance of the basolateral membrane is independent of the electrical potential difference across that barrier over the range −8 to 67 mV and averaged 195 Ωcm2. The electromotive force across this barrier averaged 50 mV under control conditions and 48 mV in the presence of amiloride. The origin of this difference is discussed.b)The resistance of the parallel pathways averaged 351 Ωcm2 and was independent of the transepithelial electrical potential difference over the range −170 to +90 mV. The conductance of these pathways can be reasonably well accounted for by the partial ionic conductances of Na, K and Cl reported previously.c)The resistance of the amiloride-insensitive pathway across the apical membrane averaged 1667 Ωcm2 and the electromotive force across this pathway averaged −51 mV. These values are in excellent agreement with those determined by others. The ionic nature of this “leak” pathway remains to be elucidated.