Sodium-coupled sugar transport: effects on intracellular sodium activities and sodium-pump activity.

Intracellular sodium activities, (Na)c, were determined in Necturus small intestine before and after addition of galactose to the mucosal bathing solution. In the absence of galactose, (Na)c averaged 12 millimoles per liter. Within 2 minutes after the addition of galactose to the mucosal solution, (Na)c increased to a mean value of 20 millimoles per liter and then declined, in parallel with an increase in transcellular sodium transport, to a value that did not differ significantly from that observed in the absence of the sugar. The final steady state in the presence of galactose was characterized by a three- to fourfold increase in the rate of transcellular Na+ transport in the absence of a significant increase in (Na)c. Thus, the increase in steady-state basolateral pump activity cannot be attributed to an increase in the intracellular sodium transport pool.

Effect of sugars on transport of alanine in intestine.

The eflect of glucose and galactose on transport of alanine by rabbit ileum has been investigated. Transmural transport and cellular ac cumulation of the amino acid were in hibited by the sugars, but alanine in flux across the mucosal border of the cells was unaltered.

Ionic permeability of epithelial tissues.

The overall permeability of epithelial tissues to solutes is generally determined by analyzing net or unidirectional transepithelial fluxes in response to transepithelial differences of concentration and/or electrical potential using relations that describe diffusional movements across a single membrane. If the solute is uncharged and diffusional movements are transcellular, the overall transepithelial permeability coefficient is determined by the permeabilities of the two limiting cell membranes combinded in series. However, if the solute is charged and the pathway for transepithelial movement involves diffusional flows across at least two membranes arranged in series (i.e. transcellular transport), the value of the overall transepithelial permeability coefficient determined using relations that describe ionic diffusion across a single membrane is not an accurate measure of the permeabilities of the two limiting membranes combined in series. Further, if ionic diffusion is transcellular, permeability coefficients determined from studies of transepithelial fluxes are not only quantitatively incorrect but can also result in grossly erroneous interpretations of changes in transepithelial permeabilities and faulty inferences regarding the route of transepithelial ionic diffusion.

Effect of hypertonicity on the increase in basolateral conductance of Necturus small intestine in response to Na+-sugar cotransport.

Exposure of Necturus small intestine to a galactose-containing perfusate that is 20% hypertonic compared to the galactose-free (control) perfusate results in a rapid depolarization of the electrical potential difference across the apical membrane, psi mc, and a decrease in the ratio of the resistance of the apical membrane to that of the basolateral membrane, (rm/rs); however, the slow repolarization of psi mc and increase in (rm/rs), observed under isotonic conditions, is blocked. These findings are consistent with the notion that the increase in the conductance of the basolateral membrane in response to Na+-coupled sugar (or amino acid) transport across the apical membrane may be a 'volume regulatory response' to cell swelling.

Cation transport in Escherichia coli. VI. K exchange.

K influx and net K flux have been measured in suspensions of chloramphenicol-arrested Escherichia coli. The rate of K exchange in the steady state was independent of the K concentration of the medium over a 200-fold range. Under a number of experimental conditions the rate of exchange may be considerably increased or decreased without changing the cellular K content. These results show that under these conditions changes in K influx are associated with equal changes in K efflux, and suggest that the latter process is, at least in part, both carrier-mediated and tightly coupled to the influx process.

Relations among transepithelial sodium transport, potassium exchange, and cell volume in rabbit ileum.

The relation between active transepithelial Na transport across rabbit ileum and 42K exchange from the serosal solution across the basolateral membranes has been explored. Although 42K influx across the basolateral membranes is inhibited by ouabain and by complete depletion of cell Na, it is not affected when transepithelial Na transport is abolished (i.e. in the presence of an Na-free mucosal solution) or stimulated (i.e. when glucose or alanine is added to the mucosal solution). We are unable to detect any relation between the ouabain-sensitive Na-K exchange mechanism responsible for the maintenance of intracellular Na and K concentrations and active transcellular Na transport. In addition, the maintenance of cell volume (water content) does not appear to be dependent upon transepithelial Na transport or the ouabain-sensitive Na-K exchange pump. Although the results of these studies cannot be considered conclusive, they raise serious questions regarding the role of the Na-K exchange pump, located at the basolateral membranes, in active transepithelial Na transport and the maintenance of cell volume.

Lysine transport across isolated rabbit ileum.

Lysine transport by in vitro distal rabbit ileum has been investigated by determining (a) transmural fluxes across short-circuited segments of the tissue; (b) accumulation by mucosal strips; and (c) influx from the mucosal solution across the brush border into the epithelium. Net transmural flux of lysine is considerably smaller than that of alanine. However, lysine influx across the brush border and lysine accumulation by mucosal strips are quantitatively comparable to alanine influx and accumulation. Evidence is presented that the "low transport capacity" of rabbit ileum for lysine is due to: (a) a carrier-mediated process responsible for efflux of lysine out of the cell across the serosal and/or lateral membranes that is characterized by a low maximal velocity; and (b) a high "backflux" of lysine out of the cell across the mucosal membrane. A possible explanation for the latter observation is discussed with reference to the relatively low Na dependence of lysine transport across the intestinal brush border.

Effects of monovalent cations on the sodium-alanine interaction in rabbit ileum. Implication of anionic groups in sodium binding.

H, K, Rb, and Li inhibit Na-dependent alanine influx across the brush border of rabbit ileum. Kinetic analysis indicates that H and K behave as competitive inhibitors of influx so that increasing the concentration of H or K in the mucosal solution is kinetically indistinguishable from decreasing the Na concentration. In addition the coupling between alanine and Na influxes is markedly reduced at pH 2.5. With the exception of H and Li, none of these monovalent cations significantly affects carrier-mediated alanine influx in the absence of Na indicating that their inhibitory effects are largely restricted to the Na-dependent fraction of influx. Increasing H concentration from 0.03 to 3 mM does not affect influx in the absence of Na but markedly inhibits influx in the presence of Na. Li significantly enhances alanine influx in the absence of Na. Ag, UO(2), and La also inhibit the Na-dependent fraction of alanine influx. These findings suggest that anionic groups having a pK(a) of approximately 4 are involved in the interaction between Na and the alanine-carrier complex; present evidence implicates carboxylate groups however, phosphoryl residues cannot be ruled out. The previously proposed kinetic model for the Na-alanine interaction has been extended to accommodate these effects of H and other monovalent cations. The mechanistic and physiological implications of these findings are discussed.

Ionic conductances of extracellular shunt pathway in rabbit ileum. Influence of shunt on transmural sodium transport and electrical potential differences.

The unidirectional influxes of Na, K, and Cl into isolated strips of rabbit ileum are comprised of movements across the mucosal membrane of the epithelial cells and ionic diffusion into an extracellular shunt pathway. A large fraction of the Na influx across the mucosal membrane alone is inhibited by Li, suggesting the participation of a carrier mechanism in the influx process. The partial ionic shunt conductances of Na, K, and Cl account for at least 82% of the total tissue conductance. The calculated shunt permeabilities (P) are (in centimeters per hour) P(K) = 0.040, P(Na) = 0.035, and P(Cl) = 0.019, so that P(K):P(Na):P(Cl) = 1.14:1.00:0.55. Diffusion potentials across the tissue resulting from isotonic replacement of NaCl in the mucosal solution with mannitol or KCl are described by the Goldman constant-field equation together with the above permeabilities of the shunt pathway. These observations are not consistent with permeation through a fixed-charge pore but can be explained by a model featuring constant ionic partition into a neutral-polar pore that traverses the tight junction. Such a pore may be lined with either fixed dipoles or fixed dipolar ions oriented such that electronegative groups influence the permselective properties of the diffusion pathway. The essential feature of both models is that electroneutrality is maintained by means of fixed membrane components and does not depend upon the presence of mobile counterions.

Studies on the electrical potential profile across rabbit ileum. Effects of sugars and amino acids on transmural and transmucosal electrical potential differences.

When isolated strips of mucosal rabbit ileum are bathed by physiological electrolyte solution the electrical potential difference (PD) across the brush border (psi(mc)) averages 36 mv, cell interior negative. Rapid replacement of Na in the mucosal solution with less permeant cations, Tris or choline, results in an immediate hyperpolarization of psi(mc). Conversely, replacement of choline in the mucosal solution with Na results in an abrupt depolarization of psi(mc). These findings indicate that Na contributes to the conductance across the brush border. The presence of actively transported sugars or amino acids in the mucosal solution brings about a marked depolarization of psi(mc) and a smaller increase in the transmural PD (Deltapsi(ms)). It appears that the Na influx that is coupled to the influxes of amino acids and sugars is electrogenic and responsible for the depolarization of psi(mc). Under control conditions Deltapsi(ms) can be attributed to the depolarization of psi(mc) together with the presence of a low resistance transepithelial shunt, possibly the lateral intercellular spaces. However, quantitatively similar effects of amino acids on psi(mc) are also seen in tissues poisoned with metabolic inhibitors or ouabain. Under these conditions Deltapsi(mc) is much smaller than under control conditions. Thus, the depolarization of psi(mc) might not account for the entire Deltapsi(ms), observed in nonpoisoned tissue. An additional electromotive force which is directly coupled to metabolic processes might contribute to the normal Deltapsi(ms).

Cation transport in Escherichia coli. VII. Potassium requirement for phosphate uptake.

When Escherichia coli K-12 is grown in media containing limiting amounts of K, growth continues normally until all the extracellular K has been consumed. Thereafter the rates of growth, glucose consumption, and oxygen consumption decrease progressively, and the cell contents of K and P fall. These changes, referred to as K limitation, are all reversed by the addition of K. By specifically altering the ionic composition of the cells it was shown that these metabolic disturbances are not due to changes in the cell content of K or Na, but are directly related to the absence of K from the extracellular medium. The cell pool of inorganic P and the uptake of PO(4) from the medium are low in K-limited cells and are immediately stimulated by the addition of K, suggesting that the primary effect of K limitation is to inhibit PO(4) uptake. All the metabolic effects of K limitation can be attributed to inhibition of PO(4) uptake. The requirement of extracellular K for PO(4) uptake may be due to a coupling between the uptake of K and PO(4).

Effect of inhibitors on alanine transport in isolated rabbit ileum.

The effects of metabolic inhibitors and ouabain on alanine transport across rabbit ileum, in vitro, have been investigated. Net transport of alanine and Na across short-circuited segments of ileum is virtually abolished by cyanide, 2,4-dinitrophenol, iodoacetate, and ouabain. However, these inhibitors do not markedly depress alanine influx from the mucosal solution, across the brush border, into the intestinal epithelium, and they do not significantly affect the Na dependence of this entry process. The results of this investigation indicate that: (a) the Na dependence of alanine influx does not reflect a mechanism in which the sole function of Na is to link metabolic energy directly to the influx process; and (b) the inhibition of net alanine transport across intestine is, in part, the result of an increased rate coefficient for alanine efflux out of the cell across the brush border. Although these findings do not exclude a direct link between metabolic energy and alanine efflux, the increased efflux may be the result of the increased intracellular Na concentration in the presence of these inhibitors. The results of these studies are qualitatively consistent with a model for alanine transport across the brush border which does not include a direct link to metabolic energy.

Kinetic relations of the Na-amino acid interaction at the mucosal border of intestine.

The relation between unidirectional influxes of Na and amino acids across the mucosal border of rabbit ileum was studied under a variety of conditions. At constant Na concentration in the mucosal bathing solution, amino acid influx followed Michaelis-Menten kinetics permitting determination of maximal influx and the apparent Michaelis constant, K(t). Reduction in Na concentration, using choline as substitute cation, caused an increase in K(t) for alanine but had no effect on maximal alanine influx. The reciprocal of K(t) was a linear function of Na concentration. Similar results were obtained for valine and leucine and these amino acids competitively inhibited alanine influx both in the presence and in the absence of Na. These results lead to a model for the transport system which involves combination of Na and amino acid with a single carrier or site leading to penetration of both solutes. The model predicts that alanine should cause an increase in Na influx and the ratio of this extra Na flux to alanine flux should vary with Na concentration. The observed relation agreed closely with predicted values for Na concentrations from 5 to 140 mM. These results support the hypothesis that interactions between Na and amino acid transport depend in part on a common entry mechanism at the mucosal border of the intestine.

Alanine and sodium fluxes across mucosal border of rabbit ileum.

Unidirectional influxes of L-alanine and Na from the mucosal solution into the epithelium of in vitro rabbit ileum have been determined. In the presence of 140 mM Na, alanine influx is approximately 2.2 micromoles/hr cm(2), but is inhibited if the NaCl in the mucosal solution is replaced by choline Cl, Tris-Cl, mannitol, LiCl, or KCl. Although alanine influx is strongly dependent upon Na in the mucosal solution, it is uninfluenced by marked reduction of intracellular Na pools. In addition, alanine influx is unaffected by intracellular alanine concentration. Na influx is markedly inhibited by the presence of Li. Evidence is presented that Na transport across the mucosal border cannot be attributed to simple diffusion even though the net flux across this surface is in the direction of the electrochemical potential difference.

Amino acid and sugar transport in rabbit ileum.

L-Alanine and 3-O-methyl-D-glucose accumulation by mucosal strips from rabbit ileum has been investigated with particular emphasis on the interaction between Na and these transport processes. L-Alanine is rapidly accumulated by mucosal tissue and intracellular concentrations of approximately 50 mM are reached within 30 min when extracellular L-alanine concentration is 5 mM. Evidence is presented that intracellular alanine exists in an unbound, osmotically active form and that accumulation is an active transport process. In the absence of extracellular Na, the final ratio of intracellular to extracellular L-alanine does not differ significantly from unity and the rate of net uptake is markedly inhibited. Amino acid accumulation is also inhibited by 5 x 10(-5)M ouabain. 3-O-methyl-D-glucose accumulation by this preparation is similarly affected by ouabain and by incubation in a Na-free medium. The effects of amino acid accumulation, of ouabain, and of incubation in a Na-free medium on cell water content and intracellular Na and K concentrations have also been investigated. These results are discussed with reference to the two hypotheses which have been suggested to explain the interaction between Na and intestinal nonelectrolyte transport.

Sodium and sugar fluxes across the mucosal border of rabbit ileum.

Unidirectional influxes of sugars and Na from muscosal solution into the cells of rabbit ileum have been examined. The influxes of glucose, galactose, and 3-0-methyl glucose (3 MG) follow Michaelis-Menten type kinetics and are markedly dependent on the presence ofNa in the mucosal solution. For 3 MG, reduction of Na concentration causes a decrease in maximal rate of influx and little change in the "apparent Michaelis constant." There appeared to be little mediated entry of 3 MG into the cells from Na-free solution. The influx of Na was increased by the presence of 3 MG in the mucosal solution and at all Na concentrations tested, there was a 1:1 ratio between sugar influx and the sugar-dependent Na influx. On the basis of these observations, a model has been developed for the sugar transport system involving a transport site that combines with both sugar and Na.

Dicarboxylic amino acid influx across brush border of rabbit ileum. Effects of amino acid charge on the sodium-amino acid interaction.

Glutamate and aspartate influxes across the brush border of rabbit intestine are saturable processes that are subject to competitive inhibition and are markedly influenced by the Na concentration in the mucosal solution. Lowering the Na concentration increases the amino acid concentration needed to elicit a half-maximal influx but does not significantly affect the maximal influx. The interaction between Na and anionic amino acid influx can be described by the same kinetic model that has been applied to the influxes of neutral amino acids and lysine. Comparison of the kinetic parameters for anionic, neutral, and cationic amino acids suggests that amino acid charge influences (a) the stability of the binary (amino acid-site) complex and (b) the affinity of this binary complex for the subsequent binding of Na. A mechanistic interpretation of these interactions is proposed.

Sodium chloride transport by rabbit gallbladder. Direct evidence for a coupled NaCl influx process.

The results of the present study that NaCl transport by in vitro rabbit gallbladder must be a consequence of a neutral coupled carrier-mediated mechanism that ultimately results in the active absorption of both ions; pure electrical coupling between the movements of Na and Cl can be excluded on the grounds of electrphysiologic considerations. Studies on the unidirectional influxes of Na and Cl have localized the site of this coupled mechanism to the mucosal membranes. Studies on the intracellular ion concentrations and the intracellular electrical potential are consistent with the notion that (a) the coupled NaCl influx process results in the movement of Cl from the mucosal solution into the cell against an apparent electrochemical potential difference; (b) the energy for the uphill movement of Cl is derived from the Na gradient across the mucosal membrane which is maintained by an active Na extrusion mechanism located at the basolateral membranes; and (c) Cl exit from the cell across the basolateral membranes is directed down an electrochemical potential gradient and may be diffusional. Finally, as for the case of rabbit ileum, the coupled NaCl influx process is inhibited by elevated intracellular levels of cyclic 3',5'-adenosine monophosphate. A working model for transcellular and paracellular NaCl transport by in vitro rabbit gallbladder is proposed.

Electrophysiological studies of sodium cotransport in epithelia: toward a cellular model.

During the past two decades, microelectrophysiological studies of small intestine and renal proximal tubule employing conventional as well as ion-selective microelectrodes have contributed significantly to our understanding of the nature of Na-coupled entry processes at the apical membrane as well as the overall workings of the simple model illustrated in FIGURE 1. These studies have unequivocally established the rheogenic and conductive nature of the Na-coupled sugar and amino-acid entry processes across the apical membrane of small intestine (and renal proximal tubule) and have, in addition, disclosed that the properties of the basolateral membrane respond to an increase in Na-coupled solute entry with an increase in the ability of the Na-K pump to extrude Na with little or no change in (Na)c32 and a parallel increase in the conductance of that barrier to K. Although these responses may be "triggered" by cell swelling, it is unclear how a cell "recognizes" minimal swelling and how this recognition, in turn, culminates in the observed changes in basolateral membrane pump-leak properties. Clearly, these findings have brought us to the interfaces between cell physiology and cell and molecular biology and have raised a number of intriguing questions that focus on the more global question: How do epithelial cells work?

The application of two-dimensional centrifugation to clinical chemistry testing.

We have developed a new clinical chemistry analyzer, the VISION System, which uses centrifugal force to separate whole blood, measure reagent and plasma volumes, and complete all steps required for a spectrophotometric analysis. The system is based on a multichambered test pack containing liquid reagents, which can be centrifuged in two planes oriented at right angles to each other. The analyzer regulates the temperature, timing and optical measurements for up to 10 different test packs in the same run. We have demonstrated good precision and accuracy on 6 clinical chemistry analytes, 2 enzymes, potassium and theophylline using this system.