Sodium ion transport in isolated intestinal epithelial cells. The effect of actively transported sugars on sodium ion efflux

A technique to measure Na⁺ efflux from isolated intestinal epithelial cells has permitted us to examine the mechanisms responsible for Na⁺ transport in absorptive cells without contamination by other cell types. We examined the effect of actively transported sugars on Na⁺ efflux from isolated rat jejunal epithelial cells to evaluate the mechanism by which actively transported non-electrolytes stimulate Na⁺ absorption. Glucose, galactose and 3-O-methylglucose, sugars known to be actively transported by the small intestine, stimulate total Na⁺ efflux from isolated epithelial cells. This stimulation results from an increase of active Na⁺ transport, since it is inhibited by ouabain. Glucose stimulation is significantly greater than that produced by galactose or 3-O-methylglucose, 2-Deoxyglucose, a sugar that is not actively transported, has no effect on total Na⁺ efflux from isolated cells. Phloridzin, which has no effect on Na⁺ efflux in a sugar-free medium, completely abolishes the effect of galactose. These findings (a) support the hypothesis that the increase in intestinal absorption of Na⁺ in the presence of actively transported non-electrolytes occurs by a transcellular route; and (b) are consistent with the ion-gradient model. The results are not compatible with the direct energy-coupling model.     

Transport of monosaccharides in kidney-cortex cells

1. The aerobic transport of d-glucose and d-galactose in rabbit kidney tissue at 25 degrees was studied. 2. In slices forming glucose from added substrates an accumulation of glucose against its concentration gradient was found. The apparent ratio of intracellular ([S](i)) and extracellular ([S](o)) glucose concentrations was increased by 0.4mm-phlorrhizin and 0.3mm-ouabain. 3. Slices and isolated renal tubules actively accumulated glucose from the saline; the apparent [S](i)/[S](o) fell below 1.0 only at [S](o) higher than 0.5mm. 4. The rate of glucose oxidation by slices was characterized by the following parameters: K(m) 1.16mm; V(max.) 4.5mumoles/g. wet wt./hr. 5. The active accumulation of glucose from the saline was decreased by 0.1mm-2,4-dinitrophenol, 0.4mm-phlorrhizin and by the absence of external Na(+). 6. The kinetic parameters of galactose entry into the cells were: K(m) 1.5mm; V(max) 10mumoles/g. wet wt./hr. 7. The efflux kinetics from slices indicated two intracellular compartments for d-galactose. The galactose efflux was greatly diminished at 0 degrees , was inhibited by 0.4mm-phlorrhizin, but was insensitive to ouabain. 8. The following mechanism of glucose and galactose transport in renal tubular cells is suggested: (a) at the tubular membrane, these sugars are actively transported into the cells by a metabolically- and Na(+)-dependent phlorrhizin-sensitive mechanism; (b) at the basal cell membrane, these sugars are transported in accordance with their concentration gradient by a phlorrhizin-sensitive Na(+)-independent facilitated diffusion. The steady-state intracellular sugar concentration is determined by the kinetic parameters of active entry, passive outflow and intracellular utilization.     

Transport of sugars or amino acids increases potassium efflux from isolated enterocytes

A technique to isolate epithelial cells from rabbit jejunum using hyaluronidase is described. The cells obtained retained their abilities to accumulate sugars and potassium (86Rb) against concentration gradients. Potassium efflux was monitored using cells preloaded with 86Rb and the rate constant of efflux was seen to increase when actively transported sugars or amino acids are added to the bathing medium. The increase is related to the transport of the non-electrolyte, but not to volume regulatory events.     

Glycodeoxycholate transport in brush border membrane vesicles isolated from rat jejunum and ileum.

The transport of the bile salt, glycodeoxycholate, was studied in vesicles derived from rat jejunal and ileal brush border membranes using a rapid filtration technique. The uptake was osmotically sensitive, linearly related to membrane protein and resembled D-glucose transport. In ileal, but not jejunal, vesicles glycodeoxycholate uptake showed a transient vesicle/medium ratio greater than 1 in the presence of an initial sodium gradient. The differences between glycodeoxycholate uptake in the presence and absence of a Na+ gradient yielded a saturable transport component. Kinetic analysis revealed a Km value similar to that described previously in everted whole intestinal segments and epithelial cells isolated from the ileum. These findings support the existence of a transport system in the brush border membrane that: (1) reflects kinetics and characteristics of bile salt transport in intact intestinal preparations, and (2) catalyzes the co-transport of Na+ and bile salt across the ileal membrane in a manner analogous to D-glucose transport.     

Rat enterocyte Na+ transport in vitro. Action of parathyroid hormone and calcitonin

Parathyroid hormone (PTH) and calcitonin exert well known effects on the renal tubule which are thought to involve specific hormone receptors and adenyl cyclase. In the intestine, it is not clear whether the action of PTH and calcitonin is only indirect or also direct, and their mechanisms of action are much less well established. In the present study, possible direct effects of PTH and calcitonin on Na+ transport in isolated intestinal epithelial cells of rats were investigated. In the presence of bovine PTH (1.2 I.U/ml) in the incubation medium, the Na+ efflux rate constant (oKNa) of isolated enterocytes was significantly reduced when compared to that in control experiments with the hormone vehicle only. The mean depression of oKNa induced by bovine PTH was 26% as compared to the control (100%) and to that induced by ouabain (4.0 mM) which was 44%. No depressant effect of bovine PTH on oKNa was observed when the isolated enterocytes were incubated with ouabain (4.0 mM). Thus, bovine PTH appeared to inhibit the ouabain-sensitive Na+ pump. When incubating the isolated epithelial cells in an EGTA-containing CA2+-free medium, bovine PTH lost its capacity to inhibit oKNa. Thus, the presence of extracellular Ca2+ appeared necessary for the inhibitory effect of bovine PTH. In contrast to its effect on oKNa, bovine PTH induced no change in net Na+ uptake by isolated enterocytes. Moreover, no significant effect on enterocyte Na+ transport could be demonstrated for salmon or porcine calcitonin at two different concentrations in the incubation medium, Neither bovine PTH nor salmon calcitonin induced significant changes in enterocyte cyclic AMP or cycle GMP concentrations. It was concluded that bovine PTH, but not calcitonin, exerted a directed inhibitory effect on the ouabain-sensitive oKNa of isolated rat enterocytes. The effect of bovine PTH occurred without measurable activation of the cyclic nucleotide system but needed the presence of Ca2+ in the incubation medium to be operative.     

Epinephrine stimulates the Na+-K+ ATPase in isolated rat jejunal crypt cells

The effect of epinephrine on the activity of the Na+-K+ ATPase was studied in isolated rat jejunal cells. The activity of the pump was assessed by measuring the ouabain inhibitable K+ accumulation by the enterocytes using 86Rb as a tracer. Epinephrine stimulated significantly the Na+-K+ ATPase in crypt cells but not in villus cells. This effect was still apparent in presence of propranolol and prazocin but disappeared in presence of yohimbine. Amiloride did not affect the epinephrine-induced stimulation. Calcium channel blockers and dibutyryl cAMP enhanced the activity of the pump, and exerted respectively overlapping and additive effects with epinephrine, when added simultaneously. The calcium ionophore A23187 inhibited the basal activity of the ATPase and the stimulatory effect of epinephrine disappeared in its presence. These results suggest that epinephrine stimulates the Na+-K+ ATPase in jejunal crypt cells by activating alpha2 receptors and decreasing intracellular calcium, and not by altering cAMP levels.     

Uridine uptake by isolated intestinal epithelial cells of guinea pig

Uptake of uridine was studied in isolated intestinal epithelial cells of guinea pig. Uptake was not severely influenced by metabolism. Free uridine was accumulated within cells 13-fold. Uptake was saturable with an apparent Km value of 46 microM and a Vmax of 0.9 nmol/mg protein per min. Uracil inhibited uptake only slightly; adenosine, guanosine and cytosine inhibited strongly. Antimycin A and ouabain inhibited almost 90%. If the extracellular Na+ concentration was decreased to 5 mM, the rate of uptake decreased 6.5-fold. The stimulatory effect of Na+ was related to the transmembraneous Na+-gradient. Cells from jejunum transported about 30% faster than cells from ileum. In conclusion, isolated enterocytes of guinea pig posses an active transport system for uridine.     

Characteristics of amino acid accumulation by isolated intestinal epithelial cells

Summary Accumulation of neutral amino acids by isolated chick epithelial cells has been studied and the results discussed in terms of the ion gradient model, and a model invoking a direct input of metabolic energy. The cells establish four- to eightfold concentration gradients of amino acids at an extracellular concentration of 1mm. The accumulation is sodium-dependent, inhibited by high extracellular potassium concentrations, and is sensitive to a variety of metabolic inhibitors. Also, amino acid uptake is depressed by actively transported sugars, and certain other amino acids, and is stimulated by phloridzin.Cells equilibrated with valine and loaded with 30 to 40mm intracellular sodium begin immediately to actively accumulate valine when suddenly introduced to media containing 20mm sodium. The cells establish a threefold gradient of amino acid during the interval when intracellular sodium is higher than extracellular sodium.Amino acid accumulation and²²Na efflux were monitored simultaneously in cells treated with phloridzin. While phloridzin causes a 30% stimulation of amino acid uptake, no variation in the rate of²²Na efflux or the steady-state level of²²Na maintained by the cells can be detected. Similarly, either 2.5mm glucose or 2.5mm 3-O-methylglucose cause approximately a 50% inhibition of 1mm valine uptake, but no detectable change in steady-state cellular²²Na content. Several aspects of the data seem inconsistent with concepts embodied in the ion gradient hypothesis, and it is suggested that a directly energized transport mechanism can better accommodate all of the data.     

D-mannose transport and metabolism in isolated enterocytes

D-mannose transport and metabolism has been studied in enterocytes isolated from chicken small intestine. In the presence of Na(+), the mannose taken up by the cells either remains free, is phosphorylated, is catabolized to H(2)O, or becomes part of membrane components. The mannose remaining free in the cytosol is released when the cells are transferred to an ice bath. The Na(+)-dependent D-mannose transport is electrogenic and inhibited by ouabain and dinitrophenol; its substrate specificity differs from SGLT-1 transporter. The Glut2 transporter inhibitors phloretin and cytochalasin B added following 30-min mannose uptake reduced the previously accumulated D-mannose, whereas these two agents increased the cell to external medium 3-O-methyl-glucose (3-OMG) concentration ratio. D-mannose efflux rate from preloaded D-[2-(3)H]-mannose enterocytes is Na(+)-independent. Phloretin did not affect D-mannose efflux rate, whereas it inhibited that of 3-OMG. Neither mannose uptake nor efflux rate were affected by fructose. It is concluded that part of the mannose taken up by the enterocytes is rapidly metabolized and that enterocytes have two D- mannose transport systems: one is concentrative and Na(+)-dependent and the other is Na(+)-independent and passive.     

Jejunal and ileal D-glucose transport in isolated brush border membranes.

D-Glucose transport was investigated in isolated brush border membranes from small intestine. The transport properties of membranes from upper jejunum were compared with those from terminal ileum. The jejunal membranes accumulate D-glucose to a greater extent than the ileal membranes when supplied with energy in the form of a NaSCN gradient. This difference in behavior is similar to that of the more intact epithelial preparations and suggests that the isolated membranes actually reflect the state present in intact cells. Ileal membranes transported D-glucose about two to three times slower than the jejunal ones, which can partially explain the lower sugar accumulation.     

Streptozotocin diabetes and sugar transport by rat ileal enterocytes: evidence for adaptation caused by an increased luminal nutrient load.

Preparations of villus enterocytes and brush border membrane vesicles have been used to study the effects of streptozotocin-induced diabetes mellitus in rats on sugar transport across the brush border and basolateral membranes of ileal epithelial cells. In isolated cells, diabetes increased Na(+)-dependent galactose transport across the brush border of mid-villus but not upper villus cells. Galactose transport across the basolateral membrane was, however, enhanced by diabetes in both cell populations. Kinetic analysis of vesicle data suggested the presence of two transporters for Na(+)-dependent glucose transport. Diabetes induced a 5-fold increase in both KT and Vmax of the high-affinity/low-capacity system together with a 2-fold increase in the Vmax of the low-affinity/high-capacity transporter. Glucose was almost undetectable in the lumen of the upper and lower ileum in control animals but was present at high levels (26.1 +/- 4.3 mM and 6.5 +/- 1.3 mM) in diabetic rats. The possible significance of these changes in luminal sugar concentration in relation to the adaptation of transport across ileal enterocytes is discussed.     

Distinct effects of various amino acids on 45Ca2+ fluxes in rat pancreatic islets.

The effects of three types of amino acids on 45Ca2+ fluxes in rat pancreatic islets have been compared. Alanine, a non-insulinotropic neutral amino acid, transported with Na+, increased 45Ca2+ efflux in the presence or in the absence of extracellular Ca2+, but not in the absence of Na+. Its effects in Na+-solutions were practically abolished by 7 mM-glucose. Alanine slightly stimulated 45Ca2+ influx (5 min uptake) only when Na+ was present. Two insulinotropic cationic amino acids (arginine and lysine) triggered similar changes in 45Ca2+ efflux. They accelerated the efflux in the presence of Ca2+ and inhibited the efflux in a Ca2+-free medium, whether glucose was present or not. In an Na+-free Ca2+-medium, arginine and lysine markedly accelerated 45Ca2+ efflux, but this effect was suppressed by 7 mM-glucose. Arginine stimulated 45Ca2+ influx irrespective of the presence or absence of glucose and Na+. Leucine, a neutral insulinotropic amino acid well metabolized by islet cells, inhibited 45Ca2+ efflux from the islets in a Ca2+-free medium; this effect was potentiated by glutamine. In the presence of Ca2+ and Na+, leucine was ineffective alone, but triggered a marked increase in 45Ca2+ efflux when combined with glutamine. In an Na+-free Ca2+-medium, leucine accelerated 45Ca2+ efflux to the same extent with or without glutamine. Leucine also stimulated 45Ca2+ influx in the presence or in the absence of Na+, but its effects were potentiated by glutamine only in the presence of Na+. The results show that amino acids of various types cause distinct changes in 45Ca2+ fluxes in pancreatic islets. Certain of these changes involve an Na+-mediated mobilization of cellular Ca2+ from sequestering sites where glucose appears to exert an opposite effect.     

Transport of 3-O-methyl D-glucose and beta-methyl D-glucoside by rabbit ileum.

The intestinal transport of three actively transported sugars has been studied in order to determine mechanistic features that, (a) can be attributed to stereo-specific affinity and (b) are common. The apparent affinity constants at the brush-border indicate that sugars are selected in the order, beta-methyl glucose greater than D-galactose greater than 3-O-methyl glucose, (the Km values are 1.23, 5.0 and 18.1 mM, respectively.) At low substrate concentrations the Kt values for Na+ activation of sugar entry across the brush-border are: 27, 25, and 140 mequiv. for beta-methyl glucose, galactose and 3-O-methyl glucose, respectively. These kinetic parameters suggest that Na+, water, sugar and membrane-binding groups are all factors which determine selective affinity. In spite of these differences in operational affinity, all three sugars show a reciprocal change in brush-border entry and exit permeability as Ringer (Na) or (sugar) is increased. Estimates of the changes in convective velocity and in the diffusive velocity when the sugar concentration in the Ringer is raised reveal that with all three sugars, the fractional reduction in convective velocity is approximately equal to the (reduction of diffusive velocity)2. This is consistent with the view that the sugars move via pores in the brush-border by convective diffusion. Theophylline reduces the serosal border permeability to beta-methyl glucose and to 3-O-methyl glucose relatively by the same extent and consequently, increase the intracellular accumulation of these sugars. The permeability of the serosal border to beta-methyl glucose entry is lower than permeability of the serosal border to beta-methyl glucose exit, which suggested that beta-methyl glucose may be convected out of the cell across the lateral serosal border.     

Effects of monensin on metabolism of isolated rat islets of Langerhans.

Monensin, a univalent ionophore, is a carboxylic acid produced by Streptomyces cinnamonensis. It will complex various alkali-metal ions, but most readily binds Na+. Because of interest in the possible role of Na+ in the regulation of insulin secretion, we examined its effects on several aspects of the metabolism of isolated rat islets of Langerhans. The ionophore inhibited glucose-stimulated insulin release in a concentration-dependent manner, completely inhibiting secretion evoked by 20 mM-glucose at concentrations as low as 0.1 microM in static incubations. In perifusion experiments, both phases of insulin release were equally affected. Monensin (0.1 microM) had no significant effect on glucose oxidation as measured by the generation of 14CO2 from [14C]glucose. Monensin increased the rate of 22Na+ efflux from preloaded islets and net 22Na+ uptake over 30 min, in the absence of changes in islet volume or extracellular space. The ionophore increased the Rb+/K+ permeability of islet cells, as shown by its inhibition of 86Rb+ retention and stimulation of 86Rb+ efflux. At 0.1 microM, monensin abolished glucose-stimulated 45Ca2+ uptake by islets during 5 min incubations, and stimulated 45Ca2+ efflux from preloaded islets perifused with Ca2+-free medium, even in the complete absence of extracellular Na+. Studies of the uptake of 14C-labelled 5,5-dimethyloxazolidine-2,4-dione showed that 0.1 microM-monensin increased net intracellular pH from 7.05 to 7.13. 7 Monensin has widespread, complex, effects on the secretory responses and ion handling by the B cells, which are difficult to interpret in terms solely of actions as a Na+ ionophore.     

Jejunal and ileal d-glucose transport in isolated brush border membranes

d-Glucose transport was investigated in isolated brush border membranes from small intestine. The transport properties of membranes from upper jejunum were compared with those from terminal ileum. The jejunal membranes accumulated d-glucose to a greater extent than the ileal membranes when supplied with energy in the form of a NASCN gradient. This difference is behavior is similar to that of the more intact epithelial preparations and suggests that the isolated membranes actually reflect the state present in intact cells. Ileal membranes transported d-glucose about two to three times slower than the jejunal ones, which can partially explain the lower sugar accumulation.     

Reversed transport of amino acids in Ehrlich cells.

Gramicidin induces a marked Na+-dependent efflux of amino acids from Ehrlich cells. In absence of Na+, gramicidin does not alter the efflux. In presence gramicidin, glycine efflux is inhibited by methionine and less so by leucine. Glycine efflux caused by HgCl2 is neither Na+ dependent nor inhibitable by amino acids. Neither efflux of inositol which is transported by an Na+-dependent route, nor efflux of several other solutes which are transported by Na+-independent routes, is affected by gramicidin. The antibiotic appears to permit a reversal in the direction of of the operation of the Na+-dependent amino acid transport system. The increased efflux is partly, but not entirely, due to an increase in the cellular Na+ concentration and a reduction of the electrochemical potential difference for Na+.     

Effect of streptozotocin-induced diabetes on the handling of phosphate by the rat small intestine.

The effect of chemically-induced diabetes on the handling of phosphate (Pi) by rat jejunal enterocytes has been investigated in the presence of a Na- or a choline-gradient. Pi uptake was significantly increased in both gradients. The Pi efflux rate constants for enterocytes from diabetic rats were similar to those of control rats. The effect of diabetes on both the protein and alkaline phosphatase isoenzymes of the rat small intestinal brush-border membranes was examined using SDS-PAGE. The patterns given by membranes from rats 14 days after the induction of diabetes were no different from those of controls.     

Characterization of Na(+)-dependent, active nucleoside transport in rat and mouse peritoneal macrophages, a mouse macrophage cell line and normal rat kidney cells

Peritoneal rat macrophages expressed solely an Na(+)-dependent, concentrative nucleoside transporter, which possesses a single Na(+)-binding site and transports purine nucleosides and uridine but not thymidine or deoxycytidine. The Michaelis-Menten constants for formycin B and Na+ were about 6 microns and 14 mM, respectively, and the estimated Na+:formycin B stoichiometry was 1:1. Rat macrophages accumulated 5 microM formycin B to a steady-state level exceeding that in the medium by about 500-fold during 60 min of incubation at 37 degrees C. Concentrative formycin B transport was resistant to inhibition by nitrobenzylthioinosine, lidoflazine, dilazep and nifedipine, but was slightly inhibited by high concentrations of dipyridamole (greater than 10 microM) and probenecid (greater than 100 microM). Mouse peritoneal macrophages and lines of mouse macrophages and normal rat kidney cells expressed Na(+)-dependent, active nucleoside transport but in addition significant Na(+)-independent, facilitated nucleoside transport. Facilitated nucleoside transport in these cells was sensitive to inhibition by nitrobenzylthioinosine, dilazep and dipyridamole. The presence of these inhibitors greatly enhanced the concentrative accumulation of formycin B by these cells by inhibiting the efflux via the facilitated transporter of the formycin B actively transported into the cells. Whereas rat macrophages lacked high-affinity nitrobenzylthioinosine-binding sites, mouse macrophages and normal rat kidney cells possessed about 10,000 such sites/cell. Rat and mouse erythrocytes, rat lymphocytes, and lines of Novikoff rat hepatoma cells, Chinese hamster ovary cells, Mus dunni cells and embryonic monkey kidney cells expressed only facilitated nucleoside transport.     

The excretion of hydrogen ion by the isolated amphibian skin: effects of antidiuretic hormone and amiloride.

H+ extrusion by the isolated skins of two amphibia, Rana ridibunda and Bufo bufo, was studied in order to test for the presence of exchange mechanisms of the type Na+/H+ and Cl-/HCO3-, which have been described in several epithelial structures. The preparations were mounted in chambers of the Ussing type, so that the short-circuit current could be used as a function of Na+ transport and the pH-stat techinique was utilize to determine the rates of H+ extrusion under different experimental conditions. The conditions were either the withdrawal of the ions intervening the mentioned exchanges (Cl- or Na+), or the addition of drugs with well-known effects on Na+ up-take and transport (antidiuretic hormone and amiloride). In the frog skin, H+ excretion was detected in solutions containing either Cl- or SO4-2-, with identical rates. Again, Na+ substitution by Mg-2+ had no effect on H+ excretion rates, neither did the suppression of Na+ influx by amiloride or its stimulation by antidiuretic hormone. These experiments were repeated with similar results in gland-free preparations of the epidermis of frog skin separated from the corion by the action of collagenase. Experiments in toad skin that H+ excretion could not be detected whan Cl- was present in the outer medium, but became apparent if an impermant anion, SO4-2-, was used. This observation is compatible with the existence of an exchange mechanism of the type Cl-/HCO3-. Secondly, in these preparations H+ extrusion increased after stimulation with antidiuretic hormone and decreased when amiloride was used or when Na+ was substituted by Mg+, suggesting that a least a fraction of the total H+ efflux is linked to Na+ influx. In the isolated frog skin this mechanism does not seem to be operative.     

Catabolite inhibition and sequential metabolism of sugars by Streptococcus lactis.

Growth of galactose-adapted cells of Streptococcus lactis ML(3) in a medium containing a mixture of glucose, galactose, and lactose was characterized initially by the simultaneous metabolism of glucose and lactose. Galactose was not significantly utilized until the latter sugars had been exhausted from the medium. The addition of glucose or lactose to a culture of S. lactis ML(3) growing exponentially on galactose caused immediate inhibition of galactose utilization and an increase in growth rate, concomitant with the preferential metabolism of the added sugar. Under nongrowing conditions, cells of S. lactis ML(3) grown previously on galactose metabolized the three separate sugars equally rapidly. However, cells suspended in buffer containing a mixture of glucose plus galactose or lactose plus galactose again consumed glucose or lactose preferentially. The rate of galactose metabolism was reduced by approximately 95% in the presence of the inhibitory sugar, but the maximum rate of metabolism was resumed upon exhaustion of glucose or lactose from the system. When presented with a mixture of glucose and lactose, the resting cells metabolized both sugars simultaneously. Lactose, glucose, and a non-metabolizable glucose analog (2-deoxy-d-glucose) prevented the phosphoenolpyruvate-dependent uptake of thiomethyl-beta-d-galactopyranoside (TMG), but the accumulation of TMG, like galactose metabolism, commenced immediately upon exhaustion of the metabolizable sugars from the medium. Growth of galactose-adapted cells of the lactose-defective variant S. lactis 7962 in the triple-sugar medium was characterized by the sequential metabolism of glucose, galactose, and lactose. Growth of S. lactis ML(3) and 7962 in the triple-sugar medium occurred without apparent diauxie, and for each strain the patterns of sequential sugar metabolism under growing and nongrowing conditions were identical. Fine control of the activities of preexisting enzyme systems by catabolite inhibition may afford a satisfactory explanation for the observed sequential utilization of sugars by these two organisms.