Evidence from studies of temperature-dependent changes of D-glucose, D-mannose and L-sorbose permeability that different states of activation of the human erythrocyte hexose transporter exist for good and bad substrates.

(1) The inhibition constant of L-sorbose flux from fresh human erythrocytes by D-glucose, Ki(sorbose) increases on cooling from 50 degrees C to 30 degrees C from 5.15 +/- 0.89 mM to 12.24 +/- 1.9 mM; the Ki(sorbose) of D-mannose increases similarly, indicating that the process is endothermic. (2) The activation energy Ea(sorbose) of net L-sorbose exit is 62.9 +/- 3.1 kJ/mol; in the co-presence of 5 mM D-glucose Ea(sorbose) is reduced to 41.7 +/- 1.6 kJ/mol (P < 0.005). (3) Cooling from 35 degrees C to 21 degrees C decreases the Ki(inf, cis) of auto-inhibition of D-glucose net exit from 5.2 +/- 0.3 mM to 1.36 +/- 0.06 mM; the Ki(inf, cis) of D-mannose falls from 10.9 +/- 1.65 mM to 5.7 +/- 0.3 mM. (4) The activation energy of D-glucose zero-trans net exit is 34.7 +/- 2.1 kJ/mol and that of D-mannose exit is 69.4 +/- 3.7 kJ/mol (P < 0.0025). (5) The exothermic and exergonic processes of auto-inhibition of D-glucose net exit are larger than those for D-mannose (P < 0.03). These data are consistent with D-glucose binding promoting an activated transporter state which following dissociation transiently remains; if an L-sorbose molecule binds within the relaxation time after D-glucose dissociation, it will have a higher mobility than otherwise. Cooling slows the relaxation time of the activated state hence raises the probability that L-sorbose will bind to the glucose-activated transporter. D-Glucose donates twice as much energy to the transporter as D-mannose, consequently produces more facilitation of flux. This view is inconsistent with the alternating carrier model of sugar transport in which net flux is considered to be rate-limited by return of the empty carrier, but is consistent with fixed two-site models.

Pre-steady-state uptake of D-glucose by the human erythrocyte is inconsistent with a circulating carrier mechanism.

Simulation shows that the four-state mobile carrier model for sugar transport in which the asymmetry arises from unequal rate constants of inward and outward translation of the free-carrier and carrier-sugar complex, does not fit with the observed data for pre-steady-state uptake recently obtained by A.G. Lowe and A.R. Walmsley [1987) Biochim. Biophys. Acta 903, 547-550). The main reason for this discrepancy is that pre-steady-state fluxes are determined mainly by the dissociation constants Ks of glucose and maltose for the external sites, rather than the Km (zero-transoi) of glucose and the Ki of maltose. The data are also inconsistent with other forms of asymmetric carrier but are fairly consistent with a symmetrical carrier with high-affinity sites for D-glucose or with a fixed site carrier model.

The thermostatics and thermodynamics of cotransport.

The thermostatics of cotransport are reviewed. A static-head equilibrium state across a cotransport system, without leaks, is thought to occur when the electrochemical potential of the driven solute, B prevents net flow of the driving solute, A. For a symport this gives the relationship (formula: see text) Where n is the stoichiometric coefficient, namely the number of moles of A transported per mole of B. (2) If either a symporter with a 2:1 stoichiometric coefficient and a 1:1 symporter, or alternatively, a 1:1 symporter and a 1:1 antiporter are placed in a series membrane array, then the predicted static-head equilibrium across the entire array conflicts with the zeroth law of thermodynamics. (3) There are two major reasons for this failure of cotransport theory; these are: (A) the thermostatic relationships derived shown in Point 1 are based on the assumption that the cotransport process takes place within a closed system. However, the membrane and the external reservoirs are open to the cotransported ligands. It follows that A and B in the external reservoirs can vary independently of the changes within the cotransport process. As no chemical reaction between A and B occurs in the external solutions, reactions within the membrane phase do not affect the equilibrium between the transported ligands in the open reservoirs. (B) It is assumed that the law of mass action can be applied to the cotransport chemical reactions within the membrane phase, without any allowance for the fact that these reactions occur within a 'small thermodynamic system'. Any proper analysis of the chemical potential of the transported intermediate must consider the effects of lower order ligand-carrier forms, which coexist and compete for space with the higher order cotransported forms on the binding matrix. If account is taken of this necessity, then a simple extension of the work of Hill and Kedem (1966) J. Theor. Biol. 10, 399-441 shows that: (a) the static-head equilibrium state cannot exist; (b) the stoichiometry of cotransport, whether symport, or antiport, does not affect the static-head distribution of cotransported ligands; (c) the hypothetical net charge of the transported ligand-carrier complex does not affect static-head equilibrium; (d) the only equilibrium state where there is zero net flow of both driving and driven transported ligand is at true equilibrium when the ligands are uniformly distributed across the membrane. (4) It is deduced that cotransport is not entirely an affinity-driven, but is partially an entropy-driven process.(ABSTRACT TRUNCATED AT 400 WORDS)

The relationship between sugar metabolism, transport and superoxide radical production in rat peritoneal macrophages.

Dexamethasone inhibits sugar-dependent phorbol myristate acetate (PMA)-stimulated superoxide production and 2-deoxy-D-glucose (2-dGlc) transport in rat peritoneal macrophages (Rist, R.J., Jones, G.E. and Naftalin, R.J. (1991) Biochem. J. 278, 119-128; Rist, R.J. and Naftalin, R.J. (1991) Biochem J. 278, 129-135). Here it is shown that with glucose as a substrate, dexamethasone (0.1 microM) acts as a non-competitive inhibitor of PMA-induced superoxide production; decreasing the maximal rate of superoxide production (P < 0.001) without altering the Km. In contrast, with 2-dGlc as a substrate, dexamethasone shows competitive inhibition of PMA-stimulated superoxide production; increasing the Km of superoxide production, (P < 0.001) without altering the Vmax. The maximal rate of PMA-stimulated superoxide production with glucose as substrate is 10-12-fold in excess of the maximal rate with 2-dGlc as substrate. Diphenylene iodonium (DPI) is a non-competitive inhibitor of PMA-stimulated glucose-dependent superoxide production in macrophages, (Ki = 1-5 microM) and significantly reduces the activity of the PMA-induced hexose monophosphate shunt, (HMPS) (P < 0.01). However, DPI (1 microM) has no significant effect on the PMA-induced increase in 2-dGlc uptake, suggesting that the stimulus for HMPS activity and superoxide production is separate from the stimulus for hexose transport. A model is described which explains the observed differences in hexose transport and glucose- and 2-dGlc-dependent superoxide production in terms of the differences in metabolism of the two sugars. Accumulation of free 2-dGlc within the cytosol leads to saturation of hexokinase and hence, the effects of PMA and dexamethasone, which alter the coupling between hexokinase and the transporter, are only observed at low concentrations of 2-dGlc, where it is accumulated to sub-saturating levels. Since glucose is completely metabolized within the cell, PMA and dexamethasone increase and decrease, respectively, net uptake of sugar and superoxide production at all glucose concentrations.

3-O-methyl-D-glucose transport in rat red cells: effects of heavy water.

Transport of 3-O-methyl-D-glucose (3-OMG) in rat red blood cells (RBCs) has been examined at 24 degrees C. The Km and Vm of zero-trans net uptake are 2.3 +/- 0.48 mM and 0.055 +/- 0.003 mumol (ml cell water)-1) min-1, whereas the Km and Vm for net exit are 2.1 +/- 0.12 mM and 0.12 +/- 0.01 mumol (ml cell water)-1 min-1. The Km and Vm for infinite-trans exchange uptake are 2.24 +/- 0.14 mM and 0.20 +/- 0.04 mumol (ml cell water)-1 min-1. In agreement with Whitesell et al. (Abumrad, N.A., Briscoe, P., Beth, A.H. and Whitesell, R.R. (1988) Biochim. Biophys. Acta 938, 222-230), we find that there is no significant acceleration of the rate of exchange exit over net exit. Substitution of D2O for water results in an increase in the Vm for zero-trans net uptake to 0.091 +/- 0.004 mumol (ml cell water)-1 min-1. There is no change in the Vm or Km for exchange uptake or net or exchange exit. Counterflow experiments indicate, in agreement with Helgerson and Carruthers (1989) Biochemistry 28, 4580-4594), that there is some compartmentalization of 3-OMG within the cells, perhaps resulting from slow complexation of the sugar with some intracellular component. The data can be simulated by assuming that transport across the membrane is mediated by either a fixed 2-site, or an alternating 1-site symmetrical transporter. With both models the observed asymmetries in net and exchange kinetics and in counterflow can be ascribed entirely to the complexation reaction of the sugar to an intracellular component. Also the D2O effects can entirely be attributed to an increase in the rate of sugar movement between bound and free compartments.

Re-examination of hexose exchanges using rat erythrocytes: evidence inconsistent with a one-site sequential exchange model, but consistent with a two-site simultaneous exchange model.

(1). The kinetic parameters of zero-trans net uptake and infinite-trans uptake of 3-O-methyl-D-glucoside, 2-deoxy-D-glucose and D-mannose into rat red cells at 24 degrees C were measured after taking account of the linear diffusion components of flux. (2). Zero-trans exists of 3-O-methyl-D-glucoside and D-mannose from rat cells were also measured. (3). After correction for linear flux via non-specific routes, the Vmax of zero-trans uptake of 3-O-methyl-D-glucoside was significantly higher, (1.25 +/- 0.06 mumol (10 min)-1 (ml cell water)-1) than the corresponding parameters of mannose or 2-deoxy-D-glucose, (0.33 +/- 0.01 and 0.39 +/- 0.01 mumol(10 min)-1 (ml cell water)-1, respectively; P < 0.001). (4). After correction for linear flux via non-specific uptake routes, the Vmax of zero-trans exit of 3-O-methyl-D-glucoside is significantly higher (1.70 +/- 0.1 mumol (10 min)-1 (ml cell water)-1) than the corresponding value for mannose exit flux, (1.10 +/- 0.1 mumol (10 min)-1 (ml cell water)-1; P < 0.001). (5). The acceleration ratio, i.e., the ratio of infinite-trans influx Vmax/zero-trans influx Vmax of mannose by mannose (9.12 +/- 0.03) is significantly higher than that of 3-O-methyl-D-glucose by 3-O-methyl-D-glucose (2.77 +/- 0.14)(P < 0.001). (6). The one-site simple carrier model of glucose transport in which sugar exchange is viewed as a sequential process, predicts that the acceleration ratio of the more rapidly moving sugar 3-O-methyl-D-glucose by 3-O-methyl-D-glucose should be greater than that of the slower sugar, mannose by mannose. Hence, the observed findings are inconsistent with the one-site model, but confirm the earlier disputed studies of Miller, D.M. (1968; Biophys. J. 8, 1329-1338). (7). A two-site model, in which sugar exchange is considered as a simultaneous process, predicts that the acceleration ratio of mannose influx by mannose should be higher than for 3-O-methyl-D-glucose by 3-O-methyl-D-glucose. The data are, therefore, consistent with a two-site model.

Interactions of sodium pentobarbital with D-glucose and L-sorbose transport in human red cells.

Pentobarbital acts as a mixed inhibitor of net D-glucose exit, as monitored photometrically from human red cells. At 30 degrees C the Ki of pentobarbital for inhibition of Vmax of zero-trans net glucose exit is 2.16+/-0.14 mM; the affinity of the external site of the transporter for D-glucose is also reduced to 50% of control by 1. 66+/-0.06 mM pentobarbital. Pentobarbital reduces the temperature coefficient of D-glucose binding to the external site. Pentobarbital (4 mM) reduces the enthalpy of D-glucose interaction from 49.3+/-9.6 to 16.24+/-5.50 kJ/mol (P<0.05). Pentobarbital (8 mM) increases the activation energy of glucose exit from control 54.7+/-2.5 kJ/mol to 114+/-13 kJ/mol (P<0.01). Pentobarbital reduces the rate of L-sorbose exit from human red cells, in the temperature range 45 degrees C-30 degrees C (P<0.001). On cooling from 45 degrees C to 30 degrees C, in the presence of pentobarbital (4 mM), the Ki (sorbose, glucose) decreases from 30.6+/-7.8 mM to 14+/-1.9 mM; whereas in control cells, Ki (sorbose, glucose) increases from 6.8+/-1.3 mM at 45 degrees C to 23.4+/-4.5 mM at 30 degrees C (P<0.002). Thus, the glucose inhibition of sorbose exit is changed from an endothermic process (enthalpy change=+60.6+/-14.7 kJ/mol) to an exothermic process (enthalpy change=-43+/-6.2 7 kJ/mol) by pentobarbital (4 mM) (P<0.005). These findings indicate that pentobarbital acts by preventing glucose-induced conformational changes in glucose transporters by binding to 'non-catalytic' sites in the transporter.

D-galactose accumulation in rabbit ileum. Effects of theophylline on serosal permeability.

The effects of theophylline and dibutyryl cyclic AMP, on in vitro unidirectional galactose fluxes across the mucosal and serosal borders of rabbit ileum have been studied. 1. When Ringer [galactose] = 2mM, theophylline and dibutyryl cyclic AMP reduce both mucosal-serosal and serosal-mucosal galactose flux by approx. 50%. The K1 for theophylline inhibition of flux in both directions is 2 mM. 1 mM dibutyryl cyclic AMP elicits a maximal inhibitory response. Concurrent with the inhibition in transmural galactose fluxes, theophylline and dibutyryl cyclic AMP increase the tissue accumulation of [galactose] and the specific-activity ratio R of 3H : 14C-labelled galactose coming from the mucosal and serosal solutions respectively. It is deduced that theophylline and dibutyryl cyclic AMP are without effect on the mucosal unidirectional permeability to galactose but cause a symmetrical reduction in serosal entry and exit permeability. 2. Reduction in the asymmetry of the mucosal border to galactose by reducing Ringer [Na], raising Ringer [galctose] or adding ouabain reduces the theophylline-dependent increase in galactose accumulation. 3. Hypertonicity in the serosal solution increases the permeability of the serosal border to galactose and reduces tissue galactose accumulation. Serosal hypertonicity partially reverses the theophylline-depedent effects on galactose transport. Replacing Ringer chloride by sulphate abolishes the theophylline-dependent effects on galactose transport. 4. It is considered that the theophylline-dependent increase in galactose accumulation results from the reduction in serosal permeability. This is shown to be a quantitatively consistent inference. 5. Further support for the view that the asymmetric transport of galactose in rabbit ileum results from convective-diffusion is presented.

Galactose transport across the serosal border of rabbit ileum and its role in intracellular accumulation.

Unidirectional fluxes of D-galactose across the brush and serosal border of rabbit ileum were determined using the method described previously (Naftalin, R. J. and Curran, P.F. (1974) J. Membrane Biol. 16, 257-278). With ringer [Na] equals 75 meguiv., the Km for galactose influx across the brush-border is 5mM, with 0.1 mM ouabain present K-m equals 50 mM, the V (2.0 munol - CM-2-H-1) remains unaltered. The Michaelis parameters for galactose influx across the serosal border are K-m equals 59 plus or minus 9 mM and V equals 4.7 plus or minus 0.24 mumol-cm-2-h-1 and for efflux K-m equals 85 plus or minus 10 mM and V equals 6.8 plus or minus 0.7 mumol-CM-2-H-1. 2. 2-Deoxy-D-glucose and methyl beta-D-glucopyranoside inhibit galactose entry exclusively at the serosal and mucosal borders respectively, while 3-O-methyl-D-glucose inhibits galactose influx at both borders. 0.1 mM ouabain increases the K1 of 3-O-methylglucose for the serosal transport system (100 mM) is unaffected by ouabain. Inhibition of mucosal galactose transport by ouabain or by competition with other sugars results in a reciprocal increase in exit permeability and decrease in entry permeability. Inhibition of serosal galactose transport results in inhibition of both the entry and exit permeability, entry is more affected. 3. There is a small degree of permeability asymetry at the serosal border to galactose which is reduced by ouabain or removel of Na+ from the Ringer. Uptake of 14C-labelled galactose from the serosal solution into the tissue is also inhibited by addition of ouabain or Na+ removal. It is therefore considered that there is a weak active transport system for galactose at the serosal border. 4. Net transepithelial galactose flux is sufficiently high and serosal permeability to galactose sufficiently low to be consistent with the view that galactose is concentrated within the tissue fluid, after conviction (Naftalin, R.J. and Holman, G.D. (1974) Biochim. Biophys. Acta., 373, 453-470) across the mucosal border because it is reflected at the serosal boundary.

Evidence of multiple operational affinities for D-glucose inside the human erythrocyte membrane.

1. The Michaelis-Menten parameters of labelled D-glucose exit from human erythrocytes at 2 degrees C into external solution containing 50 mM D-galactose were obtained. The Km is 3.4 +/0 0.4 mM, V 17.3 +/- 1.4 MMOL . 1(-1) cell water . min-1 for this infinite-trans exit procedure. 2. The kinetic parameters of equilibrium exchange of D-glucose at 2 degrees C are Km = 25 +/- 3.4 mM, V 30 +/- 4.1 mmol . 1(-1) cell water . min-1. 3. The Km for net exit of D-glucose into solutions containing zero sugar is 15.8 +/- 1.7 mM, V 9.3 +/- 3.3 mmol . 1(-1) cell water . min-1. 4. This experimental evidence corroborates the previous finding of Hankin, B.L., Lieb, W.R. and Stein, W.D. [(1972) Biochim. Biophys. Acta 255, 126--132] that there are sites with both high and low operational affinities for D-glucose at the inner surface of the human erythrocyte membrane. This result is inconsistent with current asymmetric carrier models of sugar transport.

Na+-dependent co-transport of alpha-methyl D-glucoside across the mucosal border of rabbit descending colon.

(1) The uptake and bidirectional fluxes of 1-alpha-methyl D-glucoside were studied in isolated rabbit colonic mucosa. (2) The uptake of alpha-methyl D-glucoside was linear over the first 30 min and reached maximum after 1 h; was a saturable function of sugar concentration and was Na+-dependent. (3) An increase in sugar uptake across the mucosal border and net transepithelial sugar flux across sheets of colon was observed in the presence of 10(-4) M amiloride. (4) Phlorizin (10(-4) M) inhibited sugar uptake into the tissue water and abolished net sugar flux. Amiloride-stimulated sugar uptake was also abolished by 10(-4) M phlorizin. (5) Ouabain (10(-4) M) prevented the effect of amiloride on sugar uptake and inhibited sugar uptake into the tissue. (6) These results corroborate the findings of Henriques de Jesus et al. (Henriques de Jesus, C., Da Gracia Emilio, M. and Santos, M.A. Gastroenterol. Clin. Biol. 3, 172-173) who found a sugar-dependent increase in short-circuit current in colonic mucosa exposed to amiloride.

A model for accelerated uptake and accumulation of sugars arising from phosphorylation at the inner surface of the cell membrane.

A model transport system for cellular accumulation of sugar coupled to phosphorylation is described. Sugar permeates the cell membrane via a passive facilitated transport system. On the inside surface of the membrane the bound sugar is either phosphorylated to form impermeable hexose phosphate, which is released into the intracellular solution, or released directly into the cytosol. Sugar may be regenerated from hexose phosphate in the cytosol via a phosphatase reaction. The reduction of the proportion of sites on the inner membrane surface occupied by permeable sugar, caused by the kinase reaction, increases both net and unidirectional passive inflow and reduces both net and unidirectional exit of sugar, thereby permitting large stationary state gradients of free sugar to be maintained between the cytosol and bathing solution. In cells where there is a high passive membrane permeability to free sugar, steady-state accumulation of free sugar within the cytosol, linked to metabolism is inexplicable in terms of conventional transport kinetics based on equilibrium thermodynamic assumptions. This phenomenon is analysed in terms of non-equilibrium stationary state flows of ligands via a probability network. The effects of metabolism on exchange transport are also examined. The model provides a framework to explain how sugar transport is loosely coupled to phosphorylation in mammalian epithelial cells, adipocytes, yeasts and bacteria, so that a high rate of substrate accumulation is maintained without requiring a reduction in the intracellular concentration of permeable substrate below that in the external solution.

The effects of removal of sodium ions from the mucosal solution on sugar absorption by rabbit ileum.

Net absorption and accumulation of D-galactose, beta-methyl D-glucose and low concentrations of 3-0-methyl-D-glucose by sheets of rabbit ileum are observed even when Na+ in the mucosal solution is replaced by choline. This indicates that active sugar transport can occur in the direction opposite to the brush-border Na+ gradient.

Membrane and intracellular modes of sugar-dependent increments in red cell stability.

Sugar-dependent increments in red cell stability under osmotic stress can be ascribed to changes either in the membrane or in the intracellular matrix. These two possible modes of action have been tested and characterized. Rheological investigation of membrane-free haemoglobin solutions has shown that D-glucose, but not D-fructose, promotes the formation of a visco-plastic gel structure. Gel strength is a function of glucose concentration, haemoglobin concentration and temperature. The ability of various sugars to promote gel formation correlates with their solution properties. The existence of gel structure reduces K+ and haemoglobin leak from red cells whose membranes were partially destroyed by gamma-radiation. Reduced osmotic swelling in the presence of glucose is also due to gel formation since the glucose effect is lost in resealed red cell ghosts. D-Fructose does not protect red cells against radiation damage; its mode of action in increasing red cell stability under osmotic stress is a membrane effect. Cell sizing using the Coulter Counter has shown that fructose, but not glucose, can increase the maximal volume at lysis. At 50 mM, D-fructose expands the red cell ghost volume by 11.2%; this represents a 7.2% increase in membrane area. Ghost expansion by fructose is fructose concentration dependent (0-100 mM) and is insensitive to temperature variation (0-37 degrees C).

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.

Factors affecting the compartmentalization of sodium ion within rabbit ileum in vitro.

(1) Net Na+ loss from rabbit ileum, stripped of its serosal muscle layers, into ice cold choline chloride is consistent with loss from two separate pools (rate constants 0.102 and 0.011 min-1). Since cell K+ is lost with a single rate constant, 0.0062 min-1) and inulin, a good extracellular marker, is lost with a single rate constant 0.082 min-1, it is inferred that the fast rate constant of Na loss characterizes loss from an extracellular pool and the slow constant, loss from an intracellular pool. (2) The [Na+] in the inulin space (extracellular) was calculated to be 180 +/- 13 (S.D.) mequiv. and the [Na+] in the intracellular space 30.4 +/- 4.1 (S.D.) medquiv., this provides evidence that the paracellular spaces are, at least 80 mosmol hypertonic to the external Ringer. (3) There is a saturable galactose-dependent increase in both the intracellular and extracellular [Na+]. Extracellular [Na+] is increased to 236 +/- 22 (S.D.) mequiv. Whilst intracellular [Na+] is increased to 42.6 +/- 8.8 (S.D.) mequiv. when Ringer [galactose] is 10 mM. Galactose-dependent increases in total tissue [Na+] can thus be attributed mainly to the increase in extracellular [Na+]. (4) Extracellular hypertonicity, both in the presence and absence of galactose, is dependent upon the [Na+] of the bathing Ringer. 0.1 mM ouabain abolishes the extracellular hypertonicity. This observed extracellular hypertonicity in normally functioning tissue may provide the driving force for transcellular convective flow of salt, water and sugars.

Bidirectional sodium ion movements via the paracellular and transcellular routes across short-circuited rabbit ileum.

1. It has been confirmed that the agent 2,3,6-triaminopyrimidine decreases Na+ conductance in the paracellular pathway of rabbit ileum. 2. Triaminopyrimidine has been used as a means of measuring transcellular bidirectional Na+ flux, and also, of assessing the contribution of the paracellular pathway to transepithlial Na+ flux. 3. Reduction of Ringer [Na+] to 25 mM or incubation with 0.1 mM ouabain reduces paracellular Na+ permeability. This effect may be due to lateral space collapse. Ringer galactose increases serosa to mucosa Na+ flux by a stimulating reflux through the tight junctions. A proportion of net Na+ flux in control tissues is due to asymmetry generated in the paracellular pathway. It is likely that this passive asymmetry results from an osmotic pressure gradient across the tight-junction. 4. Measurement of the tissue isotope specific activity ratio together with bidirectional transcellular Na+ fluxes allows calculation of the four unidirectional fluxes across the mucosal and serosal boundaries. Values obtained for Na+ entry (J12) and exit (J21) across the mucosal boundary are 7.97 alnd 7.13 mumol-cm(-2)-h(-1) respectively. Entry flux (J12) is a saturable function of Ringer [Na+]. The calculated Km is 295 mM and the V is 17.6 mumul-cm(-2)-h(-1). Na+ entry flux is insensitive to ouabain (0.1 mM). Ouabain results in elevation of exit (J21) flux of Na+ across the brush border. D-Galactose causes a saturable increase in Na+ flux (J12) across the mucosal boundary; the Km for this relationship is 1.2 mM and the V 2.17 mumol-cm(-2)-h(-1). The stoichiometry between sugar and Na+ entry is applixmately 1:1. In contrast to the effect of galactose on entry flux, no change in Na+ efflux across the mucosal boundary is observed when Ringer [galactose] is raised. This finding is dissonant with the prediction of the Na+ -gradient hypothesis. The calculated values of exit (J23) and entry (J32) Na+ fluxes across the serosal border are 16.74 and 15.90 mumol-cm(-2)-h(-1). 0.1 mM ouabain markedly reduces both these unidirectional fluxes. This result is consistent with a serosal location of the Na+-pump. Serosal Na+ exit flux J23 increases as a hyperbolic function of Ringer [galactose]. A small galactose-dependent decrease in entry (J32) is also observed. 0.1 mM ouabain abolishes these galactose-dependent changes. 5. The present findings together with those in the previous paper are discussed in relation to the convective-diffusion model for sugar transport.

Effects of secretagogues on the K+ permeability of mucosal and serosal borders of rabbit colonic mucosa.

(1) K+ efflux rates from the mucosal and serosal surfaces of sheets of rabbit colonic mucosa have been determined by measuring net K+ loss into K+-free Ringer solution bathing each side of the tissue. (2) Initially, there is a high rate of K+ loss from the tissue, this falls to a lower steady-state rate after 20 min. Loss of K+ from the tissue into the serosal bath is 6-8 fold faster than loss to the mucosal bath. (3) A number of intestinal secretagogues, e.g. theophylline, cyclic AMP, carbachol, ionophore A23187, as well as the laxative bisacodyl, raise the K+ efflux rate across the mucosal border by 200-300%. In the case of K+ efflux induced by carbachol the effect is shown to be dependent on raised levels of intracellular Ca2+. Ca2+-calmodulin complex does not appear to be be involved in activation of K+ efflux across the mucosal border. (4) Amiloride does not block mucosal K+ efflux, but tetraethyl-ammonium does inhibit K+ efflux across the mucosal border, induced by either bisacodyl or raised intracellular Ca2+. (5) The results suggest that laxatives may increase the rate of K+ secretion into the colonic lumen by raising the K+ permeability of the mucosal border.

Evidence for non-uniform distribution of D-glucose within human red cells during net exit and counterflow.

The kinetic parameters of net exit of D-glucose from human red blood cells have been measured after the cells were loaded to 18 mM, 75 mM and 120 mM at 2 degrees C and 75 mM and 120 mM at 20 degrees C. Reducing the temperature, or raising the loading concentration raises the apparent Km for net exit. Deoxygenation also reduces the Km for D-glucose exit from red blood cells loaded initially to 120 mM at 20 degrees C from 32.9 +/- 2.3 mM (13) with oxygenated blood to 20.5 +/- 1.3 mM (17) (P less than 0.01). Deoxygenation increases the ratio Vmax/Km from 5.29 +/- 0.26 min-1 (13) for oxygenated blood to 7.13 +/- 0.29 min-1 (17) for deoxygenated blood (P less than 0.001). The counterflow of D-glucose from solutions containing 1 mM 14C-labelled D-glucose was measured at 2 degrees C and 20 degrees C. Reduction in temperature, reduced the maximal level to which labelled D-glucose was accumulated and altered the course of equilibration of the specific activity of intracellular D-glucose from a single exponential to a more complex form. Raising the internal concentration from 18 mM to 90 mM at 2 degrees C also alters the course of equilibration of labelled D-glucose within the cell to a complex form. The apparent asymmetry of the transport system may be estimated from the intracellular concentrations of labelled and unlabelled sugar at the turning point of the counterflow transient. The estimates of asymmetry obtained from this approach indicate that there is no significant asymmetry at 20 degrees C and at 2 degrees C asymmetry is between 3 and 6. This is at least 20-fold less than predicted from the kinetic parameter asymmetries for net exit and entry. None of the above results fit a kinetic scheme in which the asymmetry of the transport system is controlled by intrinsic differences in the kinetic parameters at the inner and outer membrane surface. These results are consistent with a model for sugar transport in which movement between sugar within bound and free intracellular compartments can become the rate-limiting step in controlling net movement into, or out of the cell.

Video enhanced imaging of the fluorescent Na+ probe SBFI indicates that colonic crypts absorb fluid by generating a hypertonic interstitial fluid.

Extracellular accumulation of Na+ detected by video-enhanced microscopic imaging of the impermeant fluorescent probe SBFI confirms the view that colonic crypts produce a hypertonic ascorbate ca 1000 mOsm.kg-1, thereby generating a large osmotic pressure across the crypt wall. This creates a high fluid tension within the crypt lumen, sufficient to dehydrate faeces. When bathed in Tyrode the SBFI.Na+ fluorescence indicates a [Na+] ca 750 mM within the interstitial space of metabolizing rat descending colon. There is no evidence of interstitial Na+ accumulation in octanol (2 mM) or in rabbit colon incubated with 1.0 mM ouabain and no evidence of Na+ secretion via the crypt lumen during absorption.