The hormonal control of uterine luminal fluid secretion and absorption.

The secretion of uterine luminal fluid initially provides a transport and support medium for spermatozoa and unimplanted embryos, while the absorption of uterine luminal fluid in early pregnancy results in the closure of the lumen and allows blastocysts to establish intimate contact with the uterine epithelium. We have established an in vivo perfusion technique of the lumen to study the hormonal control of the events in the peri-implantation period. Fluorescein-labelled dextran was included in the perfusion medium to monitor fluid movements and the concentrations of Na(+) and CI(-) ions in the effluent were monitored. Using an established regimen of steroid treatment of ovariectomized rats mimicking early pregnancy, oestradiol caused fluid secretion, while progesterone resulted in an amiloride-sensitive fluid absorption. Fluid absorption peaked at about the expected time of implantation. The effect of progesterone could be inhibited by treatment with a high dose of oestradiol, by the anti-progestin RU486, and by the presence of an intra-uterine contraceptive device. Studies of expression of Na(+) and CI(-) channels (ENaC, CFTR) indicated that these channels were subject to tissue-specific regulation within the uterus, but more work is required to determine their role and the factors controlling their abundance and localization in early pregnancy.

Aldosterone reduces crypt colon permeability during low-sodium adaptation.

Fluid and electrolyte absorption by colonic crypts depends on the transport properties of crypt cellular and paracellular routes and of the pericryptal sheath. As a low-Na(+) diet increases aldosterone and angiotensin II secretion, either hormone could affect absorption. Control and adrenalectomized (ADX) Sprague-Dawley rats were kept at a high-NaCl (HS) diet and then switched to low-NaCl (LS) diet for 3 days. Aldosterone or angiotensin II plasma concentrations were maintained using implanted osmotic mini-pumps. The extracellular Na(+) concentration in isolated rat distal colonic mucosa was determined by confocal microscopy using a low-affinity Na(+) -sensitive fluorescent dye (Sodium red, and Na(+) -insensitive BODIPY) bound to polystyrene beads. Crypt permeability to FITC-labelled dextran (10 kDa) was monitored by its rate of escape from the crypt lumen into the pericryptal space. Mucosal ion permeability was estimated by transepithelial electrical resistance (TER) and amiloride-sensitive short-circuit current (SCC). The epithelial Na(+) channel, ENaC, was determined by immunolocalization. LS diet decreased crypt wall permeability to dextran by 10-fold and doubled TER. Following ADX, aldosterone decreased crypt wall dextran permeability, increased TER, increased Na(+) accumulation in the pericryptal sheath and ENaC expression even in HS. Infusion of angiotensin II to ADX rats did not reverse the effects of aldosterone deprivation. These findings indicate that aldosterone alone is responsible for both the increase in Na(+) absorption and the decreased paracellular and pericryptal sheath permeability.

Pericryptal myofibroblast growth in rat descending colon induced by low-sodium diets is mediated by aldosterone and not by angiotensin II.

Pericryptal myofibroblast growth in descending colonic crypts correlates with the activation of the renin-angiotensin-aldosterone system. Earlier work showed that during the transition from a high-Na(+) (HS) to low-Na(+) (LS) diet there are changes in the colonic crypt wall and pericryptal sheath. As LS diet increases both aldosterone and angiotensin II, the aim here was to determine their individual contributions to the trophic changes in colonic crypts. Experiments were conducted on control and adrenalectomized Sprague-Dawley rats fed an HS diet and then switched to LS diet for 3 days and supplemented with aldosterone or angiotensin II. The actions of the angiotensin-converting enzyme inhibitor captopril, the angiotensin receptor antagonist losartan and the aldosterone antagonist spironolactone on extracellular matrix proteins, claudin 4 and E-cadherin myofibroblast proteins, alpha-smooth muscle actin (alpha-SMA) and OB-cadherin (cadherin 11), angiotensin type 1 and TGFbetar1 membrane receptors were determined by immunolocalization in fixed distal colonic mucosa. The LS diet or aldosterone supplementation following ADX in HS or LS increased extracellular matrix, membrane receptors and myofibroblast proteins, but angiotensin alone had no trophic effect on alpha-SMA. These results show that aldosterone stimulates myofibroblast growth in the distal colon independently of dietary Na(+) intake and of angiotensin levels. This stimulus could be a genomic response or secondary to stretch of the pericryptal sheath myofibroblasts accompanying enhanced rates of crypt fluid absorption.

Effects of anti-GLUT antibodies on glucose transport into human erythrocyte ghosts.

We have studied the effects of anti-GLUT1 antibodies on the uptake of glucose into erythrocytes. Glucose transport into human erythrocyte ghosts was measured directly using 3H-2-deoxy-glucose, or indirectly by monitoring associated volume changes using light scattering. The uptake of glucose was significantly inhibited in ghosts resealed in solutions containing specific antibodies against GLUT1. Such an effect was not observed when an antibody against the oestrogen receptor, lacking specificity towards GLUT1, was employed instead. The antibodies were also without effect on the efflux of preloaded glucose from erythrocyte ghosts. The demonstration that anti-GLUT antibodies can inhibit glucose uptake is support for the hypothesis that they exaggerate the cytoplasmic barrier to glucose uptake created by endofacial segments of GLUT1.

Effects of high pressure on glucose transport in the human erythrocyte.

The effects of raised hydraulic pressure on D-glucose exit from human red cells at 25 degrees C were determined using light scattering measurements in a sealed pressurized spectrofluorimeter cuvette. The reduction in the rates of glucose exit with raised pressure provides an index of the activation volume, deltaV++ (delta ln k/deltaP)(T) = -deltaV++/RT. Raised pressure decreased the rate constant of glucose exit from 0.077 +/- 0.003 s(-1) to 0.050 +/- 0.002 s(-1) (n = 5, P < 0.003). The Ki for glucose binding to the external site was 2.7 +/- 0.4 mm (0.1 MPa) and was reduced to 1.45 +/- 0.15 mm (40 MPa), (P < 0.01, Student's t test). Maltose had a biphasic effect on deltaV++. At [maltose] <250 microM, deltaV++ of glucose exit increased above that with [maltose = 0 mM], at >1 mm maltose, deltaV++ was reduced below that with [maltose = 0 mM]. Pentobarbital (2 mM) decreased the deltaV++ of net glucose exit into glucose-free solution from 30 +/- 5 ml mol(-1) (control) to 2 +/- 0.5 ml mol(-1) (P < 0.01). Raised pressure had a negligible effect on L-sorbose exit. These findings suggest that stable hydrated and liganded forms of GLUT with lower affinity towards glucose permit higher glucose mobilities across the transporter and are modelled equally well with one-alternating or a two-fixed-site kinetic models.

Progesterone stimulation of fluid absorption by the rat uterine gland.

The purpose of this study was to test the hypothesis that the steroid environment affects fluid absorption by the uterine glands. Laser scanning confocal microscopy of the distribution of an extracellular marker (fluorescein isothiocyanate-labelled dextran) within rat uterine glands showed that the endometrial glands change their fluid handling characteristics under different hormonal conditions. Under progesterone dominance, the glands showed an amiloride-sensitive dextran accumulation indicating sodium-dependent fluid absorption; however, this was absent in the oestrogen-dominated state. The rate of fluid uptake in the progesterone-stimulated gland opening was estimated to be approximately 1 x 10(-4) cm s(-1), requiring a suction pressure of between 10 and 20 mm Hg at the mucosal surface. This study provides the first direct evidence of fluid absorption by the uterine glands. Such absorption may provide the mechanism for closure of the uterine lumen and immobilization of the blastocyst necessary for implantation.

In vivo fluorescence measurement of Na(+) concentration in the pericryptal space of mouse descending colon.

A method involving surgical exposure of the colonic mucosa, fluorescent dye addition, and confocal microscopy has been developed for monitoring colonic crypt function in vivo in mice. Na(+) concentration in the extracellular pericryptal space of descending colon was measured using a low-affinity Na(+)-sensitive fluorescent indicator consisting of an Na(+)-sensitive chromophore (sodium red) and an Na(+)-insensitive chromophore (Bodipy-fl) immobilized on 200-nm-diameter polystyrene beads. The Na(+) indicator beads accumulated in the pericryptal spaces surrounding the colonic crypts after a 1-h exposure of the colonic luminal surface to the bead suspension. Na(+) concentration ([Na(+)]) in the pericryptal space was 491 +/- 62 mM (n = 4). After a 70-min exposure to amiloride (0.25 mM), pericryptal [Na(+)] was reduced to 152 +/- 21 mM. Blockage of the crypt lumen with mineral oil droplets reduced pericryptal [Na(+)] to 204 +/- 44 mM. Exposure of the colonic mucosa to FITC-dextran (4.5 kDa) led to rapid accumulation of the dye into the crypt lumen with a half time of 19.8 +/- 1.0 s, which was increased to 77.9 +/- 6.0 s after amiloride treatment. These results establish an in vivo fluorescence method to measure colonic crypt function and provide direct evidence for accumulation of a hypertonic absorbate in the pericryptal space of descending colon. The pericryptal space represents the first example of a hypertonic extracellular compartment in mammals that is not created by a countercurrent amplification mechanism.

Evidence of amiloride-sensitive fluid absorption in rat descending colonic crypts from fluorescence recovery of FITC-labelled dextran after photobleaching.

1. Fluorescence recovery after photobleaching (FRAP) of fluorescein isothiocyanate (FITC)-labelled 10 and 250 kDa dextran (FITC dextran) in isolated rat descending colonic crypts was measured at 35 degrees C using laser scanning confocal microscopy. 2. FRAP of either 10 or 250 kDa FITC dextran in crypt lumens was almost complete within 2-3 min. 3. In the presence of amiloride (0.1 mM), or in the absence of Na+, the rate of FITC dextran uptake into the crypt lumens was reduced by 70-80 %. 4. The rate of fluid uptake into the crypt lumen, as estimated from the rate of total FITC dextran uptake into the crypt lumen and its adjacent pericryptal region after FRAP, was between 1.3 x 10(-3) and 1.7 x 10(-3) cm x s(-1). 5. Convective flow during FRAP was also determined from the initial rate of FITC dextran advance along the crypt lumen. This effect was almost completely blocked by amiloride (0.1 mM). 6. The permeability of 10 kDa FITC dextran across the descending colonic crypt wall was found to be higher than that of 250 kDa FITC dextran (3.7 (+/- 0.6) x 10(-5) and 1.8 (+/- 0.3) x 10(-6) cm x s(-1), respectively; n = 3 for both, P < 0.01). The permeability of the caecal crypt wall to 10 kDa dextran was higher than that of the descending crypt wall (2.03 (+/- 0.21) x 10(-5) cm x s(-1); n = 3, P < 0.025). 7. Simulation of the flow of Na+, water and FITC dextran into the crypt lumen and across the crypt wall and pericryptal sheath corroborates the observed parameters of water and Na+ flows.

Mechanisms of glucose transport at the blood-brain barrier: an in vitro study.

How the brain meets its continuous high metabolic demand in light of varying plasma glucose levels and a functional blood-brain barrier (BBB) is poorly understood. GLUT-1, found in high density at the BBB appears to maintain the continuous shuttling of glucose across the blood-brain barrier irrespective of the plasma concentration. We examined the process of glucose transport across a quasi-physiological in vitro blood-brain barrier model. Radiolabeled tracer permeability studies revealed a concentration ratio of abluminal to luminal glucose in this blood-brain barrier model of approximately 0.85. Under conditions where [glucose](lumen) was higher than [glucose](ablumen), influx of radiolabeled 2-deoxyglucose from lumen to the abluminal compartment was approximately 35% higher than efflux from the abluminal side to the lumen. However, when compartmental [glucose] were maintained equal, a reversal of this trend was seen (approximately 19% higher efflux towards the lumen), favoring establishment of a luminal to abluminal concentration gradient. Immunocytochemical experiments revealed that in addition to segregation of GLUT-1 (luminal>abluminal), the intracellular enzyme hexokinase was also asymmetrically distributed (abluminal>luminal). We conclude that glucose transport at the CNS/blood interface appears to be dependent on and regulated by a serial chain of membrane-bound and intracellular transporters and enzymes.

Radiation induced cytochrome c release causes loss of rat colonic fluid absorption by damage to crypts and pericryptal myofibroblasts.

BACKGROUND: Therapeutic or accidental exposure to radiation commonly causes gastrointestinal disturbances, including diarrhoea. Rats subjected to whole body ionising radiation at a dose of 8 Gy lose their capacity to absorb fluid via the descending colon after four days. After seven days, fluid absorption recovers to control levels. AIMS: To investigate the effect of ionising radiation on colonic permeability together with its effect on mitochondria dependent apoptotic signals and intercellular adhesion molecules. METHODS: Rats were irradiated with doses of 0-12 Gy. Colonic permeability was measured by accumulation of fluorescein isothiocyanate (FITC) dextran in crypt lumens. Changes in levels of cytochrome c, caspase 3, E and OB cadherin, beta-catenin smooth muscle actin, and collagen IV were assessed using immunocytochemistry with confocal microscopy. RESULTS: Cytosolic cytochrome c increased after 8 Gy (t(1/2) 1.4 (0.6) hours) and peaked at approximately six hours. Caspase 3 increased more slowly, particularly in crypt epithelial cells (t(1/2) 57 (14.5) hours). Pericryptal myofibroblasts disintegrated within 24 hours as was evident from loss of OB cadherin and smooth muscle actin. This coincided with increased crypt permeability to dextran. Intercellular adhesion between crypt luminal cells was not lost until day 4 when both beta-catenin and E-cadherin were minimal. The half maximal dose-response for these effects was in the range 2-4 Gy. Recovery of colonic transport was concurrent with recovery of pericryptal smooth muscle actin and OB cadherin. The pan caspase inhibitor Z-Val-Ala-Asp.fluoromethylketone (1 mg/kg per day) had a small effect in conserving the pericryptal sheath myofibroblasts and sheath permeability but had no systemic therapeutic effects. CONCLUSIONS: These data suggest that radiation damage to the colon may be initiated by mitochondrial events. Loss of crypt fluid absorption and increased permeability coincided with decreased intercellular adhesion between crypt epithelial cells and loss of pericryptal sheath barrier function.

Comparison of cattle and sheep colonic permeabilities to horseradish peroxidase and hamster scrapie prion protein in vitro.

BACKGROUND: Paracellular permeability to solutes across the descending colon is much higher in cattle than sheep. This is a possible route for transmission of infective materials, such as scrapie prion. AIMS: To compare the permeabilities of labelled scrapie prion protein and other macromolecules in bovine and ovine descending colons in vitro. METHODS: Using fresh slaughterhouse material, transepithelial fluxes of macromolecules across colonic mucosae mounted in Ussing chambers were measured by monitoring transport of either enzyme activity or radioactivity. RESULTS: The comparative bovine to ovine permeability ratio of the probes increased with molecular weight: from 3.1 (0.13) for PEG400 to 10.67 (0.20) (p<0.001) for PEG4000; and from 1.64 (0.17) for microperoxidase to 7.03 (0.20) (p<0.001) for horseradish peroxidase (HRP). The permeability of (125)I-labelled inactivated Syrian hamster scrapie prion protein (ShaPrP(sc)) was 7.02 (0.33)-fold higher in bovine than ovine colon (p<0.0025). In each species, the probe permeabilities decreased according to the formula: P = P(o). exp(-K.ra). The "ideal" permeabilities, P(o) are similar, however, K((ovine)) = 2.46 (0.20) cm/h/nm exceeds K((bovine)) = 0.85 (0.15) cm/h/nm (p<0.001) indicating that bovine colon has a higher proportion of wide pores than ovine. Image analysis confirmed that HRP permeated through the bovine mucosal layer via a pericryptal paracellular route much more rapidly than in sheep. CONCLUSIONS: These data may imply that scrapie prion is transmitted in vivo more easily across the low resistance bovine colonic barrier than in other species.

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.

Regional differences in rat large intestinal crypt function in relation to dehydrating capacity in vivo.

1. Rat descending colon absorbed fluid against a large hydraulic resistance, imposed by 10 % agarose (w/v) gel plugs inserted in the lumen, by raising the tonicity of the absorbate from the gel to 880 +/- 54 mosmol kg-1; the tonicity of the absorbate from 2.5 % gels was 352 +/- 38 mosmol kg-1. The hypertonic absorbate generated an osmotic pressure which created a fluid tension in the crypt lumen. This was monitored as a suction tension in colonic luminal gels of 45.3 +/- 3 cmH2O with 2.5 % gels and 725 +/- 145 cmH2O with 10 % gels. The caecum was unable to absorb fluid against a significant hydraulic resistance. 2. Fluorescein isothiocyanate-labelled dextran (FITC dextran; molecular mass 10000 Da) accumulated within descending colonic crypt lumens by concentration polarization. Maximal accumulation at a depth of 20-40 micrometer below the mucosal surface was 5.68 +/- 0.2-fold above control levels. Caecal crypts accumulated dextran to a maximum of 1.8 +/- 0.17-fold above control levels. 3. The relationship between crypt luminal tension and suction tension of the distal colon was also demonstrated using paraffin, which occluded the crypt lumens with microscopic droplets and completely inhibited fluid absorption from high resistance luminal gels. 4. Reduction in dietary Na+ intake raised plasma aldosterone and the capacity of the distal colon to dehydrate against a high luminal hydraulic resistance. The caecum did not respond in this way to varied Na+ intake.

Regional crypt function in rat large intestine in relation to fluid absorption and growth of the pericryptal sheath.

1. Confocal microscopic studies of rat colonic mucosa showed that the pericryptal sheath surrounding distal colonic crypts is an effective barrier both to dextran and NaCl movement, whereas no such structure surrounds the caecal crypts. 2. The distal colonic pericryptal barrier was functionally demonstrated by accumulation of Sodium Green within the pericryptal space. After exposure to benzamil, Sodium Green accumulation was decreased. Fluorescein isocyanate-labelled dextran (FITC dextran; molecular mass 10000 Da) was accumulated in the crypt lumens and pericryptal spaces. Both dextran and Sodium Green accumulation were absent from the pericryptal zone surrounding caecal crypts. 3. Low dietary Na+ intake raised rat plasma aldosterone and stimulated distal pericryptal sheath growth and adhesiveness as shown by increased amounts of F-actin, smooth muscle actin, beta-catenin and E-cadherins in the pericryptal zone. It also raised the capacity of the distal colon to dehydrate against a high luminal hydraulic resistance. This linkage indicates that trophic effects on the colon resulting from a low Na+ diet are not confined solely to effects on transepithelial Na+ transport, but are observed in the pericryptal sheath. 4. A computer model of crypt function confirms that a pericryptal sheath with low permeability to NaCl is an essential component of the crypt dehydrating mechanism.

Evidence from temperature studies that the human erythrocyte hexose transporter has a transient memory of its dissociated ligands.

The inhibition constant of L-sorbose efflux (Ki(sorbose)) from human erythrocytes for inhibition by D-glucose increases from 5.15 +/- 0.89 to 12.24 +/- 1.9 mM on cooling from 50 degrees C to 30 degrees C; the Ki(sorbose) of D-mannose increases similarly on cooling. The activation energy Ea(sorbose) of net L-sorbose exit from human erythrocytes is 62.9 +/- 3.1 kJ mol-1; but in the co-presence of 5 mM D-glucose Ea(sorbose) is reduced to 41.7 +/- 1.6 kJ mol-1 (P < 0.005). These data are consistent with the view that when D-glucose binds to the hexose transporter it leads to an activated transporter state which remains transiently activated after glucose dissociates; if L-sorbose binds to the excited state it is more mobile than otherwise and consequently the apparent Ki(sorbose) of D-glucose is raised. Cooling prolongs the decay time of the activated state; hence the Ki(sorbose) of D-glucose rises as temperature is reduced.

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.

Concentration polarization of fluorescent dyes in rat descending colonic crypts: evidence of crypt fluid absorption.

1. Using confocal microscopy, the rate of fluid absorption into isolated perifused descending rat colonic crypt lumens is estimated from the concentration polarization and distribution of fluorescein sulphonate (FS) and fluorescein isothiocyanate-dextran (FITC-dextran; molecular weight, 10,000) within the crypt lumens and pericryptal fluid. 2. The probe dyes enter the crypt via the luminal opening, are concentrated in the lumen, then escape into the pericryptal space via the paracellular spaces spanning the crypt wall. 3. FITC-dextran is maximally accumulated at a luminal depth of 60 microns to 5 times the concentration at the crypt opening (p < 0.001) and penetrates 150-200 microns along the lumen. FS is maximally accumulated within crypt lumen close to the opening. At crypt luminal depths 10-60 microns from the opening FS is accumulated by a factor of 1.5-2.0 above that found in HgCl2-treated tissue (p < 0.001). 4. Dye enters the crypt lumen slowly from the basal side, but from this side does not accumulate above the bathing solution concentration. 5. HgCl2 (20 microM) or theophylline (10 mM) completely inhibit concentrative accumulation of FITC-dextran and FS within the crypts and pericryptal space (p < 0.001). 6. Computer simulation of the dye uptake indicates that the rate of water flow into the crypt luminal opening is 1 x 10(-3) cm s-1 which is equivalent to 15 microliters (cm mucosa)-2 h-1. Approximately 75% of the fluid entering the crypt is abosrbed across the proximal 50 microns of crypt wall as a consequence of the large osmotic pressure gradient between the pericryptal and crypt luminal solutions. A pericryptal diffusion barrier with lower permeability than that across the crypt wall is required to simulate dye accumulation in the pericryptal space. Differences between FITC-dextran and FS accumulation are explained by the lower diffusion coefficient within the crypt lumen, and lower crypt wall permeability of FITC-dextran.

Effects on fluid and Na+ flux of varying luminal hydraulic resistance in rat colon in vivo.

1. A new method of measuring fluid and ionic movements and the dehydrating power of the colon in vivo is described. A range of agarose gel cylinders, with calibrated hydraulic conductivities (Lp), were inserted into the lumen of the descending colon of anaesthetized rats. Fluxes of fluid, Na+ and K+ out of the gels were measured over a period of 60-110 min. 2. Fluid absorption by the colon from 2.5% agarose gels was not slower than from solution without gel. Fluid absorption was inhibited by 66% when the agarose concentration was raised to 10%. In contrast 2.5% agarose gels caused a 73% (P < 0.001) reduction in water flow from rat ileum. 3. Increasing gel concentration to 10% or above caused the absorbate from the gels to become hypertonic (P < 0.001). 4. The measured suction pressure applied by the colonic hypertonic absorbate to the gels increased from 44 +/- 2.3 cmH2O (n = 23) with 2.5% agarose gels to 6713 +/- 960 cmH2O (n = 13) with 15% (P < 0.001). 5. Deoxycholate (2 mM) produced a decrease in fluid and Na+ absorption and reduced the suction pressure and power exerted by the colon.

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.