The effect of tanning agents on the permeability of the toad bladder to water and solutes

Summary Previous studies with phloretin have shown that the movement of urea and other solutes across the toad bladder can be inhitited with no effect on osmotic water flow, active sodium transport, or the movement of ethanol and ethylene glycol. These findings have suggested that a vasopressin-sensitive carrier is involved in the transport of solutes such as urea across the luminal membrane of the epithelial cell. The present paper describes the effect of two agents other than phloretin: tannic acid and chromate, on water and solute movement across the bladder. The pattern of action of these two agents resembles that of phloretin, and supports our earlier findings of the independence of solute and water movement. The effect of chromate on urea movement is seen only in the presence of vasopressin, and only if chromate is added prior to vasopressin. Chromate also proves to be an irreversible inhibitor of urea movement. The implications of these findings are discussed. In view of the known interactions of both agents with proteins, it is suggested that carrier-mediated transport of urea proceeds across a protein component of the membrane.Presented in part at the 57th annual meeting, Federation of American Societies for Experimental Biology, Atlantic City, April 1973.     

Serosal and mucosal facilitated transport of urea in urinary bladder of of Bufo bufo: evidence for an alleged water uptake

In Bufo bufo urinary bladder an urea facilitated transport has been localised on the luminal membrane. The transport fulfils the criteria for such a mechanism, i.e. is saturable and is inhibited by phloretin, a specific inhibitor for urea transport. Similarly to that of Bufo marinus and Rana esculenta the luminal membrane of Bufo bufo urinary bladder shows an ADH stimulated facilitated transport. Experiments wtih Amphotericin B, serosal phloretin (with and without ADH), have demonstrated the presence of a facilitated urea transport localised on basolateral membrane. Urea uptake on the isolated epithelial cells of Bufo bufo urinary bladder shows a characteristic feature, different from molecules passively transported such as glycerol yet inhibited by phloretin. Allegedly with urea, water flows in to the cells by a dragging or osmotic effect.     

Effects of sulfhydryl and other reagents on the diffusional permeability of dog erythrocytes to small solutes

The effects of p-chloromercuriphenylsulfonic acid (PCMBS), 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), phloretin and thiourea on the diffusional permeability of dog erythrocytes to tritiated water and to small 14C-labeled lipophilic and hydrophilic solutes were measured at 37 degrees C by means of the linear diffusion technique. Permeability to 3HHO was significantly decreased by PCMBS but was not affected by the other reagents. The permeability to the small hydrophilic solutes acetamide and urea was decreased by phloretin and thiourea but only the permeability to acetamide was reduced to a statistically significant extent by PCMBS. The permeability to the lipophilic solutes methanol, ethanol and antipyrine was not affected by any of these agents. We interpret these results as an indication that the small lipophilic solutes probably move through lipid areas, that the small hydrophilic solutes probably move through protein associated areas in the erythrocyte membrane and that pathways for the small hydrophilic solutes are distinct from those for water. While the pathways for water may be associated with membrane protein they do not appear to be associated specifically with band 3 protein as has been suggested for human erythrocytes. Diffusional water movement through the dog erythrocyte occurs by two distinct pathways.     

Membrane proteins and urea and acetamide transport in the human erythrocyte

The Journal of Membrane Biology - Previous studies have shown that urea and acetamide traverse the erythrocyte membrane by way of facilitated diffusion. The nature of this selective pathway is...     

Amide transport channels across toad urinary bladder

Summary Urea and other small amides cross the toad urinary bladder by a vasopressinsensitive pathway which is independent of somotic water flow. Amide transport has characteristics of facilitated transport: saturation, mutual inhibition between amides, and selective depression by agents such as phloretin. The present studies were designed to distinguish among several types of transport including (1) movement thought a fixed selective membrane channel and (2) movement via a mobile carrier. The former wold be characterized by co-transport (acceleration of labele amide flow in the direction of net flow in the opposite direction). Mucosal to serosal (MS) and serosal to mucosal (SM) permeabilities of labeled amides were determined in paired bladers. Unlabeled methylurea, a particularly potent inhibitor of amide movement, was added to either the M or S bath, while osmotic water flow was eliminated by addition of ethylene glycol to the opposite bat. Co-transport of labeled methylurea and, to a lesser degree, acetamide and urea with unlabeled methylurea was observed. Co-transport of the nonamides ethylene glycol and ethanol could not be demonstrated. Methylurea did not alter water permeability or transmembrane electrical resistance. The demonstration of co-transport is consistent with the presence of ADH-sensitive amide-selective channcels rather than a mobile carrier.     

Urea and water permeability in dogfish (Squalus acanthias) gills

We used a perfused gill preparation from dogfish to investigate the origin of low branchial permeability to urea. Urea permeability (14C-urea) was measured simultaneously with diffusional water permeability (3H2O). Permeability coefficients for urea and ammonia in the perfused preparation were almost identical to in vivo values. The permeability coefficient of urea was 0.032 x 10(-6) cm/sec and of 3H2O 6.55 x 10(-6) cm/sec. Adrenalin (1 x 10(-6) M) increased water and ammonia effluxes by a factor of 1.5 and urea efflux by a factor of 3.1. Urea efflux was almost independent of the urea concentration in the perfusion medium. The urea analogue thiourea in the perfusate had no effect on urea efflux, whereas the non-competitive inhibitor of urea transport, phloretin, increased efflux markedly. The basolateral membrane is approximately 14 times more permeable to urea than the apical membrane. We conclude that the dogfish apical membrane is extremely tight to urea, but the low apparent branchial permeability may also relate to the presence of an active urea transporter on the basolateral membrane that returns urea to the blood and hence reduces the apical urea gradient.     

Modulation of water and urea transport in human red cells: Effects of pH and phloretin

Summary It has previously been shown by Macey and Farmer (Biochim. Biophys. Acta 211:104–106, 1970) that phloretin inhibits urea transport across the human red cell membrane yet has no effect on water transport. Jennings and Solomon (J. Gen. Physiol. 67:381–397, 1976) have shown that there are separate lipid and protein binding sites for phloretin on the red cell membrane. We have now found that urea transport is inhibited by phloretin binding to the lipids with aK ₁ of 25±8 m in reason-able agreement with theK _D of 54±5 m for lipid binding. These experiments show that lipid/protein interactions can alter the conformational state of the urea transport protein. Phloretin binding to the protein site also modulates red cell urea transport, but the modulation is opposed by the specific stilbene anion transport inhibitor, DIDS (4,4-diisothiocyano-2,2-stilbene disulfonate), suggesting a linkage between the urea transport protein and band 3. Neither the lipid nor the protein phloretin binding site has any significant effect on water transport. Water transport is, however, inhibited by up to 30% in a pH-dependent manner by DIDS binding, which suggests that the DIDS/band 3 complex can modulate water transport.     

Inhibition of glucose transport into brain by phlorizin, phloretin and glucose analogues.

An indicator dilution technique with 22Na+ as the intravascular marker was used to measure unidirectional transport of D-[6-3H]glucose from blood into the isolated, perfused dog brain. 18 compounds which are structurally related to glucose were tested for their ability to inhibit glucose transport. The data suggest that no single hydroxyl group is absolutely required for glucose transport, but rather that glucose binding to the carrier probably occurs through hydrogen bonding at several sites (hydroxyls on carbons 1, 3, 4 and 6). In addition, alpha-D-glucose has higher affinity for the carrier than does beta-D-glucose. A separate series of experiments demonstrated that phlorizin and phloretin are competitive inhibitors of glucose transport into brain; however, phloretin is partially competitive and inhibits at lower concentrations than does phlorizin. Inhibition by phlorizin and phloretin is mutually competitive, indicating that these compounds compete for binding to the glucose carrier. Comparison with the results reported in the literature for similar studies using the human erythrocyte demonstrates a fundamental similarity between glucose transport systems in the blood-brain barrier and erythrocyte.     

Target analysis studies of red cell water and urea transport

Radiation inactivation was used to determine the nature and molecular weight of water and urea transporters in the human red cell. Red cells were frozen to -50 degrees C in a cryoprotectant solution, irradiated with 1.5 MeV electrons, thawed, washed and assayed for osmotic water and urea permeability by stopped-flow light scattering. The freezing and thawing process did not affect the rates of water or urea transport or the inhibitory potency of p-chloromercuribenzenesulfonate (pCMBS) on water transport and of phloretin on urea transport. Red cell urea transport inactivated with radiation (0-4 Mrad) with a single target size of 469 +/- 36 kDa. 40 microM phloretin inhibited urea flux by approx. 50% at each radiation dose, indicating that urea transporters surviving radiation were inhibitable. Water transport did not inactivate with radiation; however, the inhibitory potency of 2.5 mM pCMBS decreased from 86 +/- 1% to 4 +/- 9% over a 0-2 Mrad dose range. These studies suggest that red cell water transport either required one or more low-molecular-weight proteins, or is lipid-mediated, and that the pCMBS-binding site which regulates water flow inactivates with radiation. These results also suggest that red cell urea transport is mediated by a specific, high-molecular-weight protein. These results do not support the hypothesis that a band 3 dimer (190 kDa) mediates red cell osmotic water and urea transport.     

Evidence for facilitated diffusion of urea across the gill basolateral membrane of the rainbow trout (Oncorhynchus mykiss)

Recent in vivo evidence suggests that the mechanism of branchial urea excretion in the ammoniotelic rainbow trout (Oncorhynchus mykiss) is carrier-mediated. Further characterization of this proposed mechanism was achieved by using an in vitro isolated basolateral membrane vesicle (BLMV) preparation in which isolated gill membranes were used to determine a variety of physiological properties of the transporter. BLMV demonstrated two components of urea uptake, a linear component at concentrations up to 17.5 mmol x l(-1) and a saturable component (K(0.5)=0.35+/-0.01 mmol x l(-1); V(max)=0.14+/-0.02 micromol mg protein(-1) h(-1)) with a Hill constant of 1.35+/-0.18 at low, physiologically relevant urea concentrations (<2 mmol x l(-1)). Saturable uptake of urea at 1 mmol x l(-1) by BLMV was reduced by 88.5% when incubated with 0.25 mmol x l(-1) phloretin, a potent blocker of UT-type facilitated diffusion urea transport mechanisms. BLMV also demonstrated differential handling of urea versus urea analogues at 1 mmol x l(-1) concentrations and total analogue/total urea uptake ratios were 32% for acetamide and 84% for thiourea. Saturable urea uptake at 1 mmol x l(-1) was significantly reduced by almost 100% in the presence of 5 mmol x l(-1) thiourea but was not affected by 5 mmol x l(-1) acetamide or 5 mmol x l(-1) N-methylurea. Lastly, total urea uptake at 1 mmol x l(-1) by BLMV was sensitive to temperatures above and below the temperature of acclimation with a Q(10)>2 suggesting a protein carrier-mediated process. Combined, this evidence indicates that a facilitated diffusion urea transport mechanism is likely present in the basolateral membrane of the rainbow trout gill.     

Comparative transport efficiencies of urea analogues through urea transporter UT-B

Expression of urea transporter UT-B confers high urea permeability to mammalian erythrocytes. Erythrocyte membranes also permeate various urea analogues, suggesting common transport pathways for urea and structurally similar solutes. In this study, we examined UT-B-facilitated passage of urea analogues and other neutral small solutes by comparing transport properties of wildtype to UT-B-deficient mouse erythrocytes. Stopped-flow light-scattering measurements indicated high UT-B permeability to urea and chemical analogues formamide, acetamide, methylurea, methylformamide, ammonium carbamate, and acrylamide, each with P(s)>5.0 x 10(-6) cm/s at 10 degrees C. UT-B genetic knockout and phloretin treatment of wildtype erythrocytes similarly reduced urea analogue permeabilities. Strong temperature dependencies of formamide, acetamide, acrylamide and butyramide transport across UT-B-null membranes (E(a)>10 kcal/mol) suggested efficient diffusion of these amides across lipid bilayers. Urea analogues dimethylurea, acryalmide, methylurea, thiourea and methylformamide inhibited UT-B-mediated urea transport by >60% in the absence of transmembrane analogue gradients, supporting a pore-blocking mechanism of UT-B inhibition. Differential transport efficiencies of urea and its analogues through UT-B provide insight into chemical interactions between neutral solutes and the UT-B pore.     

Interaction of phloretin with the anion transport protein of the red blood cell membrane

Phloretin is an inhibitor of anion exchange and glucose and urea transport in human red cells. Equilibrium binding and kinetic studies indicate that phloretin binds to band 3, a major integral protein of the red cell membrane. Equilibrium phloretin binding has been found to be competitive with the binding of the anion transport inhibitor, 4,4′-dibenzamido-2,2′-disulfonic stilbene (DBDS), which binds specifically to band 3. The apparent binding (dissociation) constant of phloretin to red cell ghost band 3 in 28.5 mM citrate buffer, pH 7.4, 25°C, determined from equilibrium binding competition, is $\text{1.8 ± 0.1}\text{μM}$. Stopped-flow kinetic studies show that phloretin decreases the rate of DBDS binding to band 3 in a purely competitive manner, with an apparent phloretin inhibition constant of $\text{1.6 ± 0.4}\text{μM}$. The pH dependence of equilibrium binding studies show that it is the charged, anionic form of phloretin that competes with DBDS binding, with an apparent phloretin inhibition constant of 1.4 μM. The phloretin binding and inhibition constants determined by equilibrium binding, kinetic and pH studies are all similar to the inhibition constant of phloretin for anion exchange. These studies suggest that phloretin inhibits anion exchange in red cells by a specific interaction between phloretin and band 3.     

Effect of phloretin on monosaccharide transport in erythrocyte ghosts

Summary ³H-labelled phloretin was shown to be bound reversibly by human erythrocyte and ghost membranes but not to penetrate across them in either direction. Kinetic parameters ofd-xylose andd-galactose transport in intact cells and in ghosts, as well as the inhibition by phloretin of these transports were found to be in fair agreement. By enclosing phloretin in ghosts, its inhibition of monosaccharide transport was found to be symmetrical and thus an equivalence of the outer and the inner membrane sides of the human erythrocyte was demonstrated.     

Inhibition of glucose transport into brain by phlorizin, phloretin and glucose analogues

An indicator dilution technique with ²²Na⁺ as the intravascular marker was used to measure unidirectional transport of d-[6-³H]glucose from blood into the isolated, perfused dog brain. 18 compounds which are structurally related to glucose were tested for their ability to inhibit glucose transport. The data suggest that no single hydroxyl group is absolutely required for glucose transport, but rather that glucose binding to the carrier probably occurs through hydrogen bonding at several sites (hydroxyls on carbons 1, 3, 4 and 6). In addition, α-d-glucose has higher affinity for the carrier than does β-d-glucose.A separate series of experiments demonstrated that phlorizin and phloretin are competitive inhibitors of glucose transport into brain; however, phloretin is partially competitive and inhibits at lower concentrations than does phlorizin. Inhibition by phlorizin and phloretin is mutually competitive, indicating that these compounds compete for binding to the glucose carrier. Comparison with the results reported in the literature for similar studies using the human erythrocyte demonstrates a fundamental similarity between glucose transport systems in the blood-brain barrier and erythrocyte.     

Differential handling of urea and its analogues suggests carrier-mediated urea excretion in freshwater rainbow trout

The possible presence of urea transport mechanisms in the gill and kidney of the freshwater rainbow trout (Oncorhynchus mykiss) was investigated in vivo by comparing the branchial and renal handling of analogues acetamide and thiourea with the handling of urea. Trout were fitted with indwelling dorsal aortic catheters and urinary catheters and injected with an isosmotic dose of [(14)C]-labeled urea analogue (acetamide or thiourea) calculated to bring plasma analogue concentrations close to plasma urea concentrations. Urea and analogue concentrations were significantly greater in the urine than in the plasma. Branchial clearance rate of acetamide was only 48% of urea clearance, whereas the clearance of thiourea was only 22%, a pattern that was also observed in branchial uptake of these substances and was similar to our previous observations in toadfish and midshipmen. The renal secretion clearance rates of urea and acetamide were similar, and on average, both substances were secreted on a net basis, although reabsorption did occur in some cases. In contrast, thiourea was neither reabsorbed nor secreted by the kidney tubule. The secretion clearance rates of both acetamide and urea were well correlated with the secretion clearance rates of Na(+), Cl(-), and water, whereas there was no relationship between thiourea and these substances. The pattern of acetamide, thiourea, and urea handling by the gill of the trout is similar to that found in the gills of the midshipman and the gulf toadfish and strongly suggests the presence of a UT-type facilitated diffusion urea transport mechanism. The pattern of differential handling in the kidney is unlike that in the gill and also unlike that in the kidney of the midshipman and the gulf toadfish, suggesting a different mechanism. In addition, renal urea secretion occurs against a concentration gradient, suggesting the involvement of an active transport mechanism.     

Effects of membrane steroid modification on human erythrocyte glucose transport

The partial removal of cholesterol from the human erythrocyte membrane, by contact with lecithin sols, had mixed effects on the transport of d-glucose. When about 8% of the cholesterol was removed, the rate of d-glucose transfer was increased, but as cholesterol was progressively further removed, the transport was inhibited. Replacement of the depleted cholesterol by 3-ketosteroids did not restore the transport activity; but with substitution of steroids containing only a 3β-hydroxy substituent, the rate of glucose transport returned to normal. In some instances, as little as a 2% replacement of the removed cholesterol by 3β-hydroxy steroids was sufficient for full restoration of d-glucose transport. Cholesterol substitution by steroids with a more planar nucleus and a more bulky side chain than cholesterol also aided in the restoration of glucose transfer. The partial removal of cholesterol had no effect on the apparent K_m for d-glucose, but excessive membrane cholesterol led to a 4-fold decrease in d-glucose affinity. The extent of transport inhibition by a fixed phloretin treatment was independent of membrane steroid content.     

Effect of phloretin on the carrier-mediated electrically silent ion fluxes through the bilayer lipid membrane: measurements of pH shifts near the membrane by pH microelectrode.

The effect of phloretin on the carrier-mediated electrically silent ion fluxes through the bilayer lipid membrane (BLM) was studied. The measurements were carried out according to our conventional technique, i.e. electrical potential recording in the presence of a protonophore, and by a new method--direct measurements of pH shifts in the unstirred layers of the BLM by pH microelectrode. Both techniques gave similar results. It was shown that the addition of phloretin increased the rate of cation/H+ exchange induced by nigericin and decreased the rate of anion/OH(-)-exchange induced by tributyltin. The effect of phloretin was higher in the presence of cholesterol in the BLM. Cholesterol decreased the nigericin- and tributyltin-induced fluxes under our experimental conditions. The application of an external voltage to the membrane had no effect on the ion fluxes thereby showing that these fluxes were electroneutral. The most probable explanation of these results bases on the effect of the membrane dipole potential on the electroneutral fluxes of ions. The possible mechanism of the dipole potential effect on the carrier-mediated electrically silent ion fluxes was discussed in terms of two competing hypotheses--the translocation through the membrane or the reactions at the membrane surface being the rate-limiting steps of the whole transport process.     

Inhibition of the process of sugar transport stimulation in the frog sartorius muscles by amines and amides

In the experiments on isolated frog sartorius muscles, amines and amides were found to inhibit the process of stimulation of D-xylose transport induced by insulin, 2,4-dinitrophenol or potassium contracture. The inhibitory action was produced by urea, acetamide, guanidine, NH4Cl, mono-, di- and trimethyl- or ethylamines, some diamines (all the substances being, applied in the concentration range equal to 100 mM). The similar effect was obtained when cystamine (20 mM), tryptamine, 5-methoxytryptamine (2 mM) and adenine, adenosine, guanosine (1-10 mM) were used. There was no inhibitory effect of acetone, glycerol, tetraethylammonium, propilamine, butylamine, aminoacids, spermine, spermidine, ATP, AMP or cAMP. It has been suggested that the inhibitory substances may interact by producing hydrogen bonds from NH-groups with the neutrally or negatively charged groups at the external surface of the muscle membrane in the region with a slow hydrophobicity. As a result, no structural changes required for activation of the sugar transport system occur in the membrane.     

Various effects of prostaglandin E2 on reabsorption of water and urea in the amphibia osmosis-regulating epithelium

Principal similarities between molecular pathways providing the enhancement of water and urea reabsorption under the action of argininvasotocin (AVT) in amphibian urinary bladder suggest that prostaglandin E2 (PGE2) could be a negative regulator of urea transport. To analyse this hypothesis, the role of PGE2 in regulation of urea transport was studied in isolated frog (Rana temporaria L.) urinary bladder. The urea permeability (Pu) was determined from the rate of efflux of (14) Curea from mucosal to serosal solution in isoosmotic conditions. The water permeability was measured in separate experiments in presence of an osmotic gradient. In contrast to water permeability, we were unable to demonstrate any inhibitory effect of 10-1000 nM PGE2 on AVT-stimulated urea transport using a variety of protocols. It was found that basolateral PGE2 exposure (10 nM-1 microM) caused an increase in Pu with no effect on osmotic water flow. The PGE2 effect was markedly inhibited by phloretin, a specific inhibitor of urea transporter. Sulprostone, an EP1/EP3 prostaglandin E2 receptor agonist, had no effect on Pu suggesting the contribution of EP2/EP4 receptor subtypes. In presence of osmotic water flow, the AVT-induced urea transport was significantly higher. This water flow-dependent urea permeability was inhibited by PGE2 although the inhibitory effect was less pronounced in comparison to the action of PGE2 on osmotic water flow. On the basis of these results we can make a conclusion that PGE2 has different role in regulation of water and urea transport in the frog urinary bladder. PGE2 could be considered as a stimulator of urea transport and an inhibitor of osmotic water flow activated by the AVT. The ability of PGE2 to regulate various types of cAMP-dependent transport by different mechanisms seems to be based on the presence of multiple basolateral PGE2 receptor subtypes in amphibian osmosis-regulatory epithelium.