Urea Permeability Research Articles

Osmotic stability of red cells in renal circulation requires rapid urea transport.

Urea transport by the human erythrocyte occurs via an asymmetric-facilitated diffusion system with high Michaelis constants and high maximal velocities; the equivalent permeability in the limit of zero urea concentration is approximately 10(-3) cm/s (J. Gen. Physiol. 81: 221-237, 239-253, 1983). A physiological role for this system is revealed by numerical integration of the appropriate equations that show that rapid urea transport is essential for red cell stability in passing through the renal medulla. The calculation compares two cells. Cell A transports urea with permeability characteristics of normal red cells; cell B has urea permeability similar to lipid bilayers. On entering the hypertonic medulla, both cells shrink, but only B swells on leaving the medulla. The osmotic stress for cell B is greater than for A. Cell B is close to hypertonic hemolysis in the medulla and to hypotonic hemolysis in the cortex. Cell B remains swollen for some time after its exit; the resulting decreased deformability presents a hazard if B reenters the microcirculation. Furthermore, cell B removes a significant fraction of the filtered load of urea and compromises the osmotic gradients in the medulla.

Protein synthesis inhibitors attenuate water flow in vasopressin-stimulated toad urinary bladder

Vasopressin stimulates the introduction of aggregated particles, which may represent pathways for water flow, into the luminal membrane of toad urinary bladder. It is not known whether water transport pathways are degraded on removal from membrane or whether they are recycled. We examined the effect of the protein synthesis inhibitors cycloheximide and puromycin using repeated 30-min cycles of vasopressin followed by washout of vasopressin, all in the presence of an osmotic gradient, a protocol that maximizes aggregate turnover. "High dose" cycloheximide (200 micrograms/ml) inhibited flow immediately. "Low dose" cycloheximide (1 microgram/ml) did not affect initial flow; however, flow was inhibited by the fourth restimulation. On further rechallenge, inhibition persisted but did not increase. In the absence of vasopressin, inhibition did not develop. Despite the inhibition of flow in vasopressin-treated tissues, the cAMP-dependent protein kinase ratio (-cAMP/+cAMP), an index of in vivo cAMP effect, was elevated in cycloheximide-treated tissues, suggesting modulation at a distal site in the stimulatory cascade. Cycloheximide inhibited flow when 10 microM forskolin or 0.2 mM 8-BrcAMP was substituted for vasopressin in the fourth period; however, MIX (4 mM)-stimulated flow was enhanced by 1 microgram/ml cycloheximide but inhibited by 200 micrograms/ml cycloheximide. [14C]urea permeability was not inhibited by cycloheximide. Puromycin (0.5 mM) also inhibited water flow by the fourth challenge with vasopressin. The data suggest that protein synthesis inhibitors attenuate flow at a site that is distal to cAMP-dependent protein kinase.(ABSTRACT TRUNCATED AT 250 WORDS)

Action of forskolin on non-electrolyte permeability across the urinary bladder of Bufo bufo as compared to that of various hormones

1. 1. Forskolin, an activator of adenyl-cyclase in a receptor-independent manner, mimics the ADH effect on the urea and thiourea permeabilities across the toad bladder. 2. 2. However, differently from ADH, forskolin increases the erythritol permeability across the tissue and this effect is not reproduced by two substances increasing the urea permeability (8-BrcAMP and isoprenaline). Most probably this effect of forskolin does not involve the cAMP generating system.

Vasopressin effects on urea and H2O transport in inner medullary collecting duct subsegments.

The inner medullary collecting duct (IMCD) is widely viewed as a single renal tubule segment with homogeneous properties. However, recent morphological and functional studies have raised the possibility that the initial and terminal parts of the IMCD may differ. To test this possibility further and to localize sites of action of arginine vasopressin (AVP) along the IMCD, we measured osmotic water permeability (Pf) and urea permeability (Purea) in isolated perfused rat IMCDs. In the initial third of the IMCD, 10 nM AVP increased Pf from 16 +/- 8 to 148 +/- 50 micron/s. The terminal two-thirds of the IMCD had a significantly higher basal Pf (70 +/- 12 micron/s), which increased to 186 +/- 25 micron/s with AVP. The initial IMCD had a relatively low basal Purea (3 +/- 1 X 10(-5) cm/s), which did not change with AVP. The terminal IMCD had a significantly higher basal Purea (17 +/- 4 X 10(-5) cm/s), which increased to a very high value (69 +/- 15 X 10(-5) cm/s) with AVP. The results support the premise that (from the point of view of vasopressin effects on water and urea transport) there are two functionally distinct parts of the inner medullary collecting duct: an initial part that resembles the cortical or outer medullary portions of the mammalian collecting duct and a terminal part that resembles the toad urinary bladder. The significance of these findings for the urinary concentrating mechanism is discussed.

Synthesis and Biomedical Properties of Poly-[(Ethylene-Vinylalcohol)-g-Acrylamide

Abstract This paper deals with the graft copolymerization of acrylamide (AM) onto ethylene-vinyl alcohol copolymer (EVAL) film initiated by cerium(IV) ion. It was found that both the chemical and diffusion factors had influences on the graft reaction. The reaction was initiated on the surface and then penetrated inward as the grafting percentage was increased. The permeability of urea through the grafted EVAL film was improved compared to that of the original film as was the blood compatibility.

Gossypol interferes selectively with water and urea permeability of toad bladder.

Gossypol, a polyphenol compound with antifertility properties in human male, has been found to interfere with the response of toad bladder to vasopressin in terms of permeability to water and to urea; on the other hand, ion movement is spared for several hours as judged from short-circuit current and conductance data. Action of the drug lies distal to the generation of cyclic AMP since bladder hydrosmotic response to the latter was affected to the same extent as when the hormone was applied.

Urease immobilized polyvinyl alcohol-g-butyl acrylate membrane for urea sensor

A copolymer with balanced hydrophilicity and hydrophobicity was synthesized by grafting butyl acrylate onto poly(vinyl alcohol) (PVA-g-BA). Films made from it had good urea permeability. After immobilization of the enzyme urease on the surface of it, the film was attached to the tip of an ammonia gas electrode to form an enzyme sensor. The sensor was able to detect urea in solution in the range of 6 to 600 mg/dL with a response time of about 5 min. It might be reused for over 100 times, and the reproducibility was very good. Therefore, it is very promising that this sensor may be commercialized for clinical use. The synthesis of PVA-g-BA was initiated with cerium ammonium nitrate, and the immobilization of urease was done by crosslinking with cyanuric chloride of glutaraldehyde. The former agent was proved to be more effective than the later. The amount of enzyme immobilized with the former were 26 and 13 times more than the one- and two-step methods with the latter, respectively.

Urea permeability of mammalian inner medullary collecting duct system and papillary surface epithelium.

To compare passive urea transport across the inner medullary collecting ducts (IMCDs) and the papillary surface epithelium (PSE) of the kidney, two determinants of passive transport were measured, namely permeability coefficient and surface area. Urea permeability was measured in isolated perfused IMCDs dissected from carefully localized sites along the inner medullas of rats and rabbits. Mean permeability coefficients (X 10(-5) cm/s) in rat IMCDs were: outer third of inner medulla (IMCD1), 1.6 +/- 0.5; middle third (IMCD2), 46.6 +/- 10.5; and inner third (IMCD3), 39.1 +/- 3.6. Mean permeability coefficients in rabbit IMCDs were: IMCD1, 1.2 +/- 0.1; IMCD2, 11.6 +/- 2.8; and IMCD3, 13.1 +/- 1.8. The rabbit PSE was dissected free from the underlying renal inner medulla and was mounted in a specially designed chamber to measure its permeability to urea. The mean value was 1 X 10(-5) cm/s both in the absence and presence of vasopressin (10 nM). Morphometry of renal papillary cross sections revealed that the total surface area of IMCDs exceeds the total area of the PSE by 10-fold in the rat and threefold in the rabbit. We conclude: the IMCD displays axial heterogeneity with respect to urea permeability, with a high permeability only in its distal two-thirds; and because the urea permeability and surface area of the PSE are relatively small, passive transport across it is unlikely to be a major source of urea to the inner medullary interstitium.

Action of forskolin on non-electrolyte permeability across the frog skin as compared to that of vasopressin and isoprenaline

Forskolin, a natural diterpene activating the adenyl cyclase in a receptor-independent manner, increases symmetrically both transepithelial fluxes of urea and erithrytol through the frog skin. The effect is dose-dependent, being 5 X 10(-6) M the dose necessary to obtain the maximal action. Forskolin-induced permeabilization is inversely proportional to the molecular weight of water soluble molecules (urea greater than erythritol greater than mannitol); also the permeability of a mainly lipid soluble molecule, i.e. antipyrine, is slightly increased by the diterpene. The permeability pattern is more similar to that induced by isoprenaline as compared to that elicited by vasopressin. Differently from what occurs in other tissues, small doses of forskolin (10(-8) M) are unable to potentiate the actions of vasopressin and isoprenaline on urea permeability across the frog skin. Moreover, the maximal action of forskolin is not additive with the maximal ones of isoprenaline and vasopressin.

Effects of glutaraldehyde fixation on renal tubular function. I. Preservation of vasopressin-stimulated water and urea pathways in rat papillary collecting duct.

Using the in vitro microperfusion technique on isolated rat papillary collecting duct (PCD), we examined whether the glutaraldehyde-fixation method can be also applied to the mammalian collecting duct for preservation of the vasopressin-stimulated water and urea transport. Arginine vasopressin (AVP) at 10(-9) mol/l increased diffusional water permeability (Pdw) from 101.9 +/- 10.76 to 283.3 +/- 16.67 X 10(-7) cm2 s-1 (n = 8, P less than 0.01) and urea permeability (Purea) from 30.3 +/- 2.24 to 83.5 +/- 7.80 X 10(-7) cm2 s-1 (n = 8, P less than 0.01). Both parameters remained elevated after fixation with 0.1 mol/l glutaraldehyde even in the absence of AVP, with the values being 265.0 +/- 14.47 and 74.5 +/- 7.15 X 10(-7) cm2 s-1, respectively. Glutaraldehyde fixation did not affect the basal levels of Pdw or Purea. Phloretin at 2.5 X 10(-4) mol/l decreased glutaraldehyde-fixed AVP-stimulated Purea from 79.0 +/- 7.96 to 29.7 +/- 3.66 X 10(-7) cm2 s-1 (n = 4, P less than 0.01) and from 73.2 +/- 7.05 to 38.7 +/- 3.53 X 10(-7) cm2 s-1 (n = 4, P less than 0.01) when the drug was added to the lumen or to the bath, respectively. Phloretin also decreased glutaraldehyde-fixed non-stimulated Purea by 25-40%. However, this drug did not affect glutaraldehyde-fixed Pdw. These findings indicate that the glutaraldehyde fixation method can be applied to mammalian collecting tubules for studying vasopressin stimulated Pdw and Purea. Purea fixed by glutaraldehyde is functionally flexible and may be distinct from the water pathway.

Amylase activity, protein and urea in saliva of the red kangaroo ( Macropus rufus)

Parotid and mandibular salivas, produced at flow rates of 0.055 ± 0.0038 to 4.45 ± 0.101 and 0.052 ± 0.0059 to 4.30 ± 0.072 ml min −1 respectively by intracarotid infusion of acetylcholine or methacholine, were Anatysed. During acetylcholine stimulation, the protein concentration ranged from 86.4 ± 10.17 to 235.0 ± 23.5 mg l −1 in parotid and from 102.9 ± 11.54 to 379.6 ± 38.01 mg l −1 in mandibular saliva. The minimum protein concentrations in parotid and mandibular salivas occurred at 0.5 and 1.0 ml min −1 respectively with the concentration rising progressively at lower and higher flow rates. Both glands maintained constant levels of protein during 90 min of sustained stimulation at constant flow rates of 0.5 and 2.0 ml min −1. The optimum pH for salivary amylase activity lay between 7.4 and 7.9 (mean 7.5–7.6). The amylase activity of parotid saliva ranged between 32.2 ± 3.81 and 94.5 ± 24.05 μkat l −1 and the activity/protein ratio was similar at all flow rates (mean, 0.39 ± 0.049 μkat amylase/mg protein). Amylase levels in arterial plasma were constant within experiments but varied from 8.0 ± 0.07 to 26.4 ± 0.24 μkat l −1 between experiments, and were always less than corresponding parotid values. Mandibular amylase activities were low, highly variable or often absent. Methacholine-evoked parotid saliva had lower protein concentrations but similar amylase/protein ratios to acetylcholine-stimulated saliva. Urea concentrations in arterial plasma were constant within experiments but varied from 6.71 ± 0.092 to 14.2 ± 0.14 mmol 1 −1 between experiments. In both salivas, urea was negatively correlated with flow rate, its parotid saliva/plasma ratio ranging from 1.04 ± 0.009 to 0.92 ± 0.015 and its mandibular saliva/plasma ratio ranging between 0.84 ± 0.055 and 0.08 ± 0.007. The low protein concentrations of both salivas are thus adaptations to their specialized functions, parotid saliva being a buffer in the forestomach as in ruminants, and mandibular saliva being used as an external coolant. The low protein levels also allow the concentration to remain constant in both salivas over long periods of parasympathetic stimulation, in contrast to other salivary glands. The parotid gland synthesizes amylase, whereas the mandibular gland may not. Parotid amylase levels are lower than in many eutherian species but the special conditions in the anterior digestive tract may make them significant. The parotid has higher urea permeability and lower fluid re-absorption than the mandibular gland, and is the main route for salivary transfer of urea into the digestive tract.

Transport of aniline, chloroaniline, nitroaniline, and urea through perfluorosulfonated ionomer membranes

The permeabilities of aniline, p-chloroaniline, p-nitroaniline, and urea were measured in a perfluorosulfonated ionomer membrane. Permeabilities for these compounds were of the order of 2 x 10 -10 m 2/sec when the external solutions were pH 7. Permeabilities for these compounds were of the order of 6 x 10 -9 m 2/sec when the sink solution was pH 1 and the source solution was pH 7. These results indicate that the rate at which these amines exit the isonomer is the rate-limiting step in the transport process.

Polycations reduce vasopressin-induced water flow by endocytic removal of water channels.

Several polycations added to the luminal solution were found to inhibit the vasopressin (ADH)-induced water flow in toad urinary bladder but not the ADH-induced increase in sodium transport or in urea permeability. Ultrastructural studies were conducted to evaluate the uptake of cationized ferritin. It was found that endocytosis of cationized ferritin by luminal cells was strikingly enhanced on exposure to ADH; this increased endocytosis was concomitant with inhibition of transepithelial ADH-induced water flow. Various maneuvers preventing endocytosis were also found to counteract the polycation-induced inhibition of the ADH effect. It is suggested that polycations are endocytosed in vesicles whose walls contain the water channels but not the urea or sodium channels.

Concentration- and time-dependence of amphotericin-B induced permeability changes across ergosterol-containing liposomes

The effect of amphotericin B on the permeability properties of liposomes prepared by reverse-phase evaporation was examined by using an osmotic method. This study has revealed that the magnitude and type of the alterations in permeability induced by amphotericin B in liposomes made of egg phosphatidylcholine and ergosterol depend not only on the amphotericin B concentration in the external aqueous solution but also on the time elapsed after mixing. Thus, low amphotericin B concentrations (from 0.2 to 1.2 μM) led to, (1) an small increment of the total extent of shrinkage of liposomes suspended in non-electrolytes such as urea or salts like KNO 3, (2) an enhancement of urea and salt permeabilities at the same time scale at which volume changes were measured (ms to s), (3) a maximal blocking by tetraethylammonium of amphotericin B-induced urea permeability and (4) an enhancement of glucose permeability but only after liposomes were incubated with amphotericin B for some minutes before mixing. The high amphotericin B concentration regime (beyond 1.2 μM) led to, (1) a decrease of the total extent of shrinkage of liposomes immediately after rapid mixing of liposomes with urea solutions containing amphotericin B and (2) a 50% reduction of the tetraethylammonium blocking of amphotericin B-induced urea permeability. These results are explained by assuming that amphotericin B may form in ergosterol-containing liposomes two types of active channel differing in internal diameter.

Disk method for measuring effects of neurohypophysial hormones on urea permeability of toad bladder.

This study describes a precise and sensitive method for the bioassay of 8-arginine vasotocin (AVT). The technique is based on the observation that AVT increases the permeability to urea of the isolated toad bladder and that this effect can be measured with minute quantities of AVT by suspending bladders in humid air and applying hormone on small paper disks. The hormone diffuses from the disk to the underlying epithelium where it increases permeability to urea. The bladder is filled with Ringer's fluid containing [14C]urea and the label diffuses across the bladder wall into the disk at the serosal surface. Disks are removed at timed intervals for scintillation counting. Disks containing 0.25 pg AVT accumulated 232 +/- 8 cpm of [14C]urea in 30 min, whereas control disks without hormone at a distance of 3-4 mm on the same bladder wall accumulated only 12 +/- 2 cpm. The minimal sensitivity during the summer was 0.1 pg AVT. Average values (+/- SE) for induction of a half-maximal urea permeability response in toads from all seasons were 1.4 +/- 0.3 X 10(-10) M for AVT, 7.4 +/- 1.5 X 10(-9) M for 8-arginine vasopressin, and 2.9 +/- 0.6 X 10(-8) M for oxytocin. Plasma from a hydrated toad (255 mosm/kg H2O) showed AVT-like activity of 1.53 X 10(-11) M and from a dehydrated toad (342 mosm/kg H2O) 4.86 X 10(-11) M. Because this bioassay is specific and as sensitive as the radioimmunoassay, it may serve as a useful tool for studying the physiology of vasotocin.

Vasopressin and the Concentrating Mechanism

The role of vasopressin in the kidney has classically been considered to result from its ability to increase water permeability in the collecting duct. Recent data, however, suggest that the hormone may also promote urinary concentration by increasing interstitial tonicity. The mechanisms whereby vasopressin could enhance interstitial tonicity include increasing urea permeability in the inner medullary collecting tubule, stimulation of solute reabsorption in the thick ascending limb of the loop of Henle, increasing the glomerular filtration rate of juxtamedullary nephrons, and decreasing vasa recta blood flow. We review experiments directed at assessing the role of vasopressin in these four processes. The multitude of effects of vasopressin appears to be well integrated and contributes to the tightly regulated urinary concentration mechanisms.

Percutaneous absorption of urea

Synopsis The effect of several variables on the in vitro permeation of urea through hairless mouse skin has been studied in order to determine the causes of an increasing permeability phenomenon found in studies with a range of hydrophilic compounds. The permeation of urea increased for a period of approximately 100 h after which a steady state permeation pattern was observed for approximately 25 h. Urea did not effect its own permeation in concentrations between 0.01 M and 1.67 M, and the same pattern of increasing permeation was followed in the presence of (N-morpholine)propanesulphonic acid and tris(hydroxyme)amino-methane buffers, as in the presence of normal saline. Urea did not affect the permeation of tritiated water. Methanol and water exhibited the same pattern of increasing permeation as urea. The continuously increasing permeation rate of urea up to 100 h is believed to be due to penetration and extensive association of water with the components of the stratum corneum, altering the ultra-structure of the stratum corneum and leading to the formation of large and extensive hydrophilic diffusion channels which do not exist in fresh, untreated skin. These presumed channels open the stratum corneum to facile permeation of highly polar substances such as urea. The physical events leading up to the ultra structural changes within the tissue at the microscopic level remain obscure and are the subject of ongoing research. L'absorption percutanée de l'urée.

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°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 μM 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.

Urea efflux from the Squalus acanthias pup: the effect of stress.

ABSTRACT The extremely high concentration of urea found in elasmobranch blood (approx. 400 mmol 1–1) is secondary to nearly complete reabsorption (90–95%) of this solute in the renal tubules (Forster, 1967), and a branchial urea permeability which is approximately 1% that of the toad bladder (<10 × 10–8cm s–1, Payan, Goldstein & Forster, 1973). Evans, Oikari, Kormanik & Mansberger (1982) have recently shown that the premature spiny dogfish (Squalus acanthias) pup is capable of osmoregulation in sea water, despite an initial reduction in plasma urea concentration (13% in 3 days). This fall could have been secondary to a reduced synthetic rate, or caused by an increased efflux across surfaces such as the gills, renal and/or rectal gland epithelia, or yolk sac. The present study was therefore undertaken to measure the rate of urea efflux from intact spiny dogfish pups, and to determine the effect of stress on that efflux. Stress was induced by the experimentally common procedure of handling, MS-222 anaesthesia, and weighing.

Hydrosmotic salt effect in toad skin: Urea permeability and glutaraldehyde fixation of water channels

The "hydrosmotic salt effect" (HSE), the reversible dependence of skin osmotic water permeability upon the ionic concentration of the outer bathing solution, is known to induce the appearance of sucrose-impermeable pathways in the apical membrane of the outermost epithelial cell layer. Diffusional 14C-urea permeability, measured in the Jv = O condition to prevent solvent drag effects, indicates that the newly formed pathways induced by HSE are narrower than the size of the urea molecule, being therefore highly selective for water molecules. After mild glutaraldehyde (2% solution) fixation of the apical membrane structures, the water channels induced by the HSE are no longer affected by the ionic strength of the outer solution. This indicates that the channel-forming membrane protein can be fixed in different configurations with the water channels in the open or closed states.