Membrane pathways for water and solutes in the toad bladder: I. Independent activation of water and urea transport

Summary Vasopressin activates a number of transport systems in the toad bladder, including the systems for water, urea, sodium, and other small solutes. Evidence from experiments with selective inhibitors indicates that these transport systems are to a large extent functionally independent. In the present study, we show that the transport systems can be separately activated. Low concentrations of vasopressin (1 mU/ml) activate urea transport with virtually no effect on water transport. This selective effect is due in part to the relatively greater inhibitory action of endogenous prostaglandins on water transport. Low concentrations of 8-bromoadenosine cyclic AMP, on the other hand, activate water, but not urea transport. In additional experiments, we found that varying the ratio of exogenous cyclic AMP to theophylline activated water or urea transport selectively. These studies support the concept of independently controlled systems for water and solute transport, and provide a basis for the study of individual luminal membrane pathways for water and solutes in the accompanying paper.     

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.     

The function of water channels in Chara: The temperature dependence of water and solute flows provides evidence for composite membrane transport and for a slippage of small organic solutes across water channels

Using the cell pressure probe, the effects of temperature on hydraulic conductivity (Lp; osmotic water permeability), solute permeability (permeability coefficient, P_s), and reflection coefficients (σ_s) were measured on internodes of Chara corallina, Klein ex Willd., em R.D.W.. For the first time, complete sets of transport coefficients were obtained in the range between 10 and 35 °C which provided evidence about pathways of water and solutes as they move across the plasma membrane (water channel and bilayer arrays). Test solutes used to check for the selectivity of water channels were monohydric alcohols of different molecular size and shape (ethanol, n-propanol, iso-propanol, and tert-butanol) and heavy water (HDO). Within the limits of accuracy, Q₁₀ values for Lp and for the diffusive water permeability (P_d) were identical (Q₁₀ for Lp = 1.29 ± 0.17 (± SD; n = 15 cells) and Q₁₀ for P_d = 1.25 ± 0.16 (n = 5 cells)). The Q₁₀ values were equivalent to activation energies of E_a = 16.8 ± 6.4 and 16.6 ± 10.0 kJ · mol⁻¹, respectively, which is similar to that of self-diffusion or of viscous flow of water. The Q₁₀ values and activation energies for P_s of the alcohols were significantly larger (ethanol: Q₁₀ = 1.68 ± 0.16, E_a = 37.1 ± 5.9 kJ · mol⁻¹; n-propanol: Q₁₀ =  1.75 ± 0.40, E_a = 43.1 ± 15.3 kJ · mol⁻¹; iso-propanol: Q₁₀ = 2.12 ± 0.42, E_a =  52.2 ± 14.6 kJ · mol⁻¹; tert-butanol: Q₁₀ = 2.13 ± 0.56, E_a = 51.6 ± 17.1 kJ · mol⁻¹; ±SD; n = 5 to 6 cells). Effects of temperature on reflection coefficients were most pronounced. With increasing temperature, σ_s values of the alcohols decreased and those of HDO increased. The data indicate that water and solutes use different pathways when crossing the membrane. Ordinary and isotopic water use water channels and the other test solutes use the bilayer array (composite transport model of membrane). Changes in σ_s values with temperature were found to be a sensitive measure for the open/closed state of water channels. The decrease of σ_s with temperature was theoretically predicted from the temperature dependence of P_s and Lp. Differences between predicted and measured values of σ_s allowed estimation of the bypass flow (slippage) of solutes through water channels which did not completely exclude test solutes. The permeability of channels depended on the structure and size of test solutes. It is concluded that water channels are much less selective than is usually thought. Since water channels represent single-file or no-pass pores, solutes drag along considerable amounts of water as they diffuse across channels. This results in low overall values of σ_s. The σ_s of HDO was extremely low. Its response to temperature was opposite to that for the σ_s of the alcohols. This suggested a stronger effect of temperature on the hydraulic (osmotic) than on the diffusive water flow across individual water channels, i.e. a differential sensitivity of different mechanisms to temperature. Received: 10 October 1996 / Accepted: 2 December 1996     

Phenamil: An irreversible inhibitor of sodium channels in the toad urinary bladder

Summary Several new amiloride analogues and two reported photoaffinity analogues were tested for irreversible inhibition of short-circuit current,I _sc, in toad bladder. Bromoamiloride, a photoaffinity analogue, induced 40% irreversible inhibition at 500 m after irradiation with ultraviolet light 320 nm. Iodoamiloride caused no irreversible inhibition. Of the new analogues tested, only 3,5-diamino-6-chloro-N-[(phenylamino) aminomethylene] pyrazinecarboxamide,phenamil, irreversibly inhibitedI _sc at concentrations of 0.05 to 5 m when added to the mucosal solution. Irreversible inhibition ofI _sc by phenamil may be attributed to specific blockage of the mucosal sodium channels, which depended on: 1) time of exposure; 2) mucosal pH: 3) mucosal sodium concentration. For example, 5 m phenamil irreversibly inhibitedI _sc by 38% in 103mm Na at pH 8.6 and nearly 75% in 30mm Na at pH 6.4 after a 40-min exposure. Irreversible inhibition occurred in two phases with time constants of 10 min and approximately 140 min. Due to its irreversible nature, phenamil may be used to measure channel density.     

Membrane structural and functional responses to vasopressin in toad bladder

Summary Freeze-fracture electronmicroscopy demonstrates that vasopressin stimulation of isolated toad bladder results in a striking morphologic alteration of epithelial membrane structure. This alteration is characterized by the aggregation of intramembranous particles in orderly linear arrays at multiple sites in the luminal membranes of granular cells specifically. The size of these aggregates varies considerably, in terms of area, over a range from 0.5 to 70×10^–3 m². The median aggregate size is about 10.5×10^–3 m². Since the extent of vasopressin-associated particle aggregation, in terms of frequency of sites per area of membrane or cumulative area of membrane occupied by them, closely correlates with induced changes in transport function, as measured by osmotic water flow, the aggregates themselves appear to be of physiologic significance in the mechanism of action of vasopressin. This hypothesis is supported by the observations that sites of aggregation occur (a) in response to serosal exposure to hormone specifically, (b) independently of an osmotic gradient, and (c) following stimulation with cyclic adenosine monophosphate.     

Independent pathways for water and solute movement across the cell membrane

Summary In published studies of the relationship between movement of nonelectrolytes across cell membranes and the lipid solubility of these test molecules, it is generally found that a number of the smaller, more water-soluble molecules deviate significantly from the general pattern relating permeability (or reflection coefficient) to lipid solubility. This is often true of the amides, for example, whose reflection coefficients are considerably lower than expected on the basis of lipid solubility. While this has been interpretep in terms of the movement of these solutes through aqueous channels in the membrane, it now appears that many of these deviant molecules may cross the membrane by means of carrier-mediated diffusion, independent of osmotic water flow. This has important implications for studies in which equivalent pore radius has been estimated from the reflection coefficients of small hydrophilic molecules, and for our present concepts of membrane structure.     

Independence of water and solute pathways in human RBCs

We have investigated the permeability of the human red blood cell to four di-hydroxy alcohols, 1,2PD (1,2 propanediol), 1,3PD (1.3 propanediol), 1,4BD (1,4 butanediol), and 2,3BD (2,3 butanediol), and to water by using a recently developed ESR stopped-flow method which is free from artifacts found in light scattering methods. Numerical solutions of the Kedem-Katchalsky equations fit to experimental data yielded the following permeability coefficients: P_1,2PD = 3.17 × 10^–5 cm sec^–1, p_1,3pd = 1.75 × 10^–5 cm sec^–1, P_1,4BD = 2.05 × 10⁵ cm sec^–1, P_2,3BD = 7.32 × 10^–5 cm sec^–1. Reflection coefficients () were evaluated by comparing data fit with assumed values of = 0.6,0.8 and 1.0. In all four cases the best fit was obtained with = 1.0. Treatment of cells with PCMBS (para-chloro mercuri-benzenesulfonate) was followed by a large (> 10-fold) decrease in water permeability with virtually no change in alcohol permeability. We conclude that these alcohols do not permeate the water channels to any significant extent, and discuss some of the problems in light scattering measurements of reflection coefficients that could lead to erroneous values for .We would like to thank Professor Lenore W. Yousef (Dept. of Biology, California State Univ., Fresno) for valuable discussions and critical comments. We thank Lidia Mannuzzu for measurements of ESR spectra in the presence and absence of alcohol. We are also indebted to Kate Van Fossen for her dedicated technical support. This work was supported by NIH grant No. HL-20985.     

Functional water channels and proton pumps are in separate populations of endocytic vesicles in toad bladder granular cells

Functional water channels are retrieved by endocytosis from the apical membrane of toad bladder granular cells in response to vasopressin [Shi, L.-B., & Verkman, A.S. (1989) J. Gen. Physiol. 94, 1101-1115]. To examine whether endocytic vesicles which contain the vasopressin-sensitive water channel fuse with acidic vesicles for entry into a lysosomal pathway, ATP-dependent acidification and osmotic water permeability were measured in endosomes from control bladders and bladders treated with vasopressin (VP) and/or phorbol myristate acetate (PMA). Endosomes were labeled with the fluid-phase markers 6-carboxyfluorescein or fluorescein-dextran. Osmotic water permeability (Pf) was measured by stopped-flow fluorescence quenching and proton ATPase activity by ATP-dependent, N-ethylmaleimide-inhibitable acidification. In a microsomal pellet, Pf was low (less than 0.002 cm/s, 20 degrees C) in labeled endocytic vesicles from control bladders but high (0.05-0.1 cm/s) in a subpopulation (50-70%) of vesicles from VP- and PMA-treated bladders. Following ATP addition, the average drop in pH was 0.1 (control), 0.3 (VP), and 0.2 (PMA) unit. Measurement of pH in individual endocytic vesicles by quantitative image analysis showed that less than 20% of vesicles from VP-treated bladders acidified by greater than 0.5 pH unit. To examine whether water channels and proton pumps were present in the same endocytic vesicles, the pH of endosomes with high and low water permeability was measured from the effect of ATP on the amplitude of the fluorescence quenching signal in response to an osmotic gradient.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aldosterone control of the density of sodium channels in the toad urinary bladder

Summary Near-instantaneous current-voltage relationships and shot-noise analysis of amiloride-induced current fluctuations were used to estimate apical membrane permeability to Na (P _Na), intraepithelial Na activity (Na _c ), single-channel Na currents (i) and the number of open (conducting) apical Na channels (N₀), in the urinary bladder of the toad (Bufo marinus). To facilitate voltageclamping of the apical membrane, the serosal plasma membranes were depolarized by substitution of a high KCl (85mm) sucrose (50mm) medium for the conventional Na-Ringer's solution on the serosal side.Aldosterone (5×10^–7 m, serosal side only) elicited proportionate increases in the Na-specific current (I _Na and inP _Na, with no significant change in the dependence ofP _Na on mucosal Na (Na _o ).P _Na and the control ofP _Na by aldosterone were substrate-dependent: In substrate-depleted bladders, pretreatment with aldosterone markedly augmented the response to pyruvate (7.5×10^–3 m) which evoked coordinate and equivalent increases inI _Na andP _Na.The aldosterone-dependent increase inP _Na was a result of an equivalent increase in the area density of conducting apical Na channels. The computed single-channel current did not change. We propose that, following aldosterone-induced protein synthesis, there is a reversible metabolically-dependent recruitment of preexisting Na channels from a reservoir of electrically undetectable channels. The results do not exclude the possibility of a complementary induction of Na-channel synthesis.     

Pathways for movement of ions and water across toad urinary bladder

Hypertonicity of the mucosal bathing medium increases the electrical conductance of toad urinary bladder by osmotic distension of the epithelial "tight" or limiting junctions. However, toad urine is not normally hypertonic to plasma. In this study, the transmural osmotic gradient was varied strictly within the physiologic range; initially hypotonic mucosal bathing media were made isotonic by addition of a variety of solutes. Mucosal NaCl increased tissue conductance substantially. This phenomenon could not have reflected soley an altered conductance of the transcellular active transport pathway since mucosal KCl also increased tissue conductance, whether or not Na+ was present in the bathing media. The effect of mucosal NaCl could not have been mediated solely by a parallel transepithelial pathway formed by damaged tissue since mucosal addition of certain nonelectrolytes also increased tissue conductance. Finally, the osmotically-induced increase in conductance could not have occurred soley in transcellular transepithelial channels in parallel with the active pathway for Na+, since the permeability to 22Na from serosa to mucosa (s to m) was also increased by mucosal addition of NaCl; a number of lines of evidence suggest that s-to-m movement of Na+ proceeds largely through paracellular transepithelial pathways. The results thus establish that the permeability of the limiting junctions is physiologically dependent on the magnitude of the transmural osmotic gradient. A major role is proposed for this mechanism, serving to conserve the body stores of NaCl from excessive urinary excretion.