Evidence for permanent water channels in the basolateral membrane of an ADH-sensitive epithelium

Summary The transepithelial water permeability in frog urinary bladder is believed to be essentially dependent on the ADH-regulated apical water permeability. To get a better understanding of the transmural water movement, the diffusional water permeability (P _d) of the basolateral membrane of urinary bladder was studied. Access to this post-luminal barrier was made possible by perforating the apical membrane with amphotericin B. The addition of this antibiotic increasedP _d from 1.12±0.10×10^–4 cm/sec (n=7) to 4.08±0.33×10^–4 cm/sec (n=7). The effect of mercuric sulfhydryl reagents, which are commonly used to characterize water channels, was tested on amphotericin B-treated bladders. HgCl₂ (10^–3 m) decreasedP _d by 52% andpara-chloromercuribenzoic acid (pCMB) (1.4×10^–4 m) by 34%. The activation energy for the diffusional water transport was found to increase from 4.52±0.23 kcal/mol (n=3), in the control situation, to 9.99±0.91 kcal/mol (n=4) in the presence of 1.4×10^–4 m pCMB. Our second approach was to measure the kinetics of water efflux, by stop-flow light scattering, on isolated epithelial cells from urinary bladders.pCMB (0.5 or 1.4×10^–4 m) was found to inhibit water exit by 91±2%. These data strongly support the existence of proteins responsible for water transport across the basolateral membrane, which are permanently present.     

Frog striated muscle is permeable to hydroxide and buffer anions

Hydroxide, bicarbonate and buffer anion permeabilities in semitendinosus muscle fibers of Rana pipiens were measured. In all experiments, the fibers were initially equilibrated in isotonic, high K₂SO₄ solutions at pH _o =7.2 buffered with phosphate. Two different methods were used to estimate permeabilities: (i) membrane potential changes were recorded in response to changes in external ion concentrations, and (ii) intracellular pH changes were recorded in response to changes in external concentrations of ions that alter intracellular pH. Constant field equations were used to calculate relative or absolute permeabilities.In the first method, to increase the size of the membrane potential change produced by a sudden change in anion entry, external K⁺ was replaced by Cs⁺ prior to changes of the anion under study. At constant external Cs⁺ activity, a hyperpolarization results from increasing external pH from 7.2 to 10.0 or higher, using either CAPS (3-[cyclohexylamino]-1-propanesulfonic acid) or CHES (2-[N-cyclohexylamino]-ethanesulfonic acid) as buffer. For each buffer, the protonated form is a zwitterion of zero net charge and the nonprotonated form is an anion. Using reported values of H⁺ permeability, calculations show that the reduction in [H⁺] _o cannot account for the hyperpolarizations produced by alkaline solutions. Membrane hyperpolarization increases with increasing total external buffer concentration at constant external pH, and with increasing external pH at constant external buffer anion concentration. Taken together, these observations indicate that both OH^– and buffer anions permeate the surface membrane. The following relative permeabilities were obtained at pH_o, 10.0± 0.3: (P_OH/P_K) = 890 ± 150, (P_CAPS/P_K) = 12 ± 2 (P_CHIES/P_K) = 5.3 ± 0.9, and (P_NO3/P_K) = 4.7 ± 0.5 P_NO/P_K was independent of pH _o up to 10.75. At pH_o = 9.6, (P_HCO3/P_K) = 0.49 ± 0.03; at pH _o = 8.9, (P_Cl/P_K) = 18± 2 and at pH _o = 7.1, (P_HEPES/P_K) = 20 ± 2.In the second method, on increasing external pH from 7.2 to 10.0, using 2.5 mm CAPS (total buffer concentration), the internal pH increases linearly with time over the next 10 min. This alkalinization is due to the entry of OH^– and the absorption of internal H⁺ by entering CAPS^– anion. The rate of CAPS^– entry was determined in experiments in which the external CAPS concentration was increased at constant external pH. Such increases invariably produced an increase in the rate of internal alkalinization, which was reversed when the CAPS concentration was reduced to its initial value. From the internal buffer power, the diameter of the fiber under study and the rates of change of internal pH, the absolute permeability for both OH^– and CAPS^– were calculated. At external pH = 10.0, the average (±sem) permeabilities were: P_OH=1.68±0.19×10^–4 cm/sec and P_CAPS=2.10±0.74×10^–6cm/sec.We conclude that OH^– is about 50 times more permeable than Cl^– at alkaline pH and that the anionic forms of commonly used buffers have significant permeabilities.This research was supported by a grant from the National Institutes of Health (AR 31814). The authors wish to thank Dr. Peter G. Shrager and Dr. Bruce C. Spalding for reading an early draft of this report and for providing helpful suggestions.     

Diffusive water permeability in isolated kidney proximal tubular cells: Nature of the cellular water pathways

Summary The diffusive water permeability (P _d ) of the plasma membrane of proximal kidney tubule cells was measured using a¹H-NMR technique. The values obtained for the exchange time (T _ex) across the membrane were independent of the cytocrit and of the Mn²⁺ concentration (in the range 2.5 to 5mm). At 25°C the calculatedP _d value was (per cm² of outer surface area without taking into account membrane invaginations) 197±17 m/sec. This value equals 22.3±1.9 m/sec when the invaginations are taken into account. Cell exposure to 2.5mm parachloromercuribenzenesulfonic acid,pCMBS, (for 20 to 35 min) reducedP _d to 45% of its control value. Fivemm dithiothreitol, DTT, reverted this effect. The activation energy for the diffusive water flux was 5.2±1.0 kcal/mol under control conditions. It increased to 9.1±2.2 kcal/mol in the presence of 2.5mm pCMBS. Using our previous values for the osmotic water permeability (P _os) in proximal straight tubular cells theP _os/P _d ratio equals 18±1, under control conditions, and 3.2±0.3 in the presence ofpCMBS. These experimental results indicate the presence of pathways for water, formed by proteins, crossing these membranes, which are closed bypCMBS. Assuming laminar flow (within the pore), fromP _os/P _d of 13 to 18 an unreasonably large pore radius of 12 to 15 Å is calculated which would not hinder cell entry of known extracellular markers. Alternatively, for a single-file pore, 11 to 20 would be the number of water molecules which would be in tandem inside the pore. The water permeability remaining in the presence ofpCMBS indicates water permeation through the lipid bilayer. There are similarities between these results and those obtained in human red blood cells and in the apical cell membrane of the toad urinary bladder.     

Redistribution of hepatocyte chloride during l-alanine uptake

We used ion-sensitive, double-barrel microelectrodes to measure changes in hepatocyte transmembrane potential (V _m), intracellular K⁺, Cl^-, and Na⁺ activities (a ⁱ _k, a _Cl ⁱ and a _Na ⁱ ), and water volume during l-alanine uptake. Mouse liver slices were superfused with control and experimental Krebs physiological salt solutions. The experimental solution contained 20 m l-alanine, and the control solution was adjusted to the same osmolality (305 mOsm) with added sucrose. Hepatocytes also were loaded with 50 mm tetramethylammonium ion (TMA⁺) for 10 min. Changes in cell water volume during l-alanine uptake were determined by changes in intracellular, steady-state TMA⁺ activity measured with the K⁺ electrode. Hepatocyte control V _m was -33±1 mV. l-alanine uptake first depolarized V _m by 2±0.2 mV and then hyperpolarized V _m by 5 mV to-38±1 mV (n = 16) over 6 to 13 min. During this hyperpolarization, a _Na ⁱ increased by 30% from 19±2 to 25±3 mm (P < 0.01), and a _K ⁱ did not change significantly from 83±3 mm. However, with added ouabain (1 mm) l-alanine caused only a 2-mV increase in V _m, but now a _K ⁱ decreased from 61±3 to 54±5 mm (P < 0.05). Hyperpolarization of V _m by l-alanine uptake also resulted in a 38% decrease of a _Cl ⁱ from 20±2 to 12±3 mm (P < 0.001). Changes in V _m and V _Cl — V _m voltage traces were parallel during the time of l-alanine hyperpolarization, which is consistent with passive distribution of intracellular Cl^– with the V _m in hepatocytes. Added Ba²⁺ abolished the l-alanineinduced hyperpolarization, and a _Cl ⁱ remained unchanged. Hepatocyte water volume during l-alanine uptake increased by 12±3%. This swelling did not account for any changes in ion activities following l-alanine uptake. We conclude that hepatocyte a _K ⁱ is regulated by increased Na⁺-K⁺ pump activity during l-alanine uptake in spite of cell swelling and increased V _m due to increased K⁺ conductance. The hyperpolarization of V _m during l-alanine uptake provides electromotive force to decrease a _Cl ⁱ . The latter may contribute to hepatocyte volume regulation during organic solute transport.This work was supported by grant AA-08867 from the Alcohol, Drug Abuse, and Mental Health Association.     

Serial permeability barriers to water transport in human placental vesicles

Summary Microvillous vesicles were prepared from term human placenta by shearing, differential centrifugation and Mg²⁺ precipitation. Vesicles were purified further on a sucrose density gradient producing two bands with densities of 1.16 to 1.18 g/ml (C1) and 1.13 to 1.15 g/ml (C2). The C2 fraction, which had a 24-fold enrichment of alkaline phosphatase and a three-fold reduction in Na⁺, K⁺-ATPase activity compared to homogenates, was used to measure osmotic water (P _f ) permeability.P _f was measured from the time course of scattered light intensity following exposure of vesicles to specified gradients of impermeant solutes.P _f decreased from 3.0×10^–3 to 0.6×10^–3 cm/sec with increasing gradient size (65 to 730mm; 23°C). Four possible causes of this behavior were examined theoretically and experimentally: an unstirred layer, saturation of water transport, large changes in the vesicle surface area with changes in volume and a structural restriction to vesicle volume change. The measured dependence ofP _f on gradient size and the effect of the channel-forming ionophore gramicidin onP _f fit best to the theoretical dependences predicted by a structural restriction mechanism. This finding was supported by experiments involving the effects onP _f of increased solution viscosity, initial vesicle volume, the magnitude of transmembrane volume flow, and the effects of gradient size on activation energy (E _a) forP _f . The decreasedP _f resulting from a structural restriction limiting vesicle volume change was modeled mathematically as a second barrier in series with the vesicle membrane.E _a measured using a 250-mm inwardly directed sucrose gradient was 5.4±0.6 kcal/mol (T>27°C) and 10.0±0.6 kcal/mol (T<27°C).E _a above 27°C is in the range normally associated with transmembrane passage of water via aqueous channels. Water transport was not inhibited byp-chloromercuribenzenesulfonate.     

Very low osmotic water permeability and membrane fluidity in isolated toad bladder granules

Summary Osmotic water permeability of the apical membrane of toad urinary epithelium is increased greatly by vasopressin (VP) and is associated with exocytic addition of granules and aggrephores at the apical surface. To determine the physiological role of granule exocytosis, we measured the osmotic water permeability and membrane fluidity of isolated granules, surface membranes and microsomes prepared from toad bladder in the presence and absence of VP.P _f was measured by stopped-flow light scattering and membrane fluidity was examined by diphenylhexatriene (DPH) fluorescence anisotropy. In response to a 75mm inward sucrose gradient, granule size decreased with a single exponential time constant of 2.3±0.1 sec (sem, seven preparations, 23°C), corresponding to aP _f of 5×10^–4 cm/sec; the activation energy (E _a ) forP _f was 17.6±0.8 kcal/mole. Under the same conditions, the volume of surface membrane vesicles decreased biexponentially with time constants of 0.13 and 1.9 sec; the fast component comprised 70% of the signal. Granule, surface membrane and microsome time constants were unaffected by VP. However, in surface membranes, there was a small decrease (6±2%) in the fraction of surface membranes with fast time constant. DPH anisotropies were 0.253 (granules), 0.224 (surface membrane fluidity is remarkably lower than that of surface and microsomal membranes, and (4) rapid water transport occurs in surface membrane vesicles. The unique physical properties of the granule suggests that apical exocytic addition of granule membrane may be responsible for the low water permeability of the unstimulated apical membrane.     

Pre-steady-state analysis of the turn-on and turn-off of water permeability in the kidney collecting tubule

Summary Water transport across the mammalian collecting tubule is regulated by vasopressin-dependent water channel insertion into and retrieval from the cell apical membrane. The time course of osmotic water permeability (P _f ) following addition and removal of vasopressin (VP) and 8-Br-cAMP was measured continuously by quantitative fluorescence microscopy using an impermeant fluorophore perfused in the lumen. Cortical collecting tubules were subjected to a 120 mOsm bath-to-lumen osmotic gradient at 37°C with 10–15 nl/min lumen perfusion and 10–20 ml/min bath exchange rate. With addition of VP (250 U/ml), there was a 23±3 sec (sem,n=16) lag in whichP _f did not change, followed by a rise inP _f (initial rate 1.4±0.2×10^–4 cm/sec²) to a maximum of 265±10×10^–4 cm/sec. With addition of 8-Br-cAMP (0.01–1mm) there was an 11±2 sec lag. For [8-Br-cAMP]=0.01, 0.1 and 1mm, the initial rate ofP _f increase following the lag was (units 10^–4 cm/sec²): 1.1±0.1, 1.2±0.1 and 1.7±0.3. MaximumP _f was (units 10^–4 cm/sec): 64±4, 199±9 and 285±11. With removal of VP,P _f decreased to baseline (12×10^–4 cm/sec) with aT _1/2 of 18 min; removal of 0.1 and 1mm 8-Br-cAMP gaveT _1/2 of 4 and 8.5 min. These results demonstrate (i) a brief lag in theP _f response, longer for stimulation by VP than by 8-Br-cAMP, representing the transient build-up of biochemical intermediates proximal to the water channel insertion step, (ii) similar initialdP _f /dt (water channel insertion) over a wide range of [8-Br-cAMP] and steady-stateP _f values, and (iii) more rapidP _f decrease with removal of 8-Br-cAMP than with VP. These pre-steady-state results define the detailed kinetics of the turn-on and turn-off of tubuleP _f and provide kinetic evidence that the rate-limiting step for turn-on ofP _f is not the step at which VP regulates steady-stateP _f . If water channel insertion is assumed to be the rate-limiting step in the turn-on ofP _f , these results raise the possibility that water channels must be activated following insertion into the apical membrane.     

The Effect of Bathing Solution Tonicity on Resting Tension in Frog Muscle Fibers

Resting tension and short-range elastic properties of isolated twitch muscle fibers of the frog have been studied while bathed by solutions of different tonicities. Resting tension in isotonic solution at 2.3-µm sarcomere spacing averaged 0.46 mN·mm^-2 and was proportional to the fiber cross-section area. Hypertonic solutions, containing 0.1–0.5 mM tetracaine to block contracture tension, caused a small sustained tension increase, which was proportional to the fiber cross-section area and which reached 0.9 mN·mm^-2 at two times normal tonicity (2T). Further increases in tonicity caused little increase in tension. Hypotonic solutions decreased tension. Thus, tension at 2.3 µm is a continuous, direct function of tonicity. The dependence of tension on tonicity lessened at greater sarcomere lengths. At 3.2 µm either a very small rise or, in some fibers, a fall in tension resulted from an increase in tonicity. Hypertonic solutions also decreased the tension of extended sarcolemma preparations. In constant-speed stretch experiments the elastic modulus, calculated from the initial part of the stretch response, rose steeply with tonicity over the whole range investigated (1–2.5T). The results show that tension and stiffness of the short-range elastic component do not increase in parallel in hypertonic solutions.     

Excitation-calcium release uncoupling in aged single human skeletal muscle fibers

The biological mechanisms underlying decline in muscle power and fatigue with age are not completely understood. The contribution of alterations in the excitation-calcium release coupling in single muscle fibers was explored in this work. Single muscle fibers were voltage-clamped using the double Vaseline gap technique. The samples were obtained by needle biopsy of the vastus lateralis (quadriceps) from 9 young (25–35 years; 25.9 ± 9.1; 5 female and 4 male) and 11 old subjects (65–75 years; 70.5 ± 2.3; 6 f, 5 m). Data were obtained from 36 and 39 fibers from young and old subjects, respectively. Subjects included in this study had similar physical activity. Denervated and slow-twitch muscle fibers were excluded from this study. A significant reduction of maximum charge movement (Q_max) and DHP-sensitive Ca current were recorded in muscle fibers from the 65–75 group. Q_max values were 7.6 ± 0.9 and 3.2 ± 0.3 nC/F for young and old muscle fibers, respectively (P < 0.01). No evidences of charge inactivation or interconversion (charge 1 to charge 2) were found. The peak Ca current was (–)4.7 ± 0.08 and (–)2.15 ± 0.11 A/F for young and old fibers, respectively (P < 0.01). The peak calcium transient studied with mag-fura-2 (400 m) was 6.3 ± 0.4 m and 4.2 ± 0.3 m for young and old muscle fibers, respectively. Caffeine (0.5 mm) induced potentiation of the peak calcium transient in both groups. The decrease in the voltage-/ Ca-dependent Ca release ratio in old fibers (0.18 ± 0.02) compared to young fibers (0.47 ± 0.03) (P < 0.01), was recorded in the absence of sarcoplasmic reticulum calcium depletion. These data support a significant reduction of the amount of Ca available for triggering mechanical responses in aged skeletal muscle and, the reduction of Ca release is due to DHPR-ryanodine receptor uncoupling in fast-twitch fibers. These alterations can account, at least partially for the skeletal muscle function impairment associated with aging.This work was supported by Grant-in-Aid from the American Heart Association (National) and Muscular Dystrophy Association, and National Institutes of Health (2-P60AG18484-06)     

Effect of osmotic gradient on ADH-induced intramembranous particle aggregates in toad bladder

Summary Paired toad urinary bladders were prepared without or with an osmotic gradient (175 mosm) across them, stimulated for 2.5 (n=6), 5 (n=6), 30 (n=6) or 60 (n=6) min with ADH (20 mU/ml), and studied by freeze-fracture electron microscopy. Water permeability at these times was assessed in additional bladders (n=6 for each case) after tissue fixation according to the technique of Eggena. After both 60 and 30 min of ADH stimulation, the presence of a gradient compared with the absence of one was associated with fewer aggregates (242±35vs. 382±14 ×235 m^–2 at 60 min,P<0.01; 279±36vs. 470±51 ×235 m^–2 at 30 min,P<0.01) and lower water permeability (8.4±1.1vs. 18.8±1.8g×min^–1×cm^–1 ×mosm ^–1 at60min,P<0.005; 9.2±1.0vs. 22.0±2.1 g ×min^–1×cm^–2×mosm ^–1 at 30 min,P<0.001). In addition, with a gradient both maximum water permeability and maximum aggregate frequency were reached nearly together; a similar correspondence occurred without a gradient. We conclude that in the presence of an osmotic gradient both the ADH-associated aggregates and the water permeability response to ADH are prevented from reaching the higher levels observed in bladders not exposed to a gradient.     

Effect of divalent cations on the potassium contracture of slow muscle fibers ofRana temporaria

Summary K contractures of single slow muscle fibers ofRana temporaria were measured isometrically in the presence of normal, reduced, and increased Ca²⁺ concentrations at 18 to 20°C. At normal Ca²⁺ concentration (1.8mm) contracture tension decreased from its peak value of 35.4±8.2 N/cm² to 59.4±23.9% within one minute, and to 48.3±27% within two minutes (30 fibers). Peak tension was virtually unaffected by changes of the Ca²⁺ concentration, but maintenance of tension was impaired by low (0.2mm), and improved by high (10mm) Ca²⁺ concentrations. When Ca²⁺ was added during the K contracture, there was practically no restoration of lost tension. Effects similar to those of Ca²⁺ were observed upon addition of foreign divalent cations to the medium. Co²⁺, Ni²⁺, and Cd²⁺ were slightly more effective than Ca²⁺ and Mn²⁺; the smallest effects were obtained with Mg²⁺, Sr²⁺, and Ba²⁺. The effects of foreign divalent cations were independent of the presence of Ca²⁺. It is concluded that in slow fibers ofRana temporaria maintenance of contracture tension is not due to an influx of Ca²⁺ ions. Instead, binding of divalent cations to superficial sites seems to be essential.     

Water permeability properties of the ovarian oocytes from Bufo arenarum and Xenopus laevis: A comparative study

The water permeability properties of ovarian oocytes from Xenopus laevis and Bufo arenarum, a toad species found in the Buenos Aires region, were studied. We report that: (i) the water osmotic permeability (P _f, cm/sec × 10^–4) was significantly higher in Bufo (6°C=12.3±2.4; 18°C = 20.8±4.8) than in Xenopus oocytes (6°C=5.3±0.3; 18°C=6.2±1.6). The corresponding water diffusion permeability values (P _d, cm/sec × 10^–4) were: Xenopus = 2.3±0.3 (6°C) and 4.8±0.7 (18°C); Bufo=2.7±0.4 (6°C) and 6.0 ±0.5 (18°C). (ii) Amphotericin B increased the P _f and P _d values. The observed P _fP _d ratio was not significantly different from the expected results (n=3), after amphotericin B incorporation in both species. This means that the influence of unstirred layers and other potential artifactual compounds did not significantly affect our experimental results, (iii) Preincubation with gramicidin during 12 hr induced a clear increase in the oocyte volume. After that, a hypotonic shock only slightly increased the oocyte volume. Conversely, a hypertonic challenge induced a volume change significantly higher than the one observed in control conditions, (iv) Mercury ions did not affect the osmotic permeability in Xenopus oocytes but clearly inhibited, in a reversible way, the osmotic permeability in oocytes from B. arenarum. (v) Mercury ions did not reduce P _d values in either species, (vi) The P _fP _d values calculated from the differences observed in these parameters between both species were 11.9±5.1 at 18°C and 15.5±2.4 at 6°C. These numbers are similar to those previously reported in the case of membranes having water channels. From these results, we propose that water channels are present in the ovarian oocyte from B. arenarum but not in the ovarian oocyte from X. laevis.This work was supported by Fundación Antorchas, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina) and Universidad de Buenos Aires (UBA). It was developed in the frame of an INSERM (France)-CONICET cooperative program.     

Muscarinic activation causes biphasic inotropic response and decreases cellular Na+ activity in canine cardiac purkinje fibers

In this study, the effects of carbachol (CCh) on twitch tension, intracellular Na⁺ activity (a _Na ⁱ ), and action potential were simultaneously measured in canine cardiac Purkinje fibers in order to examine the regulation of inotropy through muscarinic receptors and its relation to a _Na ⁱ . In fibers driven at 1 Hz, CCh (10 µM) initially and transiently decreased and then increased the twitch tension by 36±8%. The action potential showed a significant elevation of the plateau and a significant shortening of the duration at 90% repolarization (APD₉₀), from 403±7 to 389±7 ms. The a _Na ⁱ decreased from 7.4±0.4 to 6.7±0.3 mM (n=23, p<0.05). Atropine (1 µM) decreased the twitch tension by 21±6% (n=7, p<0.05) without significant effects on the action potential and a _Na ⁱ , and inhibited the effects of CCh. Cs⁺ (20 mM) increased the plateau height and APD₉₀, enhanced the twitch tension by 66±24%, but decreased a _Na ⁱ from 7.3±0.3 to 6.3±0.4 mM (n=6, p<0.05). In the presence of 20 mM Cs⁺, some fibers generated slow responses. The addition of 10 µM CCh further increased the twitch tension and APD₉₀, and decreased a _Na ⁱ from 6.3±0.4 to 5.3±0.3 mM. Ouabain (0.3 µM) increased the twitch tension and a _Na ⁱ , and inhibited the CCh-induced decrease of a _Na ⁱ . In the presence of ouabain, 20 mM Cs⁺ depolarized the fiber and generated slow responses with a decreased a _Na ⁱ . The addition of 10 µM CCh enhanced the slow action potential, and increased a _Na ⁱ although there was a transient decrease during early exposure. These results suggest that activation of muscarinic receptors in canine Purkinje fibers results in an enhancement of the Na⁺-K⁺ pump activity and a biphasic inotropic response, probably via different receptor subtypes. The inhibitory effect, most likely through M₂ receptors, is associated with the activation of K⁺ channels. The stimulatory effect, on the other hand, is probably due to the action on the M₁ receptors, resulting in increases in Ca²⁺ currents.     

Kinetics of Water Transport in Eel Intestinal Vesicles

Brush border membrane vesicles, BBMV, from eel intestinal cells or kidney proximal tubule cells were prepared in a low osmolarity cellobiose buffer. The osmotic water permeability coefficient P _f for eel vesicles was not affected by pCMBS and was measured at 1.6 × 10⁻³ cm sec⁻¹ at 23°C, a value lower than 3.6 × 10⁻³ cm sec⁻¹ exhibited by the kidney vesicles and similar to published values for lipid bilayers. An activation energy E _a of 14.7 Kcal mol⁻¹ for water transport was obtained for eel intestine, contrasting with 4.8 Kcal mol⁻¹ determined for rabbit kidney proximal tubule vesicles using the same method of analysis. The high value of E _a , as well as the low P _f for the eel intestine is compatible with the absence of water channels in these membrane vesicles and is consistent with the view that water permeates by dissolution and diffusion in the membrane. Further, the initial transient observed in the osmotic response of kidney vesicles, which is presumed to reflect the inhibition of water channels by membrane stress, could not be observed in the eel intestinal vesicles. The P _f dependence on the tonicity of the osmotic shock, described for kidney vesicles and related to the dissipation of pressure and stress at low tonicity shocks, was not seen with eel vesicles. These results indicate that the membranes from two volume transporter epithelia have different mechanisms of water permeation. Presumably the functional water channels observed in kidney vesicles are not present in eel intestine vesicles. The elastic modulus of the membrane was estimated by analysis of swelling kinetics of eel vesicles following hypotonic shock. The value obtained, 0.79 × 10⁻³ N cm⁻¹, compares favorably with the corresponding value, 0.87 × 10⁻³ N cm⁻¹, estimated from measurements at osmotic equilibrium. Received: 28 January 1999/Revised: 15 June 1999     

Water permeability ofNecturus gallbladder epithelial cell membranes measured by nuclear magnetic resonance

Summary In order to assess the contribution of transcellular water flow to isosmotic fluid transport acrossNecturus gallbladder epithelium, we have measured the water permeability of the epithelial cell membranes using a nuclear magnetic resonance method. Spin-lattice (T ₁) relaxation of water protons in samples of gallbladder tissue where the extracellular fluid contained 10 to 20mm Mn²⁺ showed two exponential components. The fraction of the total water population responsible for the slower of the two was 24±2%. Both the size of the slow component, and the fact that it disappeared when the epithelial layer was removed from the tissue, suggest that it was due to water efflux from the epithelial cells. The rate constant of efflux was estimated to be 15.6±1.0 sec¹ which would be consistent with a diffusive membrane water permeabilityP _d of 1.6×10³ cm sec¹ and an osmotic permeabilityP _os of between 0.3×10⁴ and 1.4×10⁴ cm sec¹ osmolar¹. Using these data and a modified version of the standing-gradient model, we have reassessed the adequacy of a fluid transport theory based purely on transcellular osmotic water flow. We find that the model accounts satisfactorily for near-isosmotic fluid transport by the unilateral gallbladder preparation, but a substantial serosal diffusion barrier has to be included in order to account for the transport of fluid against opposing osmotic gradients.     

Pre-and post-branchial blood respiratory status during acute hypercapnia or hypoxia in rainbow trout,Oncorhynchus mykiss

Simultaneous venous (pre-branchial) and arterial (post-branchial) extracorporeal blood circulations were utilized to monitor continuously the rapid and progressive effects of acute environmental hypercapnia (water partial pressure of CO₂ 4.8±0.2 torr) or hypoxia (water partial pressure of O₂ 25±2 torr) on oxygen and carbon dioxide tensions and pH in the blood of rainbow trout (Oncorhynchus mykiss). During hypercapnia, the CO₂ tension in the arterial blood increased from 1.7±0.1 to 6.2±0.2 torr within 20 min and this was associated with a decrease of arterial extracellular pH from 7.95±0.03 to 7.38±0.03; the acid-base status of the mixed venous blood changed in a similar fashion. The decrease in blood pH in vivo was greater than in blood equilibrated in vitro with a similar CO₂ tension indicating a significant metabolic component to the acidosis in vivo. Under normocapnic conditions, venous blood CO₂ tension was slightly higher than arterial blood CO₂ tension difference was abolished or reversed during the initial 25 min of hypercapnia indicating that CO₂ was absorbed from the water during this period. Arterial O₂ tension remained constant during hypercapnia; however, venous blood O₂ tension decreased significantly (from 22.0±2.6 to 9.0±1.0 torr) during the initial 10 min. Hypercapnia elicited the release of catecholamines (adrenaline and noradrenaline) into the blood. The adrenaline concentration increased from 6±3 to 418±141 nmol · l^-1 within 25 min; noradrenaline concentration increased from 3±0.5 to 50±21 nmol · l^-1 within 15 min. During hypoxia arterial blood O₂ tension declined progressively from 108.4±9.9 to 12.8±1.7 torr within 30 min. Venous blood O₂ tension initially was stable but then decreased abruptly as catecholamines were released into the circulation. The release of catecholamines occurred concomitantly with a sudden metabolic acidosis in both blood compartments and a rise in CO₂ tension in the mixed venous blood only.Abbreviations CCO₂ plasmatotal carbondioxide - CtO₂ blood oxygen content - PO₂ partial pressure of oxygen - PCO₂ partial pressure of carbon dioxide - PaO₂ arterial bloodPO₂ - PaCO₂ arterial bloodPCO₂ - PvCO₂ venous bloodPCO₂ - PwO₂ waterPO₂ - PwCO₂ waterPCO₂ - Hb haemoglobin - SHbO₂ haemoglobin oxygen saturation - HPLC high-performance liquid chromatography - rbc red blood cell(s) - Hct haematocrit     

Electrical effects of potassium and bicarbonate on proximal tubule cells ofNecturus

Summary The effects of stepwise concentration changes of K⁺ and HCO ₃ ^– in the basolateral solution on the basolateral membrane potential (V _bl) of proximal tubule cells of the doubly-perfusedNecturus kidney were examined using conventional microelectrodes. Apparent transference numbers were calculated from changes inV _bl after alterations in external K⁺ concentration from 1.0 to 2.5mm (t _{K, 1.0–2.5}), 2.5 to 10, and in external HCO ₃ ^– concentration (at constant pH) from 5 to 10mm (t _{HCO3, 5–10}), 10 to 20, or 10 to 50.t _{K, 2.5–10} was 0.38±0.02 under control conditions but was sharply reduced to 0.08±0.03 (P>0.001) by 4mm Ba⁺⁺. This concentration of Ba⁺⁺ reducedV _bl by 9±1 mV (at 2.5 external K⁺). Perfusion with SITS (5×10^–4 m) for 1 hr hyperpolarizedV _bl by 10±3 mV and increasedt _{K, 2.5–10} significantly to 0.52±0.01 (P<0.001). Ba⁺⁺ application in the presence of SITS depolarizedV _bl by 22±3 mV. In control conditionst _{HCO3, 10–50} was 0.63±0.05 and was increased to 0.89±0.07 (P<0.01) by Ba⁺⁺ but was decreased to 0.14±0.02 (P<0.001) by SITS. In the absence of apical and basolateral chloride, the response ofV _bl to bicarbonate was diminished but still present (t _{HCO3, 10–20} was 0.35±0.03). Intracellular pH, measured with liquid ion-exchange microelectrodes, increased from 7.42±0.19 to 7.57±0.17 (P<0.02) when basolateral bicarbonate was increased from 10 to 20mm at constant pH. These data show that the effects of bicarbonate onV _bl are largely independent of effects on the K⁺ conductance and that there is a significant current-carrying bicarbonate pathway in the basolateral membrane. Hence, both K⁺ and HCO ₃ ^– gradients are important in the generation ofV _bl, and their relative effects vary reciprocally.     

Analysis of aquaporin-mediated diffusional water permeability by coherent anti-stokes Raman scattering microscopy

Water can pass through biological membranes via two pathways: simple diffusion through the lipid bilayer, or water-selective facilitated diffusion through aquaporins (AQPs). Although AQPs play an important role in osmotic water permeability (P_f), the role of AQPs in diffusional water permeability remains unclear because of the difficulty of measuring diffusional water permeability (P_d). Here, we report an accurate and instantaneous method for measuring the P_d of a single HeLa S3 cell using coherent anti-Stokes Raman scattering (CARS) microscopy with a quick perfusion device for H₂O/D₂O exchange. Ultra-high-speed line-scan CARS images were obtained every 0.488 ms. The average decay time constant of CARS intensities (τ_CARS) for the external solution H₂O/D₂O exchange was 16.1 ms, whereas the intracellular H₂O/D₂O exchange was 100.7 ± 19.6 ms. To evaluate the roles of AQP in diffusional water permeability, AQP4 fused with enhanced green fluorescent protein (AQP4-EGFP) was transiently expressed in HeLa S3 cells. The average τ_CARS for the intracellular H₂O/D₂O exchange in the AQP4-EGFP-HeLa S3 cells was 43.1 ± 15.8 ms. We also assessed the cell volume and the cell surface area to calculate P_d. The average P_d values for the AQP4-EGFP-HeLa S3 cells and the control EGFP-HeLa S3 cells were 2.7 ± 1.0 × 10⁻³ and 8.3 ± 2.6 × 10⁻⁴ cm/s, respectively. AQP4-mediated water diffusion was independent of the temperature but was dependent on the expression level of the protein at the plasma membrane. These results suggest the possibility of using CARS imaging to investigate the hydrodynamics of single mammalian cells as well as the regulation of AQPs.     

Effects of Cannabinoids on Caffeine Contractures in Slow and Fast Skeletal Muscle Fibers of the Frog

The effect of cannabinoids on caffeine contractures was investigated in slow and fast skeletal muscle fibers using isometric tension recording. In slow muscle fibers, WIN 55,212-2 (10 and 5 μM) caused a decrease in tension. These doses reduced maximum tension to 67.43 ± 8.07% (P = 0.02, n = 5) and 79.4 ± 14.11% (P = 0.007, n = 5) compared to control, respectively. Tension-time integral was reduced to 58.37 ± 7.17% and 75.10 ± 3.60% (P = 0.002, n = 5), respectively. Using the CB₁ cannabinoid receptor agonist ACPA (1 μM) reduced the maximum tension of caffeine contractures by 68.70 ± 11.63% (P = 0.01, n = 5); tension-time integral was reduced by 66.82 ± 6.89% (P = 0.02, n = 5) compared to controls. When the CB₁ receptor antagonist AM281 was coapplied with ACPA, it reversed the effect of ACPA on caffeine-evoked tension. In slow and fast muscle fibers incubated with the pertussis toxin, ACPA had no effect on tension evoked by caffeine. In fast muscle fibers, ACPA (1 μM) also decreased tension; the maximum tension was reduced by 56.48 ± 3.4% (P = 0.001, n = 4), and tension-time integral was reduced by 57.81 ± 2.6% (P = 0.006, n = 4). This ACPA effect was not statistically significant with respect to the reduction in tension in slow muscle fibers. Moreover, we detected the presence of mRNA for the cannabinoid CB₁ receptor on fast and slow skeletal muscle fibers, which was significantly higher in fast compared to slow muscle fiber expression. In conclusion, our results suggest that in the slow and fast muscle fibers of the frog cannabinoids diminish caffeine-evoked tension through a receptor-mediated mechanism.     

Comparative Permeabilities of the Paracellular and Transcellular Pathways of Corneal Endothelial Layers

Layers of rabbit corneal endothelial cells were cultured on permeable inserts. We characterized the diffusional permeability of the cell layer to nonelectrolyte and charged molecules and compared the diffusional and filtration permeabilities of the paracellular and transcellular pathways. We determined the rates of diffusion of ³H- and ¹⁴C-labeled nonelectrolyte test molecules and estimated the equivalent pore radius of the tight junction. Negatively charged molecules permeate slower than neutral molecules, while positively charged molecules permeate faster. Palmitoyl-dl-carnitine, which opens tight junctions, caused an increase of permeability and equivalent pore radius. Diffusional water permeability was determined with ³H-labeled water; the permeabilities of the tight junction and lateral intercellular space were calculated using tissue geometry and the Renkin equation. The diffusional permeability (P _d ) of the paracellular pathway to water is 0.57 μm s⁻¹ and that of the transcellular path is 2.52 μm s⁻¹. From the P _d data we calculated the filtration permeabilities (P _f ) for the paracellular and transcellular pathways as 41.3 and 30.2 μm s⁻¹, respectively. In conclusion, the movement of hydrophilic molecules through tight junctions corresponds to diffusion through negatively charged pores (r = 2.1 ± 0.35 nm). The paracellular water permeability represents 58% of the filtration permeability of the layer, which points to that route as the site of sizable water transport. In addition, we calculated for NaCl a reflection coefficient of 0.16 ≤ σ_NaCl ≤ 0.33, which militates against osmosis through the junctions and, hence, indirectly supports the electro-osmosis hypothesis.