Protamine reversibly decreases paracellular cation permeability inNecturus gallbladder

Summary Protamine, a naturally occurring arginine-rich polycationic protein (pI 9.7 to 12), was tested inNecturus gallbladder using a transepithelial AC-impedance technique. Protamine sulfate or hydrochloride (100 g/ml=20 m), dissolved in the mucosal bath, increased transepithelial resistance by 89% without affecting the resistance of subepithelial layers. At the same time, transepithelial voltage ( ^ms ) turned from slightly mucosapositive values to mucosa-negative values of approximately +1 to –5 mV. The effect of protamine on transepithelial resistance was minimal at concentrations below 5 g/ml but a maximum response was achieved between 10 and 20 g/ml. Resistance started to increase within 1 min and was maximal after 10 min. These effects were not inhibited by serosal ouabain (5×10^–4 m) but could be readily reversed by mucosal heparin. The sequence of protamine effect and heparin reversal could be repeated several times in the same gallbladder. Mucosal heparin, a strong negatively charged mucopolysaccharide, or serosal protamine were without effect. Mucosal protamine reversibly decreased the partial ionic conductance of K and Na by a factor of 3, but did not affect Cl conductance. Net water transport from mucosa to serosa was reversibly increased by 60% by protamine. We conclude that protamine reversibly decreases the conductance of the cation-selective pathway through the tight junction. Although this effect is similar to that reported for 2,4,6-triamino-pyrimidinium (TAP), the mechanism of action may differ. We propose that protamine binds to the apical cell membrane and induces a series of intracellular events which leads to a conformational alteration of the tight junction structure resulting in decreased cationic permeability.     

Noise analysis of the K+ current through the apical membrane ofNecturus gallbladder

Summary Current noise power spectra of the voltage-clamped (V=0)Necturus gallbladder, exposed to NaCl-Ringer's on both sides contained a relaxation noise component, which overlapped with a 1/f noise component, with being about 2. Substitution of all Na⁺ by K⁺ on either the serosal or mucosal side increased the relaxation as well as the 1/f noise component considerably. InNecturus gallbladder both noise components are reduced by addition of 10mm 2,4,6-triaminopyrimidine (TAP) or 5mm of tetraethylammonium (TEA⁺) added to ification of the mucosal solution to pH 5 and lower. Fivemm of tetraethylammonium (TEA⁺) added to the mucosal solution, abolished K⁺ relaxation noise and decreased the 1/f noise component. Applying a Cs⁺ concentration gradient across the epithelium did not yield relaxation noise. However, if Rb⁺ was substituted for all Na⁺ on one side, a Lorentzian noise component appeared in the spectrum. Its plateau was smaller than with KCl-Ringer's on the respective side. These data confirm the existence of fluctuating K⁺ channels in the apical membrane of theNecturus gallbladder. Furthermore it can be concluded that these channels have the permeability sequence K⁺>Rb⁺>Sc⁺. The inhibition of the fluctuations by mucosal acidification indicates the existence of acidic sites in the channel. The single-channel conductance was estimated to be between 6.5 and 40 pS.     

Cyclic AMP-induced changes in membrane conductance ofNecturus gallbladder epithelial cells

Summary Enhanced cellular cAMP levels have been shown to increase apical membrane Cl^– and HCO ₃ ^– conductances in epithelia. We found that the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) increases cAMP levels inNecturus gallbladder. We used conventional open-tip and double-barreled Cl^–-selective microelectrodes to study the effects of IBMX on membrane conductances and intracellular Cl^– activities in gallbladders mounted in a divided chamber and bathed with Ringer's solutions at 23°C and pH 7.4. In HCO ₃ ^– -free media, 0.1mM IBMX added to the mucosal medium depolarized the apical membrane potentialV _a , decreased the fractional resistanceF _R , and significantly reduced intracellular Cl^– activity (a _Cl ⁱ ). Under control conditions,a _Cl ⁱ was above the value corresponding to passive distribution across the apical cell membrane. In media containing 25mM HCO ₃ ^– , IBMX caused a small transient hyperpolarization ofV _a followed by a depolarization not significantly different from that observed in HCO ₃ ^– -free Ringer's. Removal of mucosal Cl^–, Na⁺ or Ca²⁺ did not affect the IBMX-induced depolarization inV _a . The basolateral membrane ofNecturus gallbladder is highly K⁺ permeable. Increasing serosal K⁺ from 2.5 to 80mM, depolarizedV _a . Mucosal IBMX significantly reduced this depolarization. Addition of 10mM Ba²⁺, a K⁺ channel blocker, to the serosal medium depolarizedV _a and, essentially, blocked the depolarization induced by IBMX. These results indicate that mucosal IBMX increases apical HCO ₃ ^– conductance and decreases basolateral K⁺ conductance in gallbladder epithelial cells via a cAMP-dependent mechanism. The latter effect, not previously reported in epithelial tissues, appears to be the major determinant of the IBMX-induced depolarization ofV _a .     

Structure ofNecturus gallbladder epithelium during transport at low external osmolarities

Summary Gallbladders transport isotonically over a wide range of osmolarities. This ability has been assumed to depend on the geometry of the lateral intercellular spaces. We report that this geometry in theNecturus gallbladder varies extensively with the external osmolarity and dependsin vitro on the integrity of the subepithelial tissues. The structure of the living epithelium was studied by Nomarski light microscopy while ultrastructural effects were revealed by electron microscopy. The short-term effects (<60 min) of low external osmolarities were: 1) the cells became bell-shaped with an increased cell height measured centrally, 2) lateral intercellular spaces lost their convoluted character; and 3) numerous membrane-bound cavities appeared in the cells. Furthermore, long-term exposure to the low external osmolarities caused an uneven density of epithelial cells. With subepithelial tissues intact, blistering of the epithelium cell layer was evident. Qualitative electron-microscopic data indicate that the membrane of the cavities was recruited from the basolateral cell membrane. This agrees well with light-microscopic observation that the cavities were initiated as invaginations of this cell membrane.     

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.     

A mechanism for isotonic fluid flow through the tight junctions of Necturus gallbladder epithelium

During isotonic fluid flow, Necturus gallbladder epithelium mediates net fluxes of paracellular probes by a convective process. We show here that the paracellular system is modeled by permeation through three populations of channels: (i) convective parallel-sided ones of width 7.7 nm (ii) small diffusive ones of radius 0.6 nm, and (ii) large diffusive ones of radius exceeding 50 nm. The reflexion coefficient of the convective channels is very low and the calculated osmotic flow rate is close to zero when compared with the observed fluid absorptive rate of 2 x 10^–6 cm/sec. Analysis reveals that the convective channels behave as though closed to back-diffusion of probes; if this is due to solvent drag then very high fluid velocities are required, acting through minute areas. There are no transjunctional gradients that could drive the flow, and so the fluid must be propelled through the channel by components of the junction.We propose a mechanism based upon an active junctional peristalsis which allows discrimination on the basis of molecular size, in which the channels are always occluded at some point and so back-diffusion cannot occur. There is no local gradient of salt distal to the junctions and therefore the osmotic permeability of the membranes is irrelevant. High fluid velocities are not required, and the flow can occur over a substantial fraction of the junction. The mechanism must involve motile and contractile elements associated with the junction for which there is already considerable evidence.Symbols A _i filtration area of channel i;i=b (big), s (small) and c (convectional) - B constant for streamline flow - C _i concentration of probe at i - D diffusion coefficient - D ^o diffusion coefficient in free solution - d width of junction - F _i diffusive drag factor in channel i - g ionic conductivity - G _i convective drag factor in channel i - J _ij probe flux from i to j - J _net net probe flux - J _v volume flow per cm² of epithelium - l linear extent of junction per cm² epithelial plane - L length of junctional channel - L _p hydraulic conductivity - N Avogadro's number - q available filtration area fraction of channel - r _s probe molecular radius - r _c channel radius or half-width - S _i steric factor in channel i - V _w,s partial molar volume of water or salt - v _i fluid velocity in channel i - _w dynamic viscosity of water - specific conductivity - ratio of solute radius to channel radius or half-width - diffusive/pressure-driven flow ratio - reflexion coefficient     

Partitioning of paracellular conductance along the ileal crypt-villus axis: A hypothesis based on structural analysis with detailed consideration of tight junction structure-function relationships

Summary Current models of intestinal transport suggest cells which absorb ions are located on the villus while secretory cells are located in the crypt and putatively have paracellular pathways which are highly conductive to Na⁺. One approach to assess possible variation in small intestinal paracellular conductance along the crypt-villus axis is to morphometrically analyze the structural aspects of crypt and villus tight junctions (TJs) which relate to paracellular resistance. Such detailed analysis of junctional structure in this heterogeneous epithelium would permit one to compare intestinal TJ structure-function relationships with those in a structurally simpler epithelium such as that of toad urinary bladder. This comparison would also be of considerable interest since previous similar comparisons have failed to consider in detail the geometric dissimilarity between these two epithelia. We applied light, electron microscopic, and freezefracture morphometric techniques to guinea pig ileal mucosa to quantitatively assess, for both crypts and villi, linear TJ density, relative surface contributions, and TJ strand counts. Mean linear TJ densities were 76.8 m/cm² for crypt cells and 21.8 m/cm² for villus absorptive cells. Mean TJ strand counts were 4.45 for undifferentiated crypt cell TJs and 6.03 for villus absorptive cell TJs. The villus constituted 87% and the crypt 13% of total surface. We utilized these data to predict paracellular conductance of cryptsvs. villi based on equations derived from those of Claude (P. Claude,J. Membrane Biol. 39:219–232, 1978). Such analysis predicts that 73% of ileal paracellular conductance is attributable to the crypt. Furthermore, we obtained literature values for paracellular resistance in mammalian ileum and toad urinary bladder and for toad bladder TJ structure and linear density and constructed a relationship which would allow us to more accurately compare TJ structure-function correlates between these two epithelia. Such a comparison, which considers both surface amplification and TJ structure and distribution in these epithelia, shows that one would predictin vitro measured values for paracellular resistance should be approximately two orders of magnitude less in mammalian ileum than in toad urinary bladder. This predicted discrepancy (115-fold) correlates well with the observed difference (100-fold). These findings suggest that highly similar TJ structure-function relationships apply to these geometrically dissimilar tissues and that, in mammalian ileum, the crypt compartment may be responsible for the majority of net ileal paracellular conductance. We speculate that high crypt linear TJ density and low crypt TJ strand counts may serve as the structural basis of massive paracellular Na⁺ movement which is coupled to active Cl^– secretion and appears to originate from the crypt following exposure to intestinal secretagogues.     

The effect of electrical gradients on current fluctuations and impedance recorded fromNecturus gallbladder

Summary Transepithelial current fluctuations were recorded inNecturus gallbladder, clamped at negative as well as positive potentials up to 64 mV. With NaCl-Ringer's (+10mm TAP) on both sides a mucosa-negative potential enhanced the relaxation noise component, present at zero potential, and produced peaking in the power spectrum at potentials above –36mV. Concomitantly at these potentials an inductive as well as a capacitive low-frequency feature appeared in the impedance locus. Clamping at positive potentials of 18 mV suppressed the relaxation noise component. At potentials above 51mV the spectral values increased predominantly at low frequencies. In this case the power spectrum showed only a 1/f noise component. The experiments confirm the previous finding that a K⁺ efflux through fluctuating apical K⁺ channels exists under normal conditions. With serosal KCl-Ringer's the initial Lorentzian component was enhanced at negative but suppressed at positive potentials. The increase at negative potentials was less pronounced than in experiments with NaCl-Ringer's on both sides, indicating saturation of the fluctuating K⁺ current component. With mucosal KCl-Ringer's a negative potential depressed the initial relaxation noise component, whereas it was enhanced at +18 mV clamp potential. In the latter case an additional Lorentzian component became apparent at higher frequencies. At potentials of 36 mV and above the low-frequency Lorentzian disappeared whereas the corner frequency of the high-frequency component increased. The latter experiments demonstrate that the relaxation noise component inNecturus gallbladder consists of two superimposed Lorentzians. As the relaxation times of these two components behave differently under an electrical field, there may exist two different types of K⁺ channels. It is demonstrated that peaking in the plateau of power spectra can be explained by frequency-dependent attenuation effects, caused by a polarization impedance.     

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.     

Epithelial cell volume modulation and regulation

Summary Epithelial cell volume is a sensitive indicator of the balance between solute entry into the cell and solute exit. Solute accumulation in the cell leads to cell swelling because the water permeability of the cell membranes is high. Similarly, solute depletion leads to cell shrinkage. The rate of volume change under a variety of experimental conditions may be utilized to study the rate and direction of solute transport by an epithelial cell. The pathways of water movement across an epithelium may also be deduced from the changes in cellular volume. A technique for the measurement of the volume of living epithelial cells is described, and a number of experiments are discussed in which cell volume determination provided significant new information about the dynamic behavior of epithelia. The mechanism of volume regulation of epithelial cells exposed to anisotonic bathing solution is discussed and shown to involve the transient stimulation of normally dormant ion exchangers in the cell membrane.     

Hydrostatic pressure regulates tight junctions, actin cytoskeleton and transcellular ion transport

In the epithelia and endothelia, tight junctions regulate the movement of several substances through the paracellular pathway, maintaining several gradients between apical and basal compartments including osmolality and hydrostatic pressure. In this study, we show that the change of hydrostatic pressure gradient affected tight junctions as well as actin cytoskeleton, cell height and transcellular ion transport. Hydrostatic pressure gradient from basolateral to apical side increased transepithelial conductance and altered claudin-1 localization within several tens of minutes. These changes were promptly restored by the elimination of hydrostatic pressure gradient. Hydrostatic pressure gradient also induced dynamic changes in the actin structure and cell height. We further found that hydrostatic pressure gradient from basolateral to apical side stimulates transcellular Cl⁻ transport. Our present findings indicate that the epithelial cell structures and functions are regulated by the hydrostatic pressure gradient which is generated and maintained by the epithelia themselves.     

A blood-brain barrier without tight junctions in the fly central nervous system in the early postembryonic stage

Summary The anatomical basis of the vertebrate blood-brain barrier is a series of tight junctions between endothelial cells of capillaries in the central nervous system. Over two decades ago, tight junctions were also proposed as the basis of the blood-brain barrier in insects. Currently there is a growing understanding that septate junctions might possess barrier properties in various invertebrate epithelial cells. We now examine these two views by studying the blood-brain barrier properties of the early postembryonic larva of a dipteran fly (Delia platura) by transmission electron microscopy. Newly hatched larvae possess a functioning blood-brain barrier that excludes the extracellular tracer, ionic lanthanum. This barrier is intact throughout the second instar stage as well. The ultrastructural correlate of this barrier is a series of extensive septate junctions that pervade the intercellular space between adjacent perineurial cells. No tight junctions were located in either nerve, glial or perineurial cell layers. We suggest that the overall barrier might involve septate junctions within extensive, meandering intercellular clefts.     

Hymenolepis diminuta: The effects of infection on transepithelial ion transport and tight junctions in rat intestines

In this study, we examine the effect of Hymenolepis diminuta on ion transport in the ileum and on tight junctions in the ileum and colon of rats. We also evaluate the effect of H. diminuta on C-fiber endings in the ileum, the direct habitat of H. diminuta, before and after mechanical stimulation and pharmacological modification by capsaicin (C-fiber irritant).Wistar rats were orally infected with five cysticercoids of H. diminuta. Using a modified Ussing chamber, electrophysiological parameters of the ileum were measured (transepithelial electrical potential difference and transepithelial electrical resistance) as well as the deposition of occludin (a tight junction protein) in the ileum and colon of the rats 8, 16, 25, 35, 40 and 60 days post infection.We observed a significant reduction in transepithelial electrical potential difference in the ileum of rats infected with H. diminuta. In both the ileum and colon of rats infected with H. diminuta we also observed a decrease in occludin deposition, which indicates leakage of tight junctions, correlating with the decrease in transepithelial electrical resistance of these tissues. The application of capsaicin confirmed the hypothesis that H. diminuta in rats affects the C-fiber sensory receptors, causing changes in ion transport in the ileum.The results of the performed electrophysiological and immunohistochemical examinations indicate hymenolepidosis-related changes in the active transport of ions and the passive movement of ions.     

博落回提取物替代抗生素对肉鸡生长性能、盲肠微生物及盲肠紧密连接的影响

【目的】本研究旨在探讨博落回提取物(Macleayacordataextract,MCE)替代促生长抗生素(Antibiotic growth promoters,AGPs)对黄羽肉鸡生长性能、盲肠微生物及紧密连接mRNA表达的影响。【方法】试验选取体重相近、体型均匀、健康状况良好的1日龄温氏新黄鸡二号公鸡300只,随机分为5组,每组6个重复,每个重复10羽。分别饲喂基础日粮(NC)、抗生素日粮(ANT,基础日粮添加50 mg/kg那西肽和50 mg/kg金霉素)和试验日粮(基础日粮中添加200、400、800 mg/kg MCE),试验期60 d。【结果】日粮添加400 mg/kg MCE替代AGPs显著降低了料肉比(P0.05),并显著增加了盲肠长度(P0.05)。日粮添加MCE显著提高了肉鸡盲肠食糜中Firmicutes细菌数量和ClostridiumclusterXIVa数量(P0.05);MCE替代AGPs显著降低了盲肠Escherichia coli数量(P0.05)。400 mg/kg和800 mg/kg MCE替代日粮中AGPs显著增加了肉鸡盲肠食糜中总短链脂肪酸、乙酸和丁酸含量(P0.05);400 mg/kg MCE替代AGPs显著提高了盲肠中支链脂肪酸异丁酸和异戊酸的浓度(P0.05)。日粮添加MCE显著上调了肉鸡盲肠Claudin-1、JAM2、ZO-1的mRNA表达量(P0.05),并降低了黏蛋白MUC2、MUC5ac和MUC13的表达量(P0.05)。【结论】MCE替代AGPs可通过提高盲肠有益菌数量和短链脂肪酸浓度,促进肠道发育,提升肠道屏障功能等途径,改善黄羽肉鸡生长性能,本研究中其最适添加量为400 mg/kg。     

The contribution of tonoplast and plasma membrane to the electrical properties of a higher-plant cell

Translocation of ¹⁴C-labelled assimilates down the petioles was studied in intact plants of Pelargonium zonale (L.) L'Hérit ex Ait. The central bundle of the petiole was dissected out and treated with solutions of various inhibitors. Whereas cytochalasin B had no effect on ¹⁴C-translocation, a distinct and localized inhibition was caused by CCCP (10^-7 M), antimycin (5×10^-5 M), atractylate (5×10^-5 M), and valinomycin (10^-5 M) without any significant change in the proportion of [¹⁴C]sucrose in the translocate. The inhibition of translocation is inferred both by accumulation of ¹⁴C distal to and a decrease in ¹⁴C concentration basal to the treated petiolar region. If valinomycin was fed into the transpiration stream by flapping the peripheral bundles of the petioles an increased labeling of sugar phosphates occurred in the ¹⁴C fed leaf. Plasmolysis tests indicated that whereas CCCP interfered with the semipermeability of phloem cell membranes, valinomycin had no such effect. The results with valinomycin suggest a compartmentation of potassium ions for the translocation process but are ambiguous as to whether or not a potassium pump is involved.Dedicated to Wilhelm Halbsguth, Kiel to his 65th birthday     

Mutated C-terminal fragments of Clostridium perfringens enterotoxin have increased affinity to claudin-4 and reversibly modulate tight junctions in vitro

Passage across epithelial cell sheets is the first step in drug absorption. Tight junctions (TJs) are located between adjacent epithelial cells and seal the intercellular space preventing leakage of solutes. Claudin, a tetra-transmembrane protein family, is a pivotal functional and structural component of the TJ barrier. Modulation of the claudin-based TJ seal is a strategy for mucosal drug absorption. We previously found that a claudin-4 binder, a C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE194), was a modulator of the TJ seal and a potent mucosal absorption enhancer. In the present study, we attempted to improve claudin-4 binders by modification of C-CPE194. Substitution of Asn at position 309 and Ser at position 313 with Ala increased the affinity to claudin-4 by 9.9-fold as compared to C-CPE194. Deletion of 10 amino acids in the N-terminal domain of the double-alanine-substituted mutant increased affinity to claudin-4 by 23.9-fold as compared to C-CPE194. These C-CPE194 mutants reversibly modulated the TJ seal in human intestinal epithelial cell sheets. The N-terminal-truncated mutant was the most potent modulator of the TJ seal. These findings indicate that the C-CPE mutant may be a promising lead for the development of a clinical TJ modulator.     

Effects of pH and light on the membrane conductance measured in the acid and basic zones ofChara

Summary The area-specific coductance of the membrane in the acid and basic zones (denoted byG _A andG _B , respectively) ofChara cells was measured in flowing solutions, containing 5mm zwitterionic buffer, as a function of the external pH(denoted by pH⁰). During illuminationG _A was 1 S/m² for pH⁰ in the range 5 to 8.5, and increased markedly to 3 to 6 S/m² at higher pH₀.G _B , however, was always larger thanG _A during illumination with a typical magnitude of 5 to 15 S/m² for pH⁰ 6 to 12. Thus under many experimental conditions it is possible that there is no single correct value for the membrane area-specific conductance. A flow of current in the external medium between the acid and basic regions was found to be associated with pH banding, and also withG _B exceedingG _A . This current could be present in flowing solutions without added HCO ₃ ^– over a wide range of pH⁰ and at high (25mm) buffer concentration. Combining measurements ofG _A andG _B with measurements of the currents in the acid and basic zones (denoted byJ _A andJ _B , respectively), it was estimated that the resting (i.e. in the absence of net current flow) potential difference (PD) across the membranes within the individual zones (denoted byU _A andU _B ) was –265±20 and –183±5 mV, respectively, during illumination. Upon the removal of illumination at pH⁰-7.5,G _A ,G _B andJ _B were found to decrease rapidly during the initial few hundred seconds. During this period (U _B –V _m ) remained relatively constant. A transient hyperpolarization ofV _m often occurred, the magnitude of which was correlated with the magnitude ofJ _B prior to the removal of illumination. After some 0.5 to 1 ksec of darkness,G _A andG _B had both decreased considerably and nowG _A G _B andU _A U _B V _m . Eventually, after 2 to 8 ksec of darkness, the membrane conductance was effectively homogeneous with a much smaller magnitude (typically<0.2S/m²) andV _m was depolarized by typically 5 to 15 mV.     

Co-translational effects of temperature on membrane insertion and orientation of P-glycoprotein sequences

P-glycoprotein (pgp) is a membrane transport protein that causes multidrug resistance (MDR) by actively extruding a wide variety of cytotoxic agents out of cells. It may also function as a peptide transporter, a volume-regulated chloride channel, and an ATP channel. Previously, it has been shown that hamster pgp1 Pgp is expressed in more than one topological form and that the generation of these structures is modulated by charged amino acids flanking the predicted transmembrane (TM) segments 3 and 4 and by soluble cytoplasmic factors. Different topological structures of Pgp may be related to its different functions. In this study, we examined the effects of translation temperature on the membrane insertion process and the topologies of Pgp. Using the rabbit reticulocyte lysate expression system, we showed that translation at different temperatures affects the membrane insertion and orientation of the putative TM3 and TM4 of hamster pgp1 Pgp in a co-translational manner. This observation suggests that the membrane insertion process of TM3 and TM4 of Pgp molecules may involve a protein conducting channel and/or the interaction between TM3 and TM4, which act in a temperature sensitive manner. We speculate that manipulating temperature may provide a way to understand the structure-function relationship of Pgp and help overcome Pgp-related multidrug resistance of cancer cells.Abbreviations Pgp P-glycoprotein - MDR multidrug resistance - ABC ATP-binding cassette - RRL rabbit reticulocyte lysate - TM transmembrane - RM rough microsomes - ER endoplasmic reticulum     

The effect of pH on the heat production and membrane resistance of Streptococcus bovis

Non-growing cultures of Streptococcus bovis JB1 which were incubated in 2-[N-moropholino] ethane-sulfonic acid (MES)-phosphate buffer (pH 6.8) and glucose (2 g/l) produced heat at a rate of 0.17 mW/mg protein, and this rate was proportional to the enthalpy change of the homolactic fermentation. Since the growth-independent heat production could be eliminated by dicyclohexylcarbodiimide (DCCD), an inhibitor of F₁F₀ ATPases, it appeared that virtually all of the energy was being used to counteract proton flux through the cell membrane. When the pH was decreased from 6.8 to 5.8, heat production and glucose consumption increased, the electrical potential () declined, the chemical gradient of protons (ZpH) increased, and there was a small increase in total protonmotive force (p). Further decreases in pH (5.8 to 4.5) caused a marked decrease in heat production and glucose consumption even though there was only a small decline in membrane voltage. Based on the enthalpy of ATP (4 kcal or 16.8 kJ/mol), it appeared that 38% of the wattage was passing through the cell membrane. The relationship between membrane voltage and membrane wattage or glucose consumption was non-linear (non-ohmic), and it appeared that the resistance of the membrane to current flow was not constant. Based on the electrical formula, resistance = voltage²/wattage and resistance = voltage/amperage, there was a marked increase in membrane resistance when the pH was less than 6.0. The increase in membrane resistance at low pH allowed S. bovis to maintain its membrane potential and expend less energy when its ability to ferment glucose was impaired.Abbreviations DCCD dicyclohexylcarbodiimide - MES 2-[N-moropholino] ethanesulfonic acid     

The coxsackie- and adenovirus receptor (CAR) is an in vivo marker for epithelial tight junctions, with a potential role in regulating permeability and tissue homeostasis

The coxsackie- and adenovirus receptor (CAR) is a transmembrane protein belonging to the immunoglobulin superfamily. The function of CAR as a virus receptor has been extensively analyzed, while its physiological role and expression pattern in adult tissues have remained less clear. CAR associates with epithelial tight junctions in vitro and mediates cell-cell adhesion. Using a set of affinity-purified antibodies, we show that CAR is predominantly expressed in epithelial cells lining the body cavities in adult mice, where it specifically co-localizes with the tight junction components ZO-1 and occludin. Notably, CAR could not be detected in endothelial cells of the vasculature, including brain capillaries. CAR expression correlated positively with the maturity of tight junctions and inversely with permeability. With a few exceptions, the two known CAR isoforms were co-expressed in most epithelial cells analyzed. A CAR mutant lacking the intracellular tail over-expressed in transgenic mice was diffusely localized over the plasma membrane, showing the importance of this domain for correct subcellular localization in vivo. We conclude that CAR is localized to epithelial tight junctions in vivo where it may play a role in the regulation of epithelial permeability and tissue homeostasis.