The basal permeability to water of human red blood cells evaluated by a nuclear magnetic resonance technique

The characteristics of water diffusional permeability (P) of human red blood cells were studied on isolated erythrocytes by a doping nuclear magnetic resonance technique. In order to estimate the basal permeability the maximal inhibition of water diffusion was induced by exposure of red blood cells to p-chloromercuribenzene sulfonate (PCMBS) under various conditions (concentration, duration, temperature). The lowest values of P were around 0.7×10^–3 cm s^–1 at 10°C, 1.2×10^–3 cm s^–1 at 15°C, 1.4×10^–3 cm s^–1 at 20°C, 1.8×10^–3 cm s^–1 at 25°C, 2.1×10^–3 cm s^–1 at 30°C and 3.5×10^–3 cm s^–1 at 37°C. The mean value of the activation energy of water diffusion (E_a,d) was 25 kJ/mol for control and 43.7 kJ/mol for PCMBS-inhibited erythrocytes. The values of P and E_a,d obtained after induction of maximal inhibition of water diffusion by PCMBS can be taken as references for the basal permeability to water of the human red blood cell membrane.     

Transient alterations in cellular permeability in cultured human proximal tubule cells: Implications for transport studies

Summary Primary cultures of human proximal tubule (HPT) cells possess the characteristics of a tight epithelium and retain the characteristics of in vivo renal function. HPT cells from confluent monolayers when grown on collagen-coated polycarbonate inserts in a hormonally defined serum-free medium. However, initial studies of transepithelial transport observed large bidirectional fluxes of the paracellular probe inulin. The present studies were designed to assess the transformation of HPT cell tight junctions to a “leaky” state and subsequent recovery. The apparent transepithelial electrical resistance of HPT cells at confluence was 952.0±70.0 ohms*cm², suggesting a well-developed tight junction-mediated paracellular pathway in this epithelium. However, replacement of the growth media produced an immediate 90% drop in the initial resistance, which was paralleld by an increased flux of inulin and of phenol red. This transient abolition of barrier function spontaneously reestablished over 1–2 h by a process that was dependent on the ionic composition of the added media. Complete recovery of cellular resistance was paralleled by markedly decreased fluxes of inulin and of phenol red. The recovery of cellular barrier function was inhibited by cytochalasin B suggesting an intracellular action, not a physical disruption of the monolayer. These results suggest that the tight junctions in these cells appear to transiently produce a leaky state during removal of the media, but rearrange to a “tight conformation” when incubated in the appropriate media.     

Time dependence of the effect ofp-chloromercuribenzoate on erythrocyte water permeability: A pulsed nuclear magnetic resonance study

Summary Pulsed nuclear magnetic resonance spectroscopy is employed to determine the time dependence of the change in erythrocyte water permeability following exposure top-chloromercuribenzoate (PCMB) orp-chloromercuribenzene sulfonic acid (PCMBS). pH variation was used to examine the environment of the sulfhydryl groups reactive to these drugs. PCMB reacted with at least two sulfhydryl groups which affect water permeability. This was shown by the double exponential character of the change in erythrocyte diffusional permeability with time after PCMB addition. However, only one inhibition rate process could be distinguished following PCMBS exposure, suggesting that one site bound by PCMB is not accessible to PCMBS. This site is postulated to be located in a hydrophobic region of the membrane, whereas the site reached by both drugs is located in the normal anion permeation channel. The effect of pH on the degree of inhibition due to each component and the inhibition rates is explained in terms of its effect on solubility of the reagents in the membrane and variation of the dissociated-to-undissociated ratio of PCMB.     

Catecholamines and angiotensinogen gene expression in kidney proximal tubular cells

To investigate the molecular mechanism(s) of action of catecholamines on the expression of the angiotensinogen (ANG) gene in kidney proximal tubular cells, we used opossum kidney (OK) cells with a fusion gene containing the 5-flanking regulatory sequence of the rat ANG gene fused with a human growth hormone (hGH) gene as a reporter, pOGH (rANG N-1498/+18), permanently integrated into their genomes. The level of expression of the ANG-GH fusion gene was quantified by the amount of immunoreactive-hGH (IR-hGH) secreted into the medium. The addition of norepinephrine (NE), isoproterenol (a ₁/₂-adrenergic receptor (AR) agonist) and iodoclonidine (an ₂-AR agonist) stimulated the expression of the ANG-GH fusion gene in a dose-dependent manner, whereas the addition of epinephrine and phenylephrine (₁-AR agonist) had no effect. The stimulatory effect of NE was blocked by the presence of propranolol (-AR blocker), atenolol (₁-AR blocker), yohimbine (₂-AR blocker), Rp-cAMP (an inhibitor of cAMP-dependent protein kinase AI & AII) and staurosporine (an inhibitor of protein kinase C), but was not blocked by ICI 118, 551 (₂-AR blocker) and prazosin (₁-AR blocker). The addition of a combination of isoproterenol and iodoclonidine or a combination of 8-Bromo-cAMP (8-Br-cAMP) and phorbol 12-myristate (PMA) synergistically stimulated the expression of the ANG-GH fusion gene as compared to the addition of isoproterenol, iodoclonidine, 8-Br-cAMP or PMA alone. Furthermore, the addition of NE, 8-Br-cAMP or PMA stimulated the expression of pOGH (rANG N-806/-779/-53/+18), a fusion gene containing the putative cAMP responsive element (CRE, ANG N-806/-779) upstream of the ANG promoter (ANG N-53/+18) in OK cells, but had no effect on the expression of fusion genes containing the mutant of the CRE. Gel mobility shift assays revealed that the ANG-CRE binds with the DNA-binding domain (bZIP 254-327) of the cAMP-responsive binding protein (CREB). The binding of the labeled ANG-CRE to CREB (bZIP254-327) was displaced by unlabeled ANG-CRE and the CRE of the somatostatin gene but not by the mutants of the ANG-CRE. Finally, NE stimulated the phosphorylation of CREB in OK cells. These studies demonstrate that the molecular mechanism(s) of NE action on the expression of the ANG gene in OK cells may be mediated via both the PKA and PKC signalling pathways and via the phosphorylation of CREB. The phosphorylated CREB then interacts with the CRE in the 5-flanking region of the ANG gene and subsequently stimulates the gene expression.     

Nuclear magnetic resonanceimaging of membrane permeability changes in plants during osmoticstress

The cell water balance of maize (Zea mays L., cv LG 11) andpearl millet (Pennisetum americanum L., cv MH 179) duringosmotic stress was studied non‐invasively using ¹H nuclearmagnetic resonance (NMR) microscopy. Single NMR parameter imagesof (i) the water content (ii) the transverse relaxation time (T₂)and (iii) the apparent diffusion coefficient (D_app)were used to follow the water status of the stem apical region duringosmotic stress. During stress there are hardly any changes in watercontent or T₂ of the stem region of maize. Incontrast, the apical tissue of pearl millet showed a ～ 30% decreaseof T₂ within 48 h of stress, whereasthe water content and D_app did not change. Thesechanges can be explained by an increase of the membrane permeabilityfor water. This conclusion is supported by results from scanningelectron microscopy, relaxation measurements of sugar solutionsand numerical simulations of the relaxation and (apparent) diffusionbehaviour of water in a plant cell.     

The role of sophisticated instruments in biological research: Examples from nuclear magnetic resonance

The development of NMR is described to illustrate the importance of new methodologies to solve biological problems.     

Regulation of water permeability of collecting ducts in mouse kidney during postnatal development

Water permeability of epithelial cells and response to vasopressin was studied on isolated fragments of collecting ducts in the kidney of C57BL/6J mice of three age groups (9, 18, and 60–90 days). The coefficient of osmotic water permeability P _f was evaluated from the rate of cell swelling after medium osmolality was changed from 300 to 200 mOsm/l. The P _f value proved to be significantly lower in mice at the age of 9 days than at the age of 18 days, i.e., after the transition to mixed feeding; although P _f at the age of 18 days does not reach the level of adult animals (58.6 ± 7.7, 94.5 ± 8.8, and 168.4 ± 11.8 μM/s, respectively). The antagonist of vasopressin V₂ receptors desmopressin at 1 nM significantly increased P _f in both 18-day-old and adult mice but induced no changes in 9-day-old animals. The inhibitor of protein kinase C Ro-31-8220 in the concentration of 100 nM inhibited the desmopressin effect on P _f in 18 day-old and adult mice but did not inhibit the effect of the analog of the vasopressin secondary messenger cAMP, N⁶,O²-dibutyryl cyclic monophosphate, on P _f of the plasma membrane in collecting duct cells. Thus, the response of collecting duct cells to vasopressin appears at the end of wining and correlates with the increase in unstimulated osmotic water permeability of the plasma membrane in collecting duct cells. The vasopressin signal transduction via V₂ receptors is proposed to require the activity of protein kinase C and calcium-dependent systems of intercellular mediators apart from the cAMP-mediated mechanism.     

Determination of the kinetics of permeation of dimethyl sulfoxide in isolated corneas

Corneal cryopreservation requires that endothelial cells remain viable and intercellular structure be preserved. High viability levels for cryopreserved endothelial cells have been achieved, but preserving intercellular structure, especially endothelial attachment to Descemet's membrane, has proved difficult. Cell detachment apparently is not caused by ice, suggesting osmotic or chemical mechanisms. Knowledge of the permeation kinetics of cryoprotectants (CPAs) into endothelial cells and stroma is essential for controlling osmotic and chemical activity and achieving adequate tissue permeation prior to cooling. Proton nuclear magnetic resonance (NMR) spectroscopy was used to assess the permeation of dimethyl sulfoxide (DMSO) into isolated rabbit corneas. Corneas with intact epithelia were exposed to isotonic medium or 2.0 mol/L DMSO for 60 min and subsequently transferred to 2.0 or 4.0 mol/L DMSO, respectively, at 22, 0, or −10°C. DMSO concentration in the cornea was measured vs time. The Kedem-Katchalsky model was fitted to the data. Hydraulic permeability (m³/N·s) is 7.1×10⁻¹³+216%-11% at 22°C, 8.2×10⁻¹³+235%−21% at 0°C, and 1.7×10⁻¹⁴+19% −16% at −10°C. The reflection coefficient is 1.0+2%−1% at 22°C and 0°C, and 0.9±5% at −10°C. Solute mobility (cm/s) is 5.9×10⁻⁶+6%–11% at 22°C, 3.1×10⁻⁶+12%−11% at 0°C, and 5.0×10⁻⁸ cm/s+59%−40% at −10°C.     

Osmotic gradient dependence of osmotic water permeability in rabbit proximal convoluted tubule

Summary To assess steady-state transepithelial osmotic water permeability (P _f ), rabbit proximal convoluted tubules were perfused in vitro with the impermeant salt, sodium isethionate at 26°C. Osmotic gradients () were established by varying the bath concentration of the impermeant solute, raffinose. When lumen osmolality was 300 mOsm and bath osmolality was 320, 360 and 400 mOsm, apparentP _f decreased from 0.5 to 0.10 to 0.08 cm/sec, respectively. Similar data were obtained when lumen osmolality was 400 mOsm. Five possible causes of the dependence of apparentP _f were considered experimentally and/or theoretically: (1) external unstirred layer (USL); (2) cytoplasmic USL; (3) change in surface area; (4) saturation of water transport; (5) down-regulation ofP _f . ApparentP _f was inhibited 83% byp-chloromercuribenzene sulfonate (pCMBS) at 20 mOsm, but not at 60 mOsm , suggesting presence of a serial barrier resistance to water transport. Increases in perfusate or bath solution flow rate and viscosity did not alter apparentP _f , ruling out an external USL. A simple cytoplasmic USL, described by a constant USL thickness and solute diffusion coefficient, could not account for the dependence of apparentP _f according to a mathematical model. The activation energy (E _a ) for apparentP _f increased from 7.0 to 12.5 kcal/mol when was increased from 20 to 60 mOsm, not consistent with a simple USL or a change in membrane surface area with transepithelial water flow. These findings are most consistent with a complex cytoplasmic USL, where the average solute diffusion coefficient and/or the area available for osmosis decrease with increasing . These results (1) indicate that trueP _f (at physiologically low ) is very high (>0.5 cm/sec) in the rabbit proximal tubule; (2) provide an explanation for the wide variation inP _f values reported in the literature using different , and (3) suggest the presence of a flow-dependent cytoplasmic barrier to water flow.     

Basement membrane and interstitial proteoglycans produced by MDCK cells correspond to those expressed in the kidney cortex

Multiple proteoglycans (PGs) are present in all basement membranes (BM) and may contribute to their structure and function, but their effects on cell behavior are not well understood. Their postulated functions include: a structural role in maintaining tissue histoarchitecture, or aid in selective filtration processes; sequestration of growth factors; and regulation of cellular differentiation. Furthermore, expression PGs has been found to vary in several disease states. In order to elucidate the role of PGs in the BM, a well-characterized model of polarized epithelium, Madin-Darby canine kidney (MDCK) cells has been utilized. Proteoglycans were prepared from conditioned medium by DEAE anion exchange chromatography. The eluted PGs were treated with heparitinase or chondroitinase ABC (cABC), separately or combined, followed by SDS-PAGE. Western blot analysis, using antibodies specific for various PG core proteins or CS stubs generated by cABC treatment, revealed that both basement membrane and interstitial PGs are secreted by MDCK cells. HSPGs expressed by MDCK cells are perlecan, agrin, and collagen XVIII. Various CSPG core proteins are made by MDCK cells and have been identified as biglycan, bamacan, and versican (PG-M). These PGs are also associated with mammalian kidney tubules in vivo.     

Role of aromatic amino acids in carbohydrate binding of plant lectins: Laser photo chemically induced dynamic nuclear polarization study of hevein domain-containing lectins

Carbohydrate recognition by lectins often involves the side chains of tyrosine, tryptophan, and histidine residues. These moieties are able to produce chemically induced dynamic nuclear polarization (CIDNP) signals after laser irradiation in the presence of a suitable radical pair-generating dye. Elicitation of such a response in proteins implies accessibility of the respective groups to the light-absorbing dye. In principle, this technique is suitable to monitor surface properties of a receptor and the effect of ligand binding if CIDNP-reactive amino acids are affected. The application of this method in glycosciences can provide insights into the protein-carbohydrate interaction process, as illustrated in this initial study. It focuses on a series of N-acetylglucosamine-binding plant lectins of increasing structural complexity (hevein, pseudohevein, Urtica dioica agglutinin and wheat germ agglutinin and its domain B), for which structural NMR- or X-ray crystallographic data permit a decision of the validity of the CIDNP method-derived conclusions. On the other hand, the CIDNP data presented in this study can be used for a rating of our molecular models of hevein, pseudohevein, and domain B obtained by various modeling techniques. Experimentally, the shape and intensity of CIDNP signals are determined in the absence and in the presence of specific glycoligands. When the carbohydrate ligand is bound, CIDNP signals of side chain protons of tyrosine, tryptophan, or histidine residues are altered, for example, they are broadened and of reduced intensity or disappear completely. In the case of UDA, the appearance of a new tryptophan signal upon ligand binding was interpreted as an indication for a conformational change of the corresponding indole ring. Therefore, CIDNP represents a suitable tool to study protein-carbohydrate interactions in solution, complementing methods such as X-ray crystallography, high-resolution multidimensional nuclear magnetic resonance, transferred nuclear Overhauser effect experiments, and molecular modeling. Proteins 28:268–284, 1997 © 1997 Wiley-Liss Inc.     

Electrolytes control flows of water across the apical barrier in toad skin: The hydrosmotic salt effect

Summary The reversible dependence of skin osmotic water permeability (L _PD ) upon the ionic concentration of the outer bathing solution — which we have called hydrosmotic salt effect (HSE) — was studied in the isolated skin of the toadBufo marinus ictericus. The skin osmotic water flow (J _V ) was measured as a function of outer bathing solution osmolality (O _e ).L _PD , calculated as (J _v /) _P=0 (where and P are the osmotic and hydrostatic pressure differences across the skin, respectively) was constant whenO _e was altered with sucrose, a nonelectrolyte. In contrast,L _PD increased continuously in the hypotonic range asO _e was raised from zero (distilled water) with NaCl or KCl. The HSE could also be evoked in the condition of reversed osmotic volume flow, with the outer bathing medium made hypertonic with sucrose.Diffusional¹⁴C-sucrose permeability, measured in theJ _v =0 condition to prevent solvent drag of sucrose in the paracellular pathways, indicate that the hydrosmotic salt effect cannot be explained by assuming a paracellular permeability increase, due to tight junction opening, but might be interpreted as due to changes in the osmotic water permeability of the apical membranes of the most superficial cells of the epithelium.The hydrosmotic salt effect can be elicited in control skins and in vasopressin-stimulated skins, on top of the hormonal response.The time course of the hydrosmotic salt effect is substantially different from that of the hydrosmotic response to vasopressin. Its half-time is 4 to 5 times faster than that of vasopressin action, with individual values as short as 1.5 min.The time courses of the hydrosmotic salt-effect onset and reversibility are exponential, clearly contrasting with the typical sigmoidal shape of vasopressin onset and washout time courses.Based on time course data and on speed of response we postulate that the mechanism underlying the hydrosmotic salt effect is due to modifications of existing water pathways in the apical membrane, rather than to incorporation and removal of water permeability units in this structure.     

Mercury chloride decreases the water permeability of aquaporin-4-reconstituted proteoliposomes

Background information. Mercurials inhibit AQPs (aquaporins), and site‐directed mutagenesis has identified Cys¹⁸⁹ as a site of the mercurial inhibition of AQP1. On the other hand, AQP4 has been considered to be a mercury‐insensitive water channel because it does not have the reactive cysteine residue corresponding to Cys¹⁸⁹ of AQP1. Indeed, the osmotic water permeability (P_f) of AQP4 expressed in various types of cells, including Xenopus oocytes, is not inhibited by HgCl₂. To examine the direct effects of mercurials on AQP4 in a proteoliposome reconstitution system, His‐tagged rAPR4 (rat AQP4) M23 was expressed in Saccharomyces cerevisiae, purified with an Ni²⁺‐nitrilotriacetate affinity column, and reconstituted into liposomes with the dilution method. Results. The water permeability of AQP4 proteoliposomes with or without HgCl₂ was measured with a stopped‐flow apparatus. Surprisingly, the P_f of AQP4 proteoliposomes was significantly decreased by 5 μM HgCl₂ within 30 s, and this effect was completely reversed by 2‐mercaptoethanol. The dose‐ and time‐dependent inhibitory effects of Hg²⁺ suggest that the sensitivity to mercury of AQP4 is different from that of AQP1. Site‐directed mutagenesis of six cysteine residues of AQP4 demonstrated that Cys¹⁷⁸, which is located at loop D facing the intracellular side, is a target responding to Hg²⁺. We confirmed that AQP4 is reconstituted into liposome in a bidirectional orientation. Conclusions. Our results suggest that mercury inhibits the P_f of AQP4 by mechanisms different from those for AQP1 and that AQP4 may be gated by modification of a cysteine residue in cytoplasmic loop D.     

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.     

Sequence-specific assignment of 1H-NMR resonance and determination of the secondary structure of Jingzhaotoxin-I

Jingzhaotoxin-I （JZTX-I） purified from the venom of the spider Chilobrachys jingzhao is a novel neurotoxin preferentially inhibiting cardiac sodium channel inactivation by binding to receptor site 3.The structure of this toxin in aqueous solution was investigated using 2-D ^1H-NMR techniques. The complete sequence-specific assignments of proton resonance in the ^1H-NMR spectra of JZTX-I were obtained by analyzing a series of 2-D spectra, including DQF-COSY, TOCSY and NOESY spectra, in n20 and D20. All the backbone protons except for Gin4 and more than 95% of the side-chain protons were identified by dαN,dαδ, dβN and dNN connectivities in the NOESY spectrum. These studies provide a basis for the furtherdeter mination of the solution conformation of JZTX-I. Furthermore, the secondary structure of JZTX-I was identified from NMR data. It consists mainly of a short triple-stranded antiparallel β-sheet with Trp7-Cys9, Phe20-Lys23 and Leu28-Trp31. The characteristics of the secondary structure of JZTX-I are similar to those of huwentoxin-I （HWTX-I） and hainantoxin-IV （HNTX-IV）, whose structures in solution havepre viously been reported.     

Solution Structure of the HIV-1 Intron Splicing Silencer and Its Interactions with the UP1 Domain of Heterogeneous Nuclear Ribonucleoprotein (hnRNP) A1

Splicing patterns in human immunodeficiency virus type 1 (HIV-1) are maintained through cis regulatory elements that recruit antagonistic host RNA-binding proteins. The activity of the 3′ acceptor site A7 is tightly regulated through a complex network of an intronic splicing silencer (ISS), a bipartite exonic splicing silencer (ESS3a/b), and an exonic splicing enhancer (ESE3). Because HIV-1 splicing depends on protein-RNA interactions, it is important to know the tertiary structures surrounding the splice sites. Herein, we present the NMR solution structure of the phylogenetically conserved ISS stem loop. ISS adopts a stable structure consisting of conserved UG wobble pairs, a folded 2X2 (GU/UA) internal loop, a UU bulge, and a flexible AGUGA apical loop. Calorimetric and biochemical titrations indicate that the UP1 domain of heterogeneous nuclear ribonucleoprotein A1 binds the ISS apical loop site-specifically and with nanomolar affinity. Collectively, this work provides additional insights into how HIV-1 uses a conserved RNA structure to commandeer a host RNA-binding protein.     

Structural characterization of the N-terminal autoregulatory sequence of phenylalanine hydroxylase

Phenylalanine hydroxylase (PAH) is activated by its substrate phenylalanine, and through phosphorylation by cAMP-dependent protein kinase at Ser16 in the N-terminal autoregulatory sequence of the enzyme. The crystal structures of phosphorylated and unphosphorylated forms of the enzyme showed that, in the absence of phenylalanine, in both cases the N-terminal 18 residues including the phosphorylation site contained no interpretable electron density. We used nuclear magnetic resonance (NMR) spectroscopy to characterize this N-terminal region of the molecule in different stages of the regulatory pathway. A number of sharp resonances are observed in PAH with an intact N-terminal region, but no sharp resonances are present in a truncation mutant lacking the N-terminal 29 residues. The N-terminal sequence therefore represents a mobile flexible region of the molecule. The resonances become weaker after the addition of phenylalanine, indicating a loss of mobility. The peptides corresponding to residues 2-20 of PAH have different structural characteristics in the phosphorylated and unphosphorylated forms, with the former showing increased secondary structure. Our results support the model whereby upon phenylalanine binding, the mobile N-terminal 18 residues of PAH associate with the folded core of the molecule; phosphorylation may facilitate this interaction.