Directly Observed Membrane Fusion Between Oppositely Charged Phospholipid Bilayers

A novel method was developed for the direct examination of pairwise encounters between positively and negatively charged phospholipid bilayer vesicles. Giant bilayer vesicles (unilamellar, 4–20 μm in diameter) prepared from 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, a new cationic phospholipid derivative, were electrophoretically maneuvered into contact with individual anionic phospholipid vesicles. Fluorescence video microscopy revealed that such vesicles commonly underwent fusion within milliseconds (1 video field) after contact, without leakage. Fusion occurred at constant volume and, since flaccid vesicles were rare, the excess membrane was not available after fusion. Hemifusion (the outer monolayers of each vesicle fused while the inner monolayers remained intact) was inferred from membrane-bound dye transfer and a change in the contact area. Hemifusion was observed as a final stable state and as an intermediate to fusion of vesicles composed of charged phospholipids plus zwitterionic phospholipids. Hemifusion occurred in one of three ways following adhesion: either delayed with an abrupt increase in area of contact, immediately with a gradual increase in area of contact, or with retraction during which adherent vesicles dissociated from a flat contact to a point contact. Phosphatidylethanolamine strongly promoted immediate hemifusion; the resultant hemifused state was stable and seldom underwent complete fusion. Although sometimes single contacts between vesicles led to rupture of both, in other cases, a single vesicle underwent multiple fusion events. Direct observation has unequivocally demonstrated the fusion of two, isolated bilayer-bounded bodies to yield a stable, non-leaky product, as occurs in cells, in the absence of proteins. Received: 25 November 1998/Revised: 23 March 1999     

Microstructure and dynamic surface properties of surfactant protein SP-B/dipalmitoylphosphatidylcholine interfacial films spread from lipid-protein bilayers

 Suspensions of dipalmitoylphosphatidylcholine (DPPC) bilayers containing 5, 10 or 20% (w/w) surfactant protein SP-B have been reconstituted and spread at air-liquid interfaces. Compression isotherms of DPPC/SP-B monolayers spread from these preparations were qualitatively comparable to the isotherms of the corresponding DPPC/SP-B monolayers spread from solvents. SP-B was squeezed-out at higher pressures from vesicle-spread films than from solvent-spread monolayers. SP-B caused a marked decrease on the rate of relaxation of DPPC collapse phases to equilibrium pressures in all the lipid/protein films assayed. This stabilizing effect was higher in vesicle-spread than in solvent-spread monolayers. Inclusion in the films of traces of the fluorescent probe NBD-PC (1 mol%) and use of a fluorescent derivative of SP-B labeled with a rhodamine derivative, Texas Red, allowed for direct observation of protein and lipid domains at the interface by epifluorescence microscopy. Upon compression, SP-B altered the packing of phospholipids in the bilayer-spread films, observed as a SP-B-induced reduction of the area of liquid-condensed domains, in a way similar to its effect in solvent-spread monolayers. SP-B was not associated with condensed regions of the films. Fluorescence images from vesicle-spread films showed discrete fluorescent aggregates that could be consistent with the existence of lipid-protein vesicles in close association with the monolayer. Both the retention of SP-B at higher surface pressures and the greater stability of collapse phases of DPPC/SP-B films prepared by spreading from liposomes in comparison to those spread from solvents can be interpreted as a consequence of formation of complex bilayer-monolayer interacting systems. Received: 1 December 1999 / Revised version: 2 March 2000 / Accepted: 2 March 2000     

Occludin Localization at the Tight Junction Requires the Second Extracellular Loop

Occludin is a transmembrane protein of the tight junction with two extracellular loops. Our previous demonstration that the extracellular loops are adhesive suggested the possibility that they contribute to localizing occludin at the tight junction. To address this question, truncated forms of occludin were generated in which one or both of the extracellular loops were deleted. These constructs were expressed in both occludin-null Rat-1 fibroblasts and in MDCK epithelial cells. The patterns of sensitivity to proteinase K suggested all constructs were present on the plasma membrane and retained the normal topology. In fibroblasts, all truncated forms of occludin colocalized with ZO-1 at regions of cell-cell contact, demonstrating that even in the absence of tight junctions cytoplasmic interactions with ZOs is sufficient to cluster occludin. In MDCK cell monolayers, both full-length and occludin lacking the first extracellular loop colocalized with ZO-1 at the tight junction. In contrast, constructs lacking the second, or both, extracellular loops were absent from tight junctions and were found only on the basolateral cell surface. By freeze-fracture electron microscopic analysis, overexpression of full length occludin induced side-to-side aggregation of fibrils within the junction, while excess occludin on the lateral membrane did not form fibrils. These results suggest that the second extracellular domain is required for stable assembly of occludin in the tight junction and that occludin influences the structural organization of the paracellular barrier. Received: 26 June 2000/Revised: 25 September 2000     

Properties of Hexadecaprenyl Monophosphate/Dioleoylphosphatidylcholine Vesicular Lipid Bilayers

In our study we investigated hemispherical phospholipid bilayer membranes and phospholipid vesicles made from hexadecaprenyl monophosphate (C₈₀-P), dioleoylphosphatidylocholine (DOPC) and their mixtures by voltammetric and transmission electron microscopy (TEM) techniques. The current-voltage characteristics, the membrane conductance-temperature relationships and the membrane breakdown voltage have been measured for different mixtures of C₈₀-P/DOPC. The membrane hydrophobic thickness and the activation energy of ion migration across the membrane have been determined. Hexadecaprenyl monophosphate decreased in comparison with DOPC bilayers, the membrane conductance, increased the activation energy and the membrane breakdown voltage for the various value of C₈₀-P/DOPC mole ratio, respectively. The TEM micrographs of C₈₀-P, DOPC and C₈₀-P/DOPC lipid vesicles showed several characteristic structures, which have been described. The data indicate that hexadecaprenyl monophosphate modulates the surface curvature of the membranes by the formation of aggregates in liquid-crystalline phospholipid membranes. We suggest that the dynamics and conformation of hexadecaprenyl monophosphate in membranes depend on the transmembrane electrical potential. The electron micrographs indicate that polyprenyl monophosphates with single isoprenyl chains form lipid vesicular bilayers. The thickness of the bilayer, evaluated from the micrographs, was 11 ± 1 nm. This property creates possibility of forming primitive bilayer lipid membranes by long single-chain polyprenyl phosphates in abiotic conditions. It can be the next step in understanding the origin of protocells. Received: 10 January 2000/Revised: 7 June 2000     

Identification of Isoforms of G Proteins and PKC that Colocalize with Tight Junctions

Recent evidence suggests that the formation and permeability of tight junctions are actively regulated by second-messenger-generating systems involving G proteins and protein kinase C (PKC). A possible specific target for these regulatory proteins is the tight junction protein ZO-1. An extensive immunocytochemical study was performed in cultured epithelial monolayers of MDCK and Caco-2 cells to identify which isoforms of G proteins and PKC are present at or near the zonula occludens complex. Antibodies against α-subunits of each one of the four major subfamilies were used for the localization of the G proteins. For the PKC localization, antibodies against eight different isoforms were used. In confluent monolayers, Gα₁₂ and PKC ζ, were the only isoforms of these proteins present at the cell borders. In subconfluent monolayers, Gα₁₂ and PKC ζ were found at the plasma membrane only along the areas of lateral cell-cell contact. These isoforms formed a pattern of distribution very similar to the ZO-1 protein. The present findings indicate that Gα₁₂ and PKC ζ may be part of the zonula occludens complex and may locally regulate formation and permeability of tight junctions. Received: 29 July 1995/Revised: 13 October 1995     

The Comparative Genomics of Polyglutamine Repeats: Extreme Difference in the Codon Organization of Repeat-Encoding Regions Between Mammals and Drosophila

Polyglutamine repeats within proteins are common in eukaryotes and are associated with neurological diseases in humans. Many are encoded by tandem repeats of the codon CAG that are likely to mutate primarily by replication slippage. However, a recent study in the yeast Saccharomyces cerevisiae has indicated that many others are encoded by mixtures of CAG and CAA which are less likely to undergo slippage. Here we attempt to estimate the proportions of polyglutamine repeats encoded by slippage-prone structures in species currently the subject of genome sequencing projects. We find a general excess over random expectation of polyglutamine repeats encoded by tandem repeats of codons. We nevertheless find many repeats encoded by nontandem codon structures. Mammals and Drosophila display extreme opposite patterns. Drosophila contains many proteins with polyglutamine tracts but these are generally encoded by interrupted structures. These structures may have been selected to be resistant to slippage. In contrast, mammals (humans and mice) have a high proportion of proteins in which repeats are encoded by tandem codon structures. In humans, these include most of the triplet expansion disease genes. Received: 17 August 2000 / Accepted: 20 November 2000     

The SU Glycoprotein 120 from HIV-1 Penetrates into Lipid Monolayers Mimicking Plasma Membranes

Increasing evidence suggests that the HIV envelope binds through its surface (SU) gp120 not only to receptors and coreceptors, but also to other components of the cellular membrane where the glycolipids appear to be good candidates. To assess the ability of HIV-1 SU gp120 to penetrate into phospholipid membranes, we carried out a study of the interactions between a recombinant SU gp120 from HIV-1/HXB2 and artificial lipid monolayers mimicking the composition of the outer leaflet of the lymphocytes and which were spread at the air-water interface. We show that the protein, in its aggregated form, has amphipathic properties and that the insertion of this amphipathic species into lipids is favored by the presence of sphingomyelin. Furthermore, cholesterol enhances the penetration into mixed phosphatidylcholine-sphingomyelin monolayers. Coexistence of different physical states of the lipids and thus of domains appears to play a major role for protein penetration independently of the presence of receptors and coreceptors. Received: 24 April 2000/Revised: 11 July 2000     

Distinct Stages of Protein Evolution as Suggested by Protein Sequence Analysis

Evolution of proteins encoded in nucleotide sequences began with the advent of the triplet code. The chronological order of the appearance of amino acids on the evolution scene and the steps in the evolution of the triplet code have been recently reconstructed (Trifonov, 2000b) on the basis of 40 different ranking criteria and hypotheses. According to the consensus chronology, the pair of complementary GGC and GCC codons for the amino acids alanine and glycine appeared first. Other codons appeared as complementary pairs as well, which divided their respective amino acids into two alphabets, encoded by triplets with either central purines or central pyrimidines: G, D, S, E, N, R, K, Q, C, H, Y, and W (Glycine alphabet G) and A, V, P, S, L, T, I, F, and M (Alanine alphabet A). It is speculated that the earliest polypeptide chains were very short, presumably of uniform length, belonging to two alphabet types encoded in the two complementary strands of the earliest mRNA duplexes. After the fusion of the minigenes, a mosaic of the alphabets would form. Traces of the predicted mosaic structure have been, indeed, detected in the protein sequences of complete prokaryotic genomes in the form of weak oscillations with the period 12 residues in the form of alteration of two types of 6 residue long units. The next stage of protein evolution corresponded to the closure of the chains in the loops of the size 25–30 residues (Berezovsky et al., 2000). Autocorrelation analysis of proteins of 23 complete archaebacterial and eubacterial genomes revealed that the preferred distances between valine, alanine, glycine, leucine, and isoleucine along the sequences are in the same range of 25–30 residues, indicating that the loops are primarily closed by hydrophobic interactions between the ends of the loops. The loop closure stage is followed by the formation of typical folds of 100–200 amino acids, via end-to-end fusion of the genes encoding the loop-size chains. This size was apparently dictated by the optimal ring closure for DNA. In both cases the closure into the ring (loop) rendered evolutionarily advantageous stability to the respective structures. Further gene fusions lead to the formation of modern multidomain proteins. Recombinational gene splicing is likely to have appeared after the DNA circularization stage. Received: 21 December 2000 / Accepted: 28 February 2001     

On the Possibility of Constructive Neutral Evolution

The neutral theory often is presented as a theory of ``noise' or silent changes at an isolated ``molecular level,' relevant to marking the steady pace of divergence, but not to the origin of biological structure, function, or complexity. Nevertheless, precisely these issues can be addressed in neutral models, such as those elaborated here with regard to scrambled ciliate genes, gRNA-mediated RNA editing, the transition from self-splicing to spliceosomal splicing, and the retention of duplicate genes. All of these are instances of a more general scheme of ``constructive neutral evolution' that invokes biased variation, epistatic interactions, and excess capacities to account for a complex series of steps giving rise to novel structures or operations. The directional and constructive outcomes of these models are due not to neutral allele fixations per se, but to these other factors. Neutral models of this type may help to clarify the poorly understood role of nonselective factors in evolutionary innovation and directionality. Received: 3 September 1998 / Accepted: 15 February 1999     

Cell pH and H+ Secretion by S3 Segment of Mammalian Kidney: Role of H+-ATPase and Cl−

The role of H⁺-ATPase in proximal tubule cell pH regulation was studied by microperfusion techniques and by confocal microscopy. In a first series of experiments, proximal S3 segments of rabbit kidney were perfused ``in vitro' while their cell pH was measured by fluorescence microscopy after loading with BCECF. In Na⁺- and Cl⁻-free medium, cell pH fell by a mean of 0.37 ± 0.051 pH units, but after a few minutes started to rise again slowly. This rise was of 0.17 ± 0.022 pH units per min, and was significantly reduced by bafilomycin and by the Cl⁻ channel blocker NPPB, but not by DIDS. In a second series of experiments, subcellular vesicles of proximal tubule cells of S3 segments of mouse kidney were studied by confocal microscopy after visualization by acridine orange or by Lucifer yellow. After superfusion with low Na⁺ solution, which is expected to cause cell acidification, vesicles originally disposed in the basolateral and perinuclear cell areas, moved toward the apical area, as detected by changes in fluorescence density measured by the NIH Image program. The variation of apical to basolateral fluorescence ratios during superfusion with NaCl Ringer with time was 0.0018 ± 0.0021 min⁻¹, not significantly different from zero (P > 0.42). For superfusion with Na⁺0 Ringer, this variation was 0.081 ± 0.015 min⁻¹, P < 0.001 against 0. These slopes were markedly reduced by the Cl⁻ channel blocker NPPB, and by vanadate at a concentration that has been shown to disrupt cytoskeleton function. These data show that the delayed alkalinization of proximal tubule cells in Na⁺-free medium is probably due to a vacuolar H⁺-ATPase, whose activity is stimulated in the presence of Cl⁻, and dependent on apical insertion of subcellular vesicles. The movement of these vesicles is also dependent on Cl⁻ and on the integrity of the cytoskeleton. Received: 11 April 2000/Revised: 14 August 2000     

The Barrier Domain for Solute Permeation Varies With Lipid Bilayer Phase Structure

The chemical selectivities of the transport barriers in lipid bilayers varying in composition and phase structure (gel-phase DPPC and DHPC bilayers and liquid-crystalline DPPC/CHOL/50:50 mol% bilayers) have been investigated by determining functional group contributions to transport of a series of α-substituted p-toluic acid analogs obtained in vesicle efflux experiments. Linear free energy relationships are established between the free energies of transfer for this series of compounds from water to the barrier domain and corresponding values for their transfer from water into six model bulk solvents (hexadecane, hexadecene, decadiene, chlorobutane, butyl ether, and octanol) determined in partitioning experiments to compare the barrier microenvironment to that in these model solvents. The barrier microenvironment in all bilayers studied is substantially more hydrophobic than octanol, thus establishing the location of the barrier beyond the hydrated headgroup interfacial region, as the interface is expected to be more hydrophilic than octanol. The chemical nature of the barrier domain microenvironment varies with bilayer phase structure. The barrier regions in non-interdigitated DPPC and interdigitated DHPC gel-phase bilayers exhibit some degree of hydrogen-bond acceptor capacity as may occur if these domains lie in the vicinity of the ester/ether linkages between the headgroups and the acyl chains. Intercalation of 50 mol% cholesterol into DPPC bilayers, which induces a phase transition to a liquid-crystalline phase, substantially increases the apparent barrier domain hydrophobicity relative to gel-phase bilayers to a nonhydrogen bonding, hydrocarbonlike environment resembling hexadecene. This result, combined with similar observations in liquid-crystalline egg-PC bilayers (J. Pharm. Sci. (1994), 83:1511–1518), supports the notion that the transition from the gel-phase to liquid-crystalline phase shifts the barrier domain further into the bilayer interior (i.e., deeper within the ordered chain region). Received: 16 September 1997/Revised: 14 May 1998     

Lateral Microheterogeneity of Diphenylhexatriene-Labeled Choline Phospholipids in the Erythrocyte Ghost Membrane as Determined by Time-Resolved Fluorescence Spectroscopy

Choline phospholipids are the major constituents of the outer layer of the erythrocyte membrane. To investigate their lateral membrane organization we determined the fluorescence lifetime properties of diphenylhexatriene analogues of phosphatidylcholine, choline plasmalogen, (the respective enolether derivative), and sphingomyelin inserted into the outer layer of hemoglobin-free ghosts. Fluorescence lifetimes were recorded by time-resolved phase and modulation fluorometry and analyzed in terms of Continuous Lorentzian distributions. To assess the influence of membrane proteins on the fluorescence lifetime of the labeled lipids in the biomembrane, lipid vesicles were used as controls. In general, the lifetime distributions in the ghost membranes are broad compared to vesicles. Phosphatidylcholine and sphingomyelin exhibit very similar lifetime distributions in contrast to an increased plasmalogen lifetime heterogeneity in both systems. Orientational effects of side chain mobilities on the observed lifetimes can be excluded. Fluorescence anisotropies revealed identical values for all three labeled phospholipids in the biomembrane. Received: 22 July 1999/Revised: 6 January 2000     

A Collagen Substrate Enhances the Sealing Capacity of Tight Junctions of A6 Cell Monolayers

A6 cells, a kidney derived epithelial cell line, when cultured either on a collagen-coated substrate or on polycarbonate substrate without collagen form confluent monolayers that are similar in cell density and overall morphology. However, the transepithelial electrical resistance (TER) of monolayers grown on the collagen-coated substrate is ninefold higher than that of monolayers grown without collagen. A comparative freeze-fracture study showed that this large difference in TER is not related to the length or number of tight junction strands but to differences in the specific conductance of individual strands. This conductance was obtained considering the TER, the linear junctional density and the mean number of tight junction strands. We estimated the specific linear conductance of the tight junction strands to be 2.56 × 10⁻⁷ S/cm for cells grown on collagen and 30.3 × 10⁻⁷ S/cm for the cells grown without collagen. We also examined changes in distribution and phosphorylation states of the zonula occludens associated protein, ZO-1, during monolayer formation. Immunocytochemistry reveals that the distribution of ZO-1 follows a similar time course and pattern independent of the presence or absence of collagen. While the amount of ZO-1 expression is identical in cells grown on both substrates, this protein is phosphorylated to a greater extent during the initial stages of confluence in cells cultured on collagen. We suggest that the phosphorylation levels of ZO-1 in A6 cells at the early stages of monolayer formation may determine the final molecular structure and specific conductance of the tight junctions strands. Received: 18 September 1996/Revised: 17 June 1997     

Selective Pressures on RNA Hairpins In Vivo and In Vitro

Comparison of the most stable potential hairpins in the sequences of natural ribozymes with those in the randomized sequences has revealed that the hairpin loop energies are lower than expected by chance. Although these hairpins are not necessarily parts of functional structures, there is a selective pressure to diminish the destabilizing free energies of the hairpin loops. In contrast, no significant bias is observed in the stacking values of the most stable stems. In the ribozymes isolated in vitro the loops of potential hairpins are closer to random values, which can result in less efficient folding rates. Furthermore, the effects of kinetic traps seem to be more significant in the folding pathways of the in vitro isolates due to a potential to form stable stacks incompatible with the functional folds. Similarly to natural ribozyme sequences, the untranslated regions of viral RNAs also form hairpins with relatively low loop free energies. These evolutionary trends suggest ways for efficient engineering of improved RNA constructs on the basis of analysis of in vitro isolates and approaches for the search of regions coding for functional RNA structures in large genome sequences. Received: 12 January 2001 / Accepted: 21 May 2001     

The Role of Membrane Lateral Tension in Calcium-Induced Membrane Fusion

Calcium-induced fusion of liposomes was studied with a view to understand the role of membrane tension in this process. Lipid mixing due to fusion was monitored by following fluorescence of rhodamine-phosphatidyl-ethanolamine incorporated into liposomal membrane at a self-quenching concentration. The extent of lipid mixing was found to depend on the rate of calcium addition: at slow rates it was significantly lower than when calcium was injected instantly. The vesicle inner volume was then made accessible to external calcium by adding calcium ionophore A23187. No effect on fusion was observed at high rates of calcium addition while at slow rates lipid mixing was eliminated. Fusion of labeled vesicles with a planar phospholipid membrane (BLM) was studied using fluorescence microscopy. Above a threshold concentration specific for each ion, Ca²⁺, Mg²⁺, Cd²⁺ and La³⁺ induce fusion of both charged and neutral membranes. The threshold calcium concentration required for fusion was found to be dependent on the vesicle charge, but not on the BLM charge. Pretreatment of vesicles with ionophore and calcium inhibited vesicle fusion with BLM. This effect was reversible: chelation of calcium prior to the application of vesicle to BLM completely restored their ability to fuse. These results support the hypothesis that tension in the outer monolayer of lipid vesicle is a primary reason for membrane destabilization promoting membrane fusion. How this may be a common mechanism for both purely lipidic and protein-mediated membrane fusion is discussed. Received: 27 September 1999/Revised: 22 March 2000     

Functional Asymmetry of the Sodium-d-Glucose Cotransporter Expressed in Yeast Secretory Vesicles

The sodium-d-glucose cotransporter (SGLT1) was expressed in a yeast mutant strain NY 17 (sec6-4) that accumulates secretory vesicles at a nonpermissive temperature because of a block in the delivery of these vesicles to the plasma membrane. By differential centrifugation a microsomal fraction enriched in secretory vesicles was prepared with a high specific activity of the vanadate-sensitive H⁺-ATPase and invertase. In this membrane fraction one protein band of an apparent molecular weight of 55 kDa representing the nonglycosylated SGLT1 protein could be detected by immunochemical analysis. In addition, higher molecular weight protein bands probably representing dimers and aggregates were found. In transport studies with the microsomes d-glucose fluxes showed asymmetric properties: efflux experiments revealed the typical properties of the SGLT1 such as sodium dependence, inhibition by phlorizin and potential dependence. Influx of d-glucose showed no dependence on sodium and was not inhibited by phlorizin. Furthermore, the transporter exhibited a striking asymmetry with regard to the d-glucose affinity and the sugar specificity. These results suggest that the orientation of the SGLT1 expressed in yeast secretory vesicles is, indeed, inverted with regard to its configuration in the plasma membrane of epithelial cells. Moreover, there are striking functional differences between the periplasmic and cytoplasmic face of the transporter. Received: 16 August 2000/Revised: 24 October 2000     

An Attempt to Measure the Patchwork Pattern Observed Among Alleles at Major Histocompatibility Complex Loci

By means of simulations and DNA sequence analyses, standardized identity excess (a measure of linkage disequilibrium) between segregating nucleotide sites was studied as an effort to quantify the patchwork pattern among alleles of the major histocompatibility complex loci. It was found that the pattern under selective neutrality, and/or no intralocus recombination does not fit the observed pattern based on DNA sequences. However, the intensity and type of selection and the rate of recombination are difficult to estimate by comparing simulation results with the observed pattern. Received: 10 December 1999 / Accepted: 2 March 2000     

Cholesterol in condensed and fluid phosphatidylcholine monolayers studied by epifluorescence microscopy

Epifluorescence microscopy was used to investigate the effect of cholesterol on monolayers of dipalmitoylphosphatidylcholine (DPPC) and 1 -palmitoyl-2-oleoyl phosphatidylcholine (POPC) at 21 +/- 2 degrees C using 1 mol% 1-palmitoyl-2-[12-[(7-nitro-2-1, 3-benzoxadizole-4-yl)amino]dodecanoyl]phosphatidylcholine (NBD-PC) as a fluorophore. Up to 30 mol% cholesterol in DPPC monolayers decreased the amounts of probe-excluded liquid-condensed (LC) phase at all surface pressures (pi), but did not effect the monolayers of POPC, which remained in the liquid-expanded (LE) phase at all pi. At low pi (2-5 mN/m), 10 mol% or more cholesterol in DPPC induced a lateral phase separation into dark probe-excluded and light probe-rich regions. In POPC monolayers, phase separation was observed at low pi when > or =40 mol% or more cholesterol was present. The lateral phase separation observed with increased cholesterol concentrations in these lipid monolayers may be a result of the segregation of cholesterol-rich domains in ordered fluid phases that preferentially exclude the fluorescent probe. With increasing pi, monolayers could be transformed from a heterogeneous dark and light appearance into a homogeneous fluorescent phase, in a manner that was dependent on pi and cholesterol content. The packing density of the acyl chains may be a determinant in the interaction of cholesterol with phosphatidylcholine (PC), because the transformations in monolayer surface texture were observed in phospholipid (PL)/sterol mixtures having similar molecular areas. At high pi (41 mN/m), elongated crystal-like structures were observed in monolayers containing 80-100 mol% cholesterol, and these structures grew in size when the monolayers were compressed after collapse. This observation could be associated with the segregation and crystallization of cholesterol after monolayer collapse.     

Vasopressin Regulates Water Flow in a Rat Cortical Collecting Duct Cell Line Not Containing Known Aquaporins

Transepithelial water movements and arginine-vasopressin (AVP)-associated ones were studied in a renal cell line established from a rat cortical collecting duct (RCCD₁). Transepithelial net water fluxes (J _w ) were recorded every minute in RCCD₁ monolayers cultured on permeable supports. Spontaneous net water secretion was observed, which was inhibited by serosal bumetanide (10⁻⁵ m), apical glibenclamide (10⁻⁴ m) and apical BaCl₂ (5 × 10⁻³ m). RT-PCR, RNAse protection and/or immunoblotting experiments demonstrated that known renal aquaporins (AQP1, AQP2, AQP3, AQP4, AQP6 and AQP7) were not expressed in RCCD₁ cells. AVP stimulates cAMP production and sodium reabsorption in RCCD₁ cells. We have now observed that AVP significantly reduces the spontaneous water secretory flux. The amiloride-sensitive AVP-induced increase in short-circuit current (I _sc ) was paralleled by a simultaneous modification of the observed J _w : both responses had similar time courses and half-times (about 4 min). On the other hand, AVP did not modify the osmotically driven J _w induced by serosal hypertonicity. We can conclude that: (i) transepithelial J _w occurs in RCCD₁ cells in the absence of known renal aquaporins; (ii) the ``water secretory component' observed could be linked to Cl⁻ and K⁺ secretion; (iii) the natriferic response to AVP, preserved in RCCD₁ cells, was associated with a change in net water flux, which was even observed in absence of AQP2, AQP3 or AQP4 and (iv) the hydro-osmotic response to AVP was completely lost. Received: 30 December 1999/Revised: 12 October 2000     

Inositol 1,4,5-Trisphosphate But Not Ryanodine-Receptor Agonists Induces Calcium Release from Rat Liver Golgi Apparatus Membrane Vesicles

We investigated the direct effect of inositol 1,4,5-trisphosphate (IP₃) and ryanodine receptor agonists on Ca²⁺ release from vesicles of a rat liver Golgi apparatus (GA) enriched fraction, which were actively loaded with ⁴⁵Ca²⁺. Results in GA were compared with those obtained in a rat liver endoplasmic reticulum (ER) enriched fraction. The addition of IP₃ at concentrations ranging from 100 nm to 100 μm, in the presence of thapsigargin, a specific inhibitor of sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPases, promoted a rapid decrease in the Ca²⁺ content of GA vesicles. The amount of Ca²⁺ released from the vesicles was a function of IP₃ concentration, reaching about 60% in both GA and ER fractions at 100 μm IP₃. Calcium release was inhibited by heparin, an antagonist of IP₃ receptors. Calcium exhibited a bell-shaped effect on IP₃-dependent Ca²⁺ released from GA vesicles: it activated Ca²⁺ release at concentrations up to 1 μm, and inhibited it at higher concentrations. In contrast to that found in the endoplasmic reticulum fraction, none of the ryanodine receptor agonists tested (cyclic ADP-ribose, caffeine and ryanodine) significantly induced Ca²⁺ release from GA fraction vesicles in the presence of thapsigargin. Our results indicate the presence of an IP₃-sensitive Ca²⁺ release mechanism in the Golgi apparatus membrane analogous to that of the ER. However, a Ca²⁺ release mechanism sensitive to ryanodine receptor agonists like that of ER is not evident in the GA membrane. Received: 13 March 2000/Revised: 13 July 2000