Plasma Membrane Water Permeability of Cultured Cells and Epithelia Measured by Light Microscopy with Spatial Filtering

A method was developed to measure the osmotic water permeability (P_f) of plasma membranes in cell layers and applied to cells and epithelia expressing molecular water channels. It was found that the integrated intensity of monochromatic light in a phase contrast or dark field microscope was dependent on relative cell volume. For cells of different size and shape (Sf9, MDCK, CHO, A549, tracheal epithelia, BHK), increased cell volume was associated with decreased signal intensity; generally the signal decreased 10–20% for a twofold increase in cell volume. A theory relating signal intensity to relative cell volume was developed based on spatial filtering and changes in optical path length associated with cell volume changes. Theory predictions were confirmed by signal measurements of cell layers bathed in solutions of various osmolarities and refractive indices. The excellent signal-to-noise ratio of the transmitted light detection permitted measurement of cell volume changes of <1%. The method was applied to characterize transfected cells and tissues that natively express water channels. P_f in control Chinese hamster ovary cells was low (0.0012 cm/s at 23°C) and increased more than fourfold upon stable transfection with aquaporins 1, 2, 4, or 5. P_f in apical and basolateral membranes in polarized epithelial cells grown on porous supports was measured. P_f ^bl and P_f ^ap were 0.0011 and 0.0024 cm/s (MDCK cells), and 0.0039 and 0.0052 cm/s (human tracheal cells) at 23°C. In intact toad urinary bladder, basolateral P_f was 0.036 cm/s and apical membrane P_f after vasopressin stimulation was 0.025 cm/s at 23°C. The results establish light microscopy with spatial filtering as a technically simple and quantitative method to measure water permeability in cell layers and provide the first measurement of the apical and basolateral membrane permeabilities of several important epithelial cell types.     

Ionic Permeability of the Inhibitory Postsynaptic Membrane of Lobster Muscle Fibers

Reversal potentials (E _IPSP) of the inhibitory postsynaptic potential and the membrane resting potentials (E_M) of lobster muscle fibers were determined with intracellular recording under a variety of ionic conditions. E _IPSP is solely dependent on the electromotive force of anionic batteries; i.e., on the electrochemical gradient for a "mobile" fraction of intracellular Cl (Cl_i) which is considerably smaller than the total intracellular Cl. The active inhibitory membrane is more permeable to certain "foreign" anions in the order NO₃ > SCN > Br > Cl. The membrane is impermeable to BrO_s, isethionate, and methylsulfate, but is slightly permeable to acetate and propionate. The level of Cl_i appears to be determined in part by some active (pump?) process and most of the anions studied appear to interfere with the steady-state level of Cl_i.     

Ionic Permeability and Electrical Potential Differences in Necturus Kidney Cells

The cellular concentrations of Na, K, and Cl have been measured in kidney slices of the amphibian, Necturus maculosus. Permeability coefficients have been determined for Na, K, Cl, Rb, Cs, and choline, from studies both of the uptake of radioactive isotopes and the rate of cell swelling in anisotonic solutions. The results of both methods were found to agree well. Measurements were also made of electrical potential differences across the peritubular face of the kidney cells using bathing solutions in which the electrolyte composition and concentrations could be varied. The data obtained are consistent with a model cell in which the potential difference arises as a result of differences in Na permeability relative to K on the two faces of the cell. The intracellular Na concentration is considered to be regulated by a Na-K coupled pump located at the peritubular face of the cell.     

The Ionic Permeability Changes during Acetylcholine-Induced Responses of Aplysia Ganglion Cells

ACh-induced depolarization (D response) in D cells markedly decreases as the external Na⁺ is reduced. However, when Na⁺ is completely replaced with Mg⁺⁺, the D response remains unchanged. When Na⁺ is replaced with Tris(hydroxymethyl)aminomethane, the D response completely disappears, except for a slight decrease in membrane resistance. ACh-induced hyperpolarization (H response) in H cells is markedly depressed as the external Cl^- is reduced. Frequently, the reversal of the H response; i.e., depolarization, is observed during perfusion with Cl^--free media. In cells which show both D and H responses superimposed, it was possible to separate these responses from each other by perfusing the cells with either Na⁺-free or Cl^--free Ringer's solution. High [K⁺]₀ often caused a marked hyperpolarization in either D or H cells. This is due to the primary effect of high [K⁺]₀ on the presynaptic inhibitory fibers. The removal of this inhibitory afferent interference by applying Nembutal readily disclosed the predicted K⁺ depolarization. In perfusates containing normal [Na⁺]₀, the effects of Ca⁺⁺ and Mg⁺⁺ on the activities of postsynaptic membrane were minimal, supporting the current theory that the effects of these ions on the synaptic transmission are mainly presynaptic. The possible mechanism of the hyperpolarization produced by simultaneous perfusion with both high [K⁺]₀ and ACh in certain H cells is explained quantitatively under the assumption that ACh induces exclusively an increase in Cl^- permeability of the H membrane.     

Studies on the Ionic Permeability of Muscle Cells and their Models

We studied the effect an alkali-metal ion exercised on the rate of entry of another alkali-metal ion into frog sartorius muscle cells and their models (i.e., ion exchange resin and sheep's wool). In the case of frog muscle, it was shown that the interaction fell into one of four categories; competition, facilitation, and two types of indifference. The observed pK value (4.6 to 4.7) of the surface anionic groups that combine with the alkali-metal ions suggests that they are beta- or gamma-carboxyl groups of proteins on the cell surface. The results were compared with four theoretical models which included three membrane models (continuous lipoid membrane with carrier; leaky membrane with carrier; membrane with fixed ionic sites) and one bulk-phase model. This comparison led to the conclusion that the only model that is self-consistent and agrees with all of the experimental facts is the one based on the concept that the entire living cell represents a proteinaceous fixed-charge system; this model correctly predicts all four types of interaction observed.     

Fluxes across Epithelia

Epithelial membranes are useful and convenient tissues in whichto study fundamental processes of ionic transport. They devotea substantial fraction of their total energy resources to transport,they serve as a convenient model for plasmalemmal transport,and they have interesting properties related to hormonal regulation.The frog's skin, in particular, has been fruitful in generatingconcepts relating to the properties of serial membrane systemsand to the use of isotopic tracers in the study of transportmechanisms.     

Membrane Potentials at Zero Current: The Significance of a Constant Ionic Permeability Ratio

The possibility has been examined that the Goldman-Hodgkin-Katz equation for V(0), the total membrane potential at zero current, can be derived with constant permeability ratios from a thermodynamic treatment. The flux equations have been integrated under zero current conditions subject only to the restriction that the total membrane potential should be independent of internal concentration profiles, which is the requirement for the premeability ratios to be phenomenological constants, independent of solution conditions. No assumptions have been made concerning the electric potential profile. It was found that a constant permeability ratio can only be characteristic of systems satisfying certain relationships between ionic conductances and chemical potentials. From these relationships it was possible to define the permeability ratio in terms of the thermodynamic properties of the membrane quite generally and to identify the permeability ratio as the product of mobility ratio and ratio of partition coefficients. Moreover, the ionic conductance ratio at any point in the membrane has been shown to be expressable explicitly in terms of the permeability ratio and the activities of an external solution which would be in equilibrium with the point under consideration. Lastly, a number of conclusions have been reached regarding the range of applicability of the Goldman-Hodgkin-Katz equation with constant permeability ratios.     

Epithelia and integration in sponges

An epithelium is important for integrity, homeostasis, communication and co-ordination, and its development must have been a fundamental step in the evolution of modern metazoan body plans. Sponges are metazoans that are often said to lack a true epithelium. We assess the properties of epithelia, and review the history of studies on sponge epithelia, focusing on their homology to bilaterian epithelia, their ultrastructure, and on their ability to seal. Electron micrographs show that adherens-type junctions are present in sponges but they can appear much slighter than equivalent junctions in other metazoans. Fine septae are seen in junctions of all sponge groups, but distinct septate junctions are only known from Calcarea. Similarly, all sponges can have collagenous sheets underlying their epithelia, but only homoscleromorphs are established to have a distinct basal lamina. The presence of most, but not all, gene families known to be involved in epithelial development and function also suggests that sponge epithelia function like, and are homologous to, bilaterian epithelia. However, physiological evidence that sponge epithelia regulate their internal environment is so far lacking. Given that up to six differentiated epithelia can be recognized in sponges, distinct physiological roles are expected. Recognition that sponges have epithelia challenges the perception that sponges are only loose associations of cells, and helps to relate the biology and physiology of the body plan of the adult sponge to the biology of other metazoans.     

Comparative Ultrastructure of Arthropod Transporting Epithelia

The general organization of arthropod epithelia is comparedto that of vertebrates. It is suggested that although ciliatedepithelia, stratified epithelia and in some cases continuousmuscle sheaths do not occur in arthropods, they have certainanalogous structures which carry out the same functions. Forexample, the arthropod cuticle is compared to the squamous layerof vertebrate stratified epithelia, and complex arthropod basementmembranes are compared to the muscle and connective tissue sheathsof certain vertebrate epithelia. The cellular organization oftransporting epithelial cells is then discussed, with particularreference to elaboration of plasma membranes, and similaritiesand differences between vertebrates and arthropods, and betweeninsects and crustaceans are pointed out. Specializations peculiarto insect cells are described, including the insertion of mitochondriainto apical membrane microvilli, and the presence along thismembrane of small particles called portasomes believed to beinvolved in active transport. Finally, it is shown that in themidgut of theinsect Manduca sexta, distinct ultrastructuralchanges accompany loss of potassium transport activity duringa larval molt and in the prepupal stage. The ultrastructuralchanges which occur include a proliferation of the basementmembrane and muscle tissue underlying the epithelium, and achange in the morphology of the potassium transporting gobletcells. Possible correlations between ultrastructural changesand loss of transport activity are discussed.     

Ionic Permeability on Isolated Mouse Liver Nuclei: Influence of ATP and Ca2+

Patch-clamp experiments on isolated nuclei revealed the existence of ionic channels on the nuclear envelope, but their exact localization and function are still unknown. Recent studies have demonstrated that ATP and calcium ions play an important role in nucleocytoplasmic protein traffic. ATP is essential to allow big molecules in and out of the nucleus. However, a cytoplasmic rise of calcium ions above 300 nm decreases both ATP-dependent transport and passive diffusion through the nuclear envelope. The use of isolated nuclei placed in a saline solution provides the possibility for testing only the compounds added in the bath or in the recording pipette. In the present study, we show that ATP is responsible for an increase of nuclear ionic permeability on isolated nuclei. This result not only confirms data previously reported in in situ nuclei, but also suggests that ATP is directly involved in the modulation of passive ionic permeability. In these particular experimental conditions, calcium ions decrease the channel current starting from a concentration of 1 μm. The parallelism in the modulation action of ATP and Ca⁺⁺ between nuclear pores and ionic channels present on the nuclear envelope contributes to the support of the idea that an ionic pathway is associated with the pore complex. Received: 5 September 1996/Revised: 13 January 1997     

Passive Intercellular Pathway in Amphibian Epithelia

PHYSIOLOGIC studies of transporting epithelia generally indicate that passive shunts (or “leak” pathways for water and ions) exist in parallel with transport systems. Most notably, Ussing^1–3 defines this pathway as an extracellular channel in amphibian skin and has shown that a hypertonic external bath decreases the transepithelial electrical resistance, whereas a hypertonic internal bath has the opposite effect. Similar results have been obtained with toad urinary bladder⁴, but in virtually all of the epithelia studied by electron microscopy, tight junctions⁵ have been found at the luminal end of intercellular spaces. Apparent fusion of adjacent plasma membranes and the inability of electron-dense tracer molecules to pass through such regions^5–8 suggest that they may be tight seals, preventing extracellular transepithelial flow. It is shown that these junctions are reproducibly altered when electrical resistance is changed by hypertonic solutions.     

Electron Spin Resonance Studies of Ionic Permeability Properties of Thylakoid Membranes of Beta vulgaris and Avicennia germinans

Measurement of intrathylakoid aqueous volumes by electron spin resonance spectroscopy was used to study ionic permeability properties of thylakoid membranes isolated from Beta vulgaris L. and Avicennia germinans L. The thylakoids behaved as perfect osmometers in the presence of sorbitol and betaine. Thylakoids exposed to hypertonic solutions of NaCl and KCl shrank and subsequently swelled, requiring 10 minutes to regain their original volume. The initial influx rate calculated from the kinetics of changes in intrathylakoid volume in response to 450 millimolar gradients of NaCl and KCl was 2.3 × 10⁻¹³ moles per square centimeter per second. These data show that the passive permeability to NaCl and KCl was low.